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WO2025054556A1 - Rna compositions for delivery of mpox antigens and related methods - Google Patents

Rna compositions for delivery of mpox antigens and related methods Download PDF

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Publication number
WO2025054556A1
WO2025054556A1 PCT/US2024/045726 US2024045726W WO2025054556A1 WO 2025054556 A1 WO2025054556 A1 WO 2025054556A1 US 2024045726 W US2024045726 W US 2024045726W WO 2025054556 A1 WO2025054556 A1 WO 2025054556A1
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WIPO (PCT)
Prior art keywords
mpox
antigen
infection
polyribonucleotides
polyribonucleotide
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PCT/US2024/045726
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French (fr)
Inventor
Alexandra WALLS
Katja Margit SCHLATTERER
Anja DOKIC
Ugur Sahin
Huitian DIAO
Gavin PALOWITCH
Adam ZUIANI
Asaf PORAN
Charles Lefco DULBERGER
John SROUJI
Daniel Abram Rothenberg
Nilushi S. DE SILVA
Ricardo SANCHEZ VELAZQUEZ
Lynda Maria STUART
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Biontech SE
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Biontech SE
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Publication of WO2025054556A1 publication Critical patent/WO2025054556A1/en
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/53—DNA (RNA) vaccination
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/70—Multivalent vaccine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/24011—Poxviridae
    • C12N2710/24111—Orthopoxvirus, e.g. vaccinia virus, variola
    • C12N2710/24134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • Orthopoxvirus is a genus encompassing a number of viral species including mpox virus
  • vaccinia virus also referred to herein as "VACV”, e.g., Vaccinia virus Ankara, Vaccinia virus Copenhagen, Vaccinia virus WR), rabbitpox, buffalopox, camelpox, Cantagalo virus, ectromelia virus (also referred to herein as “ECTV” and “mousepox”), borealpox virus (previously known as "Alaskapox”; also referred to herein as “AKPV” or “BRPV”), cowpox virus (also referred to herein as “CPXV”), and variola virus (also referred to herein as "VARV”).
  • Orthopoxviruses are restricted in the hosts they infect, while others have been identified in a broad range of host species. Orthopoxviruses share a number of biological phenotypes including: a lack of a specific receptor required for infection of mammalian cells, a relatively low mutations rate, environmental stability of virion, and the ability to infect hosts via a number of routes (e.g., mucosal, respiratory, parenteral, etc.).
  • Mpox was first discovered in 1958 when two outbreaks of a pox-like disease occurred in colonies of monkeys kept for research, hence the name "monkeypox.”
  • the first human case of mpox was recorded in 1970 in the Democratic Republic of Congo during a period of intensified effort to eliminate smallpox. Since then, mpox has been reported in humans in other central and western African countries.
  • the WHO declared a new PHEIC as a result of the upsurge of mpox and the emergence of a new clade (clade lb) in the Democratic Republic of the Congo and other African countries.
  • the recent mpox outbreak illustrates the potential for orthopoxvirus re-emergence and spread and highlights limitations in the current vaccine supply.
  • compositions e.g., immunogenic compositions, e.g., vaccines
  • a pathogen e.g., virus
  • a pathogen e.g., virus
  • compositions e.g., immunogenic compositions, e.g., vaccines
  • RNA platform e.g., RNA binders
  • prioritization must take place.
  • compositions e.g., immunogenic compositions, e.g., vaccines
  • Pharmaceutical compositions are designed to encode multiple mpox antigens because of the many surface proteins and complex replication cycle of orthopoxviruses.
  • Orthopoxviruses have two distinct infectious forms, mature virions (MV) and extracellular virions (EV). Each viral form has a unique set of surface antigens. While immune responses raised to any single target can offer some protection from infection, both subunit vaccine and monoclonal antibody prophylaxis studies in animals illustrated that the value of combining antibodies to multiple EV and MV targets.
  • exemplary vaccine compositions of the present disclosure elicit immune responses to a combination of mpox proteins from both EVs and MVs: A35 and B6, which are EV surface glycoproteins, and Ml and H3, which are displayed on the surface of MVs.
  • A35 and B6, which are EV surface glycoproteins, and Ml and H3, which are displayed on the surface of MVs are displayed on the surface of MVs.
  • selection of this subset of antigens from the surface proteomes of mpox virions involved a consideration, in part, of the fact that A35, B6, Ml and H3 are conserved across many orthopoxviruses, including VARV and VACV. All four proteins have been observed to be highly immunogenic following VACV immunization or in MPXV convalescent subjects.
  • A35, B6, and Ml have been shown to protect mice and macaques from lethal orthopoxvirus disease. Additionally, antibodies to all four antigens have previously been shown to protect animals in monoclonal and/or polyclonal antibody prophylaxis studies. While prior studies have shown that A35, B6, Ml and H3 can elicit immune responses and provide some degree of protection against orthopoxviruses, challenges remain with the use of pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) that include the antigens as polypeptides.
  • pharmaceutical compositions e.g., immunogenic compositions, e.g., vaccines
  • polypeptide-based therapies provide a recognition of several challenges involved in development and administration of polypeptide-based therapies.
  • development of polypeptide-based therapies can be time consuming and expensive.
  • polypeptide development is challenged by demanding and costly production, including purification and formulation methods.
  • polypeptide-based therapies present an increased number of regulatory challenges.
  • there is tight regulation over manufacture of polypeptide-based therapies e.g., assessing post- translational modifications, etc.
  • quality control during storage and administration complicate the use of protein therapies.
  • administration of polypeptide-based therapies to a subject can be painful and time consuming.
  • polypeptide-based therapies are administered intravenously over a longer period of time, increasing patient discomfort.
  • polypeptide-based therapies can have a relatively short serum half-life.
  • the present disclosure provides insights that address these challenges, making it possible to deliver one or more therapies, as described herein, to a subject safely, reliably, and with strong potency.
  • the present disclosure describes one or more orthopoxvirus (e.g., mpox) antigens, antigenic fragments, or variants thereof that are delivered to a subject via one or more polyribonucleotides.
  • mpox orthopoxvirus
  • Utilizing one or more polyribonucleotides as a therapeutic agent (in contrast to administering a polypeptide itself) involves simpler and less expensive manufacturing processes.
  • the less complex production of polyribonucleotide encoding antigens can streamline manufacture, mitigating regulatory and production challenges associated with developing and using an antigen themselves.
  • polyribonucleotides are effective at producing similar effects to recombinant polypeptides, but tend to require much lower volumes be administered to a subject.
  • polyribonucleotides encoding an antigen characteristic portion, or variant thereof can be administered to a subject and the subject's body produces the polypeptide itself.
  • a polyribonucleotide encoding antigens as a therapeutic agent, has higher therapeutic efficacy (in contrast to administering a polypeptide itself) due to its continuous translation into encoded antigen to trigger long-lasting expression compared to transient traditional antigen polypeptide delivery.
  • the present disclosure also provides technologies that address certain limitations of recombinant polypeptide technologies, including for example, expression of antigens by utilizing RNA technologies as a modality to express antigens directly in the patient's cells.
  • compositions e.g., immunogenic compositions, e.g., vaccines
  • pharmaceutical compositions for delivering particular orthopox antigen constructs to a subject (e.g., a patient) and related technologies (e.g., methods).
  • pharmaceutical compositions e.g., immunogenic compositions, e.g., vaccines
  • polyribonucleotide constructs encoding mpox antigens and related technologies (e.g., methods).
  • mpox antigens, and antigenic fragments thereof, provided herein are particularly advantageous for use in preventing or treating mpox infection and infections by related orthopoxviruses (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, modified vaccinia virus Ankara, or vaccinia viruses).
  • related orthopoxviruses e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, modified vaccinia virus Ankara, or vaccinia viruses.
  • an mpox antigen construct includes and/or encodes one or more mpox antigens or fragments thereof (e.g., one or more B cell antigens for mpox and/or one or more T cell antigens for mpox, or fragments thereof).
  • T cell antigens include, e.g., CD4 T cell antigens and/or CD8 T cells.
  • any reference herein to an antigen as a "B cell antigen” or “T cell antigen” or the like does not exclude that any given antigen, or any given agent when exposed to an immune system, can activate, induce, and/or cause a diversity of immunological responses that can include, regardless of labels applied for expediency of description, one or both of a B cell response and a T cell response.
  • an mpox vaccine may cross-protect against other orthopoxviruses (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara)).
  • orthopoxviruses e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara)
  • vaccinia virus e.g., vaccinia virus Ankara, etc.
  • provided polyribonucleotide constructs encoding mpox antigens are effective for the prevention or treatment of a rabbitpox infection when administered to a subject. In some embodiments, provided polyribonucleotide constructs encoding mpox antigens are effective for the prevention or treatment of a variola virus infection when administered to a subject. In some embodiments, provided polyribonucleotide constructs encoding mpox antigens are effective for the prevention or treatment against a cowpox virus when administered to a subject.
  • provided polyribonucleotide constructs encoding mpox antigens are effective for the prevention or treatment of a ectromelia virus infection when administered to a subject. In some embodiments, provided polyribonucleotide constructs encoding mpox antigens are effective for vaccination against mpox and one or more other orthopoxviruses. In some embodiments, provided polyribonucleotide constructs encoding mpox antigens are effective for vaccination against mpox and variola virus. In some embodiments, provided polyribonucleotide constructs encoding mpox antigens are effective for vaccination against mpox and vaccinia virus.
  • provided polyribonucleotide constructs encoding mpox antigens are effective for vaccination against mpox and cowpox virus. In some embodiments, provided polyribonucleotide constructs encoding mpox antigens are effective for vaccination against mpox and ectromelia virus. In some embodiments, provided polyribonucleotide constructs encoding mpox antigens are effective for vaccination against mpox and a novel orthopoxvirus.
  • polyribonucleotide constructs encoding mpox antigens of the present disclosure are effective for the prevention or treatment of a lethal mpox virus infection. In some embodiments, polyribonucleotide constructs encoding mpox antigens of the present disclosure are effective for the prevention or treatment of a lethal clade I mpox virus infection. In some embodiments, polyribonucleotide constructs encoding mpox antigens of the present disclosure are effective for the prevention or treatment of a strain V79-I- 005 mpox virus infection.
  • a composition of the present disclosure comprises a therapeutically effective amount of one or more polyribonucleotides encoding one or more mpox virus antigens or fragments thereof; and a pharmaceutically acceptable carrier, wherein each of the one or more polyribonucleotides or fragments thereof is fully or partially encapsulated within a lipid nanoparticle.
  • the one or more polyribonucleotides are purified.
  • the one or more polyribonucleotides are singlestranded.
  • the one or more polyribonucleotides comprise a 5'-capped Nl- methylpseudouridine.
  • mpox virus antigens or fragments thereof comprise: (i) B6R or a fragment of B6R; (ii) MIR or a fragment of MIR; (iii) A35R or a fragment of A35R; or (iv) H3L or a fragment of H3L.
  • composition of the present disclosure comprises at least three or four polyribonucleotides, and wherein the at least three or four polyribonucleotides each encode a different mpox virus antigen or fragment thereof.
  • a composition of the present disclosure comprises: (i) a polyribonucleotide encoding B6R or a fragment of B6R; (ii) a polyribonucleotide encoding MIR or a fragment of MIR; and (iii) a polyribonucleotide encoding A35R or a fragment of A35R.
  • a composition of the present disclosure comprises: (i) a polyribonucleotide encoding B6R or a fragment of B6R; (ii) a polyribonucleotide encoding MIR or a fragment of MIR; (iii) a polyribonucleotide encoding A35R or a fragment of A35R; and (iv) a polyribonucleotide encoding H3L or a fragment of H3L.
  • the lipid nanoparticle targets liver cells and/or secondary lymphoid organ cells.
  • the lipid nanoparticle is a cationic lipid nanoparticle.
  • the lipid nanoparticle comprises: (a) a polymer-conjugated lipid; (b) a cationic lipid; and (c) one or more neutral lipids.
  • the polymer-conjugated lipid comprises a PEG-conjugated lipid or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide.
  • the one or more neutral lipids comprise 1,2- Distearoyl-sn-glycero-3-phosphocholine (DPSC) or cholesterol.
  • the cationic lipid comprises ((3-hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2-butyloctanoate).
  • a lipid nanoparticle of the present disclosure comprises: (a) 2-
  • a lipid nanoparticle of the present disclosure comprises: (a) the polymer-conjugated lipid at about 1-2.5 mol% of the total lipids; (b) the cationic lipid at 35-65 mol% of the total lipids; and (c) the one or more neutral lipids are present in 35-65 mol% of the total lipids.
  • lipid nanoparticles of the present disclosure have an average diameter of about 50-150 nm.
  • a therapeutically effective amount of the one or more polyribonucleotides is 1 ⁇ g to 250 ⁇ g, 5 ⁇ g to 200 ⁇ g, 10 ⁇ g to 100 ⁇ g, or 10 ⁇ g to 60 ⁇ g. In some embodiments, a therapeutically effective amount of the one or more polyribonucleotides is about 10 ⁇ g, about 30 ⁇ g, or about 60 ⁇ g.
  • the present disclosure provides a method of preventing an orthopoxvirus infection in a subject.
  • a method of preventing an orthopoxvirus infection in a subject comprises administering a composition to the subject.
  • a composition comprises: a therapeutically effective amount of one or more polyribonucleotides encoding one or more mpox virus antigens or antigenic fragments thereof; and a pharmaceutically acceptable carrier.
  • a composition comprises at least three polyribonucleotides, wherein each of the at least three polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof.
  • a method of preventing an orthopoxvirus infection in a subject provided herein comprises administering a composition to the subject, wherein the composition comprises: a first polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof; a second polyribonucleotide encoding an MIR antigen, or antigenic fragment thereof; and a third polyribonucleotide encoding an A35 antigen, or antigenic fragment thereof.
  • a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182.
  • an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158.
  • an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174.
  • a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182;
  • an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158;
  • an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs:
  • a composition comprises at least four polyribonucleotides, wherein each of the at least four polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof.
  • a method of preventing an orthopoxvirus infection in a subject comprises administering a composition to the subject, wherein the composition comprises: a first polyribonucleotide encodes a B6R antigen, or antigenic fragment thereof; a second polyribonucleotide encodes an MIR antigen, or antigenic fragment thereof; a third polyribonucleotide encodes an A35 antigen, or antigenic fragment thereof; and a fourth polyribonucleotide encodes an H3L antigen, or antigenic fragment thereof.
  • a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182.
  • an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31- 40, and 158.
  • an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174.
  • an H3L antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
  • a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182;
  • an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158;
  • an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs:
  • a method of preventing an orthopoxvirus infection comprises an mpox infection, a vaccinia infection, an ectromelia infection, a variola infection, a borealpox infection, a cowpox infection, a volepox infection, a buffalopox infection, a camelpox infection, a rabbitpox infection, or a modified vaccinia virus Ankara infection.
  • an orthopoxvirus infection is an mpox infection.
  • an orthopoxvirus infection is a clade I mpox infection.
  • a method provided herein is a method of treating an orthopoxvirus infection in a subject.
  • a method of treating an orthopoxvirus infection in a subject comprises administering a composition to the subject.
  • a composition comprises: a therapeutically effective amount of one or more polyribonucleotides encoding one or more mpox virus antigens or antigenic fragments thereof; and a pharmaceutically acceptable carrier.
  • a composition comprises at least three polyribonucleotides, wherein each of the at least three polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof.
  • a method of treating an orthopoxvirus infection in a subject provided herein comprises administering a composition to the subject, wherein the composition comprises: a first polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof; a second polyribonucleotide encoding an MIR antigen, or antigenic fragment thereof; and a third polyribonucleotide encoding an A35 antigen, or antigenic fragment thereof.
  • a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182.
  • an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158.
  • an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174.
  • a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182;
  • an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158;
  • an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs:
  • a composition comprises at least four polyribonucleotides, wherein each of the at least four polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof.
  • a method of treating an orthopoxvirus infection in a subject comprises administering a composition to the subject, wherein the composition comprises: a first polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof; a second polyribonucleotide encoding an MIR antigen, or antigenic fragment thereof; a third polyribonucleotide encoding an A35 antigen, or antigenic fragment thereof; and a fourth polyribonucleotide encoding an H3L antigen, or antigenic fragment thereof.
  • a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182.
  • an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31- 40, and 158.
  • an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174.
  • an H3L antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
  • a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182;
  • an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158;
  • an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs:
  • the present disclosure provides a method of treating an orthopoxvirus infection, wherein the orthopoxvirus infection comprises an mpox infection, a vaccinia infection, an ectromelia infection, a variola infection, a borealpox infection, a cowpox infection, a volepox infection, a buffalopox infection, a camelpox infection, a rabbitpox infection, or a modified vaccinia virus Ankara infection.
  • an orthopoxvirus infection is an mpox infection.
  • an orthopoxvirus infection is a clade I mpox infection.
  • an orthopoxvirus infection is a clade la or clade lb mpox infection. In some embodiments, an orthopoxvirus infection is a clade II mpox infection. In some embodiments, an orthopoxvirus infection is a clade Ila or clade lib mpox infection.
  • the present disclosure provides a method of preventing an orthopoxvirus infection in a subject.
  • a method of preventing an orthopoxvirus infection in a subject comprises administering a combination to the subject.
  • a combination comprises: a therapeutically effective amount of one or more polyribonucleotides encoding one or more mpox virus antigens or antigenic fragments thereof; and a pharmaceutically acceptable carrier.
  • a combination comprises at least three polyribonucleotides, wherein each of the at least three polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof.
  • a method of preventing an orthopoxvirus infection in a subject provided herein comprises administering a combination to the subject, wherein the combination comprises: a first polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof; a second polyribonucleotide encoding an MIR antigen, or antigenic fragment thereof; and a third polyribonucleotide encoding an A35 antigen, or antigenic fragment thereof.
  • a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182.
  • an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158.
  • an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174.
  • a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182;
  • an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158;
  • an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs:
  • a combination comprises at least four polyribonucleotides, wherein each of the at least four polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof.
  • a method of preventing an orthopoxvirus infection in a subject comprises administering a combination to the subject, wherein the combination comprises: a first polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof; a second polyribonucleotide encoding an MIR antigen, or antigenic fragment thereof; a third polyribonucleotide encoding an A35 antigen, or antigenic fragment thereof; and a fourth polyribonucleotide encoding an H3L antigen, or antigenic fragment thereof.
  • a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182.
  • an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31- 40, and 158.
  • an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174.
  • an H3L antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
  • the present disclosure provides a method of preventing an orthopoxvirus infection, wherein the orthopoxvirus infection comprises an mpox infection, a vaccinia infection, an ectromelia infection, a variola infection, a borealpox infection, a cowpox infection, a volepox infection, a buffalopox infection, a camelpox infection, a rabbitpox infection, or a modified vaccinia virus Ankara infection.
  • an orthopoxvirus infection is an mpox infection.
  • an orthopoxvirus infection is a clade I mpox infection.
  • an orthopoxvirus infection is a clade la or clade lb mpox infection. In some embodiments, an orthopoxvirus infection is a clade II mpox infection. In some embodiments, an orthopoxvirus infection is a clade Ila or clade lib mpox infection.
  • a method provided herein is a method of treating an orthopoxvirus infection in a subject.
  • a method of treating an orthopoxvirus infection in a subject comprises administering a combination to the subject.
  • a combination comprises: a therapeutically effective amount of one or more polyribonucleotides encoding one or more mpox virus antigens or antigenic fragments thereof; and a pharmaceutically acceptable carrier.
  • a combination comprises at least three polyribonucleotides, wherein each of the at least three polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof.
  • a method of treating an orthopoxvirus infection in a subject comprises administering a combination to the subject, wherein the combination comprises: a first polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof; a second polyribonucleotide encoding an MIR antigen, or antigenic fragment thereof; and a third polyribonucleotide encoding an A35 antigen, or antigenic fragment thereof.
  • a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182.
  • an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158.
  • an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174.
  • a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182;
  • an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158;
  • an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs:
  • a combination comprises at least four polyribonucleotides, wherein each of the at least four polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof.
  • a method of treating an orthopoxvirus infection in a subject comprises administering a combination to the subject, wherein the combination comprises: a first polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof; a second polyribonucleotide encoding an MIR antigen, or antigenic fragment thereof; a third polyribonucleotide encoding an A35 antigen, or antigenic fragment thereof; and a fourth polyribonucleotide encoding an H3L antigen, or antigenic fragment thereof.
  • a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182.
  • an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31- 40, and 158.
  • an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174.
  • an H3L antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
  • a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182;
  • an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158;
  • an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs:
  • the present disclosure provides a method of treating an orthopoxvirus infection, wherein the orthopoxvirus infection comprises an mpox infection, a vaccinia infection, an ectromelia infection, a variola infection, a borealpox infection, a cowpox infection, a volepox infection, a buffalopox infection, a camelpox infection, a rabbitpox infection, or a modified vaccinia virus Ankara infection.
  • an orthopoxvirus infection is an mpox infection.
  • an orthopoxvirus infection is a clade I mpox infection.
  • an orthopoxvirus infection is a clade la or clade lb mpox infection. In some embodiments, an orthopoxvirus infection is a clade II mpox infection. In some embodiments, an orthopoxvirus infection is a clade Ila or clade lib mpox infection.
  • a composition of the present disclosure comprises one or more polyribonucleotides that are purified. In some embodiments, a combination of the present disclosure comprises one or more polyribonucleotides that are purified.
  • a composition of the present disclosure comprises one or more singlestranded polyribonucleotides. In some embodiments, a combination of the present disclosure comprises one or more single-stranded polyribonucleotides.
  • a composition of the present disclosure comprises one or more polyribonucleotides comprising a 5'-capped Nl-methylpseudouridine. In some embodiments, a combination of the present disclosure comprises one or more polyribonucleotides comprising a 5'-capped Nl- methylpseudouridine.
  • a composition of the present disclosure comprises one or more polyribonucleotides that are fully or partially encapsulated within a lipid nanoparticle.
  • a combination of the present disclosure comprises one or more polyribonucleotides that are fully or partially encapsulated within a lipid nanoparticle.
  • a lipid nanoparticle targets liver cells.
  • a lipid nanoparticle targets secondary lymphoid organ cells.
  • a lipid nanoparticle is a cationic lipid nanoparticle.
  • a lipid nanoparticle comprises: (a) a polymer-conjugated lipid; (b) a cationic lipid; and (c) one or more neutral lipids.
  • a polymer-conjugated lipid comprises a PEG-conjugated lipid.
  • one or more neutral lipids comprise 1,2-Distearoyl-sn-glycero-3-phosphocholine (DPSC).
  • DPSC 1,2-Distearoyl-sn-glycero-3-phosphocholine
  • one or more neutral lipids comprise cholesterol.
  • a lipid nanoparticle comprises: (a) the polymer-conjugated lipid at about 1-2.5 mol% of the total lipids; (b) the cationic lipid at 35-65 mol% of the total lipids; and (c) the one or more neutral lipids are present in 35-65 mol% of the total lipids.
  • a lipid nanoparticle has an average diameter of about 50-150 nm.
  • a method provided herein is a method of preventing an orthopoxvirus infection in a subject comprising administering a composition to the subject in one or more doses.
  • one or more doses comprises about 10 ⁇ g, about 30 ⁇ g, or about 60 ⁇ g of the one or more polyribonucleotides.
  • administering one or more doses comprises administering a first dose of a composition and a second dose of a composition.
  • a second dose of a composition is administered to a subject about 31 days after a first dose of a composition is administered to the subject.
  • one or more doses are administered to the subject intramuscularly.
  • a method provided herein is a method of treating an orthopoxvirus infection in a subject comprising administering a composition to the subject in one or more doses.
  • one or more doses comprises about 10 ⁇ g, about 30 ⁇ g, or about 60 ⁇ g of the one or more polyribonucleotides.
  • administering one or more doses comprises administering a first dose of a composition and a second dose of a composition.
  • a second dose of a composition is administered to a subject about 31 days after a first dose of a composition is administered to the subject.
  • one or more doses are administered to the subject intramuscularly.
  • a method provided herein is a method of preventing an orthopoxvirus infection in a subject comprising administering a combination to the subject in one or more doses.
  • one or more doses comprises about 10 ⁇ g, about 30 ⁇ g, or about 60 ⁇ g of the one or more polyribonucleotides.
  • administering one or more doses comprises administering a first dose of a combination and a second dose of a combination.
  • a second dose of a combination is administered to a subject about 31 days after a first dose of a combination is administered to the subject.
  • one or more doses are administered to the subject intramuscularly.
  • a method provided herein is a method of treating an orthopoxvirus infection in a subject comprising administering a combination to the subject in one or more doses.
  • one or more doses comprises about 10 ⁇ g, about 30 ⁇ g, or about 60 ⁇ g of the one or more polyribonucleotides.
  • administering one or more doses comprises administering a first dose of a combination and a second dose of a combination.
  • a second dose of a combination is administered to a subject about 31 days after a first dose of a combination is administered to the subject.
  • one or more doses are administered to the subject intramuscularly.
  • a method of preventing an orthopoxvirus infection in a subject comprising administering a composition, wherein administering the composition comprises mixing the one or more polyribonucleotides and wherein each of the one or more polyribonucleotides is administered as a single injection dose.
  • a method of treating an orthopoxvirus infection in a subject comprising administering a composition, wherein administering the composition comprises mixing the one or more polyribonucleotides and wherein each of the one or more sub-doses is administered as a single injection dose.
  • a method of preventing an orthopoxvirus infection in a subject comprising administering a combination, wherein administering the combination comprises mixing the one or more polyribonucleotides and wherein each of the one or more polyribonucleotides is administered as a single injection dose.
  • a method of treating an orthopoxvirus infection in a subject comprising administering a combination, wherein administering the combination comprises mixing the one or more polyribonucleotides and wherein each of the one or more polyribonucleotides is administered as a single injection dose.
  • the present disclosure provides a method of preventing an orthopoxvirus infection in a subject comprising administering a composition to the subject, wherein administering results in an increased serum level of one or more orthopoxvirus-neutralizing antibodies in the subject.
  • the present disclosure provides a method of treating an orthopoxvirus infection in a subject comprising administering a composition to the subject, wherein administering results in an increased serum level of one or more orthopoxvirus-neutralizing antibodies in the subject.
  • the present disclosure provides a method of preventing an orthopoxvirus infection in a subject comprising administering a combination to the subject, wherein administering results in an increased serum level of one or more orthopoxvirus-neutralizing antibodies in the subject.
  • the present disclosure provides a method of treating an orthopoxvirus infection in a subject comprising administering a combination to the subject, wherein administering results in an increased serum level of one or more orthopoxvirus-neutralizing antibodies in the subject.
  • compositions comprising: a therapeutically effective amount of one or more polyribonucleotides encoding one or more MPXV antigens or fragments thereof; and a pharmaceutically acceptable carrier.
  • each of the one or more polyribonucleotides or fragments thereof is fully or partially encapsulated within a lipid nanoparticle.
  • the present disclosure provides a method of treating an mpox virus infection in a subject comprising administering one or more doses of a composition to the subject, wherein the composition comprises: a therapeutically effective amount of one or more polyribonucleotides encoding one or more MPXV antigens or fragments thereof; and a pharmaceutically acceptable carrier.
  • each of the one or more polyribonucleotides or fragments thereof is fully or partially encapsulated within a lipid nanoparticle.
  • compositions e.g., pharmaceutical compositions, e.g., immunogenic compositions, e.g., vaccines
  • polyribonucleotides are prepared, formulated, and/or utilized in particular LNP compositions, e.g., as described herein.
  • the present disclosure provides technologies for rapid development of a pharmaceutical composition (e.g., immunogenic composition, e.g., mpox vaccine) for delivering particular mpox antigen constructs to a subject.
  • a pharmaceutical composition e.g., immunogenic composition, e.g., mpox vaccine
  • the present disclosure provides, for example, nucleic acid constructs encoding mpox antigens or fragments thereof disclosed herein, expressing mpox antigens or fragments thereof disclosed herein, and various methods of production and/or use relating thereto, as well as compositions developed therewith and methods relating thereto.
  • the present disclosure provides technologies for preventing, characterizing, treating, and/or monitoring mpox outbreaks of orthopoxviruses (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara) viruses) and/or infections including, e.g., various nucleic acid constructs and encoded proteins, as well as agents (e.g., antibodies) that bind to such proteins, and compositions that comprise and/or deliver them.
  • orthopoxviruses e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara) viruses
  • infections including
  • compositions and methods for augmenting, inducing, promoting, enhancing and/or improving an immune response against orthopoxviruses (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara) viruses) or components thereof (e.g., a protein or portion thereof).
  • orthopoxviruses e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara) viruses
  • vaccinia e.g., modified vaccinia virus Ankara
  • technologies provided herein are designed to augment, induce, promote, enhance and/or improve immunological memory against orthopoxviruses (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara) viruses) or a component thereof (e.g., a protein or portion thereof).
  • orthopoxviruses e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara) viruses
  • vaccinia e.g., modified vaccinia virus Ankara
  • compositions of the present disclosure comprise one or more polynucleotide constructs (e.g., one or more string constructs) that encode one or more epitopes from mpox.
  • the present disclosure provides vaccines or other compositions comprising nucleic acids encoding such mpox epitopes; those skilled in the art will appreciate from context when reference to a particular polynucleotide (e.g., a DNA or RNA) as "encoding" such epitopes in fact is referencing a coding strand or its complement.
  • a particular polynucleotide e.g., a DNA or RNA
  • FIG. 1 depicts a structural representation of exemplary components of compositions provided by the present disclosure; the exemplary tetravalent Combo 4 and exemplary trivalent Combo 2.
  • the B6 structure is predicted by AlphaFold.
  • the A35, Ml and H3 structures shown are of the VACV orthologs (Protein Data Bank [PDB] IDs 3K7B, 2I9L, and 5EJ0).
  • FIGs. 2A-D depict in vitro expression of MPXV antigens from exemplary polyribonucleotide constructs of the present disclosure.
  • HEK293T cells were transfected with 200 ng of polyribonucleotide.
  • Cells transfected with the exemplary multivalent polyribonucleotide compositions Combo 4 and Combo 2 were compared to non-transfected cells and cells transfected with a monovalent polyribonucleotide-LNP composition.
  • Antigen expression levels were measured by flow cytometry 18 hours post- transfection for A35 (FIG. 2A), B6 (FIG. 2B), Ml (FIG. 2C), and H3 (FIG. 2D).
  • FIG. 2A A35
  • B6 FIG. 2B
  • Ml FIG. 2C
  • H3 FIG. 2D
  • H3 analysis for exemplary Combo 2 is excluded as H3 is not included in this exemplary polyribonucleotide composition.
  • Antigen expression is shown as median fluorescence intensity (MFI) of total live cells for both intracellular (total protein) and surface (surface protein) staining conditions. The mean of technical triplicates is shown (+SEM).
  • Exemplary Combo 2 comprised A35, B6, and Ml antigens
  • exemplary Combo 4 comprised A35, B6, Ml, and H3 antigens.
  • FIGs. 3A-D are line graphs depicting serum antibody levels produced by mice administered with exemplary polyribonucleotide constructs of the present disclosure.
  • BALB/c mice were administered with 4 ⁇ g exemplary Combo 4 (squares), 4 ⁇ g exemplary Combo 2 (triangles) or with 1 ⁇ g of a polyribonucleotide encoding one antigen (circles) on days 0 and 21.
  • Serum antibody levels for A35 (FIG. 3A), B6 (FIG. 3B), Ml (FIG. 3C), and H3 (FIG. 3D) were measured by ELISA.
  • Geometric mean values of IgG ng/mL equivalents ( ⁇ SEM) are shown. The dashed line indicates the lower limit of detection.
  • FIGs. 4A-E depict induction of germinal centers (GCs) following administration with exemplary polyribonucleotide constructs of the present disclosure encoding a single (e.g., monovalent) MPXV antigen.
  • IM intramuscular
  • LNs inguinal lymph nodes
  • FIG. 4A is a representative dot plot depicting the gating strategy for total GC B cells (Live Dump).
  • FIG. 4B is a representative dot plot depicting the gating strategy for B220+CD19+ double positive GC B cells.
  • FIG. 4C is a representative dot plot depicting the gating strategy for GL7+CD95+ double positive GC B cells. Sequential gating events are shown left to right.
  • FIG. 4D is a bar graph depicting the measurement of the percentage of GC B cells in the LN.
  • FIG. 4E is a bar graph depicting the measurement of the fraction of antigen-positive cells among total GC B cells. Mean ⁇ SEM is shown.
  • FIGs. 5A-D depict measurements of T cell responses to MPXV antigens after administration with exemplary polyribonucleotide constructs described herein. Mice were administered a single administration of 4 ⁇ g exemplary Combo 4 or 1 ⁇ g of single polyribonucleotides encoding one MPXV antigen.
  • mice were sacrificed and antigen-specific T cell levels were measured by IFN-y ELISpot with mouse splenocytes stimulated with peptide pools spanning the antigen(s) relevant to each experimental group. Data for individual animals with median bar are shown for A35 (FIG. 5A), B6 (FIG. 5B), Ml (FIG. 5C), and H3 (FIG. 5D).
  • Exemplary Combo 2 comprised A35, B6, and Ml antigens
  • exemplary Combo 4 comprised A35, B6, Ml, and H3 antigens.
  • FIGs. 7A-D depict cluster plots of neutralizing antibody titers following administrations with exemplary polyribonucleotide constructs of the present disclosure encoding MPXV antigens.
  • Mice were administered IM with exemplary Combo 4 (4 ⁇ g) or exemplary Combo 2 (4 ⁇ g) compositions, or polyribonucleotides encoding one MPXV antigen (1 ⁇ g), on days 0 and 21.
  • Neutralizing antibody activities for MPXV determined via PRNT; FIG. 7A and FIG. 7B) and for VACV (determined via FRNT; FIG. 7C and FIG. 7D) were measured both in the absence of baby rabbit complement (FIG. 7A and FIG.
  • Exemplary Combo 2 comprised A35, B6, and Ml antigens
  • exemplary Combo 4 comprised A35, B6, Ml, and H3 antigens.
  • FIG. 8 is a cluster plot depicting neutralizing antibody responses following prime and boost administrations with exemplary polyribonucleotide constructs of the present disclosure encoding MPXV antigens.
  • BALB/c mice were administered with exemplary Combo 4 or exemplary Combo 2 polyribonucleotide compositions on days 0 and 21 and serum VACV-neutralizing antibodies measured on day 21 and day 35 by FRNT. The lower limit of detection is indicated with a dashed line and is set at half the lowest test serum dilution tested.
  • Exemplary Combo 2 comprised A35, B6, and Ml antigens
  • exemplary Combo 4 comprised A35, B6, Ml, and H3 antigens.
  • FIGs. 9A-I depict in vivo studies of protection against MPXV challenge from administration with exemplary polyribonucleotide constructs of the present disclosure encoding MPXV antigens.
  • FIG. 9A is a schematic illustrating an exemplary dosing schedule where CAST/Ei mice were administered with a combination of A35 and B6 polyribonucleotides, exemplary Combo 4, exemplary Combo 2, or negative control (mock treated with saline) on days 0 and 21. Animals were challenged intranasally (IN) with 9 x 10 5 PFU of clade lib MPXV (high dose) 5 weeks after the last administration.
  • FIG. 9C depict MPXV titers in the lungs of CAST/Ei mice sacrificed on day 3 or 7 post-challenge, respectively. Lungs were collected and analyzed for viral load by TCID50 assay. The lower limit of detection is indicated by a dashed line.
  • FIG. 9D is a schematic illustrating an exemplary dosing schedule where CAST/Ei mice were administered with a combination of A35 and B6 polyribonucleotides, exemplary Combo 4, exemplary Combo 2, or negative control (mock treated with saline) on days 0 and 21. Animals were challenged intranasally (IN) with 3 x 10 5 PFU of clade lib MPXV (low dose) 5 weeks after the last administration.
  • FIG. 9D is a schematic illustrating an exemplary dosing schedule where CAST/Ei mice were administered with a combination of A35 and B6 polyribonucleotides, exemplary Combo 4, exemplary Combo 2, or negative control (mock treated
  • FIG. 9E and FIG. 9F depict MPXV titers in the lungs of CAST/Ei mice sacrificed on day 3 or 7 post-challenge, respectively.
  • FIG. 9G is a schematic illustrating an exemplary dosing schedule where CAST/Ei mice were administered with polyribonucleotides encoding A35, B6, Ml, or H3 antigens of exemplary Combo 4, exemplary Combo 2, or negative control (mock treated with saline) on days 0 and 21. Animals were challenged IN with 1 x 10 5 PFU of clade I MPXV 5 weeks after the last administration. Mice were monitored for weight loss and survival for two weeks post-challenge.
  • 91 depict weight loss and survival results, respectively, of CAST/Ei mice administered as in FIG. 9G.
  • Statistical significance was assessed using a Kruskal-Wallis test with Dunn's multiple comparisons test: *p ⁇ 0.05, ** ⁇ 0.01, *** ⁇ 0.001; ns, not significant.
  • statistical significance was assessed using Mantel-Cox log-rank test: *p ⁇ 0.05, * m p ⁇ 0.0001; ns, not significant.
  • Combo 2 comprises A35, B6, and Ml antigens
  • Combo 4 comprises A35, B6, Ml, and H3 antigens.
  • FIGs. 10A-C depict exemplary in vivo studies of protection against MPXV challenge.
  • FIG. 1OA is a schematic illustrating an exemplary dosing schedule where BALB/c mice were administered with polyribonucleotides encoding A35, B6, Ml, or H3 antigens or exemplary Combo 4, exemplary Combo 2, or negative control (mock treated with saline) on days 0 and 21. Animals were challenged IN with 5 x 10 4 PFU of VACV-WR on day 42.
  • FIG. 1OB and FIG. IOC depict weight loss and survival results, respectively.
  • Combo 2 comprises A35, B6, and Ml antigens
  • Combo 4 comprises A35, B6, Ml, and H3 antigens.
  • FIGs. 11A-C depict exemplary in vivo studies of protection against ECTV challenge.
  • FIG. 11A is a schematic illustrating an exemplary dosing schedule where BALB/c mice were administered with polyribonucleotides encoding Ml, A35, B6, or H3 antigens or exemplary Combo 4, or negative control (mock treated with PBS) on days 0 and 21. Animals were challenged IN with ECTV (10 4 PFU) on day 42.
  • FIG. 11B and FIG. 11C depict weight loss and survival results, respectively.
  • Combo 4 comprises A35, B6, Ml, and H3 antigens. Statistical significance was assessed using Mantel-Cox log-rank test **** ⁇ 0.0001.
  • FIGs. 12A-E depict exemplary in vivo studies of protection against MPXV challenge.
  • FIG. 12A is a schematic illustrating an exemplary dosing schedule in cynomolgus macaques. Cynomolgus macaques were administered with 30 ⁇ g of exemplary Combo 4 or mock treated with saline on days 0 and 28. An intratracheal (IT) challenge with clade I MPXV was performed on day 60.
  • FIG. 12B, FIG. 12C, FIG. 12D, and FIG. 12E depict the measurement of serum antibody levels against A35, B6, Ml, and H3 MPXV antigens, respectively, two weeks following boost were measured by ELISA.
  • FIG. 12A is a schematic illustrating an exemplary dosing schedule in cynomolgus macaques. Cynomolgus macaques were administered with 30 ⁇ g of exemplary Combo 4 or mock treated with saline on days 0 and 28. An intratracheal (IT) challenge
  • FIG. 12F is a Kaplan-Meier survival plot depicting survival of macaques administered with exemplary Combo 4 or mock-administered with saline for 28 days post clade I MPXV challenge.
  • FIG. 12G is a line graph depicting the weight loss of individual macaques mock-administered with saline, at 28 days post clade I MPXV challenge.
  • FIG. 12H and FIG. 121 are line graphs depicting the weight loss of individual macaques (FIG. 12H) and mean weight loss (FIG. 121) of macaques administered with exemplary Combo 4 at 28 days post clade I MPXV challenge.
  • FIG. 12H is a Kaplan-Meier survival plot depicting survival of macaques administered with exemplary Combo 4 or mock-administered with saline for 28 days post clade I MPXV challenge.
  • FIG. 12G is a line graph depicting the weight loss of individual macaques mock-administered with
  • FIG. 12J is a line graph depicting lesion counts of individual macaques mock-administered with saline, at 28 days post clade I MPXV challenge.
  • FIG. 12K and FIG. 12L are line graphs depicting the lesion counts of individual macaques (FIG. 12K) and mean lesion counts (FIG. 12L) of macaques administered with exemplary Combo 4 at 28 days post clade I MPXV challenge.
  • plots for individual animals and averaged plots are given, with the end of observation for each animal due to death or study completion marked by a diamond and dashed lines indicated the baseline value.
  • FIGs. 13A-13C depict exemplary in vivo studies of protection against MPXV challenge.
  • FIG. 13A-13C depict exemplary in vivo studies of protection against MPXV challenge.
  • FIG. 13A is a schematic illustrating an exemplary dosing schedule in CAST/Ei mice.
  • CAST/Ei mice were immunized with 4 ⁇ g of exemplary Combo 2 polyribonucleotide composition, 4 ⁇ g of exemplary Combo 4 polyribonucleotide composition, an LNP incorporating a combination of polyribonucleotides encoding A35 and B6, or saline, on days 0 and 21.
  • Mice were intranasally challenged with clade I MPXV isolate (Zaire 79 strain) on day 56.
  • FIG. 13B is a line graph depicting the percent body weight change of individual mice over a 12-day period post-MPXV challenge.
  • FIG. 13B is a line graph depicting the percent body weight change of individual mice over a 12-day period post-MPXV challenge.
  • 13C is a Kaplan-Meier plot depicting the percent survival of exemplary Combo 2 and Combo 4 polyribonucleotide composition vaccinated animals compared to animals treated with saline or LNP incorporating polyribonucleotides encoding A35 and B6.
  • FIGs. 14A-14C depict exemplary in vivo studies of protection against VACV challenge.
  • FIG. 14A-14C depict exemplary in vivo studies of protection against VACV challenge.
  • FIG. 14A is a schematic illustrating an exemplary dosing schedule in Balb/C mice.
  • Balb/C mice were immunized with 4 ⁇ g of exemplary Combo 4 polyribonucleotide composition, an LNP incorporating 1 ⁇ g of polyribonucleotide encoding Ml, an LNP incorporating 1 ⁇ g of polyribonucleotide encoding A35, an LNP incorporating 1 ⁇ g of polyribonucleotide encoding B6, an LNP incorporating 1 ⁇ g of polyribonucleotide encoding H3, or saline on days 0 and 21.
  • FIG. 14B is a line graph depicting the percent body weight change of individual mice over a 10-day period post-VACV challenge.
  • FIG. 14C is a Kaplan-Meier plot depicting the percent survival of mice immunized as described in FIG. 14A.
  • FIGs. 15A-15C are schematic diagrams of the safety and immunogenicity clinical trial design of Example 10.
  • FIG. 15A describes a staggered dosing process for substudies A, B, C, and D of Example 10.
  • FIG. 15B describes the dosing and sample collection for substudies A and B.
  • FIG. 15C describes the dosing and sample collection for substudies C and D of Example 10.
  • FIGs. 16A-16B are line graphs depicting the post-MPXV challenge blood viral loads of cynomolgus macaques vaccinated with exemplary Combo 4 polyribonucleotide composition (FIG. 16B) or saline (FIG. 16A).
  • FIG. 17 is a cluster plot depicting the 50% neutralization serum titers (NT50) of cynomolgus macaques vaccinated with exemplary Combo 4 polyribonucleotide composition or saline pre- and post-MPXV challenge.
  • FIGs. 18A-18D are line graphs depicting the percentage of cells expressing A35 (FIG. 18A),
  • B6 (FIG. 18B), Ml (FIG. 18C), or H3 (FIG. 18D) MPXV antigens in HEK293T cells transfected with various concentrations of modified polyribonucleotide constructs encoding A35, B6, Ml, or H3 MPXV antigens, respectively.
  • FIGs. 19A-19D are cluster plots depicting serum anti-A35 (FIG. 19A), anti-B6 (FIG. 19B), anti-Ml (FIG. 19C), and anti-H3 (FIG. 19D) IgG 50% binding titers in BALB/c mice following administrations with exemplary Combo 4 modified polyribonucleotide formulations of the present disclosure.
  • mice were administered IM with Combo 4 (incorporating modified polyribonucleotides encoding A35, B6, Ml, and H3 MPXV antigens) (i) co-formulated as a combined preparation of LNPs incorporating modified polyribonucleotides in Tris buffer and sucrose (Combo 4 [T]); (ii) co-formulated as a combined preparation of LNPs incorporating modified polyribonucleotides in phosphate buffered saline (PBS) and sucrose (Combo 4 [P]); or (iii) formulated as four single LNP preparations incorporating modified polyribonucleotides, in PBS and sucrose then mixed before administration (Combo 4 (single mix)).
  • Combo 4 incorporating modified polyribonucleotides encoding A35, B6, Ml, and H3 MPXV antigens
  • enantiomeric or diastereomeric forms of the structure as well as all geometric or conformational isomeric forms of the structure.
  • R and S configurations of each stereocenter are contemplated as part of the disclosure. Therefore, single stereochemical isomers, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of provided compounds are within the scope of the disclosure.
  • provided compounds show one or more stereoisomers of a compound, and unless otherwise indicated, represents each stereoisomer alone and/or as a mixture. Unless otherwise stated, all tautomeric forms of provided compounds are within the scope of the disclosure.
  • structures depicted herein are meant to include compounds that differ only in the presence of one or more isotopically enriched atoms.
  • compounds having the present structures including replacement of hydrogen by deuterium or tritium, or replacement of a carbon by 13C- or 14C -enriched carbon are within the scope of this disclosure.
  • exemplary refers to an example of an embodiment. Unless otherwise indicated, the term “exemplary” is not intended to indicate that an embodiment is a “preferred” or “best” version of an embodiment.
  • agent may refer to a physical entity.
  • an agent may be characterized by a particular feature and/or effect.
  • therapeutic agent refers to a physical entity has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect.
  • an agent may be a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof.
  • aliphatic refers to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as "cycloaliphatic"), that has a single point or more than one points of attachment to the rest of the molecule.
  • aliphatic groups contain 1-12 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms (e.g., C1- 6 ).
  • aliphatic groups contain 1-5 aliphatic carbon atoms (e.g., C1-5). In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms (e.g., In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms (e.g., C1-3), and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms (e.g., C1-2). Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, or alkynyl groups and hybrids thereof. A preferred aliphatic group is C1-6 alkyl.
  • Alkyl refers to a saturated, optionally substituted straight or branched chain hydrocarbon group having (unless otherwise specified) 1-12, 1- 10, 1-8, 1-6, 1-4, 1-3, or 1-2 carbon atoms (e.g., C1-12, C1-3, or C1-2).
  • exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl.
  • alkylene refers to a bivalent alkyl group. In some embodiments,
  • alkylene is a bivalent straight or branched alkyl group.
  • an "alkylene chain” is a polymethylene group, i.e., -(CH2) n -, wherein n is a positive integer, e.g., from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3.
  • An optionally substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms is optionally replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group and also include those described in the specification herein. It will be appreciated that two substituents of the alkylene group may be taken together to form a ring system.
  • two substituents can be taken together to form a 3- to 7-membered ring.
  • the substituents can be on the same or different atoms.
  • the suffix "-ene” or “-enyl” when appended to certain groups herein are intended to refer to a bifunctional moiety of said group.
  • alkenyl used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain or cyclic hydrocarbon group having at least one double bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C2-12, C2-10, C2-8, C2-6, C2-4, or C2- 3).
  • alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl.
  • cycloalkenyl refers to an optionally substituted non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms.
  • Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.
  • alkynyl refers to an optionally substituted straight or branched chain hydrocarbon group having at least one triple bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3).
  • exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl.
  • amino acid refers to a compound and/or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds.
  • an amino acid has the general structure H2N- C(H)I-COOH.
  • an amino acid is a naturally-occurring amino acid.
  • an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid.
  • Standard amino acid refers to any of the twenty standard L- amino acids commonly found in naturally occurring peptides.
  • Nonstandard amino acid refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source.
  • an amino acid including a carboxy- and/or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above.
  • an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and/or the hydroxyl group) as compared with the general structure.
  • such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid.
  • amino acid may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.
  • aryl refers to monocyclic and bicyclic ring systems having a total of six to fourteen ring members (e.g., C6-C14), wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. In some embodiments, an "aryl” group contains between six and twelve total ring members (e.g., C6-C12). The term “aryl” may be used interchangeably with the term “aryl ring”. In some embodiments, "aryl” refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents.
  • aryl groups are hydrocarbons.
  • an "aryl” ring system is an aromatic ring (e.g., phenyl) that is fused to a non-aromatic ring (e.g., cycloalkyl). Examples of aryl rings include that are fused include
  • Two events or entities are "associated" with one another, as that term is used herein, if the presence, level, degree, type and/or form of one is correlated with that of the other.
  • a particular entity e.g., polypeptide, genetic signature, metabolite, microbe, etc.
  • two or more entities are physically "associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another.
  • two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.
  • Co-adminlstratlon refers to use of a composition (e.g., a pharmaceutical composition) described herein and one or more additional therapeutic agents.
  • one or more additional therapeutic agents comprises at least one polyribonucleotide.
  • compositions described herein and an additional therapeutic agent may be performed concurrently or separately (e.g., sequentially in any order).
  • a composition (e.g., a pharmaceutical composition) described herein and an additional therapeutic agent may be combined in one pharmaceutically-acceptable excipient, or they may be placed in separate excipient and delivered to a target cell or administered to a subject at different times.
  • compositions described herein and an additional therapeutic agent are delivered or administered sufficiently close in time that there is at least some temporal overlap in biological effect(s) generated by each on a target cell or a subject being treated.
  • Combination therapy refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents).
  • the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens.
  • administration of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination.
  • combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition.
  • Comparable refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed.
  • comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features.
  • the term “corresponding to” refers to a relationship between two or more entities.
  • the term “corresponding to” may be used to designate the position/identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition).
  • a monomeric residue in a polymer e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide
  • a residue in an appropriate reference polymer may be identified as “corresponding to” a residue in an appropriate reference polymer.
  • residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190 th amino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify "corresponding" amino acids.
  • sequence alignment strategies including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify "corresponding" residues in polypeptides and/or nucleic acids in accordance with the present disclosure.
  • software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, Scala
  • corresponding to may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity).
  • a gene or protein in one organism may be described as "corresponding to" a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and/or that it shows a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.
  • Cycloaliphatic refers to a monocyclic C3-8 hydrocarbon or a bicyclic C 5 -io hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point or more than one points of attachment to the rest of the molecule.
  • Cycloalkyh refers to an optionally substituted saturated ring monocyclic or polycyclic system of about 3 to about 10 ring carbon atoms.
  • Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
  • amino acid sequence derived from a designated amino acid sequence (peptide or polypeptide)
  • amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof.
  • Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof.
  • detecting- is used broadly herein to include appropriate means of determining the presence or absence of an entity of interest or any form of measurement of an entity of interest in a sample. Thus, “detecting” may include determining, measuring, assessing, or assaying the presence or absence, level, amount, and/or location of an entity of interest. Quantitative and qualitative determinations, measurements or assessments are included, including semi-quantitative. Such determinations, measurements or assessments may be relative, for example when an entity of interest is being detected relative to a control reference, or absolute. As such, the term “quantifying” when used in the context of quantifying an entity of interest can refer to absolute or to relative quantification.
  • Dosing regimen- Those skilled in the art will appreciate that the term "dosing regimen" (or
  • therapeutic regimen may be used to refer to a set of unit doses (e.g., one or more than one) that are administered individually to a subject, typically separated by periods of time.
  • a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
  • a gene, a cDNA, or an RNA molecule encodes a polypeptide if transcription and translation of RNA corresponding to that gene produces the polypeptide in a cell or other biological system.
  • a coding region of a polyribonucleotide encoding a target antigen refers to a coding strand, the nucleotide sequence of which is identical to the polyribonucleotide sequence of such a target antigen.
  • a coding region of a polyribonucleotide encoding a target antigen refers to a non-coding strand of such a target antigen, which may be used as a template for transcription of a gene or cDNA.
  • Engineered- refers to the aspect of having been manipulated by the hand of man.
  • a polynucleotide is considered to be “engineered” when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and/or when a particular residue in a polynucleotide is non-naturally occurring and/or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature.
  • Epitope- refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component.
  • an epitope may be recognized by a T cell, a B cell, or an antibody.
  • an epitope is comprised of a plurality of chemical atoms or groups on an antigen.
  • such chemical atoms or groups are surface- exposed when the antigen adopts a relevant three-dimensional conformation.
  • such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation.
  • an epitope of an antigen may include a continuous or discontinuous portion of the antigen.
  • an epitope is or comprises a T cell epitope.
  • an epitope is or comprises a B cell epitope.
  • an epitope may have a length of about 5 to about 30 amino acids, or about 10 to about 25 amino acids, or about 5 to about 15 amino acids, or about 5 to 12 amino acids, or about 6 to about 9 amino acids.
  • a gene product can be a transcript, e.g., a polyribonucleotide as provided herein.
  • a gene product can be a polypeptide.
  • expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc.); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
  • fragment refers a structure that is or includes a discrete portion of a reference agent (sometimes referred to as the "parent" agent). In some embodiments, a fragment lacks one or more moieties found in the reference agent. In some embodiments, a fragment is or includes one or more moieties found in the reference agent. In some embodiments, the reference agent is a polymer such as a polynucleotide or polypeptide.
  • a fragment of a polymer is or includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., residues) of the reference polymer.
  • monomeric units e.g., residues
  • a fragment of a polymer is or includes at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the reference polymer.
  • a fragment of a reference polymer is not necessarily identical to a corresponding portion of the reference polymer.
  • a fragment of a reference polymer can be a polymer having a sequence of residues having at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to the reference polymer.
  • a fragment may, or may not, be generated by physical fragmentation of a reference agent. In some instances a fragment is generated by physical fragmentation of a reference agent. In some instances, a fragment is not generated by physical fragmentation of a reference agent and can be instead, for example, produced by de novo synthesis or other means.
  • heteroaliphatic or “heteroaliphatic group,” as used herein, denotes an optionally substituted hydrocarbon moiety having, in addition to carbon atoms, from one to five heteroatoms, that may be straight-chain (i.e., unbranched), branched, or cyclic ("heterocyclic") and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic.
  • heteroatom refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen.
  • nitrogen also includes a substituted nitrogen.
  • heteroaliphatic groups contain 1-10 carbon atoms wherein 1-3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroaliphatic groups contain 1-4 carbon atoms, wherein 1-2 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In yet other embodiments, heteroaliphatic groups contain 1-3 carbon atoms, wherein 1 carbon atom is optionally and independently replaced with a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroaliphatic groups include, but are not limited to, linear or branched, heteroalkyl, heteroalkenyl, and heteroalkynyl groups. For example, a 1- to 10 atom heteroaliphatic group includes the following exemplary groups: -O-CH3, -CH2-O-CH3, -O-CH2-CH2- O-CH2-CH2-O-CH3, and the like.
  • Heteroaryl and “heteroar-", used alone or as part of a larger moiety, e.g., “heteroaralkyl,” or “heteroaralkoxy,” refer to monocyclic or bicyclic ring groups having 5 to 10 ring atoms (e.g., 5- to 6-membered monocyclic heteroaryl or 9- to 10-membered bicyclic heteroaryl); having 6, 10, or 14 n- electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms.
  • Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-a]pyridyl, imidazo[4,5- b]pyridyl, imidazo[4,5-c]pyridyl, pyrrolopyridyl, pyrrolopyrazinyl, thienopyrimidinyl, triazolopyridyl, and benzoisox
  • heteroaryl and “heteroar-”, as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms).
  • Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4/7— quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3-b]-l,4-oxazin-3(4H)-one, 4H-thieno[3,2- b]pyrrole, and benzoisoxazolyl.
  • heteroaryl may be used interchangeably with the terms “heteroaryl ring,” “heteroaryl group,” or “heteroaromatic,” any of which terms include rings that are optionally substituted.
  • Heteroatom- refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen.
  • heterocycle As used herein, the terms “heterocycle,” “heterocyclyl,” “heterocyclic radical,” and “heterocyclic ring” are used interchangeably and refer to a stable 3- to 8-membered monocyclic, a 6- to 10- membered bicyclic, or a 10- to 16-membered polycyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, such as one to four, heteroatoms, as defined above.
  • nitrogen includes a substituted nitrogen.
  • a bicyclic heterocyclic ring can also be a spirocyclic ring system (e.g., 7- to 11-membered spirocyclic fused heterocyclic ring having, in addition to carbon atoms, one or more heteroatoms as defined above (e.g., one, two, three or four heteroatoms)).
  • a bicyclic heterocyclic ring can also be a bridged ring system (e.g., 7- to 11-membered bridged heterocyclic ring having one, two, or three bridging atoms.
  • homolog refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polynucleotide molecules e.g., DNA molecules and/or RNA molecules
  • polypeptide molecules are considered to be “homologous” to one another if their sequences are at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least
  • polynucleotide molecules are identical to polynucleotide molecules.
  • polypeptide molecules are considered to be "homologous" to one another if their sequences are at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similar (e.g., containing residues with related chemical properties at corresponding positions).
  • identity refers to the overall relatedness between polynucleotide molecules e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules.
  • polynucleotide molecules e.g., DNA molecules and/or RNA molecules
  • polypeptide molecules are considered to be "substantially identical" to one another if their sequences are at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical.
  • Calculation of the percent identity of two nucleic acid or polypeptide sequences for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes).
  • the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of a reference sequence.
  • the nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0).
  • nucleic acid sequence comparisons made with the ALIGN program use a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.
  • Increased, Induced, or Reduced As used herein, these terms or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with a provided composition (e.g., a pharmaceutical composition) may be "increased" relative to that obtained with a comparable reference composition.
  • a provided composition e.g., a pharmaceutical composition
  • an assessed value achieved in a subject may be "increased" relative to that obtained in the same subject under different conditions (e.g., prior to or after an event; or presence or absence of an event such as administration of a composition (e.g., a pharmaceutical composition) as described herein, or in a different, comparable subject (e.g., in a comparable subject that differs from the subject of interest in prior exposure to a condition, e.g., absence of administration of a composition (e.g., a pharmaceutical composition) as described herein.).
  • comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and/or magnitude sufficient to achieve statistical relevance).
  • the term “reduced” or equivalent terms refers to a reduction in the level of an assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or higher, as compared to a comparable reference. In some embodiments, the term “reduced” or equivalent terms refers to a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero.
  • the term "increased” or “induced” refers to an increase in the level of an assessed value by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or higher, as compared to a comparable reference.
  • in order refers to the order of features from 5' to 3' along the polynucleotide or polyribonucleotide.
  • in order refers to the order of features moving from the N-terminal-most of the features to the C-terminal-most of the features along the polypeptide. "In order” does not mean that no additional features can be present among the listed features.
  • Ionizable- refers to a compound or group or atom that is charged at a certain pH.
  • an ionizable amino lipid such a lipid or a function group or atom thereof bears a positive charge at a certain pH.
  • an ionizable amino lipid is positively charged at an acidic pH.
  • an ionizable amino lipid is predominately neutral at physiological pH values, e.g., in some embodiments about 7.0-7.4, but becomes positively charged at lower pH values.
  • an ionizable amino lipid may have a pKa within a range of about 5 to about 7.
  • Isolated- means altered or removed from the natural state.
  • a nucleic acid or a peptide naturally present in a living animal is not “isolated,” but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is “isolated.”
  • An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
  • Lipid- As used herein, the terms “lipid” and “lipid-like material” are broadly defined as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also typically denoted as amphiphiles.
  • RNA lipid nanoparticle- refers to a nanoparticle comprising at least one lipid and RNA molecule(s), e.g., one or more polyribonucleotides as provided herein.
  • an RNA lipid nanoparticle comprises at least one cationic amino lipid.
  • an RNA lipid nanoparticle comprises at least one cationic amino lipid, at least one helper lipid, and at least one polymer-conjugated lipid (e.g., PEG-conjugated lipid).
  • RNA lipid nanoparticles as described herein can have an average size e.g., Z-average) of about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm.
  • Z-average average size of about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm.
  • RNA lipid nanoparticles can have a particle size (e.g., Z-average) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm.
  • an average size of lipid nanoparticles is determined by measuring the average particle diameter.
  • RNA lipid nanoparticles may be prepared by mixing lipids with RNA molecules described herein.
  • nucleic acid/ Polynucleotide refers to a polymer of at least 10 nucleotides or more.
  • a nucleic acid is or comprises DNA.
  • a nucleic acid is or comprises RNA.
  • a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages.
  • PNA peptide nucleic acid
  • a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N- phosphoramidite linkages and/or one or more peptide bonds, e.g., as in a "peptide nucleic acid".
  • a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil).
  • a nucleic acid comprises one or more, or all, non-natural residues.
  • a nonnatural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo- pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof).
  • a nucleoside analog e.g.
  • a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'- deoxyribose, arabinose, and hexose) as compared to those in natural residues.
  • a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide.
  • a nucleic acid has a nucleotide sequence that comprises one or more introns.
  • a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro ⁇ , reproduction in a recombinant cell or system, or chemical synthesis.
  • enzymatic synthesis e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro ⁇ , reproduction in a recombinant cell or system, or chemical synthesis.
  • a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides long.
  • operably linked refers to the association of at least a first element and a second element such that the component elements are in a relationship permitting them to function in their intended manner.
  • a nucleic acid sequence or amino acid sequence is operably linked with another sequence if it modifies the expression, structure, or activity of the linked sequence, e.g., in an intended manner.
  • two nucleic acid sequences are operably linked if they contribute to the expression, structure, or activity of a gene or encoded polypeptide.
  • a nucleic acid regulatory sequence is "operably linked" to a nucleic acid coding sequence if the regulatory sequence and coding sequence are associated in a manner that permits control of expression of the coding sequence by the regulatory sequence.
  • an "operably linked" regulatory sequence is directly or indirectly covalently associated with a coding sequence (e.g., in a single nucleic acid).
  • a regulatory sequence controls expression of a coding sequence in trans and inclusion of the regulatory sequence in the same nucleic acid as the coding sequence is a requirement of operable linkage.
  • two amino acid sequences are operably linked if they are expressed as a single polypeptide.
  • compositions comprising: [0122] Pharmaceutically effective amount.
  • pharmaceutically effective amount or “therapeutically effective amount” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses.
  • a desired reaction in some embodiments relates to inhibition of the course of the disease (e.g., orthopoxvirus infection, e.g., mpox infection).
  • a desired reaction in a treatment of a disease may be or comprise delay or prevention of the onset of a disease (e.g., orthopoxvirus infection, e.g., mpox infection) or a condition (e.g., an orthopoxvirus infection (e.g., mpox infection) associated condition).
  • a disease e.g., orthopoxvirus infection, e.g., mpox infection
  • a condition e.g., an orthopoxvirus infection (e.g., mpox infection) associated condition.
  • doses of a composition may depend on various such parameters.
  • a composition e.g., a pharmaceutical composition
  • higher doses or effectively higher doses achieved by a different, more localized route of administration may be used.
  • polypeptide- refers to a polymeric chain of amino acids.
  • a polypeptide has an amino acid sequence that occurs in nature.
  • a polypeptide has an amino acid sequence that does not occur in nature.
  • a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man.
  • a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both.
  • a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids.
  • a polypeptide may comprise D- amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide's N-terminus, at the polypeptide's C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications comprise acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof.
  • a polypeptide may be cyclic, and/or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and/or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides.
  • exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family.
  • a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class).
  • a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%.
  • a conserved region that may in some embodiments be or comprise a characteristic sequence element
  • Such a conserved region usually encompasses at least 3-4 and often up to 35 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more contiguous amino acids.
  • a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide.
  • prevent when used in connection with the occurrence of a disease, disorder, and/or condition, refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.
  • Reference- describes a standard or control relative to which a comparison is performed.
  • an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value.
  • a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest.
  • a reference or control is a historical reference or control, optionally embodied in a tangible medium.
  • a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment.
  • RNA Ribonucleic acid
  • RNA Polyribonucleotide-.
  • ribonucleic acid refers to a polymer of ribonucleotides.
  • an RNA is single stranded.
  • an RNA is double stranded.
  • an RNA comprises both single and double stranded portions.
  • an RNA can comprise a backbone structure as described in the definition of "Nucleic acid / Polynucleotide" above.
  • RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA).
  • mRNA messenger RNA
  • an RNA is an mRNA.
  • an RNA typically comprises at its 3' end a poly(A) region.
  • an RNA typically comprises at its 5' end an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation.
  • an RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and/or by chemical synthesis methods).
  • Ribonucleotide- encompasses unmodified ribonucleotides and modified ribonucleotides.
  • unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U).
  • Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g., replacement with modified bases, stabilizing bases, destabilizing bases, bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages.
  • end modifications e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.)
  • base modifications e.
  • risk of a disease, disorder, and/or condition refers to a likelihood that a particular individual will develop the disease, disorder, and/or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and/or event. In some embodiments, a reference sample or group of reference samples are from individuals comparable to a particular individual.
  • risk may reflect one or more genetic attributes, e.g., which may predispose an individual toward development (or not) of a particular disease, disorder and/or condition.
  • risk may reflect one or more epigenetic events or attributes and/or one or more lifestyle or environmental events or attributes.
  • secretory signal As used herein, the terms “secretory signal” or “signal peptide” or “signal sequence” refer to an amino acid sequence motif that targets associated polypeptides for translocation to a secretory pathway.
  • Selective or specific when used herein in reference to an agent having an activity, is understood by those skilled in the art to mean that the agent discriminates between potential target entities, states, or cells.
  • an agent is said to bind "specifically" to its target if it binds preferentially with that target in the presence of one or more competing alternative targets.
  • specific interaction is dependent upon the presence of a particular structural feature of the target entity (e.g., an epitope, a cleft, a binding site). It is to be understood that specificity need not be absolute.
  • specificity may be evaluated relative to that of a targetbinding moiety for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to that of a reference specific binding moiety. In some embodiments, specificity is evaluated relative to that of a reference non-specific binding moiety.
  • Stability may refer to physicochemical stability of a product (e.g., a pharmaceutical composition described herein). In some embodiments, “stability” refers to physicochemical stability of a product (e.g., a pharmaceutical composition described herein), in unopened thawed vials for up to 24 hours at 30 °C, and in syringes for up to 24 hours at 2- 8 °C and 12 hours at 30 °C.
  • “stability” refers to shelf-life of a product (e.g., a pharmaceutical composition described herein) for 6 months or more when stored at about -90 °C to about 4°C.
  • Substituted or optionally substituted' As described herein, compounds of the invention may contain "optionally substituted” moieties. In general, the term “substituted,” whether preceded by the term “optionally” or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent.
  • an "optionally substituted” group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position.
  • Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds.
  • stable refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in some embodiments, their recovery, purification, and use for one or more of the purposes provided herein.
  • Groups described as being “substituted” preferably have between 1 and 4 substituents, more preferably 1 or 2 substituents. Groups described as being “optionally substituted” may be unsubstituted or be “substituted” as described above.
  • Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are independently halogen; -(CH 2 )o-4R°; -(CH 2 )o-40R°; -0(CH 2 )o- 4 R°, -0-(CH 2 )OMC(0)OR°; -(CH2)O-
  • each R° may be substituted as defined below and is independently hydrogen, Ci-e aliphatic, -CH 2 Ph, -0(CH2)o-iPh, -CH2-(5- to 6-membered heteroaryl ring), or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3- to 12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4
  • Suitable monovalent substituents on R° are independently halogen, -(CH 2 )o- 2 R*, -(haloR*), - (CH 2 )o- 2 OH, -(CH 2 )O- 2 OR*, -(CH 2 )(WCH(OR*)2, -O(haloR*), -CN, -N 3 , -(CH 2 )(wC(0)R*, -(CH 2 )M 2 C(O)OH, - (CH 2 ) ⁇ 2C(O)OR*, -(CH 2 )O- 2 SR*, -(CH 2 )Q- 2 SH, -(CH2K2NH2, -(CH 2 ) ⁇ 2NHR*, -(CH 2 )O-2NR*2, -NO2, -SiR* 3 , - OSiR* 3 , -
  • Suitable divalent substituents that are bound to vicinal substitutable carbons of an "optionally substituted” group include: -O(CR* 2 ) 2-3 O-, wherein each independent occurrence of R* is selected from hydrogen, Ci-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable substituents on the aliphatic group of R* include halogen, -R*, -(haloR*), -OH, -OR*,
  • each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently Ci ⁇ aliphatic, - CH 2 Ph, -0(CH 2 )o-iPh, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include -
  • each R + is independently hydrogen, Ci-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R + , taken together with their intervening atom(s) form an unsubstituted 3- to 12-membered saturated, partially unsaturated,
  • Suitable substituents on the aliphatic group of R + are independently halogen, -R*, -(haloR*), -
  • each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently CM aliphatic, -CH 2 Ph, -0(CH 2 )o-iPh, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
  • Subject- refers to an organism to be administered with a composition described herein, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes.
  • Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans.
  • a subject is a human subject.
  • a subject is suffering from a disease, disorder, or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, rabbitpox, etc.) infection).
  • orthopoxvirus infection e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, rabbitpox, etc.)
  • a subject is susceptible to a disease, disorder, or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection).
  • a disease, disorder, or condition e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection).
  • orthopoxvirus infection e.g., mpox, variola, borealpox, ectromelia, cowpox, vol
  • a subject displays one or more symptoms or characteristics of a disease, disorder, or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection).
  • orthopoxvirus infection e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection.
  • vaccinia e.g., modified vaccinia virus Ankara, etc.
  • a subject displays one or more non-specific symptoms of a disease, disorder, or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection).
  • orthopoxvirus infection e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection.
  • vaccinia e.g., modified vaccinia virus Ankara, etc.
  • a subject does not display any symptom or characteristic of a disease, disorder, or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection).
  • orthopoxvirus infection e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection.
  • a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection).
  • a subject is a patient.
  • a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
  • vaccinia e.g., modified vaccinia virus Ankara, etc. infection
  • orthopoxvirus infection e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection
  • vaccinia e.g., modified vaccinia virus Ankara, etc.
  • orthopoxvirus infection e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection
  • vaccinia e.g., modified vaccinia virus Ankara, etc.
  • orthopoxvirus infection e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection
  • vaccinia e.g., modified vaccinia virus Ankara, etc.
  • an individual who is susceptible to a disease, disorder and/or condition e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection
  • a disease, disorder and/or condition e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection).
  • an individual who is susceptible to a disease, disorder, and/or condition may exhibit symptoms of the disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection) may exhibit symptoms of the disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection).
  • orthopoxvirus infection e.g., mpox, variola, bore
  • an individual who is susceptible to a disease, disorder, and/or condition may not exhibit symptoms of the disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection) may not exhibit symptoms of the disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection).
  • orthopoxvirus infection e.g., mpox, variola
  • an individual who is susceptible to a disease, disorder, and/or condition e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection
  • a disease, disorder, and/or condition e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection).
  • orthopoxvirus infection e.g., mpox, variola, borealpox, ectromelia, cowpo
  • an individual who is susceptible to a disease, disorder, and/or condition e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection
  • a disease, disorder, and/or condition e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection).
  • orthopoxvirus infection e.g., mpox, variola, borealpox, ectromelia, cowpo
  • the term "therapy” refers to an administration or delivery of an agent or intervention that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect (e.g., has been demonstrated to be statistically likely to have such effect when administered to a relevant population).
  • a therapeutic agent or therapy is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection).
  • orthopoxvirus infection e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection.
  • a therapeutic agent or therapy is a medical intervention (e.g., surgery, radiation, phototherapy) that can be performed to alleviate, relieve, inhibit, present, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
  • a medical intervention e.g., surgery, radiation, phototherapy
  • treatment refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection).
  • orthopoxvirus infection e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection.
  • vaccinia e.g., modified
  • Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection).
  • a disease, disorder, and/or condition e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection).
  • orthopoxvirus infection e.g., mpox, variola, borealpox,
  • treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection), for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition.
  • orthopoxvirus infection e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection
  • vaccinia e.g., modified vaccinia virus Ankara, etc
  • treatment may be administered to a subject at a later-stage of disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection).
  • orthopoxvirus infection e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection.
  • vaccinia e.g., modified vaccinia virus Ankara, etc.
  • compositions for delivering particular polyribonucleotide constructs encoding mpox antigens to a subject (e.g., a patient) and related methods for inducing protective immunity to mpox viruses belonging to other clades and other orthopoxviruses (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) viruses).
  • orthopoxviruses e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) viruses.
  • vaccinia e.g., modified vaccinia virus Ankara, etc
  • Mpox virus the causative agent of mpox disease
  • VARV variola virus
  • Mpox disease generally manifests as a selflimiting infection with symptoms including fever, headache, fatigue, lymphadenopathy and skin lesions. Illness is most severe in children, pregnant women, and individuals with underlying immune deficiencies and can be associated with high case fatality rates. However, lethal disease is most frequently caused by clade I MPXV infections (case fatality rates between 6 and 15%). The 2022 international outbreak was driven by clade lib MPXV and was associated with less severe disease.
  • Orthopox viruses are DNA encoded viruses, yielding a high level of similarity between orthopoxvirus antigens, which allows immune responses raised to certain proteins from one virus to potentially provide cross-protective immunity to other viruses within the genus.
  • Smallpox was eradicated by exploiting this potential for cross-protection through global immunization campaigns using another orthopoxvirus, vaccinia virus (VACV), as a live virus vaccine.
  • VACV vaccinia virus
  • First generation VACV immunization provided robust and durable protection against smallpox, but its unfavorable side effects and exclusion from use in certain populations encouraged the development of vaccines with an improved safety profile.
  • VACV-derivatives with improved safety profiles but more modest durability of immune response were available for use prior to May 2022, but stockpiles and existing manufacturing capacity did not meet the demand driven by the 2022 outbreak.
  • novel, potent and safe mpox vaccines is needed, especially vaccines that can be rapidly manufactured at scale and distributed globally.
  • the design of polyribonucleotide constructs encoding mpox antigens of the present disclosure draws from the study of immune responses to live VACV vaccines that formed the basis of the successful global campaign to eradicate smallpox.
  • VACV replication in a host exposes the immune system to the complete proteome of the virus, including greater than 30 virion surface proteins.
  • VACV-mediated adapted immune protection could involve antibody and/or T cell responses to all or many VACV proteins.
  • VACV proteins have differential immunogenicity in a host, suggesting that responses to a subset of antigens are needed for immunity.
  • Envelope proteins can be the targets of both protective antibody and T cell responses since they are accessible to antibodies in intact virions and infected cells.
  • narrowing to mpox envelope proteins may be insufficient to define antigen inclusion, as orthopoxviruses encode dozens of envelope proteins in their large DNA genomes. This feature stands in stark contrast to other viruses like SARS-CoV-2 for which there are only one or a few potential envelope protein targets.
  • Further complicating antigen selection are the two distinct viral forms of orthopoxviruses, extracellular virions (EVs) and mature virions (MVs).
  • the present disclosure provides nucleic acids (e.g., polyribonucleotides) encoding orthopox antigens (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)), antigen constructs thereof, and/or pharmaceutical compositions thereof that are effective for vaccination against one or more orthopox viruses (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)), antigen constructs thereof, and/or pharmaceutical compositions thereof that are effective for vaccination against one or more orthopox viruses (e.g.,
  • the present disclosure also provides the insight that polyribonucleotides, antigen constructs thereof, and pharmaceutical compositions thereof targeting one orthopox species may cross-protect against other orthopoxviruses.
  • provided polyribonucleotides encoding mpox proteins, antigen constructs thereof, and/or pharmaceutical compositions thereof are effective for vaccination against mpox and one or more other orthopox viruses.
  • provided polyribonucleotides encoding mpox proteins, antigen constructs thereof, and/or pharmaceutical compositions thereof are effective for vaccination against mpox and one or more of variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) viruses.
  • provided polyribonucleotides encoding mpox proteins, antigen constructs thereof, and/or pharmaceutical compositions thereof are effective for vaccination against mpox and variola virus.
  • provided polyribonucleotides encoding mpox proteins, antigen constructs thereof, and/or pharmaceutical compositions thereof are effective for vaccination against mpox and rabbitpox. In some embodiments, provided polyribonucleotides encoding mpox proteins, antigen constructs thereof, and/or pharmaceutical compositions thereof are effective for vaccination against mpox and vaccinia virus (e.g., modified vaccinia virus Ankara, etc.). In some embodiments, provided polyribonucleotides encoding mpox proteins, antigen constructs thereof, and/or pharmaceutical compositions thereof are effective for vaccination against mpox and ectromelia virus. In some embodiments, provided polyribonucleotides encoding mpox proteins, antigen constructs thereof, and/or pharmaceutical compositions thereof are effective for vaccination against mpox and a novel orthopox virus.
  • Mpox virions are ovoid or brick-shaped particles which are enclosed by geometrically corrugated lipoprotein outer membrane. Mature mpox virions have a densely packed core containing enzymes, a double-stranded DNA genome, and transcription factors that are protected by a protein core.
  • the mpox genome consists of a linear double-stranded DNA (about 197 kb) covalently joined at its ends by palindromic hairpins, and the inverted terminal repeats (ITRs) are made up of a hairpin loop, tandem repeats, and some open reading frames (ORF).
  • ITRs inverted terminal repeats
  • ORF open reading frames
  • vaccinia virus and most likely in mpox
  • intracellular mature virus MV
  • extracellular- enveloped virus EV
  • MV is released upon cell lysis
  • EV is released from cells via interaction with actin tails, and this is said to be the cause of rapid long distance spread of the virus within the infected host.
  • actin tails the aforementioned features are for VACV, it is likely that these features are common to all orthopoxviruses.
  • CEVs Cell-associated virions
  • IEV intracellular enveloped virus
  • TGN trans-Golgi network
  • virion morphogenesis can be defective resulting in non-infectious dense particles (DPs), but this has not yet been reported for mpox.
  • DPs non-infectious dense particles
  • mpox does not form ATIs or sequester MVs into ATIs because of truncation in the ATIP gene.
  • mpox transmission The two possible means of mpox transmission are animal-to-human transmission and human- to-human transmission. Respiratory droplets and contact with body fluids, contaminated patient's environment or items, and skin lesion of an infected person have been found to be associated with inter-human transmission. Contact between broken skin or mucous membranes and an infected patient's body fluids, respiratory droplets, or scabs is considered a "high risk" exposure that warrants post-exposure vaccination as soon as possible.
  • Congo Basin (CB) clade Central Africa clade
  • WA West Africa
  • Animal-to-human transmission occurs via direct contact with any of the aforementioned natural viral hosts or consumption of these hosts.
  • zoonotic transmission could occur by direct contact with the blood, body fluids, and inoculation from mucocutaneous lesions of an infected animal.
  • Nosocomial transmission has been reported for CB and WA clades of mpox while sexual transmission has been speculated for infected individuals with groin and genital lesions. At present, human-to-animal transmission has not been reported.
  • the reproduction number (Ro) for the CB clade is estimated to be in the range of 0.6-1.0.
  • the Ro has not been estimated for the WA clade of mpox viruses, but it is presumed to be lower than that of the CB clade.
  • the upper limit R o of 1.0 in the CB clade indicates that the viruses will not only sustain human-to-human transmission but may persist in the human population.
  • Exemplary polyribonucleotide constructs of the present disclosure include nucleic acids encoding A35, B6, Ml and/or H3.
  • antigens may be from a certain mpox clade, e.g., from clade I (clade la or clade lb) MPXV, or clade II (e.g., clade Ila or clade lib) MPXV.
  • Wild-type (WT) versions of mpox antigens provided herein can be surface-exposed, single-pass membrane proteins. Recognition of membrane proteins by the immune system can be achieved through trafficking of these proteins to the cell surface in polyribonucleotide-transfected cells.
  • WT versions of A35 and B6 are expected to traffic to the cell surface in polyribonucleotide-transfected cells.
  • WT versions of MV targets e.g., Ml and H3 are not efficiently delivered to the endoplasmic reticulum for eventual transit to the plasma membrane.
  • embedding of MV proteins into the MV envelope may be distinct from the normal host cell biosynthesis pathway for integral membrane proteins and they lack native secretory signal.
  • Mpox B6R (also referred to as MPXVgpl67) is a -35 kD polypeptide.
  • B6R is a type I integral membrane protein with an N-terminal ectodomain, a C-terminal transmembrane helix, and two sushi domains.
  • B6R is classified as a membrane glycoprotein that is a component of the mpox EV envelope.
  • B6R is also classified as being involved in negative regulation of complement activation.
  • B6R polypeptide sequences include, e.g., UniProt accession numbers Q8V4S2, V9NQJ0, A0A0F6N8B7, each of which is incorporated herein by reference in its entirety.
  • Exemplary B6R amino acid sequences are provided in Table 1 and Table 6 below.
  • exemplary polyribonucleotide constructs of the present disclosure comprise one or more polyribonucleotides that encode B6R.
  • the encoded B6R antigen is fully WT.
  • the sequence of the fully WT B6R antigen is derived from early cases in the 2022 mpox outbreak.
  • a B6R antigen is associated with a native secretory signal.
  • Mpox B6R is homologous to vaccinia B5R.
  • Vaccinia B5R see, e.g., accession number
  • AAN78219.1 is a membrane protein that is essential in packaging the intracellular mature virion form intracellular enveloped virions, and is EV-specific.
  • exemplary polyribonucleotide constructs of the present disclosure comprise one or more polyribonucleotides that encode a B6R antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 21-30, 180, or 182, or an antigenic fragment thereof.
  • Mpox A35R (also referred to as MPXV-COP-139, MPXV-SL-139, MPXV-WRAIR139) is a -20 kD polypeptide.
  • A35R is a type II, disulfide-bonded homodimer with a C-terminal ectodomain and a N-terminal transmembrane domain.
  • A35R is classified as a membrane protein, specifically a bifunctional EV membrane phosphoglycoprotein.
  • A35R polypeptide sequences include, e.g., UniProt accession numbers Q8V4U4 and Q80KX2, each of which is incorporated herein by reference in its entirety. Exemplary A35R amino acid sequences are provided in Table 1 and Table 13 below.
  • exemplary polyribonucleotide compositions of the present disclosure comprise one or more polyribonucleotides that encode A35R.
  • the encoded A35R antigen is fully WT.
  • the A35R antigen sequence is derived from early cases in the 2022 mpox outbreak.
  • an A35R antigen is associated with a native secretory signal.
  • Mpox A35R is homologous to vaccinia A33R.
  • Vaccinia A33R see, e.g., accession number
  • AAF63733 incorporated herein by reference in its entirety is a type II integral membrane protein found in EV (extracellular enveloped virus) but not MV, and is highly conserved among orthopoxviruses. See, e.g., DOIs: 10.1128/jvi.72.5.4192-4204.1998.
  • exemplary polyribonucleotide constructs of the present disclosure comprise one or more polyribonucleotides that encode a A35R antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 11-20, 172, or 174, or an antigenic fragment thereof.
  • MPXV MIR (also referred to as MV membrane protein, MPXV-COP-074, MPXV-SL-074) is a -27 kD polypeptide.
  • MIR is a type I integral membrane protein with an N-terminal ectodomain and a C-terminal transmembrane helix.
  • MIR polypeptide sequences include, e.g., UniProt accession numbers Q8V502, Q80KX3, Q5IXU5, each of which is incorporated herein by reference in its entirety. Exemplary MIR amino acid sequences are provided in Table 1 and Table 13 below.
  • exemplary polyribonucleotide compositions of the present disclosure comprise one or more polyribonucleotides that encode MIR.
  • a secretory signal from HSV-1 gD can be added to the N-terminus of MIR.
  • the secretory signal can participate in trafficking MIR to the cell surface for display.
  • MPXV MIR is homologous to vaccinia L1R.
  • Vaccinia L1R see, e.g., accession number
  • AAF63732 incorporated herein by reference in its entirety
  • exemplary polyribonucleotide constructs of the present disclosure comprise one or more polyribonucleotides that encode a MIR antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 31-40, or 158, or an antigenic fragment thereof.
  • H3L also referred to as MV heparin binding surface protein, Envelope protein H3, MPXV-COP-
  • H3L is a type I integral membrane protein with an N-terminal ectodomain and a C-terminal transmembrane helix.
  • H3L polypeptide sequences include, e.g., UniProt accession numbers, Q8V4Z2, Q3I8S1, Q5IXT2, A0A0F6N9X0, each of which is incorporated herein by reference in its entirety). Exemplary H3L amino acid sequences are provided in Table 1 and Table 13 below.
  • exemplary polyribonucleotide compositions of the present disclosure comprise one or more polyribonucleotides that encode H3L.
  • a secretory signal from HSV-1 gD can be added to the N-terminus of H3L.
  • the secretory signal can participate in trafficking H3L to the cell surface for display. Mpox H3L was found to bear high sequence similarity to vaccinia H3L.
  • exemplary polyribonucleotide constructs of the present disclosure comprise one or more polyribonucleotides that encode a H3L antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 51-60, 184, 188, or 190, or an antigenic fragment thereof.
  • the present disclosure utilizes polyribonucleotide (e.g., RNA) technologies as a modality to express one or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) viruses) polypeptide constructs that includes one or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) viruses) antigens, or one or more portions thereof, described herein.
  • orthopoxvirus e.g., mpox, variola, boreal
  • the present disclosure provides polyribonucleotides that encode one or more mpox antigens or fragments thereof.
  • the present disclosure includes the unexpected discovery that mpox antigens provided in Table 1, and fragments thereof, are particularly advantageous for use in preventing or treating mpox infection and other orthopoxvirus infections (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) viruses), e.g., in mpox antigen constructs and/or mpox vaccines as further disclosed herein.
  • orthopoxvirus infections e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified
  • multiple polyribonucleotides are designed such that the multiple polyribonucleotides together encode multiple mpox antigens because of the many surface proteins and complex replication cycle of orthopoxviruses.
  • Orthopoxviruses have two distinct infectious forms, mature virions (MV) and extracellular virions (EV). Each viral form has a unique set of surface antigens. While immune responses raised to any single target can offer some protection from infection, both subunit vaccine and monoclonal antibody prophylaxis studies in animals illustrate the value of combining antibodies to multiple EV and MV targets.
  • exemplary polyribonucleotide constructs of the present disclosure target MPXV proteins from both EVs and MVs: A35 and B6 which are EV surface glycoproteins and Ml and H3 which are displayed on the surface of MVs.
  • A35 and B6 which are EV surface glycoproteins
  • Ml and H3 which are displayed on the surface of MVs.
  • a polyribonucleotide of the present disclosure encodes a single mpox antigen of Table 1 or antigenic fragment thereof (e.g., an A35R polypeptide or antigenic fragment thereof, B6R polypeptide or antigenic fragment thereof, MIR polypeptide or antigenic fragment thereof, or H3L polypeptide or antigenic fragment thereof).
  • an exemplary polyribonucleotide of the present disclosure encodes an A35R polypeptide having a sequence of any one of the A35R sequences of Table 1, or an antigenic fragment thereof.
  • exemplary polyribonucleotide constructs of the present disclosure comprise one or more polyribonucleotides that encode an A35R antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 11-20, 172, or 174, or an antigenic fragment thereof.
  • a polyribonucleotide of the present disclosure encodes a B6R polypeptide having a sequence of any one of the B6R sequences of Table 1, or an antigenic fragment thereof.
  • exemplary polyribonucleotide constructs of the present disclosure comprise one or more polyribonucleotides that encode a B6R antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 21-30, 180, or 182, or an antigenic fragment thereof.
  • a polyribonucleotide of the present disclosure encodes an MIR polypeptide having a sequence of any one of the MIR sequences of Table 1, or an antigenic fragment thereof.
  • exemplary polyribonucleotide constructs of the present disclosure comprise one or more polyribonucleotides that encode a MIR antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 31-40, or 158, or an antigenic fragment thereof.
  • a polyribonucleotide of the present disclosure encodes an H3L polypeptide having a sequence of any one of the H3L sequences of Table 1, or an antigenic fragment thereof.
  • exemplary polyribonucleotide constructs of the present disclosure comprise one or more polyribonucleotides that encode a H3L antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 51-60, 184, 188, or 190, or an antigenic fragment thereof.
  • a polyribonucleotide of the present disclosure encodes two, three, or four mpox antigens of Table 1 or antigenic fragments thereof. In some embodiments, a polyribonucleotide of the present disclosure encodes two or more mpox polypeptides selected from: an A35R polypeptide or antigenic fragment thereof, a B6R polypeptide or antigenic fragment thereof, an MIR polypeptide or antigenic fragment thereof, and an H3L polypeptide or antigenic fragment thereof.
  • a polyribonucleotide of the present disclosure encodes a B6R polypeptide having a sequence of any one of the B6R sequences of Table 1, and an MIR polypeptide having a sequence of any one of the MIR sequences of Table 1.
  • a polyribonucleotide of the present disclosure encodes a B6R antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 21-30, 180, or 182, or an antigenic fragment thereof, and an MIR antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 31-40, or 158, or an antigenic fragment thereof.
  • a polyribonucleotide of the present disclosure encodes an A35R polypeptide having a sequence of any one of the A35R sequences of Table 1, a B6R polypeptide having a sequence of any one of the B6R sequences of Table 1, and an MIR polypeptide having a sequence of any one of the MIR sequences of Table 1.
  • a polyribonucleotide of the present disclosure encodes: (i) an A35R antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 11-20, 172, or 174, or an antigenic fragment thereof; (ii) a B6R antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 21-30, 180, or 182, or an antigenic fragment thereof; and (iii) an MIR antigen comprising an amino acid sequence at least about 85%, at least about 90%
  • a polyribonucleotide of the present disclosure encodes an A35R polypeptide having a sequence of any one of the A35R sequences of Table 1, a B6R polypeptide having a sequence of any one of the B6R sequences of Table 1, an MIR polypeptide having a sequence of any one of the MIR sequences of Table 1, and an H3L polypeptide having a sequence of any one of the H3L sequences of Table 1.
  • a polyribonucleotide of the present disclosure encodes: (i) an A35R antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 11-20, 172, or 174, or an antigenic fragment thereof; (ii) a B6R antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 21-30, 180, or 182, or an antigenic fragment thereof; (iii) an MIR antigen comprising an amino acid sequence at least about 85%, at least about 90%,
  • a polyribonucleotide of the present disclosure encodes one or more MV antigens. In some embodiments, one or more MV antigens are selected from H3L and MIR. In some embodiments, a polyribonucleotide of the present disclosure encodes one or more MV-specific antigens. In some embodiments, one or more MV-specific antigens are selected from H3L and MIR.
  • a polyribonucleotide of the present disclosure encodes one or more EV antigens. In some embodiments, one or more EV antigens are selected from A35R and B6R. In some embodiments, a polyribonucleotide of the present disclosure encodes one or more EV-specific antigens. In some embodiments, one or more EV-specific antigens are selected from A35R and B6R.
  • a polyribonucleotide of the present disclosure encodes one or more
  • A35R polypeptides (e.g., an A35R antigen or one or more antigenic fragments thereof).
  • a polyribonucleotide of the present disclosure encodes one or more B6R polypeptides (e.g., a B6R antigen or one or more antigenic fragments thereof).
  • a polyribonucleotide of the present disclosure encodes one or more MIR polypeptides (e.g., an MIR antigen or one or more antigenic fragments thereof).
  • a polyribonucleotide of the present disclosure encodes one or more H3L polypeptides (e.g., an H3L antigen or one or more antigenic fragments thereof).
  • provided technologies involve administration of a plurality of antigens to the same subject.
  • multiple antigens are administered at the same time (e.g., in a single dose).
  • different antigens may be administered at different times (for example in different doses - e.g., a prime dose vs a boost dose).
  • multiple antigens are administered via the same composition.
  • a single "antigen" polypeptide may include multiple “epitopes", which in turn may or may not be linked with one another in nature.
  • a single string construct antigen includes multiple epitopes, which may be from different parts of the same mpox protein and/or from different mpox proteins, linked together as described herein in a single polypeptide.
  • a single pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as described herein may include or deliver (e.g., because the pharmaceutical composition includes a nucleic acid, such as a polyribonucleotide, that encodes the antigen and is expressed upon administration) a single antigen, which itself may comprise multiple epitopes (either in their natural arrangement relative to one another or in an engineered or constructed arrangement as described herein), or may comprise or deliver a plurality of antigens, each of which similarly may be or comprise a single epitope or multiple epitopes (either in their natural arrangement relative to one another or in an engineered or constructed arrangement as described herein).
  • a single antigen which itself may comprise multiple epitopes (either in their natural arrangement relative to one another or in an engineered or constructed arrangement as described herein), or may comprise or deliver a plurality of antigens, each of which similarly may be or comprise a single epitope or multiple epitopes (either in their natural arrangement relative to
  • a single pharmaceutical composition may, for example, include multiple distinct nucleic acids (e.g., polyribonucleotides) that each encode different antigen(s) or, in some embodiments, may include a single nucleic acid that encodes (and expresses) multiple antigens.
  • nucleic acids e.g., polyribonucleotides
  • a single pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • multiple distinct nucleic acids e.g., polyribonucleotides
  • a single pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • mixtures may include the relevant polyribonucleotides in 1: 1 ratio, or in other ratios as may be preferred (e.g., to achieve a desired relative presentation of antigens or epitopes) in a subject to whom the composition is administered.
  • two or more polyribonucleotide molecules each encoding a different polypeptide can be mixed with particle-forming agents to form nucleic acid containing particles as described above.
  • two or more polyribonucleotide molecules each encoding a different polypeptide can be formulated into separate particle compositions, which are then mixed together.
  • individual populations of nucleic acid containing particles each population comprising a polyribonucleotide molecule encoding a different immunogenic polypeptide or antigenic fragment thereof (e.g., an mpox antigen or antigenic fragment thereof as described herein), can be separately formed and then mixed together, for example, prior to filling into vials during a manufacturing process, or immediately prior to administration (e.g., by an administering health-care professional).
  • a polyribonucleotide molecule encoding a different immunogenic polypeptide or antigenic fragment thereof (e.g., an mpox antigen or antigenic fragment thereof as described herein)
  • an mpox antigen or antigenic fragment thereof as described herein can be separately formed and then mixed together, for example, prior to filling into vials during a manufacturing process, or immediately prior to administration (e.g., by an administering health-care professional).
  • compositions comprising two or more populations of particles (e.g., in some embodiments, lipid nanoparticles), each population comprising at least one polyribonucleotide molecule encoding a different immunogenic polypeptide or antigenic fragment thereof (e.g., an mpox antigen or antigenic fragment thereof).
  • each population may be provided in a composition at a desirable proportion (e.g., in some embodiments, each population may be provided in a composition in an amount that provides the same amount of polyribonucleotide molecules).
  • one or more pharmaceutical compositions may comprise or deliver a combination comprising a polypeptide or portion thereof encoded by all or part of A35R, B6R, MIR, H3L, and/or antigenic fragments of any thereof.
  • one or more pharmaceutical compositions comprise or deliver a combination comprising one or more MV antigens (e.g., MV- specific antigens) and one or more EV antigens (e.g., EV-specific antigens).
  • MV antigens e.g., MV- specific antigens
  • EV antigens e.g., EV-specific antigens
  • one or more pharmaceutical compositions comprise or deliver a combination of mpox antigens that includes (i) one or more MV antigens (e.g., MV-specific antigens) selected from H3L, MIR, and antigenic fragments of any thereof; and (ii) one or more EV antigens (e.g., EV-specific antigens) selected from A35R, B6R, and antigenic fragments of any thereof.
  • MV antigens e.g., MV-specific antigens
  • EV-specific antigens selected from A35R, B6R, and antigenic fragments of any thereof.
  • one or more pharmaceutical compositions may comprise or deliver a combination comprising a polypeptide or portion thereof encoded by all or part of B6R, MIR, and/or antigenic fragments of any thereof.
  • one or more pharmaceutical compositions may comprise or deliver a combination comprising a polypeptide or portion thereof encoded by all or part of B6R, A35R, and/or antigenic fragments of any thereof.
  • one or more pharmaceutical compositions may comprise or deliver a combination comprising a polypeptide or portion thereof encoded by all or part of B6R, A35R, MIR, and/or antigenic fragments of any thereof.
  • one or more pharmaceutical compositions may comprise or deliver a combination comprising a polypeptide or portion thereof encoded by all or part of B6R, A35R, MIR, H3L, and/or antigenic fragments of any thereof.
  • a provided composition includes or delivers an mpox envelope glycoprotein antigen (e.g., a full-length mpox envelope glycoprotein, a fragment thereof, or one or more epitopes thereof, for example in a string construct).
  • a provided composition includes or delivers such an mpox envelope glycoprotein antigen together with one or more B cell targets (e.g., epitopes) which may, for example, be or comprise one or more other mpox proteins (or fragments or epitopes thereof).
  • B cell target is or comprises an mpox protein (or fragment or epitope thereof) that is predicted or known to induce a B cell response in infected humans.
  • a B cell target is or comprises an mpox protein (or fragment or B cell epitope thereof) against which sera from infected individual(s) is reactive.
  • a B cell target is or comprises an mpox envelope glycoprotein, or other relevant mpox protein, or a fragment or epitope thereof.
  • a provided composition comprises or delivers a string construct antigen that includes a plurality of T cell epitopes, optionally from more than one mpox protein.
  • a provided composition further comprises or delivers one or more B cell targets.
  • a string construct antigen so utilized includes mpox sequences (e.g., one or more fragments or epitopes, e.g., T cell epitopes and/or B cell epitopes, but in some embodiments specifically T cell epitopes).
  • a string construct antigen includes both B cell epitopes and T cell epitopes (optionally from the same mpox protein or from different mpox proteins).
  • different antigens may be delivered by administration of different compositions, which in turn may, in some embodiments, be administered at the same time (e.g., as an admixture or otherwise substantially simultaneously) and, in some embodiments, may be administered at different times.
  • a particular antigen or antigen(s) may be delivered via an initial pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) dose, and one or more other antigen(s) may be delivered via one or more booster dose(s).
  • an antigen utilized i.e., included in and/or otherwise delivered by
  • a pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • an antigen may comprise two or more epitopes from the same mpox protein and in their natural configuration relative to one another (e.g., in a fragment if the relevant protein). In some embodiments, however, an antigen may comprise at least two epitopes configured in a non-natural relationship relative to one another (e.g., included in a string construct).
  • polyribonucleotide e.g., RNA
  • RNA polyribonucleotide
  • the present disclosure provides an insight that various features of nucleic acid formats including, for example their flexibility and amenability to rapid design and modification, including incorporation of a variety of insights (e.g., bioinformatics inputs etc.), renders them particularly attractive for use in an mpox vaccine.
  • one or more polyribonucleotides utilized in a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as described herein encodes a plurality of epitopes (e.g., including one or more, or two or more, sequences provided in Table 1, or antigenic fragments thereof), optionally wherein each of the plurality is predicted by an H LA binding and presentation prediction algorithm to be of high likelihood to be presented by a protein encoded by an H LA to a T cell for eliciting an immune response.
  • the plurality of epitopes comprises epitopes from a single mpox protein.
  • the plurality of epitopes comprises epitopes from multiple mpox proteins.
  • one or more polyribonucleotides e.g., mRNAs
  • a pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • a pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • an mpox antigen encoded by a first polyribonucleotide is or comprises an mpox antigen, fragment, or epitope, e.g., an A35R, B6R, MIR, and/or H3L, or antigenic fragments thereof, epitopes thereof, and/or a combination thereof.
  • one or more polyribonucleotides e.g., mRNAs
  • a pharmaceutical composition e.g., immunogenic composition, e.g., vaccine
  • a second polyribonucleotide that encodes a multi-epitopic (e.g., polyepitopic) antigen.
  • a polyribonucleotide construct can include an internal ribosome entry site (IRES), e.g., between two sequences encoding antigens or fragments thereof engineered for expression from a polyribonucleotide as distinct polypeptides.
  • IRSs internal ribosome entry sites
  • IVSs Internal ribosome entry sites are cis-acting elements that can recruit the small ribosomal subunits to an internal initiator codon in a polyribonucleotide in conjunction with cellular trans-acting factors.
  • IRES internal ribosome entry site
  • EMCV encephalomyocarditis virus
  • a multi-epitope polyribonucleotide encoding a super-motif-bearing or motif-bearing polypeptide, together with a helper epitope (e.g., a heterologous helper epitope) and an endoplasmic reticulum-translocating signal sequence.
  • a helper epitope e.g., a heterologous helper epitope
  • an endoplasmic reticulum-translocating signal sequence See, for example, in An & Whitton J. Virol. 71:2292, 1997; Thomson, eta/., J. Immuno! 157:822, 1996; Whitton, eta!., J. Virol 67 :348, 1993; Hanke, et a!., Vaccine 16:426, 1998.
  • polyribonucleotides described herein include other elements such as described below, including, a secretion signal-encoding region, a 5' Cap, a Cap proximal sequence, a 5' UTR, a 3' UTR, and/or a polyA tail.
  • polyribonucleotides described herein can comprise a secretion signal-encoding region.
  • epitopes encoded in a string construct may be flanked by a secretory signal sequence, e.g., SP1 sequence (HSV-1 gD secretory signal /secretory domain, SEQ ID NO: 143).
  • polyribonucleotides described herein can comprise a nucleotide sequence that encodes a 5' UTR of interest and/or a 3' UTR of interest.
  • a polynucleotide comprises a dEarl-hAg sequence (SEQ ID NO: 155).
  • the polyribonucleotide e.g., mRNA
  • the polyribonucleotide comprises a 5 'UTR and a 3' UTR.
  • a 3' UTR comprises a poly A sequence.
  • a poly A sequence comprises between 50-200 nucleotides.
  • a poly A tail of a string construct may comprise about 150 A residues.
  • a poly A tail may comprise 120 residues or less. In some embodiments, a poly A tail of a string construct may comprise about 120 A residues. In some embodiments, a poly A tail of a string construct may comprise about 100 A residues. In some embodiments, a poly A tail of a string construct comprises a "split" or "interrupted" poly A tail (e.g., as described in W02016/005324). In some embodiments, polyribonucleotides described herein may comprise a 5' cap, which may be incorporated during transcription, or joined to a polyribonucleotide posttranscription.
  • a polyribonucleotide described herein comprises a sequence encoding a human secretory signal.
  • a human secretory signal may be or comprises the amino acid sequence of MDWIWRILFLVGAATGAHS (husec2; SEQ ID NO: 142).
  • a ribonucleic acid sequence encoding a secretory signal included in a polyribonucleotide consists of or comprises a nucleotide sequence that encodes a non-human secretory signal.
  • a polyribonucleotide encodes a human secretory signal where the secretory signal comprises the amino acid sequence MDWIWRILFLVGAATGAHS (husec2; SEQ ID NO: 142).
  • an RNA sequence encodes an orthopoxvirus (e.g., mpox) antigen or antigenic fragment thereof that may comprise or otherwise be linked to a secretory signal (e.g., a secretory sequence), such as those listed in Table 2, or a sequence having 1, 2, 3, 4, or 5 amino acid differences relative thereto.
  • a secretory signal such as MRVMAPRTLILLLSGALALTETWAGS (SEQ ID NO: 157), or a sequence having 1, 2, 3, 4, or at the most 5 amino acid differences relative thereto is utilized.
  • a secretory signal is selected from those included in Table 2 below:
  • a structural feature of mRNAs is a cap structure at the five-prime end (S').
  • Natural eukaryotic mRNA comprises a 7-methylguanosine cap linked to the mRNA via a 5 ' to 5 '-triphosphate bridge resulting in capO structure (m7GpppN).
  • capO structure m7GpppN
  • further modifications can occur at the 2'-hydroxy-group (2'-0H) (e.g., the 2'-hydroxyl group may be methylated to form 2'-0-Me) of the first and subsequent nucleotides producing "capl" and "cap2" five-prime ends, respectively).
  • RNA capping is well researched and is described, e.g., in Decroly E., et al. (2012) Nature
  • a 5'-cap structure which may be suitable in the context of the present invention is a capO (methylation of the first nucleobase, e.g.
  • capl additional methylation of the ribose of the adjacent nucleotide of m7GpppN
  • cap2 additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN
  • cap3 additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN
  • cap4 additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN
  • ARCA anti-reverse cap analogue
  • modified ARCA e.g.
  • RNA e.g., mRNA
  • 5'-cap refers to a structure found on the 5'-end of an RNA, e.g., mRNA, and generally includes a guanosine nucleotide connected to an RNA, e.g., mRNA, via a 5'- to 5'- triphosphate linkage (also referred to as Gppp or G(5')ppp(5')).
  • a guanosine nucleoside included in a 5' cap may be modified, for example, by methylation at one or more positions (e.g., at the 7- position) on a base (guanine), and/or by methylation at one or more positions of a ribose.
  • a guanosine nucleoside included in a 5' cap comprises a 3'0 methylation at a ribose (3'0MeG).
  • a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine (m7G).
  • a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine and a 3' 0 methylation at a ribose (m7(3'OMeG)).
  • m7(3'OMeG) a 3' 0 methylation at a ribose
  • providing an RNA with a 5'-cap disclosed herein may be achieved by in vitro transcription, in which a 5'-cap is co-transcriptionally expressed into an RNA strand, or may be attached to an RNA post-transcriptionally using capping enzymes.
  • co-transcriptional capping with a cap disclosed improves the capping efficiency of an RNA compared to co-transcriptional capping with an appropriate reference comparator.
  • improving capping efficiency can increase a translation efficiency and/or translation rate of an RNA, and/or increase expression of an encoded polypeptide.
  • alterations to polynucleotides generates a non-hydrolyzable cap structure which can, for example, prevent de-capping and increase RNA half-life.
  • T7 RNA polymerase prefers G as the initial site. Accordingly, in some such embodiments, the present disclosure provides caps (e.g., trinucleotide and tetranucleotide caps described herein) wherein the 3'end of the trinucleotide (e.g., N2) or tetranucleotide cap (e.g., N3) is G.
  • caps e.g., trinucleotide and tetranucleotide caps described herein
  • a utilized 5' cap is a capO, a capl, or cap2 structure. See, e.g., Fig. 1 of
  • an RNA described herein comprises a capl structure. In some embodiments, an RNA described herein comprises a cap2.
  • an RNA described herein comprises a capO structure.
  • a capO structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m 7 )G).
  • such a capO structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as (m 7 )Gppp.
  • a capO structure comprises a guanosine nucleoside methylated at the 2'-position of the ribose of guanosine.
  • a capO structure comprises a guanosine nucleoside methylated at the 3'-position of the ribose of guanosine.
  • a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine and at the 2'-position of the ribose ((m2 72 °)G).
  • a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine and at the 2'-position of the ribose ((m2 73 °)G).
  • a capl structure comprises a guanosine nucleoside methylated at the
  • a capl structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m 7 )G) and the 3' position of the ribose, and a 2'0 methylated first nucleotide in an RNA ((m 2 ' °)Ni).
  • a capl structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as, e.g., ((m 7 )Gppp( 2 ' °)Ni) or
  • a capl structure comprises a second nucleotide, N 2 , which is at position 2 and is chosen from A, G, C, or U, e.g.,
  • a cap2 structure comprises a guanosine nucleoside methylated at the
  • a cap2 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m 7 )G) and the 3' position of the ribose, and a 2'0 methylated first and second nucleotide in an RNA.
  • a cap2 structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as, e.g.,
  • a 5' cap is a dinucleotide cap structure. In some embodiments, a 5' cap is a dinucleotide cap structure comprising Ni, wherein Ni is as defined and described herein. In some embodiments, a 5' cap is a dinucleotide cap G*Ni, wherein Ni is as defined above and herein, and:
  • G* comprises a structure of formula (I): or a salt thereof, wherein each R 2 and R 3 is -OH or -OCH3; and X is 0 or S.
  • R 2 is -OH. In some embodiments, R 2 is -OCH3. In some embodiments,
  • R 3 is -OH. In some embodiments, R 3 is -OCH3. In some embodiments, R 2 is -OH and R 3 is -OH. In some embodiments, R 2 is -OH and R 3 is -CH3. In some embodiments, R 2 is -CH3 and R 3 is -OH. In some embodiments, R 2 is -CH3 and R 3 is -CH3.
  • X is 0. In some embodiments, X is S.
  • a 5' cap is a dinucleotide capO structure (e.g., (m 7 )GpppNi,
  • the 5' cap is a dinucleotide capO structure (e.g., (m 7 )GpppNi, (m2 7 ' 2 ' °)GpppNi, (m2 7 ' 3 ' °)GpppNi, (m 7 )GppSpNi, (m2 7 ' 2 ' °)GppSpNi, or (m2 7 ' 3 ' °)GppSpNi), wherein Ni is G.
  • a dinucleotide capO structure e.g., (m 7 )GpppNi, (m2 7 ' 2 ' °)GpppNi, (m2 7 ' 3 ' °)GpppNi
  • Ni is G.
  • the 5' cap is a dinucleotide capO structure (e.g., (m 7 )GpppNi, (m2 7 ' 2 ' °)GpppNi, (m2 7 ' 3 ' °)GpppNi, (m 7 )GppSpNi, (m2 7 ' 2 ' °)GppSpNi, or (m2 7 ' 3 ' °)GppSpNi), wherein Ni is A, U, or C.
  • a dinucleotide capO structure e.g., (m 7 )GpppNi, (m2 7 ' 2 ' °)GpppNi, (m2 7 ' 3 ' °)GpppNi
  • Ni is A, U, or C.
  • a 5' cap is a dinucleotide capl structure (e.g., (m 7 )Gppp(m 2 ' °)Ni, (m2 7 ' 2 ' °)Gppp(m 2 ' °)Ni, (m2 7 ' 3 '- °)Gppp(m 2 ' °)Ni, (m 7 )GppSp(m 2 ' °)Ni, (m2 7 ' 2 ' °)GppSp(m 2 ' °)Ni, or (m2 7 ' 3 ' °)GppSp(m 2 ' °)Ni), wherein Ni is as defined and described herein.
  • Ni is as defined and described herein.
  • the 5' cap is selected from the group consisting of (m 7 )GpppG C'EcapO"), (m 7 )Gppp(m 2 ' °)G (“Ecapl”), (m 2 7 - 3 ' °)GpppG ("ARCA” or “DI”), and (m 2 7 - 2 ' °)GppSpG
  • the 5' cap is (m 7 )GpppG (“EcapO”), having a structure: or a salt thereof.
  • a 5' cap is (m 7 )Gppp(m 2 ' °)G ("Ecapl”), having a structure: or a salt thereof.
  • a 5' cap is (m 2 7 - 3 ' °)GpppG ("ARCA” or "DI”), having a structure: or a salt thereof.
  • a 5' cap is (m 2 7 ' 2 ' °)GppSpG ("beta-S-ARCA"), having a structure: or a salt thereof.
  • a 5' cap is a trinucleotide cap structure.
  • a 5' cap is a trinucleotide cap structure comprising NipN 2 , wherein Ni and N 2 are as defined and described herein.
  • a 5' cap is a trinucleotide cap G*NipN 2 , wherein Ni and N 2 are as defined above and herein, and: G* comprises a structure of formula (I): or a salt thereof, wherein R 2 , R 3 , and X are as defined and described herein.
  • a 5' cap is a trinucleotide capO structure (e.g. (m 7 )GpppNipN2, (m2 7 2 -
  • a 5' cap is a trinucleotide capl structure (e.g., (m 7 )Gppp(m 2 ' °)NipN2, (m2 7 ' 2 O )Gppp(m 2 ' °)NipN2, (m2 7 ' 3 ' °)Gppp(m 2 ' °)NipN2), wherein Ni and N2 are as defined and described herein.
  • a 5' cap is a trinucleotide cap2 structure (e.g., (m 7 )Gppp(m 2 ' °)Nip(m 2 ' °)N2, (m2 7 ' 2 ' °)Gppp(m 2 ' 0 )Nip(m 2 ' °)N2, (m2 7 ' 3 '- °)Gppp(m 2 ' 0 )Nip(m 2 ' °)N2), wherein Ni and N2 are as defined and described herein.
  • a 5' cap is selected from the group consisting of (m2 7 ' 3 ' °)Gppp(m 2 ' °)ApG ("CleanCap AG 3' OMe", “CC413"), (m2 7 ' 3 '- °)Gppp(m 2 ' °)GpG (“CleanCap GG"), (m 7 )Gppp(m 2 ' °)ApG, (m 7 )Gppp(m 2 ' °)GpG, (m2 7 ' 3 ' °)Gppp(m2 5 ' 2 ' °)ApG, and (m 7 )Gppp(m 2 ' °)ApU.
  • a 5' cap is selected from the group consisting of (m2 7 ' 3 ' °)Gppp(m 2 ' _ °)ApG ("CleanCap AG", “CC413"), (m2 7 - 3 ' 0 )Gppp(m 2 '- 0 )GpG (“CleanCap GG”), (m 7 )Gppp(m 2 ' °)ApG, and (m 2 7 - 3 '- °)Gppp(m2 5 ' 2 ' °)ApG, (m 7 )Gppp(m 2 ' °)ApU, and (m2 7 ' 3 ' °)Gppp(m 2 ' °)CpG.
  • a 5' cap is (m2 7 ' 3 ' °)Gppp(m 2 ' °)ApG ("CleanCap AG 3' OMe", "CC413”), having a structure: or a salt thereof.
  • a 5' cap is (m2 7 ' 3 ' °)Gppp(m 2 ' °)GpG ("CleanCap GG”), having a structure:
  • a 5' cap is (m 7 )Gppp(m 2 ' °)ApG, having a structure: or a salt thereof.
  • a 5' cap is (m 7 )Gppp(m 2 ' °)GpG, having a structure: or a salt thereof.
  • a 5' cap is (m2 7 ' 3 ' °)Gppp(m2 5 ' 2 ' °)ApG, having a structure:
  • a 5' cap is (m2 7 ' 3 ' °)Gppp(m 2 ' °)CpG, having a structure: or a salt thereof.
  • a 5' cap is a tetranucleotide cap structure.
  • a 5' cap is a tetranucleotide cap structure comprising NipN 2 pN3, wherein Ni, N 2 , and N3 are as defined and described herein.
  • a 5' cap is a tetranucleotide cap G*NipN2pN3, wherein Ni, N 2 , and N3 are as defined above and herein, and:
  • G* comprises a structure of formula (I): or a salt thereof, wherein R 2 , R 3 , and X are as defined and described herein.
  • a 5' cap is a tetranucleotide capO structure (e.g., (m 7 )GpppNipN2pN3,
  • a 5' cap is a tetranucleotide Capl structure (e.g., (m 7 )Gppp(m 2 ' °)NipN2pN3, (m2 7 ' 2 ’ °)Gppp(m 2 ' °)NipN2pN3, (m2 7 ' 3 ' °)Gppp(m 2 ' °)NipN2N3), wherein Ni, N 2 , and N3 are as defined and described herein.
  • a 5' cap is a tetranucleotide Cap2 structure (e.g., (m 7 )Gppp(m 2 ' °)Nip(m 2 ' _ °)N 2 pN 3 , (m2 7 ' 2 ' 0 )Gppp(m 2 ' °)Nip(m 2 ' °)N2pN3, (m2 7 ' 3 ' 0 )Gppp(m 2 ' °)Nip(m 2 ' °)N2pN3), wherein Ni, N 2 , and N3 are as defined and described herein.
  • a 5' cap is selected from the group consisting of (m2 7 3 > 0 )Gppp(m 2 ' °)Ap(m 2 ' °)GpG, (m2 7 ' 3 ' 0 )Gppp(m 2 ' °)Gp(m 2 ' °)GpC, (m 7 )Gppp(m 2 ' °)Ap(m 2 ' °)UpA, and (m 7 )Gppp(m 2 - °)Ap(m 2 ' 0 )GpG.
  • a 5' cap is (m2 7 ' 3 ' 0 )Gppp(m 2 ' °)Ap(m 2 ' °)GpG, having a structure: or a salt thereof.
  • a 5' cap is (m2 7 ' 3 ' 0 )Gppp(m 2 ' °)Gp(m 2 ' °)GpC, having a structure:
  • a 5' cap is (m 7 )Gppp(m 2 ' °)Ap(m 2 ' °)UpA, having a structure: or a salt thereof.
  • a 5' cap is (m 7 )Gppp(m 2 ' °)Ap(m 2 ' °)GpG, having a structure:
  • Ni is A or an analog thereof. In some embodiments, Ni is adenosine.
  • Ni is modified adenosine. In some embodiments, Ni is 6-methyladenosine. In some embodiments, Ni is: wherein % represents the point of attachment to G*.
  • N 2 is U or an analog thereof. In some embodiments, N 2 is a modified
  • N 2 is 3-methyl-uridine (m 3 U), 5-methoxy-uridine (mo 5 U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s 2 U), 4-thio-uridine (s 4 U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxy-uridine (ho 5 U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5- oxyacetic acid (cmo 5 U), uridine 5-oxyacetic acid methyl ester (mcmo 5 U), 5-carboxymethyl-uridine (cm 5 U), 1- carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm 5 U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm 5 U), 5-methoxycarbon
  • Y 1 is 0 or S
  • Y 2 is N, C, or CH
  • Y 3 is N, NR al , CR al , or CHR al ;
  • Y 4 is NR a2 or CHR a2 ; each of R al or R a2 is independently hydrogen or Ci-6 aliphatic;
  • R 4 is -OH or -OMe
  • # represents the point of attachment to p of Nip.
  • Y 1 is 0. In some embodiments, Y 1 is S.
  • Y 2 is N. In some embodiments, Y 2 is C or CH. In some embodiments,
  • Y 2 is C. In some embodiments, Y 2 is CH.
  • Y 3 is N or CR al . In some embodiments, Y 3 is N. In some embodiments,
  • Y 3 is CR al . In some embodiments, Y 3 is CH or C(CH3). . In some embodiments, Y 3 is CH. In some embodiments, Y 3 is C(CH3). In some embodiments, Y 3 is NR al or CHR al . In some embodiments, Y 3 is NH or N(CH3). In some embodiments, Y 3 is NH, In some embodiments, Y 3 is N(CHs). In some embodiments, Y 3 is CH 2 or CH(CH3). In some embodiments, Y 3 is CH 2 . In some embodiments, Y 3 is CH(CH3).
  • Y 4 is NR a2 . In some embodiments, Y 4 is NH or NCH3. In some embodiments, Y 4 is NH. In some embodiments, Y 4 is NCH3. In some embodiments, Y 4 is CHR a2 . In some embodiments, Y 4 is CH 2 or CH(CH3). In some embodiments, Y 4 is CH 2 . In some embodiments, Y 4 is CH(CH3). [0243] In some embodiments, R al is hydrogen. In some embodiments, R al is C1-6 aliphatic. In some embodiments, R al is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, R al is methyl.
  • R a2 is hydrogen. In some embodiments, R a2 is C1-6 aliphatic. In some embodiments, R a2 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, R a2 is methyl.
  • R 4 is -OH. In some embodiments, R 4 is -OMe.
  • N 2 is of formula (Ila): or a salt thereof, wherein each of Y 1 , Y 3 , R 4 , and # is as defined above and described herein.
  • N 2 is of formula (lib) : or a salt thereof, wherein each of Y 1 , Y 3 , R 4 , and # is as defined above and described herein.
  • N 2 is uridine, 1-methylpsuedouridine, 2-thio-uridine, or 5-methyluridine.
  • N2 is: or a salt thereof, wherein # represents the point of attachment to p of Nip.
  • N 2 is:
  • a 5' cap is (m 7 - 2 ' 0 )Gppp(m 2 ' °)AipU2, (m 7 ' 3 ' °)Gppp(m 2 ' °)AipU2, (m 7 - 2 '-
  • a 5' cap is (m 7 ' 2 ' °)Gppp(m 5 ' 2 ' °)AipU2, (m 7 ' 3 ' °)Gppp(m 5 ' 2 ' °)AipU2, (m 7 ' 2 ' 0 )Gppp(m 5 ' 2 ' °)A1p ⁇ 2, (m 7 ' 3 ' 0 )Gppp(m 5 ' 2 ' °)Aip4J2, (m 7 ' 2 ' °)Gppp(m 5 ' 2 ' °)Aip(m 1 )4J2, (m 7 ' 3 ' °)Gppp(m 5 ' 2 ' _ °)Aip(m 1 ) ⁇ 2, (m 7 ' 2 ' °)Gppp(m 5 ' 2 ' °)AipS 2 U2, (m 7 ' 3 ' °)G
  • a 5' cap is (m 7 ' 2 ' °)Gppp(m 2 ' °)AipU2 having a structure: or a salt thereo f.
  • a 5' cap is (m 7 ' 3 ' °)Gppp(m 2 ' °)AipU2,
  • a 5' cap is (m 7 - 3 ' 0 )Gppp(m 2 '°)Aip4J2, or a salt thereof.
  • a 5' cap is (m 7 - 2 ' 0 )Gppp(m 2 '°)Aip4J2,
  • a 5' cap is (m 7 ' 2 ' 0 )Gppp(m 2 ' 0 )Aip(m 1 ) ⁇ 2, or a salt thereof.
  • a 5' cap is (m 7 ' 3 ' 0 )Gppp(m 2 ' 0 )Aip(m 1 ) ⁇ 2,
  • a 5' cap is (m 7 ' 3 '°)Gppp(m 2 '°)AipS 2 U2, or a salt thereof.
  • a 5' cap is (m 7 ' 2 '°)Gppp(m 2 '°)AipS 2 U2, or a salt thereof.
  • a 5' cap is (m 7 ' 3 ' °)Gppp(m 2 ' °)Aip(m 5 )U2, or a salt thereof.
  • a 5' cap is (m 7 ' 2 ' °)Gppp(m 2 ' °)Aip(m 5 )U2, or a salt there of.
  • 5' caps themselves or as part of a larger molecule (e.g., an RNA).
  • a larger molecule e.g., an RNA
  • the structures drawn above encompass a 3' ether linkage to the next nucleotide or as a free -OH.
  • a 5' UTR utilized in accordance with the present disclosure comprises a cap proximal sequence, e.g., as disclosed herein.
  • a cap proximal sequence comprises a sequence adjacent to a 5' cap.
  • a cap proximal sequence comprises nucleotides in positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.
  • a cap structure comprises one or more polynucleotides of a cap proximal sequence.
  • a cap structure comprises an m 7 Guanosine cap and nucleotide +1 (N i) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m 7 Guanosine cap and nucleotide +2 (N2) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m 7 Guanosine cap and nucleotides +1 and +2 (Ni and N2) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m 7 Guanosine cap and nucleotides +1, +2, and +3 (Ni, N2, and N3) of an RNA polynucleotide.
  • one or more residues of a cap proximal sequence may be included in an RNA by virtue of having been included in a cap entity (e.g., a capl or cap2 structure, etc); alternatively, in some embodiments, at least some of the residues in a cap proximal sequence may be enzymatically added (e.g., by a polymerase such as a T7 polymerase).
  • +1 i.e., Ni
  • +2 i.e., N2
  • +3, +4, and +5 are added by polymerase (e.g., T7 polymerase).
  • a 5' cap is a dinucleotide cap structure, wherein the cap proximal sequence comprises Ni of the 5' cap, where Ni is any nucleotide, e.g., A, C, G or U.
  • a 5' cap is a trinucleotide cap structure (e.g., the trinucleotide cap structures described above and herein), wherein the cap proximal sequence comprises Ni and N2 of the 5' cap, wherein Ni and N2 are independently any nucleotide, e.g., k, C, G or U.
  • a 5' cap is a tetranucleotide cap structure (e.g., the trinucleotide cap structures described above and herein), wherein the cap proximal sequence comprises Ni, N2, and N3 of the 5' cap, wherein Ni, N2, and N3 are any nucleotide, e.g., A, C, G or U.
  • a cap proximal sequence comprises Ni of a the 5' cap, and N2, N3, N4 and N5, wherein Ni to Ns correspond to positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.
  • a cap proximal sequence comprises Ni and N2 of a the 5' cap, and N3, N4 and Ns, wherein Ni to Ns correspond to positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.
  • a cap proximal sequence comprises Ni, N2, and N3 of a the 5' cap, and N 4 and Ns, wherein Ni to Ns correspond to positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.
  • Ni is A. In some embodiments, Ni is C. In some embodiments, Ni is
  • Ni is U.
  • N2 is A.
  • N2 is C.
  • N2 is G.
  • N2 is U.
  • N3 is A.
  • N3 is C.
  • N3 is G.
  • N3 is U.
  • N4 is A.
  • N4 is C.
  • N4 is G.
  • N4 is U.
  • N 5 is A.
  • N5 is C.
  • Ns is G. In some embodiments, N 5 is U.
  • a 5'-UTR may comprise a plurality of distinct sequence elements; in some embodiments, such plurality may be or comprise multiple copies of one or more particular sequence elements (e.g., as may be from a particular source or otherwise known as a functional or characteristic sequence element).
  • a 5' UTR comprises multiple different sequence elements.
  • UTR untranslated region
  • RNA polynucleotide DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA polynucleotide, such as an mRNA molecule.
  • An untranslated region can be present 5' (upstream) of an open reading frame (5'-UTR) and/or 3' (downstream) of an open reading frame (3'-UTR).
  • the terms "five prime untranslated region” or "5' UTR” refer to a sequence of a polyribonucleotide between the 5' end of the polyribonucleotide (e.g., a transcription start site) and a start codon of a coding region of the polyribonucleotide.
  • 5' UTR refers to a sequence of a polyribonucleotide that begins at the 5' end of the polyribonucleotide (e.g., a transcription start site) and ends one nucleotide (nt) before a start codon (usually AUG) of a coding region of the polyribonucleotide, e.g., in its natural context.
  • a 5' UTR comprises a Kozak sequence.
  • a 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap.
  • a 5' UTR disclosed herein comprises a cap proximal sequence, e.g., as defined and described herein.
  • a cap proximal sequence comprises a sequence adjacent to a 5' cap.
  • Exemplary 5' UTRs include a human alpha globin (hAg) 5'UTR or a fragment thereof, a TEV 5'
  • an RNA disclosed herein comprises a hAg 5' UTR or a fragment thereof.
  • an RNA disclosed herein comprises a 5' UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 5' UTR with the sequence according to SEQ ID NO: 155 (AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC).
  • an RNA disclosed herein comprises a 5' UTR provided in SEQ ID NO: 155.
  • an RNA disclosed herein comprises a 5' UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 5' UTR with the sequence AACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 272)(hAg-Kozak/5'UTR).
  • an RNA disclosed herein comprises a 5' UTR provided in SEQ ID NO: 272.
  • a polynucleotide e.g., DNA, RNA
  • a polyadenylate sequence e.g., as described herein.
  • a polyA sequence is situated downstream of a 3'-UTR, e.g., adjacent to a 3'-UTR.
  • poly(A) sequence or "poly-A tail” refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an RNA polynucleotide.
  • Poly(A) sequences are known to those of skill in the art and may follow the 3'-UTR in the RNAs described herein.
  • An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical.
  • polynucleotides disclosed herein comprise an uninterrupted Poly(A) sequence.
  • polynucleotides disclosed herein comprise interrupted Poly(A) sequence.
  • RNAs disclosed herein can have a poly(A) sequence attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase.
  • a poly(A) sequence of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (S') of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017, which is herein incorporated by reference).
  • a poly(A) sequence in accordance with the present disclosure is not limited to a particular length; in some embodiments, a poly(A) sequence is any length. In some embodiments, a poly(A) sequence comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides.
  • nucleotides in the poly(A) sequence typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly(A) sequence are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate).
  • consists of means that all nucleotides in the poly(A) sequence, i.e., 100% by number of nucleotides in the poly(A) sequence, are A nucleotides.
  • a nucleotide or “A” refers to adenylate.
  • a poly(A) sequence is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand.
  • the DNA sequence encoding a poly(A) sequence (coding strand) is referred to as a poly(A) cassette.
  • a poly(A) cassette present in a coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • a cassette is disclosed in WO 2016/005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 Al may be used in accordance with the present disclosure.
  • a poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on a DNA level, constant propagation of plasmid DNA in £ co// and is still associated, on an RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed.
  • a poly(A) sequence contained in an RNA polynucleotide described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
  • no nucleotides other than A nucleotides flank a poly(A) sequence at its
  • 3'-end i.e., the poly(A) sequence is not masked or followed at its 3'-end by a nucleotide other than A.
  • a poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, a poly(A) sequence may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, a poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides.
  • a poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, a poly(A) sequence comprises about 120 nucleotides. [0286] In some embodiments, a poly A tail comprises a specific number of adenosines, such as about
  • a poly A tail of a string construct may comprise 200 A residues or less. In some embodiments, a poly A tail of a string construct may comprise about 200 A residues. In some embodiments, a poly A tail of a string construct may comprise 180 A residues or less. In some embodiments, a poly A tail of a string construct may comprise about 180 A residues. In some embodiments, a poly A tail may comprise 150 residues or less.
  • a poly(A) tail comprises a plurality of A residues interrupted by a linker.
  • a linker comprises the nucleotide sequence GCATATGAC (SEQ ID NO: 156).
  • a polyribonucleotide of the present disclosure comprises a poly(A) sequence comprising the nucleotide sequence of SEQ ID NO: 268, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 268.
  • a polyribonucleotide utilized in accordance with the present disclosure comprises a 3'-UTR.
  • the terms "three prime untranslated region,” “3' untranslated region,” or “3' UTR” refer to a sequence of an mRNA molecule that begins following a stop codon of a coding region of an open reading frame sequence.
  • a 3' UTR begins immediately after a stop codon of a coding region of an open reading frame sequence, e.g., in its natural context.
  • a 3' UTR does not begin immediately after the stop codon of the coding region of an open reading frame sequence, e.g., in its natural context.
  • the term "3'-UTR" preferably does not include the poly(A) sequence.
  • a 3'-UTR is upstream of the poly(A) sequence (if present), e.g., directly adjacent to the poly(A) sequence.
  • an RNA disclosed herein comprises a 3' UTR comprising an F element and/or an I element.
  • a 3' UTR or a proximal sequence thereto comprises a restriction site.
  • a restriction site is a BamHI site.
  • a restriction site is a Xhol site.
  • an RNA construct comprises an F element.
  • a F element sequence is a 3'-UTR of amino-terminal enhancer of split (AES).
  • an RNA disclosed herein comprises a 3' UTR.
  • a 3'UTR is an FI element as described in W02017/060314, which is herein incorporated by reference in its entirety.
  • an mpox antigen utilized as described herein includes a multimerization element (e.g., a heterologous multimerization element).
  • a heterologous multimerization element comprises a dimerization, trimerization or tetramerization element.
  • a multimerization element is one described in W02017/081082 (e.g., sequences of SEQ ID NOs: 1116-1167 of W02017/081082, or fragments or variants thereof).
  • trimerization and tetramerization elements include, but are not limited to, engineered leucine zippers, fibritin foldon domain from enterobacteria phage T4, GCN4pll, GCN4-pll, and p53.
  • an antigen construct of the present disclosure includes, and/or a polyribonucleotide of the present disclosure encodes, an antigen operably linked with a multimerization element such as a foldon domain.
  • an antigen construct of the present disclosure includes, and/or a polyribonucleotide of the present disclosure encodes, an antigen operably linked with a foldon domain according to SEQ ID NO: 256 and/or encoded by a sequence according to SEQ ID NO: 257.
  • a provided antigen is able to form a trimeric complex.
  • a utilized antigen may comprise a domain allowing formation of a multimeric complex, such as for example a trimeric complex of an amino acid sequence comprising an orthopoxvirus (e.g., mpox) antigen as described herein.
  • a domain allowing formation of a multimeric complex comprises a trimerization domain, for example, a trimerization domain as described herein.
  • an orthopoxvirus (e.g., mpox) antigen can be modified by addition of a
  • T4-fibritin-derived "foldon” trimerization domain for example, to increase its immunogenicity.
  • an orthopoxvirus (e.g., mpox) antigen as described herein includes a membrane association element (e.g., a heterologous membrane association element), such as a transmembrane domain.
  • a membrane association element e.g., a heterologous membrane association element
  • a transmembrane domain can be N-terminal, C-terminal, or internal to an antigen.
  • a coding sequence of a transmembrane element is typically placed in frame (i.e., in the same reading frame), 5', 3', or internal to coding sequences (e.g., mpox antigen coding sequences) with which it is to be linked.
  • a transmembrane domain comprises or is a transmembrane domain of
  • Hemagglutinin (HA) of Influenza virus Env of HIV-1, equine infectious anaemia virus (EIAV), murine leukaemia virus (MLV), mouse mammary tumor virus, G protein of vesicular stomatitis virus (VSV), Rabies virus, or a seven transmembrane domain receptor.
  • EIAV equine infectious anaemia virus
  • MLV murine leukaemia virus
  • VSV G protein of vesicular stomatitis virus
  • Rabies virus Rabies virus
  • an antigen construct of the present disclosure includes, and/or a polyribonucleotide of the present disclosure encodes, an antigen operably linked with a transmembrane domain.
  • an antigen construct of the present disclosure includes, and/or a polyribonucleotide of the present disclosure encodes, an antigen operably linked with a HSV-1 gD transmembrane domain (TM) domain according to SEQ ID NO: 254 and/or encoded by a sequence according to SEQ ID NO: 255.
  • TM transmembrane domain
  • RNA compositions e.g., pharmaceutical compositions
  • uRNA non-modified uridine containing mRNA
  • modRNA nucleoside-modified mRNA
  • saRNA self-amplifying mRNA
  • RNA is capped, contains open reading frames (ORFs) flanked by untranslated regions (UTR), and have a polyA-tail at the 3' end.
  • ORFs open reading frames flanked by untranslated regions
  • An ORF of an uRNA and modRNA vector encodes an antigen or fragment thereof.
  • An saRNA has multiple ORFs.
  • an RNA described herein may have modified nucleosides.
  • the RNA comprises a modified nucleoside in place of at least one (e.g., every) uridine.
  • uracil describes one of the nucleobases that can occur in the nucleic acid of RNA.
  • the structure of uracil is: [0307]
  • uridine describes one of the nucleosides that can occur in RNA.
  • uridine The structure of uridine is:
  • UTP uridine 5'-triphosphate
  • Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure:
  • Pseudouridine is one example of a modified nucleoside that is an isomer of uridine, where the uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.
  • mlQJ Nl-methyl-pseudouridine
  • Nl-methyl-pseudo-UTP has the following structure:
  • m5U 5-methyl-uridine
  • one or more uridine in an RNA described herein is replaced by a modified nucleoside.
  • the modified nucleoside is a modified uridine.
  • an RNA described herein comprises a modified nucleoside in place of at least one uridine. In some embodiments, an RNA described herein comprises a modified nucleoside in place of each uridine.
  • a modified nucleoside is independently selected from pseudouridine
  • a modified nucleoside comprises pseudouridine (ip). In some embodiments, a modified nucleoside comprises Nl-methyl-pseudouridine (mlip). In some embodiments, a modified nucleoside comprises 5-methyl-uridine (m5U). In some embodiments, RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine (ip), Nl-methyl-pseudouridine (mlip), and 5-methyl-uridine (m5U).
  • modified nucleosides comprise pseudouridine (ip) and Nl-methyl-pseudouridine (mlip). In some embodiments, modified nucleosides comprise pseudouridine (ip) and 5-methyl-uridine (m5U). In some embodiments, modified nucleosides comprise Nl-methyl-pseudouridine (mlip) and 5-methyl-uridine (m5U). In some embodiments, modified nucleosides comprise pseudouridine (ip), Nl-methyl-pseudouridine (mlip), and 5- methyl-uridine (m5U).
  • a modified nucleoside replacing one or more, e.g., all, uridine in the
  • RNA may be any one or more of 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza- uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio- pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo- uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl- uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5- carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl
  • an RNA of the present disclosure comprises other modified nucleosides or comprises further modified nucleosides, e.g., modified cytidine.
  • 5-methylcytidine is substituted partially or completely, preferably completely, for cytidine.
  • an RNA of the present disclosure comprises 5-methylcytidine and one or more selected from pseudouridine ( ⁇ ), Nl-methyl-pseudouridine (m1 ⁇ ), and 5-methyl-uridine (m5U).
  • an RNA of the present disclosure comprises 5-methylcytidine and Nl-methyl-pseudouridine (mlip).
  • an RNA of the present disclosure comprises 5-methylcytidine in place of each cytidine and Nl-methyl-pseudouridine (m1 ⁇ ) in place of each uridine.
  • an RNA is "replicon RNA" or simply a
  • the replicon or self-replicating RNA is derived from or comprises elements derived from a single- stranded (ss) RNA virus, in particular a positive-stranded ssRNA virus, such as an alphavirus.
  • ss single- stranded
  • Alphaviruses are typical representatives of positive-stranded RNA viruses.
  • Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphaviral life cycle see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856, which is incorporated herein by reference in its entirety).
  • the total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5'-cap, and a 3' poly(A) tail.
  • the genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome.
  • the four non-structural proteins (nsPl-nsP4) are typically encoded together by a first ORF beginning near the 5' terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3' terminus of the genome.
  • the first ORF is larger than the second ORF, the ratio being roughly 2: 1.
  • the genomic RNA In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124, herein incorporated by reference in its entirety). Following infection, i.e., at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly-protein (nsP1234).
  • mRNA eukaryotic messenger RNA
  • Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms.
  • a first ORF encodes an alphavirus-derived RNA-dependent RNA polymerase (replicase), which upon translation mediates self-amplification of the RNA.
  • a second ORF encoding alphaviral structural proteins is replaced by an open reading frame encoding a protein of interest, e.g., an antigen or fragment thereof.
  • Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system).
  • Trans-replication requires the presence of both these nucleic acid molecules in a given host cell.
  • the nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow for recognition and RNA synthesis by the alphaviral replicase.
  • non-modified uridine platform may include, for example, one or more of an intrinsic adjuvant effect, good tolerability, and improved safety.
  • modified uridine (e.g., pseudouridine) platform may include a reduced adjuvant effect, blunted immune innate immune sensor activating capacity, good tolerability and improved safety.
  • features of a self-amplifying platform may include, for example, long duration of protein expression, good tolerability and safety, and a higher likelihood for efficacy with a very low vaccine dose.
  • RNA constructs optimized for example, for improved manufacturability, encapsulation, expression level (and/or timing), etc. Certain components are discussed below, and certain preferred embodiments are exemplified herein.
  • coding regions are codon-optimized for optimal expression in a subject to be treated using the RNA molecules described herein.
  • codon-optimization may be performed such that codons for which frequently occurring tRNAs are available are inserted in place of "rare codons.”
  • codon-optimization may include increasing guanosine/cytosine (G/C) content of a coding region of RNA described herein as compared to the G/C content of the corresponding coding sequence of a wild-type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence.
  • G/C guanosine/cytosine
  • a coding sequence (also referred to as a "coding region") is codon optimized for expression in the subject to whom a composition (e.g., a pharmaceutical composition) is to be administered (e.g., a human).
  • a composition e.g., a pharmaceutical composition
  • sequences in such a polynucleotide may differ from wild type sequences encoding the relevant antigen or fragment or epitope thereof, even when the amino acid sequence of the antigen or fragment or epitope thereof is wild type.
  • a coding sequence is codon optimized for expression in a relevant subject (e.g., a human), and even, in some cases, for expression in a particular cell or tissue.
  • codon bias differs in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • the predominance of selected tRNAs in a cell may generally be a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes may be tailored for optimal gene expression in a given organism based on codon optimization.
  • Codon usage tables are available, for example, at the "Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables may be adapted in a number of ways.
  • Computer algorithms for codon optimizing a particular sequence for expression in a particular subject or its cells are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available.
  • a polynucleotide (e.g., a polyribonucleotide) of the present disclosure is codon optimized, wherein the codons in the polynucleotide (e.g., the polyribonucleotide) are adapted to human codon usage (herein referred to as "human codon optimized polynucleotide"). Codons encoding the same amino acid occur at different frequencies in a subject, e.g., a human.
  • the coding sequence of a polynucleotide of the present disclosure is modified such that the frequency of the codons encoding the same amino acid corresponds to the naturally occurring frequency of that codon according to the human codon usage, e.g., as shown in Table 3.
  • the wild type coding sequence is preferably adapted in a way that the codon "GCC” is used with a frequency of 0.40, the codon “GCT” is used with a frequency of 0.28, the codon “GCA” is used with a frequency of 0.22 and the codon “GCG” is used with 30 a frequency of 0.10 etc. (see Table 3).
  • such a procedure (as exemplified for Ala) is applied for each amino acid encoded by the coding sequence of a polynucleotide to obtain sequences adapted to human codon usage.
  • Table 3 Human codon usage table with frequencies indicated for each amino acid.
  • a coding sequence may be optimized using a multiparametric optimization strategy.
  • optimization parameters may include parameters that influence protein expression, which can be, for example, impacted on a transcription level, an mRNA level, and/or a translational level.
  • exemplary optimization parameters include, but are not limited to transcription-level parameters (including, e.g., GC content, consensus splice sites, cryptic splice sites, SD sequences, TATA boxes, termination signals, artificial recombination sites, and combinations thereof); mRNA-level parameters (including, e.g., RNA instability motifs, ribosomal entry sites, repetitive sequences, and combinations thereof); translation-level parameters (including, e.g., codon usage, premature poly(A) sites, ribosomal entry sites, secondary structures, and combinations thereof); or combinations thereof.
  • a coding sequence may be optimized by a GeneOptimizer algorithm as described in Fath et al.
  • a coding sequence may be optimized by Eurofins' adaption and optimization algorithm "GENEius” as described in Eurofins' Application Notes: Eurofins' adaption and optimization software “GENEius” in comparison to other optimization algorithms, the entire content of which is incorporated by reference for the purposes described herein.
  • a coding sequence utilized in accordance with the present disclosure has G/C content of which increased compared to a wild type coding sequence.
  • GC enrichment may improve translation of a payload sequence.
  • sequences having an increased G (guanosine)/C (cytidine) content are more stable than sequences having an increased A (adenosine)/U (uridine) content.
  • G guanosine
  • C cytidine
  • A adenosine
  • U uridine
  • codons which contain A and/or U nucleosides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and/or U or contain a lower content of A and/or U nucleosides.
  • G/C content of a coding region of a polyribonucleotide described herein is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, or even more compared to the G/C content of the coding region prior to codon optimization, e.g., of the wild-type RNA.
  • G/C content of a coding region of a polyribonucleotide described herein is decreased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, or even more compared to the G/C content of the coding region prior to codon optimization, e.g., of the wild-type RNA.
  • stability and translation efficiency of a polyribonucleotide may incorporate one or more elements established to contribute to stability and/or translation efficiency of the polyribonucleotide; exemplary such elements are described, for example, in PCT/EP2006/009448 incorporated herein by reference.
  • a polyribonucleotide may be modified within the coding region, i.e., the sequence encoding the expressed peptide or protein, without altering the sequence of the expressed peptide or protein, for example so as to increase the GC-content to increase mRNA stability and/or to perform a codon optimization and, thus, enhance translation in cells.
  • the present disclosure includes certain exemplary antigen constructs and polyribonucleotides useful, e.g., in vaccination against orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) and/or treatment of orthopoxvirus infection, that encode and/or express one or more mpox antigens.
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus
  • orthopoxvirus e.g., mpox, variola
  • a polyribonucleotide as described herein has one of the following structures: cap-hAg-Kozak-Antigen-FI-A30L70 cap-hAg-Kozak-sec-Antigen-FI-A30L70 where cap refers to a 5' cap as described above; hAg-Kozak refers to a 5' UTR human alpha-globin; sec refers to a secretory signal; Antigen refers to a nucleotide sequence comprising a sequence that encodes an mpox antigen described herein; FI refers to a 3'-UTR as described above, and A30L70 refers to a polyA sequence.
  • hAg 5' UTR comprises a nucleotide sequence of SEQ ID NO: 155.
  • A30L70 comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.
  • the present disclosure includes certain exemplary antigen constructs and polyribonucleotides useful, e.g., in vaccination against orthopox virus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus), that encode and/or express one or more antigens according to Table 1 or antigenic fragments thereof.
  • orthopox virus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus
  • vaccinia e.g., modified vaccinia virus Ankara, etc.
  • a polyribonucleotide of the present disclosure encodes an orthopoxvirus antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to an orthopoxvirus antigen amino acid sequence in Table 1.
  • a polyribonucleotide of the present disclosure encodes an antigen polypeptide operably linked to an N-terminal viral secretory signal.
  • inclusion of a viral secretory signal can be useful, e.g., because mpox antigens do not naturally include conventional secretory signals and/or because inclusion of the viral secretory signal may allow for enhanced surface expression of the operably linked antigen on vaccinated cells.
  • Exemplary antigens of the present disclosure can further include substitution of unpaired cysteine residues present in corresponding reference sequences.
  • the present disclosure includes that such cysteines, if left unpaired, carry a high risk of causing protein misfolding and/or aggregation and that this risk is mitigated by alanine substitutions.
  • Exemplary substitutions can include positions C71A and/or C72A of A29L, C140A of B6R, and/or C86A and/or C90A of H3L.
  • the present disclosure includes exemplary polypeptide sequences and polyribonucleic acid sequences as described herein and/or as set forth in sequence identification numbers of the present disclosure, as well as sequences having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.
  • a polyribonucleotide of the present disclosure encodes a wild-type MIR polypeptide.
  • a wild-type MIR polypeptide has or includes a sequence according to SEQ ID NO: 158 and/or the polyribonucleotide has or includes a sequence according to SEQ ID NO: 159.
  • a polyribonucleotide of the present disclosure encodes a wild-type MIR polypeptide operably linked with a secretory signal such as an HSV/gD secretory signal (HSV/gD se c) according to SEQ ID NO: 160, optionally encoded by a sequence according to SEQ ID NO: 161.
  • a polyribonucleotide of the present disclosure encodes a wild-type MIR polypeptide.
  • a wild-type MIR polypeptide has or includes a sequence according to SEQ ID NO: 258 and/or the polyribonucleotide encoding the wild-type MIR polypeptide has or includes a sequence according to SEQ ID NO: 259.
  • a polyribonucleotide of the present disclosure encodes a wild-type MIR polypeptide operably linked with a secretory signal such as an HSV/gD secretory signal (HSV/gDsec) according to SEQ ID NO: 160, optionally encoded by a sequence according to SEQ ID NO: 161.
  • a secretory signal such as an HSV/gD secretory signal (HSV/gDsec)
  • a polyribonucleotide of the present disclosure encodes a soluble form of the ectodomain of A35R.
  • a soluble form of the ectodomain of A35R would function independently of membrane insertion, reducing the potential need or requirement for membrane insertion of an A35R antigen or fragment thereof.
  • the A35R ectodomain disclosed herein can have or include a sequence according to SEQ ID NO: 174. The boundaries of the ectodomain were informed by two X-ray crystallography studies defining the structure of this region of the protein.
  • the present inventors selected amino acids 89-181 for use in this design at least in part because the selected amino acids span the resolved region of the protein elucidated by these studies.
  • A35R forms a dimer that is partially dependent on a disulfide bond between residues not included in this ectodomain.
  • the present inventors engineered a construct that includes two copies of the ectodomain sufficient to form the dimer with a linker between them.
  • linker size (10 amino acids) was selected based on the measured distance between the C-terminus of one ectodomain and the N-terminus of its binding partner in the crystal structure of the ectodomain, while those of skill in the art will appreciate that the linker could be larger or smaller, and any linker disclosed herein could be used.
  • a polyribonucleotide of the present disclosure encodes a wild-type
  • a wild-type A35R polypeptide has or includes a sequence according to SEQ ID NO: 172 and/or the polyribonucleotide encoding said A35R polypeptide has or includes a sequence according to SEQ ID NO: 173.
  • a polyribonucleotide of the present disclosure encodes a wild-type A35R ectodomain (ECD) fragment that has or includes a sequence according to SEQ ID NO: 174 and/or the encoding polyribonucleotide has or includes a sequence according to SEQ ID NO: 175.
  • ECD wild-type A35R ectodomain
  • a polyribonucleotide of the present disclosure encodes a first wild-type A35R ECD fragment and a second wild-type A35R ECD fragment, where the first wild-type A35R ECD fragment has or includes a sequence according to SEQ ID NO: 174 and/or is encoded by a sequence according to SEQ ID NO: 175 and the second wild-type A35R ECD fragment has or includes a sequence according to SEQ ID NO: 174 and/or is encoded by a sequence according to SEQ ID NO: 175, optionally wherein the first wild-type A35R fragment and the second wild-type A35R fragment are operably linked via linker (e.g., a linker according to SEQ ID NO: 176 and/or encoded by SEQ ID NO: 177).
  • linker e.g., a linker according to SEQ ID NO: 176 and/or encoded by SEQ ID NO: 177.
  • a polyribonucleotide of the present disclosure encodes a first wild-type A35R fragment and a second wild-type A35R fragment, where the first wild-type A35R fragment and the second wild-type A35R fragment are operably linked by a linker, and where the first and second wildtype A35R fragments are operably linked with a secretory signal such as an HSV/gD secretory signal (HSV/gD se c) according to SEQ ID NO: 160, optionally encoded by a sequence according to SEQ ID NO: 161.
  • a secretory signal such as an HSV/gD secretory signal (HSV/gD se c) according to SEQ ID NO: 160, optionally encoded by a sequence according to SEQ ID NO: 161.
  • a polypeptide encoded by a polyribonucleotide has or includes a sequence according to SEQ ID NO: 178 and/or the encoding polyribonucleotide has or includes a sequence according to SEQ ID NO: 179.
  • a polyribonucleotide of the present disclosure encodes a wild-type B6R polypeptide.
  • a wild-type B6R polypeptide has or includes a sequence according to SEQ ID NO: 180 and/or the polyribonucleotide encoding said B6R polypeptide has or includes a sequence according to SEQ ID NO: 181.
  • a polyribonucleotide of the present disclosure encodes an B6R polypeptide that includes a substitution of C to A at position 140 corresponding to SEQ ID NO: 182 (substitution C140A as compared to a corresponding reference sequence).
  • a B6R polypeptide including a C140A substitution has or includes a sequence according to SEQ ID NO: 182 and/or the encoding polyribonucleotide has or includes a sequence according to SEQ ID NO: 183.
  • a polyribonucleotide of the present disclosure encodes a wild-type H3L polypeptide.
  • a wild-type H3L polypeptide has or includes a sequence according to SEQ ID NO: 184 and/or the polyribonucleotide encoding said H3L polypeptide has or includes a sequence according to SEQ ID NO: 185.
  • a polyribonucleotide of the present disclosure encodes a wild-type H3L polypeptide operably linked with a secretory signal such as an HSV/gD secretory signal (HSV/gD se c) according to SEQ ID NO: 160, optionally encoded by a sequence according to SEQ ID NO: 161.
  • a wild-type H3L polypeptide operably linked with an HSV/gD secretory signal has or includes a sequence according to SEQ ID NO: 186 and/or the encoding polyribonucleotide has or includes a sequence according to SEQ ID NO: 187.
  • a polyribonucleotide of the present disclosure encodes an H3L polypeptide that includes a substitution of C to A at positions 86 and 90 corresponding to SEQ ID NO: 188 (substitutions C86A and C90A as compared to a corresponding reference sequence).
  • an H3L polypeptide including C86A and C90A substitutions has or includes a sequence according to SEQ ID NO: 188 and/or the polyribonucleotide encoding said H3L polypeptide has or includes a sequence according to SEQ ID NO: 189.
  • a polyribonucleotide of the present disclosure encodes an H3L polypeptide including C86A and C90A substitutions operably linked with a secretory signal such as an HSV/gD secretory signal (HSV/gDsec) according to SEQ ID NO: 160, optionally encoded by a sequence according to SEQ ID NO: 161.
  • a secretory signal such as an HSV/gD secretory signal (HSV/gDsec) according to SEQ ID NO: 160
  • HSV/gD secretory signal HSV/gD secretory signal
  • an H3L polypeptide including C86A and C90A substitutions operably linked with an HSV/gD secretory signal sequence has or includes a sequence according to SEQ ID NO: 190 and/or the encoding polyribonucleotide has or includes a sequence according to SEQ ID NO: 191.
  • a polyribonucleotide of the present disclosure encodes a wild-type H3L polypeptide.
  • a wild-type H3L polypeptide has or includes a sequence according to SEQ ID NO: 260 and/or the polyribonucleotide encoding said H3L polypeptide has or includes a sequence according to SEQ ID NO: 261.
  • a polyribonucleotide of the present disclosure encodes a wild-type H3L polypeptide operably linked with a secretory signal such as an HSV/gD secretory sequence (HSV/gD se c) according to SEQ ID NO: 160, optionally encoded by a sequence according to SEQ ID NO: 161.
  • a secretory signal such as an HSV/gD secretory sequence (HSV/gD se c) according to SEQ ID NO: 160, optionally encoded by a sequence according to SEQ ID NO: 161.
  • a polyribonucleotide of the present disclosure encodes an H3L polypeptide that includes a substitution of C to A at positions 86 and 90 corresponding to SEQ ID NO: 262 (substitutions C86A and C90A as compared to a corresponding reference sequence).
  • an H3L polypeptide including C86A and C90A substitutions has or includes a sequence according to SEQ ID NO: 262 and/or the polyribonucleotide encoding said H3L polypeptide has or includes a sequence according to SEQ ID NO: 263.
  • a polyribonucleotide of the present disclosure encodes an H3L polypeptide including C86A and C90A substitutions operably linked with a secretory signal such as an HSV/gD secretory sequence (HSV/gD se c) according to SEQ ID NO: 160, optionally encoded by a sequence according to SEQ ID NO: 161.
  • a secretory signal such as an HSV/gD secretory sequence (HSV/gD se c) according to SEQ ID NO: 160, optionally encoded by a sequence according to SEQ ID NO: 161.
  • polyribonucleotides may be delivered for therapeutic applications described herein using any appropriate methods known in the art, including, e.g., delivery as naked polyribonucleotides (e.g., RNAs), or delivery mediated by viral and/or non-viral vectors, polymer-based vectors, lipid-based vectors, nanoparticles (e.g., lipid nanoparticles, polymeric nanoparticles, lipid-polymer hybrid nanoparticles, etc.), and/or peptide-based vectors. See, e.g., Wadhwa et al.
  • one or more polyribonucleotides can be formulated with lipid nanoparticles for delivery (e.g., administration).
  • lipid nanoparticles can be designed to protect polyribonucleotides from extracellular RNases and/or engineered for systemic delivery of the RNA to target cells (e.g., liver cells). In some embodiments, such lipid nanoparticles may be particularly useful to deliver polyribonucleotides when polyribonucleotides are intravenously or intramuscularly administered to a subject.
  • lipid and "lipid-like material” are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually poorly soluble in water. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment.
  • Hydrophobicity can be conferred by the inclusion of a polar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s).
  • the hydrophilic groups may comprise polar and/or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups.
  • an amphiphilic compound has a polar head attached to a long hydrophobic tail.
  • the polar portion is soluble in water, while the non-polar portion is insoluble in water.
  • the polar portion may have either a formal positive charge, or a formal negative charge.
  • the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt.
  • the amphiphilic compound can be, but is not limited to, one or a plurality of natural or non-natural lipids and lipid-like compounds.
  • a "lipid-like material” is a substance that is structurally and/or functionally related to a lipid but may not be considered a lipid in a strict sense.
  • the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties.
  • the term refers to molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids.
  • amphiphilic compounds that may be included in an amphiphilic layer include, but are not limited to, phospholipids, aminolipids and sphingolipids.
  • lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterols and prenol lipids (derived from condensation of isoprene subunits).
  • lipid is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides.
  • Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as sterol- containing metabolites such as cholesterol.
  • Fatty acids are a diverse group of molecules made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water.
  • the carbon chain typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more double bonds in the chain.
  • Other major lipid classes in the fatty acid category are the fatty esters and fatty amides.
  • Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best-known being the fatty acid triesters of glycerol, called triglycerides.
  • triacylglycerol is sometimes used synonymously with "triglyceride”.
  • the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids.
  • Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage.
  • Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" by ester linkages and to one "head” group by a phosphate ester linkage.
  • Examples of glycerophospholipids usually referred to as phospholipids (though sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).
  • Sphingolipids are members of a complex family of compounds that share a common structural feature, a sphingoid base backbone.
  • the major sphingoid base in mammals is commonly referred to as sphingosine.
  • Ceramides N-acyl-sphingoid bases
  • the fatty acids are typically saturated or mono-unsaturated with chain lengths from 16 to 26 carbon atoms.
  • the major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannosecontaining headgroups.
  • the glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.
  • Sterols such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, along with the glycerophospholipids and sphingomyelins.
  • Saccharolipids are compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers.
  • a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids.
  • the most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria.
  • Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E.
  • Kdo2-Lipid A a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.
  • Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.
  • Lipids and lipid-like materials may be cationic, anionic or neutral.
  • Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.
  • suitable lipids or lipid-like materials for use in the present disclosure include those described in W02020/128031 and US20200163878, the entire contents of each of which are incorporated herein by reference for the purposes described herein.
  • cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein include any cationic or cationically ionizable lipids or lipid-like materials which are able to electrostatically bind nucleic acid.
  • cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein can be associated with nucleic acid, e.g., by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.
  • Cationic lipids or lipid-like materials are characterized in that they have a net positive charge
  • Cationic lipids or lipid-like materials bind negatively charged nucleic acid by electrostatic interaction.
  • cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge.
  • a cationic lipid or lipid-like material has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH. This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH.
  • a cationic or cationically ionizable lipid or lipid-like material comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated.
  • cationic lipids include, but are not limited to 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA), 3-(N— (N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3- dimethylammonium propanes; 1,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropan
  • DODAC 1,
  • Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-K- XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31- tetraen-19-yl-4-(dimethyl
  • Suitable cationic lipids for use in the present disclosure include those described in W02020/128031 and US20200163878, the entire contents of each of which are incorporated herein by reference for the purposes described herein.
  • Further suitable cationic lipids for use in the present disclosure include those described in W02010/053572 (including Cl 2-200 described at paragraph [00225]) and W02012/170930, both of which are incorporated herein by reference for the purposes described herein.
  • Additional suitable cationic lipids for use in the present disclosure include HGT4003, HGT5000, HGTS001, HGT5001, HGT5002 (see US20150140070A1, which is incorporated herein by reference in its entirety).
  • formulations that are useful for pharmaceutical compositions can comprise at least one cationic lipid.
  • Representative cationic lipids include, but are not limited to, 1 ,2-dilinoleyoxy-3- (dimethylamino)acetoxypropane (DLin-DAC), 1 ,2-dilinoleyoxy-3morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3- dimethylaminopropane (DLinDAP), 1 ,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1 -linoleoyl-2- linoleyloxy-3dimethylaminopropane (DLin-2-DMAP), 1 ,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.CI), 1 ,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.CI), 1
  • amino or cationic lipids useful in accordance with the present disclosure have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH.
  • physiological pH e.g., pH 7.4
  • second pH preferably at or above physiological pH.
  • a protonatable lipid has a pKa of the protonatable group in the range of about 4 to about 11, e.g., a pKa of about 5 to about 7.
  • a cationic lipid may comprise from about 10 mol % to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % to about 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % to about 100 mol % of total lipid present in a lipid composition utilized in accordance with the present disclosure.
  • formulations utilized in accordance with the present disclosure may comprise lipids or lipid-like materials other than cationic or cationically ionizable lipids or lipid-like materials, i.e., non-cationic lipids or lipid-like materials (including non-cationically ionizable lipids or lipid-like materials).
  • non-cationic lipids or lipid-like materials including non-cationically ionizable lipids or lipid-like materials.
  • anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids or lipid-like materials.
  • optimizing a formulation of nucleic acid particles by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to an ionizable/cationic lipid or lipid-like material may, for example, enhance particle stability and efficacy of nucleic acid delivery.
  • a lipid or lipid-like material may be incorporated which may or may not affect the overall charge of particles.
  • such lipid or lipid-like material is a non-cationic lipid or lipid-like material.
  • a non-cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids.
  • An "anionic lipid” is negatively charged (e.g., at a selected pH).
  • a "neutral lipid” exists either in an uncharged or neutral zwitterionic form (e.g., at a selected pH).
  • a formulation comprises one of the following neutral lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.
  • cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.
  • Specific exemplary phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin.
  • Such phospholipids include in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoy
  • a formulation utilized in accordance with the present disclosure includes
  • formulations utilized in accordance with the present disclosure include both a cationic lipid and an additional (non-cationic) lipid.
  • formulations herein include a polymer conjugated lipid such as a pegylated lipid.
  • a polymer conjugated lipid such as a pegylated lipid.
  • Pegylated lipids comprise both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art.
  • the amount of (total) cationic lipid compared to the amount of other lipid(s) in formulation may affect important characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the nucleic acid.
  • the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4: 1 to about 1:2, or about 3: 1 to about 1: 1.
  • a non-cationic lipid in particular a neutral lipid, (e.g., one or more phospholipids and/or cholesterol) may comprise from about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol %, from about 0 mol % to about 70 mol %, from about 0 mol % to about 60 mol %, or from about 0 mol % to about 50 mol %, of the total lipid present in a formulation.
  • a neutral lipid e.g., one or more phospholipids and/or cholesterol
  • RNA described herein may be present in certain embodiments of the present disclosure.
  • RNA lipoplex particle contains lipid, in particular cationic lipid, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA results in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic lipid, such as DOTMA, and additional lipids, such as DOPE. In one embodiment, an RNA lipoplex particle is a nanoparticle.
  • RNA lipoplex particles include both a cationic lipid and an additional lipid.
  • the cationic lipid is DOTMA and the additional lipid is DOPE.
  • the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm.
  • the RNA lipoplex particles have an average diameter that ranges from about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter that ranges from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.
  • the liposomes and RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid.
  • the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA) and/or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP).
  • the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (DOPE), cholesterol (Choi) and/or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC).
  • DOPE 1,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine
  • DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine
  • the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (DOPE).
  • the liposomes and RNA lipoplex particles comprise 1,2-di-O- octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (DOPE).
  • RNA lipoplex particles having a net negative charge may be used to preferentially target spleen tissue or spleen cells such as antigen-presenting cells, in particular dendritic cells. Accordingly, following administration of the RNA lipoplex particles, RNA accumulation and/or RNA expression in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in the spleen. In an embodiment, after administration of the RNA lipoplex particles, no or essentially no RNA accumulation and/or RNA expression in the lung and/or liver occurs.
  • RNA lipoplex particles of the disclosure may be used for expressing RNA in such antigen presenting cells.
  • the antigen presenting cells are dendritic cells and/or macrophages.
  • LNPs Lipid Nanoparticles
  • nucleic acid such as RNA described herein is administered in the form of lipid nanoparticles (LNPs).
  • LNPs may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.
  • an LNP comprises one or more cationic lipids, and one or more stabilizing lipids.
  • Stabilizing lipids include neutral lipids and pegylated lipids.
  • a neutral lipid is selected from the group consisting of DSPC, DPPC,
  • the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC.
  • a sterol is cholesterol
  • a polymer conjugated lipid is a pegylated lipid.
  • a pegylated lipid has the following structure: or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:
  • R 12 and R 13 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60.
  • R 12 and R 13 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms.
  • w has a mean value ranging from 40 to 55.
  • the average w is about 45.
  • R 12 and R 13 are each independently a straight, saturated alkyl chain containing about 14 carbon atoms, and w has a mean value of about 45.
  • a pegylated lipid is DMG-PEG 2000, e.g., having the following structure:
  • G 1 and G 2 are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;
  • G 3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;
  • R a is H or C1-C12 alkyl
  • R 4 is C1-C12 alkyl
  • the lipid has one of the following structures (IIIA) or (IIIB): wherein:
  • A is a 3 to 8-membered cycloalkyl or cycloalkylene ring
  • the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).
  • the lipid has one of the following structures (IIIC) or
  • the lipid has one of the following structures
  • the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ):
  • y and z are each independently an integer ranging from 2 to 10.
  • y and z are each independently an integer ranging from 4 to 9 or from 4 to 6.
  • R 5 is H.
  • R 5 is C1-C24 alkyl.
  • R 5 is OH.
  • G 3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G 3 is linear C1-C24 alkylene or linear C1-C24 alkenylene.
  • R 1 or R 2 is C6-C24 alkenyl.
  • R 1 and R 2 each, independently have the following structure: wherein:
  • R 7a and R 7b are, at each occurrence, independently H or C1-C12 alkyl; and a is an integer from 2 to 12, wherein R 7a , R 7b and a are each selected such that R 1 and R 2 each independently comprise from 6 to 20 carbon atoms.
  • a is an integer ranging from 5 to 9 or from 8 to 12.
  • At least one occurrence of R 7a is H.
  • R 7a is H at each occurrence.
  • at least one occurrence of R 7b is C1-C8 alkyl.
  • C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
  • R 1 or R 2 has one of the following structures:
  • R 4 is methyl or ethyl.
  • the cationic lipid of Formula (III) has one of the structures set forth in in Table 4 below.
  • a cationic lipid has one of the structures set forth in Table 5 below.
  • an LNP comprises a cationic lipid that is an ionizable lipid-like material
  • lipid nanoparticles can have an average size (e.g., mean diameter) of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 70 to about 90 nm, or about 70 nm to about 80 nm.
  • lipid nanoparticles in accordance with the present disclosure can have an average size (e.g., mean diameter) of about 50 nm to about 100 nm.
  • lipid nanoparticles may have an average size (e.g., mean diameter) of about 50 nm to about 150 nm. In some embodiments, lipid nanoparticles may have an average size (e.g., mean diameter) of about 60 nm to about 120 nm.
  • average diameter refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321, which is herein incorporated by reference).
  • average diameter “mean diameter,” “diameter,” or “size” for particles is used synonymously with this value of the Z-average.
  • lipid nanoparticles described herein may exhibit a polydispersity index less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less.
  • lipid nanoparticles can exhibit a polydispersity index in a range of about 0.1 to about 0.3 or about 0.2 to about 0.3.
  • the "polydispersity index” is preferably calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the "average diameter.” Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of ribonucleic acid nanoparticles (e.g., ribonucleic acid nanoparticles).
  • a cationic group is one that is either in cationic form (e.g., N + ), or one that is ionizable to become cationic.
  • Use of a single number in an N/P ratio (e.g., an N/P ratio of about 5) is intended to refer to that number over 1, e.g., an N/P ratio of about 5 is intended to mean 5: 1.
  • a lipid nanoparticle described herein has an N/P ratio greater than or equal to 5.
  • a lipid nanoparticle described herein has an N/P ratio that is about 5, 6, 7, 8, 9, or 10.
  • cationic lipids, neutral lipids (e.g., DSPC, and/or cholesterol) and polymer-conjugated lipids can be solubilized in ethanol at a pre-determined molar ratio (e.g., ones described herein).
  • lipid nanoparticles are prepared at a total lipid to polyribonucleotides weight ratio of approximately 10: 1 to 30: 1. In some embodiments, such polyribonucleotides can be diluted to 0.2 mg/mL in acetate buffer.
  • a colloidal lipid dispersion comprising polyribonucleotides can be formed as follows: an ethanol solution comprising lipids, such as cationic lipids, neutral lipids, and polymer-conjugated lipids, is injected into an aqueous solution comprising polyribonucleotides (e.g., ones described herein).
  • lipids such as cationic lipids, neutral lipids, and polymer-conjugated lipids
  • lipid and polyribonucleotide solutions can be mixed at room temperature by pumping each solution at controlled flow rates into a mixing unit, for example, using piston pumps.
  • the flow rates of a lipid solution and an RNA solution into a mixing unit are maintained at a ratio of 1:3.
  • nucleic acid-lipid particles are formed as the ethanolic lipid solution is diluted with aqueous polyribonucleotides. The lipid solubility is decreased, while cationic lipids bearing a positive charge interact with the negatively charged RNA.
  • a solution comprising RNA-encapsulated lipid nanoparticles can be processed by one or more of concentration adjustment, buffer exchange, formulation, and/or filtration.
  • compositions e.g., pharmaceutical compositions, comprising one or more polyribonucleotides described herein.
  • Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • a pharmaceutically acceptable excipient includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired.
  • Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.
  • an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by the United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
  • USP United States Pharmacopoeia
  • EP European Pharmacopoeia
  • British Pharmacopoeia the British Pharmacopoeia
  • International Pharmacopoeia International Pharmacopoeia
  • compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.
  • compositions provided herein may be formulated with one or more pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).
  • a buffer system may include a 2-amino-2- (hydroxymethyl)propate-l, 3-dial (Tris), phosphate, 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES), acetate, or histidine buffer system.
  • a pharmaceutical composition disclosed herein includes a Tris buffer.
  • a pharmaceutical composition disclosed herein includes a phosphate buffer (e.g., phosphate buffered saline (PBS)).
  • PBS phosphate buffered saline
  • a pharmaceutical composition provided herein may have a pH from about 6.0 to 8.0.
  • a pharmaceutical composition provided herein may have a pH from about 6.0 to 8.0, about 6.2 to 8.0, about 6.4 to 8.0, about 6.6 to 8.0, about 6.8 to 8.0, about 7.0 to 8.0, about 7.2 to 8.0, about 7.4 to 8.0, about 7.6 to 8.0, about 7.8 to 8.0, about 6.0 to 7.8, about 6.2 to 7.8, about 6.4 to 7.8, about 6.6 to 7.8, about 6.8 to 7.8, about 7.0 to 7.8, about 7.2 to 7.8, about 7.4 to 7.8, about 7.6 to
  • compositions described herein may also comprise a cryoprotectant and/or a surfactant as a stabilizer to avoid substantial loss of the product quality and, in particular, substantial loss of mRNA activity during storage, freezing, and/or lyophilization, for example to reduce or prevent aggregation, particle collapse, mRNA degradation and/or other types of damage.
  • a cryoprotectant and/or a surfactant as a stabilizer to avoid substantial loss of the product quality and, in particular, substantial loss of mRNA activity during storage, freezing, and/or lyophilization, for example to reduce or prevent aggregation, particle collapse, mRNA degradation and/or other types of damage.
  • a cryoprotectant comprises a carbohydrate.
  • carbohydrate refers to and encompasses monosaccharides, disaccharides, trisaccharides, 5 oligosaccharides and polysaccharides.
  • a cryoprotectant comprises a monosaccharide.
  • a cryoprotectant comprises a disaccharide.
  • disaccharide refers to a compound or a chemical moiety formed by 2 monosaccharide units that are bonded together through a glycosidic linkage, for example through 1-4 linkages or 1-6 linkages. A disaccharide may be hydrolyzed into two monosaccharides.
  • a disaccharide cryoprotectant includes sucrose, trehalose, lactose, maltose and the like.
  • a cryoprotectant comprises a trisaccharide.
  • trisaccharide refers to a compound or a chemical moiety formed by 3 monosaccharide units that are bonded together through glycosidic linkages.
  • a trisaccharide cryoprotectant includes raffinose, melezitose and the like.
  • a cryoprotectant comprises an oligosaccharide.
  • oligosaccharide refers to a compound or a chemical moiety formed by 3 to about 15, such as 3 to about 10 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a linear, branched or cyclic structure.
  • an oligosaccharide cryoprotectant includes cyclodextrins, raffinose, melezitose, maltotriose, stachyose, acarbose, and the like.
  • an oligosaccharide can be oxidized or reduced.
  • a cryoprotectant may be a cyclic oligosaccharide.
  • cyclic oligosaccharide refers to a compound or a chemical moiety formed by 3 to 25 about 15, such as 6, 7, 8, 9, or 10 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a cyclic structure.
  • a cyclic oligosaccharide cryoprotectant includes cyclic oligosaccharides that are discrete compounds, such as a cyclodextrin, p cyclodextrin, or y cyclodextrin.
  • a cyclic oligosaccharide cryoprotectant includes compounds which having a cyclodextrin moiety in a larger molecular structure, such as a polymer that contains a cyclic oligosaccharide moiety.
  • a cyclic oligosaccharide can be oxidized or reduced, for example, oxidized to dicarbonyl forms.
  • cyclodextrin moiety refers to a cyclodextrin (e.g., an a, , or y cyclodextrin) radical that is incorporated into, or a part of, a larger molecular structure, such as a polymer.
  • a cyclodextrin moiety can be bonded to one or more other moieties directly, or through an optional linker.
  • a cyclodextrin moiety can be oxidized or reduced, for example, oxidized to dicarbonyl forms.
  • carbohydrate cryoprotectants e.g., cyclic oligosaccharide cryoprotectants, can be derivatized carbohydrates.
  • a cryoprotectant is a derivatized cyclic oligosaccharide, e.g., a derivatized cyclodextrin, e.g., 2-hydroxypropyl-p- cyclodextrin, e.g., partially etherified cyclodextrins (e.g., partially etherified p cyclodextrins).
  • a derivatized cyclic oligosaccharide e.g., a derivatized cyclodextrin, e.g., 2-hydroxypropyl-p- cyclodextrin, e.g., partially etherified cyclodextrins (e.g., partially etherified p cyclodextrins).
  • a cryoprotectant comprises a polysaccharide.
  • polysaccharide refers to a compound or a chemical moiety formed by at least 16 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a linear, branched or cyclic structure, and includes polymers that comprise polysaccharides as part of their backbone structure.
  • a polysaccharide backbone structure can be linear or cyclic.
  • a polysaccharide cryoprotectants includes glycogen, amylase, cellulose, dextran, maltodextrin and the like.
  • compositions disclosed herein may include sucrose.
  • sucrose functions to promote cryoprotection of the compositions, thereby preventing polyribonucleotide (especially mRNA) particle aggregation and maintaining chemical and physical stability of the composition.
  • pharmaceutical compositions described herein may include alternative cryoprotectants to sucrose.
  • Alternative stabilizers include, without limitation, trehalose and glucose.
  • a pharmaceutical composition includes a LNP incorporating a polyribonucleotide encoding a MIR antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31-40, and 158.
  • a pharmaceutical composition includes a LNP incorporating a polyribonucleotide encoding an A35R antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 11-20, 172, and 174.
  • compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into pharmaceutical compositions described herein. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
  • adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to
  • Formulations of pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing active ingredient(s) into association with a diluent or another excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.
  • a physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician could start doses of active ingredients (e.g., polyribonucleotides encapsulated in lipid nanoparticles) employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • active ingredients e.g., polyribonucleotides encapsulated in lipid nanoparticles
  • compositions e.g., pharmaceutical compositions, e.g., immunogenic compositions, e.g., vaccines.
  • the present disclosure also provides combinations.
  • combinations provided herein comprise combinations of polyribonucleotides provided herein.
  • combinations provided herein comprise combinations of LNP-polyribonucleotides provided herein.
  • Combinations provided herein also comprise combinations of compositions provided herein. Nonlimiting, exemplary compositions and combinations are provided below.
  • compositions including lipid nanoparticles (LNPs) as described herein that incorporate at least one (e.g., one, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more) polyribonucleotide(s), where the at least one polyribonucleotide encodes at least one (e.g., one, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more) MPXV antigen(s).
  • LNPs lipid nanoparticles
  • LNPs lipid nanoparticles
  • the present disclosure provides combinations, including lipid nanoparticles (LNPs) as described herein that incorporate at least one (e.g., one, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more) polyribonucleotide(s), where the at least one polyribonucleotide encodes at least one (e.g., one, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more) MPXV antigen(s).
  • LNPs lipid nanoparticles
  • a composition includes a polyribonucleotide encoding a MIR antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31-40, and 158.
  • a composition includes a polyribonucleotide encoding an A35R antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 11-20, 172, and 174.
  • a composition includes a polyribonucleotide encoding an H3L antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
  • amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
  • a combination of the present disclosure includes a LNP incorporating one polyribonucleotide.
  • a combination includes a LNP incorporating a polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21-30, 180, and 182.
  • a combination includes a polyribonucleotide encoding a MIR antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31-40, and 158.
  • a combination includes a polyribonucleotide encoding an A35R antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 11-20, 172, and 174.
  • a combination includes a polyribonucleotide encoding an H3L antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
  • amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
  • composition of the present disclosure includes two, three, or four
  • LNPs where each LNP incorporates one polyribonucleotide encoding a different mpox virus antigen.
  • a combination of the present disclosure includes two, three, or four LNPs, where each LNP incorporates one polyribonucleotide encoding a different mpox virus antigen.
  • a composition of the present disclosure includes three LNPs.
  • a combination of the present disclosure includes three LNPs.
  • three LNPs comprise an LNP encompassing a polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof, an LNP encompassing a polyribonucleotide encoding an MIR antigen, or antigenic fragment thereof, and an LNP encompassing a polyribonucleotide encoding an A35L antigen, or antigenic fragment thereof.
  • a B6R antigen, or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 1-30, 180, and 182.
  • an MIR antigen, or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31- 40, and 158.
  • an A35R antigen, or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 11-20, 172, and 174.
  • a composition of the present disclosure includes four LNPs.
  • a combination of the present disclosure includes four LNPs.
  • four LNPs comprise an LNP encompassing a a polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof, an LNP encompassing a polyribonucleotide encoding an MIR antigen, or antigenic fragment thereof, an LNP encompassing a polyribonucleotide encoding an A35L antigen, or antigenic fragment thereof, and an LNP encompassing a polyribonucleotide encoding an H3L antigen, or antigenic fragment thereof.
  • a B6R antigen, or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21-30, 180, and 182.
  • an MIR antigen, or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31- 40, and 158.
  • an A35R antigen, or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 11-20, 172, and 174.
  • an H3L antigen, or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
  • a composition of the present disclosure includes a LNP incorporating two polyribonucleotides.
  • a combination of the present disclosure includes an LNP incorporating two polyribonucleotides.
  • two LNPs comprise an LNP encompassing a polyribonucleotide encoding a B6R antigen, or an antigenic fragment thereof, and an LNP encompassing a polyribonucleotide encoding an MIR antigen.
  • B6R antigen, or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21-30, 180, and 182.
  • an MIR antigen, or antigenic fragment thereof having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31-40, and 158.
  • a composition of the present disclosure includes a LNP incorporating three polyribonucleotides.
  • a combination of the present disclosure includes a LNP incorporating three polyribonucleotides.
  • an LNP encompasses a polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof, a polyribonucleotide encoding a MIR antigen, or antigenic fragment thereof, and a polyribonucleotide encoding a A35R antigen, or antigenic fragment thereof.
  • a B6R antigen, or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21-30, 180 and 189.
  • an MIR antigen, or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31-40, and 158.
  • an A35R antigen, or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 11-20, 179 and 174.
  • a B6R antigen, or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21-30, 180 and 189;
  • an MIR antigen, or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31-40, and 158; and an A35R antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 9
  • a composition of the present disclosure includes a LNP incorporating four polyribonucleotides.
  • a combination of the present disclosure includes a LNP incorporating four polyribonucleotides.
  • four polyribonucleotides are encompassed in an LNP.
  • an LNP encompasses a polyribonucleotide encoding a B6R antigen, or an antigenic fragment thereof, a polyribonucleotide encoding a MIR antigen, or an antigenic fragment thereof, a polyribonucleotide encoding an A35R antigen, or an antigenic fragment thereof, and a polyribonucleotide encoding an H3L antigen, or an antigenic fragment thereof
  • a B6R antigen or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21-30, 180, and 182.
  • an MIR antigen, or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31-40, and 158.
  • an A35R antigen, or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 11-20, 172, and 174.
  • an H3L antigen, or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
  • a B6R antigen or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21- 30, 180, and 182; an MIR antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least
  • an A35R antigen, or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least
  • an H3L antigen, or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
  • ability to induce an antibody response may be important to effectiveness of a composition for treatment and/or prevention of orthopox (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, or vaccinia (e.g., modified vaccinia virus Ankara, rabbitpox, etc.)) infection, (e.g., a pharmaceutical composition, an immunogenic composition, or a vaccine).
  • orthopox e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, or vaccinia (e.g., modified vaccinia virus Ankara, rabbitpox, etc.)
  • infection e.g., a pharmaceutical composition, an immunogenic composition, or a vaccine.
  • provided technologies are characterized by an ability to induce (e.g., when administered to a model system and/or to a human, for example by parenteral administration such as by intramuscular administration) an antibody response targeting one or more mpox antigen(s) described herein.
  • provided technologies are characterized in that, when administered (e.g., by parenteral administration such as by intramuscular administration) to an organism (e.g., a model organism or an animal or human organism in need of protection), provided technologies induce a robust antibody response targeting one or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)) antigens.
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)
  • provided technologies are characterized in that they induce antibody titers to a level that provides sufficient protective response against an orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)), when administered to a relevant population.
  • an orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)
  • provided technologies are characterized in that they induce antibody titers to one or more mpox antigen(s) in a range of 10 3 -10 5 after at 5 days, 10 days, 21 days, or 28 days post immunization.
  • composition described herein comprising one or more polyribonucleotides each encoding one or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)) antigens or fragments thereof is characterized in that it can induce antibody titers of at least 10 3 by 5 days post immunization.
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)
  • vaccinia e.g., modified vaccinia virus Ankara, etc.
  • compositions described herein comprising one or more polyribonucleotides each encoding one or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)) antigens or fragments thereof is characterized in that it can induce antibody titers of at least 10 4 by 10 days post immunization.
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)
  • vaccinia e.g., modified vaccinia virus Ankara, etc.
  • compositions described herein comprising one or more polyribonucleotides each encoding one or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)) antigens or fragments thereof is characterized in that it can induce antibody titers of at least 10 5 by 21 days post immunization.
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)
  • vaccinia e.g., modified vaccinia virus Ankara, etc.
  • a second dose of a composition described herein induces a further increase in antibody titers.
  • a second dose is administered at least 21 days after a first dose.
  • a second dose is characterized in that it can induce antibody titers of at least 10 5 by at least 5 days after administration of the second dose.
  • a second dose is characterized in that it can induce antibody titers of at least 10 5 by at least 10 days after administration of the second dose.
  • composition described herein that delivers two or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)) antigens in combination induces similar antibody levels to single antigen immunization.
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)
  • vaccinia e.g., modified vaccinia virus Ankara, etc.
  • administration of a composition described herein that delivers two or more orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)
  • vaccinia e.g., modified vaccinia virus Ankara, etc.
  • administration of a composition described herein that delivers two or more orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)
  • vaccinia e.g., modified vaccinia virus Ankara, etc.
  • administration of a composition described herein that delivers two or more orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)
  • vaccinia e.g., modified vaccinia virus Ankara, etc.
  • administration of a composition described herein that delivers two or more orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)
  • vaccinia e.g., modified vaccinia virus Ankara, etc.
  • administration of a composition described herein to a subject induces antibody production (e.g., IgG, IgA, IgM, IgE).
  • administration of a composition described herein to a subject induces production of one or more of IgGl, IgG2A, IgG2B, and IgG3.
  • administration of a composition described herein to a subject induces the production of orthopoxvirus neutralizing antibodies.
  • administration of a composition described herein to a subject induces the production of B6R-binidng, MIR-binding, A35R-binding, and/or H3L- binding antibodies in the subject.
  • administration of a composition described herein to a subject induces enhanced orthopoxvirus neutralizing antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a composition described herein to a subject induces enhanced B6R-binding antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a composition described herein to a subject induces enhanced MIR-binding antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a composition described herein to a subject induces enhanced A35R-binding antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a composition described herein to a subject induces enhanced A35R-binding antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a composition described herein to a subject induces enhanced H3L-binding antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a composition described herein to a subject induces antibody Fc-effector functional activities (e.g., antibody-mediated complement activation, antibody-mediated NK cell activation, antibody-mediated NK cell degranulation, antibody-mediated IFNy release, antibody-mediated MIPlb release, antibody-dependent cellular monocyte phagocytosis).
  • antibody Fc-effector functional activities e.g., antibody-mediated complement activation, antibody-mediated NK cell activation, antibody-mediated NK cell degranulation, antibody-mediated IFNy release, antibody-mediated MIPlb release, antibody-dependent cellular monocyte phagocytosis.
  • administration of a composition described herein to a subject induces enhanced antibody Fc-effector functional activities (e.g., antibody-mediated complement activation, antibody-mediated NK cell activation, antibody-mediated NK cell degranulation, antibody-mediated IFNy release, antibody-mediated MIPlb release, antibody-dependent cellular monocyte phagocytosis) as compared to functional activities induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a composition described herein to a subject reduces the number of orthopoxvirus lesions in the subject.
  • administration of a composition described herein to a subject reduces the severity of orthopoxvirus lesions in the subject. In some embodiments, administration of a composition described herein to a subject reduces or prevents the onset of orthopoxvirus lesions in the subject. In some embodiments, administration of a composition described herein to a subject reduces the duration of orthopoxvirus lesion onset to resolution in the subject. In some embodiments, administration of a composition described herein to a subject results in greater reduction in the number of orthopoxvirus lesions in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • a reference contemporary modified vaccinia Ankara-based vaccine e.g., JYNEOS®
  • administration of a composition described herein to a subject results in enhanced reduction in the severity of orthopoxvirus lesions in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a composition described herein to a subject results in enhanced reduction or prevention of the onset of orthopoxvirus lesions in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a composition described herein to a subject results in enhanced reduction in the duration of orthopoxvirus lesion onset to resolution in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • a reference contemporary modified vaccinia Ankara-based vaccine e.g., JYNEOS®
  • administration of a composition described herein to a subject reduces the orthopoxvirus viral load in the subject. In some embodiments, administration of a composition described herein to a subject reduces the orthopoxvirus viral load in the throat of the subject. In some embodiments, administration of a composition described herein to a subject reduces the orthopoxvirus viral load in the blood of the subject. In some embodiments, administration of a composition described herein to a subject results in an enhanced reduction in the orthopoxvirus viral load in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • a reference contemporary modified vaccinia Ankara-based vaccine e.g., JYNEOS®
  • administration of a composition described herein to a subject results in an enhanced reduction in the orthopoxvirus viral load in the throat of the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a composition described herein to a subject results in an enhanced reduction in the orthopoxvirus viral load in the blood of the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a composition described herein to a subject reduces morbidity associated with orthopoxvirus infection in the subject.
  • administration of a composition described herein to a subject results in an enhanced reduction in morbidity associated with orthopoxvirus infection in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a composition described herein to a subject reduces morbidity associated with a lethal orthopoxvirus challenge in the subject.
  • administration of a composition described herein to a subject results in an enhanced reduction in morbidity associated with a lethal orthopoxvirus challenge in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a composition described herein to a subject induces the production of orthopoxvirus neutralizing antibodies.
  • administration of a composition described herein to a subject induces the production of B6R-binidng, MIR-binding, A35R-binding, and/or H3L- binding antibodies in the subject.
  • administration of a composition described herein to a subject induces enhanced MIR-binding antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a composition described herein to a subject induces enhanced A35R-binding antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administering induces antibody Fc-effector functional activities (e.g., antibody-mediated complement activation, antibody-mediated NK cell activation, antibody-mediated NK cell degranulation, antibody-mediated IFNy release, antibody-mediated MIPlb release, antibody-dependent cellular monocyte phagocytosis).
  • antibody Fc-effector functional activities e.g., antibody-mediated complement activation, antibody-mediated NK cell activation, antibody-mediated NK cell degranulation, antibody-mediated IFNy release, antibody-mediated MIPlb release, antibody-dependent cellular monocyte phagocytosis.
  • administration of a combination described herein to a subject reduces the number of orthopoxvirus lesions in the subject. In some embodiments, administration of a combination described herein to a subject reduces the severity of orthopoxvirus lesions in the subject. In some embodiments, administration of a combination described herein to a subject reduces or prevents the onset of orthopoxvirus lesions in the subject. In some embodiments, administration of a combination described herein to a subject reduces the duration of orthopoxvirus lesion onset to resolution in the subject.
  • administration of a combination described herein to a subject results in greater reduction in the number of orthopoxvirus lesions in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a combination described herein to a subject results in enhanced reduction in the severity of orthopoxvirus lesions in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankarabased vaccine (e.g., JYNEOS®).
  • administration of a combination described herein to a subject results in enhanced reduction or prevention of the onset of orthopoxvirus lesions in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a combination described herein to a subject results in enhanced reduction in the duration of orthopoxvirus lesion onset to resolution in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a combination described herein to a subject reduces the orthopoxvirus viral load in the subject. In some embodiments, administration of a combination described herein to a subject reduces the orthopoxvirus viral load in the throat of the subject. In some embodiments, administration of a combination described herein to a subject reduces the orthopoxvirus viral load in the blood of the subject. In some embodiments, administration of a combination described herein to a subject results in an enhanced reduction in the orthopoxvirus viral load in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • a reference contemporary modified vaccinia Ankara-based vaccine e.g., JYNEOS®
  • administration of a combination described herein to a subject results in an enhanced reduction in the orthopoxvirus viral load in the throat of the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a combination described herein to a subject results in an enhanced reduction in the orthopoxvirus viral load in the blood of the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a combination described herein to a subject reduces orthopoxvirus virus replication in the subject. In some embodiments, administration of a combination described herein to a subject reduces orthopoxvirus virus replication in the throat of the subject. In some embodiments, administration of a combination described herein to a subject reduces orthopoxvirus virus replication in the blood of the subject. In some embodiments, administration of a combination described herein to a subject results in an enhanced reduction in orthopoxvirus virus replication in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • a reference contemporary modified vaccinia Ankara-based vaccine e.g., JYNEOS®
  • administration of a combination described herein to a subject results in an enhanced reduction in orthopoxvirus virus replication in the throat of the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a combination described herein to a subject results in an enhanced reduction in orthopoxvirus virus replication in the blood of the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a combination described herein to a subject reduces weight loss associated with orthopoxvirus infection in the subject.
  • administration of a combination described herein to a subject results in an enhanced reduction in weight loss associated with orthopoxvirus infection in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a combination described herein to a subject reduces morbidity associated with orthopoxvirus infection in the subject.
  • administration of a combination described herein to a subject results in an enhanced reduction in morbidity associated with orthopoxvirus infection in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • administration of a combination described herein to a subject reduces morbidity associated with a lethal orthopoxvirus challenge in the subject.
  • administration of a combination described herein to a subject results in an enhanced reduction in morbidity associated with a lethal orthopoxvirus challenge in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
  • technologies include polyribonucleotides.
  • compositions for treatment and or prevention of an orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)
  • an orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)
  • vaccinia e.g., modified vaccinia virus Ankara, etc.
  • a subject is one suffering from and/or is susceptible to an orthopox infection.
  • a subject is one suffering from and/or is susceptible to mpox infection (e.g., a clade la, clade lb, clade Ila, or clade lib mpox infection).
  • a subject is one suffering from and/or is susceptible to variola infection.
  • a subject is one suffering from and/or is susceptible to vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection.
  • a subject may be defined by one or more criterion such as age group, gender, genetic background, preexisting clinical conditions, and/or prior exposure to therapy.
  • a subject is a model organism. In preferred embodiments, a subject is a human. In some embodiments, a subject is between 18-65 years of age. In some embodiments, a subject is an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old,
  • a subject is a human infant. In some embodiments, a subject is a human toddler. In some embodiments, a subject is a human child. In some embodiments, a subject is a human adult. In some embodiments, a subject is an elderly human. In some embodiments, a subject is a pregnant woman. In some embodiments, a subject is a human adolescent. In some embodiments, a subject is immunocompromised. In some embodiments, a subject is HIV-positive.
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, modified vaccinia virus Ankara, or vaccinia
  • methods and compositions are not subject to certain limitations that characterize other vaccine technologies.
  • live attenuated virus vaccines are contraindicated for subjects who are immunocompromised (e.g., severely immunocompromised) at least in part because such subjects are at increased risk for serious adverse reactions.
  • Subjects with most forms of altered immunocompetence should not receive live vaccines.
  • the present disclosure includes that methods and compositions provided herein can be used to treat (e.g., to vaccinate) subjects who are immunocompromised (e.g., severely immunocompromised) and/or subjects with altered immunocompetence (including without limitation primary and/or secondary immunosuppression, immunodeficiency, and immunocompromised).
  • a subject has no prior history of known or suspected smallpox vaccination prior to administration of one or more doses of a composition as disclosed herein (also referred to as "vaccinia naive").
  • a subject has a prior history of a smallpox vaccination (also referred to as "vaccinia-experienced")-
  • a subject's smallpox vaccination status is determined by the presence or absence of a smallpox vaccination scar.
  • a subject does not have febrile illness prior to administration of one or more doses of a composition as disclosed herein. In some embodiments, a subject does not have febrile illness within about 72 hours, about 48 hours, about 36 hours, about 24 hours, or about 12 hours prior to administration of one or more doses of a composition as disclosed herein.
  • a subject does not have an acute illness prior to administration of one or more doses of a composition as disclosed herein. In some embodiments, a subject does not have an acute illness within about 72 hours, about 48 hours, about 36 hours, about 24 hours, or about 12 hours prior to administration of one or more doses of a composition as disclosed herein.
  • a subject has not received a vaccine within 7 days to 56 days, 14 days to 56 days, 21 days to 56 days, 28 days to 56 days, 35 days to 56 days, 42 days to 56 days, 49 to 56 days, 7 days to 49 days, 14 days to 49 days, 21 days to 49 days, 28 days to 49 days, 35 days to 49 days, 42 days to 49 days, 7 days to 42 days, 14 days to 42 days, 21 days to 42 days, 28 days to 42 days, 35 days to 42 days, 7 days to 35 days, 14 days to 35 days, 21 days to 35 days, 28 days to 35 days, 7 days to 28 days, 14 days to 28 days, 21 days to 28 days, 7 days to 21 days, 14 days to 21 days, 14 days to 21 days, or 7 days to 14 days, before being administered one or more doses of a therapeutically effective amount of a composition as disclosed herein.
  • a subject has not received a vaccine within about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, or about 56 days before being administered one or more doses of a therapeutically effective amount of a composition as disclosed herein.
  • a subject has not received a vaccine within about 28 days, before being administered one or more doses of a therapeutically effective amount of a composition as disclosed herein.
  • the vaccine is not a seasonal influenza vaccine or a medically indicated vaccine.
  • a subject does not receive a vaccine within at least 2 weeks to 10 weeks, at least 4 weeks to 10 weeks, at least 6 weeks to 10 weeks, at least 8 weeks to 10 weeks, at least 2 weeks to 8 weeks, at least 4 weeks to 8 weeks, at least 6 weeks to 8 weeks, at least 2 weeks to 6 weeks, at least 4 weeks to 6 weeks, or at least 2 weeks to 4 weeks after administration of one or more doses of a therapeutically effective amount of a composition as disclosed herein.
  • a subject does not receive a vaccine within at least about 2 weeks, at least about 4 weeks, at least about 6 weeks, at least about 8 weeks, or at least about 10 weeks after administration of one or more doses of a therapeutically effective amount of a composition as disclosed herein.
  • a subject has not received an allergy treatment within 8 to 20 days, 10 to 20 days, 12 to 20 days, 14 to 20 days, 16 to 20 days, 18 to 20 days, 8 to 18 days, 10 to 18 days, 12 to 18 days, 14 to 18 days, 16 to 18 days, 8 to 16 days, 10 to 16 days, 12 to 16 days, 14 to 16 days, 8 to 14 days, 10 to 14 days, 12 to 14 days, 8 to 12 days, 10 to 12 days, or about 8 to 10 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein.
  • a subject has not received an allergy treatment within about 8 days, about 10 days, about 12 days, about 14 days, about 16 days, about 18 days, or about 20 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein. In some embodiments, a subject has not received an allergy treatment within about 14 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein. In some embodiments, an allergy treatment comprises antigen injections.
  • a subject has not received an immunosuppressive medication within about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, or about 56 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein. In some embodiments, a subject has not received an immunosuppressive medication within about 28 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein.
  • a subject has not received a prophylactic antipyretic and/or an analgesic medication within 7 to 56 days, 14 to 56 days, 21 to 56 days, 28 to 56 days, 35 to 56 days, 42 to 56 days, 49 to 56 days, 7 to 49 days, 14 to 49 days, 21 to 49 days, 28 to 49 days, 35 to 49 days, 42 to 49 days, 7 to 42 days, 14 to 42 days, 21 to 42 days, 28 to 42 days, 35 to 42 days, 7 to 35 days, 14 to 35 days, 21 to 35 days, 28 to 35 days, 7 to 28 days, 14 to 28 days, 21 to 28 days, 7 to 21 days, 14 to 21 days, or 7 to 14 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein.
  • a subject has not received a prophylactic antipyretic and/or an analgesic medication within about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, or about 56 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein. In some embodiments, a subject has not received a prophylactic antipyretic and/or an analgesic medication within about 14 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein.
  • a prophylactic antipyretic medication is selected from, but not limited to, acetaminophen, a non-steroidal anti-inflammatory drug (NSAID), salicylamide, salicyl salicylate, methyl salicylate, magnesium salicylate, fatelamine, ethenzamide, diflunisal, choline magnesium salicylate, benorylate/benorilatem and amoxiprin, acetylsalicylate, ceclofenac, acemetacin, alclofenac, bromfenac, diclofenac, etodolac, indomethacin, nabumetone, oxametacin, proglumetacin, sulindac, tolmetin, iminoprofen, benoxaprofen, carprofen, dexibuprofen, dexketoprofen, fenbufen, fenoprofen, flunoxaprofen,
  • NSAID non-ster
  • a prophylactic analgesic medication is selected from, but not limited to, acetaminophen, salicylamide, salicyl salicylate, methyl salicylate, magnesium salicylate, fatelamine, ethenzamide, diflunisal, choline magnesium salicylate, benorylate/benorilatem and amoxiprin, acetylsalicylate, ceclofenac, acemetacin, alclofenac, bromfenac, diclofenac, etodolac, indomethacin, nabumetone, oxametacin, proglumetacin, sulindac, tolmetin, iminoprofen, benoxaprofen, carprofen, dexibuprofen, dexketoprofen, fenbufen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprof
  • a subject administered with one or more doses of a therapeutically effective amount of a composition disclosed herein is negative for HIV-1 and/or HIV-2. In some embodiments, a subject administered with one or more doses of a therapeutically effective amount of a composition disclosed herein is negative for hepatitis B and/or hepatitis C.
  • a pharmaceutical composition described herein can be taken up by cells for production of an encoded agent at therapeutically relevant serum concentrations. Accordingly, the present disclosure provides methods of using pharmaceutical compositions described herein. For example, in some embodiments, a method provided herein comprises administering a pharmaceutical composition described herein to a subject.
  • a pharmaceutical composition described herein is for the prophylactic protection and/or prevention against orthopoxvirus infection.
  • a pharmaceutical composition described herein is for the prophylactic protection and/or prevention against mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus infection.
  • a pharmaceutical composition described herein is for the prophylactic protection and/or prevention against an mpox clade la, clade lb, clade Ila, or clade lib infection.
  • a pharmaceutical composition described herein is for treatment of a subject having mild orthopoxvirus infection and/or mild orthopoxvirus-related disease. In some embodiments, a pharmaceutical composition described herein is for treatment of a subject having mild orthopoxvirus infection and/or moderate orthopoxvirus-related disease. In some embodiments, a pharmaceutical composition described herein is for treatment of a subject having severe orthopoxvirus infection and/or mild orthopoxvirus-related disease.
  • a pharmaceutical composition described herein is for post-exposure prevention of orthopoxvirus transmission to others.
  • a pharmaceutical composition described herein is for post-exposure prevention of mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus transmission to others.
  • a pharmaceutical composition described herein is for post-exposure prevention of mpox clade la, clade lb, clade Ila, or clade lib virus transmission to others.
  • a pharmaceutical composition described herein is for post-exposure prevention of mpox clade la virus transmission to others. In some embodiments, a pharmaceutical composition described herein is for post-exposure prevention of mpox clade lb virus transmission to others.
  • a pharmaceutical composition described herein is for protection and/or prevention of post-exposure orthopoxvirus disease progression.
  • a pharmaceutical composition described herein is for post-exposure protection and/or prevention of mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus disease progression.
  • a pharmaceutical composition described herein is for post-exposure protection and/or prevention of mpox clade la, clade lb, clade Ila, or clade lib virus disease progression. In some embodiments, a pharmaceutical composition described herein is for post-exposure protection and/or prevention of mpox clade la virus disease progression. In some embodiments, a pharmaceutical composition described herein is for post-exposure protection and/or prevention of mpox clade lb virus disease progression.
  • administering typically refers to the administration of a composition to a subject to achieve delivery of an agent ⁇ e.g., at least one polyribonucleotide encoding an antigen or fragment thereof described herein) that is, or is included in, a composition to a target site or a site to be treated.
  • an agent e.g., at least one polyribonucleotide encoding an antigen or fragment thereof described herein
  • Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human.
  • Administration may be, for example, bronchial e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal e.g., by intratracheal instillation), vaginal, vitreal, etc.
  • administration may be intramuscular, intravenous, or subcutaneous.
  • administration of a pharmaceutical composition results in delivery of one or more polyribonucleotides as described herein ⁇ e.g., encoding an antigen or antigenic fragment thereof) to a subject.
  • administering a pharmaceutical composition to a subject results in expression in the subject of an antigen or fragment thereof encoded by an administered polyribonucleotide.
  • administering a pharmaceutical composition to a subject results in expression in the subject of an antigen or antigenic fragment thereof encoded by an administered polyribonucleotide.
  • a pharmaceutical composition for administration to a subject is provided as two or more separate particle compositions each comprising one or more polyribonucleotides of the present disclosure (e.g., encoding an mpox antigen as described herein), which are then mixed together prior to administration.
  • individual populations of nucleic acid containing particles, each population comprising an RNA molecule encoding a different immunogenic polypeptide or antigenic fragment thereof (e.g., an mpox antigen as described herein) can be separately formed and then mixed together, for example, prior to filling into vials during a manufacturing process, or immediately prior to administration).
  • compositions comprising two or more populations of particles (e.g., in some embodiments, lipid nanoparticles), each population comprising at least one RNA molecule encoding a different immunogenic polypeptide or antigenic fragment thereof (e.g., mpox antigen or antigenic fragment thereof).
  • each population may be provided in a composition at a desirable proportion (e.g., in some embodiments, each population may be provided in a composition in an amount that provides the same amount of RNA molecules).
  • administered pharmaceutical compositions e.g., immunogenic compositions, e.g., vaccines
  • polyribonucleotides that encode one or more mpox antigens are administered in RNA doses of from about 0.1 ⁇ g to about 300 ⁇ g, about 0.5 ⁇ g to about 200 ⁇ g, or about 1 ⁇ g to about 100 ⁇ g, such as about 1 ⁇ g, about 3 ⁇ g, about 10 ⁇ g, about 30 ⁇ g, about 50 ⁇ g, or about 100 ⁇ g.
  • a pharmaceutical composition comprises one polyribonucleotide encoding one, two, or more mpox antigens, wherein each polyribonucleotide is administered at a dose of from about 0.5 ⁇ g to about 2 ⁇ g.
  • a pharmaceutical composition comprises one polyribonucleotide encoding one, two, or more mpox antigens, wherein the polyribonucleotide is administered at a dose of about 1 ⁇ g.
  • the one, two or more mpox antigens are a B cell antigen from Table 1 or an antigenic fragment thereof.
  • a pharmaceutical composition comprises two or more polyribonucleotides each encoding an mpox antigen (e.g., from Table 1 or an antigenic fragment thereof), wherein each RNA construct is administered at a dose of from about 0.5 ⁇ g to about 2 ⁇ g.
  • a pharmaceutical composition comprises two or more polyribonucleotide each encoding an mpox antigen (e.g., from Table 1 or an antigenic fragment thereof), wherein each RNA construct is administered at a dose of about 1 ⁇ g.
  • one or more pharmaceutical compositions are administered, comprising two or more RNA mpox constructs (e.g., two, three, four, five, six or more RNA constructs), wherein each RNA construct is administered at a dose of about 1 ⁇ g.
  • two or more pharmaceutical compositions are administered, together comprising two or more RNA constructs each encoding one or more mpox antigens (e.g., from Table 1 or an antigenic fragment thereof), wherein each RNA construct is administered at a dose of about 1 ⁇ g.
  • two or more pharmaceutical compositions are administered, together comprising two, three, four, five, six or more polyribonucleotides encoding mpox antigens, wherein each polyribonucleotide is administered at a dose of about 1 ⁇ g.
  • one or more pharmaceutical compositions are administered, together comprising one or more polyribonucleotides encoding two to four mpox antigens (e.g., from Table 1 or an antigenic fragment thereof), wherein each polyribonucleotide is administered at a dose of about 1 ⁇ g.
  • one or more pharmaceutical compositions are administered, together comprising one or more polyribonucleotides encoding two to four mpox B cell antigens (e.g., from Table 1 or an antigenic fragment thereof), wherein each polyribonucleotide is administered at a dose of about 1 ⁇ g.
  • one or more pharmaceutical compositions are administered, together comprising one or more polyribonucleotides encoding two to four mpox B cell antigens selected from A35R, B6R, MIR, H3L, and/or antigenic fragments of any thereof, wherein each polyribonucleotide is administered at a dose of about 1 ⁇ g.
  • one or more pharmaceutical compositions are administered, together comprising one or more polyribonucleotides encoding two to four mpox B cell antigens selected from B6R, A35R, MIR, H3L, and/or antigenic fragments of any thereof.
  • one or more pharmaceutical compositions are administered, together comprising one or more polyribonucleotides encoding four mpox B cell antigens that are B6R, A35R, MIR, and H3L, (or antigenic fragments of any thereof). In some embodiments, one or more pharmaceutical compositions are administered, together comprising one or more polyribonucleotides encoding four mpox B cell antigens that are B6R, A35R, MIR, (or antigenic fragments of any thereof).
  • one or more pharmaceutical compositions are administered, together comprising one or more polyribonucleotides encoding three mpox B cell antigens that are B6R, A35R, and MIR, (or antigenic fragments of any thereof).
  • a pharmaceutical composition is administered, comprising two polyribonucleotides each encoding one or more mpox antigens, wherein a total polyribonucleotide dose administered is about 2 to 4 ⁇ g.
  • two or more pharmaceutical compositions are administered, together comprising two polyribonucleotides each encoding one or more mpox antigens, wherein a total polyribonucleotide dose administered is about 2 to 4 ⁇ g.
  • a pharmaceutical composition is administered, comprising one or more polyribonucleotides encoding one or more mpox antigens, wherein a total polyribonucleotide dose administered is about 10 ⁇ g, 30 ⁇ g, or 60 ⁇ g.
  • a pharmaceutical composition is administered, comprising two polyribonucleotides each encoding one or more mpox antigens, wherein a total polyribonucleotide dose administered is about 10 ⁇ g, 30 ⁇ g, or 60 ⁇ g.
  • two or more pharmaceutical compositions are administered, together comprising two polyribonucleotides each encoding one or more mpox antigens, wherein a total polyribonucleotide dose administered is about 10 ⁇ g, 30 ⁇ g, or 60 ⁇ g.
  • the one or more mpox antigens are B cell antigens selected from E8L, A35R, B6R, MIR, H3L, A28L, A29L, and/or antigenic fragments of any thereof.
  • the one or more mpox antigens are selected from B6R, A35R, MIR, H3L, E8L, and/or antigenic fragments of any thereof.
  • a pharmaceutical composition is administered, comprising three polyribonucleotides each encoding a different mpox antigen, or antigenic fragment thereof, wherein a total polyribonucleotide dose administered is about 10 ⁇ g, 30 ⁇ g, or 60 ⁇ g.
  • the three polyribonucleotides encode B6R, A35R, and MIR, and/or antigenic fragments of any thereof.
  • a pharmaceutical composition is administered, comprising four polyribonucleotides each encoding a different mpox antigen, or antigenic fragment thereof, wherein a total polyribonucleotide dose administered is about 10 ⁇ g, 30 ⁇ g, or 60 ⁇ g.
  • the four polyribonucleotides encode B6R, A35R, MIR, and H3L and/or antigenic fragments of any thereof.
  • administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses.
  • administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing.
  • administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
  • a dosing regimen comprises a plurality of doses each of which is separated in time from other doses.
  • individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses.
  • all doses within a dosing regimen are of the same unit dose amount.
  • different doses within a dosing regimen are of different amounts.
  • a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount.
  • a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount.
  • a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).
  • therapies can be administered in dosing cycles.
  • pharmaceutical compositions described herein are administered in one or more dosing cycles.
  • one dosing cycle is at least 3 or more days (including, e.g., at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 days.
  • one dosing cycle is at least 21 days.
  • one dosing cycle may involve multiple doses, e.g., according to a pattern such as, for example, a dose may be administered daily within a dosing cycle, or a dose may be administered every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 2 weeks, monthly, every 2 months within a cycle.
  • a dosing cycle is at least 4 weeks. In some certain embodiments, a dosing cycle is about 4 weeks.
  • multiple dosing cycles may be administered.
  • at least 2 dosing cycles including, e.g., at least 3 dosing cycles, at least 4 dosing cycles, at least 5 dosing cycles, at least 6 dosing cycles, at least 7 dosing cycles, at least 8 dosing cycles, at least 9 dosing cycles, at least 10 dosing cycles, or more
  • the number of dosing cycles to be administered may vary with types of treatment (e.g., monotherapy vs. combination therapy).
  • at least 3-8 dosing cycles may be administered.
  • a rest period may have a length within a range of several days to several months.
  • a rest period may have a length of at least 3 days or more, including, e.g., at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days or more.
  • a rest period may have a length of at least 1 week or more, including, e.g., at least 2 weeks, at least 3 weeks, at least 4 weeks, or more.
  • a rest period is at least 4 weeks. In certain embodiments, a rest period is about 4 weeks.
  • Dosage of pharmaceutical compositions described herein may vary with a number of factors including, e.g., but not limited to body weight of a subject to be treated, cancer types and/or cancer stages, and/or monotherapy or combination therapy.
  • a dosing cycle involves administration of a set number and/or pattern of doses.
  • a pharmaceutical composition described herein is administered at least one dose per dosing cycle, including, e.g., at least two doses per dosing cycle, at least three doses per dosing cycle, at least four doses per dosing cycle, or more.
  • a dosing cycle involves administration of a set cumulative dose, e.g., over a particular period of time, and optionally via multiple doses, which may be administered, for example, at set interval(s) and/or according to a set pattern.
  • a set cumulative dose may be administered via multiple doses at set intervals such that there is at least some temporal overlap in biological and/or pharmacokinetics effects generated by such multiple doses on a target cell or on a subject being treated.
  • a set cumulative dose may be administered via multiple doses at set intervals such that biological and/or pharmacokinetics effects generated by such multiple doses on a target cell or on a subject being treated may be additive.
  • a set cumulative dose of X mg may be administered via two doses with each dose of X/2 mg, wherein such two doses are administered sufficiently close in time such that biological and/or pharmacokinetics effects generated by each X/2-mg dose on a target cell or on a subject being treated may be additive.
  • dosing may be adjusted based on response of a subject receiving the therapy. For example, in some embodiments, dosing may involve administration of a higher dose followed later by administration of a lower dose if one or more parameters for safety pharmacology assessment indicates that the prior dose may not satisfy the medical safety requirement according to a physician. In some embodiments, dose escalation may be performed at one or more of the levels. Without wishing to be bound by any particular theory, the present disclosure, among other things, provides an insight that a pharmaceutically guided dose escalation (PGDE) method may be applied to determine an appropriate dose of pharmaceutical compositions described herein.
  • PGDE pharmaceutically guided dose escalation
  • compositions described herein can be administered to subjects as monotherapy.
  • a pharmaceutical composition provided herein may be administered as part of combination therapy.
  • subjects receiving a pharmaceutical composition provided herein may be monitored periodically over a dosing regimen to assess efficacy of the administered treatment.
  • efficacy of an administered treatment may be assessed periodically, e.g., weekly, biweekly, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, or longer.
  • polyribonucleotides or pharmaceutical compositions of the present disclosure are administered to a subject in need thereof to induce an immune response against an orthopoxvirus.
  • the orthopoxvirus is an mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus.
  • the orthopoxvirus is an mpox clade la, clade lb, clade Ila, or clade lib virus.
  • the orthopoxvirus is an mpox clade la virus.
  • the orthopoxvirus is an mpox clade lb virus.
  • compositions described herein are administered to a subject in need thereof to induce production of orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) neutralizing antibodies in the subject.
  • pharmaceutical compositions described herein are administered to a subject in need thereof to induce production of B6R-binidng, MIR-binding, A35R-binding, and/or H3L-binding antibodies in the subject.
  • compositions described herein are administered to a subject in need thereof to enhance orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) neutralizing antibody titers in the subject.
  • pharmaceutical compositions described herein are administered to a subject in need thereof to enhance B6R-binidng, MIR-binding, A35R- binding, and/or H3L-binding antibody titers in the subject.
  • compositions described herein are administered to a subject in need thereof to induce antibody Fc-effector functional activities (e.g., antibody-mediated complement activation, antibody-mediated NK cell activation, antibody-mediated NK cell degranulation, antibody-mediated IFNy release, antibody-mediated MIPlb release, antibody-dependent cellular monocyte phagocytosis) in the subject.
  • antibody Fc-effector functional activities e.g., antibody-mediated complement activation, antibody-mediated NK cell activation, antibody-mediated NK cell degranulation, antibody-mediated IFNy release, antibody-mediated MIPlb release, antibody-dependent cellular monocyte phagocytosis
  • compositions described herein are administered to a subject in need thereof to reduce the number of orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) lesions in the subject.
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus
  • compositions described herein are administered to a subject in need thereof to reduce the severity of orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) lesions in the subject.
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus
  • compositions described herein are administered to a subject in need thereof to reduce or prevent the onset of orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) lesions in the subject.
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus
  • compositions described herein are administered to a subject in need thereof to reduce the duration of orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) lesion onset to resolution in the subject.
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus
  • compositions described herein are administered to a subject in need thereof to reduce the orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) viral load in the subject.
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus
  • compositions described herein are administered to a subject in need thereof to reduce the orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) viral load in the throat of the subject.
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus
  • compositions described herein are administered to a subject in need thereof to reduce the orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) viral load in the blood of the subject.
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus
  • compositions described herein are administered to a subject in need thereof to reduce orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) virus replication in the subject.
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus
  • compositions described herein are administered to a subject in need thereof to reduce orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) virus replication in the throat of the subject.
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus
  • compositions described herein are administered to a subject in need thereof to reduce orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) virus replication in the blood of the subject.
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus
  • compositions described herein are administered to a subject in need thereof to reduce weight loss associated with orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) infection in the subject.
  • orthopoxvirus e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus
  • compositions described herein are administered to a subject in need thereof to reduce morbidity associated with (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) infection in the subject.
  • morbidity associated with e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus
  • polyribonucleotides can be produced by methods known in the art.
  • polyribonucleotides can be produced by in vitro transcription, for example, using a DNA template.
  • a plasmid DNA used as a template for in vitro transcription to generate a polyribonucleotide described herein is also within the scope of the present disclosure.
  • a DNA template is used for in vitro RNA synthesis in the presence of an appropriate RNA polymerase (e.g., a recombinant RNA-polymerase such as a T7 RNA-polymerase) with ribonucleotide triphosphates (e.g., ATP, CTP, GTP, UTP).
  • an appropriate RNA polymerase e.g., a recombinant RNA-polymerase such as a T7 RNA-polymerase
  • ribonucleotide triphosphates e.g., ATP, CTP, GTP, UTP.
  • polyribonucleotides e.g., ones described herein
  • pseudouridine (ip), Nl-methyl-pseudouridine (mlip), or 5-methyl-uridine (m5U) can be used to replace uridine triphosphate (UTP).
  • pseudouridine (ip) can be used to replace uridine triphosphate (UTP).
  • Nl-methyl-pseudouridine (mlip) can be used to replace uridine triphosphate (UTP).
  • 5-methyl-uridine (m5U) can be used to replace uridine triphosphate (UTP).

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Abstract

The present disclosure provides pharmaceutical compositions for delivery of mpox virus antigens (e.g., an mpox vaccine) and related technologies (e.g., components thereof and/or methods relating thereto). For example, the present disclosure provides polyribonucleotides encoding one or more mpox antigens or antigenic fragments thereof.

Description

RNA COMPOSITIONS FOR DELIVERY OF MPOX ANTIGENS AND RELATED METHODS CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application claims priority to United States Provisional Application No. 63/581,265, filed September 7, 2023, United States Provisional Application No. 63/588,961, filed October 9, 2023, United States Provisional Application No. 63/604,159, filed November 29, 2023, United States Provisional Application No. 63/639,545, filed April 26, 2024, and United States Provisional Application No. 63/646,826, filed May 13, 2024, the entireties of which are incorporated herein by reference.
SEQUENCE LISTING
[0002] The present application contains a Sequence Listing which has been submitted electronically in
XML format and is hereby incorporated by reference in its entirety. Said XML file copy, created on August 30, 2024, is named 2013237-1163_SL.xml and is 657,817 bytes in size.
BACKGROUND
[0003] Orthopoxvirus is a genus encompassing a number of viral species including mpox virus
(previously known as "monkeypox"; also referred to herein as"MPXV"), vaccinia virus (also referred to herein as "VACV", e.g., Vaccinia virus Ankara, Vaccinia virus Copenhagen, Vaccinia virus WR), rabbitpox, buffalopox, camelpox, Cantagalo virus, ectromelia virus (also referred to herein as "ECTV" and "mousepox"), borealpox virus (previously known as "Alaskapox"; also referred to herein as "AKPV" or "BRPV"), cowpox virus (also referred to herein as "CPXV"), and variola virus (also referred to herein as "VARV"). Some orthopoxviruses are restricted in the hosts they infect, while others have been identified in a broad range of host species. Orthopoxviruses share a number of biological phenotypes including: a lack of a specific receptor required for infection of mammalian cells, a relatively low mutations rate, environmental stability of virion, and the ability to infect hosts via a number of routes (e.g., mucosal, respiratory, parenteral, etc.).
[0004] Mpox was first discovered in 1958 when two outbreaks of a pox-like disease occurred in colonies of monkeys kept for research, hence the name "monkeypox." The first human case of mpox was recorded in 1970 in the Democratic Republic of Congo during a period of intensified effort to eliminate smallpox. Since then, mpox has been reported in humans in other central and western African countries.
[0005] Beginning in May 2022, a multinational outbreak of mpox led to over 85,000 cases worldwide spanning 114 countries, many of which have not been previously considered endemic. Transmission of this zoonotic infection has sustained endemicity in West and Central Africa for many years, but the scale of the 2022 outbreak was unprecedented leading to its declaration as a public health emergency of international concern (PHEIC) by the World Health Organization (WHO) in July 2022. The PHEIC was discontinued in May 2023 due to a significant decline in the number of reported cases and no changes in the severity of the disease (WHO Emergency Committee Meeting 2023). In 2024, the WHO declared a new PHEIC as a result of the upsurge of mpox and the emergence of a new clade (clade lb) in the Democratic Republic of the Congo and other African countries. The recent mpox outbreak illustrates the potential for orthopoxvirus re-emergence and spread and highlights limitations in the current vaccine supply.
SUMMARY
[0006] Pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) that deliver portions or components of a pathogen (e.g., virus) have advantages to live pathogen approaches by focusing the vaccine response only on targets with a high potential for eliciting protective responses. While specific antigens have been shown to be immunogenic following orthopoxvirus (e.g., VACV) administration, the minimal set of antigens and the types of immunity to them that are essential for protection from disease are not fully understood. Therefore, despite the multivalency enabled by current RNA vaccine platforms, antigen selection for subunit vaccines against orthopoxviruses is still being explored. Pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) that deliver portions or components of orthopoxviruses therefore face a key question around antigen selection as, despite the multivalency enabled by the RNA platform, prioritization must take place.
[0007] Pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) of the present disclosure are designed to encode multiple mpox antigens because of the many surface proteins and complex replication cycle of orthopoxviruses. Orthopoxviruses have two distinct infectious forms, mature virions (MV) and extracellular virions (EV). Each viral form has a unique set of surface antigens. While immune responses raised to any single target can offer some protection from infection, both subunit vaccine and monoclonal antibody prophylaxis studies in animals illustrated that the value of combining antibodies to multiple EV and MV targets. Therefore, to effectively reduce viral replication and infectivity, exemplary vaccine compositions of the present disclosure elicit immune responses to a combination of mpox proteins from both EVs and MVs: A35 and B6, which are EV surface glycoproteins, and Ml and H3, which are displayed on the surface of MVs. Without wishing to be bound by theory, selection of this subset of antigens from the surface proteomes of mpox virions involved a consideration, in part, of the fact that A35, B6, Ml and H3 are conserved across many orthopoxviruses, including VARV and VACV. All four proteins have been observed to be highly immunogenic following VACV immunization or in MPXV convalescent subjects. A35, B6, and Ml have been shown to protect mice and macaques from lethal orthopoxvirus disease. Additionally, antibodies to all four antigens have previously been shown to protect animals in monoclonal and/or polyclonal antibody prophylaxis studies. While prior studies have shown that A35, B6, Ml and H3 can elicit immune responses and provide some degree of protection against orthopoxviruses, challenges remain with the use of pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) that include the antigens as polypeptides.
[0008] The present disclosure provides a recognition of several challenges involved in development and administration of polypeptide-based therapies. First, development of polypeptide-based therapies can be time consuming and expensive. For example, polypeptide development is challenged by demanding and costly production, including purification and formulation methods. Second, polypeptide-based therapies present an increased number of regulatory challenges. In addition to demonstrating that a protein therapy will be safe and efficacious, there is tight regulation over manufacture of polypeptide-based therapies (e.g., assessing post- translational modifications, etc.) and quality control during storage and administration complicate the use of protein therapies. Third, administration of polypeptide-based therapies to a subject can be painful and time consuming. Generally, polypeptide-based therapies are administered intravenously over a longer period of time, increasing patient discomfort. Finally, polypeptide-based therapies can have a relatively short serum half-life. The present disclosure provides insights that address these challenges, making it possible to deliver one or more therapies, as described herein, to a subject safely, reliably, and with strong potency.
[0009] Among other things, the present disclosure describes one or more orthopoxvirus (e.g., mpox) antigens, antigenic fragments, or variants thereof that are delivered to a subject via one or more polyribonucleotides. Utilizing one or more polyribonucleotides as a therapeutic agent (in contrast to administering a polypeptide itself) involves simpler and less expensive manufacturing processes. The less complex production of polyribonucleotide encoding antigens can streamline manufacture, mitigating regulatory and production challenges associated with developing and using an antigen themselves. Additionally, polyribonucleotides are effective at producing similar effects to recombinant polypeptides, but tend to require much lower volumes be administered to a subject. This is because polyribonucleotides encoding an antigen characteristic portion, or variant thereof, can be administered to a subject and the subject's body produces the polypeptide itself. Without wishing to be bound by any particular theory, a polyribonucleotide encoding antigens, as a therapeutic agent, has higher therapeutic efficacy (in contrast to administering a polypeptide itself) due to its continuous translation into encoded antigen to trigger long-lasting expression compared to transient traditional antigen polypeptide delivery. The present disclosure also provides technologies that address certain limitations of recombinant polypeptide technologies, including for example, expression of antigens by utilizing RNA technologies as a modality to express antigens directly in the patient's cells.
[OO1O] The present disclosure provides pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) for delivering particular orthopox antigen constructs to a subject (e.g., a patient) and related technologies (e.g., methods). More specifically, the present disclosure provides pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) for delivering particular polyribonucleotide constructs encoding mpox antigens to a subject (e.g., a patient) and related technologies (e.g., methods). In particular, the present disclosure provides polyribonucleotide constructs encoding mpox antigens and related technologies (e.g., methods). The present disclosure includes the unexpected discovery that mpox antigens, and antigenic fragments thereof, provided herein are particularly advantageous for use in preventing or treating mpox infection and infections by related orthopoxviruses (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, modified vaccinia virus Ankara, or vaccinia viruses).
[0011] In some embodiments, the present disclosure provides certain polyribonucleotide constructs encoding mpox antigens particularly useful in effective vaccination. In some embodiments, provided polyribonucleotide constructs encoding mpox antigens are effective for vaccination against mpox. In various embodiments, an mpox antigen construct includes and/or encodes one or more mpox antigens or fragments thereof (e.g., one or more B cell antigens for mpox and/or one or more T cell antigens for mpox, or fragments thereof). As disclosed herein, T cell antigens include, e.g., CD4 T cell antigens and/or CD8 T cells. For the avoidance of doubt, as will be appreciated by those of skill in the art, any reference herein to an antigen as a "B cell antigen" or "T cell antigen" or the like does not exclude that any given antigen, or any given agent when exposed to an immune system, can activate, induce, and/or cause a diversity of immunological responses that can include, regardless of labels applied for expediency of description, one or both of a B cell response and a T cell response.
[0012] The present disclosure also provides the insight that an mpox vaccine may cross-protect against other orthopoxviruses (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara)). In some embodiments, provided polyribonucleotide constructs encoding mpox antigens are effective for the prevention or treatment of a vaccinia virus (e.g., vaccinia virus Ankara, etc.) infection when administered to a subject. In some embodiments, provided polyribonucleotide constructs encoding mpox antigens are effective for the prevention or treatment of a rabbitpox infection when administered to a subject. In some embodiments, provided polyribonucleotide constructs encoding mpox antigens are effective for the prevention or treatment of a variola virus infection when administered to a subject. In some embodiments, provided polyribonucleotide constructs encoding mpox antigens are effective for the prevention or treatment against a cowpox virus when administered to a subject. In some embodiments, provided polyribonucleotide constructs encoding mpox antigens are effective for the prevention or treatment of a ectromelia virus infection when administered to a subject. In some embodiments, provided polyribonucleotide constructs encoding mpox antigens are effective for vaccination against mpox and one or more other orthopoxviruses. In some embodiments, provided polyribonucleotide constructs encoding mpox antigens are effective for vaccination against mpox and variola virus. In some embodiments, provided polyribonucleotide constructs encoding mpox antigens are effective for vaccination against mpox and vaccinia virus. In some embodiments, provided polyribonucleotide constructs encoding mpox antigens are effective for vaccination against mpox and cowpox virus. In some embodiments, provided polyribonucleotide constructs encoding mpox antigens are effective for vaccination against mpox and ectromelia virus. In some embodiments, provided polyribonucleotide constructs encoding mpox antigens are effective for vaccination against mpox and a novel orthopoxvirus.
[0013] In some embodiments, polyribonucleotide constructs encoding mpox antigens of the present disclosure are effective for the prevention or treatment of a lethal mpox virus infection. In some embodiments, polyribonucleotide constructs encoding mpox antigens of the present disclosure are effective for the prevention or treatment of a lethal clade I mpox virus infection. In some embodiments, polyribonucleotide constructs encoding mpox antigens of the present disclosure are effective for the prevention or treatment of a strain V79-I- 005 mpox virus infection.
[0014] In some embodiments, a composition of the present disclosure comprises a therapeutically effective amount of one or more polyribonucleotides encoding one or more mpox virus antigens or fragments thereof; and a pharmaceutically acceptable carrier, wherein each of the one or more polyribonucleotides or fragments thereof is fully or partially encapsulated within a lipid nanoparticle. In some embodiments, the one or more polyribonucleotides are purified. In some embodiments, the one or more polyribonucleotides are singlestranded. In some embodiments, the one or more polyribonucleotides comprise a 5'-capped Nl- methylpseudouridine.
[0015] In some embodiments, mpox virus antigens or fragments thereof comprise: (i) B6R or a fragment of B6R; (ii) MIR or a fragment of MIR; (iii) A35R or a fragment of A35R; or (iv) H3L or a fragment of H3L.
[0016] In some embodiments, a composition of the present disclosure comprises at least three or four polyribonucleotides, and wherein the at least three or four polyribonucleotides each encode a different mpox virus antigen or fragment thereof.
[0017] In some embodiments, a composition of the present disclosure comprises: (i) a polyribonucleotide encoding B6R or a fragment of B6R; (ii) a polyribonucleotide encoding MIR or a fragment of MIR; and (iii) a polyribonucleotide encoding A35R or a fragment of A35R.
[0018] In some embodiments, a composition of the present disclosure comprises: (i) a polyribonucleotide encoding B6R or a fragment of B6R; (ii) a polyribonucleotide encoding MIR or a fragment of MIR; (iii) a polyribonucleotide encoding A35R or a fragment of A35R; and (iv) a polyribonucleotide encoding H3L or a fragment of H3L.
[0019] In some embodiments, the lipid nanoparticle targets liver cells and/or secondary lymphoid organ cells. In some embodiments, the lipid nanoparticle is a cationic lipid nanoparticle. In some embodiments, the lipid nanoparticle comprises: (a) a polymer-conjugated lipid; (b) a cationic lipid; and (c) one or more neutral lipids. In some embodiments, the polymer-conjugated lipid comprises a PEG-conjugated lipid or 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide. In some embodiments, the one or more neutral lipids comprise 1,2- Distearoyl-sn-glycero-3-phosphocholine (DPSC) or cholesterol. In some embodiments, the cationic lipid comprises ((3-hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2-butyloctanoate). [0020] In some embodiments, a lipid nanoparticle of the present disclosure comprises: (a) 2-
[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide; (b) DPSC; (c) cholesterol; and (d) ((3- hydroxypropyl)azanediyl)bis(nonane-9,l-diyl) bis(2-butyloctanoate).In some embodiments, a lipid nanoparticle of the present disclosure comprises: (a) the polymer-conjugated lipid at about 1-2.5 mol% of the total lipids; (b) the cationic lipid at 35-65 mol% of the total lipids; and (c) the one or more neutral lipids are present in 35-65 mol% of the total lipids.
[0021] In some embodiments, lipid nanoparticles of the present disclosure have an average diameter of about 50-150 nm.
[0022] In some embodiments, a therapeutically effective amount of the one or more polyribonucleotides is 1 μg to 250 μg, 5 μg to 200 μg, 10 μg to 100 μg, or 10 μg to 60 μg. In some embodiments, a therapeutically effective amount of the one or more polyribonucleotides is about 10 μg, about 30 μg, or about 60 μg.
[0023] In some embodiments, the present disclosure provides a method of preventing an orthopoxvirus infection in a subject. In some embodiments, a method of preventing an orthopoxvirus infection in a subject comprises administering a composition to the subject. In some embodiments, a composition comprises: a therapeutically effective amount of one or more polyribonucleotides encoding one or more mpox virus antigens or antigenic fragments thereof; and a pharmaceutically acceptable carrier.
[0024] In some embodiments, a composition comprises at least three polyribonucleotides, wherein each of the at least three polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof. In some embodiments, a method of preventing an orthopoxvirus infection in a subject provided herein comprises administering a composition to the subject, wherein the composition comprises: a first polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof; a second polyribonucleotide encoding an MIR antigen, or antigenic fragment thereof; and a third polyribonucleotide encoding an A35 antigen, or antigenic fragment thereof. In some embodiments, a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182. In some embodiments, an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158. In some embodiments, an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174. In some embodiments, a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182; an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158; and an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174.
[0025] In some embodiments, a composition comprises at least four polyribonucleotides, wherein each of the at least four polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof. In some embodiments, a method of preventing an orthopoxvirus infection in a subject comprises administering a composition to the subject, wherein the composition comprises: a first polyribonucleotide encodes a B6R antigen, or antigenic fragment thereof; a second polyribonucleotide encodes an MIR antigen, or antigenic fragment thereof; a third polyribonucleotide encodes an A35 antigen, or antigenic fragment thereof; and a fourth polyribonucleotide encodes an H3L antigen, or antigenic fragment thereof. In some embodiments, a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182. In some embodiments, an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31- 40, and 158. In some embodiments, an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174. In some embodiments, an H3L antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 51-60, 184, 188, and 190. In some embodiments, a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182; an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158; an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174; and an H3L antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
[0026] In some embodiments, a method of preventing an orthopoxvirus infection, wherein the orthopoxvirus infection comprises an mpox infection, a vaccinia infection, an ectromelia infection, a variola infection, a borealpox infection, a cowpox infection, a volepox infection, a buffalopox infection, a camelpox infection, a rabbitpox infection, or a modified vaccinia virus Ankara infection. In some embodiments, an orthopoxvirus infection is an mpox infection. In some embodiments, an orthopoxvirus infection is a clade I mpox infection. In some embodiments, an orthopoxvirus infection is a clade la or clade lb mpox infection. In some embodiments, an orthopoxvirus infection is a clade II mpox infection. In some embodiments, an orthopoxvirus infection is a clade Ila or clade lib mpox infection.
[0027] In some embodiments, a method provided herein is a method of treating an orthopoxvirus infection in a subject. In some embodiments, a method of treating an orthopoxvirus infection in a subject comprises administering a composition to the subject. In some embodiments, a composition comprises: a therapeutically effective amount of one or more polyribonucleotides encoding one or more mpox virus antigens or antigenic fragments thereof; and a pharmaceutically acceptable carrier.
[0028] In some embodiments, a composition comprises at least three polyribonucleotides, wherein each of the at least three polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof. In some embodiments, a method of treating an orthopoxvirus infection in a subject provided herein comprises administering a composition to the subject, wherein the composition comprises: a first polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof; a second polyribonucleotide encoding an MIR antigen, or antigenic fragment thereof; and a third polyribonucleotide encoding an A35 antigen, or antigenic fragment thereof. In some embodiments, a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182. In some embodiments, an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158. In some embodiments, an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174. In some embodiments, a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182; an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158; and an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174.
[0029] In some embodiments, a composition comprises at least four polyribonucleotides, wherein each of the at least four polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof. In some embodiments, a method of treating an orthopoxvirus infection in a subject comprises administering a composition to the subject, wherein the composition comprises: a first polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof; a second polyribonucleotide encoding an MIR antigen, or antigenic fragment thereof; a third polyribonucleotide encoding an A35 antigen, or antigenic fragment thereof; and a fourth polyribonucleotide encoding an H3L antigen, or antigenic fragment thereof. In some embodiments, a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182. In some embodiments, an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31- 40, and 158. In some embodiments, an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174. In some embodiments, an H3L antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 51-60, 184, 188, and 190. In some embodiments, a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182; an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158; an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174; and an H3L antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
[0030] In some embodiments, the present disclosure provides a method of treating an orthopoxvirus infection, wherein the orthopoxvirus infection comprises an mpox infection, a vaccinia infection, an ectromelia infection, a variola infection, a borealpox infection, a cowpox infection, a volepox infection, a buffalopox infection, a camelpox infection, a rabbitpox infection, or a modified vaccinia virus Ankara infection. In some embodiments, an orthopoxvirus infection is an mpox infection. In some embodiments, an orthopoxvirus infection is a clade I mpox infection. In some embodiments, an orthopoxvirus infection is a clade la or clade lb mpox infection. In some embodiments, an orthopoxvirus infection is a clade II mpox infection. In some embodiments, an orthopoxvirus infection is a clade Ila or clade lib mpox infection.
[0031] In some embodiments, the present disclosure provides a method of preventing an orthopoxvirus infection in a subject. In some embodiments, a method of preventing an orthopoxvirus infection in a subject comprises administering a combination to the subject. In some embodiments, a combination comprises: a therapeutically effective amount of one or more polyribonucleotides encoding one or more mpox virus antigens or antigenic fragments thereof; and a pharmaceutically acceptable carrier.
[0032] In some embodiments, a combination comprises at least three polyribonucleotides, wherein each of the at least three polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof. In some embodiments, a method of preventing an orthopoxvirus infection in a subject provided herein comprises administering a combination to the subject, wherein the combination comprises: a first polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof; a second polyribonucleotide encoding an MIR antigen, or antigenic fragment thereof; and a third polyribonucleotide encoding an A35 antigen, or antigenic fragment thereof. In some embodiments, a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182. In some embodiments, an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158. In some embodiments, an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174. In some embodiments, a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182; an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158; and an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174. [0033] In some embodiments, a combination comprises at least four polyribonucleotides, wherein each of the at least four polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof. In some embodiments, a method of preventing an orthopoxvirus infection in a subject comprises administering a combination to the subject, wherein the combination comprises: a first polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof; a second polyribonucleotide encoding an MIR antigen, or antigenic fragment thereof; a third polyribonucleotide encoding an A35 antigen, or antigenic fragment thereof; and a fourth polyribonucleotide encoding an H3L antigen, or antigenic fragment thereof. In some embodiments, a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182. In some embodiments, an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31- 40, and 158. In some embodiments, an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174. In some embodiments, an H3L antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 51-60, 184, 188, and 190. In some embodiments, a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182; an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158; an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174; and an H3L antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
[0034] In some embodiments, the present disclosure provides a method of preventing an orthopoxvirus infection, wherein the orthopoxvirus infection comprises an mpox infection, a vaccinia infection, an ectromelia infection, a variola infection, a borealpox infection, a cowpox infection, a volepox infection, a buffalopox infection, a camelpox infection, a rabbitpox infection, or a modified vaccinia virus Ankara infection. In some embodiments, an orthopoxvirus infection is an mpox infection. In some embodiments, an orthopoxvirus infection is a clade I mpox infection. In some embodiments, an orthopoxvirus infection is a clade la or clade lb mpox infection. In some embodiments, an orthopoxvirus infection is a clade II mpox infection. In some embodiments, an orthopoxvirus infection is a clade Ila or clade lib mpox infection.
[0035] In some embodiments, a method provided herein is a method of treating an orthopoxvirus infection in a subject. In some embodiments, a method of treating an orthopoxvirus infection in a subject comprises administering a combination to the subject. In some embodiments, a combination comprises: a therapeutically effective amount of one or more polyribonucleotides encoding one or more mpox virus antigens or antigenic fragments thereof; and a pharmaceutically acceptable carrier. [0036] In some embodiments, a combination comprises at least three polyribonucleotides, wherein each of the at least three polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof. In some embodiments, a method of treating an orthopoxvirus infection in a subject provided herein comprises administering a combination to the subject, wherein the combination comprises: a first polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof; a second polyribonucleotide encoding an MIR antigen, or antigenic fragment thereof; and a third polyribonucleotide encoding an A35 antigen, or antigenic fragment thereof. In some embodiments, a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182. In some embodiments, an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158. In some embodiments, an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174. In some embodiments, a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182; an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158; and an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174.
[0037] In some embodiments, a combination comprises at least four polyribonucleotides, wherein each of the at least four polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof. In some embodiments, a method of treating an orthopoxvirus infection in a subject comprises administering a combination to the subject, wherein the combination comprises: a first polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof; a second polyribonucleotide encoding an MIR antigen, or antigenic fragment thereof; a third polyribonucleotide encoding an A35 antigen, or antigenic fragment thereof; and a fourth polyribonucleotide encoding an H3L antigen, or antigenic fragment thereof. In some embodiments, a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182. In some embodiments, an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31- 40, and 158. In some embodiments, an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174. In some embodiments, an H3L antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 51-60, 184, 188, and 190. In some embodiments, a B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182; an MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158; an A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174; and an H3L antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
[0038] In some embodiments, the present disclosure provides a method of treating an orthopoxvirus infection, wherein the orthopoxvirus infection comprises an mpox infection, a vaccinia infection, an ectromelia infection, a variola infection, a borealpox infection, a cowpox infection, a volepox infection, a buffalopox infection, a camelpox infection, a rabbitpox infection, or a modified vaccinia virus Ankara infection. In some embodiments, an orthopoxvirus infection is an mpox infection. In some embodiments, an orthopoxvirus infection is a clade I mpox infection. In some embodiments, an orthopoxvirus infection is a clade la or clade lb mpox infection. In some embodiments, an orthopoxvirus infection is a clade II mpox infection. In some embodiments, an orthopoxvirus infection is a clade Ila or clade lib mpox infection.
[0039] In some embodiments, a composition of the present disclosure comprises one or more polyribonucleotides that are purified. In some embodiments, a combination of the present disclosure comprises one or more polyribonucleotides that are purified.
[0040] In some embodiments, a composition of the present disclosure comprises one or more singlestranded polyribonucleotides. In some embodiments, a combination of the present disclosure comprises one or more single-stranded polyribonucleotides.
[0041] In some embodiments, a composition of the present disclosure comprises one or more polyribonucleotides comprising a 5'-capped Nl-methylpseudouridine. In some embodiments, a combination of the present disclosure comprises one or more polyribonucleotides comprising a 5'-capped Nl- methylpseudouridine.
[0042] In some embodiments, a composition of the present disclosure comprises one or more polyribonucleotides that are fully or partially encapsulated within a lipid nanoparticle. In some embodiments, a combination of the present disclosure comprises one or more polyribonucleotides that are fully or partially encapsulated within a lipid nanoparticle. In some embodiments, a lipid nanoparticle targets liver cells. In some embodiments, a lipid nanoparticle targets secondary lymphoid organ cells. In some embodiments, a lipid nanoparticle is a cationic lipid nanoparticle. In some embodiments, a lipid nanoparticle comprises: (a) a polymer-conjugated lipid; (b) a cationic lipid; and (c) one or more neutral lipids. In some embodiments, a polymer-conjugated lipid comprises a PEG-conjugated lipid. In some embodiments, one or more neutral lipids comprise 1,2-Distearoyl-sn-glycero-3-phosphocholine (DPSC). In some embodiments, one or more neutral lipids comprise cholesterol. In some embodiments, a lipid nanoparticle comprises: (a) the polymer-conjugated lipid at about 1-2.5 mol% of the total lipids; (b) the cationic lipid at 35-65 mol% of the total lipids; and (c) the one or more neutral lipids are present in 35-65 mol% of the total lipids. In some embodiments, a lipid nanoparticle has an average diameter of about 50-150 nm. [0043] In some embodiments, a method provided herein is a method of preventing an orthopoxvirus infection in a subject comprising administering a composition to the subject in one or more doses. In some embodiments, one or more doses comprises about 10 μg, about 30 μg, or about 60 μg of the one or more polyribonucleotides. In some embodiments, administering one or more doses comprises administering a first dose of a composition and a second dose of a composition. In some embodiments, a second dose of a composition is administered to a subject about 31 days after a first dose of a composition is administered to the subject. In some embodiments, one or more doses are administered to the subject intramuscularly.
[0044] In some embodiments, a method provided herein is a method of treating an orthopoxvirus infection in a subject comprising administering a composition to the subject in one or more doses. In some embodiments, one or more doses comprises about 10 μg, about 30 μg, or about 60 μg of the one or more polyribonucleotides. In some embodiments, administering one or more doses comprises administering a first dose of a composition and a second dose of a composition. In some embodiments, a second dose of a composition is administered to a subject about 31 days after a first dose of a composition is administered to the subject. In some embodiments, one or more doses are administered to the subject intramuscularly.
[0045] In some embodiments, a method provided herein is a method of preventing an orthopoxvirus infection in a subject comprising administering a combination to the subject in one or more doses. In some embodiments, one or more doses comprises about 10 μg, about 30 μg, or about 60 μg of the one or more polyribonucleotides. In some embodiments, administering one or more doses comprises administering a first dose of a combination and a second dose of a combination. In some embodiments, a second dose of a combination is administered to a subject about 31 days after a first dose of a combination is administered to the subject. In some embodiments, one or more doses are administered to the subject intramuscularly.
[0046] In some embodiments, a method provided herein is a method of treating an orthopoxvirus infection in a subject comprising administering a combination to the subject in one or more doses. In some embodiments, one or more doses comprises about 10 μg, about 30 μg, or about 60 μg of the one or more polyribonucleotides. In some embodiments, administering one or more doses comprises administering a first dose of a combination and a second dose of a combination. In some embodiments, a second dose of a combination is administered to a subject about 31 days after a first dose of a combination is administered to the subject. In some embodiments, one or more doses are administered to the subject intramuscularly.
[0047] In some embodiments, a method of preventing an orthopoxvirus infection in a subject comprising administering a composition, wherein administering the composition comprises mixing the one or more polyribonucleotides and wherein each of the one or more polyribonucleotides is administered as a single injection dose.
[0048] In some embodiments, a method of treating an orthopoxvirus infection in a subject comprising administering a composition, wherein administering the composition comprises mixing the one or more polyribonucleotides and wherein each of the one or more sub-doses is administered as a single injection dose.
[0049] In some embodiments, a method of preventing an orthopoxvirus infection in a subject comprising administering a combination, wherein administering the combination comprises mixing the one or more polyribonucleotides and wherein each of the one or more polyribonucleotides is administered as a single injection dose. [0050] In some embodiments, a method of treating an orthopoxvirus infection in a subject comprising administering a combination, wherein administering the combination comprises mixing the one or more polyribonucleotides and wherein each of the one or more polyribonucleotides is administered as a single injection dose.
[0051] In some embodiments, the present disclosure provides a method of preventing an orthopoxvirus infection in a subject comprising administering a composition to the subject, wherein administering results in an increased serum level of one or more orthopoxvirus-neutralizing antibodies in the subject.
[0052] In some embodiments, the present disclosure provides a method of treating an orthopoxvirus infection in a subject comprising administering a composition to the subject, wherein administering results in an increased serum level of one or more orthopoxvirus-neutralizing antibodies in the subject.
[0053] In some embodiments, the present disclosure provides a method of preventing an orthopoxvirus infection in a subject comprising administering a combination to the subject, wherein administering results in an increased serum level of one or more orthopoxvirus-neutralizing antibodies in the subject.
[0054] In some embodiments, the present disclosure provides a method of treating an orthopoxvirus infection in a subject comprising administering a combination to the subject, wherein administering results in an increased serum level of one or more orthopoxvirus-neutralizing antibodies in the subject.
[0055] Also provided herein is a method of preventing an mpox virus infection in a subject comprising administering one or more doses of a composition to the subject, wherein the composition comprises: a therapeutically effective amount of one or more polyribonucleotides encoding one or more MPXV antigens or fragments thereof; and a pharmaceutically acceptable carrier. In some embodiments, each of the one or more polyribonucleotides or fragments thereof is fully or partially encapsulated within a lipid nanoparticle. In some embodiments, the present disclosure provides a method of treating an mpox virus infection in a subject comprising administering one or more doses of a composition to the subject, wherein the composition comprises: a therapeutically effective amount of one or more polyribonucleotides encoding one or more MPXV antigens or fragments thereof; and a pharmaceutically acceptable carrier. In some embodiments, each of the one or more polyribonucleotides or fragments thereof is fully or partially encapsulated within a lipid nanoparticle.
[0056] In some embodiments, provided compositions (e.g., pharmaceutical compositions, e.g., immunogenic compositions, e.g., vaccines) that include one or more polyribonucleotides are prepared, formulated, and/or utilized in particular LNP compositions, e.g., as described herein.
[0057] Among other things, the present disclosure provides technologies for rapid development of a pharmaceutical composition (e.g., immunogenic composition, e.g., mpox vaccine) for delivering particular mpox antigen constructs to a subject.
[0058] Additionally, the present disclosure provides, for example, nucleic acid constructs encoding mpox antigens or fragments thereof disclosed herein, expressing mpox antigens or fragments thereof disclosed herein, and various methods of production and/or use relating thereto, as well as compositions developed therewith and methods relating thereto.
[0059] The present disclosure provides technologies for preventing, characterizing, treating, and/or monitoring mpox outbreaks of orthopoxviruses (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara) viruses) and/or infections including, e.g., various nucleic acid constructs and encoded proteins, as well as agents (e.g., antibodies) that bind to such proteins, and compositions that comprise and/or deliver them. In some embodiments, provided herein are technologies (e.g., compositions and methods) for augmenting, inducing, promoting, enhancing and/or improving an immune response against orthopoxviruses (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara) viruses) or components thereof (e.g., a protein or portion thereof). In some embodiments, technologies provided herein are designed to augment, induce, promote, enhance and/or improve immunological memory against orthopoxviruses (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara) viruses) or a component thereof (e.g., a protein or portion thereof). In some embodiments, technologies described herein are designed to act as an immunological boost to a primary vaccine, such as a vaccine directed to an epitope and/or epitopes of orthopoxviruses (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara) viruses). In some embodiments, compositions of the present disclosure comprise one or more polynucleotide constructs (e.g., one or more string constructs) that encode one or more epitopes from mpox. In some embodiments, the present disclosure provides vaccines or other compositions comprising nucleic acids encoding such mpox epitopes; those skilled in the art will appreciate from context when reference to a particular polynucleotide (e.g., a DNA or RNA) as "encoding" such epitopes in fact is referencing a coding strand or its complement.
BRIEF DESCRIPTION OF THE DRAWING
[0060] FIG. 1 depicts a structural representation of exemplary components of compositions provided by the present disclosure; the exemplary tetravalent Combo 4 and exemplary trivalent Combo 2. The B6 structure is predicted by AlphaFold. The A35, Ml and H3 structures shown are of the VACV orthologs (Protein Data Bank [PDB] IDs 3K7B, 2I9L, and 5EJ0).
[0061] FIGs. 2A-D depict in vitro expression of MPXV antigens from exemplary polyribonucleotide constructs of the present disclosure. HEK293T cells were transfected with 200 ng of polyribonucleotide. Cells transfected with the exemplary multivalent polyribonucleotide compositions Combo 4 and Combo 2 were compared to non-transfected cells and cells transfected with a monovalent polyribonucleotide-LNP composition. Antigen expression levels were measured by flow cytometry 18 hours post- transfection for A35 (FIG. 2A), B6 (FIG. 2B), Ml (FIG. 2C), and H3 (FIG. 2D). In (FIG. 2D), H3 analysis for exemplary Combo 2 is excluded as H3 is not included in this exemplary polyribonucleotide composition. Antigen expression is shown as median fluorescence intensity (MFI) of total live cells for both intracellular (total protein) and surface (surface protein) staining conditions. The mean of technical triplicates is shown (+SEM). Exemplary Combo 2 comprised A35, B6, and Ml antigens, and exemplary Combo 4 comprised A35, B6, Ml, and H3 antigens.
[0062] FIGs. 3A-D are line graphs depicting serum antibody levels produced by mice administered with exemplary polyribonucleotide constructs of the present disclosure. BALB/c mice were administered with 4 μg exemplary Combo 4 (squares), 4 μg exemplary Combo 2 (triangles) or with 1 μg of a polyribonucleotide encoding one antigen (circles) on days 0 and 21. Serum antibody levels for A35 (FIG. 3A), B6 (FIG. 3B), Ml (FIG. 3C), and H3 (FIG. 3D) were measured by ELISA. Geometric mean values of IgG ng/mL equivalents (±SEM) are shown. The dashed line indicates the lower limit of detection.
[0063] FIGs. 4A-E depict induction of germinal centers (GCs) following administration with exemplary polyribonucleotide constructs of the present disclosure encoding a single (e.g., monovalent) MPXV antigen. Following an intramuscular (IM) prime and boost of BALB/c mice, inguinal lymph nodes (LNs) were harvested and analyzed for the presence of GC B cells by flow cytometry. FIG. 4A is a representative dot plot depicting the gating strategy for total GC B cells (Live Dump). FIG. 4B is a representative dot plot depicting the gating strategy for B220+CD19+ double positive GC B cells. FIG. 4C is a representative dot plot depicting the gating strategy for GL7+CD95+ double positive GC B cells. Sequential gating events are shown left to right. FIG. 4D is a bar graph depicting the measurement of the percentage of GC B cells in the LN. FIG. 4E is a bar graph depicting the measurement of the fraction of antigen-positive cells among total GC B cells. Mean ±SEM is shown. [0064] FIGs. 5A-D depict measurements of T cell responses to MPXV antigens after administration with exemplary polyribonucleotide constructs described herein. Mice were administered a single administration of 4 μg exemplary Combo 4 or 1 μg of single polyribonucleotides encoding one MPXV antigen. On day 7 postadministration, mice were sacrificed and antigen-specific T cell levels were measured by IFN-y ELISpot with mouse splenocytes stimulated with peptide pools spanning the antigen(s) relevant to each experimental group. Data for individual animals with median bar are shown for A35 (FIG. 5A), B6 (FIG. 5B), Ml (FIG. 5C), and H3 (FIG. 5D). For FIGs. 5A-D, statistical significance was assessed using a Kruskal-Wallis test with Dunn's multiple comparisons test: n = 4 - 5, -p < 0.05, "p < 0.01, *** <0.001; ns, not significant. Exemplary Combo 2 comprised A35, B6, and Ml antigens, and exemplary Combo 4 comprised A35, B6, Ml, and H3 antigens.
[0065] FIGs. 6A-D depict IgG responses in rats administered exemplary polyribonucleotide constructs of the present disclosure encoding a single (e.g., monovalent) MPXV antigen. Wistar rats were administered with 10 μg of an individual MPXV-antigen encoding polyribonucleotide (e.g., mRNA) on days 0 and 28. Serum antibody levels for A35 (FIG. 6A), B6 (FIG. 6B), Ml (FIG. 6C), and H3 (FIG. 6D) were measured by ELISA. Geometric mean values of IgG ng/mL equivalents (±SEM) are shown. The lower limit of detection is indicated with a dashed line and is set at half the lowest test serum dilution tested.
[0066] FIGs. 7A-D depict cluster plots of neutralizing antibody titers following administrations with exemplary polyribonucleotide constructs of the present disclosure encoding MPXV antigens. Mice were administered IM with exemplary Combo 4 (4 μg) or exemplary Combo 2 (4 μg) compositions, or polyribonucleotides encoding one MPXV antigen (1 μg), on days 0 and 21. Neutralizing antibody activities for MPXV (determined via PRNT; FIG. 7A and FIG. 7B) and for VACV (determined via FRNT; FIG. 7C and FIG. 7D) were measured both in the absence of baby rabbit complement (FIG. 7A and FIG. 7D) and with the addition of baby rabbit complement (FIG. 7B and FIG. 7C). The lower limit of detection is indicated with a dashed line and is set at half the lowest test serum dilution tested. The upper limit of detection for MPXV neutralization is shown at twice the highest dilution tested. Exemplary Combo 2 comprised A35, B6, and Ml antigens, and exemplary Combo 4 comprised A35, B6, Ml, and H3 antigens.
[0067] FIG. 8 is a cluster plot depicting neutralizing antibody responses following prime and boost administrations with exemplary polyribonucleotide constructs of the present disclosure encoding MPXV antigens. BALB/c mice were administered with exemplary Combo 4 or exemplary Combo 2 polyribonucleotide compositions on days 0 and 21 and serum VACV-neutralizing antibodies measured on day 21 and day 35 by FRNT. The lower limit of detection is indicated with a dashed line and is set at half the lowest test serum dilution tested. Exemplary Combo 2 comprised A35, B6, and Ml antigens, and exemplary Combo 4 comprised A35, B6, Ml, and H3 antigens.
[0068] FIGs. 9A-I depict in vivo studies of protection against MPXV challenge from administration with exemplary polyribonucleotide constructs of the present disclosure encoding MPXV antigens. FIG. 9A is a schematic illustrating an exemplary dosing schedule where CAST/Ei mice were administered with a combination of A35 and B6 polyribonucleotides, exemplary Combo 4, exemplary Combo 2, or negative control (mock treated with saline) on days 0 and 21. Animals were challenged intranasally (IN) with 9 x 105 PFU of clade lib MPXV (high dose) 5 weeks after the last administration. FIG. 9B and FIG. 9C depict MPXV titers in the lungs of CAST/Ei mice sacrificed on day 3 or 7 post-challenge, respectively. Lungs were collected and analyzed for viral load by TCID50 assay. The lower limit of detection is indicated by a dashed line. FIG. 9D is a schematic illustrating an exemplary dosing schedule where CAST/Ei mice were administered with a combination of A35 and B6 polyribonucleotides, exemplary Combo 4, exemplary Combo 2, or negative control (mock treated with saline) on days 0 and 21. Animals were challenged intranasally (IN) with 3 x 105 PFU of clade lib MPXV (low dose) 5 weeks after the last administration. FIG. 9E and FIG. 9F depict MPXV titers in the lungs of CAST/Ei mice sacrificed on day 3 or 7 post-challenge, respectively. FIG. 9G is a schematic illustrating an exemplary dosing schedule where CAST/Ei mice were administered with polyribonucleotides encoding A35, B6, Ml, or H3 antigens of exemplary Combo 4, exemplary Combo 2, or negative control (mock treated with saline) on days 0 and 21. Animals were challenged IN with 1 x 105 PFU of clade I MPXV 5 weeks after the last administration. Mice were monitored for weight loss and survival for two weeks post-challenge. FIG. 9H and FIG. 91 depict weight loss and survival results, respectively, of CAST/Ei mice administered as in FIG. 9G. Statistical significance was assessed using a Kruskal-Wallis test with Dunn's multiple comparisons test: *p < 0.05, ** < 0.01, *** < 0.001; ns, not significant. For survival data (n = 9 - 10), statistical significance was assessed using Mantel-Cox log-rank test: *p < 0.05, *mp < 0.0001; ns, not significant. Combo 2 comprises A35, B6, and Ml antigens, and Combo 4 comprises A35, B6, Ml, and H3 antigens.
[0069] FIGs. 10A-C depict exemplary in vivo studies of protection against MPXV challenge. FIG. 1OA is a schematic illustrating an exemplary dosing schedule where BALB/c mice were administered with polyribonucleotides encoding A35, B6, Ml, or H3 antigens or exemplary Combo 4, exemplary Combo 2, or negative control (mock treated with saline) on days 0 and 21. Animals were challenged IN with 5 x 104 PFU of VACV-WR on day 42. FIG. 1OB and FIG. IOC depict weight loss and survival results, respectively. Statistical significance was assessed using a Kruskal-Wallis test with Dunn's multiple comparisons test: p < 0.05, "p < 0.01, *** < 0.001; ns, not significant. For survival data (n = 15 - 16), statistical significance was assessed using Mantel-Cox log-rank test: *p < 0.05, **** < 0.0001; ns, not significant. Combo 2 comprises A35, B6, and Ml antigens, and Combo 4 comprises A35, B6, Ml, and H3 antigens.
[0070] FIGs. 11A-C depict exemplary in vivo studies of protection against ECTV challenge. FIG. 11A is a schematic illustrating an exemplary dosing schedule where BALB/c mice were administered with polyribonucleotides encoding Ml, A35, B6, or H3 antigens or exemplary Combo 4, or negative control (mock treated with PBS) on days 0 and 21. Animals were challenged IN with ECTV (104 PFU) on day 42. FIG. 11B and FIG. 11C depict weight loss and survival results, respectively. Combo 4 comprises A35, B6, Ml, and H3 antigens. Statistical significance was assessed using Mantel-Cox log-rank test **** < 0.0001.
[0071] FIGs. 12A-E depict exemplary in vivo studies of protection against MPXV challenge. FIG. 12A is a schematic illustrating an exemplary dosing schedule in cynomolgus macaques. Cynomolgus macaques were administered with 30 μg of exemplary Combo 4 or mock treated with saline on days 0 and 28. An intratracheal (IT) challenge with clade I MPXV was performed on day 60. FIG. 12B, FIG. 12C, FIG. 12D, and FIG. 12E, depict the measurement of serum antibody levels against A35, B6, Ml, and H3 MPXV antigens, respectively, two weeks following boost were measured by ELISA. FIG. 12F is a Kaplan-Meier survival plot depicting survival of macaques administered with exemplary Combo 4 or mock-administered with saline for 28 days post clade I MPXV challenge. FIG. 12G is a line graph depicting the weight loss of individual macaques mock-administered with saline, at 28 days post clade I MPXV challenge. FIG. 12H and FIG. 121 are line graphs depicting the weight loss of individual macaques (FIG. 12H) and mean weight loss (FIG. 121) of macaques administered with exemplary Combo 4 at 28 days post clade I MPXV challenge. FIG. 12J is a line graph depicting lesion counts of individual macaques mock-administered with saline, at 28 days post clade I MPXV challenge. FIG. 12K and FIG. 12L are line graphs depicting the lesion counts of individual macaques (FIG. 12K) and mean lesion counts (FIG. 12L) of macaques administered with exemplary Combo 4 at 28 days post clade I MPXV challenge. For weight loss and lesion data, plots for individual animals and averaged plots are given, with the end of observation for each animal due to death or study completion marked by a diamond and dashed lines indicated the baseline value.
[0072] FIGs. 13A-13C depict exemplary in vivo studies of protection against MPXV challenge. FIG.
13A is a schematic illustrating an exemplary dosing schedule in CAST/Ei mice. CAST/Ei mice were immunized with 4 μg of exemplary Combo 2 polyribonucleotide composition, 4 μg of exemplary Combo 4 polyribonucleotide composition, an LNP incorporating a combination of polyribonucleotides encoding A35 and B6, or saline, on days 0 and 21. Mice were intranasally challenged with clade I MPXV isolate (Zaire 79 strain) on day 56. FIG. 13B is a line graph depicting the percent body weight change of individual mice over a 12-day period post-MPXV challenge. FIG. 13C is a Kaplan-Meier plot depicting the percent survival of exemplary Combo 2 and Combo 4 polyribonucleotide composition vaccinated animals compared to animals treated with saline or LNP incorporating polyribonucleotides encoding A35 and B6.
[0073] FIGs. 14A-14C depict exemplary in vivo studies of protection against VACV challenge. FIG.
14A is a schematic illustrating an exemplary dosing schedule in Balb/C mice. Balb/C mice were immunized with 4 μg of exemplary Combo 4 polyribonucleotide composition, an LNP incorporating 1 μg of polyribonucleotide encoding Ml, an LNP incorporating 1 μg of polyribonucleotide encoding A35, an LNP incorporating 1 μg of polyribonucleotide encoding B6, an LNP incorporating 1 μg of polyribonucleotide encoding H3, or saline on days 0 and 21. Mice were intranasally challenged with a lethal dose of vaccinia virus (VACV) isolate 21 days following the boost dose at day 21. FIG. 14B is a line graph depicting the percent body weight change of individual mice over a 10-day period post-VACV challenge. FIG. 14C is a Kaplan-Meier plot depicting the percent survival of mice immunized as described in FIG. 14A.
[0074] FIGs. 15A-15C are schematic diagrams of the safety and immunogenicity clinical trial design of Example 10. FIG. 15A describes a staggered dosing process for substudies A, B, C, and D of Example 10. FIG. 15B describes the dosing and sample collection for substudies A and B. FIG. 15C describes the dosing and sample collection for substudies C and D of Example 10.
[0075] FIGs. 16A-16B are line graphs depicting the post-MPXV challenge blood viral loads of cynomolgus macaques vaccinated with exemplary Combo 4 polyribonucleotide composition (FIG. 16B) or saline (FIG. 16A).
[0076] FIG. 17 is a cluster plot depicting the 50% neutralization serum titers (NT50) of cynomolgus macaques vaccinated with exemplary Combo 4 polyribonucleotide composition or saline pre- and post-MPXV challenge.
[0077] FIGs. 18A-18D are line graphs depicting the percentage of cells expressing A35 (FIG. 18A),
B6 (FIG. 18B), Ml (FIG. 18C), or H3 (FIG. 18D) MPXV antigens in HEK293T cells transfected with various concentrations of modified polyribonucleotide constructs encoding A35, B6, Ml, or H3 MPXV antigens, respectively.
[0078] FIGs. 19A-19D are cluster plots depicting serum anti-A35 (FIG. 19A), anti-B6 (FIG. 19B), anti-Ml (FIG. 19C), and anti-H3 (FIG. 19D) IgG 50% binding titers in BALB/c mice following administrations with exemplary Combo 4 modified polyribonucleotide formulations of the present disclosure. Mice were administered IM with Combo 4 (incorporating modified polyribonucleotides encoding A35, B6, Ml, and H3 MPXV antigens) (i) co-formulated as a combined preparation of LNPs incorporating modified polyribonucleotides in Tris buffer and sucrose (Combo 4 [T]); (ii) co-formulated as a combined preparation of LNPs incorporating modified polyribonucleotides in phosphate buffered saline (PBS) and sucrose (Combo 4 [P]); or (iii) formulated as four single LNP preparations incorporating modified polyribonucleotides, in PBS and sucrose then mixed before administration (Combo 4 (single mix)).
DEFINITIONS
[0079] Compounds, compositions and constructs of this disclosure include those described generally above and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, the chemical elements are identified in accordance with the Periodic Table of Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.
[0080] Unless otherwise stated, structures depicted herein are meant to include all stereoisomeric
(e.g., enantiomeric or diastereomeric) forms of the structure, as well as all geometric or conformational isomeric forms of the structure. For example, the R and S configurations of each stereocenter are contemplated as part of the disclosure. Therefore, single stereochemical isomers, as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of provided compounds are within the scope of the disclosure. For example, in some cases, provided compounds show one or more stereoisomers of a compound, and unless otherwise indicated, represents each stereoisomer alone and/or as a mixture. Unless otherwise stated, all tautomeric forms of provided compounds are within the scope of the disclosure.
[0081] Unless otherwise indicated, structures depicted herein are meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including replacement of hydrogen by deuterium or tritium, or replacement of a carbon by 13C- or 14C -enriched carbon are within the scope of this disclosure.
[0082] As used herein, the term "exemplary" refers to an example of an embodiment. Unless otherwise indicated, the term "exemplary" is not intended to indicate that an embodiment is a "preferred" or "best" version of an embodiment.
[0083] About. The term "about", when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by "about" in that context. For example, in some embodiments, the term "about" may encompass a range of values that are within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.
[0084] Agent. As used herein, the term "agent," may refer to a physical entity. In some embodiments, an agent may be characterized by a particular feature and/or effect. For example, as used herein, the term "therapeutic agent" refers to a physical entity has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect. In some embodiments, an agent may be a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, nucleic acid, saccharide, lipid, metal, or a combination or complex thereof. [0085] Aliphatic. The term "aliphatic" refers to a straight-chain (i.e., unbranched) or branched, substituted or unsubstituted hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic hydrocarbon or bicyclic hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as "cycloaliphatic"), that has a single point or more than one points of attachment to the rest of the molecule. Unless otherwise specified, aliphatic groups contain 1-12 aliphatic carbon atoms. In some embodiments, aliphatic groups contain 1-6 aliphatic carbon atoms (e.g., C1-6). In some embodiments, aliphatic groups contain 1-5 aliphatic carbon atoms (e.g., C1-5). In other embodiments, aliphatic groups contain 1-4 aliphatic carbon atoms (e.g.,
Figure imgf000021_0001
In still other embodiments, aliphatic groups contain 1-3 aliphatic carbon atoms (e.g., C1-3), and in yet other embodiments, aliphatic groups contain 1-2 aliphatic carbon atoms (e.g., C1-2). Suitable aliphatic groups include, but are not limited to, linear or branched, substituted or unsubstituted alkyl, alkenyl, or alkynyl groups and hybrids thereof. A preferred aliphatic group is C1-6 alkyl.
[0086] Alkyl'. The term "alkyl," used alone or as part of a larger moiety, refers to a saturated, optionally substituted straight or branched chain hydrocarbon group having (unless otherwise specified) 1-12, 1- 10, 1-8, 1-6, 1-4, 1-3, or 1-2 carbon atoms (e.g., C1-12,
Figure imgf000021_0002
C1-3, or C1-2). Exemplary alkyl groups include methyl, ethyl, propyl, butyl, pentyl, hexyl, and heptyl.
[0087] Alkylene'. The term "alkylene" refers to a bivalent alkyl group. In some embodiments,
"alkylene" is a bivalent straight or branched alkyl group. In some embodiments, an "alkylene chain" is a polymethylene group, i.e., -(CH2)n-, wherein n is a positive integer, e.g., from 1 to 6, from 1 to 4, from 1 to 3, from 1 to 2, or from 2 to 3. An optionally substituted alkylene chain is a polymethylene group in which one or more methylene hydrogen atoms is optionally replaced with a substituent. Suitable substituents include those described below for a substituted aliphatic group and also include those described in the specification herein. It will be appreciated that two substituents of the alkylene group may be taken together to form a ring system. In some embodiments, two substituents can be taken together to form a 3- to 7-membered ring. The substituents can be on the same or different atoms. The suffix "-ene" or "-enyl" when appended to certain groups herein are intended to refer to a bifunctional moiety of said group. For example, "-ene" or "-enyl", when appended to "cyclopropyl" becomes "cyclopropylene" or "cyclopropylenyl" and is intended to refer to a bifunctional cyclopropyl group, e.g.,
Figure imgf000021_0003
[0088] Aikenyh The term "alkenyl", used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain or cyclic hydrocarbon group having at least one double bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C2-12, C2-10, C2-8, C2-6, C2-4, or C2- 3). Exemplary alkenyl groups include ethenyl, propenyl, butenyl, pentenyl, hexenyl, and heptenyl. The term "cycloalkenyl" refers to an optionally substituted non-aromatic monocyclic or multicyclic ring system containing at least one carbon-carbon double bond and having about 3 to about 10 carbon atoms. Exemplary monocyclic cycloalkenyl rings include cyclopentenyl, cyclohexenyl, and cycloheptenyl.
[0089] Alkynyh The term "alkynyl", used alone or as part of a larger moiety, refers to an optionally substituted straight or branched chain hydrocarbon group having at least one triple bond and having (unless otherwise specified) 2-12, 2-10, 2-8, 2-6, 2-4, or 2-3 carbon atoms (e.g., C2-12, C2-10, C2-8, C2-6, C2-4, or C2-3). Exemplary alkynyl groups include ethynyl, propynyl, butynyl, pentynyl, hexynyl, and heptynyl. [0090] Amino acid'. In its broadest sense, as used herein, the term "amino acid" refers to a compound and/or substance that can be, is, or has been incorporated into a polypeptide chain, e.g., through formation of one or more peptide bonds. In some embodiments, an amino acid has the general structure H2N- C(H)I-COOH. In some embodiments, an amino acid is a naturally-occurring amino acid. In some embodiments, an amino acid is a non-natural amino acid; in some embodiments, an amino acid is a D-amino acid; in some embodiments, an amino acid is an L-amino acid. "Standard amino acid" refers to any of the twenty standard L- amino acids commonly found in naturally occurring peptides. "Nonstandard amino acid" refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. In some embodiments, an amino acid, including a carboxy- and/or amino-terminal amino acid in a polypeptide, can contain a structural modification as compared with the general structure above. For example, in some embodiments, an amino acid may be modified by methylation, amidation, acetylation, pegylation, glycosylation, phosphorylation, and/or substitution (e.g., of the amino group, the carboxylic acid group, one or more protons, and/or the hydroxyl group) as compared with the general structure. In some embodiments, such modification may, for example, alter the circulating half-life of a polypeptide containing the modified amino acid as compared with one containing an otherwise identical unmodified amino acid. In some embodiments, such modification does not significantly alter a relevant activity of a polypeptide containing the modified amino acid, as compared with one containing an otherwise identical unmodified amino acid. As will be clear from context, in some embodiments, the term "amino acid" may be used to refer to a free amino acid; in some embodiments it may be used to refer to an amino acid residue of a polypeptide.
[0091] Aryi\ The term "aryl" refers to monocyclic and bicyclic ring systems having a total of six to fourteen ring members (e.g., C6-C14), wherein at least one ring in the system is aromatic and wherein each ring in the system contains three to seven ring members. In some embodiments, an "aryl" group contains between six and twelve total ring members (e.g., C6-C12). The term "aryl" may be used interchangeably with the term "aryl ring". In some embodiments, "aryl" refers to an aromatic ring system which includes, but not limited to, phenyl, biphenyl, naphthyl, anthracyl and the like, which may bear one or more substituents. Unless otherwise specified, "aryl" groups are hydrocarbons. In some embodiments, an "aryl" ring system is an aromatic ring (e.g., phenyl) that is fused to a non-aromatic ring (e.g., cycloalkyl). Examples of aryl rings include that are fused include
Figure imgf000022_0001
[0092] Associated'. Two events or entities are "associated" with one another, as that term is used herein, if the presence, level, degree, type and/or form of one is correlated with that of the other. For example, a particular entity (e.g., polypeptide, genetic signature, metabolite, microbe, etc.) is considered to be associated with a particular disease, disorder, or condition, if its presence, level and/or form correlates with incidence of, susceptibility to, severity of, stage of, etc. the disease, disorder, or condition (e.g., across a relevant population). In some embodiments, two or more entities are physically "associated" with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof. [0093] Co-adminlstratlon: As used herein, the term "co-administration" refers to use of a composition (e.g., a pharmaceutical composition) described herein and one or more additional therapeutic agents. In some embodiments, one or more additional therapeutic agents comprises at least one polyribonucleotide. The combined use of a composition (e.g., a pharmaceutical composition) described herein and an additional therapeutic agent may be performed concurrently or separately (e.g., sequentially in any order). In some embodiments, a composition (e.g., a pharmaceutical composition) described herein and an additional therapeutic agent may be combined in one pharmaceutically-acceptable excipient, or they may be placed in separate excipient and delivered to a target cell or administered to a subject at different times. Each of these situations is contemplated as falling within the meaning of "co-administration" or "combination," provided that a composition (e.g., a pharmaceutical composition) described herein and an additional therapeutic agent are delivered or administered sufficiently close in time that there is at least some temporal overlap in biological effect(s) generated by each on a target cell or a subject being treated.
[0094] Combination therapy: As used herein, the term "combination therapy" refers to those situations in which a subject is simultaneously exposed to two or more therapeutic regimens (e.g., two or more therapeutic agents). In some embodiments, the two or more regimens may be administered simultaneously; in some embodiments, such regimens may be administered sequentially (e.g., all "doses" of a first regimen are administered prior to administration of any doses of a second regimen); in some embodiments, such agents are administered in overlapping dosing regimens. In some embodiments, administration of combination therapy may involve administration of one or more agent(s) or modality(ies) to a subject receiving the other agent(s) or modality(ies) in the combination. For clarity, combination therapy does not require that individual agents be administered together in a single composition (or even necessarily at the same time), although in some embodiments, two or more agents, or active moieties thereof, may be administered together in a combination composition.
[0095] Comparable: As used herein, the term "comparable" refers to two or more agents, entities, situations, sets of conditions, etc., that may not be identical to one another but that are sufficiently similar to permit comparison there between so that one skilled in the art will appreciate that conclusions may reasonably be drawn based on differences or similarities observed. In some embodiments, comparable sets of conditions, circumstances, individuals, or populations are characterized by a plurality of substantially identical features and one or a small number of varied features. Those of ordinary skill in the art will understand, in context, what degree of identity is required in any given circumstance for two or more such agents, entities, situations, sets of conditions, etc. to be considered comparable. For example, those of ordinary skill in the art will appreciate that sets of circumstances, individuals, or populations are comparable to one another when characterized by a sufficient number and type of substantially identical features to warrant a reasonable conclusion that differences in results obtained or phenomena observed under or with different sets of circumstances, individuals, or populations are caused by or indicative of the variation in those features that are varied.
[0096] Corresponding to: As used herein, the term "corresponding to" refers to a relationship between two or more entities. For example, the term "corresponding to" may be used to designate the position/identity of a structural element in a compound or composition relative to another compound or composition (e.g., to an appropriate reference compound or composition). For example, in some embodiments, a monomeric residue in a polymer (e.g., an amino acid residue in a polypeptide or a nucleic acid residue in a polynucleotide) may be identified as "corresponding to" a residue in an appropriate reference polymer. For example, those of ordinary skill will appreciate that, for purposes of simplicity, residues in a polypeptide are often designated using a canonical numbering system based on a reference related polypeptide, so that an amino acid "corresponding to" a residue at position 190, for example, need not actually be the 190th amino acid in a particular amino acid chain but rather corresponds to the residue found at 190 in the reference polypeptide; those of ordinary skill in the art readily appreciate how to identify "corresponding" amino acids. For example, those skilled in the art will be aware of various sequence alignment strategies, including software programs such as, for example, BLAST, CS-BLAST, CUSASW++, DIAMOND, FASTA, GGSEARCH/GLSEARCH, Genoogle, HMMER, HHpred/HHsearch, IDF, Infernal, KLAST, USEARCH, parasail, PSI-BLAST, PSI-Search, ScalaBLAST, Sequilab, SAM, SSEARCH, SWAPHI, SWAPHI-LS, SWIMM, or SWIPE that can be utilized, for example, to identify "corresponding" residues in polypeptides and/or nucleic acids in accordance with the present disclosure. Those of skill in the art will also appreciate that, in some instances, the term "corresponding to" may be used to describe an event or entity that shares a relevant similarity with another event or entity (e.g., an appropriate reference event or entity). To give but one example, a gene or protein in one organism may be described as "corresponding to" a gene or protein from another organism in order to indicate, in some embodiments, that it plays an analogous role or performs an analogous function and/or that it shows a particular degree of sequence identity or homology, or shares a particular characteristic sequence element.
[0097] Cycloaliphatic. As used herein, the term "cycloaliphatic" refers to a monocyclic C3-8 hydrocarbon or a bicyclic C5-io hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic, that has a single point or more than one points of attachment to the rest of the molecule.
[0098] Cycloalkyh As used herein, the term "cycloalkyl" refers to an optionally substituted saturated ring monocyclic or polycyclic system of about 3 to about 10 ring carbon atoms. Exemplary monocyclic cycloalkyl rings include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and cycloheptyl.
[0099] Derived'. In the context of an amino acid sequence (peptide or polypeptide) "derived from" a designated amino acid sequence (peptide or polypeptide), it refers to a structural analogue of a designated amino acid sequence. In some embodiments, an amino acid sequence which is derived from a particular amino acid sequence has an amino acid sequence that is identical, essentially identical or homologous to that particular sequence or a fragment thereof. Amino acid sequences derived from a particular amino acid sequence may be variants of that particular sequence or a fragment thereof.
[0100] Detecting-. The term "detecting" is used broadly herein to include appropriate means of determining the presence or absence of an entity of interest or any form of measurement of an entity of interest in a sample. Thus, "detecting" may include determining, measuring, assessing, or assaying the presence or absence, level, amount, and/or location of an entity of interest. Quantitative and qualitative determinations, measurements or assessments are included, including semi-quantitative. Such determinations, measurements or assessments may be relative, for example when an entity of interest is being detected relative to a control reference, or absolute. As such, the term "quantifying" when used in the context of quantifying an entity of interest can refer to absolute or to relative quantification. Absolute quantification may be accomplished by correlating a detected level of an entity of interest to known control standards (e.g., through generation of a standard curve). Alternatively, relative quantification can be accomplished by comparison of detected levels or amounts between two or more different entities of interest to provide a relative quantification of each of the two or more different entities of interest, i.e., relative to each other.
[0101] Dosing regimen-. Those skilled in the art will appreciate that the term "dosing regimen" (or
"therapeutic regimen") may be used to refer to a set of unit doses (e.g., one or more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses.
[0102] Encode-. As used herein, the term "encode" or "encoding" refers to sequence information of a first molecule that guides production of a second molecule having a defined sequence of nucleotides (e.g., a polyribonucleotide) or a defined sequence of amino acids. For example, a DNA molecule can encode an RNA molecule (e.g., by a transcription process that includes a DNA-dependent RNA polymerase enzyme). An RNA molecule can encode a polypeptide (e.g., by a translation process). Thus, a gene, a cDNA, or an RNA molecule encodes a polypeptide if transcription and translation of RNA corresponding to that gene produces the polypeptide in a cell or other biological system. In some embodiments, a coding region of a polyribonucleotide encoding a target antigen refers to a coding strand, the nucleotide sequence of which is identical to the polyribonucleotide sequence of such a target antigen. In some embodiments, a coding region of a polyribonucleotide encoding a target antigen refers to a non-coding strand of such a target antigen, which may be used as a template for transcription of a gene or cDNA.
[0103] Engineered-. In general, the term "engineered" refers to the aspect of having been manipulated by the hand of man. For example, a polynucleotide is considered to be "engineered" when two or more sequences that are not linked together in that order in nature are manipulated by the hand of man to be directly linked to one another in the engineered polynucleotide and/or when a particular residue in a polynucleotide is non-naturally occurring and/or is caused through action of the hand of man to be linked with an entity or moiety with which it is not linked in nature.
[0104] Epitope-. As used herein, the term "epitope" refers to a moiety that is specifically recognized by an immunoglobulin (e.g., antibody or receptor) binding component. For example, an epitope may be recognized by a T cell, a B cell, or an antibody. In some embodiments, an epitope is comprised of a plurality of chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface- exposed when the antigen adopts a relevant three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically near to each other in space when the antigen adopts such a conformation. In some embodiments, at least some such chemical atoms are groups are physically separated from one another when the antigen adopts an alternative conformation (e.g., is linearized). Accordingly, in some embodiments, an epitope of an antigen may include a continuous or discontinuous portion of the antigen. In some embodiments, an epitope is or comprises a T cell epitope. In some embodiments, an epitope is or comprises a B cell epitope. In some embodiments, an epitope may have a length of about 5 to about 30 amino acids, or about 10 to about 25 amino acids, or about 5 to about 15 amino acids, or about 5 to 12 amino acids, or about 6 to about 9 amino acids.
[0105] Expression-. As used herein, the term "expression" of a nucleic acid sequence refers to the generation of a gene product from the nucleic acid sequence. In some embodiments, a gene product can be a transcript, e.g., a polyribonucleotide as provided herein. In some embodiments, a gene product can be a polypeptide. In some embodiments, expression of a nucleic acid sequence involves one or more of the following: (1) production of an RNA template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, etc.); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.
[0106] Fragment: As used herein, "fragment" refers a structure that is or includes a discrete portion of a reference agent (sometimes referred to as the "parent" agent). In some embodiments, a fragment lacks one or more moieties found in the reference agent. In some embodiments, a fragment is or includes one or more moieties found in the reference agent. In some embodiments, the reference agent is a polymer such as a polynucleotide or polypeptide. In some embodiments, a fragment of a polymer is or includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more monomeric units (e.g., residues) of the reference polymer. In some embodiments, a fragment of a polymer is or includes at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more of the monomeric units (e.g., residues) found in the reference polymer. A fragment of a reference polymer is not necessarily identical to a corresponding portion of the reference polymer. For example, a fragment of a reference polymer can be a polymer having a sequence of residues having at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more identity to the reference polymer. A fragment may, or may not, be generated by physical fragmentation of a reference agent. In some instances a fragment is generated by physical fragmentation of a reference agent. In some instances, a fragment is not generated by physical fragmentation of a reference agent and can be instead, for example, produced by de novo synthesis or other means.
[0107] Heteroallphatlc. The term "heteroaliphatic" or "heteroaliphatic group," as used herein, denotes an optionally substituted hydrocarbon moiety having, in addition to carbon atoms, from one to five heteroatoms, that may be straight-chain (i.e., unbranched), branched, or cyclic ("heterocyclic") and may be completely saturated or may contain one or more units of unsaturation, but which is not aromatic. The term "heteroatom" refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen. The term "nitrogen" also includes a substituted nitrogen. Unless otherwise specified, heteroaliphatic groups contain 1-10 carbon atoms wherein 1-3 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In some embodiments, heteroaliphatic groups contain 1-4 carbon atoms, wherein 1-2 carbon atoms are optionally and independently replaced with heteroatoms selected from oxygen, nitrogen, and sulfur. In yet other embodiments, heteroaliphatic groups contain 1-3 carbon atoms, wherein 1 carbon atom is optionally and independently replaced with a heteroatom selected from oxygen, nitrogen, and sulfur. Suitable heteroaliphatic groups include, but are not limited to, linear or branched, heteroalkyl, heteroalkenyl, and heteroalkynyl groups. For example, a 1- to 10 atom heteroaliphatic group includes the following exemplary groups: -O-CH3, -CH2-O-CH3, -O-CH2-CH2- O-CH2-CH2-O-CH3, and the like.
[0108] Heteroaryl: The terms "heteroaryl" and "heteroar-", used alone or as part of a larger moiety, e.g., "heteroaralkyl," or "heteroaralkoxy," refer to monocyclic or bicyclic ring groups having 5 to 10 ring atoms (e.g., 5- to 6-membered monocyclic heteroaryl or 9- to 10-membered bicyclic heteroaryl); having 6, 10, or 14 n- electrons shared in a cyclic array; and having, in addition to carbon atoms, from one to five heteroatoms. Heteroaryl groups include, without limitation, thienyl, furanyl, pyrrolyl, imidazolyl, pyrazolyl, triazolyl, tetrazolyl, oxazolyl, isoxazolyl, oxadiazolyl, thiazolyl, isothiazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, indolizinyl, purinyl, naphthyridinyl, pteridinyl, imidazo[1,2-a]pyrimidinyl, imidazo[1,2-a]pyridyl, imidazo[4,5- b]pyridyl, imidazo[4,5-c]pyridyl, pyrrolopyridyl, pyrrolopyrazinyl, thienopyrimidinyl, triazolopyridyl, and benzoisoxazolyl. The terms "heteroaryl" and "heteroar-", as used herein, also include groups in which a heteroaromatic ring is fused to one or more aryl, cycloaliphatic, or heterocyclyl rings, where the radical or point of attachment is on the heteroaromatic ring (i.e., a bicyclic heteroaryl ring having 1 to 3 heteroatoms). Nonlimiting examples include indolyl, isoindolyl, benzothienyl, benzofuranyl, dibenzofuranyl, indazolyl, benzimidazolyl, benzotriazolyl, benzothiazolyl, benzothiadiazolyl, benzoxazolyl, quinolyl, isoquinolyl, cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, 4/7— quinolizinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl, phenoxazinyl, tetrahydroquinolinyl, tetrahydroisoquinolinyl, pyrido[2,3-b]-l,4-oxazin-3(4H)-one, 4H-thieno[3,2- b]pyrrole, and benzoisoxazolyl. The term "heteroaryl" may be used interchangeably with the terms "heteroaryl ring," "heteroaryl group," or "heteroaromatic," any of which terms include rings that are optionally substituted. [0109] Heteroatom-. The term "heteroatom" as used herein refers to nitrogen, oxygen, or sulfur, and includes any oxidized form of nitrogen or sulfur, and any quaternized form of a basic nitrogen.
[0110] Heterocycle-. As used herein, the terms "heterocycle," "heterocyclyl," "heterocyclic radical," and "heterocyclic ring" are used interchangeably and refer to a stable 3- to 8-membered monocyclic, a 6- to 10- membered bicyclic, or a 10- to 16-membered polycyclic heterocyclic moiety that is either saturated or partially unsaturated, and having, in addition to carbon atoms, one or more, such as one to four, heteroatoms, as defined above. When used in reference to a ring atom of a heterocycle, the term "nitrogen" includes a substituted nitrogen. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl), or NR+ (as in N-substituted pyrrolidinyl). A heterocyclic ring can be attached to its pendant group at any heteroatom or carbon atom that results in a stable structure and any of the ring atoms can be optionally substituted. Examples of such saturated or partially unsaturated heterocyclic radicals include, without limitation, azetidinyl, oxetanyl, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperidinyl, decahydroquinolinyl, oxazolidinyl, piperazinyl, dioxanyl, dioxolanyl, diazepinyl, oxazepinyl, thiazepinyl, morpholinyl, and thiamorpholinyl. A heterocyclyl group may be mono-, bi-, tri-, or polycyclic, preferably mono-, bi-, or tricyclic, more preferably mono- or bicyclic. A bicyclic heterocyclic ring also includes groups in which the heterocyclic ring is fused to one or more aryl rings. Exemplary bicyclic heterocyclic groups include indolinyl, isoindolinyl, benzodioxolyl, 1,3-dihydroisobenzofuranyl, 2,3-dihydrobenzofuranyl, and tetrahydroquinolinyl. A bicyclic heterocyclic ring can also be a spirocyclic ring system (e.g., 7- to 11-membered spirocyclic fused heterocyclic ring having, in addition to carbon atoms, one or more heteroatoms as defined above (e.g., one, two, three or four heteroatoms)). A bicyclic heterocyclic ring can also be a bridged ring system (e.g., 7- to 11-membered bridged heterocyclic ring having one, two, or three bridging atoms.
[0111] Homology. As used herein, the term "homology" or "homolog" refers to the overall relatedness between polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polynucleotide molecules (e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules are considered to be "homologous" to one another if their sequences are at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least
85%, at least 90%, at least 95%, or at least 99% identical. In some embodiments, polynucleotide molecules
(e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules are considered to be "homologous" to one another if their sequences are at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% similar (e.g., containing residues with related chemical properties at corresponding positions). For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as similar to one another as "hydrophobic" or "hydrophilic" amino acids, and/or as having "polar" or "non-polar" side chains. Substitution of one amino acid for another of the same type may often be considered a "homologous" substitution. [0112] Identity. As used herein, the term "identity" refers to the overall relatedness between polynucleotide molecules e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polynucleotide molecules e.g., DNA molecules and/or RNA molecules) and/or polypeptide molecules are considered to be "substantially identical" to one another if their sequences are at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical. Calculation of the percent identity of two nucleic acid or polypeptide sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second sequence for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In some embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or substantially 100% of the length of a reference sequence. The nucleotides at corresponding positions are then compared. When a position in the first sequence is occupied by the same residue (e.g., nucleotide or amino acid) as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller, 1989, which has been incorporated into the ALIGN program (version 2.0). In some embodiments, nucleic acid sequence comparisons made with the ALIGN program use a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can, alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.
[0113] Increased, Induced, or Reduced: As used herein, these terms or grammatically comparable comparative terms, indicate values that are relative to a comparable reference measurement. For example, in some embodiments, an assessed value achieved with a provided composition (e.g., a pharmaceutical composition) may be "increased" relative to that obtained with a comparable reference composition.
Alternatively or additionally, in some embodiments, an assessed value achieved in a subject may be "increased" relative to that obtained in the same subject under different conditions (e.g., prior to or after an event; or presence or absence of an event such as administration of a composition (e.g., a pharmaceutical composition) as described herein, or in a different, comparable subject (e.g., in a comparable subject that differs from the subject of interest in prior exposure to a condition, e.g., absence of administration of a composition (e.g., a pharmaceutical composition) as described herein.). In some embodiments, comparative terms refer to statistically relevant differences (e.g., that are of a prevalence and/or magnitude sufficient to achieve statistical relevance). Those skilled in the art will be aware, or will readily be able to determine, in a given context, a degree and/or prevalence of difference that is required or sufficient to achieve such statistical significance. In some embodiments, the term "reduced" or equivalent terms refers to a reduction in the level of an assessed value by at least 5%, at least 10%, at least 20%, at least 50%, at least 75% or higher, as compared to a comparable reference. In some embodiments, the term "reduced" or equivalent terms refers to a complete or essentially complete inhibition, i.e., a reduction to zero or essentially to zero. In some embodiments, the term "increased" or "induced" refers to an increase in the level of an assessed value by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 80%, at least 100%, at least 200%, at least 500%, or higher, as compared to a comparable reference.
[0114] In order: As used herein with reference to a polynucleotide or polyribonucleotide, "in order" refers to the order of features from 5' to 3' along the polynucleotide or polyribonucleotide. As used herein with reference to a polypeptide, "in order" refers to the order of features moving from the N-terminal-most of the features to the C-terminal-most of the features along the polypeptide. "In order" does not mean that no additional features can be present among the listed features. For example, if Features A, B, and C of a polynucleotide are described herein as being "in order, Feature A, Feature B, and Feature C," this description does not exclude, e.g., Feature D being located between Features A and B.
[0115] Ionizable-. The term "ionizable" refers to a compound or group or atom that is charged at a certain pH. In the context of an ionizable amino lipid, such a lipid or a function group or atom thereof bears a positive charge at a certain pH. In some embodiments, an ionizable amino lipid is positively charged at an acidic pH. In some embodiments, an ionizable amino lipid is predominately neutral at physiological pH values, e.g., in some embodiments about 7.0-7.4, but becomes positively charged at lower pH values. In some embodiments, an ionizable amino lipid may have a pKa within a range of about 5 to about 7.
[0116] Isolated-. The term "isolated" means altered or removed from the natural state. For example, a nucleic acid or a peptide naturally present in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural state is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment such as, for example, a host cell.
[0117] Lipid-. As used herein, the terms "lipid" and "lipid-like material" are broadly defined as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also typically denoted as amphiphiles.
[0118] RNA lipid nanoparticle-, As used herein, the term "RNA lipid nanoparticle" refers to a nanoparticle comprising at least one lipid and RNA molecule(s), e.g., one or more polyribonucleotides as provided herein. In some embodiments, an RNA lipid nanoparticle comprises at least one cationic amino lipid. In some embodiments, an RNA lipid nanoparticle comprises at least one cationic amino lipid, at least one helper lipid, and at least one polymer-conjugated lipid (e.g., PEG-conjugated lipid). In various embodiments, RNA lipid nanoparticles as described herein can have an average size e.g., Z-average) of about 100 nm to 1000 nm, or about 200 nm to 900 nm, or about 200 nm to 800 nm, or about 250 nm to about 700 nm. In some embodiments of the present disclosure, RNA lipid nanoparticles can have a particle size (e.g., Z-average) of about 30 nm to about 200 nm, or about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 80 nm to about 100 nm, about 90 nm to about 100 nm, about 70 to about 90 nm, about 80 nm to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, an average size of lipid nanoparticles is determined by measuring the average particle diameter. In some embodiments, RNA lipid nanoparticles may be prepared by mixing lipids with RNA molecules described herein.
[0119] Neutralization-. As used herein, the term "neutralization" refers to an event in which binding agents such as antibodies bind to a biological active site of a virus such as a receptor binding protein, thereby inhibiting the parasitic infection of cells. In some embodiments, the term "neutralization" refers to an event in which binding agents eliminate or significantly reduce ability of infecting cells. [0120] Nucleic acid/ Polynucleotide: As used herein, the term "nucleic acid" refers to a polymer of at least 10 nucleotides or more. In some embodiments, a nucleic acid is or comprises DNA. In some embodiments, a nucleic acid is or comprises RNA. In some embodiments, a nucleic acid is or comprises peptide nucleic acid (PNA). In some embodiments, a nucleic acid is or comprises a single stranded nucleic acid. In some embodiments, a nucleic acid is or comprises a double-stranded nucleic acid. In some embodiments, a nucleic acid comprises both single and double-stranded portions. In some embodiments, a nucleic acid comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, a nucleic acid comprises a backbone that comprises both phosphodiester and non-phosphodiester linkages. For example, in some embodiments, a nucleic acid may comprise a backbone that comprises one or more phosphorothioate or 5'-N- phosphoramidite linkages and/or one or more peptide bonds, e.g., as in a "peptide nucleic acid". In some embodiments, a nucleic acid comprises one or more, or all, natural residues (e.g., adenine, cytosine, deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine, guanine, thymine, uracil). In some embodiments, a nucleic acid comprises one or more, or all, non-natural residues. In some embodiments, a nonnatural residue comprises a nucleoside analog (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo- pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2- aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, 6-O-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a non-natural residue comprises one or more modified sugars (e.g., 2'-fluororibose, ribose, 2'- deoxyribose, arabinose, and hexose) as compared to those in natural residues. In some embodiments, a nucleic acid has a nucleotide sequence that encodes a functional gene product such as an RNA or polypeptide. In some embodiments, a nucleic acid has a nucleotide sequence that comprises one or more introns. In some embodiments, a nucleic acid may be prepared by isolation from a natural source, enzymatic synthesis (e.g., by polymerization based on a complementary template, e.g., in vivo or in vitro}, reproduction in a recombinant cell or system, or chemical synthesis. In some embodiments, a nucleic acid is at least 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, or 20,000 or more residues or nucleotides long.
[0121] Operably linked: As used herein, "operably linked" refers to the association of at least a first element and a second element such that the component elements are in a relationship permitting them to function in their intended manner. For example, a nucleic acid sequence or amino acid sequence is operably linked with another sequence if it modifies the expression, structure, or activity of the linked sequence, e.g., in an intended manner. In many cases, two nucleic acid sequences are operably linked if they contribute to the expression, structure, or activity of a gene or encoded polypeptide. For example, a nucleic acid regulatory sequence is "operably linked" to a nucleic acid coding sequence if the regulatory sequence and coding sequence are associated in a manner that permits control of expression of the coding sequence by the regulatory sequence. In some embodiments, an "operably linked" regulatory sequence is directly or indirectly covalently associated with a coding sequence (e.g., in a single nucleic acid). In some embodiments, a regulatory sequence controls expression of a coding sequence in trans and inclusion of the regulatory sequence in the same nucleic acid as the coding sequence is a requirement of operable linkage. In many cases, two amino acid sequences are operably linked if they are expressed as a single polypeptide.
[0122] Pharmaceutically effective amount. The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of the treatment of a particular disease (e.g., orthopoxvirus infection, e.g., mpox infection), a desired reaction in some embodiments relates to inhibition of the course of the disease (e.g., orthopoxvirus infection, e.g., mpox infection). In some embodiments, such inhibition may comprise slowing down the progress of a disease (e.g., orthopoxvirus infection, e.g., mpox infection) and/or interrupting or reversing the progress of the disease (e.g., orthopoxvirus infection, e.g., mpox infection). In some embodiments, a desired reaction in a treatment of a disease (e.g., orthopoxvirus infection, e.g., mpox infection) may be or comprise delay or prevention of the onset of a disease (e.g., orthopoxvirus infection, e.g., mpox infection) or a condition (e.g., an orthopoxvirus infection (e.g., mpox infection) associated condition). An effective amount of a composition (e.g., a pharmaceutical composition) described herein will depend, for example, on a disease (e.g., orthopoxvirus infection, e.g., mpox infection) or a condition (e.g., an orthopoxvirus (e.g., mpox infection) associated condition) to be treated, the severity of such a disease (e.g., orthopoxvirus infection, e.g., mpox infection) or a condition, individual parameters of the patient, including, e.g., age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, doses of a composition (e.g., a pharmaceutical composition) described herein may depend on various such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.
[0123] Polypeptide-. As used herein, the term "polypeptide" refers to a polymeric chain of amino acids. In some embodiments, a polypeptide has an amino acid sequence that occurs in nature. In some embodiments, a polypeptide has an amino acid sequence that does not occur in nature. In some embodiments, a polypeptide has an amino acid sequence that is engineered in that it is designed and/or produced through action of the hand of man. In some embodiments, a polypeptide may comprise or consist of natural amino acids, non-natural amino acids, or both. In some embodiments, a polypeptide may comprise or consist of only natural amino acids or only non-natural amino acids. In some embodiments, a polypeptide may comprise D- amino acids, L-amino acids, or both. In some embodiments, a polypeptide may comprise only D-amino acids. In some embodiments, a polypeptide may comprise only L-amino acids. In some embodiments, a polypeptide may include one or more pendant groups or other modifications, e.g., modifying or attached to one or more amino acid side chains, at the polypeptide's N-terminus, at the polypeptide's C-terminus, or any combination thereof. In some embodiments, such pendant groups or modifications comprise acetylation, amidation, lipidation, methylation, pegylation, etc., including combinations thereof. In some embodiments, a polypeptide may be cyclic, and/or may comprise a cyclic portion. In some embodiments, a polypeptide is not cyclic and/or does not comprise any cyclic portion. In some embodiments, a polypeptide is linear. In some embodiments, a polypeptide may be or comprise a stapled polypeptide. In some embodiments, the term "polypeptide" may be appended to a name of a reference polypeptide, activity, or structure; in such instances it is used herein to refer to polypeptides that share the relevant activity or structure and thus can be considered to be members of the same class or family of polypeptides. For each such class, the present specification provides and/or those skilled in the art will be aware of exemplary polypeptides within the class whose amino acid sequences and/or functions are known; in some embodiments, such exemplary polypeptides are reference polypeptides for the polypeptide class or family. In some embodiments, a member of a polypeptide class or family shows significant sequence homology or identity with, shares a common sequence motif (e.g., a characteristic sequence element) with, and/or shares a common activity (in some embodiments at a comparable level or within a designated range) with a reference polypeptide of the class; in some embodiments with all polypeptides within the class). For example, in some embodiments, a member polypeptide shows an overall degree of sequence homology or identity with a reference polypeptide that is at least about 30-40%, and is often greater than about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more and/or includes at least one region (e.g., a conserved region that may in some embodiments be or comprise a characteristic sequence element) that shows very high sequence identity, often greater than 90% or even 95%, 96%, 97%, 98%, or 99%. Such a conserved region usually encompasses at least 3-4 and often up to 35 or more amino acids; in some embodiments, a conserved region encompasses at least one stretch of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more contiguous amino acids. In some embodiments, a relevant polypeptide may comprise or consist of a fragment of a parent polypeptide. [0124] Prevent. As used herein, the terms "prevent" or "prevention" when used in connection with the occurrence of a disease, disorder, and/or condition, refers to reducing the risk of developing the disease, disorder and/or condition and/or to delaying onset of one or more characteristics or symptoms of the disease, disorder or condition. Prevention may be considered complete when onset of a disease, disorder or condition has been delayed for a predefined period of time.
[0125] Reference-. As used herein, the term "reference" describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control.
[0126] Ribonucleic acid (RNA) or Polyribonucleotide-. As used herein, the terms "ribonucleic acid," "RNA," or "polyribonucleotide" refers to a polymer of ribonucleotides. In some embodiments, an RNA is single stranded. In some embodiments, an RNA is double stranded. In some embodiments, an RNA comprises both single and double stranded portions. In some embodiments, an RNA can comprise a backbone structure as described in the definition of "Nucleic acid / Polynucleotide" above. An RNA can be a regulatory RNA (e.g., siRNA, microRNA, etc.), or a messenger RNA (mRNA). In some embodiments, an RNA is an mRNA. In some embodiments, where an RNA is a mRNA, an RNA typically comprises at its 3' end a poly(A) region. In some embodiments, where an RNA is a mRNA, an RNA typically comprises at its 5' end an art-recognized cap structure, e.g., for recognizing and attachment of a mRNA to a ribosome to initiate translation. In some embodiments, an RNA is a synthetic RNA. Synthetic RNAs include RNAs that are synthesized in vitro (e.g., by enzymatic synthesis methods and/or by chemical synthesis methods).
[0127] Ribonucleotide-. As used herein, the term "ribonucleotide" encompasses unmodified ribonucleotides and modified ribonucleotides. For example, unmodified ribonucleotides include the purine bases adenine (A) and guanine (G), and the pyrimidine bases cytosine (C) and uracil (U). Modified ribonucleotides may include one or more modifications including, but not limited to, for example, (a) end modifications, e.g., 5' end modifications (e.g., phosphorylation, dephosphorylation, conjugation, inverted linkages, etc.), 3' end modifications (e.g., conjugation, inverted linkages, etc.), (b) base modifications, e.g., replacement with modified bases, stabilizing bases, destabilizing bases, bases that base pair with an expanded repertoire of partners, or conjugated bases, (c) sugar modifications (e.g., at the 2' position or 4' position) or replacement of the sugar, and (d) internucleoside linkage modifications, including modification or replacement of the phosphodiester linkages. The term "ribonucleotide" also encompasses ribonucleotide triphosphates including modified and non-modified ribonucleotide triphosphates.
[0128] Rlsle. As will be understood from context, "risk" of a disease, disorder, and/or condition refers to a likelihood that a particular individual will develop the disease, disorder, and/or condition. In some embodiments, risk is expressed as a percentage. In some embodiments, risk is expressed as a risk relative to a risk associated with a reference sample or group of reference samples. In some embodiments, a reference sample or group of reference samples have a known risk of a disease, disorder, condition and/or event. In some embodiments, a reference sample or group of reference samples are from individuals comparable to a particular individual. In some embodiments, risk may reflect one or more genetic attributes, e.g., which may predispose an individual toward development (or not) of a particular disease, disorder and/or condition. In some embodiments, risk may reflect one or more epigenetic events or attributes and/or one or more lifestyle or environmental events or attributes.
[0129] Secretory signal'. As used herein, the terms "secretory signal" or "signal peptide" or "signal sequence" refer to an amino acid sequence motif that targets associated polypeptides for translocation to a secretory pathway.
[0130] Selective or specific. The terms "selective" or "specific," when used herein in reference to an agent having an activity, is understood by those skilled in the art to mean that the agent discriminates between potential target entities, states, or cells. For example, in some embodiments, an agent is said to bind "specifically" to its target if it binds preferentially with that target in the presence of one or more competing alternative targets. In many embodiments, specific interaction is dependent upon the presence of a particular structural feature of the target entity (e.g., an epitope, a cleft, a binding site). It is to be understood that specificity need not be absolute. In some embodiments, specificity may be evaluated relative to that of a targetbinding moiety for one or more other potential target entities (e.g., competitors). In some embodiments, specificity is evaluated relative to that of a reference specific binding moiety. In some embodiments, specificity is evaluated relative to that of a reference non-specific binding moiety.
[0131] Stability. The terms "stability" or "desired storage stability" as used herein may refer to physicochemical stability of a product (e.g., a pharmaceutical composition described herein). In some embodiments, "stability" refers to physicochemical stability of a product (e.g., a pharmaceutical composition described herein), in unopened thawed vials for up to 24 hours at 30 °C, and in syringes for up to 24 hours at 2- 8 °C and 12 hours at 30 °C. In some embodiments, "stability" refers to shelf-life of a product (e.g., a pharmaceutical composition described herein) for 6 months or more when stored at about -90 °C to about 4°C. [0132] Substituted or optionally substituted'. As described herein, compounds of the invention may contain "optionally substituted" moieties. In general, the term "substituted," whether preceded by the term "optionally" or not, means that one or more hydrogens of the designated moiety are replaced with a suitable substituent. "Substituted" applies to one or more hydrogens that are either explicit or implicit from the structure
Figure imgf000034_0001
otherwise indicated, an "optionally substituted" group may have a suitable substituent at each substitutable position of the group, and when more than one position in any given structure may be substituted with more than one substituent selected from a specified group, the substituent may be either the same or different at every position. Combinations of substituents envisioned by this invention are preferably those that result in the formation of stable or chemically feasible compounds. The term "stable," as used herein, refers to compounds that are not substantially altered when subjected to conditions to allow for their production, detection, and, in some embodiments, their recovery, purification, and use for one or more of the purposes provided herein. Groups described as being "substituted" preferably have between 1 and 4 substituents, more preferably 1 or 2 substituents. Groups described as being "optionally substituted" may be unsubstituted or be "substituted" as described above.
[0133] Suitable monovalent substituents on a substitutable carbon atom of an "optionally substituted" group are independently halogen; -(CH2)o-4R°; -(CH2)o-40R°; -0(CH2)o-4R°, -0-(CH2)OMC(0)OR°; -(CH2)O-
4CH(OR°)2; -(CH2)CMSR°; -(CH2)o-4Ph, which may be substituted with R°; -(CH2)o^O(CH2)o-iPh which may be substituted with R°; -CH=CHPh, which may be substituted with R°; -(CH2)o^iO(CH2)o-i-pyridyl which may be substituted with R°; -NO2; -CN; -N3; -(CH2)(MN(RO)2; -(CH2)OMN(R°)C(0)R°; -N(R°)C(S)R°; -(CH2)O- 4N(R°)C(O)NR°2; -N(RO)C(S)NR°2; -(CH2)OMN(R°)C(0)OR°; - N(R°)N(R°)C(O)R°; -N(R°)N(R°)C(O)NR°2; -N(R°)N(R°)C(O)OR°; -(CH2)(MC(O)R°; C(S)R°; -(CH2)CMC(O)OR°; - (CH2)(MC(O)SR°; -(CH2)o-4C(o)OSiR0 3; -(CH2)(MOC(O)R°; -OC(O)(CH2)CMSR°; -(CH2)(MSC(O)R°; -(CH2)O- 4C(O)NR°2; -C(S)NRO 2; -C(S)SR°; -SC(S)SR°, -(CH2)0MOC(O)NR°2; -C(O)N(OR°)R°; -C(O)C(O)R°; - C(O)CH2C(O)R°; -C(NOR°)R°; -(CH2)(MSSR°; -(CH2)0MS(O)2R°; -(CH2)(MS(O)2OR°; -(CH2)CMOS(O)2R°; - S(O)2NR°2; -(CH2)O-4S(0)R°; -N(R°)S(O)2NR°2; -N(R°)S(O)2R°; -N(OR°)R°; -C(NH)NR°2; -
P(O)2R°; -P(0)RO2; -OP(O)R°2; -OP(O)(OR°)2; SiR°3; - (CIM straight or branched alkylene)O-N(R°)2; or - (CIM straight or branched alkylene)C(O)O-N(R°)2, wherein each R° may be substituted as defined below and is independently hydrogen, Ci-e aliphatic, -CH2Ph, -0(CH2)o-iPh, -CH2-(5- to 6-membered heteroaryl ring), or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R°, taken together with their intervening atom(s), form a 3- to 12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, which may be substituted as defined below.
[0134] Suitable monovalent substituents on R° (or the ring formed by taking two independent occurrences of R° together with their intervening atoms), are independently halogen, -(CH2)o-2R*, -(haloR*), - (CH2)o-2OH, -(CH2)O-2OR*, -(CH2)(WCH(OR*)2, -O(haloR*), -CN, -N3, -(CH2)(wC(0)R*, -(CH2)M2C(O)OH, - (CH2)^2C(O)OR*, -(CH2)O-2SR*, -(CH2)Q-2SH, -(CH2K2NH2, -(CH2)^2NHR*, -(CH2)O-2NR*2, -NO2, -SiR*3, - OSiR*3, -C(O)SR*, -(CM straight or branched alkylene)C(O)OR*, or -SSR* wherein each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently selected from Ci- 4 aliphatic, -CH2Ph, -0(CH2)o-iPh, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents on a saturated carbon atom of R° include =0 and =S.
[0135] Suitable divalent substituents on a saturated carbon atom of an "optionally substituted" group include the following: =0 ("oxo"), =S, = NNR*2, = NNHC(O)R*, =NNHC(O)OR*, = NNHS(O)2R*, = NR*, =NOR*, - O(C(R*2))2-3O-, or -S(C(R*2))2-3S-, wherein each independent occurrence of R* is selected from hydrogen, Ci- 6 aliphatic which may be substituted as defined below, or an unsubstituted 5- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur. Suitable divalent substituents that are bound to vicinal substitutable carbons of an "optionally substituted" group include: -O(CR*2)2-3O-, wherein each independent occurrence of R* is selected from hydrogen, Ci-6 aliphatic which may be substituted as defined below, or an unsubstituted 5-6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
[0136] Suitable substituents on the aliphatic group of R* include halogen, -R*, -(haloR*), -OH, -OR*,
-O(haloR*), -CN, -C(O)OH, -C(O)OR*, -NH2, -NHR*, -NR*2, or -NO2, wherein each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently Ci^ aliphatic, - CH2Ph, -0(CH2)o-iPh, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
[0137] Suitable substituents on a substitutable nitrogen of an "optionally substituted" group include -
R+, -NR+ 2, -C(O)R+, -C(O)OR+, -C(O)C(O)R+, -C(O)CH2C(O)R+, -S(O)2R+, -S(O)2NR+ 2, -C(S)NR+ 2, -C(NH)NR+ 2, or -N(R+)S(O)2R+; wherein each R+ is independently hydrogen, Ci-6 aliphatic which may be substituted as defined below, unsubstituted -OPh, or an unsubstituted 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, or, notwithstanding the definition above, two independent occurrences of R+, taken together with their intervening atom(s) form an unsubstituted 3- to 12-membered saturated, partially unsaturated, or aryl mono- or bicyclic ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
[0138] Suitable substituents on the aliphatic group of R+ are independently halogen, -R*, -(haloR*), -
OH, -OR*, -O(haloR*), -CN, -C(O)OH, -C(O)OR*, -NH2, -NHR*, -NR*2, or -NO2, wherein each R* is unsubstituted or where preceded by "halo" is substituted only with one or more halogens, and is independently CM aliphatic, -CH2Ph, -0(CH2)o-iPh, or a 3- to 6-membered saturated, partially unsaturated, or aryl ring having 0-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur.
[0139] Subject-. As used herein, the term "subject" refers to an organism to be administered with a composition described herein, e.g., for experimental, diagnostic, prophylactic, and/or therapeutic purposes.
Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, domestic pets, etc.) and humans. In some embodiments, a subject is a human subject. In some embodiments, a subject is suffering from a disease, disorder, or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, rabbitpox, etc.) infection). In some embodiments, a subject is susceptible to a disease, disorder, or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection). In some embodiments, a subject displays one or more symptoms or characteristics of a disease, disorder, or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection). In some embodiments, a subject displays one or more non-specific symptoms of a disease, disorder, or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection). In some embodiments, a subject does not display any symptom or characteristic of a disease, disorder, or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection). In some embodiments, a subject is someone with one or more features characteristic of susceptibility to or risk of a disease, disorder, or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection). In some embodiments, a subject is a patient. In some embodiments, a subject is an individual to whom diagnosis and/or therapy is and/or has been administered.
[0140] Suffering fronr. An individual who is "suffering from" a disease, disorder, and/or condition
(e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection) has been diagnosed with and/or displays one or more symptoms of a disease, disorder, and/or condition.
[0141] Susceptible to An individual who is "susceptible to" a disease, disorder, and/or condition
(e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection) is one who has a higher risk of developing the disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection) than does a member of the general public. In some embodiments, an individual who is susceptible to a disease, disorder and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection) may not have been diagnosed with the disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection). In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection) may exhibit symptoms of the disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection). In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection) may not exhibit symptoms of the disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection). In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection) will develop the disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection). In some embodiments, an individual who is susceptible to a disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection) will not develop the disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection).
[0142] Therapy. The term "therapy" refers to an administration or delivery of an agent or intervention that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect (e.g., has been demonstrated to be statistically likely to have such effect when administered to a relevant population). In some embodiments, a therapeutic agent or therapy is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection). In some embodiments, a therapeutic agent or therapy is a medical intervention (e.g., surgery, radiation, phototherapy) that can be performed to alleviate, relieve, inhibit, present, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition.
[0143] Treat. As used herein, the term "treat," "treatment," or "treating" refers to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay onset of, reduce severity of, and/or reduce incidence of one or more symptoms or features of a disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection). Treatment may be administered to a subject who does not exhibit signs of a disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection). In some embodiments, treatment may be administered to a subject who exhibits only early signs of the disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection), for example for the purpose of decreasing the risk of developing pathology associated with the disease, disorder, and/or condition. In some embodiments, treatment may be administered to a subject at a later-stage of disease, disorder, and/or condition (e.g., orthopoxvirus infection, e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection).
DETAILED DESCRIPTION
[0144] The unprecedented rise in MPXV transmission in 2022 revealed an unexpected need for rapidly scalable orthopoxvirus vaccines and/or treatments to meet the demands of even a moderate size outbreak. The recent 2024 outbreak has further underscored this need. Provided herein are exemplary polyribonucleotide constructs (alone or in combination) that have the potential to fulfill this need. The successful global response to the COVID-19 pandemic utilizing polyribonucleotide constructs illustrates the power of polyribonucleotide technology to deliver an efficacious, safe vaccine and/or treatment swiftly during a fast-moving viral pandemic. [0145] The present disclosure provides pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) for delivering particular polyribonucleotide constructs encoding mpox antigens to a subject (e.g., a patient) and related methods for inducing protective immunity to mpox viruses belonging to other clades and other orthopoxviruses (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) viruses). I, MPOX and Other Orthopoxyiruses
[0146] Mpox virus, the causative agent of mpox disease, is a member of the orthopoxvirus genus and is related to variola virus (VARV), the causative agent of smallpox. Mpox disease generally manifests as a selflimiting infection with symptoms including fever, headache, fatigue, lymphadenopathy and skin lesions. Illness is most severe in children, pregnant women, and individuals with underlying immune deficiencies and can be associated with high case fatality rates. However, lethal disease is most frequently caused by clade I MPXV infections (case fatality rates between 6 and 15%). The 2022 international outbreak was driven by clade lib MPXV and was associated with less severe disease. The recent 2024 outbreak resulted from an upsurge of a new clade (clade lb). Orthopox viruses are DNA encoded viruses, yielding a high level of similarity between orthopoxvirus antigens, which allows immune responses raised to certain proteins from one virus to potentially provide cross-protective immunity to other viruses within the genus. Smallpox was eradicated by exploiting this potential for cross-protection through global immunization campaigns using another orthopoxvirus, vaccinia virus (VACV), as a live virus vaccine. First generation VACV immunization provided robust and durable protection against smallpox, but its unfavorable side effects and exclusion from use in certain populations encouraged the development of vaccines with an improved safety profile. VACV-derivatives with improved safety profiles but more modest durability of immune response were available for use prior to May 2022, but stockpiles and existing manufacturing capacity did not meet the demand driven by the 2022 outbreak. The development of novel, potent and safe mpox vaccines is needed, especially vaccines that can be rapidly manufactured at scale and distributed globally.
[0147] Without wishing to be bound by any theory, the design of polyribonucleotide constructs encoding mpox antigens of the present disclosure draws from the study of immune responses to live VACV vaccines that formed the basis of the successful global campaign to eradicate smallpox. VACV replication in a host exposes the immune system to the complete proteome of the virus, including greater than 30 virion surface proteins. In principle, VACV-mediated adapted immune protection could involve antibody and/or T cell responses to all or many VACV proteins. However, VACV proteins have differential immunogenicity in a host, suggesting that responses to a subset of antigens are needed for immunity.
[0148] Envelope proteins can be the targets of both protective antibody and T cell responses since they are accessible to antibodies in intact virions and infected cells. However, narrowing to mpox envelope proteins may be insufficient to define antigen inclusion, as orthopoxviruses encode dozens of envelope proteins in their large DNA genomes. This feature stands in stark contrast to other viruses like SARS-CoV-2 for which there are only one or a few potential envelope protein targets. Further complicating antigen selection are the two distinct viral forms of orthopoxviruses, extracellular virions (EVs) and mature virions (MVs).
[0149] The specific mechanisms of protection for immune responses raised to MV and EV targets vary; immune responses raised to EV and MV targets have different functional profiles in vitro and in vivo. Antibodies against MV targets like Ml can generate neutralizing antibody responses in which pre-incubation of anti-MV antibodies with MV particles can prevent infection of cells in vitro independent of Fc-mediated antibody effector functions. In contrast, in vitro neutralization of EV particles through pre-incubation with anti-A35 or anti- 66 antibodies depends on the presence of complement proteins. However, comparison of active or passive immunization against EV or MV antigens in mice demonstrates that EV antigens are targets for in vivo protection from severe disease in VACV infection models. In vivo protection provided by anti-EV antibodies depends on Fc- effector functions, including both complement cascade activation and Fc receptor-mediated functions, consistent with results from in vitro EV neutralization assays. [0150] The present disclosure provides nucleic acids (e.g., polyribonucleotides) encoding orthopox antigens (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)), antigen constructs thereof, and/or pharmaceutical compositions thereof that are effective for vaccination against one or more orthopox viruses (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, rabbitpox, etc.)). The present disclosure also provides the insight that polyribonucleotides, antigen constructs thereof, and pharmaceutical compositions thereof targeting one orthopox species may cross-protect against other orthopoxviruses. For example, in some embodiments, provided polyribonucleotides encoding mpox proteins, antigen constructs thereof, and/or pharmaceutical compositions thereof, are effective for vaccination against mpox and one or more other orthopox viruses. In some embodiments, provided polyribonucleotides encoding mpox proteins, antigen constructs thereof, and/or pharmaceutical compositions thereof are effective for vaccination against mpox and one or more of variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) viruses. In some embodiments, provided polyribonucleotides encoding mpox proteins, antigen constructs thereof, and/or pharmaceutical compositions thereof are effective for vaccination against mpox and variola virus. In some embodiments, provided polyribonucleotides encoding mpox proteins, antigen constructs thereof, and/or pharmaceutical compositions thereof are effective for vaccination against mpox and rabbitpox. In some embodiments, provided polyribonucleotides encoding mpox proteins, antigen constructs thereof, and/or pharmaceutical compositions thereof are effective for vaccination against mpox and vaccinia virus (e.g., modified vaccinia virus Ankara, etc.). In some embodiments, provided polyribonucleotides encoding mpox proteins, antigen constructs thereof, and/or pharmaceutical compositions thereof are effective for vaccination against mpox and ectromelia virus. In some embodiments, provided polyribonucleotides encoding mpox proteins, antigen constructs thereof, and/or pharmaceutical compositions thereof are effective for vaccination against mpox and a novel orthopox virus.
Mpox Structure
[0151] Mpox virions are ovoid or brick-shaped particles which are enclosed by geometrically corrugated lipoprotein outer membrane. Mature mpox virions have a densely packed core containing enzymes, a double-stranded DNA genome, and transcription factors that are protected by a protein core.
[0152] The mpox genome consists of a linear double-stranded DNA (about 197 kb) covalently joined at its ends by palindromic hairpins, and the inverted terminal repeats (ITRs) are made up of a hairpin loop, tandem repeats, and some open reading frames (ORF). Although mpox virus is a DNA virus, its entire life cycle occurs in the cytoplasm of infected cells. All the proteins required for viral DNA replication, transcription, virion assembly, and egress are encoded by the mpox genome. The genes encoding for housekeeping functions are highly conserved among orthopoxviruses and are present in the central region of the genome while those that encode for the genes mediating virus-host interactions are less conserved and located in the terminal regions of the genome.
[0153] In vaccinia virus (and most likely in mpox) intracellular mature virus (MV) and extracellular- enveloped virus (EV) are two forms of infectious virions produced in poxvirus-infected cells. MV is released upon cell lysis, while EV is released from cells via interaction with actin tails, and this is said to be the cause of rapid long distance spread of the virus within the infected host. Although the aforementioned features are for VACV, it is likely that these features are common to all orthopoxviruses. Cell-associated virions (CEVs) are formed following the microtubule-mediated transport of intracellular enveloped virus (IEV) to the cell periphery, in which the outer membrane of IEV fuses with the plasma membrane and remains attached to the cell surface. CEVs are mostly responsible for cell-to-cell spread. IEV is formed when an MV is wrapped by a double membrane derived from early endosomal component or the trans-Golgi network (TGN). However, apart from IEV exocytosis, an alternative route for the formation of an EV is by the budding of the MV through the plasma membrane. In the prototype vaccinia virus, virion morphogenesis can be defective resulting in non-infectious dense particles (DPs), but this has not yet been reported for mpox. In addition, unlike some strains of cowpox virus in which MVs are occluded within A-type inclusions (ATI), mpox does not form ATIs or sequester MVs into ATIs because of truncation in the ATIP gene.
Mpox Transmission
[0154] The two possible means of mpox transmission are animal-to-human transmission and human- to-human transmission. Respiratory droplets and contact with body fluids, contaminated patient's environment or items, and skin lesion of an infected person have been found to be associated with inter-human transmission. Contact between broken skin or mucous membranes and an infected patient's body fluids, respiratory droplets, or scabs is considered a "high risk" exposure that warrants post-exposure vaccination as soon as possible.
Congo Basin (CB) clade (Central Africa clade) is reported to be more virulent than West Africa (WA) clade and thereby contributes more to inter-human transmission. Animal-to-human transmission, which is also known as zoonotic transmission, occurs via direct contact with any of the aforementioned natural viral hosts or consumption of these hosts. In addition, zoonotic transmission could occur by direct contact with the blood, body fluids, and inoculation from mucocutaneous lesions of an infected animal. Nosocomial transmission has been reported for CB and WA clades of mpox while sexual transmission has been speculated for infected individuals with groin and genital lesions. At present, human-to-animal transmission has not been reported. Human-to-human transmission, secondary attack rates (SARS), and serial transmission events is much higher with the CB clade compared to the WA clade. The reproduction number (Ro) for the CB clade is estimated to be in the range of 0.6-1.0. The Ro has not been estimated for the WA clade of mpox viruses, but it is presumed to be lower than that of the CB clade. The upper limit Ro of 1.0 in the CB clade indicates that the viruses will not only sustain human-to-human transmission but may persist in the human population. Presumably, if as expected the RQ of the WA clade is much lower than what was estimated for the CB clade, then sustained human-to- human transmission and persistence in a human population are highly unlikely and outbreaks will be largely due to spillover events from zoonotic hosts.
Mpox Treatment
[0155] Currently, there are no specific clinically proven treatments for mpox infection. As with most viral illnesses, the treatment is supportive symptom management. There are, however, prevention measures that can help prevent and/or reduce severity of an outbreak. Infected individuals should remain in isolation, wear a surgical mask, and keep lesions covered as much as reasonably possible until all lesion crusts have naturally fallen off and a new skin layer has formed. For severe cases, investigational use can be considered for compounds with demonstrated benefit against orthopoxviruses in animal studies and severe vaccinia vaccine complications. The oral DNA polymerase inhibitor brincidofovir, oral intracellular viral release inhibitor tecovirimat, and intravenous vaccinia immune globulin have unknown efficacy against the mpox virus. For individuals exposed to the virus, temperature and symptoms should be monitored twice per day for 21 days because that is the accepted upper limit of the mpox incubation period. Infectiousness aligns with symptom onset; therefore, close contacts need not isolate while asymptomatic. According to the Centers for Disease Control and Prevention (CDC), vaccination within four days of exposure may prevent disease onset, and vaccination within 14 days may reduce disease severity. Exemplary Mpox Polypeptides
[0156] Exemplary polyribonucleotide constructs of the present disclosure include nucleic acids encoding A35, B6, Ml and/or H3. In some embodiments, antigens may be from a certain mpox clade, e.g., from clade I (clade la or clade lb) MPXV, or clade II (e.g., clade Ila or clade lib) MPXV. Wild-type (WT) versions of mpox antigens provided herein can be surface-exposed, single-pass membrane proteins. Recognition of membrane proteins by the immune system can be achieved through trafficking of these proteins to the cell surface in polyribonucleotide-transfected cells. For example, WT versions of A35 and B6 are expected to traffic to the cell surface in polyribonucleotide-transfected cells. However, WT versions of MV targets, e.g., Ml and H3 are not efficiently delivered to the endoplasmic reticulum for eventual transit to the plasma membrane. During viral replication, embedding of MV proteins into the MV envelope may be distinct from the normal host cell biosynthesis pathway for integral membrane proteins and they lack native secretory signal.
B6R
[0157] Mpox B6R (also referred to as MPXVgpl67) is a -35 kD polypeptide. B6R, is a type I integral membrane protein with an N-terminal ectodomain, a C-terminal transmembrane helix, and two sushi domains. B6R is classified as a membrane glycoprotein that is a component of the mpox EV envelope. B6R is also classified as being involved in negative regulation of complement activation. B6R polypeptide sequences include, e.g., UniProt accession numbers Q8V4S2, V9NQJ0, A0A0F6N8B7, each of which is incorporated herein by reference in its entirety. Exemplary B6R amino acid sequences are provided in Table 1 and Table 6 below. In some embodiments, exemplary polyribonucleotide constructs of the present disclosure comprise one or more polyribonucleotides that encode B6R. For example, in some embodiments, the encoded B6R antigen is fully WT. In some embodiments, the sequence of the fully WT B6R antigen is derived from early cases in the 2022 mpox outbreak. In some embodiments, a B6R antigen is associated with a native secretory signal.
[0158] Mpox B6R is homologous to vaccinia B5R. Vaccinia B5R (see, e.g., accession number
AAN78219.1) is a membrane protein that is essential in packaging the intracellular mature virion form intracellular enveloped virions, and is EV-specific.
DOIs: https://doi.org/10.1083/jcb.200104124, https://doi.org/10.1128/jvi.68.1.130- 147.1994, https://doi.org/10.1099/0022-1317-83-12-2915.
[0159] In some embodiments, exemplary polyribonucleotide constructs of the present disclosure comprise one or more polyribonucleotides that encode a B6R antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 21-30, 180, or 182, or an antigenic fragment thereof.
A35R
[0160] Mpox A35R (also referred to as MPXV-COP-139, MPXV-SL-139, MPXV-WRAIR139) is a -20 kD polypeptide. A35R is a type II, disulfide-bonded homodimer with a C-terminal ectodomain and a N-terminal transmembrane domain. A35R is classified as a membrane protein, specifically a bifunctional EV membrane phosphoglycoprotein. A35R polypeptide sequences include, e.g., UniProt accession numbers Q8V4U4 and Q80KX2, each of which is incorporated herein by reference in its entirety. Exemplary A35R amino acid sequences are provided in Table 1 and Table 13 below. In some embodiments, exemplary polyribonucleotide compositions of the present disclosure comprise one or more polyribonucleotides that encode A35R. For example, in some embodiments, the encoded A35R antigen is fully WT. In some embodiments, the A35R antigen sequence is derived from early cases in the 2022 mpox outbreak. In some embodiments, an A35R antigen is associated with a native secretory signal.
[0161] Mpox A35R is homologous to vaccinia A33R. Vaccinia A33R (see, e.g., accession number
AAF63733, incorporated herein by reference in its entirety) is a type II integral membrane protein found in EV (extracellular enveloped virus) but not MV, and is highly conserved among orthopoxviruses. See, e.g., DOIs: 10.1128/jvi.72.5.4192-4204.1998.
[0162] In some embodiments, exemplary polyribonucleotide constructs of the present disclosure comprise one or more polyribonucleotides that encode a A35R antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 11-20, 172, or 174, or an antigenic fragment thereof.
MIR
[0163] MPXV MIR (also referred to as MV membrane protein, MPXV-COP-074, MPXV-SL-074) is a -27 kD polypeptide. MIR is a type I integral membrane protein with an N-terminal ectodomain and a C-terminal transmembrane helix. MIR polypeptide sequences include, e.g., UniProt accession numbers Q8V502, Q80KX3, Q5IXU5, each of which is incorporated herein by reference in its entirety. Exemplary MIR amino acid sequences are provided in Table 1 and Table 13 below. In some embodiments, exemplary polyribonucleotide compositions of the present disclosure comprise one or more polyribonucleotides that encode MIR. In some embodiments, a secretory signal from HSV-1 gD can be added to the N-terminus of MIR. In some embodiments, the secretory signal can participate in trafficking MIR to the cell surface for display.
[0164] MPXV MIR is homologous to vaccinia L1R. Vaccinia L1R (see, e.g., accession number
AAF63732, incorporated herein by reference in its entirety) is a myristoylated transmembrane protein of about 250 residues that is expressed on the surface of the MVs. It is considered essential at least in that genetic deletion of L1R renders vaccinia viruses incapable of maturation. L1R appears to be required for maturation of viral particles. See, e.g., DOIs: https://doi.org/10.1128/jvi.68.10.6401-6410.1994, 10.1073/pnas.062163799. [0165] In some embodiments, exemplary polyribonucleotide constructs of the present disclosure comprise one or more polyribonucleotides that encode a MIR antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 31-40, or 158, or an antigenic fragment thereof.
H3L
[0166] H3L (also referred to as MV heparin binding surface protein, Envelope protein H3, MPXV-COP-
087, MPXV-SL-087, MPXV-WRAIR087, MPXVgpO93) is a -37.5 kD polypeptide that localizes to the mpox viral envelope. H3L is a type I integral membrane protein with an N-terminal ectodomain and a C-terminal transmembrane helix. H3L polypeptide sequences include, e.g., UniProt accession numbers, Q8V4Z2, Q3I8S1, Q5IXT2, A0A0F6N9X0, each of which is incorporated herein by reference in its entirety). Exemplary H3L amino acid sequences are provided in Table 1 and Table 13 below. In some embodiments, exemplary polyribonucleotide compositions of the present disclosure comprise one or more polyribonucleotides that encode H3L. In some embodiments, a secretory signal from HSV-1 gD can be added to the N-terminus of H3L. In some embodiments, the secretory signal can participate in trafficking H3L to the cell surface for display. Mpox H3L was found to bear high sequence similarity to vaccinia H3L. [0167] In some embodiments, exemplary polyribonucleotide constructs of the present disclosure comprise one or more polyribonucleotides that encode a H3L antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 51-60, 184, 188, or 190, or an antigenic fragment thereof.
II, Polyribonucleotides
A. Exemplary Polyribonucleotide Constructs
[0168] The present disclosure, among other things, utilizes polyribonucleotide (e.g., RNA) technologies as a modality to express one or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) viruses) polypeptide constructs that includes one or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) viruses) antigens, or one or more portions thereof, described herein.
[0169] In some embodiments, the present disclosure provides polyribonucleotides that encode one or more mpox antigens or fragments thereof. The present disclosure includes the unexpected discovery that mpox antigens provided in Table 1, and fragments thereof, are particularly advantageous for use in preventing or treating mpox infection and other orthopoxvirus infections (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) viruses), e.g., in mpox antigen constructs and/or mpox vaccines as further disclosed herein.
Figure imgf000043_0001
[0170] In some polyribonucleotide constructs of the present disclosure, multiple polyribonucleotides are designed such that the multiple polyribonucleotides together encode multiple mpox antigens because of the many surface proteins and complex replication cycle of orthopoxviruses. Orthopoxviruses have two distinct infectious forms, mature virions (MV) and extracellular virions (EV). Each viral form has a unique set of surface antigens. While immune responses raised to any single target can offer some protection from infection, both subunit vaccine and monoclonal antibody prophylaxis studies in animals illustrate the value of combining antibodies to multiple EV and MV targets. Therefore, to effectively reduce viral replication and infectivity, exemplary polyribonucleotide constructs of the present disclosure target MPXV proteins from both EVs and MVs: A35 and B6 which are EV surface glycoproteins and Ml and H3 which are displayed on the surface of MVs. Without wishing to be bound by any particular theory, selection of this subset of antigens from the surface proteomes of MPXV virions may be guided by two factors. First, A35, B6, Ml and H3 are conserved across many orthopoxviruses, including VARV and VACV. Second, the use of these targets in protein and DNA-vectored subunit vaccines has been demonstrated to elicit protective immunity in mouse and non-human primate systems; however, protein and DNA based compositions have associated challenges. As discussed herein, polyribonucleotides and compositions provided herein can address and overcome the prior challenges. [0171] In various embodiments, a polyribonucleotide of the present disclosure encodes a single mpox antigen of Table 1 or antigenic fragment thereof (e.g., an A35R polypeptide or antigenic fragment thereof, B6R polypeptide or antigenic fragment thereof, MIR polypeptide or antigenic fragment thereof, or H3L polypeptide or antigenic fragment thereof). In some embodiments, an exemplary polyribonucleotide of the present disclosure encodes an A35R polypeptide having a sequence of any one of the A35R sequences of Table 1, or an antigenic fragment thereof. In some embodiments, exemplary polyribonucleotide constructs of the present disclosure comprise one or more polyribonucleotides that encode an A35R antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 11-20, 172, or 174, or an antigenic fragment thereof.
[0172] In some embodiments, a polyribonucleotide of the present disclosure encodes a B6R polypeptide having a sequence of any one of the B6R sequences of Table 1, or an antigenic fragment thereof. In some embodiments, exemplary polyribonucleotide constructs of the present disclosure comprise one or more polyribonucleotides that encode a B6R antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 21-30, 180, or 182, or an antigenic fragment thereof.
[0173] In some embodiments, a polyribonucleotide of the present disclosure encodes an MIR polypeptide having a sequence of any one of the MIR sequences of Table 1, or an antigenic fragment thereof. In some embodiments, exemplary polyribonucleotide constructs of the present disclosure comprise one or more polyribonucleotides that encode a MIR antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 31-40, or 158, or an antigenic fragment thereof.
[0174] In some embodiments, a polyribonucleotide of the present disclosure encodes an H3L polypeptide having a sequence of any one of the H3L sequences of Table 1, or an antigenic fragment thereof. In some embodiments, exemplary polyribonucleotide constructs of the present disclosure comprise one or more polyribonucleotides that encode a H3L antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 51-60, 184, 188, or 190, or an antigenic fragment thereof.
[0175] In some embodiments, a polyribonucleotide of the present disclosure encodes two, three, or four mpox antigens of Table 1 or antigenic fragments thereof. In some embodiments, a polyribonucleotide of the present disclosure encodes two or more mpox polypeptides selected from: an A35R polypeptide or antigenic fragment thereof, a B6R polypeptide or antigenic fragment thereof, an MIR polypeptide or antigenic fragment thereof, and an H3L polypeptide or antigenic fragment thereof.
[0176] In some embodiments, a polyribonucleotide of the present disclosure encodes a B6R polypeptide having a sequence of any one of the B6R sequences of Table 1, and an MIR polypeptide having a sequence of any one of the MIR sequences of Table 1. In some embodiments, a polyribonucleotide of the present disclosure encodes a B6R antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 21-30, 180, or 182, or an antigenic fragment thereof, and an MIR antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 31-40, or 158, or an antigenic fragment thereof.
[0177] In some embodiments, a polyribonucleotide of the present disclosure encodes an A35R polypeptide having a sequence of any one of the A35R sequences of Table 1, a B6R polypeptide having a sequence of any one of the B6R sequences of Table 1, and an MIR polypeptide having a sequence of any one of the MIR sequences of Table 1. In some embodiments, a polyribonucleotide of the present disclosure encodes: (i) an A35R antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 11-20, 172, or 174, or an antigenic fragment thereof; (ii) a B6R antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 21-30, 180, or 182, or an antigenic fragment thereof; and (iii) an MIR antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 31-40, or 158, or an antigenic fragment thereof.
[0178] In some embodiments, a polyribonucleotide of the present disclosure encodes an A35R polypeptide having a sequence of any one of the A35R sequences of Table 1, a B6R polypeptide having a sequence of any one of the B6R sequences of Table 1, an MIR polypeptide having a sequence of any one of the MIR sequences of Table 1, and an H3L polypeptide having a sequence of any one of the H3L sequences of Table 1. In some embodiments, a polyribonucleotide of the present disclosure encodes: (i) an A35R antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 11-20, 172, or 174, or an antigenic fragment thereof; (ii) a B6R antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 21-30, 180, or 182, or an antigenic fragment thereof; (iii) an MIR antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 31-40, or 158, or an antigenic fragment thereof; and (iv) a H3L antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to any one of SEQ ID NOs: 51-60, 184, 188, or 190, or an antigenic fragment thereof.
[0179] In some embodiments, a polyribonucleotide of the present disclosure encodes one or more MV antigens. In some embodiments, one or more MV antigens are selected from H3L and MIR. In some embodiments, a polyribonucleotide of the present disclosure encodes one or more MV-specific antigens. In some embodiments, one or more MV-specific antigens are selected from H3L and MIR.
[0180] In some embodiments, a polyribonucleotide of the present disclosure encodes one or more EV antigens. In some embodiments, one or more EV antigens are selected from A35R and B6R. In some embodiments, a polyribonucleotide of the present disclosure encodes one or more EV-specific antigens. In some embodiments, one or more EV-specific antigens are selected from A35R and B6R.
[0181] In some embodiments, a polyribonucleotide of the present disclosure encodes one or more
A35R polypeptides (e.g., an A35R antigen or one or more antigenic fragments thereof). In some embodiments, a polyribonucleotide of the present disclosure encodes one or more B6R polypeptides (e.g., a B6R antigen or one or more antigenic fragments thereof). In some embodiments, a polyribonucleotide of the present disclosure encodes one or more MIR polypeptides (e.g., an MIR antigen or one or more antigenic fragments thereof). In some embodiments, a polyribonucleotide of the present disclosure encodes one or more H3L polypeptides (e.g., an H3L antigen or one or more antigenic fragments thereof).
[0182] As described herein, in some embodiments, provided technologies involve administration of a plurality of antigens to the same subject. In some embodiments, multiple antigens are administered at the same time (e.g., in a single dose). In some embodiments, different antigens may be administered at different times (for example in different doses - e.g., a prime dose vs a boost dose). In some embodiments, multiple antigens are administered via the same composition.
[0183] For clarity, a single "antigen" polypeptide may include multiple "epitopes", which in turn may or may not be linked with one another in nature. For example, a single string construct antigen includes multiple epitopes, which may be from different parts of the same mpox protein and/or from different mpox proteins, linked together as described herein in a single polypeptide.
[0184] Thus, a single pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as described herein may include or deliver (e.g., because the pharmaceutical composition includes a nucleic acid, such as a polyribonucleotide, that encodes the antigen and is expressed upon administration) a single antigen, which itself may comprise multiple epitopes (either in their natural arrangement relative to one another or in an engineered or constructed arrangement as described herein), or may comprise or deliver a plurality of antigens, each of which similarly may be or comprise a single epitope or multiple epitopes (either in their natural arrangement relative to one another or in an engineered or constructed arrangement as described herein). Still further, a single pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) may, for example, include multiple distinct nucleic acids (e.g., polyribonucleotides) that each encode different antigen(s) or, in some embodiments, may include a single nucleic acid that encodes (and expresses) multiple antigens. Yet further, a single pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) that includes multiple distinct nucleic acids (e.g., polyribonucleotides) encoding antigens may, in some embodiments, be prepared by mixing the polyribonucleotides and then incorporating the mixture into LNPs, or alternatively by formulating individual polyribonucleotides into LNPs and then mixing the LNPs. In some embodiments, mixtures (whether of polyribonucleotides pre-LNP preparation or of LNPs) may include the relevant polyribonucleotides in 1: 1 ratio, or in other ratios as may be preferred (e.g., to achieve a desired relative presentation of antigens or epitopes) in a subject to whom the composition is administered.
[0185] In some embodiments, two or more polyribonucleotide molecules each encoding a different polypeptide (e.g., an mpox antigen as described herein) can be mixed with particle-forming agents to form nucleic acid containing particles as described above. In alternative embodiments, two or more polyribonucleotide molecules each encoding a different polypeptide (e.g., an mpox antigen, or antigenic fragment thereof, as described herein) can be formulated into separate particle compositions, which are then mixed together. For example, in some embodiments, individual populations of nucleic acid containing particles, each population comprising a polyribonucleotide molecule encoding a different immunogenic polypeptide or antigenic fragment thereof (e.g., an mpox antigen or antigenic fragment thereof as described herein), can be separately formed and then mixed together, for example, prior to filling into vials during a manufacturing process, or immediately prior to administration (e.g., by an administering health-care professional). Accordingly, in some embodiments, described herein is a composition comprising two or more populations of particles (e.g., in some embodiments, lipid nanoparticles), each population comprising at least one polyribonucleotide molecule encoding a different immunogenic polypeptide or antigenic fragment thereof (e.g., an mpox antigen or antigenic fragment thereof). In some embodiments, each population may be provided in a composition at a desirable proportion (e.g., in some embodiments, each population may be provided in a composition in an amount that provides the same amount of polyribonucleotide molecules).
[0186] In some embodiments, one or more pharmaceutical compositions (e.g., immunogenic composition, e.g., vaccine) may comprise or deliver a combination comprising a polypeptide or portion thereof encoded by all or part of A35R, B6R, MIR, H3L, and/or antigenic fragments of any thereof.
[0187] In some embodiments, one or more pharmaceutical compositions (e.g., immunogenic composition, e.g., vaccine) comprise or deliver a combination comprising one or more MV antigens (e.g., MV- specific antigens) and one or more EV antigens (e.g., EV-specific antigens). In some embodiments, one or more pharmaceutical compositions comprise or deliver a combination of mpox antigens that includes (i) one or more MV antigens (e.g., MV-specific antigens) selected from H3L, MIR, and antigenic fragments of any thereof; and (ii) one or more EV antigens (e.g., EV-specific antigens) selected from A35R, B6R, and antigenic fragments of any thereof.
[0188] In some embodiments, one or more pharmaceutical compositions (e.g., immunogenic composition, e.g., vaccine) may comprise or deliver a combination comprising a polypeptide or portion thereof encoded by all or part of B6R, MIR, and/or antigenic fragments of any thereof. In some embodiments, one or more pharmaceutical compositions (e.g., immunogenic composition, e.g., vaccine) may comprise or deliver a combination comprising a polypeptide or portion thereof encoded by all or part of B6R, A35R, and/or antigenic fragments of any thereof. In some embodiments, one or more pharmaceutical compositions (e.g., immunogenic composition, e.g., vaccine) may comprise or deliver a combination comprising a polypeptide or portion thereof encoded by all or part of B6R, A35R, MIR, and/or antigenic fragments of any thereof. In some embodiments, one or more pharmaceutical compositions (e.g., immunogenic composition, e.g., vaccine) may comprise or deliver a combination comprising a polypeptide or portion thereof encoded by all or part of B6R, A35R, MIR, H3L, and/or antigenic fragments of any thereof.
[0189] In some embodiments, a provided composition includes or delivers an mpox envelope glycoprotein antigen (e.g., a full-length mpox envelope glycoprotein, a fragment thereof, or one or more epitopes thereof, for example in a string construct). In some embodiments, a provided composition includes or delivers such an mpox envelope glycoprotein antigen together with one or more B cell targets (e.g., epitopes) which may, for example, be or comprise one or more other mpox proteins (or fragments or epitopes thereof). In some embodiments, such a B cell target is or comprises an mpox protein (or fragment or epitope thereof) that is predicted or known to induce a B cell response in infected humans. For example, in some embodiments, a B cell target is or comprises an mpox protein (or fragment or B cell epitope thereof) against which sera from infected individual(s) is reactive. In some particular embodiments, a B cell target is or comprises an mpox envelope glycoprotein, or other relevant mpox protein, or a fragment or epitope thereof.
[0190] In some embodiments, a provided composition comprises or delivers a string construct antigen that includes a plurality of T cell epitopes, optionally from more than one mpox protein. In some such embodiments, a provided composition further comprises or delivers one or more B cell targets. Alternatively or additionally, in some embodiments, a string construct antigen so utilized includes mpox sequences (e.g., one or more fragments or epitopes, e.g., T cell epitopes and/or B cell epitopes, but in some embodiments specifically T cell epitopes).
[0191] In some embodiments, a string construct antigen includes both B cell epitopes and T cell epitopes (optionally from the same mpox protein or from different mpox proteins).
[0192] In some embodiments, different antigens may be delivered by administration of different compositions, which in turn may, in some embodiments, be administered at the same time (e.g., as an admixture or otherwise substantially simultaneously) and, in some embodiments, may be administered at different times. To give but one example, in some embodiments, a particular antigen or antigen(s) may be delivered via an initial pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) dose, and one or more other antigen(s) may be delivered via one or more booster dose(s).
[0193] In some embodiments, an antigen utilized (i.e., included in and/or otherwise delivered by) a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) described herein comprises multiple epitopes, e.g., of a single mpox protein or of multiple proteins.
[0194] In some embodiments, an antigen may comprise two or more epitopes from the same mpox protein and in their natural configuration relative to one another (e.g., in a fragment if the relevant protein). In some embodiments, however, an antigen may comprise at least two epitopes configured in a non-natural relationship relative to one another (e.g., included in a string construct).
[0195] Experience with SARS-CoV-2 has demonstrated that polyribonucleotide (e.g., RNA) administration can be a particularly effective way to deliver an infectious disease antigen. Furthermore, the present disclosure provides an insight that various features of nucleic acid formats including, for example their flexibility and amenability to rapid design and modification, including incorporation of a variety of insights (e.g., bioinformatics inputs etc.), renders them particularly attractive for use in an mpox vaccine.
[0196] In some embodiments, one or more polyribonucleotides (e.g., mRNAs) utilized in a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as described herein encodes a plurality of epitopes (e.g., including one or more, or two or more, sequences provided in Table 1, or antigenic fragments thereof), optionally wherein each of the plurality is predicted by an H LA binding and presentation prediction algorithm to be of high likelihood to be presented by a protein encoded by an H LA to a T cell for eliciting an immune response. In some embodiments, the plurality of epitopes comprises epitopes from a single mpox protein. In some embodiments, the plurality of epitopes comprises epitopes from multiple mpox proteins. [0197] In some embodiments, one or more polyribonucleotides (e.g., mRNAs) utilized in a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as described herein include a first polyribonucleotide that encodes an mpox antigen expressed prior to cell infiltration or infection and includes one or more portions expected or known to interface with host cytoplasm. In some embodiments, an mpox antigen encoded by a first polyribonucleotide is or comprises an mpox antigen, fragment, or epitope, e.g., an A35R, B6R, MIR, and/or H3L, or antigenic fragments thereof, epitopes thereof, and/or a combination thereof. In some embodiments, one or more polyribonucleotides (e.g., mRNAs) utilized in a pharmaceutical composition (e.g., immunogenic composition, e.g., vaccine) as described herein includes a second polyribonucleotide that encodes a multi-epitopic (e.g., polyepitopic) antigen. In some embodiments, a multi-epitopic antigen comprises two or more antigens found in Table 1 or antigenic fragments thereof or epitopes thereof. In some embodiments, a multi-epitopic antigen comprises two or more antigens listed in Table 1, and/or antigenic fragments and/or epitopes thereof.
[0198] In some embodiments, a polyribonucleotide construct can include an internal ribosome entry site (IRES), e.g., between two sequences encoding antigens or fragments thereof engineered for expression from a polyribonucleotide as distinct polypeptides. Internal ribosome entry sites (IRESs) are cis-acting elements that can recruit the small ribosomal subunits to an internal initiator codon in a polyribonucleotide in conjunction with cellular trans-acting factors. The ability of internal ribosome entry site (IRES) elements to promote internal initiation of translation of polyribonucleotide sequences can facilitate or permit expression of two or more polypeptides from a polycistronic polyribonucleic acid. An exemplary IRES is the encephalomyocarditis virus (EMCV) IRES.
[0199] In some embodiments, a multi-epitope polyribonucleotide encoding a super-motif-bearing or motif-bearing polypeptide, together with a helper epitope (e.g., a heterologous helper epitope) and an endoplasmic reticulum-translocating signal sequence. See, for example, in An & Whitton J. Virol. 71:2292, 1997; Thomson, eta/., J. Immuno! 157:822, 1996; Whitton, eta!., J. Virol 67 :348, 1993; Hanke, et a!., Vaccine 16:426, 1998.
[0200] Additionally, polyribonucleotides described herein, in some embodiments, include other elements such as described below, including, a secretion signal-encoding region, a 5' Cap, a Cap proximal sequence, a 5' UTR, a 3' UTR, and/or a polyA tail. In some embodiments, polyribonucleotides described herein can comprise a secretion signal-encoding region. In some embodiments, epitopes encoded in a string construct may be flanked by a secretory signal sequence, e.g., SP1 sequence (HSV-1 gD secretory signal /secretory domain, SEQ ID NO: 143). In some embodiments, polyribonucleotides described herein can comprise a nucleotide sequence that encodes a 5' UTR of interest and/or a 3' UTR of interest. In some embodiments, a polynucleotide comprises a dEarl-hAg sequence (SEQ ID NO: 155). In some embodiments, the polyribonucleotide (e.g., mRNA) comprises a 5 'UTR and a 3' UTR. In some embodiments, a 3' UTR comprises a poly A sequence. In some embodiments, a poly A sequence comprises between 50-200 nucleotides. In some embodiments, a poly A tail of a string construct may comprise about 150 A residues. In some embodiments, a poly A tail may comprise 120 residues or less. In some embodiments, a poly A tail of a string construct may comprise about 120 A residues. In some embodiments, a poly A tail of a string construct may comprise about 100 A residues. In some embodiments, a poly A tail of a string construct comprises a "split" or "interrupted" poly A tail (e.g., as described in W02016/005324). In some embodiments, polyribonucleotides described herein may comprise a 5' cap, which may be incorporated during transcription, or joined to a polyribonucleotide posttranscription.
1. Secretory Signals
[0201] In some embodiments, a polyribonucleotide described herein comprises a sequence encoding a human secretory signal. For example, in some embodiments, such a human secretory signal may be or comprises the amino acid sequence of MDWIWRILFLVGAATGAHS (husec2; SEQ ID NO: 142). In some embodiments, a ribonucleic acid sequence encoding a secretory signal included in a polyribonucleotide consists of or comprises a nucleotide sequence that encodes a non-human secretory signal. In some embodiments, a polyribonucleotide encodes a human secretory signal where the secretory signal comprises the amino acid sequence MDWIWRILFLVGAATGAHS (husec2; SEQ ID NO: 142).
[0202] In some embodiments, an RNA sequence encodes an orthopoxvirus (e.g., mpox) antigen or antigenic fragment thereof that may comprise or otherwise be linked to a secretory signal (e.g., a secretory sequence), such as those listed in Table 2, or a sequence having 1, 2, 3, 4, or 5 amino acid differences relative thereto. In some embodiments, a secretory signal such as MRVMAPRTLILLLSGALALTETWAGS (SEQ ID NO: 157), or a sequence having 1, 2, 3, 4, or at the most 5 amino acid differences relative thereto is utilized.
[0203] In some embodiments, a secretory signal is selected from those included in Table 2 below:
Table 2: Exemplary Secretory Signal Sequences
Figure imgf000050_0001
2. 5' Cap
[0204] A structural feature of mRNAs is a cap structure at the five-prime end (S'). Natural eukaryotic mRNA comprises a 7-methylguanosine cap linked to the mRNA via a 5 ' to 5 '-triphosphate bridge resulting in capO structure (m7GpppN). In most eukaryotic mRNA and some viral mRNA, further modifications can occur at the 2'-hydroxy-group (2'-0H) (e.g., the 2'-hydroxyl group may be methylated to form 2'-0-Me) of the first and subsequent nucleotides producing "capl" and "cap2" five-prime ends, respectively). Diamond, et al., (2014) Cytokine & Growth Factor Reviews, 25:543-550 reported that capO-mRNA cannot be translated as efficiently as capl-mRNA in which the role of 2'-O-Me in the penultimate position at the mRNA 5' end is determinant. Lack of the 2'-O-met has been shown to trigger innate immunity and activate an IFN response. Daffis, et al. (2010) Nature, 468:452-456; and Ziist et al. (2011) Nature Immunology, 12: 137-143.
[0205] RNA capping is well researched and is described, e.g., in Decroly E., et al. (2012) Nature
Reviews 10: 51-65; and in Ramanathan A., et al., (2016) Nucleic Acids Res, 44(16): 7511-7526, the entire contents of each of which is hereby incorporated by reference. For example, in some embodiments, a 5'-cap structure which may be suitable in the context of the present invention is a capO (methylation of the first nucleobase, e.g. m7GpppN), capl (additional methylation of the ribose of the adjacent nucleotide of m7GpppN), cap2 (additional methylation of the ribose of the 2nd nucleotide downstream of the m7GpppN), cap3 (additional methylation of the ribose of the 3rd nucleotide downstream of the m7GpppN), cap4 (additional methylation of the ribose of the 4th nucleotide downstream of the m7GpppN), ARCA ("anti-reverse cap analogue"), modified ARCA (e.g. phosphothioate modified ARCA), inosine, N1 -methyl-guanosine, 2'-fluoro-guanosine, 7-deaza- guanosine, 8-oxo-guanosine, 2-amino-guanosine, LNA-guanosine, and 2-azido-guanosine. [0206] The term "5'-cap" as used herein refers to a structure found on the 5'-end of an RNA, e.g., mRNA, and generally includes a guanosine nucleotide connected to an RNA, e.g., mRNA, via a 5'- to 5'- triphosphate linkage (also referred to as Gppp or G(5')ppp(5')). In some embodiments, a guanosine nucleoside included in a 5' cap may be modified, for example, by methylation at one or more positions (e.g., at the 7- position) on a base (guanine), and/or by methylation at one or more positions of a ribose. In some embodiments, a guanosine nucleoside included in a 5' cap comprises a 3'0 methylation at a ribose (3'0MeG). In some embodiments, a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine (m7G). In some embodiments, a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine and a 3' 0 methylation at a ribose (m7(3'OMeG)). It will be understood that the notation used in the above paragraph, e.g., "(m27'3 O)G" or "m7(3'OMeG)", applies to other structures described herein. [0207] In some embodiments, providing an RNA with a 5'-cap disclosed herein may be achieved by in vitro transcription, in which a 5'-cap is co-transcriptionally expressed into an RNA strand, or may be attached to an RNA post-transcriptionally using capping enzymes. In some embodiments, co-transcriptional capping with a cap disclosed improves the capping efficiency of an RNA compared to co-transcriptional capping with an appropriate reference comparator. In some embodiments, improving capping efficiency can increase a translation efficiency and/or translation rate of an RNA, and/or increase expression of an encoded polypeptide. In some embodiments, alterations to polynucleotides generates a non-hydrolyzable cap structure which can, for example, prevent de-capping and increase RNA half-life.
[0208] In some embodiments, T7 RNA polymerase prefers G as the initial site. Accordingly, in some such embodiments, the present disclosure provides caps (e.g., trinucleotide and tetranucleotide caps described herein) wherein the 3'end of the trinucleotide (e.g., N2) or tetranucleotide cap (e.g., N3) is G.
[0209] In some embodiments, it will be appreciated that all compounds or structures (e.g., 5' caps) provided herein encompass the free base or salt form (e.g., an Na+ salt) comprising a suitable counterion (e.g., Na+). Compounds or structures (e.g., 5' caps) depicted as a salt also encompass the free base and include suitable counterions (e.g., Na+).
[0210] In some embodiments, a utilized 5' cap is a capO, a capl, or cap2 structure. See, e.g., Fig. 1 of
Ramanathan A., et al., and Fig. 1 of Decroly E., et al., each of which is incorporated herein by reference in its entirety. See, e.g., Fig. 1 of Ramanathan A., et al., and Fig. 1 of Decroly E., et al., each of which is incorporated herein by reference in its entirety. In some embodiments, an RNA described herein comprises a capl structure. In some embodiments, an RNA described herein comprises a cap2.
[0211] In some embodiments, an RNA described herein comprises a capO structure. In some embodiments, a capO structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m7)G). In some embodiments, such a capO structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as (m7)Gppp. In some embodiments, a capO structure comprises a guanosine nucleoside methylated at the 2'-position of the ribose of guanosine. In some embodiments, a capO structure comprises a guanosine nucleoside methylated at the 3'-position of the ribose of guanosine. In some embodiments, a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine and at the 2'-position of the ribose ((m272 °)G). In some embodiments, a guanosine nucleoside included in a 5' cap comprises methylation at the 7-position of guanine and at the 2'-position of the ribose ((m273 °)G).
[0212] In some embodiments, a capl structure comprises a guanosine nucleoside methylated at the
7-position of guanine ((m7)G) and optionally methylated at the 2' or 3' position pf the ribose, and a 2'0 methylated first nucleotide in an RNA ((m2' °)Ni). In some embodiments, a capl structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m7)G) and the 3' position of the ribose, and a 2'0 methylated first nucleotide in an RNA ((m2' °)Ni). In some embodiments, a capl structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as, e.g., ((m7)Gppp(2' °)Ni) or
(m27'3 O)Gppp(2' °)Ni), wherein Ni is as defined and described herein. In some embodiments, a capl structure comprises a second nucleotide, N2, which is at position 2 and is chosen from A, G, C, or U, e.g.,
(m7)Gppp(2' °)NipN2 or (m27'3' °)Gppp(2' 0)NipN2, wherein each of Ni and N2 is as defined and described herein. [0213] In some embodiments, a cap2 structure comprises a guanosine nucleoside methylated at the
7-position of guanine ((m7)G) and optionally methylated at the 2' or 3' position of the ribose, and a 2'0 methylated first and second nucleotides in an RNA ((m2' °)Nip(m2' °)N2). In some embodiments, a cap2 structure comprises a guanosine nucleoside methylated at the 7-position of guanine ((m7)G) and the 3' position of the ribose, and a 2'0 methylated first and second nucleotide in an RNA. In some embodiments, a cap2 structure is connected to an RNA via a 5'- to 5'-triphosphate linkage and is also referred to herein as, e.g.,
((m7)Gppp(2' 0)Nip(2' 0)N2) or (m27'3' 0)Gppp(2' 0)Nip(2, 0)N2), wherein each of Ni and N2 is as defined and described herein.
[0214] In some embodiments, a 5' cap is a dinucleotide cap structure. In some embodiments, a 5' cap is a dinucleotide cap structure comprising Ni, wherein Ni is as defined and described herein. In some embodiments, a 5' cap is a dinucleotide cap G*Ni, wherein Ni is as defined above and herein, and:
G* comprises a structure of formula (I):
Figure imgf000052_0001
or a salt thereof, wherein each R2 and R3 is -OH or -OCH3; and X is 0 or S.
[0215] In some embodiments, R2 is -OH. In some embodiments, R2 is -OCH3. In some embodiments,
R3 is -OH. In some embodiments, R3 is -OCH3. In some embodiments, R2 is -OH and R3 is -OH. In some embodiments, R2 is -OH and R3 is -CH3. In some embodiments, R2 is -CH3 and R3 is -OH. In some embodiments, R2 is -CH3 and R3 is -CH3.
[0216] In some embodiments, X is 0. In some embodiments, X is S.
[0217] In some embodiments, a 5' cap is a dinucleotide capO structure (e.g., (m7)GpppNi,
(m27'2' °)GpppNi, (m27'3' °)GpppNi, (m7)GppSpNi, (m27'2' °)GppSpNi, or (m27'3' °)GppSpNi), wherein Ni is as defined and described herein. In some embodiments, the 5' cap is a dinucleotide capO structure (e.g., (m7)GpppNi, (m27'2' °)GpppNi, (m27'3' °)GpppNi, (m7)GppSpNi, (m27'2' °)GppSpNi, or (m27'3' °)GppSpNi), wherein Ni is G. In some embodiments, the 5' cap is a dinucleotide capO structure (e.g., (m7)GpppNi, (m27'2' °)GpppNi, (m27'3' °)GpppNi, (m7)GppSpNi, (m27'2' °)GppSpNi, or (m27'3' °)GppSpNi), wherein Ni is A, U, or C. In some embodiments, a 5' cap is a dinucleotide capl structure (e.g., (m7)Gppp(m2' °)Ni, (m27'2' °)Gppp(m2' °)Ni, (m27'3'- °)Gppp(m2' °)Ni, (m7)GppSp(m2' °)Ni, (m27'2' °)GppSp(m2' °)Ni, or (m27'3' °)GppSp(m2' °)Ni), wherein Ni is as defined and described herein. In some embodiments, the 5' cap is selected from the group consisting of (m7)GpppG C'EcapO"), (m7)Gppp(m2' °)G ("Ecapl"), (m2 7-3' °)GpppG ("ARCA" or "DI"), and (m2 7-2' °)GppSpG
("beta-S-ARCA"). In some embodiments, the 5' cap is (m7)GpppG ("EcapO"), having a structure:
Figure imgf000053_0001
or a salt thereof.
[0218] In some embodiments, a 5' cap is (m7)Gppp(m2' °)G ("Ecapl"), having a structure:
Figure imgf000053_0002
or a salt thereof.
[0219] In some embodiments, a 5' cap is (m2 7-3' °)GpppG ("ARCA" or "DI"), having a structure:
Figure imgf000053_0003
or a salt thereof.
[0220] In some embodiments, a 5' cap is (m2 7'2' °)GppSpG ("beta-S-ARCA"), having a structure:
Figure imgf000053_0004
or a salt thereof.
[0221] In some embodiments, a 5' cap is a trinucleotide cap structure. In some embodiments, a 5' cap is a trinucleotide cap structure comprising NipN2, wherein Ni and N2 are as defined and described herein. In some embodiments, a 5' cap is a trinucleotide cap G*NipN2, wherein Ni and N2 are as defined above and herein, and: G* comprises a structure of formula (I):
Figure imgf000054_0001
or a salt thereof, wherein R2, R3, and X are as defined and described herein.
[0222] In some embodiments, a 5' cap is a trinucleotide capO structure (e.g. (m7)GpppNipN2, (m27 2 -
°)GpppNipN2, or (m27'3' °)GpppNipN2), wherein Ni and N2 are as defined and described herein). In some embodiments, a 5' cap is a trinucleotide capl structure (e.g., (m7)Gppp(m2' °)NipN2, (m27'2 O)Gppp(m2' °)NipN2, (m27'3' °)Gppp(m2' °)NipN2), wherein Ni and N2 are as defined and described herein. In some embodiments, a 5' cap is a trinucleotide cap2 structure (e.g., (m7)Gppp(m2' °)Nip(m2' °)N2, (m27'2' °)Gppp(m2' 0)Nip(m2' °)N2, (m27'3'- °)Gppp(m2' 0)Nip(m2' °)N2), wherein Ni and N2 are as defined and described herein. In some embodiments, a 5' cap is selected from the group consisting of (m27'3' °)Gppp(m2' °)ApG ("CleanCap AG 3' OMe", "CC413"), (m27'3'- °)Gppp(m2' °)GpG ("CleanCap GG"), (m7)Gppp(m2' °)ApG, (m7)Gppp(m2' °)GpG, (m27'3' °)Gppp(m25'2' °)ApG, and (m7)Gppp(m2' °)ApU. In some embodiments, a 5' cap is selected from the group consisting of (m27'3' °)Gppp(m2'_ °)ApG ("CleanCap AG", "CC413"), (m27-3' 0)Gppp(m2'-0)GpG ("CleanCap GG"), (m7)Gppp(m2' °)ApG, and (m2 7-3'- °)Gppp(m25'2' °)ApG, (m7)Gppp(m2' °)ApU, and (m27'3' °)Gppp(m2' °)CpG.
[0223] In some embodiments, a 5' cap is (m27'3' °)Gppp(m2' °)ApG ("CleanCap AG 3' OMe", "CC413"), having a structure:
Figure imgf000054_0002
or a salt thereof.
[0224] In some embodiments, a 5' cap is (m27'3' °)Gppp(m2' °)GpG ("CleanCap GG"), having a structure:
Figure imgf000055_0001
or a salt thereof.
[0225] In some embodiments, a 5' cap is (m7)Gppp(m2' °)ApG, having a structure:
Figure imgf000055_0002
or a salt thereof.
[0226] In some embodiments, a 5' cap is (m7)Gppp(m2' °)GpG, having a structure:
Figure imgf000055_0003
or a salt thereof.
[0227] In some embodiments, a 5' cap is (m27'3' °)Gppp(m25'2' °)ApG, having a structure:
Figure imgf000056_0001
or a salt thereof.
[0228] In some embodiments, a 5' cap is (m7)Gppp(m2' °)ApU, having a structure:
Figure imgf000056_0002
or a salt thereof.
[0229] In some embodiments, a 5' cap is (m27'3' °)Gppp(m2' °)CpG, having a structure:
Figure imgf000056_0003
or a salt thereof. [0230] In some embodiments, a 5' cap is a tetranucleotide cap structure. In some embodiments, a 5' cap is a tetranucleotide cap structure comprising NipN2pN3, wherein Ni, N2, and N3 are as defined and described herein. In some embodiments, a 5' cap is a tetranucleotide cap G*NipN2pN3, wherein Ni, N2, and N3 are as defined above and herein, and:
G* comprises a structure of formula (I):
Figure imgf000057_0002
or a salt thereof, wherein R2, R3, and X are as defined and described herein.
[0231] In some embodiments, a 5' cap is a tetranucleotide capO structure (e.g., (m7)GpppNipN2pN3,
(m2 7'2' °)GpppNipN2pN3, or (m27'3' °)GpppNiN2pN3), wherein Ni, N2, and N3 are as defined and described herein). In some embodiments, a 5' cap is a tetranucleotide Capl structure (e.g., (m7)Gppp(m2' °)NipN2pN3, (m27'2’ °)Gppp(m2' °)NipN2pN3, (m27'3' °)Gppp(m2' °)NipN2N3), wherein Ni, N2, and N3 are as defined and described herein. In some embodiments, a 5' cap is a tetranucleotide Cap2 structure (e.g., (m7)Gppp(m2' °)Nip(m2'_ °)N2pN3, (m27'2' 0)Gppp(m2' °)Nip(m2' °)N2pN3, (m27'3' 0)Gppp(m2' °)Nip(m2' °)N2pN3), wherein Ni, N2, and N3 are as defined and described herein. In some embodiments, a 5' cap is selected from the group consisting of (m27 3 > 0)Gppp(m2' °)Ap(m2' °)GpG, (m27'3' 0)Gppp(m2' °)Gp(m2' °)GpC, (m7)Gppp(m2' °)Ap(m2' °)UpA, and (m7)Gppp(m2 - °)Ap(m2' 0)GpG.
[0232] In some embodiments, a 5' cap is (m27'3' 0)Gppp(m2' °)Ap(m2' °)GpG, having a structure:
Figure imgf000057_0001
or a salt thereof.
[0233] In some embodiments, a 5' cap is (m27'3' 0)Gppp(m2' °)Gp(m2' °)GpC, having a structure:
Figure imgf000058_0001
or a salt thereof.
[0234] In some embodiments, a 5' cap is (m7)Gppp(m2' °)Ap(m2' °)UpA, having a structure:
Figure imgf000058_0002
or a salt thereof.
[0235] In some embodiments, a 5' cap is (m7)Gppp(m2' °)Ap(m2' °)GpG, having a structure:
Figure imgf000059_0001
or a salt thereof.
[0236] In some embodiments, Ni is A or an analog thereof. In some embodiments, Ni is adenosine.
In some embodiments, Ni is modified adenosine. In some embodiments, Ni is 6-methyladenosine. In some embodiments, Ni is:
Figure imgf000059_0002
wherein % represents the point of attachment to G*.
[0237] In some embodiments, N2 is U or an analog thereof. In some embodiments, N2 is a modified
U. In some embodiments, N2 is 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo-uridine), uridine 5- oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl-uridine (cm5U), 1- carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5-carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5-methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5-methylaminomethyl-uridine (mnm5U), 1-ethyl- pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5-carboxymethylaminomethyl-uridine (cmnm5U), 5- carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5-propynyl-uridine, 1-propynyl-pseudouridine, 5- taurinomethyl-uridine (Tm5U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(Tm5s2U), 1- taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m5s2U), l-methyl-4-thio-pseudouridine (m1s4ψ), 4- thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ ), 2-thio-l-methyl-pseudouridine, 1-methyl-l-deaza- pseudouridine, 2-thio-l-methyl-l-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6- dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2-methoxy- uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2-thio-pseudouridine, Nl-methyl- pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), l-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp3 i ), 5-(isopentenylaminomethyl)uridine (inm5U), 5-(isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a- thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl-uridine (m5Um), 2'-O-methyl-pseudouridine (ipm), 2-thio- 2'-O-methyl-uridine (s2Um), 5-methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O- methyl-uridine (ncm5Um), 5-carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl- uridine (m3Um), 5-(isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F- ara-uridine, 2'-F-uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(l-E-propenylamino)uridine, or any other modified uridine known in the art.
[0238] In some embodiments, N2 is of formula (II):
Figure imgf000060_0001
or a salt thereof, wherein: each = is independently a single or double bond, as allowed by valency;
Y1 is 0 or S;
Y2 is N, C, or CH;
Y3 is N, NRal, CRal, or CHRal;
Y4 is NRa2 or CHRa2; each of Ral or Ra2 is independently hydrogen or Ci-6 aliphatic;
R4 is -OH or -OMe; and
# represents the point of attachment to p of Nip.
[0239] In some embodiments, Y1 is 0. In some embodiments, Y1 is S.
[0240] In some embodiments, Y2 is N. In some embodiments, Y2 is C or CH. In some embodiments,
Y2 is C. In some embodiments, Y2 is CH.
[0241] In some embodiments, Y3 is N or CRal. In some embodiments, Y3 is N. In some embodiments,
Y3 is CRal. In some embodiments, Y3 is CH or C(CH3). . In some embodiments, Y3 is CH. In some embodiments, Y3 is C(CH3). In some embodiments, Y3 is NRal or CHRal. In some embodiments, Y3 is NH or N(CH3). In some embodiments, Y3 is NH, In some embodiments, Y3 is N(CHs). In some embodiments, Y3 is CH2 or CH(CH3). In some embodiments, Y3 is CH2. In some embodiments, Y3 is CH(CH3).
[0242] In some embodiments, Y4 is NRa2. In some embodiments, Y4 is NH or NCH3. In some embodiments, Y4 is NH. In some embodiments, Y4 is NCH3. In some embodiments, Y4 is CHRa2. In some embodiments, Y4 is CH2 or CH(CH3). In some embodiments, Y4 is CH2. In some embodiments, Y4 is CH(CH3). [0243] In some embodiments, Ral is hydrogen. In some embodiments, Ral is C1-6 aliphatic. In some embodiments, Ral is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, Ral is methyl.
[0244] In some embodiments, Ra2 is hydrogen. In some embodiments, Ra2 is C1-6 aliphatic. In some embodiments, Ra2 is methyl, ethyl, n-propyl, or isopropyl. In some embodiments, Ra2 is methyl.
[0245] In some embodiments, R4 is -OH. In some embodiments, R4 is -OMe.
[0246] In some embodiments, N2 is of formula (Ila):
Figure imgf000061_0001
or a salt thereof, wherein each of Y1, Y3, R4, and # is as defined above and described herein.
[0247] In some embodiments, N2 is of formula (lib) :
Figure imgf000061_0002
or a salt thereof, wherein each of Y1, Y3, R4, and # is as defined above and described herein.
[0248] In some embodiments, N2 is uridine, 1-methylpsuedouridine, 2-thio-uridine, or 5-methyluridine.
[0249] In some embodiments N2 is:
Figure imgf000061_0003
or a salt thereof, wherein # represents the point of attachment to p of Nip.
[0250] In some embodiments N2 is:
Figure imgf000062_0001
or a salt thereof, wherein # represents the point of attachment to p of Nip.
[0251] In some embodiments, p is -P(=O)(OH)-, or a salt thereof.
[0252] In some embodiments, a 5' cap is (m7-2' 0)Gppp(m2' °)AipU2, (m7'3' °)Gppp(m2' °)AipU2, (m7-2'-
°)Gppp(m2' °)Aip4J2, (m7'3' 0)Gppp(m2' °)Aip4J2, (m7'2' °)Gppp(m2' °)Aip(m1)4J2, (m7'3' °)Gppp(m2' °)Aip(m1)4J2, (m7'2' °)Gppp(m2' °)AipS2U2, (m7'3' °)Gppp(m2' °)AipS2U2, (m7'2' °)Gppp(m2' °)Aip(m5)U2, or (m7'3' °)Gppp(m2'_ °)Aip(m5)U2.
[0253] In some embodiments, a 5' cap is (m7'2' °)Gppp(m5'2' °)AipU2, (m7'3' °)Gppp(m5'2' °)AipU2, (m7'2' 0)Gppp(m5'2' °)A1pψ 2, (m7'3' 0)Gppp(m5'2' °)Aip4J2, (m7'2' °)Gppp(m5'2' °)Aip(m1)4J2, (m7'3' °)Gppp(m5'2'_ °)Aip(m1)ψ 2, (m7'2' °)Gppp(m5'2' °)AipS2U2, (m7'3' °)Gppp(m5'2' °)AipS2U2, (m7'2' °)Gppp(m5'2' °)Aip(m5)U2, or (m7'3' °)Gppp(m5'2' °)Aip(m5)U2.
[0254] In some embodiments a 5' cap is (m7'2' °)Gppp(m2' °)AipU2 having a structure: or a salt thereo
Figure imgf000062_0002
f.
[0255] In some embodiments, a 5' cap is (m7'3' °)Gppp(m2' °)AipU2,
Figure imgf000063_0001
or a salt thereof.
[0256] In some embodiments, a 5' cap is (m7-3'0)Gppp(m2'°)Aip4J2,
Figure imgf000063_0002
or a salt thereof.
[0257] In some embodiments, a 5' cap is (m7-2'0)Gppp(m2'°)Aip4J2,
Figure imgf000064_0001
or a salt thereof.
[0258] In some embodiments, a 5' cap is (m7'2'0)Gppp(m2'0)Aip(m1)ψ2,
Figure imgf000064_0002
or a salt thereof.
[0259] In some embodiments, a 5' cap is (m7'3'0)Gppp(m2'0)Aip(m1)ψ2,
Figure imgf000065_0001
or a salt thereof.
[0260] In some embodiments, a 5' cap is (m7'3'°)Gppp(m2'°)AipS2U2,
Figure imgf000065_0002
or a salt thereof.
[0261] In some embodiments, a 5' cap is (m7'2'°)Gppp(m2'°)AipS2U2,
Figure imgf000065_0003
or a salt thereof.
[0262] In some embodiments, a 5' cap is (m7'3' °)Gppp(m2' °)Aip(m5)U2,
Figure imgf000066_0002
or a salt thereof.
[0263] In some embodiments, a 5' cap is (m7'2' °)Gppp(m2' °)Aip(m5)U2, or a salt there
Figure imgf000066_0001
of.
[0264] In some embodiments, it will be appreciated that the disclosure of 5' caps above and herein encompasses 5' caps themselves or as part of a larger molecule (e.g., an RNA). For example, the structures drawn above encompass a 3' ether linkage to the next nucleotide or as a free -OH.
[0265] In some embodiments, it will be appreciated that any of the structures above may exist in a salt form. For example, each of the phosphate groups may be deprotonated, e.g., -0P(=0)(0 )-, and be associated with an appropriate counterion.
3. Cap Proximal Sequences
[0266] In some embodiments, a 5' UTR utilized in accordance with the present disclosure comprises a cap proximal sequence, e.g., as disclosed herein. In some embodiments, a cap proximal sequence comprises a sequence adjacent to a 5' cap. In some embodiments, a cap proximal sequence comprises nucleotides in positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide. [0267] In some embodiments, a cap structure comprises one or more polynucleotides of a cap proximal sequence. In some embodiments, a cap structure comprises an m7 Guanosine cap and nucleotide +1 (N i) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m7 Guanosine cap and nucleotide +2 (N2) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m7 Guanosine cap and nucleotides +1 and +2 (Ni and N2) of an RNA polynucleotide. In some embodiments, a cap structure comprises an m7 Guanosine cap and nucleotides +1, +2, and +3 (Ni, N2, and N3) of an RNA polynucleotide. [0268] Those skilled in the art, reading the present disclosure, will appreciate that, in some embodiments, one or more residues of a cap proximal sequence (e.g., one or more of residues +1, +2, +3, +4, and/or +5) may be included in an RNA by virtue of having been included in a cap entity (e.g., a capl or cap2 structure, etc); alternatively, in some embodiments, at least some of the residues in a cap proximal sequence may be enzymatically added (e.g., by a polymerase such as a T7 polymerase). For example, in certain exemplified embodiments where a m27'3' °Gppp(mi2' °)ApG cap is utilized, +1 (i.e., Ni) and +2 (i.e., N2) are the (mi2' °)A and G residues of the cap, and +3, +4, and +5 are added by polymerase (e.g., T7 polymerase).
[0269] In some embodiments, a 5' cap is a dinucleotide cap structure, wherein the cap proximal sequence comprises Ni of the 5' cap, where Ni is any nucleotide, e.g., A, C, G or U. In some embodiments, a 5' cap is a trinucleotide cap structure (e.g., the trinucleotide cap structures described above and herein), wherein the cap proximal sequence comprises Ni and N2 of the 5' cap, wherein Ni and N2 are independently any nucleotide, e.g., k, C, G or U. In some embodiments, a 5' cap is a tetranucleotide cap structure (e.g., the trinucleotide cap structures described above and herein), wherein the cap proximal sequence comprises Ni, N2, and N3 of the 5' cap, wherein Ni, N2, and N3 are any nucleotide, e.g., A, C, G or U.
[0270] In some embodiments, e.g., where a 5' cap is a dinucleotide cap structure, a cap proximal sequence comprises Ni of a the 5' cap, and N2, N3, N4 and N5, wherein Ni to Ns correspond to positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide. In some embodiments, e.g., where a 5' cap is a trinucleotide cap structure, a cap proximal sequence comprises Ni and N2 of a the 5' cap, and N3, N4 and Ns, wherein Ni to Ns correspond to positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide. In some embodiments, e.g., where a 5' cap is a tetranucleotide cap structure, a cap proximal sequence comprises Ni, N2, and N3 of a the 5' cap, and N4 and Ns, wherein Ni to Ns correspond to positions +1, +2, +3, +4, and/or +5 of an RNA polynucleotide.
[0271] In some embodiments, Ni is A. In some embodiments, Ni is C. In some embodiments, Ni is
G. In some embodiments, Ni is U. In some embodiments, N2 is A. In some embodiments, N2 is C. In some embodiments, N2 is G. In some embodiments, N2 is U. In some embodiments, N3 is A. In some embodiments, N3 is C. In some embodiments, N3 is G. In some embodiments, N3 is U. In some embodiments, N4 is A. In some embodiments, N4 is C. In some embodiments, N4 is G. In some embodiments, N4 is U. In some embodiments, N5 is A. In some embodiments, N5 is C. In some embodiments, Ns is G. In some embodiments, N5 is U. It will be understood that, each of the embodiments described above and herein (e.g., for Ni through N5) may be taken singly or in combination and/or may be combined with other embodiments of variables described above and herein (e.g., 5' caps).
4. 5' UTR
[0272] In some embodiments, a nucleic acid (e.g., DNA, RNA) utilized in accordance with the present disclosure comprises a 5'-UTR. In some embodiments, a 5'-UTR may comprise a plurality of distinct sequence elements; in some embodiments, such plurality may be or comprise multiple copies of one or more particular sequence elements (e.g., as may be from a particular source or otherwise known as a functional or characteristic sequence element). In some embodiments, a 5' UTR comprises multiple different sequence elements.
[0273] The term "untranslated region" or "UTR" is commonly used in the art to refer to a region in a
DNA molecule which is transcribed but is not translated into an amino acid sequence, or to the corresponding region in an RNA polynucleotide, such as an mRNA molecule. An untranslated region (UTR) can be present 5' (upstream) of an open reading frame (5'-UTR) and/or 3' (downstream) of an open reading frame (3'-UTR). As used herein, the terms "five prime untranslated region" or "5' UTR" refer to a sequence of a polyribonucleotide between the 5' end of the polyribonucleotide (e.g., a transcription start site) and a start codon of a coding region of the polyribonucleotide. In some embodiments, "5' UTR" refers to a sequence of a polyribonucleotide that begins at the 5' end of the polyribonucleotide (e.g., a transcription start site) and ends one nucleotide (nt) before a start codon (usually AUG) of a coding region of the polyribonucleotide, e.g., in its natural context. In some embodiments, a 5' UTR comprises a Kozak sequence. A 5'-UTR is downstream of the 5'-cap (if present), e.g., directly adjacent to the 5'-cap. In some embodiments, a 5' UTR disclosed herein comprises a cap proximal sequence, e.g., as defined and described herein. In some embodiments, a cap proximal sequence comprises a sequence adjacent to a 5' cap.
[0274] Exemplary 5' UTRs include a human alpha globin (hAg) 5'UTR or a fragment thereof, a TEV 5'
UTR or a fragment thereof, a HSP70 5' UTR or a fragment thereof, or a c-Jun 5' UTR or a fragment thereof. [0275] In some embodiments, an RNA disclosed herein comprises a hAg 5' UTR or a fragment thereof.
[0276] In some embodiments, an RNA disclosed herein comprises a 5' UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 5' UTR with the sequence according to SEQ ID NO: 155 (AGAATAAACTAGTATTCTTCTGGTCCCCACAGACTCAGAGAGAACCCGCCACC). In some embodiments, an RNA disclosed herein comprises a 5' UTR provided in SEQ ID NO: 155.
[0277] In some embodiments, an RNA disclosed herein comprises a 5' UTR having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a 5' UTR with the sequence AACUAGUAUUCUUCUGGUCCCCACAGACUCAGAGAGAACCCGCCACC (SEQ ID NO: 272)(hAg-Kozak/5'UTR). In some embodiments, an RNA disclosed herein comprises a 5' UTR provided in SEQ ID NO: 272.
5. PolyA Tail
[0278] In some embodiments, a polynucleotide (e.g., DNA, RNA) disclosed herein comprises a polyadenylate (polyA) sequence, e.g., as described herein. In some embodiments, a polyA sequence is situated downstream of a 3'-UTR, e.g., adjacent to a 3'-UTR.
[0279] As used herein, the term "poly(A) sequence" or "poly-A tail" refers to an uninterrupted or interrupted sequence of adenylate residues which is typically located at the 3'-end of an RNA polynucleotide. Poly(A) sequences are known to those of skill in the art and may follow the 3'-UTR in the RNAs described herein. An uninterrupted poly(A) sequence is characterized by consecutive adenylate residues. In nature, an uninterrupted poly(A) sequence is typical. In some embodiments, polynucleotides disclosed herein comprise an uninterrupted Poly(A) sequence. In some embodiments, polynucleotides disclosed herein comprise interrupted Poly(A) sequence. In some embodiments, RNAs disclosed herein can have a poly(A) sequence attached to the free 3'-end of the RNA by a template-independent RNA polymerase after transcription or a poly(A) sequence encoded by DNA and transcribed by a template-dependent RNA polymerase. [0280] It has been demonstrated that a poly(A) sequence of about 120 A nucleotides has a beneficial influence on the levels of RNA in transfected eukaryotic cells, as well as on the levels of protein that is translated from an open reading frame that is present upstream (S') of the poly(A) sequence (Holtkamp et al., 2006, Blood, vol. 108, pp. 4009-4017, which is herein incorporated by reference).
[0281] In some embodiments, a poly(A) sequence in accordance with the present disclosure is not limited to a particular length; in some embodiments, a poly(A) sequence is any length. In some embodiments, a poly(A) sequence comprises, essentially consists of, or consists of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and, in particular, about 120 A nucleotides. In this context, "essentially consists of" means that most nucleotides in the poly(A) sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% by number of nucleotides in the poly(A) sequence are A nucleotides, but permits that remaining nucleotides are nucleotides other than A nucleotides, such as U nucleotides (uridylate), G nucleotides (guanylate), or C nucleotides (cytidylate). In this context, "consists of" means that all nucleotides in the poly(A) sequence, i.e., 100% by number of nucleotides in the poly(A) sequence, are A nucleotides. The term "A nucleotide" or "A" refers to adenylate.
[0282] In some embodiments, a poly(A) sequence is attached during RNA transcription, e.g., during preparation of in vitro transcribed RNA, based on a DNA template comprising repeated dT nucleotides (deoxythymidylate) in the strand complementary to the coding strand. The DNA sequence encoding a poly(A) sequence (coding strand) is referred to as a poly(A) cassette.
[0283] In some embodiments, a poly(A) cassette present in a coding strand of DNA essentially consists of dA nucleotides, but is interrupted by a random sequence of the four nucleotides (dA, dC, dG, and dT). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length. Such a cassette is disclosed in WO 2016/005324 Al, hereby incorporated by reference. Any poly(A) cassette disclosed in WO 2016/005324 Al may be used in accordance with the present disclosure. A poly(A) cassette that essentially consists of dA nucleotides, but is interrupted by a random sequence having an equal distribution of the four nucleotides (dA, dC, dG, dT) and having a length of e.g., 5 to 50 nucleotides shows, on a DNA level, constant propagation of plasmid DNA in £ co// and is still associated, on an RNA level, with the beneficial properties with respect to supporting RNA stability and translational efficiency is encompassed. In some embodiments, a poly(A) sequence contained in an RNA polynucleotide described herein essentially consists of A nucleotides, but is interrupted by a random sequence of the four nucleotides (A, C, G, U). Such random sequence may be 5 to 50, 10 to 30, or 10 to 20 nucleotides in length.
[0284] In some embodiments, no nucleotides other than A nucleotides flank a poly(A) sequence at its
3'-end, i.e., the poly(A) sequence is not masked or followed at its 3'-end by a nucleotide other than A.
[0285] In some embodiments, a poly(A) sequence may comprise at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, a poly(A) sequence may essentially consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, a poly(A) sequence may consist of at least 20, at least 30, at least 40, at least 80, or at least 100 and up to 500, up to 400, up to 300, up to 200, or up to 150 nucleotides. In some embodiments, a poly(A) sequence comprises at least 100 nucleotides. In some embodiments, the poly(A) sequence comprises about 150 nucleotides. In some embodiments, a poly(A) sequence comprises about 120 nucleotides. [0286] In some embodiments, a poly A tail comprises a specific number of adenosines, such as about
50 or more, about 60 or more, about 70 or more, about 80 or more, about 90 or more, about 100 or more, about 120, or about 150 or about 200. In some embodiments a poly A tail of a string construct may comprise 200 A residues or less. In some embodiments, a poly A tail of a string construct may comprise about 200 A residues. In some embodiments, a poly A tail of a string construct may comprise 180 A residues or less. In some embodiments, a poly A tail of a string construct may comprise about 180 A residues. In some embodiments, a poly A tail may comprise 150 residues or less.
[0287] In some embodiments, a poly(A) tail comprises a plurality of A residues interrupted by a linker.
In some embodiments, a linker comprises the nucleotide sequence GCATATGAC (SEQ ID NO: 156).
[0288] In some embodiments, a polyribonucleotide of the present disclosure comprises a poly(A) sequence comprising the nucleotide sequence of SEQ ID NO: 268, or a nucleotide sequence having at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, or 80% identity to the nucleotide sequence of SEQ ID NO: 268.
6. 3' UTR
[0289] In some embodiments, a polyribonucleotide utilized in accordance with the present disclosure comprises a 3'-UTR. As used herein, the terms "three prime untranslated region," "3' untranslated region," or "3' UTR" refer to a sequence of an mRNA molecule that begins following a stop codon of a coding region of an open reading frame sequence. In some embodiments, a 3' UTR begins immediately after a stop codon of a coding region of an open reading frame sequence, e.g., in its natural context. In other embodiments, a 3' UTR does not begin immediately after the stop codon of the coding region of an open reading frame sequence, e.g., in its natural context. The term "3'-UTR" preferably does not include the poly(A) sequence. Thus, a 3'-UTR is upstream of the poly(A) sequence (if present), e.g., directly adjacent to the poly(A) sequence.
[0290] In some embodiments, an RNA disclosed herein comprises a 3' UTR comprising an F element and/or an I element. In some embodiments, a 3' UTR or a proximal sequence thereto comprises a restriction site. In some embodiments, a restriction site is a BamHI site. In some embodiments, a restriction site is a Xhol site.
[0291] In some embodiments, an RNA construct comprises an F element. In some embodiments, a F element sequence is a 3'-UTR of amino-terminal enhancer of split (AES).
[0292] In some embodiments, an RNA disclosed herein comprises a 3' UTR.
[0293] In some embodiments, a 3'UTR is an FI element as described in W02017/060314, which is herein incorporated by reference in its entirety.
7. Multimerization Elements
[0294] In some embodiments, an mpox antigen utilized as described herein includes a multimerization element (e.g., a heterologous multimerization element). In some embodiments, a heterologous multimerization element comprises a dimerization, trimerization or tetramerization element.
[0295] In some embodiments, a multimerization element is one described in W02017/081082 (e.g., sequences of SEQ ID NOs: 1116-1167 of W02017/081082, or fragments or variants thereof).
[0296] Exemplary trimerization and tetramerization elements include, but are not limited to, engineered leucine zippers, fibritin foldon domain from enterobacteria phage T4, GCN4pll, GCN4-pll, and p53. [0297] In various embodiments, an antigen construct of the present disclosure includes, and/or a polyribonucleotide of the present disclosure encodes, an antigen operably linked with a multimerization element such as a foldon domain. In various embodiments an antigen construct of the present disclosure includes, and/or a polyribonucleotide of the present disclosure encodes, an antigen operably linked with a foldon domain according to SEQ ID NO: 256 and/or encoded by a sequence according to SEQ ID NO: 257.
[0298] In some embodiments, a provided antigen is able to form a trimeric complex. For example, a utilized antigen may comprise a domain allowing formation of a multimeric complex, such as for example a trimeric complex of an amino acid sequence comprising an orthopoxvirus (e.g., mpox) antigen as described herein. In some embodiments, a domain allowing formation of a multimeric complex comprises a trimerization domain, for example, a trimerization domain as described herein.
[0299] In some embodiments, an orthopoxvirus (e.g., mpox) antigen can be modified by addition of a
T4-fibritin-derived "foldon" trimerization domain, for example, to increase its immunogenicity.
8. Membrane Association Elements
[0300] In some embodiments, an orthopoxvirus (e.g., mpox) antigen as described herein includes a membrane association element (e.g., a heterologous membrane association element), such as a transmembrane domain.
[0301] A transmembrane domain can be N-terminal, C-terminal, or internal to an antigen. A coding sequence of a transmembrane element is typically placed in frame (i.e., in the same reading frame), 5', 3', or internal to coding sequences (e.g., mpox antigen coding sequences) with which it is to be linked.
[0302] In some embodiments, a transmembrane domain comprises or is a transmembrane domain of
Hemagglutinin (HA) of Influenza virus, Env of HIV-1, equine infectious anaemia virus (EIAV), murine leukaemia virus (MLV), mouse mammary tumor virus, G protein of vesicular stomatitis virus (VSV), Rabies virus, or a seven transmembrane domain receptor.
[0303] In some embodiments, an antigen construct of the present disclosure includes, and/or a polyribonucleotide of the present disclosure encodes, an antigen operably linked with a transmembrane domain. In various embodiments an antigen construct of the present disclosure includes, and/or a polyribonucleotide of the present disclosure encodes, an antigen operably linked with a HSV-1 gD transmembrane domain (TM) domain according to SEQ ID NO: 254 and/or encoded by a sequence according to SEQ ID NO: 255.
B. RNA Formats
[0304] At least three distinct formats useful for RNA compositions (e.g., pharmaceutical compositions) have been developed, namely non-modified uridine containing mRNA (uRNA), nucleoside-modified mRNA (modRNA), and self-amplifying mRNA (saRNA). Each of these platforms displays unique features. In general, in all three formats, RNA is capped, contains open reading frames (ORFs) flanked by untranslated regions (UTR), and have a polyA-tail at the 3' end. An ORF of an uRNA and modRNA vector encodes an antigen or fragment thereof. An saRNA has multiple ORFs.
[0305] In some embodiments, an RNA described herein may have modified nucleosides. In some embodiments, the RNA comprises a modified nucleoside in place of at least one (e.g., every) uridine.
[0306] The term "uracil," as used herein, describes one of the nucleobases that can occur in the nucleic acid of RNA. The structure of uracil is:
Figure imgf000071_0001
[0307] The term "uridine," as used herein, describes one of the nucleosides that can occur in RNA.
The structure of uridine is:
Figure imgf000072_0001
[0308] UTP (uridine 5'-triphosphate) has the following structure:
Figure imgf000072_0002
[0309] Pseudo-UTP (pseudouridine 5'-triphosphate) has the following structure:
Figure imgf000072_0003
[0310] "Pseudouridine" is one example of a modified nucleoside that is an isomer of uridine, where the uracil is attached to the pentose ring via a carbon-carbon bond instead of a nitrogen-carbon glycosidic bond.
[0311] Another exemplary modified nucleoside is Nl-methyl-pseudouridine (mlQJ), which has the structure:
Figure imgf000072_0004
[0312] Nl-methyl-pseudo-UTP has the following structure:
Figure imgf000073_0001
[0313] Another exemplary modified nucleoside is 5-methyl-uridine (m5U), which has the structure:
Figure imgf000073_0002
[0314] In some embodiments, one or more uridine in an RNA described herein is replaced by a modified nucleoside. In some embodiments, the modified nucleoside is a modified uridine.
[0315] In some embodiments, an RNA described herein comprises a modified nucleoside in place of at least one uridine. In some embodiments, an RNA described herein comprises a modified nucleoside in place of each uridine.
[0316] In some embodiments, a modified nucleoside is independently selected from pseudouridine
(ip), Nl-methyl-pseudouridine (mlip), and 5-methyl-uridine (m5U). In some embodiments, a modified nucleoside comprises pseudouridine (ip). In some embodiments, a modified nucleoside comprises Nl-methyl-pseudouridine (mlip). In some embodiments, a modified nucleoside comprises 5-methyl-uridine (m5U). In some embodiments, RNA may comprise more than one type of modified nucleoside, and the modified nucleosides are independently selected from pseudouridine (ip), Nl-methyl-pseudouridine (mlip), and 5-methyl-uridine (m5U). In some embodiments, modified nucleosides comprise pseudouridine (ip) and Nl-methyl-pseudouridine (mlip). In some embodiments, modified nucleosides comprise pseudouridine (ip) and 5-methyl-uridine (m5U). In some embodiments, modified nucleosides comprise Nl-methyl-pseudouridine (mlip) and 5-methyl-uridine (m5U). In some embodiments, modified nucleosides comprise pseudouridine (ip), Nl-methyl-pseudouridine (mlip), and 5- methyl-uridine (m5U).
[0317] In some embodiments, a modified nucleoside replacing one or more, e.g., all, uridine in the
RNA may be any one or more of 3-methyl-uridine (m3U), 5-methoxy-uridine (mo5U), 5-aza-uridine, 6-aza- uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s2U), 4-thio-uridine (s4U), 4-thio-pseudouridine, 2-thio- pseudouridine, 5-hydroxy-uridine (ho5U), 5-aminoallyl-uridine, 5-halo-uridine (e.g., 5-iodo-uridine or 5-bromo- uridine), uridine 5-oxyacetic acid (cmo5U), uridine 5-oxyacetic acid methyl ester (mcmo5U), 5-carboxymethyl- uridine (cm5U), 1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm5U), 5- carboxyhydroxymethyl-uridine methyl ester (mchm5U), 5-methoxycarbonylmethyl-uridine (mcm5U), 5- methoxycarbonylmethyl-2-thio-uridine (mcm5s2U), 5-aminomethyl-2-thio-uridine (nm5s2U), 5- methylaminomethyl-uridine (mnm5U), 1-ethyl-pseudouridine, 5-methylaminomethyl-2-thio-uridine (mnm5s2U), 5-methylaminomethyl-2-seleno-uridine (mnm5se2U), 5-carbamoylmethyl-uridine (ncm5U), 5- carboxymethylaminomethyl-uridine (cmnm5U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm5s2U), 5- propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine (Tm5U), 1-taurinomethyl-pseudouridine, 5- taurinomethyl-2-thio-uridine(Tm5s2U), l-taurinomethyl-4-thio-pseudouridine), 5-methyl-2-thio-uridine (m5s2U), l-methyl-4-thio-pseudouridine (mls4ψ ), 4-thio-l-methyl-pseudouridine, 3-methyl-pseudouridine (m3ψ ), 2-thio- 1-methyl-pseudouridine, 1-methyl-l-deaza-pseudouridine, 2-thio-l-methyl-l-deaza-pseudouridine, dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine, 5-methyl-dihydrouridine (m5D), 2-thio-dihydrouridine, 2-thio- dihydropseudouridine, 2-methoxy-uridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, 4-methoxy-2- thio-pseudouridine, Nl-methyl-pseudouridine, 3-(3-amino-3-carboxypropyl)uridine (acp3U), l-methyl-3-(3- amino-3-carboxypropyl)pseudouridine (acp3 ψ ), 5-(isopentenylaminomethyl)uridine (inm5U), 5- (isopentenylaminomethyl)-2-thio-uridine (inm5s2U), a-thio-uridine, 2'-O-methyl-uridine (Um), 5,2'-O-dimethyl- uridine (m5Um), 2'-O-methyl-pseudouridine (ψ m), 2-thio-2'-O-methyl-uridine (s2Um), 5- methoxycarbonylmethyl-2'-O-methyl-uridine (mcm5Um), 5-carbamoylmethyl-2'-O-methyl-uridine (ncm5Um), 5- carboxymethylaminomethyl-2'-O-methyl-uridine (cmnm5Um), 3,2'-O-dimethyl-uridine (m3Um), 5- (isopentenylaminomethyl)-2'-O-methyl-uridine (inm5Um), 1-thio-uridine, deoxythymidine, 2'-F-ara-uridine, 2'-F- uridine, 2'-OH-ara-uridine, 5-(2-carbomethoxyvinyl) uridine, 5-[3-(l-E-propenylamino)uridine, or any other modified uridine known in the art.
[0318] In some embodiments, an RNA of the present disclosure comprises other modified nucleosides or comprises further modified nucleosides, e.g., modified cytidine. For example, in some embodiments, in an RNA of the present disclosure 5-methylcytidine is substituted partially or completely, preferably completely, for cytidine. In some embodiments, an RNA of the present disclosure comprises 5-methylcytidine and one or more selected from pseudouridine (ψ ), Nl-methyl-pseudouridine (m1ψ ), and 5-methyl-uridine (m5U). In some embodiments, an RNA of the present disclosure comprises 5-methylcytidine and Nl-methyl-pseudouridine (mlip). In some embodiments, an RNA of the present disclosure comprises 5-methylcytidine in place of each cytidine and Nl-methyl-pseudouridine (m1ψ ) in place of each uridine.
[0319] In some embodiments of the present disclosure, an RNA is "replicon RNA" or simply a
"replicon," in particular "self-replicating RNA" or "self-amplifying RNA." In one particularly preferred embodiment, the replicon or self-replicating RNA is derived from or comprises elements derived from a single- stranded (ss) RNA virus, in particular a positive-stranded ssRNA virus, such as an alphavirus. Alphaviruses are typical representatives of positive-stranded RNA viruses. Alphaviruses replicate in the cytoplasm of infected cells (for a review of the alphaviral life cycle see Jose et al., Future Microbiol., 2009, vol. 4, pp. 837-856, which is incorporated herein by reference in its entirety). The total genome length of many alphaviruses typically ranges between 11,000 and 12,000 nucleotides, and the genomic RNA typically has a 5'-cap, and a 3' poly(A) tail. The genome of alphaviruses encodes non-structural proteins (involved in transcription, modification and replication of viral RNA and in protein modification) and structural proteins (forming the virus particle). There are typically two open reading frames (ORFs) in the genome. The four non-structural proteins (nsPl-nsP4) are typically encoded together by a first ORF beginning near the 5' terminus of the genome, while alphavirus structural proteins are encoded together by a second ORF which is found downstream of the first ORF and extends near the 3' terminus of the genome. Typically, the first ORF is larger than the second ORF, the ratio being roughly 2: 1. In cells infected by an alphavirus, only the nucleic acid sequence encoding non-structural proteins is translated from the genomic RNA, while the genetic information encoding structural proteins is translatable from a subgenomic transcript, which is an RNA molecule that resembles eukaryotic messenger RNA (mRNA; Gould et al., 2010, Antiviral Res., vol. 87 pp. 111-124, herein incorporated by reference in its entirety). Following infection, i.e., at early stages of the viral life cycle, the (+) stranded genomic RNA directly acts like a messenger RNA for the translation of the open reading frame encoding the non-structural poly-protein (nsP1234).
[0320] Alphavirus-derived vectors have been proposed for delivery of foreign genetic information into target cells or target organisms. In simple approaches, a first ORF encodes an alphavirus-derived RNA- dependent RNA polymerase (replicase), which upon translation mediates self-amplification of the RNA. A second ORF encoding alphaviral structural proteins is replaced by an open reading frame encoding a protein of interest, e.g., an antigen or fragment thereof. Alphavirus-based trans-replication systems rely on alphavirus nucleotide sequence elements on two separate nucleic acid molecules: one nucleic acid molecule encodes a viral replicase, and the other nucleic acid molecule is capable of being replicated by said replicase in trans (hence the designation trans-replication system). Trans-replication requires the presence of both these nucleic acid molecules in a given host cell. The nucleic acid molecule capable of being replicated by the replicase in trans must comprise certain alphaviral sequence elements to allow for recognition and RNA synthesis by the alphaviral replicase.
[0321] Features of a non-modified uridine platform may include, for example, one or more of an intrinsic adjuvant effect, good tolerability, and improved safety. Features of modified uridine (e.g., pseudouridine) platform may include a reduced adjuvant effect, blunted immune innate immune sensor activating capacity, good tolerability and improved safety. Features of a self-amplifying platform may include, for example, long duration of protein expression, good tolerability and safety, and a higher likelihood for efficacy with a very low vaccine dose.
[0322] The present disclosure provides particular RNA constructs optimized, for example, for improved manufacturability, encapsulation, expression level (and/or timing), etc. Certain components are discussed below, and certain preferred embodiments are exemplified herein.
C. Codon Optimization and GC Enrichment
[0323] As used herein, the term "codon-optimized" refers to alteration of codons in a coding region of a nucleic acid molecule (e.g., a polyribonucleotide) to reflect the typical codon usage of a host organism (e.g., a subject receiving a nucleic acid molecule (e.g., a polyribonucleotide)) without preferably altering the amino acid sequence encoded by the nucleic acid molecule. Within the context of the present disclosure, in some embodiments, coding regions are codon-optimized for optimal expression in a subject to be treated using the RNA molecules described herein. In some embodiments, codon-optimization may be performed such that codons for which frequently occurring tRNAs are available are inserted in place of "rare codons." In some embodiments, codon-optimization may include increasing guanosine/cytosine (G/C) content of a coding region of RNA described herein as compared to the G/C content of the corresponding coding sequence of a wild-type RNA, wherein the amino acid sequence encoded by the RNA is preferably not modified compared to the amino acid sequence. [0324] In some embodiments, a coding sequence (also referred to as a "coding region") is codon optimized for expression in the subject to whom a composition (e.g., a pharmaceutical composition) is to be administered (e.g., a human). Thus, in some embodiments, sequences in such a polynucleotide (e.g., a polyribonucleotide) may differ from wild type sequences encoding the relevant antigen or fragment or epitope thereof, even when the amino acid sequence of the antigen or fragment or epitope thereof is wild type.
[0325] In some embodiments, a coding sequence is codon optimized for expression in a relevant subject (e.g., a human), and even, in some cases, for expression in a particular cell or tissue.
[0326] Various species exhibit particular bias for certain codons of a particular amino acid. Without wishing to be bound by any one theory, codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell may generally be a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes may be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are available, for example, at the "Codon Usage Database" available at www.kazusa.orjp/codon/ and these tables may be adapted in a number of ways. Computer algorithms for codon optimizing a particular sequence for expression in a particular subject or its cells are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available.
[0327] In some embodiments, a polynucleotide (e.g., a polyribonucleotide) of the present disclosure is codon optimized, wherein the codons in the polynucleotide (e.g., the polyribonucleotide) are adapted to human codon usage (herein referred to as "human codon optimized polynucleotide"). Codons encoding the same amino acid occur at different frequencies in a subject, e.g., a human. Accordingly, in some embodiments, the coding sequence of a polynucleotide of the present disclosure is modified such that the frequency of the codons encoding the same amino acid corresponds to the naturally occurring frequency of that codon according to the human codon usage, e.g., as shown in Table 3. For example, in the case of the amino acid Ala, the wild type coding sequence is preferably adapted in a way that the codon "GCC" is used with a frequency of 0.40, the codon "GCT" is used with a frequency of 0.28, the codon "GCA" is used with a frequency of 0.22 and the codon "GCG" is used with 30 a frequency of 0.10 etc. (see Table 3). Accordingly, in some embodiments, such a procedure (as exemplified for Ala) is applied for each amino acid encoded by the coding sequence of a polynucleotide to obtain sequences adapted to human codon usage.
Table 3: Human codon usage table with frequencies indicated for each amino acid.
Figure imgf000076_0001
Figure imgf000077_0001
[0328] Certain strategies for codon optimization and/or G/C enrichment for human expression are described in W02002/098443, which is incorporated by reference herein in its entirety. In some embodiments, a coding sequence may be optimized using a multiparametric optimization strategy. In some embodiments, optimization parameters may include parameters that influence protein expression, which can be, for example, impacted on a transcription level, an mRNA level, and/or a translational level. In some embodiments, exemplary optimization parameters include, but are not limited to transcription-level parameters (including, e.g., GC content, consensus splice sites, cryptic splice sites, SD sequences, TATA boxes, termination signals, artificial recombination sites, and combinations thereof); mRNA-level parameters (including, e.g., RNA instability motifs, ribosomal entry sites, repetitive sequences, and combinations thereof); translation-level parameters (including, e.g., codon usage, premature poly(A) sites, ribosomal entry sites, secondary structures, and combinations thereof); or combinations thereof. In some embodiments, a coding sequence may be optimized by a GeneOptimizer algorithm as described in Fath et al. "Multiparameter RNA and Codon Optimization: A Standardized Tool to Assess and Enhance Autologous Mammalian Gene Expression" PLoS ONE^ySy. el7596; Rabb et al., "The GeneOptimizer Algorithm: using a sliding window approach to cope with the vast sequence space in multiparameter DNA sequence optimization" Systems and Synthetic Biology (2010) 4:215-225; and Graft et al. "Codon-optimized genes that enable increased heterologous expression in mammalian cells and elicit efficient immune responses in mice after vaccination of naked DNA" Methods Moi Med (2004) 94: 197-210, the entire content of each of which is incorporated herein for the purposes described herein. In some embodiments, a coding sequence may be optimized by Eurofins' adaption and optimization algorithm "GENEius" as described in Eurofins' Application Notes: Eurofins' adaption and optimization software "GENEius" in comparison to other optimization algorithms, the entire content of which is incorporated by reference for the purposes described herein.
[0329] In some embodiments, a coding sequence utilized in accordance with the present disclosure has G/C content of which increased compared to a wild type coding sequence.
[0330] Without wishing to be bound by any particular theory, it is proposed that GC enrichment may improve translation of a payload sequence. Typically, sequences having an increased G (guanosine)/C (cytidine) content are more stable than sequences having an increased A (adenosine)/U (uridine) content. In respect to the fact that several codons code for one and the same amino acid (so-called degeneration of the genetic code), the most favorable codons for the stability can be determined (so-called alternative codon usage). Depending on the amino acid to be encoded by a polyribonucleotide, there are various possibilities for modification of the ribonucleic acid sequence, compared to its wild-type sequence. In particular, codons which contain A and/or U nucleosides can be modified by substituting these codons by other codons, which code for the same amino acids but contain no A and/or U or contain a lower content of A and/or U nucleosides.
[0331] In some embodiments, G/C content of a coding region of a polyribonucleotide described herein is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, or even more compared to the G/C content of the coding region prior to codon optimization, e.g., of the wild-type RNA. In some embodiments, G/C content of a coding region of a polyribonucleotide described herein is decreased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, or even more compared to the G/C content of the coding region prior to codon optimization, e.g., of the wild-type RNA. [0332] In some embodiments, stability and translation efficiency of a polyribonucleotide may incorporate one or more elements established to contribute to stability and/or translation efficiency of the polyribonucleotide; exemplary such elements are described, for example, in PCT/EP2006/009448 incorporated herein by reference. In some embodiments, to increase expression of a polyribonucleotide used according to the present disclosure, a polyribonucleotide may be modified within the coding region, i.e., the sequence encoding the expressed peptide or protein, without altering the sequence of the expressed peptide or protein, for example so as to increase the GC-content to increase mRNA stability and/or to perform a codon optimization and, thus, enhance translation in cells.
D. Exemplary Polyribonucleotide Sequences
[0333] The present disclosure includes certain exemplary antigen constructs and polyribonucleotides useful, e.g., in vaccination against orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) and/or treatment of orthopoxvirus infection, that encode and/or express one or more mpox antigens. In some embodiments, a polyribonucleotide, as described herein has one of the following structures: cap-hAg-Kozak-Antigen-FI-A30L70 cap-hAg-Kozak-sec-Antigen-FI-A30L70 where cap refers to a 5' cap as described above; hAg-Kozak refers to a 5' UTR human alpha-globin; sec refers to a secretory signal; Antigen refers to a nucleotide sequence comprising a sequence that encodes an mpox antigen described herein; FI refers to a 3'-UTR as described above, and A30L70 refers to a polyA sequence. In some embodiments, hAg 5' UTR comprises a nucleotide sequence of SEQ ID NO: 155. In some embodiments, A30L70 comprises 30 adenine nucleotides followed by 70 adenine nucleotides, wherein the 30 adenine nucleotides and 70 adenine nucleotides are separated by a linker sequence.
1. Exemplary B Cell Antigen Polyribonucleotide Sequences
[0334] The present disclosure includes certain exemplary antigen constructs and polyribonucleotides useful, e.g., in vaccination against orthopox virus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus), that encode and/or express one or more antigens according to Table 1 or antigenic fragments thereof. In some embodiments, a polyribonucleotide of the present disclosure encodes a wild-type antigen sequence. In some embodiments, a polyribonucleotide of the present disclosure encodes an orthopoxvirus antigen comprising an amino acid sequence at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to an orthopoxvirus antigen amino acid sequence in Table 1. In some embodiments, a polyribonucleotide of the present disclosure encodes an antigen polypeptide operably linked to an N-terminal viral secretory signal. Without wishing to be bound by any particular theory, inclusion of a viral secretory signal can be useful, e.g., because mpox antigens do not naturally include conventional secretory signals and/or because inclusion of the viral secretory signal may allow for enhanced surface expression of the operably linked antigen on vaccinated cells.
[0335] Exemplary antigens of the present disclosure (e.g., H3L, and B6R or fragments thereof) can further include substitution of unpaired cysteine residues present in corresponding reference sequences. Without wishing to be bound by any particular scientific theory, the present disclosure includes that such cysteines, if left unpaired, carry a high risk of causing protein misfolding and/or aggregation and that this risk is mitigated by alanine substitutions. Exemplary substitutions can include positions C71A and/or C72A of A29L, C140A of B6R, and/or C86A and/or C90A of H3L.
[0336] For the avoidance of doubt, the present disclosure includes exemplary polypeptide sequences and polyribonucleic acid sequences as described herein and/or as set forth in sequence identification numbers of the present disclosure, as well as sequences having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto. [0337] In some embodiments, a polyribonucleotide of the present disclosure encodes a wild-type MIR polypeptide. In some embodiments, a wild-type MIR polypeptide has or includes a sequence according to SEQ ID NO: 158 and/or the polyribonucleotide has or includes a sequence according to SEQ ID NO: 159. In some embodiments, a polyribonucleotide of the present disclosure encodes a wild-type MIR polypeptide operably linked with a secretory signal such as an HSV/gD secretory signal (HSV/gDsec) according to SEQ ID NO: 160, optionally encoded by a sequence according to SEQ ID NO: 161. In some embodiments, a wild-type MIR polypeptide operably linked with an HSV/gD secretory signal has or includes a sequence according to SEQ ID NO: 162 and/or the encoding polyribonucleotide has or includes a sequence according to SEQ ID NO: 162.
[0338] In some embodiments, a polyribonucleotide of the present disclosure encodes a wild-type MIR polypeptide. In some embodiments, a wild-type MIR polypeptide has or includes a sequence according to SEQ ID NO: 258 and/or the polyribonucleotide encoding the wild-type MIR polypeptide has or includes a sequence according to SEQ ID NO: 259. In some embodiments, a polyribonucleotide of the present disclosure encodes a wild-type MIR polypeptide operably linked with a secretory signal such as an HSV/gD secretory signal (HSV/gDsec) according to SEQ ID NO: 160, optionally encoded by a sequence according to SEQ ID NO: 161. [0339] In some embodiments, a polyribonucleotide of the present disclosure encodes a soluble form of the ectodomain of A35R. Without wishing to be bound by any particular scientific theory, a soluble form of the ectodomain of A35R would function independently of membrane insertion, reducing the potential need or requirement for membrane insertion of an A35R antigen or fragment thereof. The A35R ectodomain disclosed herein can have or include a sequence according to SEQ ID NO: 174. The boundaries of the ectodomain were informed by two X-ray crystallography studies defining the structure of this region of the protein. The present inventors selected amino acids 89-181 for use in this design at least in part because the selected amino acids span the resolved region of the protein elucidated by these studies. A35R forms a dimer that is partially dependent on a disulfide bond between residues not included in this ectodomain. To compensate for the absence of this disulfide, the present inventors engineered a construct that includes two copies of the ectodomain sufficient to form the dimer with a linker between them. Without wishing to be limited or bound by any particular scientific theory, linker size (10 amino acids) was selected based on the measured distance between the C-terminus of one ectodomain and the N-terminus of its binding partner in the crystal structure of the ectodomain, while those of skill in the art will appreciate that the linker could be larger or smaller, and any linker disclosed herein could be used.
[0340] In some embodiments, a polyribonucleotide of the present disclosure encodes a wild-type
A35R polypeptide. In some embodiments, a wild-type A35R polypeptide has or includes a sequence according to SEQ ID NO: 172 and/or the polyribonucleotide encoding said A35R polypeptide has or includes a sequence according to SEQ ID NO: 173. In some embodiments, a polyribonucleotide of the present disclosure encodes a wild-type A35R ectodomain (ECD) fragment that has or includes a sequence according to SEQ ID NO: 174 and/or the encoding polyribonucleotide has or includes a sequence according to SEQ ID NO: 175. In some embodiments, a polyribonucleotide of the present disclosure encodes a first wild-type A35R ECD fragment and a second wild-type A35R ECD fragment, where the first wild-type A35R ECD fragment has or includes a sequence according to SEQ ID NO: 174 and/or is encoded by a sequence according to SEQ ID NO: 175 and the second wild-type A35R ECD fragment has or includes a sequence according to SEQ ID NO: 174 and/or is encoded by a sequence according to SEQ ID NO: 175, optionally wherein the first wild-type A35R fragment and the second wild-type A35R fragment are operably linked via linker (e.g., a linker according to SEQ ID NO: 176 and/or encoded by SEQ ID NO: 177). In some embodiments, a polyribonucleotide of the present disclosure encodes a first wild-type A35R fragment and a second wild-type A35R fragment, where the first wild-type A35R fragment and the second wild-type A35R fragment are operably linked by a linker, and where the first and second wildtype A35R fragments are operably linked with a secretory signal such as an HSV/gD secretory signal (HSV/gDsec) according to SEQ ID NO: 160, optionally encoded by a sequence according to SEQ ID NO: 161. In certain such embodiments, a polypeptide encoded by a polyribonucleotide has or includes a sequence according to SEQ ID NO: 178 and/or the encoding polyribonucleotide has or includes a sequence according to SEQ ID NO: 179. [0341] In some embodiments, a polyribonucleotide of the present disclosure encodes a wild-type B6R polypeptide. In some embodiments, a wild-type B6R polypeptide has or includes a sequence according to SEQ ID NO: 180 and/or the polyribonucleotide encoding said B6R polypeptide has or includes a sequence according to SEQ ID NO: 181. In some embodiments, a polyribonucleotide of the present disclosure encodes an B6R polypeptide that includes a substitution of C to A at position 140 corresponding to SEQ ID NO: 182 (substitution C140A as compared to a corresponding reference sequence). In some embodiments, a B6R polypeptide including a C140A substitution has or includes a sequence according to SEQ ID NO: 182 and/or the encoding polyribonucleotide has or includes a sequence according to SEQ ID NO: 183.
[0342] In some embodiments, a polyribonucleotide of the present disclosure encodes a wild-type H3L polypeptide. In some embodiments, a wild-type H3L polypeptide has or includes a sequence according to SEQ ID NO: 184 and/or the polyribonucleotide encoding said H3L polypeptide has or includes a sequence according to SEQ ID NO: 185. In some embodiments, a polyribonucleotide of the present disclosure encodes a wild-type H3L polypeptide operably linked with a secretory signal such as an HSV/gD secretory signal (HSV/gDsec) according to SEQ ID NO: 160, optionally encoded by a sequence according to SEQ ID NO: 161. In some embodiments, a wild-type H3L polypeptide operably linked with an HSV/gD secretory signal has or includes a sequence according to SEQ ID NO: 186 and/or the encoding polyribonucleotide has or includes a sequence according to SEQ ID NO: 187.
[0343] In some embodiments, a polyribonucleotide of the present disclosure encodes an H3L polypeptide that includes a substitution of C to A at positions 86 and 90 corresponding to SEQ ID NO: 188 (substitutions C86A and C90A as compared to a corresponding reference sequence). In some embodiments, an H3L polypeptide including C86A and C90A substitutions has or includes a sequence according to SEQ ID NO: 188 and/or the polyribonucleotide encoding said H3L polypeptide has or includes a sequence according to SEQ ID NO: 189. In some embodiments, a polyribonucleotide of the present disclosure encodes an H3L polypeptide including C86A and C90A substitutions operably linked with a secretory signal such as an HSV/gD secretory signal (HSV/gDsec) according to SEQ ID NO: 160, optionally encoded by a sequence according to SEQ ID NO: 161. In some embodiments, an H3L polypeptide including C86A and C90A substitutions operably linked with an HSV/gD secretory signal sequence has or includes a sequence according to SEQ ID NO: 190 and/or the encoding polyribonucleotide has or includes a sequence according to SEQ ID NO: 191. [0344] In some embodiments, a polyribonucleotide of the present disclosure encodes a wild-type H3L polypeptide. In some embodiments, a wild-type H3L polypeptide has or includes a sequence according to SEQ ID NO: 260 and/or the polyribonucleotide encoding said H3L polypeptide has or includes a sequence according to SEQ ID NO: 261. In some embodiments, a polyribonucleotide of the present disclosure encodes a wild-type H3L polypeptide operably linked with a secretory signal such as an HSV/gD secretory sequence (HSV/gDsec) according to SEQ ID NO: 160, optionally encoded by a sequence according to SEQ ID NO: 161.
[0345] In some embodiments, a polyribonucleotide of the present disclosure encodes an H3L polypeptide that includes a substitution of C to A at positions 86 and 90 corresponding to SEQ ID NO: 262 (substitutions C86A and C90A as compared to a corresponding reference sequence). In some embodiments, an H3L polypeptide including C86A and C90A substitutions has or includes a sequence according to SEQ ID NO: 262 and/or the polyribonucleotide encoding said H3L polypeptide has or includes a sequence according to SEQ ID NO: 263. In some embodiments, a polyribonucleotide of the present disclosure encodes an H3L polypeptide including C86A and C90A substitutions operably linked with a secretory signal such as an HSV/gD secretory sequence (HSV/gDsec) according to SEQ ID NO: 160, optionally encoded by a sequence according to SEQ ID NO: 161.
III. RNA Delivery Technologies
[0346] Provided polyribonucleotides may be delivered for therapeutic applications described herein using any appropriate methods known in the art, including, e.g., delivery as naked polyribonucleotides (e.g., RNAs), or delivery mediated by viral and/or non-viral vectors, polymer-based vectors, lipid-based vectors, nanoparticles (e.g., lipid nanoparticles, polymeric nanoparticles, lipid-polymer hybrid nanoparticles, etc.), and/or peptide-based vectors. See, e.g., Wadhwa et al. "Opportunities and Challenges in the Delivery of mRNA-Based Vaccines" Pharmaceutics (2020) 102 (27 pages), the content of which is incorporated herein by reference, for information on various approaches that may be useful for delivery of polyribonucleotides described herein.
[0347] In some embodiments, one or more polyribonucleotides can be formulated with lipid nanoparticles for delivery (e.g., administration).
[0348] In some embodiments, lipid nanoparticles can be designed to protect polyribonucleotides from extracellular RNases and/or engineered for systemic delivery of the RNA to target cells (e.g., liver cells). In some embodiments, such lipid nanoparticles may be particularly useful to deliver polyribonucleotides when polyribonucleotides are intravenously or intramuscularly administered to a subject.
A. Lipid Compositions
1. Lipids and Lipid-Like Materials
[0349] The terms "lipid" and "lipid-like material" are broadly defined herein as molecules which comprise one or more hydrophobic moieties or groups and optionally also one or more hydrophilic moieties or groups. Molecules comprising hydrophobic moieties and hydrophilic moieties are also frequently denoted as amphiphiles. Lipids are usually poorly soluble in water. In an aqueous environment, the amphiphilic nature allows the molecules to self-assemble into organized structures and different phases. One of those phases consists of lipid bilayers, as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment. Hydrophobicity can be conferred by the inclusion of a polar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). The hydrophilic groups may comprise polar and/or charged groups and include carbohydrates, phosphate, carboxylic, sulfate, amino, sulfhydryl, nitro, hydroxyl, and other like groups. [0350] Often, an amphiphilic compound has a polar head attached to a long hydrophobic tail. In some embodiments, the polar portion is soluble in water, while the non-polar portion is insoluble in water. In addition, the polar portion may have either a formal positive charge, or a formal negative charge. Alternatively, the polar portion may have both a formal positive and a negative charge, and be a zwitterion or inner salt. For purposes of the disclosure, the amphiphilic compound can be, but is not limited to, one or a plurality of natural or non-natural lipids and lipid-like compounds.
[0351] A "lipid-like material" is a substance that is structurally and/or functionally related to a lipid but may not be considered a lipid in a strict sense. For example, the term includes compounds that are able to form amphiphilic layers as they are present in vesicles, multilamellar/unilamellar liposomes, or membranes in an aqueous environment and includes surfactants, or synthesized compounds with both hydrophilic and hydrophobic moieties. Generally speaking, the term refers to molecules, which comprise hydrophilic and hydrophobic moieties with different structural organization, which may or may not be similar to that of lipids.
[0352] Specific examples of amphiphilic compounds that may be included in an amphiphilic layer include, but are not limited to, phospholipids, aminolipids and sphingolipids.
[0353] Generally, lipids may be divided into eight categories: fatty acids, glycerolipids, glycerophospholipids, sphingolipids, saccharolipids, polyketides (derived from condensation of ketoacyl subunits), sterols and prenol lipids (derived from condensation of isoprene subunits). Although the term "lipid" is sometimes used as a synonym for fats, fats are a subgroup of lipids called triglycerides. Lipids also encompass molecules such as fatty acids and their derivatives (including tri-, di-, monoglycerides, and phospholipids), as well as sterol- containing metabolites such as cholesterol.
[0354] Fatty acids are a diverse group of molecules made of a hydrocarbon chain that terminates with a carboxylic acid group; this arrangement confers the molecule with a polar, hydrophilic end, and a nonpolar, hydrophobic end that is insoluble in water. The carbon chain, typically between four and 24 carbons long, may be saturated or unsaturated, and may be attached to functional groups containing oxygen, halogens, nitrogen, and sulfur. If a fatty acid contains a double bond, there is the possibility of either a cis or trans geometric isomerism, which significantly affects the molecule's configuration. Cis-double bonds cause the fatty acid chain to bend, an effect that is compounded with more double bonds in the chain. Other major lipid classes in the fatty acid category are the fatty esters and fatty amides.
[0355] Glycerolipids are composed of mono-, di-, and tri-substituted glycerols, the best-known being the fatty acid triesters of glycerol, called triglycerides. The word "triacylglycerol" is sometimes used synonymously with "triglyceride". In these compounds, the three hydroxyl groups of glycerol are each esterified, typically by different fatty acids. Additional subclasses of glycerolipids are represented by glycosylglycerols, which are characterized by the presence of one or more sugar residues attached to glycerol via a glycosidic linkage.
[0356] Glycerophospholipids are amphipathic molecules (containing both hydrophobic and hydrophilic regions) that contain a glycerol core linked to two fatty acid-derived "tails" by ester linkages and to one "head" group by a phosphate ester linkage. Examples of glycerophospholipids, usually referred to as phospholipids (though sphingomyelins are also classified as phospholipids) are phosphatidylcholine (also known as PC, GPCho or lecithin), phosphatidylethanolamine (PE or GPEtn) and phosphatidylserine (PS or GPSer).
[0357] Sphingolipids are members of a complex family of compounds that share a common structural feature, a sphingoid base backbone. The major sphingoid base in mammals is commonly referred to as sphingosine. Ceramides (N-acyl-sphingoid bases) are a major subclass of sphingoid base derivatives with an amide-linked fatty acid. The fatty acids are typically saturated or mono-unsaturated with chain lengths from 16 to 26 carbon atoms. The major phosphosphingolipids of mammals are sphingomyelins (ceramide phosphocholines), whereas insects contain mainly ceramide phosphoethanolamines and fungi have phytoceramide phosphoinositols and mannosecontaining headgroups. The glycosphingolipids are a diverse family of molecules composed of one or more sugar residues linked via a glycosidic bond to the sphingoid base. Examples of these are the simple and complex glycosphingolipids such as cerebrosides and gangliosides.
[0358] Sterols, such as cholesterol and its derivatives, or tocopherol and its derivatives, are important components of membrane lipids, along with the glycerophospholipids and sphingomyelins.
[0359] Saccharolipids are compounds in which fatty acids are linked directly to a sugar backbone, forming structures that are compatible with membrane bilayers. In the saccharolipids, a monosaccharide substitutes for the glycerol backbone present in glycerolipids and glycerophospholipids. The most familiar saccharolipids are the acylated glucosamine precursors of the Lipid A component of the lipopolysaccharides in Gram-negative bacteria. Typical lipid A molecules are disaccharides of glucosamine, which are derivatized with as many as seven fatty-acyl chains. The minimal lipopolysaccharide required for growth in E. coli is Kdo2-Lipid A, a hexa-acylated disaccharide of glucosamine that is glycosylated with two 3-deoxy-D-manno-octulosonic acid (Kdo) residues.
[0360] Polyketides are synthesized by polymerization of acetyl and propionyl subunits by classic enzymes as well as iterative and multimodular enzymes that share mechanistic features with the fatty acid synthases. They comprise a large number of secondary metabolites and natural products from animal, plant, bacterial, fungal and marine sources, and have great structural diversity. Many polyketides are cyclic molecules whose backbones are often further modified by glycosylation, methylation, hydroxylation, oxidation, or other processes.
[0361] Lipids and lipid-like materials may be cationic, anionic or neutral. Neutral lipids or lipid-like materials exist in an uncharged or neutral zwitterionic form at a selected pH.
[0362] In some embodiments, suitable lipids or lipid-like materials for use in the present disclosure include those described in W02020/128031 and US20200163878, the entire contents of each of which are incorporated herein by reference for the purposes described herein.
2. Cationic or cationically ionizable lipids or lipid-like materials
[0363] In some embodiments cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein include any cationic or cationically ionizable lipids or lipid-like materials which are able to electrostatically bind nucleic acid. In one embodiment, cationic or cationically ionizable lipids or lipid-like materials contemplated for use herein can be associated with nucleic acid, e.g., by forming complexes with the nucleic acid or forming vesicles in which the nucleic acid is enclosed or encapsulated.
[0364] Cationic lipids or lipid-like materials are characterized in that they have a net positive charge
(e.g., at a relevant pH). Cationic lipids or lipid-like materials bind negatively charged nucleic acid by electrostatic interaction. Generally, cationic lipids possess a lipophilic moiety, such as a sterol, an acyl chain, a diacyl or more acyl chains, and the head group of the lipid typically carries the positive charge.
[0365] In certain embodiments, a cationic lipid or lipid-like material has a net positive charge only at certain pH, in particular acidic pH, while it has preferably no net positive charge, preferably has no charge, i.e., it is neutral, at a different, preferably higher pH such as physiological pH. This ionizable behavior is thought to enhance efficacy through helping with endosomal escape and reducing toxicity as compared with particles that remain cationic at physiological pH. [0366] In some embodiments, a cationic or cationically ionizable lipid or lipid-like material comprises a head group which includes at least one nitrogen atom (N) which is positive charged or capable of being protonated.
[0367] Examples of cationic lipids include, but are not limited to 1,2-dioleoyl-3-trimethylammonium propane (DOTAP); N,N-dimethyl-2,3-dioleyloxypropylamine (DODMA), 1,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA), 3-(N— (N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), dimethyldioctadecylammonium (DDAB); 1,2-dioleoyl-3-dimethylammonium-propane (DODAP); 1,2-diacyloxy-3- dimethylammonium propanes; 1,2-dialkyloxy-3-dimethylammonium propanes; dioctadecyldimethyl ammonium chloride (DODAC), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 2,3-di(tetradecoxy)propyl-(2- hydroxyethyl)-dimethylazanium (DMRIE), 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (DMEPC), 1,2- dimyristoyl-3-trimethylammonium propane (DMTAP), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethyl ammonium bromide (DORIE), and 2,3-dioleoyloxy- N-[2(spermine carboxamide)ethyl]-N,N-dimethyl-l-propanamium trifluoroacetate (DOSPA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N- dimethylaminopropane (DLenDMA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-(cholest-5-en- 3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-oc-tadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'- oxapentoxy)-3-dimethyl-l-(cis,cis-9',12'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4- dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-
Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-Dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4- dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-K- XTC2-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31- tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), N-(2-Hydroxyethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-l-propanaminium bromide (DMRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3-bis(cis-9- tetradecenyloxy)-l-propanaminium bromide (GAP-DMORIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3- bis(dodecyloxy)-l-propanaminium bromide (GAP-DLRIE), (±)-N-(3-aminopropyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-l-propanaminium bromide (GAP-DMRIE), N-(2-Aminoethyl)-N,N-dimethyl-2,3- bis(tetradecyloxy)-l-propanaminium bromide (PAE-DMRIE), N-(4-carboxybenzyl)-N,N-dimethyl-2,3- bis(oleoyloxy)propan-l-aminium (DOBAQ), 2-({8-[(3P)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-
[(9Z,12Z)-octadeca-9,12-dien-l-yloxy]propan-l-amine (Octyl-CLinDMA), 1,2-dimyristoyl-3-dimethylammonium- propane (DMDAP), 1,2-dipalmitoyl-3-dimethylammonium-propane (DPDAP), Nl-[2-((lS)-1-[(3- aminopropyl)amino]-4-[di(3-amino-propyl)amino]butylcarboxamido)ethyl]-3,4-di[oleyloxy]-benzamide (MVL5), 1,2-dioleoyl-sn-glycero-3-ethylphosphocholine (DOEPC), 2,3-bis(dodecyloxy)-N-(2-hydroxyethyl)-N,N- dimethylpropan-l-amonium bromide (DLRIE), N-(2-aminoethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-l- aminium bromide (DMORIE), di((Z)-non-2-en-l-yl) 8,8'-
((((2(dimethylamino)ethyl)thio)carbonyl)azanediyl)dioctanoate (ATX), N,N-dimethyl-2,3-bis(dodecyloxy)propan-1- amine (DLDMA), N,N-dimethyl-2,3-bis(tetradecyloxy)propan-l-amine (DMDMA), Di((Z)-non-2-en-l-yl)-9-((4- (dimethylaminobutanoyl)oxy)heptadecanedioate (L319), N-Dodecyl-3-((2-dodecylcarbamoyl-ethyl)-{2-[(2- dodecylcarbamoyl-ethyl)-2-{(2-dodecylcarbamoyl-ethyl)-[2-(2-dodecylcarbamoyl-ethylamino)-ethyl]-amino}- ethylamino)propionamide (lipidoid 98N12-5), l-[2-[bis(2-hydroxydodecyl)amino]ethyl-[2-[4-[2-[bis(2 hydroxydodecyl)amino]ethyl]piperazin-l-yl]ethyl]amino]dodecan-2-ol (lipidoid C12-200), LIPOFECTIN® (commercially available cationic liposomes comprising DOTMA and 1 ,2-dioleoyl-sn-3phosphoethanolamine (DOPE), from GIBCO/BRL, Grand Island, N.Y.); LIPOFECTAMINE® (commercially available cationic liposomes comprising N-(1 -(2,3dioleyloxy)propyl)-N-(2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE), from GIBCO/BRL); and TRANSFECTAM® (commercially available cationic lipids comprising dioctadecylamidoglycyl carboxyspermine (DOGS) in ethanol from Promega Corp., Madison, Wis.) or any combination of any of the foregoing. Further suitable cationic lipids for use in the present disclosure include those described in W02020/128031 and US20200163878, the entire contents of each of which are incorporated herein by reference for the purposes described herein. Further suitable cationic lipids for use in the present disclosure include those described in W02010/053572 (including Cl 2-200 described at paragraph [00225]) and W02012/170930, both of which are incorporated herein by reference for the purposes described herein. Additional suitable cationic lipids for use in the present disclosure include HGT4003, HGT5000, HGTS001, HGT5001, HGT5002 (see US20150140070A1, which is incorporated herein by reference in its entirety).
[0368] In some embodiments, formulations that are useful for pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) compositions as described herein can comprise at least one cationic lipid. Representative cationic lipids include, but are not limited to, 1 ,2-dilinoleyoxy-3- (dimethylamino)acetoxypropane (DLin-DAC), 1 ,2-dilinoleyoxy-3morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3- dimethylaminopropane (DLinDAP), 1 ,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1 -linoleoyl-2- linoleyloxy-3dimethylaminopropane (DLin-2-DMAP), 1 ,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.CI), 1 ,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.CI), 1 ,2-dilinoleyloxy-3-(N- methylpiperazino)propane (DLin-MPZ), 3-(N,Ndilinoleylamino)-l ,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-l ,2-propanediol (DOAP), 1 ,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), and 2,2- dilinoleyl-4-dimethylaminomethyl-[l ,3]-dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l ,3]- dioxolane (DLin-KC2-DMA); dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA); MC3 (US20100324120, which is incorporated herein by reference in its entirety).
[0369] In some embodiments, amino or cationic lipids useful in accordance with the present disclosure have at least one protonatable or deprotonatable group, such that the lipid is positively charged at a pH at or below physiological pH (e.g., pH 7.4), and neutral at a second pH, preferably at or above physiological pH. It will, of course, be understood that the addition or removal of protons as a function of pH is an equilibrium process, and that the reference to a charged or a neutral lipid refers to the nature of the predominant species and does not require that all of lipids have to be present in the charged or neutral form. Lipids having more than one protonatable or deprotonatable group, or which are zwitterionic, are not excluded and may likewise be suitable in the context of the present invention.
[0370] In some embodiments, a protonatable lipid has a pKa of the protonatable group in the range of about 4 to about 11, e.g., a pKa of about 5 to about 7.
[0371] In some embodiments, a cationic lipid may comprise from about 10 mol % to about 100 mol %, about 20 mol % to about 100 mol %, about 30 mol % to about 100 mol %, about 40 mol % to about 100 mol %, or about 50 mol % to about 100 mol % of total lipid present in a lipid composition utilized in accordance with the present disclosure.
3. Additional lipids or lipid-like materials
[0372] In some embodiments, formulations utilized in accordance with the present disclosure may comprise lipids or lipid-like materials other than cationic or cationically ionizable lipids or lipid-like materials, i.e., non-cationic lipids or lipid-like materials (including non-cationically ionizable lipids or lipid-like materials). Collectively, anionic and neutral lipids or lipid-like materials are referred to herein as non-cationic lipids or lipid-like materials. In some embodiments, optimizing a formulation of nucleic acid particles by addition of other hydrophobic moieties, such as cholesterol and lipids, in addition to an ionizable/cationic lipid or lipid-like material may, for example, enhance particle stability and efficacy of nucleic acid delivery.
[0373] In some embodiments, a lipid or lipid-like material may be incorporated which may or may not affect the overall charge of particles. In certain embodiments, such lipid or lipid-like material is a non-cationic lipid or lipid-like material.
[0374] In some embodiments, a non-cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids. An "anionic lipid" is negatively charged (e.g., at a selected pH).
[0375] A "neutral lipid" exists either in an uncharged or neutral zwitterionic form (e.g., at a selected pH). In some embodiments, a formulation comprises one of the following neutral lipid components: (1) a phospholipid, (2) cholesterol or a derivative thereof; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof. Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, tocopherol and derivatives thereof, and mixtures thereof.
[0376] Specific exemplary phospholipids that can be used include, but are not limited to, phosphatidylcholines, phosphatidylethanolamines, phosphatidylglycerols, phosphatidic acids, phosphatidylserines or sphingomyelin. Such phospholipids include in particular diacylphosphatidylcholines, such as distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dimyristoylphosphatidylcholine (DMPC), dipentadecanoylphosphatidylcholine, dilauroylphosphatidylcholine, dipalmitoylphosphatidylcholine (DPPC), diarachidoylphosphatidylcholine (DAPC), dibehenoylphosphatidylcholine (DBPC), ditricosanoylphosphatidylcholine (DTPC), dilignoceroylphatidylcholine (DLPC), palmitoyloleoyl-phosphatidylcholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 Diether PC), l-oleoyl-2- cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), l-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC) and phosphatidylethanolamines, in particular diacylphosphatidylethanolamines, such as dioleoylphosphatidylethanolamine (DOPE), distearoyl-phosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoyl-phosphatidylethanolamine (DMPE), dilauroylphosphatidylethanolamine (DLPE), diphytanoyl-phosphatidylethanolamine (DPyPE), and further phosphatidylethanolamine lipids with different hydrophobic chains.
[0377] In certain embodiments, a formulation utilized in accordance with the present disclosure includes
DSPC or DSPC and cholesterol.
[0378] In certain embodiments, formulations utilized in accordance with the present disclosure include both a cationic lipid and an additional (non-cationic) lipid.
[0379] In some embodiments, formulations herein include a polymer conjugated lipid such as a pegylated lipid. "Pegylated lipids" comprise both a lipid portion and a polyethylene glycol portion. Pegylated lipids are known in the art.
[0380] Without wishing to be bound by theory, the amount of (total) cationic lipid compared to the amount of other lipid(s) in formulation may affect important characteristics, such as charge, particle size, stability, tissue selectivity, and bioactivity of the nucleic acid. In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4: 1 to about 1:2, or about 3: 1 to about 1: 1.
[0381] In some embodiments, a non-cationic lipid, in particular a neutral lipid, (e.g., one or more phospholipids and/or cholesterol) may comprise from about 0 mol % to about 90 mol %, from about 0 mol % to about 80 mol %, from about 0 mol % to about 70 mol %, from about 0 mol % to about 60 mol %, or from about 0 mol % to about 50 mol %, of the total lipid present in a formulation.
4. Lipoplex Particles
[0382] In certain embodiments of the present disclosure, the RNA described herein may be present in
RNA lipoplex particles.
[0383] An "RNA lipoplex particle" contains lipid, in particular cationic lipid, and RNA. Electrostatic interactions between positively charged liposomes and negatively charged RNA results in complexation and spontaneous formation of RNA lipoplex particles. Positively charged liposomes may be generally synthesized using a cationic lipid, such as DOTMA, and additional lipids, such as DOPE. In one embodiment, an RNA lipoplex particle is a nanoparticle.
[0384] In certain embodiments, RNA lipoplex particles include both a cationic lipid and an additional lipid. In an exemplary embodiment, the cationic lipid is DOTMA and the additional lipid is DOPE.
[0385] In some embodiments, the molar ratio of the at least one cationic lipid to the at least one additional lipid is from about 10:0 to about 1:9, about 4: 1 to about 1:2, or about 3: 1 to about 1: 1. In specific embodiments, the molar ratio may be about 3: 1, about 2.75: 1, about 2.5: 1, about 2.25: 1, about 2: 1, about 1.75: 1, about 1.5: 1, about 1.25: 1, or about 1: 1. In an exemplary embodiment, the molar ratio of the at least one cationic lipid to the at least one additional lipid is about 2: 1.
[0386] In some embodiments, RNA lipoplex particles have an average diameter that in one embodiment ranges from about 200 nm to about 1000 nm, from about 200 nm to about 800 nm, from about 250 to about 700 nm, from about 400 to about 600 nm, from about 300 nm to about 500 nm, or from about 350 nm to about 400 nm. In specific embodiments, the RNA lipoplex particles have an average diameter of about 200 nm, about 225 nm, about 250 nm, about 275 nm, about 300 nm, about 325 nm, about 350 nm, about 375 nm, about 400 nm, about 425 nm, about 450 nm, about 475 nm, about 500 nm, about 525 nm, about 550 nm, about 575 nm, about 600 nm, about 625 nm, about 650 nm, about 700 nm, about 725 nm, about 750 nm, about 775 nm, about 800 nm, about 825 nm, about 850 nm, about 875 nm, about 900 nm, about 925 nm, about 950 nm, about 975 nm, or about 1000 nm. In an embodiment, the RNA lipoplex particles have an average diameter that ranges from about 250 nm to about 700 nm. In another embodiment, the RNA lipoplex particles have an average diameter that ranges from about 300 nm to about 500 nm. In an exemplary embodiment, the RNA lipoplex particles have an average diameter of about 400 nm.
[0387] RNA lipoplex particles and compositions comprising RNA lipoplex particles described herein are useful for delivery of RNA to a target tissue after parenteral administration, in particular after intravenous administration. The RNA lipoplex particles may be prepared using liposomes that may be obtained by injecting a solution of the lipids in ethanol into water or a suitable aqueous phase. In one embodiment, the aqueous phase has an acidic pH. In one embodiment, the aqueous phase comprises acetic acid, e.g., in an amount of about 5 mM. Liposomes may be used for preparing RNA lipoplex particles by mixing the liposomes with RNA. In one embodiment, the liposomes and RNA lipoplex particles comprise at least one cationic lipid and at least one additional lipid. In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3- trimethylammonium propane (DOTMA) and/or 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). In one embodiment, the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (DOPE), cholesterol (Choi) and/or 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). In one embodiment, the at least one cationic lipid comprises 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) and the at least one additional lipid comprises 1,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (DOPE). In one embodiment, the liposomes and RNA lipoplex particles comprise 1,2-di-O- octadecenyl-3-trimethylammonium propane (DOTMA) and 1,2-di-(9Z-octadecenoyl)-sn-glycero-3- phosphoethanolamine (DOPE).
[0388] Spleen targeting RNA lipoplex particles are described in WO 2013/143683, herein incorporated by reference. It has been found that RNA lipoplex particles having a net negative charge may be used to preferentially target spleen tissue or spleen cells such as antigen-presenting cells, in particular dendritic cells. Accordingly, following administration of the RNA lipoplex particles, RNA accumulation and/or RNA expression in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in the spleen. In an embodiment, after administration of the RNA lipoplex particles, no or essentially no RNA accumulation and/or RNA expression in the lung and/or liver occurs. In one embodiment, after administration of the RNA lipoplex particles, RNA accumulation and/or RNA expression in antigen presenting cells, such as professional antigen presenting cells in the spleen occurs. Thus, RNA lipoplex particles of the disclosure may be used for expressing RNA in such antigen presenting cells. In one embodiment, the antigen presenting cells are dendritic cells and/or macrophages.
5. Lipid Nanoparticles (LNPs)
[0389] In some embodiments, nucleic acid such as RNA described herein is administered in the form of lipid nanoparticles (LNPs). In some embodiments, LNPs may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.
[0390] In some embodiments, an LNP comprises one or more cationic lipids, and one or more stabilizing lipids. Stabilizing lipids include neutral lipids and pegylated lipids.
[0391] In some embodiments, an LNP comprises a cationic lipid, a neutral lipid, a sterol, a polymer conjugated lipid; and an RNA, encapsulated within or associated with the lipid nanoparticle.
[0392] In some embodiments, a neutral lipid is selected from the group consisting of DSPC, DPPC,
DMPC, DOPC, POPC, DOPE, DOPG, DPPG, POPE, DPPE, DMPE, DSPE, and SM. In some embodiments, the neutral lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE and SM. In some embodiments, the neutral lipid is DSPC.
[0393] In some embodiments, a sterol is cholesterol.
[0394] In some embodiments, a polymer conjugated lipid is a pegylated lipid. In some embodiments, a pegylated lipid has the following structure:
Figure imgf000088_0001
or a pharmaceutically acceptable salt, tautomer or stereoisomer thereof, wherein:
R12 and R13 are each independently a straight or branched, saturated or unsaturated alkyl chain containing from 10 to 30 carbon atoms, wherein the alkyl chain is optionally interrupted by one or more ester bonds; and w has a mean value ranging from 30 to 60. In some embodiments, R12 and R13 are each independently straight, saturated alkyl chains containing from 12 to 16 carbon atoms. In some embodiments, w has a mean value ranging from 40 to 55. In some embodiments, the average w is about 45. In some embodiments, R12 and R13 are each independently a straight, saturated alkyl chain containing about 14 carbon atoms, and w has a mean value of about 45. [0395] In some embodiments, a pegylated lipid is DMG-PEG 2000, e.g., having the following structure:
Figure imgf000089_0003
[0396] In some embodiments, a cationic lipid component of LNPs has the structure of Formula (III):
Figure imgf000089_0002
or a pharmaceutically acceptable salt, tautomer, prodrug or stereoisomer thereof, wherein: one of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, -S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa- , NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O-, and the other of L1 or L2 is -O(C=O)-, -(C=O)O-, -C(=O)-, -O-, - S(O)X-, -S-S-, -C(=O)S-, SC(=O)-, -NRaC(=O)-, -C(=O)NRa-, NRaC(=O)NRa-, -OC(=O)NRa- or -NRaC(=O)O- or a direct bond;
G1 and G2 are each independently unsubstituted C1-C12 alkylene or C1-C12 alkenylene;
G3 is C1-C24 alkylene, C1-C24 alkenylene, C3-C8 cycloalkylene, C3-C8 cycloalkenylene;
Ra is H or C1-C12 alkyl;
R1 and R2 are each independently C6-C24 alkyl or C6-C24 alkenyl;
R3 is H, OR5, CN, -C(=O)OR4, -OC(=O)R4 or -NR5C(=O)R4;
R4 is C1-C12 alkyl;
R5 is H or C Ce alkyl; and x is 0, 1 or 2.
[0397] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIA) or (IIIB): wherein:
Figure imgf000089_0001
A is a 3 to 8-membered cycloalkyl or cycloalkylene ring;
R5 is, at each occurrence, independently H, OH or C1-C24 alkyl; n is an integer ranging from 1 to 15.
[0398] In some of the foregoing embodiments of Formula (III), the lipid has structure (IIIA), and in other embodiments, the lipid has structure (IIIB).
[0399] In other embodiments of Formula (III), the lipid has one of the following structures (IIIC) or
(HID):
Figure imgf000090_0001
(IIIC) (HID) wherein y and z are each independently integers ranging from 1 to 12.
[0400] In any of the foregoing embodiments of Formula (III), one of L1 or L2 is -0(C=0)-. For example, in some embodiments each of L1 and L2 are -0(C=0)-. In some different embodiments of any of the foregoing, L1 and L2 are each independently -(C=0)0- or -0(C=0)-. For example, in some embodiments each of L1 and L2 is -(C=0)0-.
[0401] In some different embodiments of Formula (III), the lipid has one of the following structures
(HIE) or (IIIF):
Figure imgf000090_0002
(HIE) (IIIF)
[0402] In some of the foregoing embodiments of Formula (III), the lipid has one of the following structures (IIIG), (IIIH), (IIII), or (IIIJ):
Figure imgf000090_0003
(IIII) (IIIJ)
[0403] In some of the foregoing embodiments of Formula (III), n is an integer ranging from 2 to 12, for example from 2 to 8 or from 2 to 4. For example, in some embodiments, n is 3, 4, 5 or 6. In some embodiments, n is 3. In some embodiments, n is 4. In some embodiments, n is 5. In some embodiments, n is 6.
[0404] In some other of the foregoing embodiments of Formula (III), y and z are each independently an integer ranging from 2 to 10. For example, in some embodiments, y and z are each independently an integer ranging from 4 to 9 or from 4 to 6. [0405] In some of the foregoing embodiments of Formula (III), R5 is H. In other of the foregoing embodiments, R5 is C1-C24 alkyl. In other embodiments, R5 is OH.
[0406] In some embodiments of Formula (III), G3 is unsubstituted. In other embodiments, G3 is substituted. In various different embodiments, G3 is linear C1-C24 alkylene or linear C1-C24 alkenylene.
[0407] In some other foregoing embodiments of Formula (III), R1 or R2, or both, is C6-C24 alkenyl. For example, in some embodiments, R1 and R2 each, independently have the following structure: wherein:
Figure imgf000091_0001
R7a and R7b are, at each occurrence, independently H or C1-C12 alkyl; and a is an integer from 2 to 12, wherein R7a, R7b and a are each selected such that R1 and R2 each independently comprise from 6 to 20 carbon atoms. For example, in some embodiments a is an integer ranging from 5 to 9 or from 8 to 12.
[0408] In some of the foregoing embodiments of Formula (III), at least one occurrence of R7a is H. For example, in some embodiments, R7a is H at each occurrence. In other different embodiments of the foregoing, at least one occurrence of R7b is C1-C8 alkyl. For example, in some embodiments, C1-C8 alkyl is methyl, ethyl, n-propyl, iso-propyl, n-butyl, iso-butyl, tert-butyl, n-hexyl or n-octyl.
[0409] In different embodiments of Formula (III), R1 or R2, or both, has one of the following structures:
Figure imgf000091_0002
[0410] In some of the foregoing embodiments of Formula (III), R3 is OH, CN, -C(=O)OR4, -OC(=O)R4 or -NHC(=O)R4. In some embodiments, R4 is methyl or ethyl.
[0411] In various different embodiments, the cationic lipid of Formula (III) has one of the structures set forth in in Table 4 below.
Table 4: Exemplar C d f F l (III)
Figure imgf000091_0003
Figure imgf000092_0001
Figure imgf000093_0001
Figure imgf000094_0001
Figure imgf000095_0001
Figure imgf000096_0002
[0412] In various different embodiments, a cationic lipid has one of the structures set forth in Table 5 below.
Table 5: Exemplary Cationic Lipid Structures
Figure imgf000096_0001
Figure imgf000097_0002
[0413] In some embodiments, an LNP comprises a cationic lipid that is an ionizable lipid-like material
(lipidoid). In some embodiments, a cationic lipid has the following structure:
Figure imgf000097_0001
[0414] In some embodiments, lipid nanoparticles can have an average size (e.g., mean diameter) of about 30 nm to about 150 nm, about 40 nm to about 150 nm, about 50 nm to about 150 nm, about 60 nm to about 130 nm, about 70 nm to about 110 nm, about 70 nm to about 100 nm, about 70 to about 90 nm, or about 70 nm to about 80 nm. In some embodiments, lipid nanoparticles in accordance with the present disclosure can have an average size (e.g., mean diameter) of about 50 nm to about 100 nm. In some embodiments, lipid nanoparticles may have an average size (e.g., mean diameter) of about 50 nm to about 150 nm. In some embodiments, lipid nanoparticles may have an average size (e.g., mean diameter) of about 60 nm to about 120 nm. In some embodiments, lipid nanoparticles in accordance with the present disclosure can have an average size (e.g., mean diameter) of about 30 nm, 35 nm, 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, 95 nm, 100 nm, 105 nm, 110 nm, 115 nm, 120 nm, 125 nm, 130 nm, 135 nm, 140 nm, 145 nm, or 150 nm. The term "average diameter" or "mean diameter" refers to the mean hydrodynamic diameter of particles as measured by dynamic laser light scattering (DLS) with data analysis using the so-called cumulant algorithm, which provides as results the so-called Z-average with the dimension of a length, and the polydispersity index (PI), which is dimensionless (Koppel, D., J. Chem. Phys. 57, 1972, pp 4814-4820, ISO 13321, which is herein incorporated by reference). Here "average diameter," "mean diameter," "diameter," or "size" for particles is used synonymously with this value of the Z-average. [0415] In some embodiments, lipid nanoparticles described herein may exhibit a polydispersity index less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less. By way of example, lipid nanoparticles can exhibit a polydispersity index in a range of about 0.1 to about 0.3 or about 0.2 to about 0.3. The "polydispersity index" is preferably calculated based on dynamic light scattering measurements by the so-called cumulant analysis as mentioned in the definition of the "average diameter." Under certain prerequisites, it can be taken as a measure of the size distribution of an ensemble of ribonucleic acid nanoparticles (e.g., ribonucleic acid nanoparticles).
[0416] Lipid nanoparticles described herein can be characterized by an "N/P ratio," which is the molar ratio of cationic (nitrogen) groups (the "N" in N/P) in the cationic polymer to the anionic (phosphate) groups (the
"P" in N/P) in RNA. It is understood that a cationic group is one that is either in cationic form (e.g., N+), or one that is ionizable to become cationic. Use of a single number in an N/P ratio (e.g., an N/P ratio of about 5) is intended to refer to that number over 1, e.g., an N/P ratio of about 5 is intended to mean 5: 1. In some embodiments, a lipid nanoparticle described herein has an N/P ratio greater than or equal to 5. In some embodiments, a lipid nanoparticle described herein has an N/P ratio that is about 5, 6, 7, 8, 9, or 10. In some embodiments, an N/P ratio for a lipid nanoparticle described herein is from about 10 to about 50. In some embodiments, an N/P ratio for a lipid nanoparticle described herein is from about 10 to about 70. In some embodiments, an N/P ratio for a lipid nanoparticle described herein is from about 10 to about 120.
B. Exemplary Methods of Making Lipid Nanoparticles
[0417] Lipids and lipid nanoparticles comprising nucleic acids and their method of preparation are known in the art, including, e.g., as described in U.S. Patent Nos. 8,569,256, 5,965,542 and U.S. Patent Publication Nos.
2016/0199485, 2016/0009637, 2015/0273068, 2015/0265708, 2015/0203446, 2015/0005363, 2014/0308304,
2014/0200257, 2013/086373, 2013/0338210, 2013/0323269, 2013/0245107, 2013/0195920, 2013/0123338,
2013/0022649, 2013/0017223, 2012/0295832, 2012/0183581, 2012/0172411, 2012/0027803, 2012/0058188,
2011/0311583, 2011/0311582, 2011/0262527, 2011/0216622, 2011/0117125, 2011/0091525, 2011/0076335,
2011/0060032, 2010/0130588, 2007/0042031, 2006/0240093, 2006/0083780, 2006/0008910, 2005/0175682,
2005/017054, 2005/0118253, 2005/0064595, 2004/0142025, 2007/0042031, 1999/009076 and PCT Pub. Nos. WO 99/39741, WO 2018/081480, WO 2017/004143, WO 2017/075531, WO 2015/199952, WO 2014/008334, WO 2013/086373, WO 2013/086322, WO 2013/016058, WO 2013/086373, W02011/141705, and WO 2001/07548, the full disclosures each of which are herein incorporated by reference in their entirety for the purposes described herein.
[0418] For example, in some embodiments, cationic lipids, neutral lipids (e.g., DSPC, and/or cholesterol) and polymer-conjugated lipids can be solubilized in ethanol at a pre-determined molar ratio (e.g., ones described herein). In some embodiments, lipid nanoparticles (lipid nanoparticle) are prepared at a total lipid to polyribonucleotides weight ratio of approximately 10: 1 to 30: 1. In some embodiments, such polyribonucleotides can be diluted to 0.2 mg/mL in acetate buffer.
[0419] In some embodiments, using an ethanol injection technique, a colloidal lipid dispersion comprising polyribonucleotides can be formed as follows: an ethanol solution comprising lipids, such as cationic lipids, neutral lipids, and polymer-conjugated lipids, is injected into an aqueous solution comprising polyribonucleotides (e.g., ones described herein).
[0420] In some embodiments, lipid and polyribonucleotide solutions can be mixed at room temperature by pumping each solution at controlled flow rates into a mixing unit, for example, using piston pumps. In some embodiments, the flow rates of a lipid solution and an RNA solution into a mixing unit are maintained at a ratio of 1:3. Upon mixing, nucleic acid-lipid particles are formed as the ethanolic lipid solution is diluted with aqueous polyribonucleotides. The lipid solubility is decreased, while cationic lipids bearing a positive charge interact with the negatively charged RNA.
[0421] In some embodiments, a solution comprising RNA-encapsulated lipid nanoparticles can be processed by one or more of concentration adjustment, buffer exchange, formulation, and/or filtration.
[0422] In some embodiments, RNA-encapsulated lipid nanoparticles can be processed through filtration.
[0423] In some embodiments, particle size and/or internal structure of lipid nanoparticles (with or without RNAs) may be monitored by appropriate techniques such as, e.g., small-angle X-ray scattering (SAXS) and/or transmission electron cryomicroscopy (CryoTEM).
IV. Pharmaceutical Compositions
[0424] In some embodiments, the present disclosure provides compositions, e.g., pharmaceutical compositions, comprising one or more polyribonucleotides described herein. Pharmaceutical formulations may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, includes any and all solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R.
Gennaro (Lippincott, Williams & Wilkins, Baltimore, MD, 2006; incorporated herein by reference) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this disclosure.
[0425] In some embodiments, an excipient is approved for use in humans and for veterinary use. In some embodiments, an excipient is approved by the United States Food and Drug Administration. In some embodiments, an excipient is pharmaceutical grade. In some embodiments, an excipient meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and/or the International Pharmacopoeia.
[0426] Pharmaceutically acceptable excipients used in the manufacture of pharmaceutical compositions include, but are not limited to, inert diluents, dispersing and/or granulating agents, surface active agents and/or emulsifiers, disintegrating agents, binding agents, preservatives, buffering agents, lubricating agents, and/or oils. Such excipients may optionally be included in pharmaceutical formulations. Excipients such as cocoa butter and suppository waxes, coloring agents, coating agents, sweetening, flavoring, and/or perfuming agents can be present in the composition, according to the judgment of the formulator.
[0427] General considerations in the formulation and/or manufacture of pharmaceutical agents may be found, for example, in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).
[0428] In some embodiments, pharmaceutical compositions provided herein may be formulated with one or more pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).
[0429] In some embodiments, pharmaceutical composition provided herein may be formulated with one or more buffer systems. In some embodiments, a buffer system may include a 2-amino-2- (hydroxymethyl)propate-l, 3-dial (Tris), phosphate, 2-[4-(2-hydroxyethyl)piperazin-l-yl]ethanesulfonic acid (HEPES), acetate, or histidine buffer system. In some embodiments, a pharmaceutical composition disclosed herein includes a Tris buffer. In some embodiments, a pharmaceutical composition disclosed herein includes a phosphate buffer (e.g., phosphate buffered saline (PBS)).
[0430] In some embodiments, a pharmaceutical composition provided herein may have a pH from about 6.0 to 8.0. For example, in some embodiments, a pharmaceutical composition provided herein may have a pH from about 6.0 to 8.0, about 6.2 to 8.0, about 6.4 to 8.0, about 6.6 to 8.0, about 6.8 to 8.0, about 7.0 to 8.0, about 7.2 to 8.0, about 7.4 to 8.0, about 7.6 to 8.0, about 7.8 to 8.0, about 6.0 to 7.8, about 6.2 to 7.8, about 6.4 to 7.8, about 6.6 to 7.8, about 6.8 to 7.8, about 7.0 to 7.8, about 7.2 to 7.8, about 7.4 to 7.8, about 7.6 to
7.8, about 6.0 to 7.6, about 6.2 to 7.6, about 6.4 to 7.6, about 6.6 to 7.6, about 6.8 to 7.6, about 7.0 to 7.6, about 7.2 to 7.6, about 7.4 to 7.6, about 6.0 to 7.4, about 6.2 to 7.4, about 6.4 to 7.4, about 6.6 to 7.4, about 6.8 to 7.4, about 7.0 to 7.4, about 7.2 to 7.4, about 6.0 to 7.2, about 6.2 to 7.2, about 6.4 to 7.2, about 6.6 to
7.2, about 6.8 to 7.2, about 7.0 to 7.2, about 6.0 to 7.0, about 6.2 to 7.0, about 6.4 to 7.0, about 6.6 to 7.0, about 6.8 to 7.0, about 6.0 to 6.8, about 6.2 to 6.8, about 6.4 to 6.8, about 6.6 to 6.8, about 6.0 to 6.6, about 6.2 to 6.6, about 6.4 to 6.6, about 6.6 to 6.6, about 6.0 to 6.4, about 6.2 to 6.4, or about 6.0 to 6.2. In some embodiments, pharmaceutical compositions described herein may also comprise a cryoprotectant and/or a surfactant as a stabilizer to avoid substantial loss of the product quality and, in particular, substantial loss of mRNA activity during storage, freezing, and/or lyophilization, for example to reduce or prevent aggregation, particle collapse, mRNA degradation and/or other types of damage.
[0431] In some embodiments, a cryoprotectant comprises a carbohydrate. The term "carbohydrate", as used herein, refers to and encompasses monosaccharides, disaccharides, trisaccharides, 5 oligosaccharides and polysaccharides.
[0432] In some embodiments, a cryoprotectant comprises a monosaccharide. The term
"monosaccharide," as used herein refers to a single carbohydrate unit (e.g., a simple sugar) that cannot be hydrolyzed to simpler carbohydrate units. In some embodiments, a monosaccharide cryoprotectant includes glucose, fructose, galactose, xylose, ribose and the like.
[0433] In some embodiments, a cryoprotectant comprises a disaccharide. The term "disaccharide," as used herein refers to a compound or a chemical moiety formed by 2 monosaccharide units that are bonded together through a glycosidic linkage, for example through 1-4 linkages or 1-6 linkages. A disaccharide may be hydrolyzed into two monosaccharides. In some embodiments, a disaccharide cryoprotectant includes sucrose, trehalose, lactose, maltose and the like.
[0434] In some embodiments, a cryoprotectant comprises a trisaccharide. The term "trisaccharide," as used herein refers to a compound or a chemical moiety formed by 3 monosaccharide units that are bonded together through glycosidic linkages. In some embodiments, a trisaccharide cryoprotectant includes raffinose, melezitose and the like.
[0435] In some embodiments, a cryoprotectant comprises an oligosaccharide. The term
"oligosaccharide," as used herein refers to a compound or a chemical moiety formed by 3 to about 15, such as 3 to about 10 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a linear, branched or cyclic structure. In some embodiments, an oligosaccharide cryoprotectant includes cyclodextrins, raffinose, melezitose, maltotriose, stachyose, acarbose, and the like. In some embodiments, an oligosaccharide can be oxidized or reduced. [0436] In some embodiments, a cryoprotectant may be a cyclic oligosaccharide. The term "cyclic oligosaccharide," as used herein refers to a compound or a chemical moiety formed by 3 to 25 about 15, such as 6, 7, 8, 9, or 10 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a cyclic structure. In some embodiments, a cyclic oligosaccharide cryoprotectant includes cyclic oligosaccharides that are discrete compounds, such as a cyclodextrin, p cyclodextrin, or y cyclodextrin. In other embodiments a cyclic oligosaccharide cryoprotectant includes compounds which having a cyclodextrin moiety in a larger molecular structure, such as a polymer that contains a cyclic oligosaccharide moiety. In some embodiments, a cyclic oligosaccharide can be oxidized or reduced, for example, oxidized to dicarbonyl forms. The term "cyclodextrin moiety," as used herein refers to a cyclodextrin (e.g., an a, , or y cyclodextrin) radical that is incorporated into, or a part of, a larger molecular structure, such as a polymer. In some embodiments, a cyclodextrin moiety can be bonded to one or more other moieties directly, or through an optional linker. In some embodiments, a cyclodextrin moiety can be oxidized or reduced, for example, oxidized to dicarbonyl forms. In some embodiments, carbohydrate cryoprotectants, e.g., cyclic oligosaccharide cryoprotectants, can be derivatized carbohydrates. For example, in some embodiments, a cryoprotectant is a derivatized cyclic oligosaccharide, e.g., a derivatized cyclodextrin, e.g., 2-hydroxypropyl-p- cyclodextrin, e.g., partially etherified cyclodextrins (e.g., partially etherified p cyclodextrins).
[0437] In some embodiments, a cryoprotectant comprises a polysaccharide. The term
"polysaccharide," as used herein refers to a compound or a chemical moiety formed by at least 16 monosaccharide units that are bonded together through glycosidic linkages, for example through 1-4 linkages or 1-6 linkages, to form a linear, branched or cyclic structure, and includes polymers that comprise polysaccharides as part of their backbone structure. In some embodiments, a polysaccharide backbone structure can be linear or cyclic. In some endpoints, a polysaccharide cryoprotectants includes glycogen, amylase, cellulose, dextran, maltodextrin and the like.
[0438] In some embodiments, pharmaceutical compositions disclosed herein may include sucrose.
Without wishing to be bound by theory, sucrose functions to promote cryoprotection of the compositions, thereby preventing polyribonucleotide (especially mRNA) particle aggregation and maintaining chemical and physical stability of the composition. In some embodiments, pharmaceutical compositions described herein may include alternative cryoprotectants to sucrose. Alternative stabilizers include, without limitation, trehalose and glucose.
[0439] In some embodiments, pharmaceutical compositions of the present disclosure may include a chelating agent. Chelating agents refer to chemical compounds that are capable of forming at least two coordinate covalent bonds with a metal ion, thereby generating a stable, water-soluble complex. Without wishing to be bound by theory, chelating agents reduce the concentration of free divalent ions, which may otherwise induce accelerated polyribonucleotide degradation. In some embodiments, a chelating agent may include, without limitation, ethylenediaminetetraacetic acid (EDTA), a salt of EDTA, desferrioxamine B, deferoxamine, dithiocarb sodium, penicillamine, pentetate calcium, a sodium salt of pentetic acid, succimer, trientine, nitrilotriacetic acid, transdiaminocyclohexanetetraacetic acid 5 (DCTA), diethylenetriaminepentaacetic acid (DTPA), and bis(aminoethyl)glycolether-N,N,N',N'-tetraacetic acid. In some embodiments, a chelating agent comprises EDTA or a salt of EDTA. In an exemplary embodiment, the chelating agent comprises EDTA. In some embodiments, a pharmaceutical composition described herein does not include a chelating agent.
[0440] In some embodiments, a pharmaceutical composition of the present disclosure may include about 0.05 mg/ml to 5 mg/ml of polyribonucleotide. For example, in some embodiments, a pharmaceutical compositions may include about 0.05 mg/ml to 5 mg/ml, about 0.1 mg/ml to 5 mg/ml, about 0.5 mg/ml to 5 mg/ml, about 1.0 mg/ml to 5 mg/ml, 0.01 mg/ml to 1 mg/ml, about 0.05 mg/ml to 1 mg/ml, about 0.1 mg/ml to 1 mg/ml, about 0.5 mg/ml to 1 mg/ml, about 0.05 mg/ml to 0.5 mg/ml, about 0.1 mg/ml to 0.5 mg/ml, about 0.05 mg/ml to 0.1 mg/ml, or about 0.01 mg/ml to 0.05 mg/ml of polyribonucleotide. In some embodiments, a pharmaceutical composition of the present disclosure includes 0.1 mg/ml of polyribonucleotide. In some embodiments, a pharmaceutical composition of the present disclosure includes 0.5 mg/ml of polyribonucleotide. [0441] In some embodiments, a pharmaceutical composition of the present disclosure is provided for administration to a subject in a vial. In some embodiments, a vial includes about a 0.6 to 1.6 ml fill volume of a pharmaceutical composition disclosed herein. For example, in some embodiments, a vial includes about a 0.6 to
1.6 ml, about a 0.8 to 1.6 ml, about a 1.0 to 1.6 ml, about a 1.2 to 1.6 ml, about a 1.4 to 1.6 ml, about a 0.6 to
1.4 ml, about a 0.8 to 1.4 ml, about a 1.0 to 1.4 ml, about a 1.2 to 1.4 ml, about a 0.6 to 1.2 ml, about a 0.8 to
1.2 ml, about a 1.0 to 1.2 ml, about a 0.6 to 1.0 ml, about a 0.8 to 1.0 ml, or about a 0.6 to 0.8 ml fill volume of a pharmaceutical composition disclosed herein.
[0442] In some embodiments, a pharmaceutical composition of the present disclosure includes a LNP incorporating one polyribonucleotide. In some embodiments, a pharmaceutical composition includes a LNP incorporating a polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21-30, 180, and 182. In some embodiments, a pharmaceutical composition includes a LNP incorporating a polyribonucleotide encoding a MIR antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31-40, and 158. In some embodiments, a pharmaceutical composition includes a LNP incorporating a polyribonucleotide encoding an A35R antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 11-20, 172, and 174. In some embodiments, a pharmaceutical composition includes a LNP incorporating a polyribonucleotide encoding an H3L antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
[0443] In some embodiments, two or more pharmaceutical compositions are mixed prior to administration to a subject, where each of the two or more pharmaceutical compositions includes a LNP incorporating a different polyribonucleotide. For example, in some embodiments, four pharmaceutical compositions are mixed prior to administration to a subject, where one pharmaceutical composition includes a LNP incorporating a polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof, one pharmaceutical composition includes a LNP incorporating a polyribonucleotide encoding a MIR antigen, or antigenic fragment thereof, one pharmaceutical composition includes a LNP incorporating a polyribonucleotide encoding a A35R antigen, or antigenic fragment thereof, and one pharmaceutical composition includes a LNP incorporating a polyribonucleotide encoding a H3L antigen, or antigenic fragment thereof.
[0444] In some embodiments, a pharmaceutical composition of the present disclosure includes two or more LNPs, where each of the two or more LNPs incorporate a different polyribonucleotide. In some embodiments, a pharmaceutical composition described herein includes two or more of: a LNP incorporating a polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21-30, 180, and 182; a LNP incorporating a polyribonucleotide encoding a MIR antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31-40, and 158; a LNP incorporating a polyribonucleotide encoding an A35R antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 11-20, 172, and 174; and/or a LNP incorporating a polyribonucleotide encoding an H3L antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
[0445] In some embodiments, a pharmaceutical composition described herein includes: a LNP incorporating a polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21-30, 180, and 182; a LNP incorporating a polyribonucleotide encoding a MIR antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31-40, and 158; a LNP incorporating a polyribonucleotide encoding an A35R antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 11-20, 172, and 174; and a LNP incorporating a polyribonucleotide encoding an H3L antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
[0446] Pharmaceutical compositions described herein can be administered by appropriate methods known in the art. As will be appreciated by a skilled artisan, the route and/or mode of administration may depend on a number of factors, including, e.g., but not limited to stability and/or pharmacokinetics and/or pharmacodynamics of pharmaceutical compositions described herein.
[0447] In some embodiments, pharmaceutical compositions described herein are formulated for parenteral administration, which includes modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, subcuticular, or intraarticular injection and infusion. In preferred embodiments, pharmaceutical compositions described herein are formulated for intravenous, intramuscular, or subcutaneous administration. [0448] In some embodiments, pharmaceutical compositions described herein are formulated for intravenous administration. In some embodiments, pharmaceutically acceptable excipients that may be useful for intravenous administration include sterile aqueous solutions or dispersions and sterile powders for preparation of sterile injectable solutions or dispersions. [0449] Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. In some embodiments, a pharmaceutical composition described herein can be formulated as a solution, microemulsion, lipid nanoparticles, or other ordered structure suitable to high drug concentration. In some embodiments, a carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of surfactants. In some embodiments, a pharmaceutical composition may include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. In some embodiments, prolonged absorption of pharmaceutical compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
[0450] Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization and/or microfiltration. In some embodiments, pharmaceutical compositions can be prepared as described herein and/or methods known in the art.
[0451] These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Prevention of the presence of microorganisms may be ensured both by sterilization procedures, and by the inclusion of various antibacterial and antifungal agents, for example, paraben, chlorobutanol, phenol sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like into pharmaceutical compositions described herein. In addition, prolonged absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as aluminum monostearate and gelatin.
[0452] Formulations of pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing active ingredient(s) into association with a diluent or another excipient and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.
[0453] A pharmaceutical composition in accordance with the present disclosure may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of at least one RNA product produced using a system and/or method described herein.
[0454] Relative amounts of polyribonucleotides encapsulated in lipid nanoparticles, a pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition can vary, depending upon the subject to be treated, target cells, diseases or disorders, and may also further depend upon the route by which the composition is to be administered.
[0455] In some embodiments, pharmaceutical compositions described herein are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Actual dosage levels of the active ingredients (e.g., polyribonucleotides encapsulated in lipid nanoparticles) in the pharmaceutical compositions described herein may be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present disclosure employed, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.
[0456] A physician having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician could start doses of active ingredients (e.g., polyribonucleotides encapsulated in lipid nanoparticles) employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
[0457] In some embodiments, a pharmaceutical composition described herein is formulated (e.g., but not limited to, for intravenous, intramuscular, or subcutaneous administration) to deliver an active dose that confers a plasma concentration of an antigen or fragment thereof encoded by at least one polyribonucleotide (e.g., ones described herein) that mediates pharmacological activity via its dominant mode of action, viral neutralization.
[0458] In some embodiments, a pharmaceutical composition is formulated (e.g., but not limited to, for intravenous, intramuscular, or subcutaneous administration) to deliver a dose of 5 mg RNA/kg.
[0459] In some embodiments, a pharmaceutical composition described herein may further comprise one or more additives, for example, in some embodiments that may enhance stability of such a composition under certain conditions. Examples of additives may include but are not limited to salts, buffer substances, preservatives, and carriers. For example, in some embodiments, a pharmaceutical composition may further comprise a cryoprotectant and/or an aqueous buffered solution, which may in some embodiments include one or more salts, including, e.g., alkali metal salts or alkaline earth metal salts such as, e.g., sodium salts, potassium salts, and/or calcium salts.
[0460] In some embodiments, a pharmaceutical composition provided herein is a preservative-free, sterile RNA-lipid nanoparticle dispersion in an aqueous buffer for intravenous or intramuscular administration. [0461] Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions that are suitable for administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and/or perform such modification with merely ordinary, if any, experimentation.
V, Exemplary Compositions or Combinations
[0462] The present disclosure provides compositions (e.g., pharmaceutical compositions, e.g., immunogenic compositions, e.g., vaccines). The present disclosure also provides combinations. In some embodiments, combinations provided herein comprise combinations of polyribonucleotides provided herein. In some embodiments, combinations provided herein comprise combinations of LNP-polyribonucleotides provided herein. Combinations provided herein also comprise combinations of compositions provided herein. Nonlimiting, exemplary compositions and combinations are provided below.
[0463] The present disclosure provides compositions, including lipid nanoparticles (LNPs) as described herein that incorporate at least one (e.g., one, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more) polyribonucleotide(s), where the at least one polyribonucleotide encodes at least one (e.g., one, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more) MPXV antigen(s). [0464] The present disclosure provides combinations, including lipid nanoparticles (LNPs) as described herein that incorporate at least one (e.g., one, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more) polyribonucleotide(s), where the at least one polyribonucleotide encodes at least one (e.g., one, 2 or more, 3 or more, 4 or more, 5 or more, or 6 or more) MPXV antigen(s).
[0465] In some embodiments, a composition of the present disclosure includes a LNP incorporating one polyribonucleotide. In some embodiments, a composition includes a LNP incorporating a polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21-30, 180, and 182. In some embodiments, a composition includes a polyribonucleotide encoding a MIR antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31-40, and 158. In some embodiments, a composition includes a polyribonucleotide encoding an A35R antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 11-20, 172, and 174. In some embodiments, a composition includes a polyribonucleotide encoding an H3L antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
[0466] In some embodiments, a combination of the present disclosure includes a LNP incorporating one polyribonucleotide. In some embodiments, a combination includes a LNP incorporating a polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21-30, 180, and 182. In some embodiments, a combination includes a polyribonucleotide encoding a MIR antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31-40, and 158. In some embodiments, a combination includes a polyribonucleotide encoding an A35R antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 11-20, 172, and 174. In some embodiments, a combination includes a polyribonucleotide encoding an H3L antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
[0467] In some embodiments, a composition of the present disclosure includes two, three, or four
LNPs, where each LNP incorporates one polyribonucleotide encoding a different mpox virus antigen. In some embodiments, a combination of the present disclosure includes two, three, or four LNPs, where each LNP incorporates one polyribonucleotide encoding a different mpox virus antigen.
[0468] In some embodiments, a composition of the present disclosure includes three LNPs. In some embodiments, a combination of the present disclosure includes three LNPs. In some embodiments, three LNPs comprise an LNP encompassing a polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof, an LNP encompassing a polyribonucleotide encoding an MIR antigen, or antigenic fragment thereof, and an LNP encompassing a polyribonucleotide encoding an A35L antigen, or antigenic fragment thereof. In some embodiments, a B6R antigen, or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 1-30, 180, and 182. In some embodiments, an MIR antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31- 40, and 158. In some embodiments, an A35R antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 11-20, 172, and 174.
[0469] In some embodiments, a composition of the present disclosure includes four LNPs. In some embodiments, a combination of the present disclosure includes four LNPs. In some embodiments, four LNPs comprise an LNP encompassing a a polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof, an LNP encompassing a polyribonucleotide encoding an MIR antigen, or antigenic fragment thereof, an LNP encompassing a polyribonucleotide encoding an A35L antigen, or antigenic fragment thereof, and an LNP encompassing a polyribonucleotide encoding an H3L antigen, or antigenic fragment thereof. In some embodiments, a B6R antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21-30, 180, and 182. In some embodiments, an MIR antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31- 40, and 158. In some embodiments, an A35R antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 11-20, 172, and 174. In some embodiments, an H3L antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 51-60, 184, 188, and 190. In some embodiments, a B6R antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21-30, 180, and 182; an MIR antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31-40, and 158; an A35R antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 11-20, 172, and 174; and an H3L antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
[0470] In some embodiments, a composition of the present disclosure includes a LNP incorporating two polyribonucleotides. In some embodiments, a combination of the present disclosure includes an LNP incorporating two polyribonucleotides. In some embodiments, two LNPs comprise an LNP encompassing a polyribonucleotide encoding a B6R antigen, or an antigenic fragment thereof, and an LNP encompassing a polyribonucleotide encoding an MIR antigen. In some embodiments, B6R antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21-30, 180, and 182. In some embodiments, an MIR antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31-40, and 158. In some embodiments, B6R antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21-30, 180, and 182; and an MIR antigen, or antigenic fragment thereof, having an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31-40, and 158.
[0471] In some embodiments, a composition of the present disclosure includes a LNP incorporating three polyribonucleotides. In some embodiments, a combination of the present disclosure includes a LNP incorporating three polyribonucleotides. In some embodiments, an LNP encompasses a polyribonucleotide encoding a B6R antigen, or antigenic fragment thereof, a polyribonucleotide encoding a MIR antigen, or antigenic fragment thereof, and a polyribonucleotide encoding a A35R antigen, or antigenic fragment thereof. In some embodiments, a B6R antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21-30, 180 and 189. In some embodiments, an MIR antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31-40, and 158. In some embodiments, an A35R antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 11-20, 179 and 174. In some embodiments, a B6R antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21-30, 180 and 189; an MIR antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31-40, and 158; and an A35R antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 11-20, 179 and 174.
[0472] In some embodiments, a composition of the present disclosure includes a LNP incorporating four polyribonucleotides. In some embodiments, a combination of the present disclosure includes a LNP incorporating four polyribonucleotides. In some embodiments, four polyribonucleotides are encompassed in an LNP. In some embodiments, an LNP encompasses a polyribonucleotide encoding a B6R antigen, or an antigenic fragment thereof, a polyribonucleotide encoding a MIR antigen, or an antigenic fragment thereof, a polyribonucleotide encoding an A35R antigen, or an antigenic fragment thereof, and a polyribonucleotide encoding an H3L antigen, or an antigenic fragment thereof In some embodiments, a B6R antigen or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21-30, 180, and 182. In some embodiments, an MIR antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31-40, and 158. In some embodiments, an A35R antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 11-20, 172, and 174. In some embodiments, an H3L antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 51-60, 184, 188, and 190. In some embodiments, a B6R antigen or antigenic fragment thereof comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 21- 30, 180, and 182; an MIR antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least
95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 31-40, and
158; an A35R antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 11-20, 172, and 174; and an H3L antigen, or antigenic fragment thereof, comprises an amino acid sequence at least 85% identical (e.g., at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
VI. Characterization
[0473] Without wishing to be bound by any particular theory, it is proposed that ability to induce an antibody response may be important to effectiveness of a composition for treatment and/or prevention of orthopox (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, or vaccinia (e.g., modified vaccinia virus Ankara, rabbitpox, etc.)) infection, (e.g., a pharmaceutical composition, an immunogenic composition, or a vaccine).
[0474] In some embodiments, provided technologies (e.g., compositions and/or dosing regimens, combinations, etc.) are characterized by an ability to induce (e.g., when administered to a model system and/or to a human, for example by parenteral administration such as by intramuscular administration) an antibody response targeting one or more mpox antigen(s) described herein. That is, in some embodiments, provided technologies are characterized in that, when administered (e.g., by parenteral administration such as by intramuscular administration) to an organism (e.g., a model organism or an animal or human organism in need of protection), provided technologies induce a robust antibody response targeting one or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)) antigens. For the purpose of this section, it should be understood that reference to a "composition" also encompasses "combinations" to the extent such reference makes technical sense.
[0475] In some embodiments, provided technologies are characterized in that they induce antibody titers to a level that provides sufficient protective response against an orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)), when administered to a relevant population.
[0476] In some embodiments, provided technologies are characterized in that they induce antibody titers to one or more mpox antigen(s) in a range of 103-105 after at 5 days, 10 days, 21 days, or 28 days post immunization.
[0477] In some embodiments, administration of a composition described herein comprising one or more polyribonucleotides each encoding one or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)) antigens or fragments thereof is characterized in that it can induce antibody titers of at least 103 by 5 days post immunization. In some embodiments, administration of a composition described herein comprising one or more polyribonucleotides each encoding one or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)) antigens or fragments thereof is characterized in that it can induce antibody titers of at least 104 by 10 days post immunization. In some embodiments, administration of a composition described herein comprising one or more polyribonucleotides each encoding one or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)) antigens or fragments thereof is characterized in that it can induce antibody titers of at least 105 by 21 days post immunization.
[0478] In some embodiments, administration of a second dose of a composition described herein induces a further increase in antibody titers. In some embodiments, a second dose is administered at least 21 days after a first dose. In some embodiments, a second dose is characterized in that it can induce antibody titers of at least 105 by at least 5 days after administration of the second dose. In some embodiments, a second dose is characterized in that it can induce antibody titers of at least 105 by at least 10 days after administration of the second dose.
[0479] In some embodiments, administration of a composition described herein that delivers two or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)) antigens in combination induces similar antibody levels to single antigen immunization.
[0480] In some embodiments, administration of a composition described herein that delivers two or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)) antigens is characterized in that it induces an antibody response in a range of 103-105 after at 5 days, 10 days, 21 days, or 28 days post immunization. [0481] In some embodiments, administration of a composition described herein that delivers two or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)) antigens is characterized in that it induces antibody titers of at least 103 by 5 days post immunization. In some embodiments, administration of a composition described herein that delivers two or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)) antigens is characterized in that it induces antibody titers of at least 104 by 10 days post immunization. In some embodiments, administration of a composition described herein that delivers two or more orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)) antigens is characterized in that it induces antibody titers of at least 105 by 21 days post immunization.
[0482] In some embodiments, administration of a composition described herein to a subject induces antibody production (e.g., IgG, IgA, IgM, IgE). For example, in some embodiments, administration of a composition described herein to a subject induces production of one or more of IgGl, IgG2A, IgG2B, and IgG3. [0483] In some embodiments, administration of a composition described herein to a subject induces the production of orthopoxvirus neutralizing antibodies. In some embodiments, administration of a composition described herein to a subject induces the production of B6R-binidng, MIR-binding, A35R-binding, and/or H3L- binding antibodies in the subject. In some embodiments, administration of a composition described herein to a subject induces enhanced orthopoxvirus neutralizing antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a composition described herein to a subject induces enhanced B6R-binding antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a composition described herein to a subject induces enhanced MIR-binding antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a composition described herein to a subject induces enhanced A35R-binding antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a composition described herein to a subject induces enhanced A35R-binding antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a composition described herein to a subject induces enhanced H3L-binding antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
[0484] In some embodiments, administration of a composition described herein to a subject induces antibody Fc-effector functional activities (e.g., antibody-mediated complement activation, antibody-mediated NK cell activation, antibody-mediated NK cell degranulation, antibody-mediated IFNy release, antibody-mediated MIPlb release, antibody-dependent cellular monocyte phagocytosis). In some embodiments, administration of a composition described herein to a subject induces enhanced antibody Fc-effector functional activities (e.g., antibody-mediated complement activation, antibody-mediated NK cell activation, antibody-mediated NK cell degranulation, antibody-mediated IFNy release, antibody-mediated MIPlb release, antibody-dependent cellular monocyte phagocytosis) as compared to functional activities induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). [0485] In some embodiments, administration of a composition described herein to a subject reduces the number of orthopoxvirus lesions in the subject. In some embodiments, administration of a composition described herein to a subject reduces the severity of orthopoxvirus lesions in the subject. In some embodiments, administration of a composition described herein to a subject reduces or prevents the onset of orthopoxvirus lesions in the subject. In some embodiments, administration of a composition described herein to a subject reduces the duration of orthopoxvirus lesion onset to resolution in the subject. In some embodiments, administration of a composition described herein to a subject results in greater reduction in the number of orthopoxvirus lesions in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a composition described herein to a subject results in enhanced reduction in the severity of orthopoxvirus lesions in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a composition described herein to a subject results in enhanced reduction or prevention of the onset of orthopoxvirus lesions in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a composition described herein to a subject results in enhanced reduction in the duration of orthopoxvirus lesion onset to resolution in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
[0486] In some embodiments, administration of a composition described herein to a subject reduces the orthopoxvirus viral load in the subject. In some embodiments, administration of a composition described herein to a subject reduces the orthopoxvirus viral load in the throat of the subject. In some embodiments, administration of a composition described herein to a subject reduces the orthopoxvirus viral load in the blood of the subject. In some embodiments, administration of a composition described herein to a subject results in an enhanced reduction in the orthopoxvirus viral load in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a composition described herein to a subject results in an enhanced reduction in the orthopoxvirus viral load in the throat of the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a composition described herein to a subject results in an enhanced reduction in the orthopoxvirus viral load in the blood of the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
[0487] In some embodiments, administration of a composition described herein to a subject reduces orthopoxvirus virus replication in the subject. In some embodiments, administration of a composition described herein to a subject reduces orthopoxvirus virus replication in the throat of the subject. In some embodiments, administration of a composition described herein to a subject reduces orthopoxvirus virus replication in the blood of the subject. In some embodiments, administration of a composition described herein to a subject results in an enhanced reduction in orthopoxvirus virus replication in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a composition described herein to a subject results in an enhanced reduction in orthopoxvirus virus replication in the throat of the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a composition described herein to a subject results in an enhanced reduction in orthopoxvirus virus replication in the blood of the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
[0488] In some embodiments, administration of a composition described herein to a subject reduces weight loss associated with orthopoxvirus infection in the subject. In some embodiments, administration of a composition described herein to a subject results in an enhanced reduction in weight loss associated with orthopoxvirus infection in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
[0489] In some embodiments, administration of a composition described herein to a subject reduces morbidity associated with orthopoxvirus infection in the subject. In some embodiments, administration of a composition described herein to a subject results in an enhanced reduction in morbidity associated with orthopoxvirus infection in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a composition described herein to a subject reduces morbidity associated with a lethal orthopoxvirus challenge in the subject. In some embodiments, administration of a composition described herein to a subject results in an enhanced reduction in morbidity associated with a lethal orthopoxvirus challenge in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). [0490] In some embodiments, administration of a composition described herein to a subject induces the production of orthopoxvirus neutralizing antibodies. In some embodiments, administration of a composition described herein to a subject induces the production of B6R-binidng, MIR-binding, A35R-binding, and/or H3L- binding antibodies in the subject. In some embodiments, administration of a composition described herein to a subject induces enhanced orthopoxvirus neutralizing antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a composition described herein to a subject induces enhanced B6R-binding antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a composition described herein to a subject induces enhanced MIR-binding antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a composition described herein to a subject induces enhanced A35R-binding antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a composition described herein to a subject induces enhanced A35R-binding antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a composition described herein to a subject induces enhanced H3L-binding antibody titers as compared to titers induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
[0491] In some embodiments, administration of a combination described herein to a subject induces antibody Fc-effector functional activities (e.g., antibody-mediated complement activation, antibody-mediated NK cell activation, antibody-mediated NK cell degranulation, antibody-mediated IFNy release, antibody-mediated MIPlb release, antibody-dependent cellular monocyte phagocytosis). In some embodiments, administration of a combination described herein to a subject induces enhanced antibody Fc-effector functional activities (e.g., antibody-mediated complement activation, antibody-mediated NK cell activation, antibody-mediated NK cell degranulation, antibody-mediated IFNy release, antibody-mediated MIPlb release, antibody-dependent cellular monocyte phagocytosis) as compared to functional activities induced by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
[0492] In some embodiments, administration of a combination described herein to a subject reduces the number of orthopoxvirus lesions in the subject. In some embodiments, administration of a combination described herein to a subject reduces the severity of orthopoxvirus lesions in the subject. In some embodiments, administration of a combination described herein to a subject reduces or prevents the onset of orthopoxvirus lesions in the subject. In some embodiments, administration of a combination described herein to a subject reduces the duration of orthopoxvirus lesion onset to resolution in the subject. In some embodiments, administration of a combination described herein to a subject results in greater reduction in the number of orthopoxvirus lesions in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a combination described herein to a subject results in enhanced reduction in the severity of orthopoxvirus lesions in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankarabased vaccine (e.g., JYNEOS®). In some embodiments, administration of a combination described herein to a subject results in enhanced reduction or prevention of the onset of orthopoxvirus lesions in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a combination described herein to a subject results in enhanced reduction in the duration of orthopoxvirus lesion onset to resolution in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
[0493] In some embodiments, administration of a combination described herein to a subject reduces the orthopoxvirus viral load in the subject. In some embodiments, administration of a combination described herein to a subject reduces the orthopoxvirus viral load in the throat of the subject. In some embodiments, administration of a combination described herein to a subject reduces the orthopoxvirus viral load in the blood of the subject. In some embodiments, administration of a combination described herein to a subject results in an enhanced reduction in the orthopoxvirus viral load in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a combination described herein to a subject results in an enhanced reduction in the orthopoxvirus viral load in the throat of the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a combination described herein to a subject results in an enhanced reduction in the orthopoxvirus viral load in the blood of the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
[0494] In some embodiments, administration of a combination described herein to a subject reduces orthopoxvirus virus replication in the subject. In some embodiments, administration of a combination described herein to a subject reduces orthopoxvirus virus replication in the throat of the subject. In some embodiments, administration of a combination described herein to a subject reduces orthopoxvirus virus replication in the blood of the subject. In some embodiments, administration of a combination described herein to a subject results in an enhanced reduction in orthopoxvirus virus replication in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a combination described herein to a subject results in an enhanced reduction in orthopoxvirus virus replication in the throat of the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a combination described herein to a subject results in an enhanced reduction in orthopoxvirus virus replication in the blood of the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
[0495] In some embodiments, administration of a combination described herein to a subject reduces weight loss associated with orthopoxvirus infection in the subject. In some embodiments, administration of a combination described herein to a subject results in an enhanced reduction in weight loss associated with orthopoxvirus infection in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®).
[0496] In some embodiments, administration of a combination described herein to a subject reduces morbidity associated with orthopoxvirus infection in the subject. In some embodiments, administration of a combination described herein to a subject results in an enhanced reduction in morbidity associated with orthopoxvirus infection in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). In some embodiments, administration of a combination described herein to a subject reduces morbidity associated with a lethal orthopoxvirus challenge in the subject. In some embodiments, administration of a combination described herein to a subject results in an enhanced reduction in morbidity associated with a lethal orthopoxvirus challenge in the subject as compared to the reduction achieved by a reference contemporary modified vaccinia Ankara-based vaccine (e.g., JYNEOS®). VII. Patient Populations
[0497] Technologies provided herein can be useful for treatment and/or prevention of an orthopox
(e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)) infection. As described herein, technologies include polyribonucleotides. Accordingly, the present disclosure provides pharmaceutical compositions for treatment and or prevention of an orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.)) infection. In some embodiments, a pharmaceutical composition comprises a polyribonucleotide as described herein.
[0498] In some embodiments, a subject is one suffering from and/or is susceptible to an orthopox infection. In some embodiments, a subject is one suffering from and/or is susceptible to mpox infection (e.g., a clade la, clade lb, clade Ila, or clade lib mpox infection). In some embodiments, a subject is one suffering from and/or is susceptible to variola infection. In some embodiments, a subject is one suffering from and/or is susceptible to vaccinia (e.g., modified vaccinia virus Ankara, etc.) infection. In some embodiments, a subject may be defined by one or more criterion such as age group, gender, genetic background, preexisting clinical conditions, and/or prior exposure to therapy.
[0499] In some embodiments, a subject is a model organism. In preferred embodiments, a subject is a human. In some embodiments, a subject is between 18-65 years of age. In some embodiments, a subject is an age in a range of from about 0 months to about 6 months old, from about 6 to about 12 months old, from about 6 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old, or from about 95 to about 100 years old.
[0500] In some embodiments, a subject is a human infant. In some embodiments, a subject is a human toddler. In some embodiments, a subject is a human child. In some embodiments, a subject is a human adult. In some embodiments, a subject is an elderly human. In some embodiments, a subject is a pregnant woman. In some embodiments, a subject is a human adolescent. In some embodiments, a subject is immunocompromised. In some embodiments, a subject is HIV-positive.
[0501] Among the various advantages of certain methods and compositions provided herein, including in particular methods and compositions of orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, modified vaccinia virus Ankara, or vaccinia) vaccination that include administration of polyribonucleotide of the present disclosure, are that such methods and compositions are not subject to certain limitations that characterize other vaccine technologies. To provide one particular example, live attenuated virus vaccines (such as Dryvax) are contraindicated for subjects who are immunocompromised (e.g., severely immunocompromised) at least in part because such subjects are at increased risk for serious adverse reactions. Subjects with most forms of altered immunocompetence should not receive live vaccines. The present disclosure includes that methods and compositions provided herein can be used to treat (e.g., to vaccinate) subjects who are immunocompromised (e.g., severely immunocompromised) and/or subjects with altered immunocompetence (including without limitation primary and/or secondary immunosuppression, immunodeficiency, and immunocompromised).
[0502] In some embodiments, a subject has no prior history of known or suspected smallpox vaccination prior to administration of one or more doses of a composition as disclosed herein (also referred to as "vaccinia naive"). In some embodiments, a subject has a prior history of a smallpox vaccination (also referred to as "vaccinia-experienced")- In some embodiments, a subject's smallpox vaccination status is determined by the presence or absence of a smallpox vaccination scar.
[0503] In some embodiments, a subject does not have febrile illness prior to administration of one or more doses of a composition as disclosed herein. In some embodiments, a subject does not have febrile illness within about 72 hours, about 48 hours, about 36 hours, about 24 hours, or about 12 hours prior to administration of one or more doses of a composition as disclosed herein.
[0504] In some embodiments, a subject does not have an acute illness prior to administration of one or more doses of a composition as disclosed herein. In some embodiments, a subject does not have an acute illness within about 72 hours, about 48 hours, about 36 hours, about 24 hours, or about 12 hours prior to administration of one or more doses of a composition as disclosed herein.
[0505] In some embodiments, a subject has not received a vaccine within 7 days to 56 days, 14 days to 56 days, 21 days to 56 days, 28 days to 56 days, 35 days to 56 days, 42 days to 56 days, 49 to 56 days, 7 days to 49 days, 14 days to 49 days, 21 days to 49 days, 28 days to 49 days, 35 days to 49 days, 42 days to 49 days, 7 days to 42 days, 14 days to 42 days, 21 days to 42 days, 28 days to 42 days, 35 days to 42 days, 7 days to 35 days, 14 days to 35 days, 21 days to 35 days, 28 days to 35 days, 7 days to 28 days, 14 days to 28 days, 21 days to 28 days, 7 days to 21 days, 14 days to 21 days, or 7 days to 14 days, before being administered one or more doses of a therapeutically effective amount of a composition as disclosed herein. In some embodiments, a subject has not received a vaccine within about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, or about 56 days before being administered one or more doses of a therapeutically effective amount of a composition as disclosed herein. In some embodiments, a subject has not received a vaccine within about 28 days, before being administered one or more doses of a therapeutically effective amount of a composition as disclosed herein. In some embodiments, the vaccine is not a seasonal influenza vaccine or a medically indicated vaccine.
[0506] In some embodiments, a subject has not received a vaccine within 1 to 10 half-lives, 2 to 10 half-lives, 3 to 10 half-lives, 4 to 10 half-lives, 5 to 10 half-lives, 6 to 10 half-lives, 7 to 10 half-lives, 8 to 10 halflives, 9 to 10 half-lives, 1 to 9 half-lives, 2 to 9 half-lives, 3 to 9 half-lives, 4 to 9 half-lives, 5 to 9 half-lives, 6 to 9 half-lives, 7 to 9 half-lives, 8 to 9 half-lives, 1 to 8 half-lives, 2 to 8 half-lives, 3 to 8 half-lives, 4 to 8 half-lives, 5 to 8 half-lives, 6 to 8 half-lives, 7 to 8 half-lives, 1 to 7 half-lives, 2 to 7 half-lives, 3 to 7 half-lives, 4 to 7 halflives, 5 to 7 half-lives, 6 to 7 half-lives, 1 to 6 half-lives, 2 to 6 half-lives, 3 to 6 half-lives, 4 to 6 half-lives, 5 to 6 half-lives, 1 to 5 half-lives, 2 to 5 half-lives, 3 to 5 half-lives, 4 to 5 half-lives, 1 to 4 half-lives, 2 to 4 half-lives, 3 to 4 half-lives, 1 to 3 half-lives, 2 to 3 half-lives, or 1 to 2 half-lives of the vaccine, before being administered one or more doses of a therapeutically effective amount of a composition as disclosed herein. In some embodiments, a subject has not received a vaccine within about 1 half-life, about 2 half-lives, about 3 half-lives, about 4 half-lives, about 5 half-lives, about 6 half-lives, about 7 half-lives, about 8 half-lives, about 9 half-lives, or about 10 half-lives of the vaccine, before being administered one or more doses of a therapeutically effective amount of a composition as disclosed herein. In some embodiments, a subject has not received a vaccine within about 5 half-lives of the vaccine, before being administered one or more doses of a therapeutically effective amount of a composition as disclosed herein. In some embodiments, the vaccine is not a seasonal influenza vaccine or a medically indicated vaccine.
[0507] In some embodiments, a subject does not receive a vaccine within at least 2 weeks to 10 weeks, at least 4 weeks to 10 weeks, at least 6 weeks to 10 weeks, at least 8 weeks to 10 weeks, at least 2 weeks to 8 weeks, at least 4 weeks to 8 weeks, at least 6 weeks to 8 weeks, at least 2 weeks to 6 weeks, at least 4 weeks to 6 weeks, or at least 2 weeks to 4 weeks after administration of one or more doses of a therapeutically effective amount of a composition as disclosed herein. In some embodiments, a subject does not receive a vaccine within at least about 2 weeks, at least about 4 weeks, at least about 6 weeks, at least about 8 weeks, or at least about 10 weeks after administration of one or more doses of a therapeutically effective amount of a composition as disclosed herein. In some embodiments, a subject does not receive a vaccine within at least about 8 weeks after administration of one or more doses of a therapeutically effective amount of a composition as disclosed herein. In some embodiments, the vaccine is not a seasonal influenza vaccine or a medically indicated vaccine.
[0508] In some embodiments, a subject has not received an orthopoxvirus-based vaccine before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein. In some embodiments, an orthopoxvirus-based vaccine is selected from, but not limited to, a vaccinia vaccine, an mpox vaccine, a variola vaccine, a borealpox vaccine, an ectromelia vaccine, a cowpox vaccine, a volepox vaccine , a buffalopox vaccine, a camelpox vaccine, rabbitpox vaccine, a vaccinia (e.g., modified vaccinia virus Ankara, etc.) vaccine, and a vector orthopoxvirus-based vaccine.
[0509] In some embodiments, a subject has not received blood, plasma products, or immunoglobulins within about 80 to 160 days, about 100 to 160 days, about 120 to 160 days, about 140 to 160 days, about 80 to 140 days, about 100 to 140 days, about 120 to 140 days, about 80 to 120 days, about 80 to 120 days, about 100 to 120 days, or about 80 to 100 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein. In some embodiments, a subject has not received blood, plasma products, or immunoglobulins within about 80 days, about 100 days, about 120 days, about 140 days, or about 160 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein. In some embodiments, a subject has not received blood, plasma products, or immunoglobulins within about 120 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein.
[0510] In some embodiments, a subject has not received an allergy treatment within 8 to 20 days, 10 to 20 days, 12 to 20 days, 14 to 20 days, 16 to 20 days, 18 to 20 days, 8 to 18 days, 10 to 18 days, 12 to 18 days, 14 to 18 days, 16 to 18 days, 8 to 16 days, 10 to 16 days, 12 to 16 days, 14 to 16 days, 8 to 14 days, 10 to 14 days, 12 to 14 days, 8 to 12 days, 10 to 12 days, or about 8 to 10 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein. In some embodiments, a subject has not received an allergy treatment within about 8 days, about 10 days, about 12 days, about 14 days, about 16 days, about 18 days, or about 20 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein. In some embodiments, a subject has not received an allergy treatment within about 14 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein. In some embodiments, an allergy treatment comprises antigen injections. [0511] In some embodiments, a subject has not received an immunosuppressive medication within 7 to 56 days, 14 to 56 days, 21 to 56 days, 28 to 56 days, 35 to 56 days, 42 to 56 days, 49 to 56 days, 7 to 49 days, 14 to 49 days, 21 to 49 days, 28 to 49 days, 35 to 49 days, 42 to 49 days, 7 to 42 days, 14 to 42 days, 21 to 42 days, 28 to 42 days, 35 to 42 days, 7 to 35 days, 14 to 35 days, 21 to 35 days, 28 to 35 days, 7 to 28 days, 14 to 28 days, 21 to 28 days, 7 to 21 days, 14 to 21 days, or 7 to 14 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein. In some embodiments, a subject has not received an immunosuppressive medication within about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, or about 56 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein. In some embodiments, a subject has not received an immunosuppressive medication within about 28 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein.
[0512] In some embodiments, an immunosuppressive medication comprises a systemic corticosteroid or radiotherapy. In some embodiments, a systemic corticosteroid is selected from, but not limited to, methylprednisolone, dexamethasone, hydrocortisone, prednisone, prednisolone, fluticasone, flumethasone, fluocinolone, budesonide, beclomethasone, ciclesonide, cortisone, triamcinolone, betamethasone, deflazacort, difluprednate, loteprednol, para methasone, tixocortol, aldosterone, cloprednol, cortivazol, deoxycortone, desonide, desoximetasone, difluorocortolone, fluclorolone, fludrocortisone, flunisolide, fluocinonide, fluocortin butyl, fluorocortisone, fluorocortolone, fluoromethoIone, flurandrenolone, halcinonide, icomethasone, meprednisone, mometasone, rofleponide, RPR 106541, and their respective pharmaceutically acceptable derivatives, such as beclomethasone dipropionate (anhydrous or monohydrate), beclomethasone monopropionate, dexamethasone 21-isonicotinate, fluticasone propionate, icomethasone enbutate, tixocortol 21- pivalate, triamcinolone acetonide, and pharmaceutically acceptable salts and/or derivatives thereof. In some embodiments, a corticosteroid is prednisone.
[0513] In some embodiments, a subject has not received a prophylactic antipyretic and/or an analgesic medication within 7 to 56 days, 14 to 56 days, 21 to 56 days, 28 to 56 days, 35 to 56 days, 42 to 56 days, 49 to 56 days, 7 to 49 days, 14 to 49 days, 21 to 49 days, 28 to 49 days, 35 to 49 days, 42 to 49 days, 7 to 42 days, 14 to 42 days, 21 to 42 days, 28 to 42 days, 35 to 42 days, 7 to 35 days, 14 to 35 days, 21 to 35 days, 28 to 35 days, 7 to 28 days, 14 to 28 days, 21 to 28 days, 7 to 21 days, 14 to 21 days, or 7 to 14 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein. In some embodiments, a subject has not received a prophylactic antipyretic and/or an analgesic medication within about 7 days, about 14 days, about 21 days, about 28 days, about 35 days, about 42 days, about 49 days, or about 56 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein. In some embodiments, a subject has not received a prophylactic antipyretic and/or an analgesic medication within about 14 days before administration of one or more doses of a therapeutically effective amount of a composition disclosed herein.
[0514] In some embodiments, a prophylactic antipyretic medication is selected from, but not limited to, acetaminophen, a non-steroidal anti-inflammatory drug (NSAID), salicylamide, salicyl salicylate, methyl salicylate, magnesium salicylate, faislamine, ethenzamide, diflunisal, choline magnesium salicylate, benorylate/benorilatem and amoxiprin, acetylsalicylate, ceclofenac, acemetacin, alclofenac, bromfenac, diclofenac, etodolac, indomethacin, nabumetone, oxametacin, proglumetacin, sulindac, tolmetin, iminoprofen, benoxaprofen, carprofen, dexibuprofen, dexketoprofen, fenbufen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, ketorolac, loxoprofen, naproxen, oxaprozin, pirprofen, suprofen, tiaprofenic acid, mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, droxicam, lornoxicam, meloxicam, piroxicam, tenoxicam, dipyrone, azapropazone, clofezone, kebuzone, metamizole, mofebutazone, oxyphenbutazone, phenazone, phenylbutazone, sulfinpyrazone, decoxib, rofecoxib, parecoxib, and etoricoxib. [0515] In some embodiments, a prophylactic analgesic medication is selected from, but not limited to, acetaminophen, salicylamide, salicyl salicylate, methyl salicylate, magnesium salicylate, faislamine, ethenzamide, diflunisal, choline magnesium salicylate, benorylate/benorilatem and amoxiprin, acetylsalicylate, ceclofenac, acemetacin, alclofenac, bromfenac, diclofenac, etodolac, indomethacin, nabumetone, oxametacin, proglumetacin, sulindac, tolmetin, iminoprofen, benoxaprofen, carprofen, dexibuprofen, dexketoprofen, fenbufen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, ketorolac, loxoprofen, naproxen, oxaprozin, pirprofen, suprofen, tiaprofenic acid, mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, droxicam, lornoxicam, meloxicam, piroxicam, tenoxicam, dipyrone, azapropazone, clofezone, kebuzone, metamizole, mofebutazone, oxyphenbutazone, phenazone, phenylbutazone, sulfinpyrazone, decoxib, rofecoxib, parecoxib, etoricoxib, codeine, dihydrocodeine, morphine or a morphine derivative or pharmaceutically acceptable salt thereof, diacetylmorphine, hydrocodone, hydromorphone, levorphanol, oxymorphone, alfentanil, buprenorphine, butorphanol, fentanyl, sufentanil, meperidine, methadone, nalbuphine, propoxyphene, pentazocine, and pharmaceutically acceptable salts thereof.
[0516] In some embodiments, a subject administered with one or more doses of a therapeutically effective amount of a composition disclosed herein is negative for HIV-1 and/or HIV-2. In some embodiments, a subject administered with one or more doses of a therapeutically effective amount of a composition disclosed herein is negative for hepatitis B and/or hepatitis C.
VIII. Treatment Methods
[0517] In some embodiments, a pharmaceutical composition described herein can be taken up by cells for production of an encoded agent at therapeutically relevant serum concentrations. Accordingly, the present disclosure provides methods of using pharmaceutical compositions described herein. For example, in some embodiments, a method provided herein comprises administering a pharmaceutical composition described herein to a subject.
[0518] In some embodiments, a pharmaceutical composition described herein is for the prophylactic protection and/or prevention against orthopoxvirus infection. In some embodiments, a pharmaceutical composition described herein is for the prophylactic protection and/or prevention against mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus infection. In some embodiments, a pharmaceutical composition described herein is for the prophylactic protection and/or prevention against an mpox clade la, clade lb, clade Ila, or clade lib infection. In some embodiments, a pharmaceutical composition described herein is for the prophylactic protection and/or prevention against an mpox clade la infection. In some embodiments, a pharmaceutical composition described herein is for the prophylactic protection and/or prevention against an mpox clade lb infection.
[0519] In some embodiments, a pharmaceutical composition described herein is for treatment of a subject having mild orthopoxvirus infection and/or mild orthopoxvirus-related disease. In some embodiments, a pharmaceutical composition described herein is for treatment of a subject having mild orthopoxvirus infection and/or moderate orthopoxvirus-related disease. In some embodiments, a pharmaceutical composition described herein is for treatment of a subject having severe orthopoxvirus infection and/or mild orthopoxvirus-related disease.
[0520] In some embodiments, a pharmaceutical composition described herein is for post-exposure prevention of orthopoxvirus transmission to others. In some embodiments, a pharmaceutical composition described herein is for post-exposure prevention of mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus transmission to others. In some embodiments, a pharmaceutical composition described herein is for post-exposure prevention of mpox clade la, clade lb, clade Ila, or clade lib virus transmission to others. In some embodiments, a pharmaceutical composition described herein is for post-exposure prevention of mpox clade la virus transmission to others. In some embodiments, a pharmaceutical composition described herein is for post-exposure prevention of mpox clade lb virus transmission to others.
[0521] In some embodiments, a pharmaceutical composition described herein is for protection and/or prevention of post-exposure orthopoxvirus disease progression. In some embodiments, a pharmaceutical composition described herein is for post-exposure protection and/or prevention of mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus disease progression. In some embodiments, a pharmaceutical composition described herein is for post-exposure protection and/or prevention of mpox clade la, clade lb, clade Ila, or clade lib virus disease progression. In some embodiments, a pharmaceutical composition described herein is for post-exposure protection and/or prevention of mpox clade la virus disease progression. In some embodiments, a pharmaceutical composition described herein is for post-exposure protection and/or prevention of mpox clade lb virus disease progression.
[0522] As used herein, the term "administering" or "administration" typically refers to the administration of a composition to a subject to achieve delivery of an agent {e.g., at least one polyribonucleotide encoding an antigen or fragment thereof described herein) that is, or is included in, a composition to a target site or a site to be treated. Those of ordinary skill in the art will be aware of a variety of routes that may, in appropriate circumstances, be utilized for administration to a subject, for example a human. Administration may be, for example, bronchial e.g., by bronchial instillation), buccal, dermal (which may be or comprise, for example, one or more of topical to the dermis, intradermal, interdermal, transdermal, etc.), enteral, intra-arterial, intradermal, intragastric, intramedullary, intramuscular, intranasal, intraperitoneal, intrathecal, intravenous, intraventricular, within a specific organ (e.g., intrahepatic), mucosal, nasal, oral, rectal, subcutaneous, sublingual, topical, tracheal e.g., by intratracheal instillation), vaginal, vitreal, etc. In preferred embodiments, administration may be intramuscular, intravenous, or subcutaneous.
[0523] In some embodiments, administration of a pharmaceutical composition results in delivery of one or more polyribonucleotides as described herein {e.g., encoding an antigen or antigenic fragment thereof) to a subject. In some embodiments, administering a pharmaceutical composition to a subject results in expression in the subject of an antigen or fragment thereof encoded by an administered polyribonucleotide. In some embodiments, administering a pharmaceutical composition to a subject results in expression in the subject of an antigen or antigenic fragment thereof encoded by an administered polyribonucleotide.
[0524] In some embodiments, a pharmaceutical composition for administration to a subject is provided as two or more separate particle compositions each comprising one or more polyribonucleotides of the present disclosure (e.g., encoding an mpox antigen as described herein), which are then mixed together prior to administration. For example, in some embodiments, individual populations of nucleic acid containing particles, each population comprising an RNA molecule encoding a different immunogenic polypeptide or antigenic fragment thereof (e.g., an mpox antigen as described herein), can be separately formed and then mixed together, for example, prior to filling into vials during a manufacturing process, or immediately prior to administration). Accordingly, in some embodiments, described herein is a composition comprising two or more populations of particles (e.g., in some embodiments, lipid nanoparticles), each population comprising at least one RNA molecule encoding a different immunogenic polypeptide or antigenic fragment thereof (e.g., mpox antigen or antigenic fragment thereof). In some embodiments, each population may be provided in a composition at a desirable proportion (e.g., in some embodiments, each population may be provided in a composition in an amount that provides the same amount of RNA molecules).
[0525] In some embodiments, administered pharmaceutical compositions (e.g., immunogenic compositions, e.g., vaccines) comprising polyribonucleotides that encode one or more mpox antigens (e.g., from Table 1 or antigenic fragments thereof) are administered in RNA doses of from about 0.1 μg to about 300 μg, about 0.5 μg to about 200 μg, or about 1 μg to about 100 μg, such as about 1 μg, about 3 μg, about 10 μg, about 30 μg, about 50 μg, or about 100 μg.
[0526] In some embodiments, a pharmaceutical composition comprising a polyribonucleotide that encodes one or more mpox antigens (e.g., from Table 1 or antigenic fragments thereof) are administered at RNA doses of from about 0.5 μg to about 10 μg. In some embodiments, a pharmaceutical composition comprises one polyribonucleotide encoding an mpox antigen that is administered at a dose of from about 0.5 μg to about 2 μg. In some embodiments, a pharmaceutical composition comprises one polyribonucleotide encoding an mpox antigen (e.g., from Table 1 or an antigenic fragment thereof) that is administered at a dose of about 1 μg. In some embodiments, a pharmaceutical composition comprises one polyribonucleotide encoding one, two, or more mpox antigens, wherein each polyribonucleotide is administered at a dose of from about 0.5 μg to about 2 μg. In some embodiments, a pharmaceutical composition comprises one polyribonucleotide encoding one, two, or more mpox antigens, wherein the polyribonucleotide is administered at a dose of about 1 μg. In some embodiments, the one, two or more mpox antigens are a B cell antigen from Table 1 or an antigenic fragment thereof.
[0527] In some embodiments, a pharmaceutical composition comprises two or more polyribonucleotides each encoding an mpox antigen (e.g., from Table 1 or an antigenic fragment thereof), wherein each RNA construct is administered at a dose of from about 0.5 μg to about 2 μg. In some embodiments, a pharmaceutical composition comprises two or more polyribonucleotide each encoding an mpox antigen (e.g., from Table 1 or an antigenic fragment thereof), wherein each RNA construct is administered at a dose of about 1 μg.
[0528] In some embodiments, one or more pharmaceutical compositions are administered, comprising two or more RNA mpox constructs (e.g., two, three, four, five, six or more RNA constructs), wherein each RNA construct is administered at a dose of about 1 μg. In some embodiments, two or more pharmaceutical compositions are administered, together comprising two or more RNA constructs each encoding one or more mpox antigens (e.g., from Table 1 or an antigenic fragment thereof), wherein each RNA construct is administered at a dose of about 1 μg. In some embodiments, two or more pharmaceutical compositions are administered, together comprising two, three, four, five, six or more polyribonucleotides encoding mpox antigens, wherein each polyribonucleotide is administered at a dose of about 1 μg. In some embodiments, one or more pharmaceutical compositions are administered, together comprising one or more polyribonucleotides encoding two to four mpox antigens (e.g., from Table 1 or an antigenic fragment thereof), wherein each polyribonucleotide is administered at a dose of about 1 μg. In some embodiments, one or more pharmaceutical compositions are administered, together comprising one or more polyribonucleotides encoding two to four mpox B cell antigens (e.g., from Table 1 or an antigenic fragment thereof), wherein each polyribonucleotide is administered at a dose of about 1 μg.
[0529] In some embodiments, one or more pharmaceutical compositions are administered, together comprising one or more polyribonucleotides encoding two to four mpox B cell antigens selected from A35R, B6R, MIR, H3L, and/or antigenic fragments of any thereof, wherein each polyribonucleotide is administered at a dose of about 1 μg. In some embodiments, one or more pharmaceutical compositions are administered, together comprising one or more polyribonucleotides encoding two to four mpox B cell antigens selected from B6R, A35R, MIR, H3L, and/or antigenic fragments of any thereof. In some embodiments, one or more pharmaceutical compositions are administered, together comprising one or more polyribonucleotides encoding four mpox B cell antigens that are B6R, A35R, MIR, and H3L, (or antigenic fragments of any thereof). In some embodiments, one or more pharmaceutical compositions are administered, together comprising one or more polyribonucleotides encoding four mpox B cell antigens that are B6R, A35R, MIR, (or antigenic fragments of any thereof). In some embodiments, one or more pharmaceutical compositions are administered, together comprising one or more polyribonucleotides encoding three mpox B cell antigens that are B6R, A35R, and MIR, (or antigenic fragments of any thereof).
[0530] In some embodiments, a pharmaceutical composition is administered, comprising two polyribonucleotides each encoding one or more mpox antigens, wherein a total polyribonucleotide dose administered is about 2 to 4 μg. In some embodiments, two or more pharmaceutical compositions are administered, together comprising two polyribonucleotides each encoding one or more mpox antigens, wherein a total polyribonucleotide dose administered is about 2 to 4 μg.
[0531] In some embodiments, a pharmaceutical composition is administered, comprising one or more polyribonucleotides encoding one or more mpox antigens, wherein a total polyribonucleotide dose administered is about 10 μg, 30 μg, or 60 μg. In some embodiments, a pharmaceutical composition is administered, comprising two polyribonucleotides each encoding one or more mpox antigens, wherein a total polyribonucleotide dose administered is about 10 μg, 30 μg, or 60 μg. In some embodiments, two or more pharmaceutical compositions are administered, together comprising two polyribonucleotides each encoding one or more mpox antigens, wherein a total polyribonucleotide dose administered is about 10 μg, 30 μg, or 60 μg. In some embodiments, the one or more mpox antigens are B cell antigens selected from E8L, A35R, B6R, MIR, H3L, A28L, A29L, and/or antigenic fragments of any thereof. In some embodiments, the one or more mpox antigens are selected from B6R, A35R, MIR, H3L, E8L, and/or antigenic fragments of any thereof.
[0532] In some embodiments, a pharmaceutical composition is administered, comprising three polyribonucleotides each encoding a different mpox antigen, or antigenic fragment thereof, wherein a total polyribonucleotide dose administered is about 10 μg, 30 μg, or 60 μg. In some embodiments, the three polyribonucleotides encode B6R, A35R, and MIR, and/or antigenic fragments of any thereof.
[0533] In some embodiments, a pharmaceutical composition is administered, comprising four polyribonucleotides each encoding a different mpox antigen, or antigenic fragment thereof, wherein a total polyribonucleotide dose administered is about 10 μg, 30 μg, or 60 μg. In some embodiments, the four polyribonucleotides encode B6R, A35R, MIR, and H3L and/or antigenic fragments of any thereof.
[0534] In some embodiments, administration may involve only a single dose. In some embodiments, administration may involve application of a fixed number of doses.
[0535] In some embodiments, administration may involve dosing that is intermittent (e.g., a plurality of doses separated in time) and/or periodic (e.g., individual doses separated by a common period of time) dosing. In some embodiments, administration may involve continuous dosing (e.g., perfusion) for at least a selected period of time.
[0536] In some embodiments, a dosing regimen comprises a plurality of doses each of which is separated in time from other doses. In some embodiments, individual doses are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e., is a therapeutic dosing regimen).
[0537] Those skilled in the art are aware that therapies can be administered in dosing cycles. In some embodiments, pharmaceutical compositions described herein are administered in one or more dosing cycles.
[0538] In some embodiments, one dosing cycle is at least 3 or more days (including, e.g., at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 28, at least 29, or at least 30 days. In some embodiments, one dosing cycle is at least 21 days.
[0539] In some embodiments, one dosing cycle may involve multiple doses, e.g., according to a pattern such as, for example, a dose may be administered daily within a dosing cycle, or a dose may be administered every 2 days, every 3 days, every 4 days, every 5 days, every 6 days, every 7 days, every 2 weeks, monthly, every 2 months within a cycle. In some certain embodiments, a dosing cycle is at least 4 weeks. In some certain embodiments, a dosing cycle is about 4 weeks.
[0540] In some embodiments, multiple dosing cycles may be administered. For example, in some embodiments, at least 2 dosing cycles (including, e.g., at least 3 dosing cycles, at least 4 dosing cycles, at least 5 dosing cycles, at least 6 dosing cycles, at least 7 dosing cycles, at least 8 dosing cycles, at least 9 dosing cycles, at least 10 dosing cycles, or more) can be administered. In some embodiments, the number of dosing cycles to be administered may vary with types of treatment (e.g., monotherapy vs. combination therapy). In some embodiments, at least 3-8 dosing cycles may be administered.
[0541] In some embodiments, there may be a "rest period" between dosing cycles; in some embodiments, there may be no rest period between dosing cycles. In some embodiments, there may be sometimes a rest period and sometimes no rest period between dosing cycles.
[0542] In some embodiments, a rest period may have a length within a range of several days to several months. For example, in some embodiments, a rest period may have a length of at least 3 days or more, including, e.g., at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days or more. In some embodiments, a rest period may have a length of at least 1 week or more, including, e.g., at least 2 weeks, at least 3 weeks, at least 4 weeks, or more. In certain embodiments, a rest period is at least 4 weeks. In certain embodiments, a rest period is about 4 weeks.
[0543] Dosage of pharmaceutical compositions described herein may vary with a number of factors including, e.g., but not limited to body weight of a subject to be treated, cancer types and/or cancer stages, and/or monotherapy or combination therapy. In some embodiments, a dosing cycle involves administration of a set number and/or pattern of doses. For example, in some embodiments, a pharmaceutical composition described herein is administered at least one dose per dosing cycle, including, e.g., at least two doses per dosing cycle, at least three doses per dosing cycle, at least four doses per dosing cycle, or more.
[0544] In some embodiments, a dosing cycle involves administration of a set cumulative dose, e.g., over a particular period of time, and optionally via multiple doses, which may be administered, for example, at set interval(s) and/or according to a set pattern. In some embodiments, a set cumulative dose may be administered via multiple doses at set intervals such that there is at least some temporal overlap in biological and/or pharmacokinetics effects generated by such multiple doses on a target cell or on a subject being treated. In some embodiments, a set cumulative dose may be administered via multiple doses at set intervals such that biological and/or pharmacokinetics effects generated by such multiple doses on a target cell or on a subject being treated may be additive. By way of example only, in some embodiments, a set cumulative dose of X mg may be administered via two doses with each dose of X/2 mg, wherein such two doses are administered sufficiently close in time such that biological and/or pharmacokinetics effects generated by each X/2-mg dose on a target cell or on a subject being treated may be additive.
[0545] In some embodiments, dosing may be adjusted based on response of a subject receiving the therapy. For example, in some embodiments, dosing may involve administration of a higher dose followed later by administration of a lower dose if one or more parameters for safety pharmacology assessment indicates that the prior dose may not satisfy the medical safety requirement according to a physician. In some embodiments, dose escalation may be performed at one or more of the levels. Without wishing to be bound by any particular theory, the present disclosure, among other things, provides an insight that a pharmaceutically guided dose escalation (PGDE) method may be applied to determine an appropriate dose of pharmaceutical compositions described herein.
[0546] In some embodiments, pharmaceutical compositions described herein can be administered to subjects as monotherapy. [0547] In some embodiments, a pharmaceutical composition provided herein may be administered as part of combination therapy.
[0548] In some embodiments, subjects receiving a pharmaceutical composition provided herein (e.g., a pharmaceutical composition) may be monitored periodically over a dosing regimen to assess efficacy of the administered treatment. For example, in some embodiments, efficacy of an administered treatment may be assessed periodically, e.g., weekly, biweekly, every 4 weeks, every 5 weeks, every 6 weeks, every 7 weeks, every 8 weeks, or longer.
[0549] In some embodiments, polyribonucleotides or pharmaceutical compositions of the present disclosure are administered to a subject in need thereof to induce an immune response against an orthopoxvirus. In some embodiments, the orthopoxvirus is an mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus. In some embodiments, the orthopoxvirus is an mpox clade la, clade lb, clade Ila, or clade lib virus. In some embodiments, the orthopoxvirus is an mpox clade la virus. In some embodiments, the orthopoxvirus is an mpox clade lb virus.
[0550] In some embodiments, pharmaceutical compositions described herein are administered to a subject in need thereof to induce production of orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) neutralizing antibodies in the subject. In some embodiments, pharmaceutical compositions described herein are administered to a subject in need thereof to induce production of B6R-binidng, MIR-binding, A35R-binding, and/or H3L-binding antibodies in the subject. In some embodiments, pharmaceutical compositions described herein are administered to a subject in need thereof to enhance orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) neutralizing antibody titers in the subject. In some embodiments, pharmaceutical compositions described herein are administered to a subject in need thereof to enhance B6R-binidng, MIR-binding, A35R- binding, and/or H3L-binding antibody titers in the subject.
[0551] In some embodiments, pharmaceutical compositions described herein are administered to a subject in need thereof to induce antibody Fc-effector functional activities (e.g., antibody-mediated complement activation, antibody-mediated NK cell activation, antibody-mediated NK cell degranulation, antibody-mediated IFNy release, antibody-mediated MIPlb release, antibody-dependent cellular monocyte phagocytosis) in the subject.
[0552] In some embodiments, pharmaceutical compositions described herein are administered to a subject in need thereof to reduce the number of orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) lesions in the subject. In some embodiments, pharmaceutical compositions described herein are administered to a subject in need thereof to reduce the severity of orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) lesions in the subject. In some embodiments, pharmaceutical compositions described herein are administered to a subject in need thereof to reduce or prevent the onset of orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) lesions in the subject. In some embodiments, pharmaceutical compositions described herein are administered to a subject in need thereof to reduce the duration of orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) lesion onset to resolution in the subject.
[0553] In some embodiments, pharmaceutical compositions described herein are administered to a subject in need thereof to reduce the orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) viral load in the subject. In some embodiments, pharmaceutical compositions described herein are administered to a subject in need thereof to reduce the orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) viral load in the throat of the subject. In some embodiments, pharmaceutical compositions described herein are administered to a subject in need thereof to reduce the orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) viral load in the blood of the subject.
[0554] In some embodiments, pharmaceutical compositions described herein are administered to a subject in need thereof to reduce orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) virus replication in the subject. In some embodiments, pharmaceutical compositions described herein are administered to a subject in need thereof to reduce orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) virus replication in the throat of the subject. In some embodiments, pharmaceutical compositions described herein are administered to a subject in need thereof to reduce orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) virus replication in the blood of the subject.
[0555] In some embodiments, pharmaceutical compositions described herein are administered to a subject in need thereof to reduce weight loss associated with orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) infection in the subject.
[0556] In some embodiments, pharmaceutical compositions described herein are administered to a subject in need thereof to reduce morbidity associated with (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) infection in the subject.
IX. Methods of Manufacture
[0557] Individual polyribonucleotides can be produced by methods known in the art. For example, in some embodiments, polyribonucleotides can be produced by in vitro transcription, for example, using a DNA template. A plasmid DNA used as a template for in vitro transcription to generate a polyribonucleotide described herein is also within the scope of the present disclosure.
[0558] A DNA template is used for in vitro RNA synthesis in the presence of an appropriate RNA polymerase (e.g., a recombinant RNA-polymerase such as a T7 RNA-polymerase) with ribonucleotide triphosphates (e.g., ATP, CTP, GTP, UTP). In some embodiments, polyribonucleotides (e.g., ones described herein) can be synthesized in the presence of modified ribonucleotide triphosphates. By way of example only, in some embodiments, pseudouridine (ip), Nl-methyl-pseudouridine (mlip), or 5-methyl-uridine (m5U) can be used to replace uridine triphosphate (UTP). In some embodiments, pseudouridine (ip) can be used to replace uridine triphosphate (UTP). In some embodiments, Nl-methyl-pseudouridine (mlip) can be used to replace uridine triphosphate (UTP). In some embodiments, 5-methyl-uridine (m5U) can be used to replace uridine triphosphate (UTP).
[0559] As will be clear to those skilled in the art, during in vitro transcription, an RNA polymerase
(e.g., as described and/or utilized herein) typically traverses at least a portion of a single-stranded DNA template in the 3'— > 5' direction to produce a single-stranded complementary RNA in the 5'— > 3' direction.
[0560] In some embodiments where a polyribonucleotide comprises a polyA tail, one of those skill in the art will appreciate that such a polyA tail may be encoded in a DNA template, e.g., by using an appropriately tailed PCR primer, or it can be added to a polyribonucleotide after in vitro transcription, e.g., by enzymatic treatment (e.g., using a poly(A) polymerase such as an E. coli Poly(A) polymerase). Suitable poly(A) tails are described herein above. In some embodiments, a poly(A) tail comprises a plurality of A residues interrupted by a linker. In some embodiments, a linker comprises the nucleotide sequence GCATATGAC (SEQ ID NO: 156).
[0561] In some embodiments, those skilled in the art will appreciate that addition of a 5' cap to an
RNA (e.g., mRNA) can facilitate recognition and attachment of the RNA to a ribosome to initiate translation and enhances translation efficiency. Those skilled in the art will also appreciate that a 5' cap can also protect an RNA product from 5' exonuclease mediated degradation and thus increases half-life. Methods for capping are known in the art; one of ordinary skill in the art will appreciate that in some embodiments, capping may be performed after in vitro transcription in the presence of a capping system (e.g., an enzyme-based capping system such as, e.g., capping enzymes of vaccinia virus). In some embodiments, a cap may be introduced during in vitro transcription, along with a plurality of ribonucleotide triphosphates such that a cap is incorporated into a polyribonucleotide during transcription (also known as co-transcriptional capping). In some embodiments, a GTP fed-batch procedure with multiple additions in the course of the reaction may be used to maintain a low concentration of GTP in order to effectively cap the RNA. Suitable 5' cap are described herein above. For example, in some embodiments, a 5' cap comprises m7(3'OmeG)(5')ppp(5')(2'OmeA)pG.
[0562] Following RNA transcription, a DNA template is digested. In some embodiments, digestion can be achieved with the use of Dnase I under appropriate conditions.
[0563] In some embodiments, in-vitro transcribed polyribonucleotides may be provided in a buffered solution, for example, in a buffer such as HEPES, a phosphate buffer solution, a citrate buffer solution, an acetate buffer solution, or Tris buffer solution; in some embodiments, such solution may be buffered to a pH within a range of, for example, about 6.5 to about 7.5; in some embodiments approximately 7.0. In some embodiments, production of polyribonucleotides may further include one or more of the following steps: purification, mixing, filtration, and/or filling.
[0564] In some embodiments, polyribonucleotides can be purified (e.g., in some embodiments after in vitro transcription reaction), for example, to remove components utilized or formed in the course of the production, like, e.g., proteins, DNA fragments, and/or or nucleotides. Various nucleic acid purifications that are known in the art can be used in accordance with the present disclosure. Certain purification steps may be or include, for example, one or more of precipitation, column chromatography (including, e.g., but not limited to anionic, cationic, hydrophobic interaction chromatography (HIC)), solid substrate-based purification (e.g., magnetic bead-based purification). In some embodiments, polyribonucleotides may be purified using magnetic bead-based purification, which in some embodiments may be or comprise magnetic bead-based chromatography. In some embodiments, polyribonucleotides may be purified using hydrophobic interaction chromatography (HIC) and/or diafiltration. In some embodiments, polyribonucleotides may be purified using HIC followed by diafiltration. [0565] In some embodiments, dsRNA may be obtained as side product during in vitro transcription. In some such embodiments, a second purification step may be performed to remove dsRNA contamination. For example, in some embodiments, cellulose materials (e.g., microcrystalline cellulose) may be used to remove dsRNA contamination, for examples in some embodiments in a chromatographic format. In some embodiments, cellulose materials (e.g., microcrystalline cellulose) can be pretreated to inactivate potential Rnase contamination, for example in some embodiments by autoclaving followed by incubation with aqueous basic solution, e.g., NaOH. In some embodiments, cellulose materials may be used to purify polyribonucleotides according to methods described in WO 2017/182524, the entire content of which is incorporated herein by reference.
[0566] In some embodiments, a batch of polyribonucleotides may be further processed by one or more steps of filtration and/or concentration. For example, in some embodiments, polyribonucleotide(s), for example, after removal of dsRNA contamination, may be further subject to diafiltration (e.g., in some embodiments by tangential flow filtration), for example, to adjust the concentration of polyribonucleotides to a desirable RNA concentration and/or to exchange buffer to a drug substance buffer.
[0567] In some embodiments, polyribonucleotides may be processed through 0.2 pm filtration before they are filled into appropriate containers.
[0568] In some embodiments, polyribonucleotides and compositions thereof may be manufactured in accordance with a process as described herein, or as otherwise known in the art.
[0569] In some embodiments, polyribonucleotides and compositions thereof may be manufactured at a large scale. For example, in some embodiments, a batch of polyribonucleotides can be manufactured at a scale of greater than 1 g, greater than 2 g, greater than 3 g, greater than 4 g, greater than 5 g, greater than 6 g, greater than 7 g, greater than 8 g, greater than 9 g, greater than 10 g, greater than 15 g, greater than 20 g, or higher.
[0570] In some embodiments, RNA quality control may be performed and/or monitored at any time during production process of polyribonucleotides and/or compositions comprising the same. For example, in some embodiments, RNA quality control parameters, including one or more of RNA identity (e.g., sequence, length, and/or RNA natures), RNA integrity, RNA concentration, residual DNA template, and residual dsRNA, may be assessed and/or monitored after each or certain steps of a polyribonucleotide manufacturing process, e.g., after in vitro transcription, and/or each purification step.
[0571] In some embodiments, the stability of polyribonucleotides (e.g., produced by in vitro transcription) and/or compositions comprising two or more RNAs can be assessed under various test storage conditions, for example, at room temperatures vs. fridge or sub-zero temperatures over a period of time (e.g., at least 3 months, at least 6 months, at least 9 months, at least 12 months, or longer). In some embodiments, polyribonucleotides (e.g., ones described herein) and/or compositions thereof may be stored stable at a fridge temperature (e.g., about 4°C to about 10°C) for at least 1 month or longer including, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or longer. In some embodiments, polyribonucleotides (e.g., ones described herein) and/or compositions thereof may be stored stable at a sub-zero temperature (e.g., -20°C or below) for at least 1 month or longer including, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, or at least 12 months or longer. In some embodiments, polyribonucleotides (e.g., ones described herein) and/or compositions thereof may be stored stable at room temperature (e.g., at about 25°C) for at least 1 month or longer.
[0572] In some embodiments, one or more assessments may be utilized during manufacture, or other preparation or use of polyribonucleotides (e.g., as a release test).
[0573] In some embodiments, one or more quality control parameters may be assessed to determine whether polyribonucleotides described herein meet or exceed acceptance criteria (e.g., for subsequent formulation and/or release for distribution). In some embodiments, such quality control parameters may include, but are not limited to RNA integrity, RNA concentration, residual DNA template and/or residual dsRNA. Certain methods for assessing RNA quality are known in the art; for example, one of skill in the art will recognize that in some embodiments, one or more analytical tests can be used for RNA quality assessment. Examples of such certain analytical tests may include but are not limited to gel electrophoresis, UV absorption, and/or PCR assay. [0574] In some embodiments, a batch of polyribonucleotides may be assessed for one or more features as described herein to determine next action step(s). For example, a batch of polyribonucleotides can be designated for one or more further steps of manufacturing and/or formulation and/or distribution if RNA quality assessment indicates that such a batch of polyribonucleotides meet or exceed the relevant acceptance criteria. Otherwise, an alternative action can be taken (e.g., discarding the batch) if such a batch of polyribonucleotides does not meet or exceed the acceptance criteria.
[0575] In some embodiments, a batch of polyribonucleotides that satisfy assessment results can be utilized for one or more further steps of manufacturing and/or formulation and/or distribution.
[0576] In some embodiments, manufacture of a vaccine composition of the present disclosure includes characterizing the efficacy of the vaccine composition by: (i) administering the vaccine composition to a CAST/Ei mouse in one or more doses; (ii) infecting the mouse with an orthopoxvirus (e.g., MPXV); and (iii) measuring a viral titer of the orthopoxvirus (e.g., MPXV) in a sample collected from the mouse. In some embodiments, the vaccine composition is characterized as efficacious if the viral titer is significantly lower than a viral titer of the orthopoxvirus in a control sample.
[0577] In some embodiments, manufacture of a vaccine composition of the present disclosure includes testing the ability of the vaccine composition to significantly lower an orthopoxvirus (e.g. MPXV) viral titer measured in a sample collected from a CAST/Ei mouse as compared to a control sample. In some embodiments, the sample is collected from a CAST/Ei mouse that was administered with the vaccine composition, in one or more doses prior to infection with the orthopoxvirus.
[0578] In some embodiments, a method of manufacture includes administering a CAST/Ei mouse with one or more doses of the vaccine composition prior to infections with the orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, modified vaccinia virus Ankara, or vaccinia virus). For example, in some embodiments, a method includes administering one, two, three, four, five, or more doses to the CAST/Ei mouse.
[0579] In some embodiments, a dose of the vaccine composition is administered to the CAST/Ei mouse 25 to 75 (e.g., 25 to 75, 30 to 75, 35 to 75, 40 to 75, 45 to 75, 50 to 75, 55 to 75, 60 to 75, 65 to 75, 70 to 75, 25 to 70, 30 to 70, 35 to 70, 40 to 70, 45 to 70, 50 to 70, 55 to 70, 60 to 70, 65 to 70, 25 to 65, 30 to 65,
35 to 65, 40 to 65, 45 to 65, 50 to 65, 55 to 65, 60 to 65, 25 to 60, 30 to 60, 35 to 60, 40 to 60, 45 to 60, 50 to
60, 55 to 60, 25 to 55, 30 to 55, 35 to 55, 40 to 55, 45 to 55, 50 to 55, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 25 to 45, 30 to 45, 35 to 45, 40 to 45, 25 to 40, 30 to 40, 35 to 40, 25 to 35, 30 to 35, 25 to 30, 50, 51,
52, 53, 54, 55, 56, 57, 58, 59, or 60) days prior to infection with the orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus). For example, in some embodiments, a dose of the vaccine composition is administered to the CAST/Ei mouse 56 days prior to infection with the orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus).
[0580] In some embodiments, a method of manufacture includes administering two doses of the vaccine composition to the CAST/Ei mouse. In some embodiments a dose is administered to the CAST/Ei mouse 25 to 75 (e.g., 25 to 75, 30 to 75, 35 to 75, 40 to 75, 45 to 75, 50 to 75, 55 to 75, 60 to 75, 65 to 75, 70 to 75, 25 to 70, 30 to 70, 35 to 70, 40 to 70, 45 to 70, 50 to 70, 55 to 70, 60 to 70, 65 to 70, 25 to 65, 30 to 65, 35 to 65, 40 to 65, 45 to 65, 50 to 65, 55 to 65, 60 to 65, 25 to 60, 30 to 60, 35 to 60, 40 to 60, 45 to 60, 50 to 60, 55 to 60, 25 to 55, 30 to 55, 35 to 55, 40 to 55, 45 to 55, 50 to 55, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 25 to 45, 30 to 45, 35 to 45, 40 to 45, 25 to 40, 30 to 40, 35 to 40, 25 to 35, 30 to 35, 25 to 30, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60) days prior to infection with the (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus), and an additional dose is administered to the CAST/Ei mouse 20 to 50 days (e.g., 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, 45 to 50, 20 to 45, 25 to 45, 30 to 45, 35 to 45, 40 to 45, 20 to 40, 25 to 40, 30 to 40, 35 to 40, 20 to 35, 25 to 35, 30 to 35, 20 to 30, 25 to 30, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40) days prior to infection with the orthopoxvirus. For example, in some embodiments, a method of manufacture includes administering the dose of the vaccine composition and the additional dose of the vaccine composition to the CAST/Ei mouse at 56 days and 35 days prior to orthopox (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) infection, respectively.
[0581] In some embodiments, the one or more doses of the vaccine composition includes 0.5 to 10 μg
(e.g., 0.5 to 10, 1 to 10, 1.5 to 10, 2 to 10, 2.5 to 10, 3 to 10, 3.5 to 10, 4 to 10, 4.5 to 10, 5 to 10, 5.5 to 10, 6 to 10, 6.5 to 10, 7 to 10, 7.5 to 10, 8 to 10, 8.5 to 10, 9 to 10, 9.5 to 10, 0.5 to 9, 1 to 9, 1.5 to 9, 2 to 9, 2.5 to 9, 3 to 9, 3.5 to 9, 4 to 9, 4.5 to 9, 5 to 9, 5.5 to 9, 6 to 9, 6.5 to 9, 7 to 9, 7.5 to 9, 8 to 9, 8.5 to 9, 0.5 to 8, 1 to 8, 1.5 to 8, 2 to 8, 2.5 to 8, 3 to 8, 3.5 to 8, 4 to 8, 4.5 to 8, 5 to 8, 5.5 to 8, 6 to 8, 6.5 to 8, 7 to 8, 7.5 to 8,
0.5 to 7, 1 to 7, 1.5 to 7, 2 to 7, 2.5 to 7, 3 to 7, 3.5 to 7, 4 to 7, 4.5 to 7, 5 to 7, 5.5 to 7, 6 to 7, 6.5 to 7, 0.5 to 6, 1 to 6, 1.5 to 6, 2 to 6, 2.5 to 6, 3 to 6, 3.5 to 6, 4 to 6, 4.5 to 6, 5 to 6, 5.5 to 6, 0.5 to 5, 1 to 5, 1.5 to 5,
2 to 5, 2.5 to 5, 3 to 5, 3.5 to 5, 4 to 5, 4.5 to 5, 0.5 to 4, 1 to 4, 1.5 to 4, 2 to 4, 2.5 to 4, 3 to 4, 3.5 to 4, 0.5 to 3, 1 to 3, 1.5 to 3, 2 to 3, 2.5 to 3, 0.5 to 2, 1 to 2, 1.5 to 2, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 μg) of one or more mpox antigen-encoding polyribonucleotides of the present disclosure. In some embodiments, one or more doses of a vaccine composition includes 1 μg of one or more mpox antigen-encoding polyribonucleotides. In some embodiments, the one or more doses of the vaccine composition includes 4 μg of the polyribonucleotide. In some embodiments, a method includes administering two doses of the vaccine composition and each of the doses includes 4 μg of the polyribonucleotide.
[0582] In some embodiments, a method of manufacture includes administering one or more doses of the vaccine composition to the CAST/Ei mouse intramuscularly, intravenously, or intranasally. For example, in some embodiments, the method of manufacture includes administering one or more doses of the vaccine composition to the CAST/Ei mouse intramuscularly.
[0583] In some embodiments, a method of manufacture includes infecting the CAST/Ei mouse with 6 x 105 PFU to 1.2 x 107 PFU (e.g., 6 x 105 PFU to 1.2 x 107 PFU, 7 x 105 PFU to 1.2 x 107 PFU, 8 x 105 PFU to 1.2 x 107 PFU, 9 x 105 PFU to 1.2 x 107 PFU, 1 x 107 PFU to 1.2 x 107 PFU, 1.1 x 107 PFU to 1.2 x 107 PFU, 6 x 105 PFU to 1.1 x 107 PFU, 7 x 105 PFU to 1.1 x 107 PFU, 8 x 105 PFU to 1.1 x 107 PFU, 9 x 105 PFU to 1.1 x 107 PFU,
1 x 107 PFU to 1.1 x 107 PFU, 6 x 105 PFU to 1 x 107 PFU, 7 x 105 PFU to 1 x 107 PFU, 8 x 105 PFU to 1 x 107
PFU, 9 x 105 PFU to 1 x 107 PFU, 6 x 105 PFU to 9 x 105 PFU, 7 x 105 PFU to 9 x 105 PFU, 8 x 105 PFU to 9 x 105
PFU, 6 x 105 PFU to 8 x 105 PFU, 7 x 105 PFU to 8 x 105 PFU, 6 x 105 PFU to 7 x 105 PFU, 6 x 105 PFU, 7 x 105
PFU, 8 x 105 PFU, 9 x 105 PFU, 1 x 107 PFU, 1.1 x 107 PFU, or 1.2 x 107 PFU) of the orthopoxvirus. For example, in some embodiments, the method of manufacture includes infecting the CAST/Ei mouse with 9 x 105 PFU of orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus).
[0584] In some embodiments, a method of manufacture includes infecting the CAST/Ei mouse with orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) intranasally.
[0585] In some embodiments, a method of manufacture includes collecting one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more) samples from the CAST/Ei mouse. In some embodiments, one or more samples from the CAST/Ei mouse is/are collected 1 to 10 days (e.g., 1 to 10, 2 to 10, 3 to 10, 4 to 10, 5 to 10, 6 to 10, 7 to 10, 8 to 10, 9 to 10, 1 to 9, 2 to 9, 3 to 9, 4 to 9, 5 to 9, 6 to 9, 7 to 9, 8 to 9, 1 to 8, 2 to 8, 3 to 8, 4 to 8, 5 to 8, 6 to 8, 7 to 8, 1 to 7, 2 to 7, 3 to 7, 4 to 7, 5 to 7, 6 to 7, 1 to 6, 2 to
6, 3 to 6, 4 to 6, 5 to 6, 1 to 5, 2 to 5, 3 to 5, 4 to 5, 1 to 4, 2 to 4, 3 to 4, 1 to 3, 2 to 3, 1 to 2, 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10 days post infection with the orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus). For example in some embodiments, two samples are collected from the CAST/Ei mouse at 3 days and 7 days post infection with orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus). In some embodiments, a method of manufacture includes collecting one or more (e.g., one, two, three, four, five, six, seven, eight, nine, ten, or more) samples from the CAST/Ei mouse post infection with orthopoxvirus, where the sample is a lung tissue sample, a bronchiolar lavage sample, a blood sample, a urine sample, or a fecal sample. For example, in some embodiments, the one or more samples is a homogenized lung tissue sample.
[0586] In some embodiments, a method of manufacture includes comparing one or more sample collected from the orthopox-infected CAST/Ei mouse with a control sample. As understood by a person of skill in the art, a control sample can include a sample collected from an orthopox-infected CAST/Ei mouse treated under the same conditions as a mouse administered with a vaccine composition, but without having been administered with the vaccine composition. For example, in some embodiments, a method includes comparing the viral titer of orthopox in a lung tissue sample collected from an orthopox-infected CAST/Ei mouse administered with one or more doses of vaccine composition, with the viral titer of orthopox in a lung tissue sample collected from an orthopox-infected CAST/Ei mouse that has not been administered with one or more doses of vaccine composition.
[0587] In some embodiments, a method of manufacture includes infecting a CAST/Ei mouse with an mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, modified vaccinia virus Ankara, or vaccinia virus. For example, in some embodiments, a method of manufacture includes infecting a CAST/Ei mouse with an mpox virus.
[0588] In some embodiments, a method of manufacture includes characterizing whether a vaccine composition significantly lowers the viral titer of orthopox (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) in a sample collected from an orthopox-infected CAST/Ei mouse as compared to a control sample. In some embodiments, a vaccine composition significantly lowers the viral titer of orthopox (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) in a sample, when the viral titer is at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% lower than the viral titer of orthopoxvirus (e.g., mpox, variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) in a control sample.
X. DNA Constructs
[0589] Among other things, the present disclosure provides DNA constructs, for example that may encode one or more antigens or fragments thereof as described herein, or components thereof. In some embodiments, DNA constructs provided by and/or utilized in accordance with the present disclosure are comprised in a vector.
[0590] Non-limiting examples of a vector include plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). In some embodiments, a vector is an expression vector. In some embodiments, a vector is a cloning vector. In general, a vector is a nucleic acid construct that can receive or otherwise become linked to a nucleic acid element of interest (e.g., a construct that is or encodes a payload, or that imparts a particular functionality, etc.).
[0591] Expression vectors, which may be plasmid or viral or other vectors, typically include an expressible sequence of interest (e.g., a coding sequence) that is functionally linked with one or more control elements (e.g., promoters, enhancers, transcription terminators, etc.). Typically, such control elements are selected for expression in a system of interest. In some embodiments, a system is ex vivo (e.g., an in vitro transcription system); in some embodiments, a system is in vivo (e.g., a bacterial, yeast, plant, insect, fish, vertebrate, mammalian cell or tissue, etc.).
[0592] Cloning vectors are generally used to modify, engineer, and/or duplicate (e.g., by replication in vivo, for example in a simple system such as bacteria or yeast, or in vitro, such as by amplification such as polymerase chain reaction or other amplification process). In some embodiments, a cloning vector may lack expression signals.
[0593] In some embodiments, a vector may include replication elements such as primer binding site(s) and/or origin(s) of replication. In many embodiments, a vector may include insertion or modification sites such as restriction endonuclease recognition sites and/or guide RNA binding sites, etc.
[0594] In some embodiments, a vector is a viral vector (e.g., an AAV vector). In some embodiments, a vector is a non-viral vector. In some embodiments, a vector is a plasmid.
[0595] Those skilled in the art are aware of a variety of technologies useful for the production of recombinant polynucleotides (e.g., DNA or RNA) as described herein. For example, restriction digestion, reverse transcription, amplification (e.g., by polymerase chain reaction), Gibson assembly, etc., are well established and useful tools and technologies. Alternatively or additionally, certain nucleic acids may be prepared or assembled by chemical and/or enzymatic synthesis. In some embodiments, a combination of known methods is utilized to prepare a recombinant polynucleotide. [0596] In some embodiments, polynucleotide(s) of the present disclosure are included in a DNA construct (e.g., a vector) amenable to transcription and/or translation.
[0597] In some embodiments, an expression vector comprises a polynucleotide that encodes proteins and/or polypeptides of the present disclosure operatively linked to a sequence or sequences that control expression (e.g., promoters, start signals, stop signals, polyadenylation signals, activators, repressors, etc.). In some embodiments, a sequence or sequences that control expression are selected to achieve a desired level of expression. In some embodiments, more than one sequence that controls expression (e.g., promoters) are utilized. In some embodiments, more than one sequence that controls expression (e.g., promoters) are utilized to achieve a desired level of expression of a plurality of polynucleotides that encode a plurality of proteins and/or polypeptides. In some embodiments, a plurality of recombinant proteins and/or polypeptides are expressed from the same vector (e.g., a bi-cistronic vector, a tri-cistronic vector, multi-cistronic). In some embodiments, a plurality of polypeptides are expressed, each of which is expressed from a separate vector.
[0598] In some embodiments, an expression vector comprising a polynucleotide of the present disclosure is used to produce a RNA and/or protein and/or polypeptide in a host cell. In some embodiments, a host cell may be in vitro (e.g., a cell line) - for example a cell or cell line (e.g., Human Embryonic Kidney (HEK cells), Chinese Hamster Ovary cells, etc.) suitable for producing polynucleotides of the present disclosure and proteins and/or polypeptides encoded by said polynucleotides.
[0599] In some embodiments, an expression vector is an RNA expression vector. In some embodiments, an RNA expression vector comprises a polynucleotide template used to produce a RNA in cell-free enzymatic mix. In some embodiments, an RNA expression vector comprising a polynucleotide template is enzymatically linearized prior to in vitro transcription. In some embodiments, a polynucleotide template is generated through PCR as a linear polynucleotide template. In some embodiments, a linearized polynucleotide is mixed with enzymes suitable for RNA synthesis, RNA capping and/or purification. In some embodiments, the resulting RNA is suitable for producing proteins encoded by the RNA.
[0600] A variety of methods are known in the art to introduce an expression vector into host cells. In some embodiments, a vector may be introduced into host cells using transfection. In some embodiments, transfection is completed, for example, using calcium phosphate transfection, lipofection, or polyethylenimine- mediated transfection. In some embodiments, a vector may be introduced into a host cell using transduction.
[0601] In some embodiments, transformed host cells are cultured following introduction of a vector into a host cell to allow for expression of said recombinant polynucleotides. In some embodiments, a transformed host cells are cultured for at least 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours 40 hours, 44 hours, 48 hours, 52 hours, 56 hours, 60 hours, 64 hours, 68 hours, 72 hours or longer. Transformed host cells are cultured in growth conditions (e.g., temperature, carbon-dioxide levels, growth medium) in accordance with the requirements of a host cell selected. A skilled artisan would recognize culture conditions for host cells selected are well known in the art.
XI, Doses
[0602] In some embodiments, the present disclosure provides a method of treating or preventing an orthopoxvirus virus (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, modified vaccinia virus Ankara, or vaccinia virus) infection comprising administering to a subject in need thereof a therapeutically effective amount of a composition or combination disclosed herein, in a treatment cycle comprising one or more doses (e.g., one dose, two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine doses, or ten doses) of the composition or combination. In some embodiments, a treatment cycle comprises two or more doses (e.g., two doses, three doses, four doses, five doses, six doses, seven doses, eight doses, nine doses, or ten doses) of a composition or combination. In some embodiments, a treatment cycle comprises two doses. In some embodiments, a first dose is a priming dose of a composition or combination disclosed herein. In some embodiments, a second dose is a booster dose of a composition or combination disclosed herein.
[0603] In some embodiments, a subject is administered one or more doses of a composition or combination disclosed herein prior to infection with and orthopoxvirus (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) viruses).
[0604] In some embodiments, a second dose of a therapeutically effective amount of a composition or combination disclosed herein is administered to a subject 1 day to 24 weeks, 3.5 days to 24 weeks, 1 week to 24 weeks, 2 weeks to 24 weeks, 4 weeks to 24 weeks, 6 weeks to 24 weeks, 8 weeks to 24 weeks, 10 weeks to 24 weeks, 12 weeks to 24 weeks, 16 weeks to 24 weeks, 20 weeks to 24 weeks, 1 day to 20 weeks, 3.5 days to 20 weeks, 1 week to 20 weeks, 2 weeks to 20 weeks, 4 weeks to 20 weeks, 6 weeks to 20 weeks, 8 weeks to 20 weeks, 10 weeks to 20 weeks, 12 weeks to 20 weeks, 16 weeks to 20 weeks, 1 day to 16 weeks, 3.5 days to 16 weeks, 1 week to 16 weeks, 2 weeks to 16 weeks, 4 weeks to 16 weeks, 6 weeks to 16 weeks, 8 weeks to 16 weeks, 10 weeks to 16 weeks, 12 weeks to 16 weeks, 1 day to 12 weeks, 3.5 days to 12 weeks, 1 week to 12 weeks, 2 weeks to 12 weeks, 4 weeks to 12 weeks, 6 weeks to 12 weeks, 8 weeks to 12 weeks, 10 weeks to 12 weeks, 1 day to 10 weeks, 3.5 days to 10 weeks, 1 week to 10 weeks, 2 week to 10 weeks, 4 weeks to 10 weeks, 6 weeks to 10 weeks, 8 weeks to 10 weeks, 1 day to 8 weeks, 3.5 days to 8 weeks, 1 week to 8 weeks, 2 weeks to 8 weeks, 4 weeks to 8 weeks, 6 weeks to 8 weeks, 1 day to 6 weeks, 3.5 days to 6 weeks, 1 week to 6 weeks, 2 weeks to 6 weeks, 4 weeks to 6 weeks, 1 day to 4 weeks, 3.5 days to 4 weeks, 1 week to 4 weeks, 2 week to 4 weeks, 1 day to 2 weeks, 3.5 days to 2 weeks, 1 week to 2 weeks, 1 day to 1 week, 3.5 days to 1 week, or 1 day to 3.5 days, after administration of a first dose of a therapeutically effective amount of the composition or combination to the subject. In some embodiments, a second dose of a therapeutically effective amount of a composition or combination disclosed herein is administered 1 week to 12 weeks after administration of a first dose of a therapeutically effective amount of the composition or combination to the subject. In some embodiments, a second dose of a therapeutically effective amount of a composition or combination disclosed herein is administered 2 weeks to 10 weeks after administration of a first dose of a therapeutically effective amount of the composition or combination to the subject.
[0605] In some embodiments, a second dose of a therapeutically effective amount of a composition or combination disclosed herein is administered about 1 week, about 2 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, about 20 weeks, or about 24 weeks after administration of a first dose of a therapeutically effective amount of a composition of combination to a subject. In some embodiments, a second dose of a therapeutically effective amount of a composition or combination disclosed herein is administered about 4 weeks after administration of a first dose of a therapeutically effective amount of a composition or combination to a subject.
[0606] In some embodiments, a second dose of a therapeutically effective amount of a composition or combination disclosed herein is administered about 1 day, about 3.5 days, about 7 days, about 14 days, about 28 days, about 29 days, about 30 days, about 31 days, about 32 days, about 33 days about 34 days, or about 35 days after administration of a first dose of a therapeutically effective amount of a composition or combination to a subject. In some embodiments, a second dose of a therapeutically effective amount of a composition or combination disclosed herein is administered about 7 days, about 14 days, about 21 days, about 28 days, about 31 days, or about 35 days after administration of a first dose of a therapeutically effective amount of a composition or combination to a subject.
[0607] In some embodiments, each of one or more doses of a therapeutically effective amount of a composition or combination disclosed herein is administered to a subject intramuscularly, subcutaneously, orally, or intranasally. In some embodiments, each of one or more doses of a therapeutically effective amount of a composition or combination disclosed herein is administered to a subject intramuscularly.
[0608] In some embodiments, each of one or more doses of a therapeutically effective amount of a composition or combination disclosed herein comprises 0.1 to 500 μg, 0.2 to 500 μg, 0.5 to 500 μg, 0.75 to 500 μg, 1 to 500 μg, 2.5 to 500 μg, 5 to 500 μg, 7.5 to 500 μg, 10 to 500 μg, 12.5 to 500 μg, 15 to 500 μg, 17.5 to 500 μg, 20 to 500 μg, 25 to 500 μg, 30 to 500 μg, 35 to 500 μg, 40 to 500 μg, 45 to 500 μg, 50 to 500 μg, 55 to 500 μg, 60 to 500 μg, 65 to 500 μg, 70 to 500 μg, 75 to 500 μg, 80 to 500 μg, 85 to 500 μg, 90 to 500 μg, 95 to 500 μg, 100 to 500 μg, 125 to 500 μg, 150 to 500 μg, 175 to 500 μg, 200 to 500 μg, 225 to 500 μg, 250 to 500 μg, 275 to 500 μg, 300 to 500 μg, 325 to 500 μg, 350 to 500 μg, 375 to 500 μg, 400 to 500 μg, 425 to 500 μg, 450 to 500 μg, 475 to 500 μg, 0.1 to 450 μg, 0.2 to 450 μg, 0.5 to 450 μg, 0.75 to 450 μg, 1 to 450 μg, 2.5 to 450 μg, 5 to 450 μg, 7.5 to 450 μg, 10 to 450 μg, 12.5 to 450 μg, 15 to 450 μg, 17.5 to 450 μg, 20 to 450 μg, 25 to 450 μg, 30 to 450 μg, 35 to 450 μg, 40 to 450 μg, 45 to 450 μg, 50 to 450 μg, 55 to 450 μg, 60 to 450 μg, 65 to 450 μg, 70 to 450 μg, 75 to 450 μg, 80 to 450 μg, 85 to 450 μg, 90 to 450 μg, 95 to 450 μg, 100 to 450 μg, 125 to 450 μg, 150 to 450 μg, 175 to 450 μg, 200 to 450 μg, 225 to 450 μg, 250 to 450 μg, 275 to 450 μg, 300 to 450 μg, 325 to 450 μg, 350 to 450 μg, 375 to 450 μg, 400 to 450 μg, 425 to 450 μg, 0.1 to 400 μg, 0.2 to 400 μg, 0.5 to 400 μg, 0.75 to 400 μg, 1 to 400 μg, 2.5 to 400 μg, 5 to 400 μg, 7.5 to 400 μg, 10 to 400 μg, 12.5 to 400 μg, 15 to 400 μg, 17.5 to 400 μg, 20 to 400 μg, 25 to 400 μg, 30 to 400 μg, 35 to 400 μg, 40 to 400 μg, 45 to 400 μg, 50 to 400 μg, 55 to 400 μg, 60 to 400 μg, 65 to 400 μg, 70 to 400 μg, 75 to 400 μg, 80 to 400 μg, 85 to 400 μg, 90 to 400 μg, 95 to 400 μg, 100 to 400 μg, 125 to 400 μg, 150 to 400 μg, 175 to 400 μg, 200 to 400 μg, 225 to 400 μg, 250 to 400 μg, 275 to 400 μg, 300 to 400 μg, 325 to 400 μg, 350 to 400 μg, 375 to 400 μg, 0.1 to 350 μg, 0.2 to 350 μg, 0.5 to 350 μg, 0.75 to 350 μg, 1 to 350 μg, 2.5 to 350 μg, 5 to 350 μg, 7.5 to 350 μg, 10 to 350 μg, 12.5 to 350 μg, 15 to 350 μg, 17.5 to 350 μg, 20 to 350 μg, 25 to 350 μg, 30 to 350 μg, 35 to 350 μg, 40 to 350 μg, 45 to 350 μg, 50 to 350 μg, 55 to 350 μg, 60 to 350 μg, 65 to 350 μg, 70 to 350 μg, 75 to 350 μg, 80 to 350 μg, 85 to 350 μg, 90 to 350 μg, 95 to 350 μg, 100 to 350 μg, 125 to 350 μg, 150 to 350 μg, 175 to 350 μg, 200 to 350 μg, 225 to 350 μg, 250 to 350 μg, 275 to 350 μg, 300 to 350 μg, 325 to 350 μg, 0.1 to 300 μg, 0.2 to 300 μg, 0.5 to 300 μg, 0.75 to 300 μg, 1 to 300 μg, 2.5 to 300 μg, 5 to 300 μg, 7.5 to 300 μg, 10 to 300 μg, 12.5 to 300 μg, 15 to 300 μg, 17.5 to 300 μg, 20 to 300 μg, 25 to 300 μg, 30 to 300 μg, 35 to 300 μg, 40 to 300 μg, 45 to 300 μg, 50 to 300 μg, 55 to 300 μg, 60 to 300 μg, 65 to 300 μg, 70 to 300 μg, 75 to 300 μg, 80 to 300 μg, 85 to 300 μg, 90 to 300 μg, 95 to 300 μg, 100 to 300 μg, 125 to 300 μg, 150 to 300 μg, 175 to 300 μg, 200 to 300 μg, 225 to 300 μg, 250 to 300 μg, 275 to 300 μg, 0.1 to 250 μg, 0.2 to 250 μg, 0.5 to 250 μg, 0.75 to 250 μg, 1 to 250 μg, 2.5 to 250 μg, 5 to 250 μg, 7.5 to 250 μg, 10 to 250 μg, 12.5 to 250 μg, 15 to 250 μg, 17.5 to 250 μg, 20 to 250 μg, 25 to 250 μg, 30 to 250 μg, 35 to 250 μg, 40 to 250 μg, 45 to 250 μg, 50 to 250 μg, 55 to 250 μg, 60 to 250 μg, 65 to 250 μg, 70 to 250 μg, 75 to 250 μg, 80 to 250 μg, 85 to
250 μg, 90 to 250 μg, 95 to 250 μg, 100 to 250 μg, 125 to 250 μg, 150 to 250 μg, 175 to 250 μg, 200 to 250 μg,
225 to 250 μg, 0.1 to 200 μg, 0.2 to 200 μg, 0.5 to 200 μg, 0.75 to 200 μg, 1 to 200 μg, 2.5 to 200 μg, 5 to 200 μg, 7.5 to 200 μg, 10 to 200 μg, 12.5 to 200 μg, 15 to 200 μg, 17.5 to 200 μg, 20 to 200 μg, 25 to 200 μg, 30 to
200 μg, 35 to 200 μg, 40 to 200 μg, 45 to 200 μg, 50 to 200 μg, 55 to 200 μg, 60 to 200 μg, 65 to 200 μg, 70 to 200 μg, 75 to 200 μg, 80 to 200 μg, 85 to 200 μg, 90 to 200 μg, 95 to 200 μg, 100 to 200 μg, 125 to 200 μg, 150 to 200 μg, 175 to 200 μg, 0.1 to 150 μg, 0.2 to 150 μg, 0.5 to 150 μg, 0.75 to 150 μg, 1 to 150 μg, 2.5 to 150 μg, 5 to 150 μg, 7.5 to 150 μg, 10 to 150 μg, 12.5 to 150 μg, 15 to 150 μg, 17.5 to 150 μg, 20 to 150 μg, 25 to 150 μg, 30 to 150 μg, 35 to 150 μg, 40 to 150 μg, 45 to 150 μg, 50 to 150 μg, 55 to 150 μg, 60 to 150 μg,
65 to 150 μg, 70 to 150 μg, 75 to 150 μg, 80 to 150 μg, 85 to 150 μg, 90 to 150 μg, 95 to 150 μg, 100 to 150 μg, 125 to 150 μg, 0.1 to 100 μg, 0.2 to 100 μg, 0.5 to 100 μg, 0.75 to 100 μg, 1 to 100 μg, 2.5 to 100 μg, 5 to 100 μg, 7.5 to 100 μg, 10 to 100 μg, 12.5 to 100 μg, 15 to 100 μg, 17.5 to 100 μg, 20 to 100 μg, 25 to 100 μg, 30 to 100 μg, 35 to 100 μg, 40 to 100 μg, 45 to 100 μg, 50 to 100 μg, 55 to 100 μg, 60 to 100 μg, 65 to 100 μg,
70 to 100 μg, 75 to 100 μg, 80 to 100 μg, 85 to 100 μg, 90 to 100 μg, 95 to 100 μg, 0.1 to 90 μg, 0.2 to 90 μg,
0.5 to 90 μg, 0.75 to 90 μg, 1 to 90 μg, 2.5 to 90 μg, 5 to 90 μg, 7.5 to 90 μg, 10 to 90 μg, 12.5 to 90 μg, 15 to 90 μg, 17.5 to 90 μg, 20 to 90 μg, 25 to 90 μg, 30 to 90 μg, 35 to 90 μg, 40 to 90 μg, 45 to 90 μg, 50 to 90 μg, 55 to 90 μg, 60 to 90 μg, 65 to 90 μg, 70 to 90 μg, 75 to 90 μg, 80 to 90 μg, 85 to 90 μg, 0.1 to 80 μg, 0.2 to 80 μg, 0.5 to 80 μg, 0.75 to 80 μg, 1 to 80 μg, 2.5 to 80 μg, 5 to 80 μg, 7.5 to 80 μg, 10 to 80 μg, 12.5 to 80 μg, 15 to 80 μg, 17.5 to 80 μg, 20 to 80 μg, 25 to 80 μg, 30 to 80 μg, 35 to 80 μg, 40 to 80 μg, 45 to 80 μg, 50 to 80 μg, 55 to 80 μg, 60 to 80 μg, 65 to 80 μg, 70 to 80 μg, 75 to 80 μg, 0.1 to 70 μg, 0.2 to 70 μg, 0.5 to 70 μg, 0.75 to 70 μg, 1 to 70 μg, 2.5 to 70 μg, 5 to 70 μg, 7.5 to 70 μg, 10 to 70 μg, 12.5 to 70 μg, 15 to 70 μg, 17.5 to 70 μg, 20 to 70 μg, 25 to 70 μg, 30 to 70 μg, 35 to 70 μg, 40 to 70 μg, 45 to 70 μg, 50 to 70 μg, 55 to 70 μg, 60 to 70 μg, 65 to 70 μg, 0.1 to 60 μg, 0.2 to 60 μg, 0.5 to 60 μg, 0.75 to 60 μg, 1 to 60 μg, 2.5 to 60 μg, 5 to 60 μg, 7.5 to 60 μg, 10 to 60 μg, 12.5 to 60 μg, 15 to 60 μg, 17.5 to 60 μg, 20 to 60 μg, 25 to 60 μg, 30 to 60 μg, 35 to 60 μg, 40 to 60 μg, 45 to 60 μg, 50 to 60 μg, 55 to 60 μg, 0.1 to 50 μg, 0.2 to 50 μg, 0.5 to 50 μg, 0.75 to 50 μg, 1 to 50 μg, 2.5 to 50 μg, 5 to 50 μg, 7.5 to 50 μg, 10 to 50 μg, 12.5 to 50 μg, 15 to 50 μg, 17.5 to 50 μg, 20 to 50 μg, 25 to 50 μg, 30 to 50 μg, 35 to 50 μg, 40 to 50 μg, 45 to 50 μg, 0.1 to 40 μg, 0.2 to 40 μg, 0.5 to 40 μg, 0.75 to 40 μg, 1 to 40 μg, 2.5 to 40 μg, 5 to 40 μg, 7.5 to 40 μg, 10 to 40 μg, 12.5 to 40 μg, 15 to 40 μg, 17.5 to 40 μg, 20 to 40 μg, 25 to 40 μg, 30 to 40 μg, 35 to 40 μg, 0.1 to 30 μg, 0.2 to 30 μg, 0.5 to 30 μg, 0.75 to 30 μg, 1 to 30 μg, 2.5 to 30 μg, 5 to 30 μg, 7.5 to 30 μg, 10 to 30 μg, 12.5 to 30 μg, 15 to 30 μg, 17.5 to 30 μg, 20 to 30 μg, 25 to 30 μg, 0.1 to 20 μg, 0.2 to 20 μg, 0.5 to 20 μg, 0.75 to 20 μg, 1 to 20 μg, 2.5 to 20 μg, 5 to 20 μg, 7.5 to 20 μg, 10 to 20 μg, 12.5 to 20 μg, 15 to 20 μg, 17.5 to 20 μg, 0.1 to 10 μg, 0.2 to 10 μg, 0.5 to 10 μg, 0.75 to 10 μg, 1 to 10 μg, 2.5 to 10 μg, 5 to 10 μg, 7.5 to 10 μg, 0.1 to 1 μg, 0.2 to 1 μg, 0.5 to 1 μg, or 0.75 to 1 μg of one or more polyribonucleotides encoding one or more MPXV antigens, or antigenic fragments thereof.
[0609] In some embodiments, each of one or more doses of a composition or combination disclosed herein comprises 1 μg to 250 μg of one or more polyribonucleotides encoding one or more MPXV antigens or antigenic fragments thereof. In some embodiments, each of one or more doses comprises 5 μg to 200 μg of one or more polyribonucleotides encoding one or more MPXV antigens or antigenic fragments thereof. In some embodiments, each of one or more doses comprises 10 μg to 100 μg of one or more polyribonucleotides encoding one or more MPXV antigens or antigenic fragments thereof. In some embodiments, each of one or more doses comprises 10 μg to 60 μg of one or more polyribonucleotides encoding one or more MPXV antigens or antigenic fragments thereof. In some embodiments, each of one or more doses comprises 10 μg of one or more polyribonucleotides encoding one or more MPXV antigens or antigenic fragments thereof. In some embodiments, each of one or more doses comprises 30 μg of one or more polyribonucleotides encoding one or more MPXV antigens or antigenic fragments thereof. In some embodiments, each of one or more doses comprises 60 μg of one or more polyribonucleotides encoding one or more MPXV antigens or antigenic fragments thereof.
[0610] In some embodiments, each of one or more doses comprises about 0.5 μg, about 0.75 μg, about 1.0 μg, about 1.25 μg, about 1.33 μg, about 1.5μg, about 1.75 μg, about 2.0 μg, about 2.25 μg or about 2.5 μg of one or more polyribonucleotides encoding one or more MPXV antigens or antigenic fragments thereof. In some embodiments, each of one or more doses comprises about 1.33 μg of each of three polyribonucleotides, wherein each polyribonucleotide encodes a different MPXV antigen or antigenic fragment thereof. In some embodiments, each of one or more doses comprises about 1 μg of each of four polyribonucleotides, wherein each polyribonucleotide encodes a different MPXV antigen or antigenic fragment thereof. In some embodiments, each of one or more doses comprises about 3.33 μg of each of three polyribonucleotides, wherein each polyribonucleotide encodes a different MPXV antigen or antigenic fragment thereof. In some embodiments, each of one or more doses comprises about 2.5 μg of each of four polyribonucleotides, wherein each polyribonucleotide encodes a different MPXV antigen or antigenic fragment thereof. In some embodiments, each of one or more doses comprises about 10 μg of each of three polyribonucleotides, wherein each polyribonucleotide encodes a different MPXV antigen or antigenic fragment thereof. In some embodiments, each of one or more doses comprises about 7.5 μg of each of four polyribonucleotides, wherein each polyribonucleotide encodes a different MPXV antigen or antigenic fragment thereof. In some embodiments, each of one or more doses comprises about 20 μg of each of three polyribonucleotides, wherein each polyribonucleotide encodes a different MPXV antigen or antigenic fragment thereof. In some embodiments, each of one or more doses comprises about 15 μg of each of four polyribonucleotides, wherein each polyribonucleotide encodes a different MPXV antigen or antigenic fragment thereof.
XII. Concomitant Therapies
[0611] In some embodiments, the present disclosure provides a method of treating or preventing an orthopoxvirus (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, modified vaccinia virus Ankara, or vaccinia virus) infection comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of a composition or combination as disclosed herein concomitantly with administration of an antipyretic medication. In some embodiments, an antipyretic medication is administered within about 60 minutes, within about 30 minutes, or within about 15 minutes after administration of one or more doses of a therapeutically effective amount of a composition or combination as disclosed herein. In some embodiments, an antipyretic medication is administered concurrently with administration of one or more doses of a therapeutically effective amount of a composition or combination as disclosed herein. In some embodiments, an antipyretic medication is administered within about 60 minutes, within about 30 minutes, or within about 15 minutes before administration of one of more doses of a therapeutically effective amount of a composition or combination as disclosed herein.
[0612] In some embodiments, an antipyretic is acetaminophen, a non-steroidal anti-inflammatory drug (NSAID), salicylamide, salicyl salicylate, methyl salicylate, magnesium salicylate, faislamine, ethenzamide, diflunisal, choline magnesium salicylate, benorylate/benorilatem and amoxiprin, acetylsalicylate, ceclofenac, acemetacin, alclofenac, bromfenac, diclofenac, etodolac, indomethacin, nabumetone, oxametacin, proglumetacin, sulindac, tolmetin, iminoprofen, benoxaprofen, carprofen, dexibuprofen, dexketoprofen, fenbufen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, ketorolac, loxoprofen, naproxen, oxaprozin, pirprofen, suprofen, tiaprofenic acid, mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, droxicam, lornoxicam, meloxicam, piroxicam, tenoxicam, dipyrone, azapropazone, clofezone, kebuzone, metamizole, mofebutazone, oxyphenbutazone, phenazone, phenylbutazone, sulfinpyrazone, decoxib, rofecoxib, parecoxib, and etoricoxib. In certain embodiments, the antipyretic is an NSAID. In certain embodiments, an antipyretic is acetaminophen.
[0613] In some embodiments, the present disclosure provides a method of treating or preventing an orthopoxvirus (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, modified vaccinia virus Ankara, or vaccinia virus) infection comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of a composition as disclosed herein concomitantly with administration of an analgesic medication. In some embodiments, an analgesic medication is administered within 60 minutes, within 30 minutes, or within 15 minutes after administration of one or more doses of a therapeutically effective amount of a composition as disclosed herein. In some embodiments, an analgesic medication is administered concurrently with administration of one or more doses of a therapeutically effective amount of a composition as disclosed herein. In some embodiments, an analgesic medication is administered within 60 minutes, within 30 minutes, or within 15 minutes before administration of one of more doses of a therapeutically effective amount of a composition as disclosed herein.
[0614] In some embodiments, the analgesic is acetaminophen, salicylamide, salicyl salicylate, methyl salicylate, magnesium salicylate, faislamine, ethenzamide, diflunisal, choline magnesium salicylate, benorylate/benorilatem and amoxiprin, acetylsalicylate, ceclofenac, acemetacin, alclofenac, bromfenac, diclofenac, etodolac, indomethacin, nabumetone, oxametacin, proglumetacin, sulindac, tolmetin, iminoprofen, benoxaprofen, carprofen, dexibuprofen, dexketoprofen, fenbufen, fenoprofen, flunoxaprofen, flurbiprofen, ibuprofen, ibuproxam, indoprofen, ketoprofen, ketorolac, loxoprofen, naproxen, oxaprozin, pirprofen, suprofen, tiaprofenic acid, mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, droxicam, lornoxicam, meloxicam, piroxicam, tenoxicam, dipyrone, azapropazone, clofezone, kebuzone, metamizole, mofebutazone, oxyphenbutazone, phenazone, phenylbutazone, sulfinpyrazone, decoxib, rofecoxib, parecoxib, etoricoxib, codeine, dihydrocodeine, morphine or a morphine derivative or pharmaceutically acceptable salt thereof, diacetylmorphine, hydrocodone, hydromorphone, levorphanol, oxymorphone, alfentanil, buprenorphine, butorphanol, fentanyl, sufentanil, meperidine, methadone, nalbuphine, propoxyphene, pentazocine, or pharmaceutically acceptable salts thereof. In some embodiments, the analgesic is acetaminophen.
[0615] In some embodiments, the present disclosure provides a method of treating or preventing an orthopoxvirus (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, modified vaccinia virus Ankara, or vaccinia virus) infection comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of a composition as disclosed herein concomitantly with administration of an antipyretic medication and an analgesic medication. In some embodiments, an antipyretic medication and analgesic medication is administered within 60 minutes, within 30 minutes, or within 15 minutes after administration of one or more doses of a therapeutically effective amount of a composition as disclosed herein. In some embodiments, an antipyretic medication and analgesic medication is administered concurrently with administration of one or more doses of a therapeutically effective amount of a composition as disclosed herein. In some embodiments, an antipyretic medication and analgesic medication is administered within 60 minutes, within 30 minutes, or within 15 minutes before administration of one of more doses of a therapeutically effective amount of a composition as disclosed herein.
[0616] In some embodiments, an antipyretic medication and/or analgesic medication is acetaminophen. In some embodiments, acetaminophen is administered to the subject at a dose of about 0.1 g/day to about 20 g/day, about 0.25 g/day to about 20 g/day, about 0.5 g/day to about 20 g/day, about 0.75 g/day to about 20 g/day, about 1.0 g/day to about 20 g/day, about 1.25 g/day to about 20 g/day, about 1.5 g/day to about 20 g/day, about 1.75 g/day to about 20 g/day, about 2.0 g/day to about 20 g/day, about 2.25 g/day to about 20 g/day, about 2.5 g/day to about 20 g/day, about 2.75 g/day to about 20 g/day, about 3.0 g/day to about 20 g/day, about 3.25 g/day to about 20 g/day, about 3.5 g/day to about 20 g/day, about 3.75 g/day to about 20 g/day, about 4.0 g/day to about 20 g/day, about 4.25 g/day to about 20 g/day, about 4.5 g/day to about 20 g/day, about 4.75 g/day to about 20 g/day, about 5.0 g/day to about 20 g/day, about 5.25 g/day to about 20 g/day, about 5.5 g/day to about 20 g/day, about 5.75 g/day to about 20 g/day, about 6.0 g/day to about 20 g/day, about 6.25 g/day to about 20 g/day, about 6.5 g/day to about 20 g/day, about 6.75 g/day to about 20 g/day, about 7.0 g/day to about 20 g/day, about 7.25 g/day to about 20 g/day, about 7.5 g/day to about 20 g/day, about 7.75 g/day to about 20 g/day, about 8.0 g/day to about 20 g/day, about 8.25 g/day to about 20 g/day, about 8.5 g/day to about 20 g/day, about 8.75 g/day to about 20 g/day, about 9.0 g/day to about 20 g/day, about 9.25 g/day to about 20 g/day, about 9.5 g/day to about 20 g/day, about 9.75 g/day to about 20 g/day, about 10 g/day to about 20 g/day, about 12.5 g/day to about 20 g/day, about 15 g/day to about 20 g/day, about 17.5 g/day to about 20 g/day, about 0.1 g/day to about 15 g/day, about 0.25 g/day to about 15 g/day, about 0.5 g/day to about 15 g/day, about 0.75 g/day to about 15 g/day, about 1.0 g/day to about 15 g/day, about 1.25 g/day to about 15 g/day, about 1.5 g/day to about 15 g/day, about 1.75 g/day to about 15 g/day, about 2.0 g/day to about 15 g/day, about 2.25 g/day to about 151 g/day, about 2.5 g/day to about 15 g/day, about 2.75 g/day to about 15 g/day, about 3.0 g/day to about 15 g/day, about 3.25 g/day to about 15 g/day, about 3.5 g/day to about 15 g/day, about 3.75 g/day to about 15 g/day, about 4.0 g/day to about 15 g/day, about 4.25 g/day to about 15 g/day, about 4.5 g/day to about 15 g/day, about 4.75 g/day to about 15 g/day, about 5.0 g/day to about 15 g/day, about 5.25 g/day to about 15 g/day, about 5.5 g/day to about 15 g/day, about 5.75 g/day to about 15 g/day, about 6.0 g/day to about 15 g/day, about 6.25 g/day to about 15 g/day, about 6.5 g/day to about 15 g/day, about 6.75 g/day to about 15 g/day, about 7.0 g/day to about 15 g/day, about 7.25 g/day to about 15 g/day, about 7.5 g/day to about 15 g/day, about 7.75 g/day to about 15 g/day, about 8.0 g/day to about 15 g/day, about 8.25 g/day to about 15 g/day, about 8.5 g/day to about 15 g/day, about 8.75 g/day to about 15 g/day, about 9.0 g/day to about 15 g/day, about 9.25 g/day to about 15 g/day, about 9.5 g/day to about 15 g/day, about 9.75 g/day to about 15 g/day, about 10 g/day to about 15 g/day, about 12.5 g/day to about 15 g/day, about 0.1 g/day to about 10 g/day, about 0.25 g/day to about 10 g/day, about 0.5 g/day to about 10 g/day, about 0.75 g/day to about 10 g/day, about 1.0 g/day to about 10 g/day, about 1.25 g/day to about 10 g/day, about 1.5 g/day to about 10 g/day, about 1.75 g/day to about 10 g/day, about 2.0 g/day to about 10 g/day, about 2.25 g/day to about 101 g/day, about 2.5 g/day to about 10 g/day, about 2.75 g/day to about 10 g/day, about 3.0 g/day to about 10 g/day, about 3.25 g/day to about 10 g/day, about 3.5 g/day to about 10 g/day, about 3.75 g/day to about 10 g/day, about 4.0 g/day to about 10 g/day, about 4.25 g/day to about 10 g/day, about 4.5 g/day to about 10 g/day, about 4.75 g/day to about 10 g/day, about 5.0 g/day to about 10 g/day, about 5.25 g/day to about 10 g/day, about 5.5 g/day to about 10 g/day, about 5.75 g/day to about 10 g/day, about 6.0 g/day to about 10 g/day, about 6.25 g/day to about 10 g/day, about 6.5 g/day to about 10 g/day, about 6.75 g/day to about 10 g/day, about 7.0 g/day to about 10 g/day, about 7.25 g/day to about 10 g/day, about 7.5 g/day to about 10 g/day, about 7.75 g/day to about 10 g/day, about 8.0 g/day to about 10 g/day, about 8.25 g/day to about 10 g/day, about 8.5 g/day to about 10 g/day, about 8.75 g/day to about 10 g/day, about 9.0 g/day to about 10 g/day, about 9.25 g/day to about 10 g/day, about 9.5 g/day to about 10 g/day, about 9.75 g/day to about 10 g/day, about 0.1 g/day to about 7.5 g/day, about 0.25 g/day to about 7.5 g/day, about 0.5 g/day to about 7.5 g/day, about 0.75 g/day to about 7.5 g/day, about 1.0 g/day to about 7.5 g/day, about 1.25 g/day to about 7.5 g/day, about 1.5 g/day to about 7.5 g/day, about 1.75 g/day to about 7.5 g/day, about 2.0 g/day to about 7.5 g/day, about 2.25 g/day to about 7.51 g/day, about 2.5 g/day to about 7.5 g/day, about 2.75 g/day to about 7.5 g/day, about 3.0 g/day to about 7.5 g/day, about 3.25 g/day to about 7.5 g/day, about 3.5 g/day to about 7.5 g/day, about 3.75 g/day to about 7.5 g/day, about 4.0 g/day to about 7.5 g/day, about 4.25 g/day to about 7.5 g/day, about 4.5 g/day to about 7.5 g/day, about 4.75 g/day to about 7.5 g/day, about 5.0 g/day to about 7.5 g/day, about 5.25 g/day to about 7.5 g/day, about 5.5 g/day to about 7.5 g/day, about 5.75 g/day to about 7.5 g/day, about 6.0 g/day to about 7.5 g/day, about 6.25 g/day to about 7.5 g/day, about 6.5 g/day to about 7.5 g/day, about 6.75 g/day to about 7.5 g/day, about 7.0 g/day to about 7.5 g/day, about 7.25 g/day to about 7.5 g/day, about 0.1 g/day to about 5.0 g/day, about 0.25 g/day to about 5.0 g/day, about 0.5 g/day to about 5.0 g/day, about 0.75 g/day to about 5.0 g/day, about 1.0 g/day to about 5.0 g/day, about 1.25 g/day to about 5.0 g/day, about 1.5 g/day to about 5.0 g/day, about 1.75 g/day to about 5.0 g/day, about 2.0 g/day to about 5.0 g/day, about 2.25 g/day to about 5.0 g/day, about 2.5 g/day to about 5.0 g/day, about 2.75 g/day to about 5.0 g/day, about 3.0 g/day to about 5.0 g/day, about 3.25 g/day to about 5.0 g/day, about 3.5 g/day to about 5.0 g/day, about 3.75 g/day to about 5.0 g/day, about 4.0 g/day to about 5.0 g/day, about 4.25 g/day to about 5.0 g/day, about 4.5 g/day to about 5.0 g/day, about 4.75 g/day to about 5.0 g/day, about 0.1 g/day to about 2.5 g/day, about 0.25 g/day to about 2.5 g/day, about 0.5 g/day to about 2.5 g/day, about 0.75 g/day to about 2.5 g/day, about 1.0 g/day to about 2.5 g/day, about 1.25 g/day to about 2.5 g/day, about 1.5 g/day to about 2.5 g/day, about 1.75 g/day to about 2.5 g/day, about 2.0 g/day to about 2.5 g/day, or about 2.25 g/day to about 2.5 g/day. In some embodiments, acetaminophen is administered to a subject at a dose of about 0.5 g/day to about 10 g/day. In some embodiments, acetaminophen is administered to a subject at a dose of about 1 g/day to about 5 g/day. [0617] In some embodiments, acetaminophen is administered to the subject at a dose of about 0.5 g/day, about 1 g/day, about, about 1.5 g/day, about 2 g/day, about 2.5 g/day, about 3 g/day, about 3.5 g/day, about 4 g/day, about 4.5 g/day, or about 5 g/day. In some embodiments, acetaminophen is administered to the subject at a dose of about 4 g/day.
XIII. Combinatorial Use with Other Vaccines
[0618] In some embodiments, the present disclosure provides a method of treating or preventing an orthopoxvirus (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) infection comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of a composition or combination as disclosed herein, wherein the subject is further administered a seasonal influenza vaccine at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 12 days, at least about 14 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, or at least about 20 days before administration of the therapeutically acceptable amount of the composition or combination. In some embodiments, a subject is further administered a seasonal influenza vaccine at least 14 days before administration of a therapeutically acceptable amount of a composition or combination as disclosed herein. [0619] In some embodiments, the present disclosure provides a method of treating or preventing an orthopoxvirus (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, modified vaccinia virus Ankara, or vaccinia virus) infection comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of a composition or combination as disclosed herein, wherein the subject is further administered a seasonal influenza vaccine at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 12 days, at least about 14 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, or at least about 20 days after administration of the therapeutically acceptable amount of the composition or combination. In some embodiments, a subject is further administered a seasonal influenza vaccine at least about 14 days after administration of a therapeutically acceptable amount of a composition or combination disclosed herein.
[0620] In some embodiments, the present disclosure provides a method of treating or preventing an orthopoxvirus (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) infection comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of a composition as disclosed herein, wherein the subject is further administered a medically indicated vaccine at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 12 days, at least 14 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, or at least 20 days before administration of the therapeutically acceptable amount of the composition. In some embodiments, the subject is further administered a medically indicated vaccine at least 14 days before the administration of the therapeutically acceptable amount of the composition.
[0621] In some embodiments, the present disclosure provides a method of treating or preventing orthopoxvirus (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) infection comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of a composition or combination as disclosed herein, wherein the subject is further administered a medically indicated vaccine at least about 1 day, at least about 2 days, at least about 3 days, at least about 4 days, at least about 5 days, at least about 6 days, at least about 7 days, at least about 8 days, at least about 9 days, at least about 10 days, at least about 12 days, at least about 14 days, at least about 16 days, at least about 17 days, at least about 18 days, at least about 19 days, or at least about 20 days after administration of the therapeutically acceptable amount of the composition or combination. In some embodiments, a subject is further administered a medically indicated vaccine at least about 14 days after administration of a therapeutically acceptable amount of a composition or combination disclosed herein.
[0622] In some embodiments, a medically indicated vaccine includes, but is not limited to, a vaccine selected from a rabies vaccine, a tetanus vaccine, a hepatitis A vaccine, a hepatitis B vaccine, a measles mumps rubella (MMR) vaccine, a polio vaccine, a diphtheria vaccine, a varicella vaccine, a pertussis vaccine, a shingles vaccine, a pneumococcal vaccine, a human papillomavirus vaccine (HPV), a meningococcal vaccine, and a rotavirus vaccine. In some embodiments, a medically indicated vaccine is a rabies vaccine or a tetanus vaccine. XIV, Combinatorial Use with Corticosteroids
[0623] In some embodiments, the present disclosure provides a method of treating or preventing an orthopoxvirus (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) infection comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of a composition or combination as disclosed herein, wherein the subject is further administered a corticosteroid at least about 2 hours, at least about 1.5 hours at least about 1 hour, at least about 45 minutes, at least about 30 minutes, at least about 15 minutes, at least about 10 minutes, or at least about 5 minutes before administration of the one or more doses of a therapeutically effective amount of a composition or combination as disclosed herein. [0624] In some embodiments, the present disclosure provides a method of treating or preventing an orthopoxvirus (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) infection comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of a composition or combination as disclosed herein, wherein the subject is further administered a corticosteroid concurrently with administration of the one or more doses of the therapeutically effective amount of a composition or combination as disclosed herein.
[0625] In some embodiments, the present disclosure provides a method of treating or preventing an orthopoxvirus (e.g., variola, borealpox, ectromelia, cowpox, volepox, buffalopox, camelpox, rabbitpox, or vaccinia (e.g., modified vaccinia virus Ankara, etc.) virus) infection comprising administering to a subject in need thereof one or more doses of a therapeutically effective amount of a composition or combination as disclosed herein, wherein the subject is further administered a corticosteroid at least about 5 minutes at least about 10 minutes, at least about 15 minutes, at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 1.5 hours, or at least about 2 hours after administration of the one or more doses of a therapeutically effective amount of a composition or combination as disclosed herein.
[0626] In some embodiments, a corticosteroid includes, but is not limited to, a coriticosteroid selected from methylprednisolone, dexamethasone, hydrocortisone, prednisone, prednisolone, fluticasone, flumethasone, fluocinolone, budesonide, beclomethasone, ciclesonide, cortisone, triamcinolone, betamethasone, deflazacort, difluprednate, loteprednol, para methasone, tixocortol, aldosterone, cloprednol, cortivazol, deoxycortone, desonide, desoximetasone, difluorocortolone, fluclorolone, fludrocortisone, flunisolide, fluocinonide, fluocortin butyl, fluorocortisone, fluorocortolone, fluoromethoIone, flurandrenolone, halcinonide, icomethasone, meprednisone, mometasone, rofleponide, RPR 106541, and their respective pharmaceutically acceptable derivatives, such as beclomethasone dipropionate (anhydrous or monohydrate), beclomethasone monopropionate, dexamethasone 21-isonicotinate, fluticasone propionate, icomethasone enbutate, tixocortol 21- pivalate, triamcinolone acetonide, and pharmaceutically acceptable salts and/or derivatives thereof.
[0627] In some embodiments, a corticosteroid is administered via inhalation, topical injection, or local injection.
EXEMPLARY EMBODIMENTS
[0628] Embodiment 1. A composition comprising a polyribonucleotide encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier.
[0629] Embodiment 2. A composition comprising a plurality of polyribonucleotides and a pharmaceutically acceptable carrier, wherein at least one polyribonucleotide of the plurality of polyribonucleotides encodes one or more MPXV antigens or fragments thereof.
[0630] Embodiment 3. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein at least two polyribonucleotides of the plurality of polyribonucleotides are not the same.
[0631] Embodiment 4. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein all of the polyribonucleotides of the plurality of polyribonucleotides encode one or more MPXV antigens or fragments thereof.
[0632] Embodiment 5. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein at least one polyribonucleotide of the plurality of polyribonucleotides encodes only one MPXV antigen or fragment thereof. [0633] Embodiment 6. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein a first polyribonucleotide of the plurality of polyribonucleotides encodes a first set of one or more MPXV antigens or fragments thereof, wherein the first set of one or more MPXV antigens or fragments thereof comprise:
(i) B6R or a fragment of B6R,
(ii) MIR or a fragment of MIR,
(iii) A35R or a fragment of A35R,
(iv) H3L or a fragment of H3L, and
(v) E8L or a fragment of E8L, or
(vi) a combination of any thereof; wherein a second polyribonucleotide of the plurality of polyribonucleotides encodes a second set of one or more MPXV antigens or fragments thereof, wherein the first set of one or more MPXV antigens or fragments thereof and the second set of one or more MPXV antigens or fragments thereof are different.
[0634] Embodiment 7. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein a first polyribonucleotide of the plurality of polyribonucleotides encodes a first set of one or more MPXV antigens or fragments thereof, wherein the first set of one or more MPXV antigens or fragments thereof comprise:
(i) A29L or a fragment of A29L,
(ii) A35R or a fragment of A35R,
(iii) B6R or a fragment of B6R,
(iv) MIR or a fragment of MIR,
(v) E8L or a fragment of E8L,
(vi) A28L or a fragment of A28L,
(vii) H3L or a fragment of H3L, or
(viii) a combination of any thereof; wherein a second polyribonucleotide of the plurality of polyribonucleotides encodes a second set of one or more MPXV antigens or fragments thereof, wherein the first set of one or more MPXV antigens or fragments thereof and the second set of one or more MPXV antigens or fragments thereof are different.
[0635] Embodiment 8. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein a first polyribonucleotide of the plurality of polyribonucleotides encodes a first set of one or more MPXV antigens or fragments thereof, wherein the first set of one or more MPXV antigens or fragments thereof comprise:
(i) A29L or a fragment of A29L,
(ii) A35R or a fragment of A35R,
(iii) B6R or a fragment of B6R,
(iv) MIR or a fragment of MIR,
(v) E8L or a fragment of E8L,
(vi) H3L or a fragment of H3L,
(vii) A45L or a fragment of A45L,
(viii) B9R or a fragment of B9R,
(ix) B16R or a fragment of B16R,
(x) C10L or a fragment of C10L, (xi) C21L or a fragment of C21L,
(xii) E7R or a fragment of E7R,
(xiii) F3L or a fragment of F3L,
(xiv) F4L or a fragment of F4L,
(xv) G6R or a fragment of G6R,
(xvi) H5R or a fragment of H5R,
(xvii) I3L or a fragment of I3L,
(xviii) O2L or a fragment of O2L,
(xix) Q1L or a fragment of Q1L,
(xx) B12R or a fragment of B12R,
(xxi) C17L or a fragment of C17L,
(xxii) A28L,or a fragment of A28L, or
(xxiii) a combination of any thereof; wherein a second polyribonucleotide of the plurality of polyribonucleotides encodes a second set of one or more MPXV antigens or fragments thereof, wherein the first set of one or more MPXV antigens or fragments thereof and the second set of one or more MPXV antigens or fragments thereof are different.
[0636] Embodiment 9. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the first set of one or more MPXV antigens or fragments thereof and the second set of one or more MPXV antigens or fragments thereof do not include any of the same MPXV antigens or fragments thereof.
[0637] Embodiment 10. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the first polyribonucleotide encodes a B6R antigen or fragment thereof, and the second polyribonucleotide encodes an MIR antigen or fragment thereof.
[0638] Embodiment 11. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the first polyribonucleotide encodes a B6R antigen or fragment thereof, comprising an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182, and the second polyribonucleotide encodes an MIR antigen or fragment thereof, comprising an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158.
[0639] Embodiment 12. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the composition comprises a polyribonucleotide encoding an A35R antigen or fragment thereof.
[0640] Embodiment 13. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the composition comprises a polyribonucleotide encoding an A35R antigen or fragment thereof, comprising an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172 and 174.
[0641] Embodiment 14. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the composition comprises a polyribonucleotide encoding an E8L antigen or fragment thereof.
[0642] Embodiment 15. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the composition comprises a polyribonucleotide encoding an E8L antigen or fragment thereof, comprising an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 41-50 and 192.
[0643] Embodiment 16. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the composition comprises a polyribonucleotide encoding an H3L antigen or fragment thereof.
[0644] Embodiment 17. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the composition comprises a polyribonucleotide encoding an H3L antigen or fragment thereof, comprising an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 51-60, 184, 188 and 190.
[0645] Embodiment 18. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the composition further comprises lipid nanoparticles, polyplexes, lipidated polyplexes, or liposomes, wherein the polyribonucleotide or plurality of polyribonucleotides are fully or partially encapsulated within the lipid nanoparticles, polyplexes, lipidated polyplexes, or liposomes.
[0646] Embodiment 19. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the composition further comprises lipid nanoparticles, wherein the polyribonucleotide or plurality of polyribonucleotides are fully or partially encapsulated within the lipid nanoparticles.
[0647] Embodiment 20. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the lipid nanoparticles target liver cells.
[0648] Embodiment 21. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the lipid nanoparticles target secondary lymphoid organ cells.
[0649] Embodiment 22. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the lipid nanoparticles are cationic lipid nanoparticles.
[0650] Embodiment 23. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the lipid nanoparticles each comprise:
(a) a polymer-conjugated lipid;
(b) a cationic lipid; and (c) one or more neutral lipids.
[0651] Embodiment 24. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the one or more neutral lipids comprise 1,2-Distearoyl-sn-glycero-3-phosphocholine (DPSC).
[0652] Embodiment 25. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the one or more neutral lipids comprise cholesterol.
[0653] Embodiment 26. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the lipid nanoparticles comprise:
(a) the polymer-conjugated lipid at about 1-2.5 mol% of the total lipids;
(b) the cationic lipid at 35-65 mol% of the total lipids; and
(c) the one or more neutral lipids are present in 35-65 mol% of the total lipids.
[0654] Embodiment 27. A composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the lipid nanoparticles have an average diameter of about 50-150 nm.
[0655] Embodiment 28 A method of inducing an immune response against an orthopoxvirus in a subject comprising administering to the subject a composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier.
[0656] Embodiment 29. A method of inducing an immune response against an orthopoxvirus in a subject comprising administering to the subject a composition comprising a plurality of polyribonucleotides encoding one or more MPXV antigens or fragments thereof and a pharmaceutically acceptable carrier, wherein the orthopoxvirus is a mpox virus, a variola virus, a vaccinia virus, or a cowpox virus.
[0657] Embodiment 30. A composition or combination comprising: a therapeutically effective amount of one or more polyribonucleotides encoding one or more MPXV antigens or fragments thereof; and a pharmaceutically acceptable carrier, wherein each of the one or more polyribonucleotides or fragments thereof is fully or partially encapsulated within a lipid nanoparticle.
[0658] Embodiment 31. A composition or combination wherein the one or more polyribonucleotides are purified.
[0659] Embodiment 32. A composition or combination wherein the one or more polyribonucleotides are single-stranded.
[0660] Embodiment 33. A composition or combination wherein the one or more polyribonucleotides comprise a 5'-capped Nl-methylpseudouridine.
[0661] Embodiment 34. A composition or combination wherein the MPXV antigens or fragments thereof comprise:
(i) B6R or a fragment of B6R;
(ii) MIR or a fragment of MIR;
(iii) A35R or a fragment of A35R; or
(iv) H3L or a fragment of H3L. [0662] Embodiment 35. A composition or combination comprising at least three polyribonucleotides, and wherein the at least three polyribonucleotides each encode a different MPXV antigen or fragment thereof.
[0663] Embodiment 36. A composition or combination comprising:
(i) a polyribonucleotide encoding B6R or a fragment of B6R;
(ii) a polyribonucleotide encoding MIR or a fragment of MIR; and
(iii) a polyribonucleotide encoding A35R or a fragment of A35R.
[0664] Embodiment 37. A composition or combination comprising at least four polyribonucleotides, and wherein the at least four polyribonucleotides each encode a different MPXV antigen or fragment thereof.
[0665] Embodiment 38. A composition or combination comprising:
(i) a polyribonucleotide encoding B6R or a fragment of B6R;
(ii) a polyribonucleotide encoding MIR or a fragment of MIR;
(iii) a polyribonucleotide encoding A35R or a fragment of A35R; and
(iv) a polyribonucleotide encoding H3L or a fragment of H3L.
[0666] Embodiment 39. A composition or combination wherein the lipid nanoparticle targets liver cells.
[0667] Embodiment 40. A composition or combination wherein the lipid nanoparticle targets secondary lymphoid organ cells.
[0668] Embodiment 41. A composition or combination wherein the lipid nanoparticle is a cationic lipid nanoparticle.
[0669] Embodiment 42. A composition or combination wherein the lipid nanoparticle comprises:
(a) a polymer-conjugated lipid;
(b) a cationic lipid; and
(c) one or more neutral lipids.
[0670] Embodiment 43. A composition or combination wherein the one or more neutral lipids comprise 1,2-Distearoyl-sn-glycero-3-phosphocholine (DPSC).
[0671] Embodiment 44. A composition or combination wherein the one or more neutral lipids comprise cholesterol.
[0672] Embodiment 45. A composition or combination wherein the lipid nanoparticle comprises:
(a) the polymer-conjugated lipid at about 1-2.5 mol% of the total lipids;
(b) the cationic lipid at 35-65 mol% of the total lipids; and
(c) the one or more neutral lipids are present in 35-65 mol% of the total lipids.
[0673] Embodiment 46. A composition or combination wherein the lipid nanoparticles have an average diameter of about 50-150 nm.
[0674] Embodiment 47. A composition or combination wherein the therapeutically effective amount of the one or more polyribonucleotides is 1 μg to 250 μg.
[0675] Embodiment 48. A composition or combination wherein the therapeutically effective amount of the one or more polyribonucleotides is 5 μg to 200 μg.
[0676] Embodiment 49. A composition or combination wherein the therapeutically effective amount of the one or more polyribonucleotides is 10 μg to 100 μg.
[0677] Embodiment 50. A composition or combination wherein the therapeutically effective amount of the one or more polyribonucleotides is 10 μg to 60 μg. [0678] Embodiment 51. A composition or combination wherein the therapeutically effective amount of the one or more polyribonucleotides is about 10 μg.
[0679] Embodiment 52. A composition or combination wherein the therapeutically effective amount of the one or more polyribonucleotides is about 30 μg.
[0680] Embodiment 53. A composition or combination wherein the therapeutically effective amount of the one or more polyribonucleotides is about 60 μg.
[0681] Embodiment 54. A method of preventing or treating an MPXV infection in a subject comprising administering one or more doses of a composition to the subject, wherein the composition comprises: a therapeutically effective amount of one or more polyribonucleotides encoding one or more MPXV antigens or fragments thereof; and a pharmaceutically acceptable carrier, wherein each of the one or more polyribonucleotides or fragments thereof is fully or partially encapsulated within a lipid nanoparticle.
[0682] Embodiment 55. A method of preventing or treating an orthopoxvirus infection in a subject comprising administering one or more doses of a composition to the subject, wherein the composition comprises: a therapeutically effective amount of one or more polyribonucleotides encoding one or more MPXV antigens or fragments thereof; and a pharmaceutically acceptable carrier, wherein each of the one or more polyribonucleotides or fragments thereof is fully or partially encapsulated within a lipid nanoparticle.
[0683] Embodiment 56. A method wherein the one or more polyribonucleotides are purified.
[0684] Embodiment 57. A method wherein the one or more polyribonucleotides are single-stranded.
[0685] Embodiment 58. A method wherein the one or more polyribonucleotides comprise a 5'-capped
Nl-methylpseudouridine.
[0686] Embodiment 59. A method wherein the MPXV antigens or fragments thereof comprise:
(i) B6R or a fragment of B6R;
(ii) MIR or a fragment of MIR;
(iii) A35R or a fragment of A35R; or
(iv) H3L or a fragment of H3L.
[0687] Embodiment 60. A method wherein the composition or combination comprises at least three polyribonucleotides, and wherein the at least three polyribonucleotides each encode a different MPXV antigen or fragment thereof.
[0688] Embodiment 61. A method wherein the composition or combination comprises:
(i) a polyribonucleotide encoding B6R or a fragment of B6R;
(ii) a polyribonucleotide encoding MIR or a fragment of MIR; and
(iii) a polyribonucleotide encoding A35R or a fragment of A35R.
[0689] Embodiment 62. A method wherein the composition or combination comprises at least four polyribonucleotides, and wherein the at least four polyribonucleotides each encode a different MPXV antigen or fragment thereof.
[0690] Embodiment 63. A method wherein the composition or combination comprises:
(i) a polyribonucleotide encoding B6R or a fragment of B6R;
(ii) a polyribonucleotide encoding MIR or a fragment of MIR; (iii) a polyribonucleotide encoding A35R or a fragment of A35R; and
(iv) a polyribonucleotide encoding H3L or a fragment of H3L.
[0691] Embodiment 64. A method wherein the lipid nanoparticle targets liver cells. [0692] Embodiment 65. A method wherein the lipid nanoparticle targets secondary lymphoid organ cells.
[0693] Embodiment 66. A method wherein the lipid nanoparticle is a cationic lipid nanoparticle.
[0694] Embodiment 67. A method wherein the lipid nanoparticle comprises:
(a) a polymer-conjugated lipid;
(b) a cationic lipid; and
(c) one or more neutral lipids.
[0695] Embodiment 68. A method wherein the one or more neutral lipids comprise 1,2-Distearoyl-sn- glycero-3-phosphocholine (DPSC).
[0696] Embodiment 69. A method wherein the one or more neutral lipids comprise cholesterol.
[0697] Embodiment 70. A method wherein the lipid nanoparticle comprises:
(a) the polymer-conjugated lipid at about 1-2.5 mol% of the total lipids;
(b) the cationic lipid at 35-65 mol% of the total lipids; and
(c) the one or more neutral lipids are present in 35-65 mol% of the total lipids.
[0698] Embodiment 71. A method wherein the lipid nanoparticles have an average diameter of about
50-150 nm.
[0699] Embodiment 72. A method wherein the therapeutically effective amount of the one or more polyribonucleotides is 1 μg to 250 μg.
[0700] Embodiment 73. A method wherein the therapeutically effective amount of the one or more polyribonucleotides is 5 μg to 200 μg.
[0701] Embodiment 74. A method wherein the therapeutically effective amount of the one or more polyribonucleotides is 10 μg to 100 μg.
[0702] Embodiment 75. A method wherein the therapeutically effective amount of the one or more polyribonucleotides is 10 μg to 60 μg.
[0703] Embodiment 76. A method wherein the therapeutically effective amount of the one or more polyribonucleotides is about 10 μg.
[0704] Embodiment 77. A method wherein the therapeutically effective amount of the one or more polyribonucleotides is about 30 μg.
[0705] Embodiment 78. A method wherein the therapeutically effective amount of the one or more polyribonucleotides is about 60 μg.
[0706] Embodiment 79. A method wherein the subject is administered two doses of the therapeutically effective amount of the composition or combination.
[0707] Embodiment 80. A method wherein a second dose of the therapeutically effective amount of the composition is administered to the subject 1 week to 12 weeks after administration of a first dose of the therapeutically effective amount of the composition is administered to the subject.
[0708] Embodiment 81. A method wherein the second dose of the therapeutically effective amount of the composition is administered to the subject 2 weeks to 10 weeks after administration of the first dose of the therapeutically effective amount of the composition is administered to the subject. [0709] Embodiment 82. A method wherein the second dose of the therapeutically effective amount of the composition is administered to the subject about 4 weeks after administration of the first dose of the therapeutically effective amount of the composition is administered to the subject.
[0710] Embodiment 83. A method wherein the second dose of the therapeutically effective amount of the composition is administered to the subject about 31 days after administration of the first dose of the therapeutically effective amount of the composition is administered to the subject.
[0711] Embodiment 84. A method wherein the one or more doses of the therapeutically effective amount of the composition are administered to the subject intramuscularly, subcutaneously, orally, or intranasally.
[0712] Embodiment 85. A method wherein the one or more doses of the therapeutically effective amount of the composition are administered to the subject intramuscularly.
[0713] Embodiment 86. A method wherein the method further comprises administering an antipyretic and/or analgesic medication to the subject after administration of the one or more doses of the therapeutically effective amount of the composition.
[0714] Embodiment 87. A method wherein the antipyretic and/or analgesic medication is acetaminophen or a non-steroidal anti-inflammatory drug (NSAID).
[0715] Embodiment 88. A method wherein acetaminophen is administered at a dose of 0.5 g/day to
10 g/day.
[0716] Embodiment 89. A method wherein acetaminophen is administered at a dose of 1 g/day to 5 g/day.
[0717] Embodiment 90. A method wherein acetaminophen is administered at a dose of about 4 g/day.
[0718] Embodiment 91. A method wherein the method further comprises administering a seasonal influenza vaccine to the subject at least 14 days before or after administration of the one or more doses of the therapeutically effective amount of the composition.
[0719] Embodiment 92. A method wherein the method further comprises administering a medically indicated vaccine to the subject at least 14 days before or after administration of the one or more doses of the therapeutically effective amount of the composition.
[0720] Embodiment 93. A method wherein the medically indicated vaccine is a rabies vaccine or a tetanus vaccine.
[0721] Embodiment 94. A method wherein the method further comprises administering a corticosteroid to the subject before or after administration of the one or more doses of the therapeutically effective amount of the composition.
[0722] Embodiment 95. A method wherein the corticosteroid is inhaled, topical, or locally injected.
[0723] Embodiment 96. A method wherein the method further comprises collecting one or more samples from the subject after administration of the one or more doses of the therapeutically effective amount of the composition.
[0724] Embodiment 97. A method wherein the one or more samples comprise a blood volume draw.
[0725] Embodiment 87. A method wherein the method further comprises measuring levels of MPXV- specific neutralizing antibodies in the one or more samples collected from the subject.
[0726] Embodiment 99. A method wherein the levels of MPXV-specific neutralizing antibodies are measured using a plaque reduction neutralization test (PRNT). [0727] Embodiment 100. A method wherein the method further comprises measuring levels of neutralizing antibodies specific for the one or more MPXV antigens or fragments thereof antibodies in the one or more samples collected from the subject.
[0728] Embodiment 101. A method wherein the levels are measured using an enzyme-linked immunosorbent assay (ELISA).
[0729] Embodiment 102. A method wherein the subject is a mammal.
[0730] Embodiment 103. A method wherein the subject is a human.
[0731] Embodiment 104. A method wherein the subject has no prior history of known or suspected smallpox vaccination.
[0732] Embodiment 105. A method wherein smallpox vaccination is determined based on the medical record or presence of a smallpox vaccination scar.
[0733] Embodiment 106. A method wherein the subject does not have febrile illness or other acute illness, or did not have febrile illness or other acute illness within 48 hours before administration of the one or more doses of the therapeutically effective amount of the composition.
[0734] Embodiment 107. A method wherein the subject has not received a vaccine within 28 days or
5 half-lives of the vaccine before administration of the one or more doses of the therapeutically effective amount of the composition, wherein the vaccine does not comprise a seasonal influenza vaccine or a medically indicated vaccine.
[0735] Embodiment 108. A method wherein the subject does not receive a vaccine until at least 8 weeks after administration of the one or more doses of the therapeutically effective amount of the composition, wherein the vaccine does not comprise a seasonal influenza vaccine or a medically indicated vaccine.
[0736] Embodiment 109. A method wherein the subject has not received an orthopoxyvirus-based vaccine before administration of the one or more doses of the therapeutically effective amount of the composition.
[0737] Embodiment 110. A method wherein the orthopoxvirus-based vaccine includes a smallpox vaccine, a vaccinia vaccine, an MPXV vaccine, or vector orthopoxvirus-based vaccine.
[0738] Embodiment 111. A method wherein the subject has not received blood, plasma products, or immunoglobulins within about 120 days before the administration of the one or more doses of the therapeutically effective amount of the composition.
[0739] Embodiment 112. A method wherein the subject has not received an allergy treatment within about 14 days before administration of the one or more doses of the therapeutically effective amount of the composition, and/or does not receive an allergy treatment within about 14 days after administration of the one or more doses of the therapeutically effective amount of the composition.
[0740] Embodiment 113. A method wherein the allergy treatment comprises antigen injections.
[0741] Embodiment 114. A method wherein the subject has not received an immunosuppressive medication within about 28 days before administration of the one or more doses of the therapeutically effective amount of the composition.
[0742] Embodiment 115. A method wherein the immunosuppressive medication comprises a systemic corticosteroid or radiotherapy.
[0743] Embodiment 116. A method wherein the corticosteroid is prednisone. [0744] Embodiment 117. A method wherein the subject has not received prophylactic antipyretics and/or analgesic medication within about 14 days before administration of the one or more doses of the therapeutically effective amount of the composition.
[0745] Embodiment 118. A method wherein the subject is negative for HIV-1 and HIV-2.
[0746] Embodiment 119. A method wherein the subject is negative for Hepatitis B and Hepatitis C.
[0747] Embodiment 120. A composition or combination wherein a therapeutically effective amount is sufficient to prevent an orthopoxvirus infection in a subject.
[0748] Embodiment 121. A composition or combination wherein a therapeutically effective amount is sufficient to treat an orthopoxvirus infection in a subject.
[0749] Embodiment 122. A composition or combination wherein the orthopoxvirus infection is an mpox virus infection.
[0750] Embodiment 123. A composition or combination wherein the mpox virus infection is a lethal clade I mpox virus infection.
[0751] Embodiment 124. A composition or combination wherein the lethal clade I mpox virus infection is strain V79-I-005.
[0752] Embodiment 125. A composition or combination, wherein the orthopoxvirus infection is a vaccinia virus infection.
[0753] Embodiment 126. A composition or combination, wherein the orthopoxvirus infection is a variola virus infection.
[0754] Embodiment 127. A composition or combination, wherein the orthopoxvirus infection is a cowpox virus infection.
[0755] Embodiment 128. A method wherein the orthopoxvirus infection is an mpox virus infection.
[0756] Embodiment 129. A method wherein an mpox virus infection is a lethal clade I mpox virus infection
[0757] Embodiment 130. A method wherein the lethal clade I mpox virus infection is strain V79-I-
005.
[0758] Embodiment 131. A method wherein the orthopoxvirus infection is a vaccinia virus infection. [0759] Embodiment 132. A method wherein the orthopoxvirus infection is a variola virus infection. [0760] Embodiment 133. A method wherein the orthopoxvirus infection is a cowpox virus infection. [0761] Embodiment 134. A method wherein the orthopoxvirus infection is an ectromelia virus infection
EXAMPLES
Example 1: Exemplary In vitro Expression of Polyribonucleotide Constructs encoding MPXV Antigens [0762] The present Example describes in vitro experiments characterizing the levels of expression achieved by exemplary individual nucleoside-modified polyribonucleotides (e.g., mRNAs) with backbone structures for optimized translational performance.
[0763] Each exemplary polyribonucleotide encodes for one of the following minimally modified clade lib MPXV antigens: A35, B6, Ml and H3. Two exemplary combination MPXV antigen compositions were evaluated: an exemplary quadrivalent combination (Combo 4) including all four antigens and an exemplary trivalent combination (Combo 2) including A35, B6 and Ml (FIG. 1). Both A35 and B6 polyribonucleotides used fully wild-type (WT) viral sequences derived from early cases in the 2022 outbreak, which included the native secretory signal of these proteins. WT versions of A35 and B6 are expected to traffic to the cell surface in polyribonucleotide transfected cells. WT versions of the MV targets Ml and H3, may not be efficiently delivered to the endoplasmic reticulum for eventual transit to the plasma membrane. During viral replication, embedding of MV proteins into the MV envelope is distinct from the normal host cell biosynthesis pathway for integral membrane proteins and they lack native secretory signals. A secretory signal from the Herpes Simplex Virus 1 gD protein was therefore added to the N-termini of WT Ml and H3 to facilitate the correct trafficking for cell surface display.
[0764] HEK293T cells were seeded in DMEM supplemented with 10% FBS in 12-well plates with a cell density of 4 x 105 cells/well 6 h prior to transfection. For transfection, LNP-formulated polyribonucleotide (RNA) was diluted to 200 ng total RNA in 100 pL Opti-MEM for individual constructs, and 200 ng per construct for the combinations (Combo 4 and Combo 2). RNA and Opti-MEM mixture was then added directly to the cells (100 pL/well). The plate was gently mixed and centrifuged at 500 x g for 5 min at room temperature before incubating at 37°C and 5% CO2 for 18 h. Following the incubation, cells were prepared for subsequent FACS analysis. Culture media was removed from the 12-well plates and the cells were gently liberated from the plate surface by pipetting in cold DPBS. Each sample was then split into two, with one half used for detection of total protein and the other for surface protein (by fixing and permeabilizing or fixing alone, respectively).
[0765] For total protein detection, cells were transferred to a 96-well plate and stained with 50 pL
Fixable Viability Dye eFluor 450 diluted 1:500 in DPBS for 15 min at room temperature. Cells were then washed with FACS buffer (lx DPBS, 1% BSA, 5 mM EDTA), centrifuged at 300 x g for 5 min at 4°C and fixed with 100 pL Fixation Buffer for 12 min at room temperature. Following fixation, cells were washed with lx Permeabilization Buffer, centrifuged at 500 x g for 5 min at 4°C and stained with an appropriate primary antibody diluted in Permeabilization Buffer. Afterwards, cells were washed twice with lx Permeabilization Buffer, centrifuged at 500 x g for 5 min at 4°C, and incubated with a secondary donkey anti-mouse or goat anti-human antibody labelled with Alexa Fluor® 647 for 30 min at 4°C. Finally, cells were washed twice with lx Permeabilization Buffer as described above and resuspended in 180 pL FACS buffer before acquisition on a BD FACS Celesta II.
[0766] For surface protein detection, the remaining cells for each sample were transferred to another
96-well plate. Cells were stained directly for 30 min at 4°C with 50 pL of pre-mixed Fixable Viability Dye eFluor™ 450, diluted 1:500, and the appropriate primary antibody diluted together in DPBS. Cells were then washed with DPBS, centrifuged at 300 x g for 5 min at 4°C, and incubated with a secondary donkey anti-mouse or goat antihuman antibody labelled with Alexa Fluor® 647, diluted 1:500 in DPBS, for 30 min on ice. Following the secondary staining, cells were again washed with DPBS, centrifuged at 300 x g for 5 min at 4°C, and fixed by the addition of 100 pL Fixation Buffer for 12 min. at room temperature. Finally, the cells were centrifuged at 500 x g for 5 min at room temperature, washed twice with FACS buffer and resuspended in 180 pL FACS buffer before acquisition with a BD FACS Celesta II.
[0767] Total protein expression analysis demonstrates that exemplary Combo 4 and Combo 2 polyribonucleotide compositions were successfully translated in cells in vitro and surface expression analysis confirmed that the antigens, all transmembrane proteins, were strongly expressed and correctly localized to the cell surface, facilitating recognition by immune cells upon administration (FIGs. 2A-2D). Expression levels for each antigen in single polyribonucleotide-transfected cells and exemplary Combo 4 and Combo 2-transfected cells were comparable. This observation suggested that co-delivery of multiple polyribonucleotide components does not interfere with the translation of each individual polyribonucleotide (e.g., mRNA). Example 2: Exemplary in vivo Immunogenicity of Polyribonucleotide Constructs encoding MPXV Antigens
[0768] In this Example, the immunogenicity of exemplary Combo 4 and Combo 2 polyribonucleotide compositions was evaluated using BALB/c mice. Antibody responses to exemplary Combo 4 composition-encoded antigens were administered to subject mice in combination or separately. Mice were immunized intramuscularly (IM) with 4 μg exemplary Combo 4 (1 μg of each of A35, B6, Ml and H3 polyribonucleotide components), 4 μg of exemplary Combo 2 (1.3 μg of each of A35, B6, and Ml components), or 1 μg of a single component antigen LNP-encapsulated polyribonucleotide on days 0 and 21. One group of mice was mock immunized with saline in parallel. Blood was collected weekly until the end of the study on day 35 and used for antibody immunoassays. [0769] Total immunoglobulin G (IgG) raised to each MPXV-antigen was measured by enzyme-linked immunosorbent assay (ELISA) for each blood collection (FIGs. 3A-3D). IgG induction was observed following the prime dose and further increased after the day 21 boost for all antigens. Importantly, comparable serum antibody levels were observed following multivalent or monovalent polyribonucleotide-LNP immunizations for each antigen. This similarity indicated that there was no reduction in response to each protein when multiple antigens were delivered simultaneously. To explore whether exemplary Combo 4 and Combo 2 polyribonucleotides had the potential to induce durable immunity, germinal center induction was explored in inguinal lymph nodes at day 35 by flow cytometry, two weeks following the second vaccine dose (FIGs. 4A-4E). [0770] For flow cytometry experiments, single cell suspensions of ipsilateral inguinal lymph nodes from immunized or control mice were washed in FACS buffer (PBS containing 1% BSA) and collected by centrifugation at 300 x g. Cell pellets were resuspended in FACS buffer containing 1 μg/mL of either A35, B6, Ml, or H3 and incubated on ice for 30 minutes. Each LN suspension was treated only with protein corresponding to the immunization of the source animal; saline treated animals were placed in Ml solution. Following the antigen stain, 100 pL of FACs buffer was added to the cells and they were centrifuged at 300 x g for 5 minutes at 4oC. The supernatants were decanted, cell pellets resuspended in 200 pL FACS buffer, and centrifugation repeated. This wash step was repeated once and then cell pellets were resuspended in stain solution, FACS buffer containing: LIVE/DEAD Fixable Near-IR Dead Cell Stain solution, anti-mouse CD19-BV605, anti-mouse/human CD45R/B220-BV785, anti-mouse CD95-PECy7, anti-mouse/human GL7-FITC, anti-mouse CD11C-BV421, antimouse F4/80-BV421, anti-mouse CD4-BV421, anti-mouse CD8a-BV421, anti-mouse Ly-6G/Ly-6C (Gr-l)-BV421, anti-Flag Tag-PE and anti-Flag Tag-APC. Antigen binding to B cells was detected via anti-Flag antibody recognition of Flag-Tag-bearing recombinant MPXV proteins bound on the cell surface. Following the antigen stain, 100 pL of FACs buffer was added to the cells and they were centrifuged at 300 x g for 5 minutes at 4oC. The cells were washed once as above, resuspended in 200 pL FACS buffer and analyzed on a BD Symphony A5. [0771] Antigen-specific germinal center B cells were observed for all polyribonucleotide treatments consistent with a durable immune response following exemplary Combo 4 and Combo 2 administrations. These observations align with the potent germinal center induction observed in human recipients of the SARS-CoV-2 vaccine (BNT162b2) suggesting that robust germinal center formation is a general feature of the polyribonucleotide (e.g., mRNA) platform.
[0772] To complement the initial study of humoral immunity, the immunogenicity of exemplary Combo
4 and Combo 2 polyribonucleotide compositions was further evaluated by measuring splenic T cell responses in BALB/c mice. T cell responses were examined following a single intramuscular (IM) immunization with 4 μg Combo 4 or 1 μg of each (i.e., A35, B6, Ml, H3) single component antigen LNP-encapsulated polyribonucleotide. One group of mice was mock immunized with saline in parallel. Mice were sacrificed 7 days after immunization and mouse splenocytes were stimulated with peptide pools spanning the antigen(s) relevant to each experimental group for 20 hours, and T cell responses were measured by IFN-y ELISpot.
[0773] ELISpot assays with fresh splenocytes were performed according to the manufacturer's protocol (with minor modifications as described below) using the R&D systems mouse IFN-y ELISpot kit. Briefly, 96-well ELISpot plates were blocked with serum-free assay media (X-VIVO + 1% Pen-Strep + 1% Glutamax) for at least 1 hour at 37°C. 100 pL of the splenocyte solution (3 x 105 cells) were transferred to the respective well of the 96-well ELISpot plate. Another 100 pL of overlapping 15mer peptide pools or controls were added at 0.3 pM final concentration per peptide. For a positive control, the splenocytes were stimulated with 0.3 pM ConA. For a non-stimulation control, medium with DMSO equivalent to the highest volume of peptide mix was added. Plates were incubated overnight in a 37°C humidified incubator with 5% CO2 and after approximately 20 h, cells were removed from the plates and detection of spots was initiated according to manufacturer's protocol. Briefly, the detection antibody was added for an overnight incubation at 4°C. Streptavidin-ALP (alkaline phosphatase), and the ready-to-use substrate were then added to the wells according to the manufacturer's protocol. After plate drying for several days, an ELISpot plate reader (ImmunoSpot® S6 Core Analyzer, CTL) was used to count and analyze spot numbers per well. Saturated wells were assigned a value of 1500 spots.
[0774] Significant polyribonucleotide composition-induced, antigen-specific responses were detected for all MPXV antigens, with A35 driving the most robust T cell response (FIGs. 5A-5D). Consistent with the ELISA results, T cell induction in subject administered with exemplary multivalent polyribonucleotide compositions was comparable to the levels observed for polyribonucleotides encoding single antigens suggesting limited or no interference resulting from delivery of multiple antigens.
[0775] To confirm that immunogenicity of exemplary Combo 4 and Combo 2-encoded MPXV antigens was not specific to the mouse model system, immunogenicity experiments in rats were also performed. Wistar rats were administered with 10 μg of exemplary Combo 4 and Combo 2 polyribonucleotides (e.g., mRNAs) on days 0 and 28 and serum anti-MPXV IgG levels were examined until day 43 (FIG. 6A to FIG. 6D). Similar to the findings in the mouse study, IgG induction was observed in Wistar rats following the prime dose and further increased after the day 28 boost for all MPXV antigens.
Example 3: MPXV Antigen Vaccines Induce Strong MPXV and VACV Neutralizing Antibody Responses [0776] In this Example, MPXV- and VACV-neutralizing antibody levels at day 35 in BALC/c mice from the initial prime and boost immunogenicity experiment were determined by plaque reduction neutralization test (PRNT) and focus reduction neutralization test (FRNT), respectively.
[0777] Both PRNT and FRNT assays measuring neutralizing antibody titers for MPXV and VACV, respectively, were conducted under two conditions: without addition of baby rabbit complement (FIGs. 7A and 7C) and in the presence of baby rabbit complement (FIGs. 7B and 7D). The complement neutralization assay was included as inhibition of infection via complement cascade activation is an established mode of action for anti-orthopoxvirus antibodies. Orthopoxvirus neutralization assays present unique complexities arising from the production of two infectious forms of virus, EV and MV.
[0778] For PRNT assays, Vero E6 cells were seeded in 24-well plates at a concentration of 2.5x l05 cells/well and incubated overnight at 37°C, 5% CO2 until the cells reached 90-100% confluency. On the day of cell seeding, serum test samples and controls were heat-inactivated at 56°C for 30 minutes and used to generate a two-fold dilution series in 96-well plates. Serum dilutions were combined with MPXV virus either in the presence or absence of 10% baby rabbit complement and incubated overnight at 4°C. The day following cell seeding, media was removed from cells and replaced with 100 pL of virus and serum dilution mixture. Following a 1 h incubation at 37°C, 5% CO2 with rocking at intervals the virus and serum solutions were removed from cells and replaced with 500 pL overlay media containing 0.5% methylcellulose. The plates were then incubated at 37°C in 5% CO2 for two days. Following this infection step, A 0.4% Crystal Violet stain solution was added to each well in a volume of 250 pL, and the plates were incubated at RT for 30-120 minutes. After staining, the Crystal Violet solution was removed from the plates by decanting and the plates were inverted and set aside to dry. Dried plates were scanned with a flatbed scanner and desktop scanning software to create digital images for each plate, which were used for manual counting of plaques in each well. 50% neutralization titers (NT50) were calculated based on the average number of plaques detected in the virus control wells. The limits of detection were set at half the lowest test serum dilution and twice the highest dilution tested.
[0779] For FRNT assays, following heat inactivation of each serum sample, a two-fold serum dilution series was generated, combined with VACV in a 96-well plate and incubated for 1 hr at 37°C. Virus and serum dilutions were then transferred to a 96-well plate containing Vero cells and further incubated for 1 h at 37 °C with 5% CO2. All media was then removed from the plate and a 0.5% methylcellulose-containing media is added to the plates. Following incubation for 18 h at 33°C with 5% CO2, infected vero cells were then fixed with 4% paraformaldehyde, stained with detection antibodies, and imaged and analyzed with an automated plate reader. The neutralizing titer of a serum sample is calculated as the reciprocal serum dilution corresponding to a 50% reduction in viral foci, the 50% neutralization antibody titer (NT50). FRNTs were conducted both in the presence and absence of 4% baby rabbit complement during the initial incubation step.
[0780] Classic PRNT assays employ virus preparations dominated by MVs, therefore sera from mice immunized with single EV targets were not expected to neutralize virus in this assay. Consistent with this expectation, sera from mice immunized with the EV proteins A35 and B6 alone did not show neutralization activity (regardless of the addition of complement) (FIG. 7A - FIG. 7D). Conversely, sera from animals immunized with the MV protein Ml (exemplary Combo 4 or exemplary Combo 2) efficiently neutralized MPXV and VACV both with and without complement. Sera from mice immunized with the MV target H3 alone also show neutralization activity with the addition of complement against both MPXV and VACV. An additional study was conducted in BALB/c mice to further examine neutralizing antibody responses following vaccine prime and boost. BALB/c mice were immunized as above with exemplary Combo 4 and Combo 2 polyribonucleotide compositions and VACV-neutralizing antibody responses in sera collected on days 21 and 35 were measured by FRNT without the addition of complement (FIG. 8).
[0781] Modest neutralizing antibody titers were observed post prime for exemplary Combo 4 and exemplary Combo 2 (GMTs 130 and 224, respectively) and strong neutralizing antibody titers were observed post boost (GMTs 7947 and 5167, respectively). These results demonstrate functional antibody responses are elicited by exemplary Combo 4 and exemplary Combo 2 polyribonucleotide compositions against MPXV with crossrecognition of a related orthopoxvirus. The component analysis studies in the VACV intranasal challenge model additionally confirm that A35, B6 and Ml have the greatest individual protective potential, while H3 alone was insufficient for protection from VACV infection. These results strongly support the inclusion of A35, B6 and Ml in MPXV polyribonucleotide compositions. However, the inclusion of H3 still provides benefits by broadening the response elicited by the exemplary Combo 4 polyribonucleotide composition, increasing the likelihood of cross- protective immunity and, by eliciting antibodies, capable of neutralizing both MPXV and VACV in the presence of complement proteins.
Example 4: MPXV Antigen Vaccines Protect Mice in Multiple Orthopoxvirus Challenge Models
[0782] In this experiment, the protective efficacy of exemplary Combo 4 and Combo 2 polyribonucleotide compositions against MPXV was evaluated in two murine challenge models. Many mouse lines, including the commonly used BALB/c and C57BL/6 lines, are resistant to MPXV infection. However, the immunocompetent inbred mouse line CAST/Ei has been identified as susceptible to intranasal (IN) MPXV challenge. Infection of CAST/Ei mice with a high pathogenicity clade I MPXV isolate resulted in a lethal infection of CAST/Ei, whereas infection with a clade II isolate from the 2022 outbreak produced a non-lethal infection with high viral replication rates in the lungs of infected animals.
[0783] First, exemplary Combo 4 and Combo 2 polyribonucleotide compositions were tested in the clade lib MPXV challenge model to match the most recent outbreak conditions. CAST/Ei mice were administered with either 4 μg of exemplary Combo 4 polyribonucleotide composition, exemplary Combo 2 polyribonucleotide composition, a combination of A35 and B6 polyribonucleotides, or mock-treated with saline on days 0 and 21 (as depicted in FIG. 9A). The A35 and B6 combination treatment was included to assess whether the EV antigens have a functional role in protection in vivo in Heuo an in vitro functional assay, as well as to assess the benefits of combining EV and MV targets in a multivalent vaccine. At 5 weeks following the second dose, mice were challenged IN with 9 x 105 plaque forming units (PFU) of MPXV (strain hMPXV/USA/MA001/2022 from the recent outbreak). The mice were sacrificed at either day 3 or day 7 post-infection and MPXV virus titers were measured in the lungs (FIG. 9B and FIG. 9C, respectively). Mock-administered animals had high viral loads in the lungs at both days 3 and 7. Exemplary Combo 4 and Combo 2-administered animals had no measurable viral replication in their lungs at either timepoint, consistent with potent polyribonucleotide composition-induced protection. Mice administered with the A35 and B6 antigens (EV) did not have a significant reduction in viral titers in the lung on day 3, but had a partial reduction (but no abrogation) of viral titers on day 7. These findings demonstrate that administration with EV antigens only contributes to a reduction in tissue viral replication in vivo, k replicate second experiment was conducted with a lower inoculum (3 x 105 PFU) from a second lower titer preparation of hMPXV/USA/MA001/2022 (FIGs. 9D-9F). These results confirm that exemplary Combo 4 and exemplary Combo 2 polyribonucleotide compositions can robustly provide protection from the 2022 outbreak MPXV strain.
[0784] While clade lib MPXV protection is a goal for polyribonucleotide constructs encoding mpox antigens, the polyribonucleotide compositions should also function in protecting against more pathogenic clade I MPXV isolates to effectively prepare for all future outbreak scenarios. To demonstrate exemplary Combo 4 and exemplary Combo 2's potential against a range of MPXV strains, a lethal clade I MPXV challenge study was conducted in CAST/Ei mice. Mice were immunized as described above and challenged IN with IxlO5 PFU of clade I MPXV (strain V79-I-005) 5 weeks post boost (as depicted in FIG. 9G). Weight loss and survival were monitored for two weeks post challenge (FIG. 9H and FIG. 91, respectively). Both exemplary Combo 4 and exemplary Combo 2 polyribonucleotide compositions provided 100% protection from weight loss and death following clade I challenge. Mice administered with a combination of A35 and B6 polyribonucleotides (e.g., mRNAs) had a small yet significant survival benefit. Together, these findings demonstrate that exemplary Combo 4 and exemplary Combo 2 polyribonucleotide compositions can provide protection to a wide range of MPXV isolates and strongly support the approach of combining EV and MV antigens for optimal and broad immunity.
[0785] VACV-based vaccines have high utility in part because they provide protection across the orthopox virus genus including against VARV and MPXV. Because of the high conservation of the antigens in polyribonucleotide constructs encoding mpox antigens across orthopoxviruses (Table 5), Exemplary Combo 4 and Combo 2 polyribonucleotide compositions have the potential to provide a similarly broad level of protection against multiple virus species. To determine whether exemplary Combo 4 polyribonucleotide composition provides cross-protective immunity to an orthopoxvirus other than MPXV, IN VACV challenge of exemplary Combo 4-immunized BALB/c mice was performed. As an additional objective, the protective potential of each exemplary Combo 4 polyribonucleotide (e.g., mRNA) component outside of combination administration was evaluated using single antigen administered groups of mice. Mice were administered with either 4 μg of the multivalent Combo 4, 1 μg of a single polyribonucleotide (e.g., mRNA) component, or mock treated with saline on days 0 and 21. A lethal challenge dose of 5 x 104 PFU of VACV-Western Reserve (WR) was administered IN 3 weeks following the second vaccination dose (as depicted in FIG. 1OA). Mice were monitored for weight loss and death (FIG. 1OB, and FIG. IOC, respectively) for 2 weeks following challenge. Mock-treated mice and mice immunized with H3 alone rapidly succumbed to infection, but mice administered with either the exemplary multivalent Combo 4 polyribonucleotide composition, or the single components A35, B6, and Ml were fully protected from death. Mice receiving Ml administration alone displayed transient weight loss post challenge in contrast to mice administered with A35 or B6, consistent with previous reports of EV antigen immunization yielding protection superior to MV antigen immunization in the VACV IN challenge model. These findings demonstrate the potential of exemplary polyribonucleotide compositions of the present disclosure to protect against a range of orthopoxviruses.
[0786] Without wishing to be bound by any particular theory, the reduced efficacy of EV immunization in the CAST/Ei may reflect differences in pathogenesis between VACV and MPXV, or perhaps differences in baseline immune profiles between CAST/Ei and BALB/c mice. CAST/Ei are moderately deficient in IFN-y production and have a reduced representation of NK cells, potentially impacting protective effector functions like ADCC. Irrespective of the specific mechanisms underlying the discrepancy between VACV and MPXV challenge results, these findings support the inclusion of both EV and MV targets for protection and illustrates the value in using multiple challenge systems when evaluating efficacy of polyribonucleotide compositions encoding MPXV antigens pre-clinically.
Table 6: Exemplary MPXV Target Protein Amino Acid Sequence Consensus and Identity with VACV and VARV Orthologs
Figure imgf000158_0001
^0787] To determine whether exemplary Combo 4 polyribonucleotide composition provides cross- protective immunity to rabbitpox virus, BALB/c mice are administered with either 4 μg of the multivalent Combo 4, 1 μg of a single polyribonucleotide (e.g., mRNA) component, or mock treated with PBS on days 0 and 21. A lethal challenge dose of 1 x 104 PFU of rabbitpoxvirus is administered IN 3 weeks following the second vaccination dose. Mice are monitored for weight loss and death for 2 weeks following challenge.
[0788] To determine whether exemplary Combo 4 polyribonucleotide composition provides cross- protective immunity to ECTV, BALB/c mice were administered with either 4 μg of the multivalent Combo 4, 1 μg of a single polyribonucleotide (e.g., mRNA) component, or mock treated with PBS on days 0 and 21. A lethal challenge dose of 1 x 104 PFU of ECTV was administered IN 3 weeks following the second vaccination dose (as depicted in FIG. 11A). Mice were monitored for weight loss and death (FIG. 11B, and FIG. 11C, respectively) for 2 weeks following challenge. Mock-treated mice and mice immunized with Ml alone, A35 alone, B6 alone, and H3 alone rapidly succumbed to infection, but mice administered with exemplary multivalent Combo 4 polyribonucleotide composition were fully protected from significant weight loss and death. Similar to VACV, these findings demonstrate the potential of exemplary polyribonucleotide compositions of the present disclosure to protect against a range of orthopoxviruses.
[0789] To determine whether exemplary Combo 4 polyribonucleotide composition provides cross- protective immunity to CPXV, BALB/c mice were administered with either 4 μg of the multivalent Combo 4, 1 μg of a single polyribonucleotide (e.g., mRNA) component, or mock treated with PBS on days 0 and 21. A lethal challenge dose of 2 x 105 PFU of CPXV (ATCC Cat# VR-302; Brighton, Lot# 70054947) was administered IN 3 weeks following the second vaccination dose (as depicted in FIG. 2OA). Mice were monitored for weight loss (FIG. 2OB) and death for 2 weeks following challenge. Mock-treated mice and mice immunized with Ml alone, A35 alone, B6 alone, and H3 alone rapidly succumbed to infection, but mice administered with exemplary multivalent Combo 4 polyribonucleotide composition were fully protected from significant weight loss. Similar to VACV and ECTV, these findings demonstrate the potential of exemplary polyribonucleotide compositions of the present disclosure to protect against a range of orthopoxviruses.
Example 5: Exemplary MPXV Antigen Vaccinated Macaques are Protected from a Lethal Clade I MPXV Challenge
[0790] In the present Example, the efficacy of exemplary polyribonucleotide compositions encoding
MPXV antigens was evaluated in a non-human primate MPXV challenge model.
[0791] Mouse models of orthopoxvirus disease are useful but do not recapitulate important facets of human mpox disease including the development of lesions. To evaluate the efficacy of exemplary Combo 4 polyribonucleotide composition in eliciting protective immunity in a model that better represents human mpox and smallpox disease, twelve cynomolgus macaques were divided into two groups of six and received either a day 0 prime and day 28 boost administration with 30 μg Combo 4 or mock saline treatment (FIG. 12A). IgG responses to each constituent antigen: A35 (FIG. 12B), B6 (FIG. 12C), Ml (FIG. 12D), and H3 (FIG. 12E), were measured up to day 56 after the first administration using ELISA. High IgG binding titers were detected to all four antigens, which were further increased by the second administration (FIGs. 12B-12E). At day 60, all animals were administered a lethal intratracheal (IT) dose of 5 x 107 PFU of clade I MPXV (strain V79-I-005) and monitored for 28 days for disease symptoms including weight loss, lesions, respiratory distress and death. In comparison to the mock treated group, where five out of six (83.3%) succumbed to infection, all exemplary Combo 4-administered animals survived until the conclusion of the challenge period (FIG. 12F). The exemplary Combo 4-administered animals exhibited relatively mild disease symptoms including rapidly-resolving weight loss (FIGs. 12G-12I). Exemplary Combo 4-administered animals also showed limited or no lesions following infection, indicating infection was well controlled in these animals despite stringent challenge conditions (FIGs.
12J-12L).
Example 6: Exemplary in vivo Characterization of Vaccine Candidates
[0792] The present Example describes non-clinical pharmacological analysis of exemplary mpox polyribonucleotide compositions (e.g., Combo 2, or Combo 4) in in vivo mouse studies. As described in the present Example, Combo 2 and Combo 4 were lipid nanoparticle (LNP)-formulated compositions incorporating modified polyribonucleotides encoding MPXV surface antigens as listed in Table 7, below. Table 7: Exemplary Mpox Combination Vaccine Candidates
Figure imgf000160_0001
IN VIVO MPXV CHALLENGE IN CAST/Ei MICE
[0793] Many mouse lines, including the commonly used BALB/c and C57BL/6 lines, are resistant to
MPXV infection. However, the immunocompetent inbred mouse line CAST/Ei has been identified as being susceptible to intranasal MPXV challenge. Infection of CAST/Ei mice with a highly pathogenic clade I MPXV isolate (Congo Basin) results in a lethal infection of CAST/Ei (Americo etaL, J Virol, 2010), whereas infection with a clade II isolate from the 2022 mpox outbreak (SP2833) produces a non-lethal infection with high viral replication rates in the lungs of infected animals (Warner etaL. Sci Trans! Med, 2022).
[0794] In the present Example, CAST/Ei mice were immunized with 4μg of exemplary Combo 2 (i.e.
1.33 μg of each polyribonucleotide), 4 μg of exemplary Combo 4 (i.e. 1 μg of each polyribonucleotide), an LNP incorporating a combination of polyribonucleotides encoding A35 and B6 (2 μg of each polyribonucleotide), or saline, on days 0 and 21. 56 days following the dose at day 21, mice were intranasally challenged with 1 x 105 plaque forming units (PFUs) of clade I MPXV isolate (Zaire 79 strain). Weight loss and survival were monitored for a 14 day period after MPXV challenge (as depicted in FIG. 13A).
[0795] As shown in FIG. 13B and FIG. 13C, all animals vaccinated with either exemplary Combo 2 or exemplary Combo 4 survived without weight loss, compared to animals receiving saline who almost all succumbed to the infection, many by days 6-7 post challenge. Only some animals immunized with LNP incorporating the combination of polyribonucleotides encoding A35 and B6 survived the challenge, and recovered from weight loss; the deaths in this group were overall delayed by a day or two compared with the saline control group. These data support the efficacy of exemplary Combo 2 and exemplary Combo 4 polyribonucleotide compositions and highlights the value of MV components in the vaccine.
Example 7: Exemplary in vivo Characterization of Broad Orthopoxvirus Protection
IN VIVO VACCINIA VIRUS CHALLENGE IN BALB/C MICE
[0796] In the present Example, Balb/C mice were immunized with 4 μg of exemplary Combo 4 (i.e., 1 μg of each polyribonucleotide), an LNP incorporating 1 μg of polyribonucleotide encoding Ml, an LNP incorporating 1 μg of polyribonucleotide encoding A35, an LNP incorporating 1 μg of polyribonucleotide encoding B6, an LNP incorporating 1 μg of polyribonucleotide encoding H3, or saline on days 0 and 21. 21 days following the dose at day 21, mice were intranasally challenged with 5 x 104 PFUs of vaccinia virus (VACV) isolate (i.e., a lethal dose). Weight loss and survival were monitored for a 14 day period after VACV challenge (as depicted in FIG. 14A).
[0797] As shown in FIG. 14B and FIG. 14C, all animals vaccinated with either exemplary Combo 4 polyribonucleotide composition, or LNP incorporating polyribonucleotide encoding A35, or B6 survived without weight loss. All animals vaccinated with LNP incorporating polyribonucleotide encoding Ml survived, with very mild weight loss. All animals receiving either LNP incorporating polyribonucleotide encoding H3 or saline succumbed to the VACV infection. These data support the efficacy of Combo 4 against VACV infection, and demonstrates that A35, B6, and Ml each contribute to protection from challenge. H3 did not protect from challenge, as was expected based on most published literature, despite its complement driven neutralization demonstrated in vitro. Example 8: Exemplary in vivo Characterization of Vaccine Candidates in Non-Human Primates [0798] The present Example describes non-clinical pharmacological analysis of an exemplary mpox polyribonucleotide composition (e.g., Combo 4) in cynomolgus macaque studies.
[0799] Prior to study initiation, serum from 22 cynomolgus macaques were prescreened with a commercially available Mpox IgG ELISA kit (Alpha Diagnostic); 14 naive Mpox-seronegative monkeys were selected for the study. All animals were randomized into three respective groups based on body weight using Provantis software.
[0800] On Days 0 and 28, animals in Groups 1 and 2 were anesthetized (ketamine HCI at approximately 10-30 mg/kg intramuscularly) and vaccinated in the BSL-2 condition according to Table 8. Group 3 was not vaccinated. Vaccination materials were administered within approximately four hours of formulation. On Day 60, animals in Groups 1 and 2 were anesthetized and challenged with 5 x 107 PFU MPXV/per animal in 1.0 mL volume via the intratracheal route. On Day 88, all remaining animals were euthanized and tissues were collected.
[0801] On Day 0, Group 3 animals were anesthetized (ketamine HCI at approximately 10-30 mg/kg intramuscularly) and challenged with 5 x 107 PFU MPXV/per animal in 1.0 mL volume via the intratracheal route in the BSL-3.
Table 8: Group Assignment
Figure imgf000161_0001
1 Group 3 was not vaccinated but received the same challenge material as Groups 1 and 2 on Day 0. [0802] All animals (6 of 6) immunized with exemplary Combo 4 polyribonucleotide composition survived the MPXV challenge and completed the in-life period of the study successfully, while only 1 of 6 animals survived in the saline control group. The remaining 5 saline-treated animals perished due to disease burden and protocol-required euthanasia. These data support the efficacy of exemplary Combo 4 polyribonucleotide composition against MPXV infection in non-human primates.
Example 9: Exemplary in vivo Characterization of Vaccine Candidates in Non-Human Primates [0803] Treated animals as described in Example 8 were additionally monitored on a continuous basis over the 28-day post-challenge period; clinical observations and evaluation of disease, to include body weight, body temperature, and lesion counts, were assessed and documented as shown in Table 10. Blood samples were collected for quantitative polymerase chain reaction (qPCR), plaque assay, Plaque Reduction Neutralization Test (PRNT), ELISpot, MSD Cytokine analysis and ELISA.
Vaccination Site Lesion Photographs
[0804] Animals in Groups 1 and 2 will have vaccination site lesion photographs collected according to the timepoints listed in Table 9.
Blood Collection and Processing [0805] Blood was collected from anesthetized animals into either serum separator tubes without anticoagulant, EDTA tubes or CPT tubes at the time points indicated in Table 9 and Table 10. Samples were taken from any accessible vein (actual bleed site will be documented in the study records). Blood was collected from moribund animals prior to euthanasia.
[0806] Blood collected into serum separator tubes was processed to serum on the day of collection for either PRNT, or ELISA testing. Serum samples were aliquoted into at least three tubes and stored at -70 °C or below until analyzed. Blood collected into EDTA tubes were mixed by inverting the tubes few times and saved at -70 °C or below until analyzed by PCR. CPT tubes were processed according to the manufacturer's instruction. PBMCs were washed in D-PBS and resuspended in RPMI1640 supplemented with FBS and L-glutamine and counted. Cells were pelleted and resuspended in CS-10 cryopreservation medium and aliquoted at 105 cells per ml in cryovials. The cells were cryopreserved in a Nunc Frosty or Corning at -70 °C. The cryovials were transferred to liquid nitrogen storage until use.
Clinical Observations
[0807] Detailed clinical observations were performed according to the Table 9 and Table 10 schedules. Clinical observations included monitoring for fever, poxvirus lesions, lethargy, appetite loss beginning three or more days after infection, lesions progressing throughout the typical stages of infection, depression, weakness, recumbency, dehydration, dyspnea, cough, eating, nasal discharge, ocular discharge, and edema. Lesion Counts and Photographs
[0808] Post-challenge, poxvirus lesion counts and photos were performed as outlined in Table 9 and
Table 10. Briefly, lesions were counted on the following areas of the body: head, neck, inside the mouth, hand, arm, foot, leg, chest, abdomen, groin, genitalia, back, perineum and tail by manipulating the hair/fur so that all areas of the skin were observed. If there was more than 200 lesions or a mass of lesions on any site, data was entered as TNTC (Too Numerous to Count). The development of pox lesions following MPXV challenge was captured by digital photography.
Necropsy and Macroscopic Observations
[0809] At the conclusion of the study, animals were euthanized over a maximum 4-day period.
Tissues were collected from all animals including in the case of early death of the animals or moribund animals. Animal ID's were collected during necropsy.
[0810] Tissues (spleen, brain, kidney, skin, lung, heart, esophagus, liver) from only one animal from
Group 3 that was euthanized prior to scheduled euthanasia or found dead were collected in three sections. One section was collected in 10% neutral buffered formalin and be used for the validation of inactivation procedures of Mpox virus infected tissue for bringing the tissues out of BSL-3 for hematoxylin and eosin (H&E) staining. A second section was collected and snap frozen to serve as control infected tissues for inactivation procedures. A third section was collected in 10% neutral buffered formalin and was used for H&E staining for this study. [0811] A limited gross necropsy was performed, including those that died on study or were euthanized in extremis (unless severely autolyzed), and all the following tissues except testes and epididymis were collected and fixed in 10% neutral buffered formalin for a minimum of 48 hours at room temperature: tongue, skin with associated lesions, epididymis, heart, esophagus, liver, brain, lungs, kidneys, spleen, oropharynx, tonsil , testes, lymph nodes (minimally bronchial, mandibular, mediastinal, mesenteric), parathyroids (if present), intestine, duodenum, jejunum, ileum, cecum, colon, and rectum. Testes and epididymis were fixed in Modified Davidson's fixative for at least 24 hours and then transferred them to the 10% neutral buffered formalin. [0812] Other abnormal tissues/gross lesions were collected as determined by a prosector. These samples underwent histopathological evaluation after H&E staining. The residual carcass was discarded without further evaluation.
Histology
[0813] Tissues collected from each animal will be processed to slides. The fixed tissues will be trimmed, processed, and sectioned using a microtome (approximately 5-pm sections). Tissue sections will be mounted on glass slides and stained with H&E. Slides will be examined microscopically. Special stains may be used at the discretion of the pathologist when necessary to establish a diagnosis. Use of any special stain will be documented.
Microscopic Observations
[0814] All slides will be submitted to a veterinary pathologist for evaluation. Records of gross findings for a specimen from postmortem observations will be available to the pathologist when examining that specimen histopathological analysis. A four-step grading system will be used to rank the severity of microscopic observations for comparison among groups.
Back-Titration of Challenge Materia! by Plague Assay
[0815] Two aliquots (one frozen just after the preparation and another frozen after the last animals is challenged) of the challenge material will be back titrated by plaque assay on Vero E6 cells to confirm the actual delivered dose. Remaining challenge material will be retained and stored at -70 °C or below until completion of the study.
Viral Load Determination by Real-Time Quantitative PCR
[0816] Viral loads were measured using a Real-Time qPCR method for detection of MPXV genomes in peripheral blood (at time points indicated in Table 9 and Table 10). DNA was extracted from blood (either fresh or frozen) and then amplified using a Pan-orthopox qPCR assay (Kulesh et a!., 2004) with the following primers and probe combination: GAACA I I I I I GGCAGAGAGAGCC (Forward primer); CAACTCTTAGCCGAAGCGTATGAG (Reverse primer); 56-FAM/CAGGCTACC/ZEN/AGTTCAA/3IABkFQ (TaqMan probe).
[0817] Briefly, whole blood was inactivated by adding an appropriate amount of ATL/Proteinase K lysis buffer (Qiagen) and incubating at around 56 °C for at least one hour. DNA extraction was performed using QIAamp 96 Virus QIAcube HT with QIAcube HT robot or manually with Qiagen QIAamp DNA Mini kit as described by Mucker etai, 2017. Quality control positive extraction controls were prepared using vaccinia virus and extracted at the same time as samples.
[0818] Samples were analyzed using the qualified Pan-Orthopox Virus E9L Gene-Specific Quantitative
PCR Assay. This Pan-Orthopox assay specifically detects and quantifies E9L (DNA polymerase) target conserved across multiple orthopox viruses, and it is able to detect but not differentiate horsepox, mpox, and vaccinia viruses. The assay was qualified using horsepox virus. Specificity, PCR efficiency and PCR Parallelism were demonstrated with DNA extracted from samples challenged with mpox virus, Zaire79, horsepox Vaccine and Smallpox (Vaccinia) scACAM2000 Vaccine. Serially diluted, quantified dsDNA gBIock standard were used to generate a standard curve that allowed for the quantitation of viral DNA in each biological sample. All qPCR reactions were performed using the TaqMan Fast Advanced Master Mix. All qPCR reactions were run on the QuantStudios 6 Flex.
[0819] As shown in FIG. 16A and FIG. 16B, all animals had peak viral loads between 4 days and 12 days post-MPXV challenge. All animals vaccinated with exemplary Combo 4 polyribonucleotide composition returned to pre-MPXV challenge viral load levels by 21 days post challenge (FIG. 16B). In contrast, all but one of the saline-treated animals perished due to disease burden or protocol-required euthanasia (FIG. 16A). ELISA
[0820] Two different types of ELISA will be performed: a) VACV Indirect ELISA where VACV will be used as coating antigen. For VACV ELISA total 84 serum samples from 7 different time points as stated in Table 9 will be analyzed. b) Monkey Pox virus recombinant ELISA will be performed using 4 different recombinant mpox virus proteins (Ml, A35, B6 and H3) as coating antigens. For MPXV recombinant protein ELISA there will be total 96 serum samples for each recombinant antigen as stated in Table 9.
MSD Cytokine Assay [0821] The Meso Scale Discovery (MSD) technology will be used to detect multiple cytokines from 36 total serum samples from 4 different timepoints as stated in Table 9 (Vaccination Phase). The panel will include cytokines GM-CSF, IL-5, IL-7, IL-12/IL-23p40, IL-15, IL-16, IL-17A, TNF-p, and VEGF-A. ELISpot [0822] ELISpot will be performed on cryopreserved PBMCs from 48 samples (4 time points, N=12) as per Table 9 using NHP IFN-y kit from Mabtech (ELISpot Pro: Monkey IFN-y (ALP)) or similar products according to the manufacturer's instruction. The cryopreserved cells will be thawed in a water bath at 37 C until small ice pieces remain. The cells will be transferred into 15ml tubes filled with 10 ml of pre-warmed complete RPMI1640 medium. Tubes will be spun at 400x g for 5 minutes at room temperature. The medium will be aspirated and the cells will be resuspended in 5 ml of fresh medium and counted. If needed, Benzonase or DNAse will be used to dissolve small cell clumps. Cells number will be adjusted to needed concentration and roughly 3.3 x 105 cells (in triplicate) will be plated for peptide stimulation and negative control, while half of the concertation will be used for positive control.
[0823] In brief, 96-well ELISpot plates will be washed with 200 pL/well sterile PBS (four times) then primed by adding the sterile media containing 10% of the same serum that was used for cell suspensions (200 pL/well) for at least 30 minutes at room temperature.
[0824] Medium will be removed and stimuli followed by cell suspension will be added. Alternatively, cells and stimuli can be mixed before addition to the plate. Plates will be incubated in a 37 °C humidified with 5% CO2 incubator for 12-48 hours. A peptide pool supplied by the sponsor will be used for stimulation of PBMCs. Con A (or similar) and media alone (unstimulated) will be used in the assay as positive and negative controls respectively. Care should be taken not to move this plate during this time and prevent evaporation (e.g., by wrapping the plate in aluminum foil). Next, plates will be washed with PBS 5 times, 200 pL/well. The 7-B6-ALP will be diluted 1:200 in PBS containing 0.5% fetal calf serum and 100 pL/well will be added, incubated for 2 hours at room temperature. Plates will be washed 5 time with PBS, 200 pL/well. The substrate solution will be filtered through 0.45pm filter and 100 pL/well substrate will be added. The plates will be incubated until the distinct spots emerge and then development will be stopped by adding tap water. Plates will be dried and spots will be counted by using an automated ELISpot reader system and ImmunoSpot software (Cellular Technology Limited).
MPXV PRNT
[0825] Following the vaccination and challenge, blood samples were collected for PRNT assay as outlined in Table 9. Briefly, serum samples were serially diluted in DMEM containing Glutamax and 2% FBS and added to an equal volume of a fixed concentration of MPXV (Zaire strain). The serum-virus was incubated at 37 ± 1°C incubator to form a neutralization mixture. Subsequently, 100 pL of each serum-virus mixture was added, in triplicate, to a fresh 24-well plate containing confluent Vero cells and incubated at 37 ± 1°C incubator. MPXV pre-incubated with normal monkey and immune serum were used as negative and positive controls, respectively.
Neutralization endpoint titers were calculated based on the reciprocal dilution of the test serum that produced
50% plaque reduction compared to the virus control.
[0826] As shown in FIG. 17, all animals vaccinated with exemplary Combo 4 polyribonucleotide composition had detectable neutralizing antibody titers by 56 days post-immunization, whereas saline-treated animals did not produce neutralizing antibodies. Following MPXV challenge, all animals vaccinated with exemplary Combo 4 polyribonucleotide composition had elevated neutralizing antibody titers as compared to saline-treated animals. As previously described, all but one of the saline-treated animals perished due to disease burden or protocol-required euthanasia (FIG. 17).
Table 9: Key In-Life Study Procedures (Vaccination Phase Groups 1-2)
Figure imgf000165_0001
Table 9 (Continued): Key In-Life Study Procedures (Vaccination Phase Groups l-2j
Figure imgf000165_0002
Animals remaining were euthanized over a 3-day period beginning on Day 88.
2 The decision to euthanize a given anima! post-challenge and prior to Day 88 was determined based upon consultation between the Study Director and Veterinary Staff.
Table 10: Key In-Life Study Procedures (Challenge Phase Group 3)
Figure imgf000165_0003
Figure imgf000166_0001
Example 10: Characterization of safety and immunogenicity of exemplary MPXV Vaccine Candidates
Objectives
[0827] This example describes a randomized, partially observer-blind, dose-escalation, Phase I/II trial evaluating the safety, tolerability, reactogenicity and immunogenicity of exemplary RNA-based multivalent vaccine candidates for active immunization against mpox (MPX).
[0828] This example additionally includes four sub-studies that may be conducted in parallel, as required by the clinical plan, within the framework of this master protocol.
[0829] Substudy A: Substudy A is an open-label, dose-escalation, Phase I substudy to assess the reactogenicity, safety and immunogenicity of up to three dose levels of two exemplary multivalent vaccine candidates.
[0830] Substudy A will enroll -64 healthy subjects with no prior history of known or suspected smallpox vaccination (vaccinia-naive subjects). Exemplary Combo 4 polyribonucleotide composition (comprising polyribonucleotides encoding A35, B6, Ml, and H3 MPXV antigens) will be evaluated at 10 (group Al), 30 (Group A2), and 60 μg (Group A4) dose levels. Exemplary Combo 2 polyribonucleotide composition (comprising polyribonucleotides encoding A35, B6, and Ml MPXV antigens) will be evaluated at 30 μg dose level (Group A3). Two doses will be given -31 d apart. The Groups A2 and A3 will be run simultaneously and, therefore, allocation to treatment for these two groups will be randomized. Group A3 may not be activated in which case there will be no randomization. This substudy will inform Substudy C on the preferred candidate and dose level. [0831] In Substudy A, two doses of each multivalent vaccine candidate (one at day 1/visit 1 and one at day 31/visit 5) are administered intramuscularly (e.g., into the deltoid muscle of the non-dominant arm). Administration of each multivalent vaccine candidate can be performed by simultaneous (i.e., one injection) intramuscular administration after mixing of each monovalent vaccine component before administration to the subject.
[0832] Outcome measures for assessing the reactogenicity after one and two doses of each multivalent vaccine candidate include determining the proportion of subjects reporting solicited local reactions at the injection site (e.g., pain, erythema/redness, induration/swelling) and/or systemic events (e.g., vomiting, diarrhea, headache, fatigue, myalgia, arthralgia, chills, fever) up to 7 days after each multivalent vaccine candidate dose.
[0833] Outcome measures for assessing the safety after one and two doses of each multivalent vaccine candidate include: determining the proportion of subjects with at least one unsolicited adverse event (AE) occurring from dose 1 to 28 days after dose 1; determining the proportion of subjects with at least one unsolicited adverse event (AE) occurring from dose 2 to 24 weeks after dose 2; and determining the proportion of subjects with worsening grading shifts in hematology and chemistry laboratory values between baseline and 1 week after dose 1, before dose 2, 1 week after dose 2, and 1 month after dose 2. [0834] An additional exploratory objective of Substudy A includes assessing neutralizing antibody responses against vaccinia and mpox viruses elicited by each multivalent vaccine candidate. Geometric mean titers for each group is measured at 2 weeks after dose 2 and compared to baseline (e.g., pre-dose 1). The proportion of subjects with seroconversion is also determined.
[0835] Exploratory objections of Substudy A also includes assessing the kinetics and specificities of humoral immune responses induced by vaccination with each multivalent vaccine candidate. Geometric mean titers for each group is measured at all timepoints.
[0836] Substudy B: Substudy B is a randomized, observer-blinded and sponsor-unblinded Phase I substudy to assess the reactogenicity, safety and immunogenicity of two exemplary multivalent vaccine candidates.
[0837] Substudy B will enroll ~32 healthy subjects with prior history of smallpox vaccination (vaccinia- experienced; also referred to as "primed healthy adults"). The smallpox vaccination status will be determined based on the medical record of vaccination before 1980 or the presence of the smallpox vaccination characteristic scar. Both exemplary multivalent vaccine candidates will be evaluated at 30 μg dose levels, two doses will be given ~31 d apart. Subjects will be randomized 1: 1. One of the groups may not be activated in which case, this substudy will be a one group open-label substudy.
[0838] In Substudy B, two doses of each multivalent vaccine candidate (one at day 1/visit 1 and one at day 31/visit 5) are administered intramuscularly (e.g., into the deltoid muscle of the non-dominant arm). Administration of each multivalent vaccine candidate can be performed by simultaneous (i.e., one injection) intramuscular administration after mixing of each monovalent vaccine component before administration to the subject.
[0839] Outcome measures for assessing the reactogenicity after one and two doses of each multivalent vaccine candidate include determining the proportion of subjects reporting solicited local reactions at the injection site (e.g., pain, erythema/redness, induration/swelling) and/or systemic events (e.g., vomiting, diarrhea, headache, fatigue, myalgia, arthralgia, chills, fever) up to 7 days after each multivalent vaccine candidate dose.
[0840] Outcome measures for assessing the safety after one and two doses of each multivalent vaccine candidate include: determining the proportion of subjects with at least one unsolicited adverse event (AE) occurring from dose 1 to 28 days after dose 1; determining the proportion of subjects with at least one unsolicited adverse event (AE) occurring from dose 2 to 28 days after dose 2; determining the proportion of subjects with at least one severe adverse event (SAE) occurring from dose 1 to 24 weeks after dose 2; and determining the proportion of subjects with worsening grading shifts in hematology and chemistry laboratory values between baseline and 1 week after dose 1, before dose 2, 1 week after dose 2, and 1 month after dose 2. [0841] An additional exploratory objective of Substudy B includes assessing neutralizing antibody responses against vaccinia and mpox viruses elicited by each multivalent vaccine candidate. Geometric mean titers for each group is measured at 2 weeks after dose 2 and compared to baseline (e.g., pre-dose 1). The proportion of subjects with seroconversion is also determined.
[0842] Exploratory objectives of Substudy B also includes assessing the kinetics and specificities of humoral immune responses induced by vaccination with each multivalent vaccine candidate. Geometric mean titers for each group is measured at all timepoints.
[0843] Substudy C: Substudy C is a randomized, observer-blinded and sponsor-unblinded (doubledummy) Phase Ila substudy to assess the reactogenicity, safety and immunogenicity of the selected exemplary multivalent vaccine candidate and MVA-BN vaccine (a heavily attenuated vaccine based on modified Vaccinia Ankara) as an active control. The exemplary multivalent vaccine candidate and its dose level will be selected based on Substudy A results.
[0844] This substudy will enroll ~100 healthy subjects with no prior history of known or suspected smallpox vaccination (vaccinia-naive subjects). The smallpox vaccination status will be determined based on the medical record of vaccination before 1980 or the presence of the smallpox vaccination characteristic scar. The exemplary multivalent vaccine candidate will be tested at a dose level of 60 μg or lower. The standard MVA-BN vaccine dose (5xl07 Infectious Units) will be used. Subjects will receive two doses of exemplary multivalent vaccine candidate or MVA-BN ~31 days apart. Because the two treatments use different administration routes (intramuscular [exemplary vaccine candidate] and subcutaneous [MVA-BN vaccine]), subjects will receive either the exemplary multivalent vaccine candidate plus MVA-BN placebo or the MVA-BN vaccine plus exemplary multivalent vaccine candidate placebo (double-dummy). Subjects will be randomized 1: 1.
[0845] In Substudy C, two doses of each multivalent vaccine candidate (one at day 1/visit 1 and one at day 31/visit 5) are administered intramuscularly (e.g., into the deltoid muscle of the non-dominant arm). Administration of each multivalent vaccine candidate can be performed by simultaneous (i.e., one injection) intramuscular administration after mixing of each monovalent vaccine component before administration to the subject.
[0846] Outcome measures for assessing the reactogenicity after one and two doses of each multivalent vaccine candidate include determining the proportion of subjects reporting solicited local reactions at the injection site (e.g., pain, erythema/redness, induration/swelling) and/or systemic events (e.g., vomiting, diarrhea, headache, fatigue, myalgia, arthralgia, chills, fever) up to 7 days after each multivalent vaccine candidate dose.
[0847] Outcome measures for assessing the safety after one and two doses of each multivalent vaccine candidate include: determining the proportion of subjects with at least one unsolicited adverse event (AE) occurring from dose 1 to 28 days after dose 1; determining the proportion of subjects with at least one unsolicited adverse event (AE) occurring from dose 2 to 28 days after dose 2; determining the proportion of subjects with at least one severe adverse event (SAE) occurring from dose 1 to 24 weeks after dose 2; and determining the proportion of subjects with worsening grading shifts in hematology and chemistry laboratory values between baseline and 1 week after dose 1, before dose 2, 1 week after dose 2, and 1 month after dose 2. [0848] Secondary objectives of Substudy C include assessing neutralizing antibody responses against vaccinia and mpox viruses elicited by each multivalent vaccine candidate. Secondary outcome measures include: measuring the geometric mean titers for each group at 2 weeks after dose 2 and compared to baseline (e.g., pre-dose 1); determining the proportion of subjects with seroconversion; comparing the ratio between geometric mean titers for multivalent vaccine candidates and MVA-BN; and determining the percentage of subjects with seroresponse to the vaccinia-specific virus between multivalent vaccine candidates and MVA-BN.
[0849] Exploratory objectives of Substudy C also include assessing the kinetics and specificities of humoral immune responses induced by vaccination with each multivalent vaccine candidate. Geometric mean titers for each group is measured at all timepoints.
[0850] Substudy D: Substudy D is an open label, Phase Ila substudy to assess the reactogenicity, safety, and immunogenicity of one dose level of the selected exemplary multivalent vaccine candidate. This substudy will enroll approximately 25 healthy subjects with no prior history of known or suspected smallpox vaccination (i.e., vaccinia-naive subjects). The exemplary multivalent vaccine candidate and its dose level (e.g., < 60 μg) will be selected based on Substudy A results.
[0851] For Substudy D, the planned genetic analyses comprise different approaches to further elucidate immune responses to the selected exemplary multivalent vaccine candidate, e.g., analysis of T cell and B cell receptor repertoire by next generation sequencing and/or single-cell RNA sequencing, transcriptomic analysis, and human leukocyte antigen (HLA) typing. Sampling will be performed at the timepoints described in FIG. 13C.
[0852] Planned additional research analyses will include using a systems serology approach to comprehensively characterize the humoral response to the exemplary multivalent vaccine candidate, including measurement of antibody isotype/subclass, affinity, and functional antibody-mediated responses. Further, transcriptomic analysis will be used to characterize the molecular and cellular networks that influence innate and adaptive immune responses t. o the exemplary multivalent vaccine candidate.
[0853] In selected subjects, future additional research analyses will be conducted using residual biological samples. For all substudies, future additional research analyses to be performed on residual samples may include, but are not limited to: phenotypic or functional characterization of antigen-specific T cells or B cells (e.g., by flow cytometry-based phenotyping including multimer staining); analysis of T cell and B cell receptor repertoire (e.g., by next generation sequencing and/or single-cell RNA sequencing); and evaluation of biophysical and functional properties of antigen-specific antibodies. Phenotypic or functional characterization of other immune cell populations that may be relevant to understanding the vaccine-induced immune responses may be included in this research. Residual samples may be used to inform the development of other vaccines or vaccine- related products, and/or for vaccine-related assay work supporting vaccine programs or further scientific research purposes.
[0854] FIG. 15A, FIG. 15B, and FIG. 15C, are schematic diagrams of the clinical trial design. FIG.
15A describes a staggered dosing process for substudies A, B, C, and D. FIG. 15B describes the dosing and sample collection for substudies A and B. FIG. 15C describes the dosing and sample collection for substudies C and D.
Exclusion criteria
[0855] The exclusion criteria for subjects enrolled in the clinical study of the present example include:
- prior administration of a non-trial vaccine within 28 days before Dose 1 and until Visit 9 inclusive;
- prior administration of any orthopoxvirus-based vaccines, including vaccines for smallpox, vaccinia disease or mpox prevention, or vector orthopoxvirus-based vaccines;
- prior administration of a non-trial investigational medicinal product;
- prior administration of blood/plasma products and/or immunoglobulins;
- prior allergy treatment with antigen injections within 14 days before each dose and during the 14 days after each dose;
- prior administration of immunosuppressive medications including systemic corticosteroids or radiotherapy; and
- prior administration of prophylactic antipyretics and other pain medication to prevent symptoms associated with trial treatment administration.
Permitted Concomitant Therapies During Tria!
[0856] The following concomitant therapies are permitted during participation in the clinical trial of the present example if medically indicated: - administration of antipyretics and other pain-relieving medication to treat symptoms after exemplary multivalent vaccine candidate administration (e.g., standard therapeutic doses of acetaminophen, preferably at doses of up to 4 g/day, or a non-steroidal anti-inflammatory drug (NSAID) if acetaminophen is contraindicated);
- administration of NSAID or pain-relieving medication for ongoing conditions;
- administration of a seasonal influenza vaccine is allowed if administered at least 14 days before or after any exemplary multivalent vaccine candidate administration;
- administration of medically indicated vaccines (e.g., rabies, tetanus); and
- administration of inhaled, topical, or locally injected corticosteroids (e.g., intraarticular or intrabursal administration).
Assessments
[0857] Perceived pain at the injection site is assessed as absent, mild, moderate or severe, according to the grading scale in Table 11.
[0858] Subjects are provided with a ruler to measure erythema / redness and induration / swelling.
Erythema / redness and induration / swelling are measured at the greatest single diameter and recorded and then categorized during analysis as absent, mild, moderate or severe, based on the grading scale in Table 11.
Table 11: Local Reaction Grading Scale
Figure imgf000170_0001
a. Investigator or medically qualified person confirmation is required.
Modified from the US FDA guidance (US FDA 2007)
[0859] Symptoms of systemic reactions are assessed as absent, mild, moderate, severe, potentially life-threatening, according to the grading scale in Table 12.
Table 12: Systemic Reaction Grading Scale
Figure imgf000170_0002
Figure imgf000171_0001
a. Investigator or medically qualified person confirmation is required.
Modified from the US FDA guidance (US FDA 2007)
Immune Responses
[0860] Immune responses are assessed at the times indicated in FIGs. 15A-15C. Immune response analyses are categorized as humoral immune responses, cell-mediated immune responses, or innate immune responses.
[0861] Humoral immune response assessments include:
• levels of vaccinia virus specific neutralizing antibodies using, e.g., plaque reduction neutralization test (PRNT);
• levels of MPXV-specific neutralizing antibodies using, e.g., PRNT or microneutralization assays; and/or
• levels of Combo 4 and Combo 2 multivalent vaccine candidate antigen-specific binding antibodies using, e.g., enzyme-linked immunosorbent assay (ELISA) or similar assay.
[0862] Cell-mediated immune response assessments include:
• evaluation of antigen-specific CD4 and CD8 T cells including functional characteristics such as the expression of cytokines after stimulation with peptide pools encoding vaccine or viral antigens.
[0863] Innate immune response assessments include:
• levels of circulating cytokines using, for example, a multiplex cytokine panel.
[0864] For the humoral immunogenicity analyses in Substudies A, B, C, and D, geometric mean titers will be computed along with associated 95% confidence intervals (CI) at each timepoint. For neutralizing antibody responses against vaccinia and mpox viruses elicited by each multivalent vaccine candidate, geometric mean fold rise, and percentage of subjects with seroconversions will also be computed with associated 95% Cis. [0865] In Substudy C, in addition to the analysis described above, the geometric mean ratio with associated 95% CI and difference of seroresponses with associated 95% Cis between MVA-BN group (C2) and multivalent vaccine candidate at 2 weeks after Dose 2 for Vaccinia-specific and MPXV-specific neutralizing antibody levels will be presented to provide further information.
Additional Characterizations
[0866] Further exploratory research analyses may be conducted using residual biological samples from subsets of subjects in order to further characterize the vaccine induced and/or innate immune responses, e.g., additional characterization of MPXV-specific antibodies including, but not limited to, for example, functional assays, MV/EV specific assays and avidity assays.
Adverse Events
[0867] An adverse event (AE) is any untoward medical occurrence in a trial subject administered a pharmaceutical product and which does not necessarily have to have a causal relationship with this treatment. An AE can therefore be any unfavorable and unintended sign (including an abnormal laboratory finding), symptom, or disease (new or exacerbated) temporally associated with the use of a medicinal product, whether or not considered related to the medicinal product. A medically attended adverse event (MAAE) is an adverse event for which the trial subjects received medical attention defined as unscheduled hospitalization, or an otherwise unscheduled visit to or from medical personnel for any reason, including visits to emergency rooms or other medical facilities.
[0868] AEs include:
• any abnormal laboratory test results (e.g., clinical chemistry results) or other safety assessments (e.g., ECG, vital signs measurements), including those that worsen from baseline, considered clinically significant in the medical and scientific judgment of the investigator;
• exacerbation of a chronic or intermittent pre-existing condition including either an increase in frequency and/or severity of the condition; and
• signs, symptoms, or the clinical sequelae of a suspected overdose of either trial treatment or a concomitant medication.
[0869] A serious adverse event (SAE) is defined as any untoward medical occurrence that, at any dose, results in death or is life threatening.
[0870] An adverse event of special interest (AESI) is defined as any untoward medical occurrence in a trial subject administered a pharmaceutical product reflecting potential risks of MPXV vaccination. AESIs include myocarditis and pericarditis.
[0871] The assessment of AE, SAE, and/or AESI intensity is consistently done for all trial subjects treated with the same treatment and dose. For further guidance on AE, SAE, and AESI assessments, see, for example US FDA 2007 guidance, incorporated herein by reference in its entirety. Where specific guidance for an AE term is not provided, the following general approach is followed:
Grade 1 - Mild; does not interfere with the trial subject's usual function;
Grade 2 - Moderate; interferes to some extent with the trial subject's usual function;
Grade 3 - Severe; interferes significantly with the trial subject's usual function; and
Grade 4 - Potentially life-threatening; life-threatening consequences, urgent intervention required.
Example 11: Exemplary in vitro Expression of Polyribonucleotide Constructs Encoding MPXV Antigens [0872] The present Example describes in vitro experiments characterizing the levels of expression achieved by various doses of exemplary individual nucleoside-modified polyribonucleotides (e.g., mRNAs) with backbone structures for optimized translational performance and formulated for encapsulation in LNPs and Tris/Sucrose buffer.
[0873] HEK293T cells were seeded in DMEM supplemented with 10% FBS in 12-well plates with a cell density of 4 x 105 cells/well 6 h prior to transfection. For transfection, various amounts of nucleoside-modified polyribonucleotide (RNA) was diluted to 200 ng total RNA in 100 pL Opti-MEM for individual MPXV antigen constructs encoding A35, B6, Ml, and H3 and added to the appropriate wells. The plates were gently mixed and centrifuged at 500 x g for 5 min at room temperature before incubating at 37°C and 5% CO2 for 18 h. Following the incubation, cells were prepared for subsequent FACS analysis. Culture media was removed from the 12-well plates and the cells were gently liberated from the plate surface by pipetting in cold DPBS. Each sample was then split into two, with one half used for detection of total protein and the other for surface protein (by fixing and permeabilizing or fixing alone, respectively).
[0874] For total protein detection, cells were transferred to a 96-well plate and stained with 50 pL
Fixable Viability Dye eFluor 450 diluted 1:500 in DPBS for 15 min at room temperature. Cells were then washed with FACS buffer (lx DPBS, 1% BSA, 5 mM EDTA), centrifuged at 300 x g for 5 min at 4°C and fixed with 100 pL Fixation Buffer for 12 min at room temperature. Following fixation, cells were washed with lx Permeabilization Buffer, centrifuged at 500 x g for 5 min at 4°C and stained with an appropriate primary antibody diluted in Permeabilization Buffer. Afterwards, cells were washed twice with lx Permeabilization Buffer, centrifuged at 500 x g for 5 min at 4°C, and incubated with a secondary donkey anti-mouse or goat anti-human antibody labelled with Alexa Fluor® 647 for 30 min at 4°C. Finally, cells were washed twice with lx Permeabilization Buffer as described above and resuspended in 180 pL FACS buffer before acquisition on a BD FACS Celesta II.
[0875] For surface protein detection, the remaining cells for each sample were transferred to another
96-well plate. Cells were stained directly for 30 min at 4°C with 50 pL of pre-mixed Fixable Viability Dye eFluor™ 450, diluted 1:500, and the appropriate primary antibody diluted together in DPBS. Cells were then washed with DPBS, centrifuged at 300 x g for 5 min at 4°C, and incubated with a secondary donkey anti-mouse or goat antihuman antibody labelled with Alexa Fluor® 647, diluted 1:500 in DPBS, for 30 min on ice. Following the secondary staining, cells were again washed with DPBS, centrifuged at 300 x g for 5 min at 4°C, and fixed by the addition of 100 pL Fixation Buffer for 12 min. at room temperature. Finally, the cells were centrifuged at 500 x g for 5 min at room temperature, washed twice with FACS buffer and resuspended in 180 pL FACS buffer before acquisition with a BD FACS Celesta II.
[0876] FIGs. 18A-18D show the dose-response curves of two independent experiments of HEK293T cells transfected with various amounts of nucleoside-modified polyribonucleotides encoding an MPXV A35 antigen construct (FIG. 18A), an MPXV B6 antigen construct (FIG. 18B), an MPXV Ml antigen construct (FIG. 18C), and an MPXV H3 antigen construct (FIG. 18D). The nucleoside-modified polyribonucleotides were formulated for LNP encapsulation and administration in Tris/Sucrose buffer. The dose response curves that nucleoside- modified polyribonucleotides formulated for LNP encapsulation and administration in Tris/sucrose buffer were expressed in a dose-dependent manner and detectable using a flow cytometry analysis.
Example 12: Exemplary in vivo Immunogenicity of Formulated Polyribonucleotide Constructs encoding MPXV Antigens
[0877] In this Example, the immunogenicity of exemplary Combo 4 formulated as (i) LNPs incorporating nucleoside-modified polyribonucleotides encoding MPXV A35, B6, Ml, and H3 in Tris/sucrose buffer; (ii) LNPs incorporating nucleoside-modified polyribonucleotides encoding MPXV A35, B6, Ml, and H3 in PBS/sucrose buffer; or (iii)_LNPs incorporating nucleoside-modified polyribonucleotides encoding MPXV A35, B6, Ml, or H3 and mixed immediately prior to administration, was evaluated using BALB/c mice.
[0878] Mice were immunized intramuscularly (IM) with: (i) 4 μg exemplary Combo 4 (1 μg of each of
A35, B6, Ml and H3 nucleoside-modified polyribonucleotide components) formulated in Tris/sucrose buffer (Combo 4 [T]); (ii) 4 μg of exemplary Combo 4 (1 μg of each of A35, B6, Ml and H3 nucleoside-modified polyribonucleotide components) formulated in PBS/sucrose buffer (Combo 4 [P]); of (iii) 4 μg of exemplary Combo 4 (1 μg of each of A35, B6, Ml and H3 nucleoside-modified polyribonucleotide components) formulated as single nucleoside-modified polyribonucleotide components in PBS/sucrose buffer and mixed immediately prior to administration (Combo 4 (single mix)). Blood was collected at day 14 post-immunization, levels of serum immunoglobulin G (IgG) raised to each MPXV antigen was measured by enzyme-linked immunosorbent assay (ELISA), and 50% binding titers for anti-A35 IgG (FIG. 19A), anti-B6 IgG (FIG. 19B), anti-Ml IgG (FIG. 19C), and anti-H3 IgG (FIG. 19D) were calculated.
[0879] As shown in FIGs. 19A to 19D, all exemplary Combo 4 formulations induced increases in serum anti-MPXV antigen IgG titers. No statistically significant differences in binding titers were observed between the formulations and/or between the four expressed MPXV antigens (ANOVA with Tukey's multiple comparisons test). Table 13: Table of Exemplary Antigen Sequences
Figure imgf000174_0001
Figure imgf000175_0001
Figure imgf000176_0001
Figure imgf000177_0001
Table 14: Table of Exemplary Construct Sequences
Figure imgf000178_0001
Figure imgf000179_0001
Figure imgf000180_0001
Figure imgf000181_0001
Figure imgf000182_0001
Figure imgf000183_0001
EQUIVALENTS
[0880] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of technologies described herein. The scope of the present disclosure is not intended to be limited to the above Description, but rather is as set forth in the following claims.

Claims

CLAIMS WHAT IS CLAIMED IS:
1. A method of preventing or treating an orthopoxvirus infection in a subject comprising administering a composition or combination to the subject, wherein the composition or combination comprises: a therapeutically effective amount of one or more polyribonucleotides encoding one or more mpox virus antigens or antigenic fragments thereof; and a pharmaceutically acceptable carrier.
2. The method of claim 1, wherein the composition or combination comprises at least three polyribonucleotides, and wherein each of the at least three polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof.
3. The method of claim 1, wherein the composition or combination comprises three polyribonucleotides, and wherein each of the three polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof.
4. The method of claim 2 or 3, wherein: a first polyribonucleotide encodes a B6R antigen, or antigenic fragment thereof; a second polyribonucleotide encodes an MIR antigen, or antigenic fragment thereof; and a third polyribonucleotide encodes an A35R antigen, or antigenic fragment thereof.
5. The method of claim 4, wherein: the B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182; the MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158; and the A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174.
6. The method of any one of claims 1 to 5, wherein the composition or combination comprises at least four polyribonucleotides, and wherein each of the at least four polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof.
7. The method of any one of claims 1 to 6, wherein the composition or combination comprises four polyribonucleotides, and wherein each of the four polyribonucleotides encodes a different mpox virus antigen or antigenic fragment thereof.
8. The method of claim 6 or 7, wherein: a first polyribonucleotide encodes a B6R antigen, or antigenic fragment thereof; a second polyribonucleotide encodes an MIR antigen, or antigenic fragment thereof; a third polyribonucleotide encodes an A35R antigen, or antigenic fragment thereof; and a fourth polyribonucleotide encodes an H3L antigen, or antigenic fragment thereof.
9. The method of claim 8, wherein: the B6R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 21-30, 180, and 182; the MIR antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 31-40, and 158; the A35R antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 11-20, 172, and 174; and the H3L antigen comprises an amino acid sequence at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 51-60, 184, 188, and 190.
10. The method of any one of claims 1 to 9, wherein the orthopoxvirus infection comprises an mpox infection, a vaccinia infection, an ectromelia infection, a variola infection, a borealpox infection, a cowpox infection, a volepox infection, a buffalopox infection, or a camelpox infection.
11. The method of any one of claims 1 to 10, wherein the orthopoxvirus infection is a clade I mpox infection.
12. The method of claim 11, wherein the orthopoxvirus infection is a clade la or clade lb mpox infection.
13. The method of any one of claims 1 to 10, wherein the orthopoxvirus infection is a clade II mpox infection.
14. The method of claim 13, wherein the orthopoxvirus infection is a clade Ila or clade lib mpox infection.
15. The method of any one of claims 1 to 14, wherein the one or more polyribonucleotides are purified.
16. The method of any one of claims 1 to 15, wherein the one or more polyribonucleotides are single-stranded.
17. The method of any one of claims 1 to 16, wherein the one or more polyribonucleotides comprise a 5'-capped
Nl-methylpseudouridine.
18. The method of any one of claims 1 to 17, wherein each of the one or more polyribonucleotides are fully or partially encapsulated within a lipid nanoparticle.
19. The method of claim 18, wherein the lipid nanoparticle targets liver cells.
20. The method of claim 18 or 19, wherein the lipid nanoparticle targets secondary lymphoid organ cells.
21. The method of any one of claims 18 to 20, wherein the lipid nanoparticle is a cationic lipid nanoparticle.
22. The method of any one of claims 18 to 21, wherein the lipid nanoparticle comprises:
(a) a polymer-conjugated lipid;
(b) a cationic lipid; and
(c) one or more neutral lipids.
23. The method of claim 22, wherein the polymer-conjugated lipid comprises a PEG-conjugated lipid.
24. The method of claim 22 or 23, wherein the one or more neutral lipids comprise 1,2-Distearoyl-sn-glycero-3- phosphocholine (DPSC).
25. The method of any one of claims 22 to 24, wherein the one or more neutral lipids comprise cholesterol.
26. The method of any one of claims 22 to 25, wherein the lipid nanoparticle comprises:
(a) the polymer-conjugated lipid at about 1-2.5 mol% of the total lipids;
(b) the cationic lipid at 35-65 mol% of the total lipids; and
(c) the one or more neutral lipids are present in 35-65 mol% of the total lipids.
27. The method of any one of claims 22 to 26, wherein the lipid nanoparticles have an average diameter of about 50-150 nm.
28. The method of any one of claims 1 to 27, comprising administering the composition or combination in one or more doses to the subject.
29. The method of claim 28, wherein the one or more doses comprises about 10 μg, about 30 μg, or about 60 μg of the one or more polyribonucleotides.
30. The method of claim 28 or 29, comprising administering two doses of the composition or combination, wherein a second dose of the composition or combination is administered to the subject about 31 days after a first dose of the composition or combination is administered to the subject.
31. The method of any one of claims 28 to 30, wherein the one or more doses are administered to the subject intramuscularly.
32. The method of any one of claims 28 to 31, wherein administering the composition or combination comprises mixing the one or more polyribonucleotides and wherein each of the one or more doses is administered as a single injection.
33. The method of any one of claims 1 to 32, wherein administering the composition or combination to the subject results in an increased serum level of one or more orthopoxvirus-neutralizing antibody in the subject.
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