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WO2021188818A1 - Constructions de vaccin et compositions et procédés d'utilisation de celles-ci - Google Patents

Constructions de vaccin et compositions et procédés d'utilisation de celles-ci Download PDF

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WO2021188818A1
WO2021188818A1 PCT/US2021/023002 US2021023002W WO2021188818A1 WO 2021188818 A1 WO2021188818 A1 WO 2021188818A1 US 2021023002 W US2021023002 W US 2021023002W WO 2021188818 A1 WO2021188818 A1 WO 2021188818A1
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phage
protein
seq
nucleic acid
cov
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Biswajit Biswas
Kimberly BISHOP-LILLY
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US Department of Navy
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US Department of Navy
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • 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
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6075—Viral proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/20011—Coronaviridae
    • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10322—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2795/10342—Use of virus, viral particle or viral elements as a vector virus or viral particle as vehicle, e.g. encapsulating small organic molecule
    • 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
    • C12N2795/00—Bacteriophages
    • C12N2795/00011—Details
    • C12N2795/10011—Details dsDNA Bacteriophages
    • C12N2795/10311—Siphoviridae
    • C12N2795/10341—Use of virus, viral particle or viral elements as a vector
    • C12N2795/10343—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • NCI 12662PCT_ST25.txt is 18 kilobytes in size.
  • the instant invention relates to nucleic acid sequences encoding one or more antigens of interest, and compositions comprising said nucleic acid sequences, for use in a phage-based display vaccine platform for creating bacteriophage (phage) vaccine particles (PVPs) expressing one or more of said antigens useful for inducing an immune response against a corona virus in a subject in need thereof.
  • the inventive subject matter also comprises immunogenic compositions comprising one or more PVPs of the instant invention useful to provide enhanced immunity against a corona virus in a subject in need thereof, and related methods of use.
  • the corona vims is SARS-CoV-2.
  • adjuvants are typically administered with many recombinant vaccines to enhance immunostimulatory effects.
  • many contemporary adjuvants not only fail to provide necessary immune- stimulation, but also require extensive resources that are not amenable to rapid vaccine development against evolving diseases like SARS-CoV-2.
  • Phages present various advantages as possible vaccine delivery agents. For example, foreign protein can be expressed on phage capsids in high copy number to produce a nanoparticle vaccine for provoking both cell mediated and humoral responses, while inherently producing adjuvant affects in vivo. Phages are also reportedly safe for human use and resulting vaccines can be propagated rapidly using conventionally bacteriological media hi addition, the stability of the phage-based vaccine platform at room temperature implies no cold-chain requirement, an ideal platform for addressing global public health concerns in austere environments. Phages also can be adapted inexpensively for large-scale production by using simple bacteriological media and do not require large-scale cell culture systems for manufacture. Thus, the incorporation of phage particles into the formulation of vaccine candidates enormously decreases the expense of producing new vaccines while increasing efficiency.
  • the present invention relates to nucleic acid sequences for use in a phage-based vaccine platform to create antigenic phage vaccine particles (PVP) against coronavirus.
  • the nucleic acid sequences comprise, consist essentially of, or consist of one or more nucleic acid sequences encoding target antigenic sequences of a coronavirus peptide or protein.
  • the target antigenic sequence comprises a peptide sequence of the receptor-binding domain (RBD) of a coronavirus spike protein, or a fragment or variant thereof.
  • RBD receptor-binding domain
  • the fragment comprises amino acids 383-592 of the peptide sequence of the RBD of the SARS-CoV-2 SI subunit protein (SEQ ID NO: 1).
  • SEQ ID NO: 1 is encoded by the nucleotide sequence set forth in SEQ ID NO: 2.
  • the target antigenic sequence comprises a peptide sequence of the Heptad repeat domain 2 (HR2) in the S2 subunit protein of a coronavirus spike protein, or a fragment or variant thereof.
  • the fragment comprises amino acids 1025-1187 of the peptide sequence of HR2 of the SARS-CoV-2 S2 subunit protein (SEQ ID NO: 3).
  • SEQ ID NO: 3 is encoded by the nucleotide sequence set forth in SEQ ID NO: 4.
  • the nucleic acid sequences comprise, consist essentially of, or consist of one or more nucleic acid sequences encoding a target antigenic sequence of a coronavirus peptide or protein operably linked to a sequence encoding a phage capsid protein or a fragment or variant thereof.
  • the invention relates to variants of at least 85% sequence identity to a nucleic acid construct or sequence disclosed herein or a fragment thereof.
  • the phage capsid protein is a native, full- length sequence of lambda phage capsid protein gpD, or a fragment, truncated version, or variant thereof.
  • the nucleic acid sequence is a construct encoding amino acids 383-592 of the peptide sequence of the RBD of the SARS-CoV-2 SI subunit protein fused to a 112 amino acid lambda gpD protein (SEQ ID NO: 5.)
  • SEQ ID NO: 5 is encoded by the nucleotide sequence set forth herein as NV101-COVID-19, “Construct 1” (SEQ ID NO: 6.)
  • the nucleic acid sequence is a construct encoding amino acids 1025-1187 of the peptide sequence of HR2 of the SARS-CoV-2 S2 subunit protein fused to full length lambda gpD protein (SEQ ID NO: 7.)
  • SEQ ID NO: 7 In a particular embodiment, SEQ ID NO:
  • the nucleic acid sequence comprising a nucleotide sequence encoding a target antigenic sequence is codon optimized for expression in bacteria.
  • the bacteria is Escherichia coli (E. colxi. in a particular embodiment, the E. coli strain is W3110.
  • the instant invention relates to vectors comprising any nucleic acid constructs and sequences disclosed herein or a fragment or variant thereof.
  • the invention relates to bacteria comprising one or more nucleic acid sequences and/or specific constructs disclosed herein, or a fragment or variant thereof.
  • the bacteria is Escherichia coli (E. coli).
  • the E. coli is E. coli strain W3110.
  • the invention relates to phages comprising one or more nucleic acid sequences and/or specific constructs disclosed herein or a fragment or variant thereof, as well as phages expressing one or more peptides or proteins encoded by- said nucleic acid sequences and/or specific constructs disclosed herein or a fragment or variant thereof.
  • the phage is selected from the group consisting of lambda, T4, T7, M13/fl QB, and MS.
  • the phage is lambda.
  • the invention relates to various compositions comprising any one or more nucleic acid sequences and constructs disclosed herein, as well as compositions comprising any one or more peptide sequences encoded by one or more nucleic acid sequences and constructs of the instant invention; compositions comprising one or more transformed bacteria, recombinant phages, and/or PVPs disclosed herein.
  • the compositions of the instant invention include immunogenic compositions against a coronavirus.
  • the immunogenic compositions of the instant invention comprise a therapeutically effective amount of PVPs against a coronavirus as disclosed herein.
  • the immunogenic composition comprises PVPs displaying a chimeric capsid protein encoded by a nucleic acid sequence or construct of the instant invention and/or a fragment or variant thereof.
  • the chimeric capsid protein comprises amino acids 383-592 of the peptide sequence of the RBD of the SARS-CoV-2 SI subunit protein (SEQ ID NO: 1).
  • the chimeric capsid protein comprises amino acids 1025-1187 of the peptide sequence of HR2 of the SARS-CoV-2 S2 subunit protein (SEQ ID NO: 3).
  • the immunogenic composition comprises recombinant phage expressing the chimeric capsid protein encoded by Construct 1 (SEQ ID NO:6) and/or recombinant phage expressing the chimeric capsid protein encoded by Construct 2 (SEQ ID NO:8).
  • compositions and immunogenic compositions of the instant invention are pharmaceutical compositions.
  • the pharmaceutical compositions comprise a therapeutically effective amount of one or more PVPs of the instant invention, a pharmaceutically acceptable excipient, and, optionally , an effective amount of one or more adjuvants.
  • the pharmaceutical composition may further comprise an effective amount of one more additional anti-viral agent and'or other active pharmaceutical ingredient.
  • the pharmaceutical composition is a vaccine.
  • the vaccine is a single subunit vaccine.
  • the single subunit vaccine comprises recombinant phage (PVPs) expressing the chimeric capsid protein encoded by Construct 1 (SEQ ID NO: 6) or recombinant phage (PVPs) expressing the chimeric capsid protein encoded by Construct 2 (SEQ ID NO: 8).
  • the vaccine formulation is a multisubunit vaccine comprising a combination of two or more PVPs, wherein said PVPs display different chimeric capsid proteins.
  • the multi-subunit vaccine comprises recombinant phage (PVPs) expressing the chimeric capsid protein encoded by Construct 1 (SEQ ID NO: 6) and recombinant phage (PVPs) expressing the chimeric capsid protein encoded by Construct 2 (SEQ ID NO: 8).
  • PVPs recombinant phage
  • Construct 1 SEQ ID NO: 6
  • PVPs recombinant phage
  • Construct 2 SEQ ID NO: 8
  • the invention relates to methods of preventing, ameliorating or treating disease caused by coronavirus in a subject in need thereof comprising administering to the subject a therapeutically effective amount of one or more compositions of the instant invention alone or in combination with one or more additional anti-viral agent and/or active pharmaceutical ingredient.
  • the instant invention relates to methods of enhancing an immune response against one or more coronaviruses in a subject in need thereof comprising administering a therapeutically effective amount of one or more compositions of the instant invention alone or in combination with one or more anti-viral agent and/or other active pharmaceutical agent.
  • the invention further relates to methods of increasing the titer of virus-specific IgG antibodies, and/or increasing the titer of IFN-g responses in a subject in need thereof comprising administering to the subject an effective amount of one or more immunogenic compositions comprising one or more types of PVPs of the instant invention, alone or in combination with one or more anti-viral agent and or other active pharmaceutical agent.
  • the coronavirus is selected from the group consisting of SARS-CoV-2, Middle East respiratory syndrome coronavirus (MERS-CoV) and Severe Acute Respiratory Syndrome (SARS).
  • the coronavirus is SARS-CoV-2.
  • the one or more additional anti-viral or other active pharmaceutical ingredient is selected from the group consisting of protease inhibitors, tenofovir, remdesivir, chloroquine, and favilavir.
  • kits as well as the use of the nucleic acid sequences, constructs, recombinant bacteriophage (PVPs) and compositions disclosed herein as research tools and in diagnostics.
  • PVPs recombinant bacteriophage
  • Figure 1 is a schematic depicting the degree of relatedness of SARS-CoV-
  • Figure 2 depicts amino acids 383-592 of the peptide sequence of the RBD of the SARS-CoV-2 SI subunit protein (SEQ ID NO: 1); the corresponding cDNA sequence based on the vims RNA sequence (SEQ ID:NO 13), and the corresponding codon optimized cDNA sequence encoding the peptide sequence (SEQ ID NO: 2).
  • Figure 3 depicts amino acids 1025-1187 of the peptide sequence of FIR2 of the SARS-CoV-2 S2 subunit protein (SEQ ID NO: 3); the corresponding cDNA sequence based on the virus RNA sequence (SEQ ID.NO 14), and the corresponding codon optimized cDNA sequence encoding the peptide sequence (SEQ ID NO: 4).
  • Figures 4(A)-4(C) are cartoons depicting the components of a nucleotide construct encoding an embodiment of a fusion protein of the instant invention.
  • the construct comprises a fusion between a nucleotide sequence encoding a foil length phage lambda gpD protein (or a functional fragment or variant thereof) and a nucleotide sequence encoding a targeted antigenic peptide sequence of a coronavirus (‘vaccine target”) and the locations of start and stop codons.
  • Figure 4(B) depicts the chimeric capsid protein comprising the targeted antigenic sequence of the RBD of the SARS-CoV-2 SI subunit protein.
  • Figure 4(C) depicts the chimeric capsid protein comprising the targeted antigenic sequence of HR2 of the SARS-CoV-2 S2 subunit protein. Regions of vector peptides (e.g., set forth as SEQ ID: NO 15 herein) are not depicted.
  • Figures 5(A)-5(D) depict the amino acid and nucleic acid sequences of the fusion proteins of Construct 1 and Construct 2.
  • Figure 5(A) depicts the amino acid sequence of the fusion protein of Construct 1 (SEQ ID NO: 5);
  • Figure 5(B) depicts the amino acid sequence of the fusion protein of Construct 2 (SEQ ID NO:7);
  • Figure 5(C) depicts the nucleic acid sequence encoding the fusion protein of Construct 1 (SEQ ID NO: 6);
  • Figure 5(D) depicts the nucleic acid sequence encoding the fusion protein of Construct 2 (SEQ ID NO: 8).
  • FIG. 5(A) and Figure 5(B) The bold text depicted in Figure 5(A) and Figure 5(B) is the amino acid sequence of the phage gpD protein; the stylized script is a 14 AA region of the vector comprising restriction sites (SEQ ID NO: 15); the italicized text represents the spike protein insert; and the underlined sequence (AR) presents amino acids generated by restriction site (Bsshll) in the cloning process.
  • the enlarged and bolded font “H” at position 150 of the spike protein sequence reflects variability at this position in nature, with most sequenced isolates having an R at position 150. H versus R here is believed to have little to no effect on protein structure.
  • Figure 6 is a cartoon summarizing key steps of the cloning strategy used to create Construct 1, N V 101 -COVID- 19 and Construct 2, NV 102-COVID- 19.
  • codon optimized cDNA encoding the targeted antigenic sequences amino acids 383-592 of the peptide sequence of the RBD of the SARS-CoV-2 SI subunit protein (NVlOl-COVID-19) or amino acids 1025-1187 of the peptide sequence of HR2 of the SARS-CoV-2 S2 subunit protein (NV102-COVID-19)
  • the synthesized DNA fragments were then PCR amplified using two modified primers for generating Nhel and BssHII restriction sites at the 5 and 3 ends of the fragments, respectively.
  • the PCR amplified products were cleaned using PCR clean kit (Invitrogen, Carlsbad, CA) and restriction digested with Nhel and BssHII restriction enzymes. After restriction digestion, these fragments were inserted at the Nhel-Bsshll site of the 3' end of a DNA segment encoding gpD under the control of the lac promoter.
  • the constructs were created in a plasmid vector (donor plasmid, pUCDcDL3-A), which also carries loxPwt and loxP511 sequences. The presence of virus-specific insert DNA in the recombinant donor plasmid was confirmed by restriction enzyme analysis.
  • Cre- expressing E.coli were transformed with these recombinant donor plasmids and infected with a recipient lambda phage that carries a stuffer DNA segment flanked by loxPwt and loxP511 sites (in house stock lambda phage).
  • the infecting lambda has an amber mutation in its gpD gene and therefore is unable to produce the gpD protein and propagate efficiently in the Cre positive E. coli strain unless the recombination event successfully transferred an external gpD, in lambda genome.
  • an ampicillin resistant gene (Beta-lactamases) was also transferred in lambda genome that was later used as a selective marker.
  • Lambda phage infected Cre-expressing E. coli was grown in LB Ampicillin (100 ug/ml) at 37° C for four hours in presence of 0.2% maltose and 0.1M CaCh. Recombination occurred in vivo at the lox sites and Amp r cointegrates were formed which spontaneously lysed the E. coli and V ' ere released in culture media. Only some of the lambda genes are depicted.
  • Figure 7 is a cartoon depicting the recombinant lambda genome comprising the NVlOl-COVID-19 construct disclosed herein. Sequence analysis verified that the recombinant lambda genome comprised the proper RBD insert in the correct orientation (darker shaded arrow labeled “NVlOl-COVID-19”). Detailed annotation information is found in co-pending priority application, U.S. provisional application, Ser. No. 63/008,412 filed April 10, 2020, the disclosure of which is hereby incorporated herein by reference.
  • Figure 8 is a cartoon depicting the recombinant lambda genome comprising the NV102-COVID-19 construct disclosed herein. Sequence analysis verified that the recombinant lambda genome comprised the proper FIR2 insert in the correct orientation (lighter shaded arrow labeled “NV102B-COVID-19”). Detailed annotation information is found in co-pending priority application, U.S. provisional application, Ser. No. 63/008,412 filed April 10, 2020, the disclosure of which is hereby incorporated herein by reference.
  • Figure 9 depicts a simple protocol of a prophetic mouse assay to assess safety, reactogenicity, and immunogenicity of SARS-CoV-2 vaccine formulations of the instant invention.
  • One or more PVPs may be assayed using the depicted protocol.
  • Figure 10 depicts a prophetic schedule of events for the clinical trials described in Examples 13 and 14. Notation therein refers to the following: a, If screening (Day -30 to Day -1) is performed within 14 days of Dose 1, then do not repeat medical history; b, Prior and new concomitant medications will be recorded at all study visits (Day 0 through study discharge); c, Full physical examination performed at screening and study discharge only, perform targeted examinations at other visits as determined by Investigator or per participant complaints, vital signs will be performed pre and post vaccination at week 0, 2, and 4 week visits; d, Sodium (Na), potassium (K), chloride (Cl), bicarbonate (HCOs), glucose, BUN, Cr, ALT, AST, CPK; e, HIV antibody or rapid test, HBsAg, HCV antibody; f, Serum pregnancy test at screening and urine pregnancy test at each subsequent vaccination day; g, Collect at least 51 mL at screening and weeks 1 for all subjects enrolled and receiving the vaccine at day 0 (6 x 8.5
  • Figure 12 depicts a genome map of “COVID_Phi_Lambda-NV102B”
  • Figures 13(A) and 13(B) depict peptides in the fusion protein encoded by
  • Figures 14(A) and 14(B) depict peptides in the fusion protein encoded by
  • an antigen can mean at least one antigen, as well as a plurality of antigens, i.e., more than one antigen.
  • the term "and '' or” when used in a list of two or more items means that any one of the listed characteristics can be present, or any combination of two or more of the listed characteristics can be present.
  • compositions of the instant invention can contain A feature alone; B alone; C alone; A and B in combination; A and C in combination; B and C in combination; or A, B, and C in combination.
  • phage display vaccine platform a phage is genetically engineered to express immunogenic antigens (epitopes) expressed on the outer phage surface (capsid). Additionally, display proteins or peptides can be selected for their specific binding affinity of antigen-presenting cells (APCs). Lambda phage has gained popularity as a phage display platform because a site-specific recombination process that facilitates rapid high efficiency cloning of foreign DNA in its genome can be employed. For example, foreign antigens may be fused to the phage capsid protein, gpD.
  • phage DNA vaccine platform foreign antigen genes are incorporated into the phage genome under the control of strong eukaryotic promoters. Similar to other vectors exploited for transferring genetic material (e.g., Modified Vaccina virus Ankara (MV A), adeno vectors), the phage acts as a passive carrier to transfer the foreign DNA into mammalian cells, often targeting dendritic and Kupffer cells where the antigen gene is expressed.
  • MV A Modified Vaccina virus Ankara
  • the phage acts as a passive carrier to transfer the foreign DNA into mammalian cells, often targeting dendritic and Kupffer cells where the antigen gene is expressed.
  • both phage display and phage DNA vaccine platforms are utilized; the platform employs phages which express the immunizing antigen on its surface (i.e., target antigen fused to a capsid protein) in the phage display format, and also incorporate foreign antigen genes to be transduced into the host cell (e.g., APCs) for expression.
  • phages which express the immunizing antigen on its surface (i.e., target antigen fused to a capsid protein) in the phage display format, and also incorporate foreign antigen genes to be transduced into the host cell (e.g., APCs) for expression.
  • Coronavirus is a genus in the Coronaviridae family of viruses and are single-stranded RNA viruses familiar to one of skill in the art. These viruses include, e.g., Middle East respiratory syndrome coronavirus (MERS-CoV), Severe Acute Respiratory Syndrome (SARS) and the recently identified virus, SARS-CoV-2.
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • SARS Severe Acute Respiratory Syndrome
  • the methods of phage-based display described herein are used with targeted antigenic sequences from the genome of SARS- CoV-2 to create compositions that may prove useful not only for developing immunogenic compositions (e.g., vaccine formulations) which elicit broad-spectrum immunity against SARS-CoV-2 but also for related diagnostic uses.
  • the present invention is directed to nucleic acid sequences and constructs encoding one or more targeted antigens of interest, or fragments or variants thereof, for use in creating the PVPs (also referred to interchangeably herein as “phage virus particles” or “phage vaccine particles”), compositions, and vaccines of the instant invention.
  • PVPs also referred to interchangeably herein as “phage virus particles” or “phage vaccine particles”
  • Such nucleic acid sequences and constructs include, e.g., DNA (including cDNA) or RNA (including mRNA).
  • the nucleic acid sequences may be isolated or synthesized using standard techniques familiar to one of skill in the art.
  • the instant invention describes selecting target coronavirus antigenic peptide sequences (epitopes), isolating and/or synthesizing nucleic acid sequences encoding these targeted antigenic viral peptides/epitopes (e.g., cDNA based on viral RNA, optionally codon optimized), and cloning this nucleic acid sequence into a phage genome, ideally for expression on the phage capsid. It is contemplated herein that the nucleic acid sequence encoding the antigenic viral peptides/epitopes is operably linked to a nucleic acid sequence encoding part or all of a phage capsid protein.
  • the recombinant phage that are created express and display these antigenic fusion proteins/peptides on the phage virion surface in the form of a fusion or chimeric capsid protein (PVPs).
  • PVPs fusion or chimeric capsid protein
  • the PVPs of the instant invention can be rapidly and inexpensively propagated in bacteriological media (e.g., Luria-Bertani (LB) media), and then purified and analyzed in various preclinical and clinical assays, e.g., to determine safety, efficacy and antibody response of candidate immunogenic compositions comprising the PVPs.
  • bacteriological media e.g., Luria-Bertani (LB) media
  • the term "antigen” comprises a compound, composition, or substance that can stimulate the production of antibodies and/or a T cell response in a subject, including compositions that are injected, absorbed, or otherwise introduced into a subject.
  • the term “antigen” includes all related antigenic epitopes.
  • epitope or “antigenic determinant” refers to a site on an antigen to which B and/or T cells respond.
  • T-cell epitope refers to an epitope that, when bound to an appropriate MHC molecule, is specifically bound by a T cell (via a T cell receptor).
  • a ”B-cell epitope is an epitope that is specifically bound by an antibody (or B cell receptor molecule).
  • target antigen refers to peptide sequences that may serve as antigenic epitopes in the phage-based display vaccine platform described herein.
  • target antigenic sequences of a coronavirus and like terms comprise antigenic epitopes of SARS-CoV-2.
  • antigenic epitopes of the instant invention comprise a peptide sequence of a coronavirus S protein, or a fragment or variant thereof.
  • variant includes peptides or polypeptides that may differ in amino acid sequence by one or more substitutions, deletions, insertions, inversions, fusions, truncations or a combination of any of these and retain relevant immunogenicity.
  • peptides, polypeptides, or chimeric or fusion polypeptides of the instant disclosure can include, for example, modified forms of naturally occurring amino acids such as D-stereoisomers, non-naturally occurring amino acids; amino acid analogs; mimetics as well as retro-inverso forms thereof.
  • SARS-CoV-2 Publicly available sequences of SARS-CoV-2 were used to assess the relatedness of SARS-CoV-2 to previously sequenced viruses belonging to the coronavirus family. Genetic sequence analysis indicates that SARS-CoV-2 is very closely related to Severe Acute Respiratory Syndrome Coronavirus (SARS CoV) and bat SARS ( Figure 1). In addition, recent reports have confirmed that SARS-CoV-2 uses the same mechanism to bind to and enter host cells as other coronaviruses.
  • Coronaviruses express a “spike’ or “S” glycoprotein on their surface that binds to angiotensin-converting enzyme 2 (ACE2), a molecule found on the surface of eukaryotic cells. Binding of coronavirus to the ACE2 viral receptor results in conformational changes that promote merger of vims and host cell membranes, followed by entry of the vims into the eukaryotic cells where the virus replicates. Post replication, progeny virions are released into the extracellular milieu and infect more cells in the host organism. In addition to host cell entry, the S protein can induce cell-cell merger to form syncytia (giant cells).
  • ACE2 angiotensin-converting enzyme 2
  • the S protein of SARS-CoV-2 contains S1 and S2 subunits; the former comprises the receptor-binding domain (RBD) specific for the ACE2 receptor and makes up the “head” region, while the latter subunit makes up the “stalk” region which plays a role in viral cell membrane fission involving the creation of a six-helical bundle involving a two-heptad repeat domain.
  • RBD receptor-binding domain
  • target antigens for display in a PVP of the instant invention include peptide sequence of a SARS-CoV-2 spike (S) protein, or a fragment or variant thereof.
  • the target antigen for display in a PVP of the instant invention comprises a peptide sequence of the SI subunit of a SARS-CoV-2 spike protein, or a fragment or variant thereof.
  • the target antigen sequence comprises the receptor binding domain (RBD) of the SI subunit of a SARS- CoV-2 spike protein, or a fragment or variant thereof.
  • the target antigen for display in a PVP of the instant invention comprises a peptide sequence of the S2 subunit of the SARS-CoV-2 spike protein, or a fragment or variant thereof.
  • targeted antigenic sequences from the Si subunit of SARS-CoV-2 include amino acids 383-592 of the peptide sequence of the RBD of the SARS-CoV-2 SI subunit protein, set forth herein as SEQ ID NO: 1.
  • Targeted antigenic sequences from the S2 subunit of SARS-CoV-2 include amino acids 1025-1187 of the peptide sequence of the Heptad repeat domain 2 (HR2) in the S2 subunit protein, set forth herein as SEQ ID NO: 3.
  • Corresponding cDNA and codon optimized cDNA sequences are set forth herein (SEQ ID NOs: 13, 14, 2, 4). See Figures 2 and 3.
  • a lambda phage construct referred to interchangeably herein as “NV102-COVID-19” or “Construct 2” has been created and is set forth herein as SEQ ID NO: 8. See Figure 5(D). Construct 2 is used to create PVPs correctly displaying amino acids 1025-1187 of the peptide sequence of the HR2 in the S2 subunit protein as a chimera with the lambda phage capsid protein, gpD (SEQ ID NO: 7). See Figure 5(B).
  • Nucleic acid sequences and constructs contemplated herein include codon optimized forms; Construct 1 and Construct 2 comprise nucleic acid sequences of the respective target antigens that are codon optimized.
  • nucleic acid sequences and constructs encoding targeted antigenic peptides and epitopes disclosed herein may be codon optimized depending on the bacterial host organism in which the PVPs of the instant t invention are to be generated. Codon optimization agorithms are available from a variety of commercial vendors and include freely available web-based programs e.g., the OPTIMIZER program. (See Puigbo P, et al. Nucleic Acids Research 2007; 35.W126- W1 31 ; Puigbo P, et al, Nucleic Acids Research 2008; 36:D524-7.)
  • sequence variants may share between about 85% to about 100 % identity with the sequences identified herein and yet retain relevant activity/immunogenicity.
  • amino acid and nucleic acid sequence variants may share 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, and 100 % identity with the amino acid and nucleic acid sequences identified herein as SEQ ID NOs; 1-15.
  • a chimera or fusion protein of the instant invention can be produced by standard recombinant DNA or RNA techniques familiar to one of skill in the art. Additional standard experimental and clinical protocols for use herein include, e.g., protocols in the fields of molecular biology, virology, bacteriology, phage biology, computational and synthetic biology, protein engineering, mutagenesis, reverse vaccinology, immunology, gene sequencing, and phage genetics.
  • the fusion proteins may be isolated directly from the PVPs and/or can be prepared with the aid of recombinant, enzymatic, or chemical techniques for use in a method of the instant invention, e.g., using the amino acid sequences set forth herein as SEQ ID NO; 5 and SEQ ID NO; 7, and/or the nucleic acid sequences set forth herein as SEQ ID NO;6 and SEQ ID NO:8.
  • the fusion protein is removed from the phage capsid and thus many of the polypeptides, nucleic acids, and other cellular material of the phage are no longer present.
  • Purified forms are contemplated herein, i.e., in particular embodiments, the isolated fusion protein is at least 60% free, at least 75% free, or at least 90% free from other components with which they are naturally associated in the phage.
  • Polypeptides that are produced outside the organism in which they naturally occur, e.g., through chemical or recombinant means, are considered to be isolated and purified by definition, since they were never present in a natural environment.
  • DNA or RNA fragments) encoding different polypeptide sequences of interest may be designed and created, i.e., ligated together in-frame in accordance with conventional techniques.
  • Nucleic acid sequences encoding such fusions can be synthesized using commercial available automated synthesizers and/or polymerase chain reaction (PCR) amplification techniques. See e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 4 th Edition (2001) the contents of which are incorporated by reference herein.
  • nucleic acid sequences encoding the targeted antigenic sequences of the instant invention comprise modified forms, e.g., the sequences may be engineered accordingly to include restriction sites to facilitate the cloning strategy employed to create a recombinant phage genome. It is also contemplated herein that the nucleic acid sequences and constructs of the instant invention may be engineered to create PVPs that display other viral epitopes of interest and/or one or more additional immunostimulatory sequences. In a particular embodiment, such sequences comprise immunostimulatory unmethylated CpG motifs of the phage genome that may be added to a nucleic acid sequence or construct to further enhance immunity of a PVP of the instant invention. In addition, as discussed above, codon optimization may be performed using conventional methods, if desired.
  • targeted antigenic peptides may be engineered to be expressed by the phage as a fusion protein with a high copy number phage capsid protein.
  • high-density display (along with good adjuvant nature of phage) enhances the presentation of immunogenic antigens to APCs and thus produce a strong immunogenic response.
  • chimeric capsid proteins may be made using the major lambda phage “D” capsid protein, gpD, e.g., by fusing the gpD protein of the phage to the amino terminus of a targeted antigenic sequence.
  • Lambda gpD protein also known, e.g., as ‘capsid decoration protein’ aka
  • auxiliary' protein D aka ‘major capsid protein D’
  • lambda phage capsid is found on the lambda phage capsid. See, e.g., Yang, F et al Nat Struct Biol. 2000 Mar; 7(3):230-7. It is required for the packaging of full-length lambda phage genomes and is typically expressed in high copy number (approximately 450 copies per phage) on the surface of lambda phage.
  • a lambda phage display vaccine platform presenting the porcine circovirus and influenza antigens fused to surface gpD was successfully used to produce neutralizing antibodies (Thomas, 2012, Vaccine 30(6): 998-1008).
  • the use of lambda phage surface gpD for a cancer vaccine therapy is disclosed in US Patent 9,744,223 and US Patent 10,702,591 the entire contents of which are incorporated by reference herein.
  • gpD forms of gpD exist in nature; it is contemplated herein that these different versions (including functional fragments or variants thereof) may be used in the instant invention. These include but are not limited to, the full length (112 A A) gpD sequence “Bacteriophage lambda head decoration protein D” designated as UNIPROT D7XM12-1:
  • PVPs expressing chimeric capsid proteins of the instant invention may be created using conventional cloning strategies familiar to one of skill in the art.
  • an appropriate cloning strategy comprises identifying and synthesizing the DNA encoding the target peptide sequence, and inserting the sequence at the 3 end of a DNA segment encoding the chosen phage capsid protein under the control of a suitable promoter.
  • the DNA may be codon optimized for bacterial expression, but it is not mandatory.
  • the synthesized DNA sequence may be modified slightly to produce restriction sites at 5 and 3 end of the construct.
  • constructs are cloned into a plasmid vector (specifically designated as the “donor plasmid”) that also carries appropriate restriction sites for homologous recombination in the phage genome.
  • Restriction enzymes, vectors, “donor plasmids” and additional materials and methods appropriate for such steps are familiar to one of skill in the art and may be obtained from a variety of commercial vendors
  • Recombinase positive bacterial cells are used to infect phage. Typically, successful recombination in vivo is evidenced by the creation of antibiotic resistant colonies. The resulting recombinant bacterial colonies are immune to superinfection and carry the recombinant phage as lysogens. The recombinant colonies containing the phage lysogens is grown in appropriate bacterial broth, e.g., at 37°C for several hours. Recombinant phages will spontaneously induce in the cultures and result in complete lysis. The cell-free supernatant may then be use to infect appropriate host bacterial cells to obtain plaques. Phage obtained from single plaques may be further amplified and purified using various methods familiar to one of skill in the art to create immunogenic compositions comprising PVPs.
  • target antigen nucleotide sequences were synthesized (cDNA) and codon optimized for optimal expression on E. coli.
  • the synthesized DNA encoding the target antigen peptide sequences were modified slightly to produce Nhe I and Bssh II restriction sites at 5 and 3 end of the construct for insertion at the Nhel-Bsshll site of the 3 end of a DNA segment encoding gpD under the control of the lac promoter.
  • the constructs were then cloned in a plasmid vector (donor plasmid pUCDcDL3), which also carries loxPwt and loxPSl l sequences. Cre -expressing cells (E.
  • coli C600 were transformed with these recombinant plasmids, and subsequently used to infect recipient lambda phage (in house stock phage) that carries a stuffer DNA segment flanked by loxPwt and loxP511 sites. Recombination occurred in vivo at the lox sites, and ampicillin resistant (Amp 1 ) colonies formed. The resulting Amp 1 colonies are immune to superinfection and carry the recombinant lambda phage as plasmid cointegrates. The Amp r colonies containing the lambda cointegrate were grown in LB Amp at 37°C for four hours. Lambda phages spontaneously induced in the cultures and resulted in complete lysis.
  • phage capsids should be verified. This can be performed using conventional methods and commercially available materials familiar to one of skill in the art, e.g.. Western blots, ELISAs, plaque- lift assays, and/or immunoelectron microscopy.
  • Western blots e.g. Western blots, ELISAs, plaque- lift assays, and/or immunoelectron microscopy.
  • PVPs are plated on LB agar plates to obtain 100-150 plaques/plate. The plates are incubated at 37°C for approximately 18 hours, until the plaques are about one mm in size.
  • a 137 mm colony/plaque screen membrane (e.g., NEN® Research products, Boston, MA) is carefully placed on the top of the phage plaques.
  • the membrane is lifted and washed three times with Tris saline to remove debris.
  • the w'ashed membranes are then blocked with 2% casein solution for 1 hour.
  • the membranes are incubated in a casein solution containing the appropriate target antigen- specific mouse polyclonal antibodies. After incubation at room temperature for two hours, the membranes are washed three times in Tris saline with 0.05% Triton X-100 for 15 minutes each.
  • the polyclonal-treated membranes are incubated with 2.0 pg/ml of alkaline phosphatase labeled rabbit anti-mouse IgG for one hour at room temperature.
  • the membranes are washed three times as previously described above, followed by a final wash with 0.9% NaCl.
  • the membranes are treated with zinc chloride salt (e.g., Fast Red salt hemi; Sigma Cat# F8764, St. Louis, MO) and naphthol substrate solution for about 10 minutes and the reaction is stopped by washing the membrane in distilled water. Pink, immunoreactive spots correspond to recombinant phages that express the desired specific peptide/protein on the phage capsid.
  • zinc chloride salt e.g., Fast Red salt hemi; Sigma Cat# F8764, St. Louis, MO
  • reagents for use in detecting the expression of chimeric capsid proteins include but are not limited to rabbit or mouse polyclonal sera raised against recombinant expressed virus target antigens as well as phage capsid protein specific polyclonal antibodies.
  • Commercial vendors of polyclonal antibodies for uses contemplated herein include but are not limited to, BioVision (Milpitas, CA); RayBiotech (Peachtree Corners, GA); Genscript (Piscataway, NJ); Norms (Centennial, CO); Sino Biological (Wayne, PA) and SeraCareLife Sciences (Gaithersburg, MD).
  • phages for use with the methods of the instant invention include lambda phage as well as T4, T7, and M13/fl phages.
  • QB and MS2 are contemplated for use herein. See, e.g., Peabody DS et al., J Mol Biol. 2008; 380(l):252-63.
  • Phage for use in the methods of the instant invention are commercially available from a variety of vendors, e.g., ATCC (Manassas, VA) and Invitrogen (Carlsbad, CA) as are materials for propagating phage, e.g., T7 70014 T7Selecf Packaging Kit (Sigma-Aldrich, Rockville, MD).
  • phage selected for use are stable under storage, and can be propagated quickly and easily using conventional methods.
  • lambda phage may be propagated in E.coli to create PVPs of the instant invention.
  • the lambda phage used herein in house stock
  • the E. coli strain W3T10 may be propagated effectively in the E. coli strain W3T10.
  • T7, T4, M13/fl phage may be propagated on various strains of E.coli.
  • Phage for use in the instant invention ideally possess one or more capsid proteins that may be used to create chimeric, target antigen/capsid fusion proteins which express in relatively high copy number on the surface of the phage.
  • capsid proteins that may be used to create chimeric, target antigen/capsid fusion proteins which express in relatively high copy number on the surface of the phage.
  • “relatively high copy number” refers to a level of expression on the phage capsid that can successfully contribute to inducing a quantifiable immune response in a subject, e.g., gpD is typically expressed at around 420 copies per lambda phage particle.
  • capsid proteins for use as proposed herein include, e.g., gpV in lambda phage; gpIII, gpVI, gpVII, gpVIII and gpIX in Ml 3; HOC and SOC in T4 phage; plOA, plOB, p8, pi 1, pl7 or pl2 in T7 phage.
  • gpV in lambda phage
  • HOC and SOC in T4 phage
  • plOA plOB, p8, pi 1, pl7 or pl2 in T7 phage.
  • fusion proteins for capsid display typically use phage capsid proteins that are not critical for phage viability.
  • PVP compositions of the instant invention may be inactivated prior to administration to a subject in need thereof. Methods of inactivating phage are familiar to one of skill in the art.
  • PVP compositions of the instant invention maybe subjected to irradiation, including but not limited to non-ionizing radiation (UV-253.7 nm) as well as ionizing radiation (gamma radiation) prior to clinical use. See e.g., Sommer R. et al, Water Res. 2001 Sep; 35(13):3109-16. Doi: 10.1016/s0043-1354(01)00030-6.
  • Small-scale growth and purification of plaque purified recombinant bacteriophage of the instant invention may be performed using conventional methods familiar to one of skill in the art, e.g., plate lysate method and liquid lysate methods. The details of these procedures are well known in the art and are described, e.g., in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 4 th Edition (2001), the contents of which are incorporated by reference herein.
  • Methods for large-scale growth are also contemplated herein, including methods that provide optimal quality and purity for vaccine manufacture. Such methods are ideally (i) scalable to large-scale production levels (GLP/GMP), (ii) robust and efficient, (iii) capable of providing lot-to-lot consistency, (iv) commercially cost effective, and (v) will be FDA regulatory compliant.
  • GLP/GMP large-scale production levels
  • bacterial lysates can be treated with DNAse prior to purification to remove host cell nucleic acids, and reduce lysate solution viscosity and filter requirements as described in detail in Example 2.
  • LPS lipopolysaccharides
  • Example 4 a process for removing lipopolysaccharides (LPS) from phage vaccine lysates using organic solvent extraction is described in detail in Example 4. It is contemplated herein that these modified purification steps may be performed in order to reduce the contaminant load in the bacterial lysate before passing the PVPs through a further purification process comprising tangential flow filtration (TFF) described in detail in Example 3.
  • TMF tangential flow filtration
  • recombinant phage from validated seed stock may be amplified on a small scale in about 3 hours, and the resulting lysate then amplified on a larger scale in about three hours.
  • the lysate from the large-scale amplification may then be subjected to TFF for about three hours, and then subjected to cesium chloride density gradient purification in an ultracentrifuge (approx. 16 hours).
  • Gradient purified recombinant phage may then be subjected to dialysis (e.g., 3 exchanges of PBS at pH 7.4 for 4 hours), and then filtered a 0.22 micron filter.
  • large-scale production of PVPs may be performed in a 10 liter fermenter, e.g., as described in Example 5. Organic solvent purification as described in Example 4 may also be performed.
  • compositions comprising
  • compositions of the instant invention comprise PVPs displaying one or more coronavirus antigens, e.g., SARS-CoV- 2 antigens.
  • Compositions of the instant invention may also comprise fusion proteins obtained from PVPs (e.g., isolated and purified) as well as fusion proteins prepared with the aid of recombinant, enzymatic, or chemical techniques as discussed above.
  • compositions of the instant invention comprising PVPs displaying viral antigens may produce coronavirus-specific, e.g., SARS-CoV-2-specific protective immunogenic responses when injected in mammalian systems.
  • the compositions of the instant invention include but are not limited to, immunogenic compositions.
  • an "immunogenic composition” and like terms encompass compositions that may be administered to a subject in need thereof (e.g., human or animal) in order to enhance an immune response against a coronavirus.
  • an immunogenic composition comprises one or more antigenic epitopes, e.g., in the form of PVPs displaying a coronavirus antigen and/or antigenic subunits as a fusion protein or other peptide on the phage capsid.
  • antigenic composition will be understood to encompass compositions that are intended for administration to a subject or population of subjects for the purpose of eliciting a protective or palliative immune response against a coronavirus, e.g., SARS-CoV-2, MERS-CoV and SARS (e.g., vaccine compositions or vaccines).
  • animal challenge models may be used to evaluate the efficacy of various candidate immunogenic compositions and vaccines of the instant invention, including single subunit and multi-subunit antigen challenge studies.
  • animal challenge models are familiar to one of skill in the art.
  • ferrets may be used, e.g., as described in detail in Shi et al, Science 29 May 2020, Vol. 368, Issue 6494, pp.1016-1020.
  • Syrian hamsters may be used, e.g., as described in Brocato, R.
  • compositions of the instant invention may be subjected to one or more animal studies as well as clinical trials in humans such as described in Examples 13 and 14.
  • a “single component” immunogenic composition of the instant invention comprises PVPs expressing a single type of target antigenic sequence/epitopes.
  • “multi-component”, “multivalent”, “multisubunit” and like terms may be used interchangeably herein and refer to an immunogenic composition of the instant invention that comprises a combination of PVPs expressing two or more types of target antigenic sequence/epitopes.
  • the compositions of the instant invention are immunogenic compositions comprising PVPs displaying one or more SARS-CoV-2 antigens.
  • the invention relates to an immunogenic composition comprising phage displaying a chimeric capsid polypeptide comprising an epitope/antigenic sequence from the RBD of the spike protein found in SARS-CoV-2.
  • the chimeric capsid polypeptide is set forth as SEQ ID NO: 5 and is encoded by the nucleic acid construct NV101 -COVID-19 (“Construct 1”) set forth herein as SEQ ID NO: 6.
  • the invention in another embodiment, relates to an immunogenic composition
  • an immunogenic composition comprising phage displaying a chimeric capsid polypeptide comprising an epitope/antigenic sequence from the stalk of the spike protein found in SARS-CoV-2.
  • the chimeric capsid polypeptide is set forth as SEQ ID NO: 7 and is encoded by the nucleic acid construct NV102-COVID-19 (“Construct 2”) set forth herein as SEQ ID NO: 8.
  • an immunogenic composition of the instant invention may comprise a combination of PVPs some of which express Construct 1 and some of which express Construct 2.
  • antibodies may be elicited that promote opsonization and/or inhibit the virus life cycle by blocking receptor binding and/or blocking conformational changes in spike that are triggered by receptor binding, therefore blocking entry of free-floating virus into host cells, and also by reducing the formation of syncytia (giant cells), therefore inhibiting cell-to-cell spread.
  • antibodies that may be generated in a subject and provide a clinical benefit include but are not limited to neutralizing antibodies.
  • Various embodiments of the disclosed immunogenic compositions and vaccines also include, e.g., use of inactivated PVPs, as well as the use of soluble spike protein, use of vims like particles expressing whole or partial S protein; and using chemical conjugation of partial or complete spike or other structural proteins of SARS- CoV -2 on any phage surface.
  • Immunogenic compositions of the instant invention also comprise pharmaceutical compositions comprising one or more pharmaceutically acceptable excipients, carriers, diluents, and/or adjuvants suitable for administration to a subject in need thereof.
  • pharmaceutical compositions may further comprise one or more other active pharmaceutical ingredients (APIs).
  • APIs active pharmaceutical ingredients
  • a pharmaceutical composition of the instant invention is a coronavirus vaccine formulation, e.g., a SARS-CoV-2 vaccine formulation.
  • a vaccine formulation of the instant invention is a multivalent formulation.
  • the vaccine formulation is a multivalent formulation against two or more coronaviruses, including but not limited to, any two or more viruses of the Coronaviridae family of viruses e.g., SARS-CoV-2, MERS-CoV and SARS.
  • a pharmaceutical composition of the instant invention may not only comprise PVPs of the instant invention but also comprise one or more DNA constructs (e.g., DNA plasmids) encoding one or more anti-viral antigen polypeptides or fragments thereof, in combination with one or more pharmaceutically acceptable excipients, carriers, or diluents.
  • DNA constructs include DNA vaccines discussed below.
  • “Pharmaceutical compositions” are familiar to one of skill in the art and typically comprise active pharmaceutical ingredients formulated in combination with inactive ingredients selected from a variety of conventional pharmaceutically acceptable excipients, carriers, buffers, diluents, etc. One of skill in the art will also appreciate that not all pharmaceutical compositions are immunogenic compositions.
  • pharmaceutically acceptable is understood herein to refer to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism.
  • a biological system such as a cell, cell culture, tissue, or organism.
  • pharmaceutically acceptable excipients, carriers and diluents are familiar to one of skill in the art and can be found, e.g., in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA 5 th Edition (1975).
  • pharmaceutically acceptable excipients include, but are not limited to, wetting or emulsifying agents, pH buffering substances, binders, stabilizers, preservatives, bulking agents, adsorbents, disinfectants, detergents, sugar alcohols, gelling or viscosity enhancing additives, flavoring agents, and colors.
  • Pharmaceutically acceptable carriers include macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, trehalose, lipid aggregates (such as oil droplets or liposomes), and inactive virus particles.
  • Pharmaceutically acceptable diluents include, but are not limited to, water, saline, and glycerol.
  • the type and amount of pharmaceutically acceptable components included in the pharmaceutical compositions of the instant invention may vary, e.g., depending upon the desired route of administration and desired physical state, solubility, stability, and rate of in vivo release of the composition.
  • a vaccine formulation is typically in the form of a pyrogen-free, parenterally acceptable aqueous solution of suitable pH and stability, and may contain an isotonic vehicle as well as pharmaceutical acceptable stabilizers, preservatives, buffers, antioxidants, or other additives familiar to one of skill in the art.
  • isotonic properties of Ringer’s solution make a suitable buffer for phage compositions while “SM buffer” is typically used by one of skill in the art for phage dilution and storage.
  • SM buffer typically used by one of skill in the art for phage dilution and storage.
  • host bacterial components from the phage preparations that may have deleterious effects on the host, which include but are not limited to LPS, peptidoglycan, bacterial toxins, and bacterial DNA.
  • Therapeutic phage preparations can be designed to contain these kinds of materials in amounts below acceptable limits.
  • the formulations and compositions of the instant invention may further comprise one or more non-immunogenic components, e.g., one or more pharmaceutically acceptable excipients, carriers, diluents, stabilizers, preservatives, buffers, and disinfectants as discussed above.
  • one or more pharmaceutically acceptable excipients e.g., one or more pharmaceutically acceptable excipients, carriers, diluents, stabilizers, preservatives, buffers, and disinfectants as discussed above.
  • one of skill in the art will appreciate that the development of a robust and stable vaccine formulation will ideally employ various excipients and formulation parameters that will provide stability to the antigen and thus prevent aggregation, loss of protein structure, and/or chemical degradation such as oxidation and deamidation.
  • compositions and compositions of the instant invention may also comprise pharmaceutically acceptable substances which can produce and/or further enhance an immune response in a subject. These substances include, but are not limited to, adjuvants familiar to one of skill in th e art.
  • an adjuvant is a substance that aids a subject’s immune response to an antigen.
  • An adjuvant can be used to increase the immunogenic efficacy of a vaccine, and may also have the ability to increase the stability of a vaccine formulation, i.e., adjuvants are agents that enhance the production of an antigen-specific immune response as compared to administration of the antigen in the absence of the agent.
  • adjuvants are agents that enhance the production of an antigen-specific immune response as compared to administration of the antigen in the absence of the agent.
  • an “effective amount” of an adjuvant is that amount which is capable of producing an enhanced immune response as described above.
  • Adjuvants suitable for use with the compositions and vaccines of the instant invention are familiar to one of skill in the art and are available from a variety of commercial vendors. These include, for example, glycolipids; chemokines; compounds that induce the production of cytokines and chemokines; interferons; inert carriers, such as alum, bentonite, latex, and acrylic particles; pluronic block polymers; depot formers; surface active materials, such as saponin, lysolecithin, retinal, liposomes, and pluronic polymer formulations; macrophage stimulators, such as bacterial lipopolysaccharide; alternate pathway complement activators, such as insulin, zymosan, endotoxin, and levamisole; non-ionic surfactants; poly(oxyethylene)-poly(oxypropylene) tri-block copolymers; trehalose dimycolate (TDM); cell wall skeleton (CWS); complete Freund's adjuvant; incomplete Freund's
  • PCPP Poly[di(carboxylatophenoxy)phosphazene] (PCPP), monophosphoryl lipid A, QS-21, cholera toxin and formyl methionyl peptide.
  • the adjuvant may be selected from the group consisting of antigen delivery systems (e.g. aluminum compounds or liposomes), immunopotenti ators (e.g. toll-like receptor ligands), or a combination thereof (e.g., AS01 or AS04.) These substances are familiar to one of skill in the art.
  • an adjuvant for use in the compositions and methods of the instant invention is selected from the group consisting of toll-like receptor ligands, aluminum phosphate, aluminum hydroxide, monophosphoryl lipid A, liposomes, and derivatives and combinations thereof. See, e.g., Alving, C.
  • the present invention is directed to methods of preventing, ameliorating or treating disease caused by a coronavirus in a subject in need thereof comprising administering to the subject an immunogenic composition of the instant invention and inducing an immune response in said subject.
  • immunogenic compositions are administered to a subject to elicit an immune response that protects the subject against symptoms or conditions induced by a pathogen.
  • symptoms or disease caused by a pathogen are prevented (or treated, e.g., reduced or ameliorated) by inhibiting replication of the pathogen following exposure of the subject to the pathogen.
  • the immunogenic compositions disclosed herein are suitable for preventing, ameliorating and/or treating disease caused by infection with a virus from the Coronaviridae family of viruses e.g., SARS-CoV-2, MERS-CoV and SARS.
  • preventing, ameliorating or treating disease caused by a coronavirus encompasses, e.g., averting or hindering the onset or development of a pathological condition associated with a coronavirus infection, e.g., a SARS-CoV-2 infection, as well as treating, curing, retarding, and/or reducing the severity of one or more pathological conditions associated with coronavirus infection, including preventing COVID-19 and/or reducing the severity or duration of COVID-19.
  • compositions, vaccine formulations, and methods of the present invention encompass administration of the immunogenic compositions and vaccine formulations disclosed herein to generate immunity in a subject if later challenged by infection w ith a coronavirus. It is further understood herein, however, that the compositions, vaccine formulations, and methods of the present invention do not necessarily provide total immunity to a coronavirus (e.g., SARS-CoV-2) and/or totally cure or eliminate all disease symptoms.
  • a coronavirus e.g., SARS-CoV-2
  • an "immune response” is a response of a cell of the immune system, such as a B cell, T cell, or monocyte, to a stimulus.
  • An immune response can be a B cell response, which results in the production of specific antibodies, such as antigen specific neutralizing antibodies.
  • An immune response can also be a T cell response, such as a CD4+ response or a CD8+ response. In some cases, the response is specific for a particular antigen (that is, an "antigen-specific response").
  • a “protective immune response” is an immune response that inhibits a detrimental function or activity of a pathogen, reduces infection by a pathogen, or decreases symptoms (including death) that result from infection by the pathogen.
  • a protective immune response can be measured, for example, by the inhibition of viral replication or plaque formation in a plaque reduction assay or ELISA-neutralization assay, or by measuring resistance to pathogen challenge in vivo.
  • enhancing an immune response in a subject provides a meaningful clinical benefit to the subject.
  • Such benefit may be, e.g., preventing, ameliorating, treating, inhibiting, and/or reducing one of more pathological conditions associated with a viral infection, e.g., coronavirus infection, or related sequelae, in the subject.
  • the methods of the present invention can be considered therapeutic methods or preventative or prophylactic methods.
  • the immunogenic compositions of the instant invention may be administered to a subject and thus treat, prevent, and/or ameliorate a disease caused by one or more corona viruses.
  • both humoral and cellular immune responses may be enhanced in a subject by the methods and compositions of the instant invention.
  • antibodies may be elicited that promote opsonization and/or inhibit the virus life cycle by blocking receptor binding and/or blocking conformational changes in spike that are triggered by receptor binding, therefore blocking entry of free-floating virus into host cells, and also by reducing the formation of syncytia (giant cells), therefore inhibiting cell-to-cell spread.
  • antibodies that may be generated in a subject and provide a clinical benefit include but are not limited to neutralizing antibodies.
  • the methods and compositions of the instant invention may be employed in order to induce or enhance an immune response in a subject in need thereof and thus treat, prevent and/or ameliorate one or more pathological conditions associated with coronavirus in the subject.
  • the terms, “induce”, “enhance”, “immune enhancing”, “enhancement of immunity”, “modulator of immune responses to antigen” and like terms encompass any increase in immunity, and any measure of immunity, including enhancement of cellular and/or humoral immunity and/or by protective efficacy of an antigen, in a subject.
  • one or more immunogenic compositions of the instant invention may be administered to a subject alone, or in combination according to an appropriate dosage regimen determined by one of skill in the art.
  • an immunogenic composition comprising PVPs expressing fusion proteins encoded by either Construct 1 or Construct 2 may be administered to a subject in need thereof, i.e., as a single subunit vaccine.
  • PVPs e.g., PVPs expressing different antigens encoded by separate nucleic acid constructs
  • a subject in need thereof e.g., in the form of separate immunogenic compositions or in the form of a single immunogenic, multi-subunit composition.
  • co-administration of PVPs expressing Construct 1 and PVPs expressing Construct 2 as separate compositions and/or as a multi subunit vaccine is contemplated herein.
  • co-administration of different viral antigens may provide protection against COVID-19 by multiple mechanisms.
  • a multi-subunit vaccine that can be injected or otherwise administered (e.g., parenteral, subcutaneous, sublingual, intradermal, intramuscular, intraperitoneal or intravenous administration) to humans in order to induce production of antibodies against SARS-CoV-2, and therefore protect against infection or reduce severity of illness if infection still does occur, is contemplated herein.
  • a “subject”, a “subject in need thereof’ and like terms are interchangeable and includes humans as well as non-humans who would benefit from the methods and compositions of the instant invention.
  • the subject is a patient.
  • the term "patient” refers to any human being or animal that is to receive an immunogenic composition, e.g., a coronavirus vaccine, described herein.
  • the patient may already be infected with the virus or may be at risk of infection.
  • the coronavirus vaccine formulations for administration to a subject in need thereof as disclosed herein may be based on one or more strains or variants of coronavirus.
  • a coronavirus vaccine may be based on one or more vims strains selected for various serotype, and may be designed by a clinician in response to the needs of the subject.
  • vims can be naturally occurring or synthetic.
  • Various strains of coronavirus are familiar to one of skill in the art and can be found, for example, in various online viral genome databases which are familiar to and easily accessible by one of skill in the art.
  • an “effective amount”, “therapeutically effective amount”, “immunologically effective amount” and like terms refer to, e.g., the amount of an immunogenic composition, e.g., vaccine formulation, comprising one or more PVPs of the instant invention, alone or in combination in a composition (as the case may be), that produces a desired enhancement in immune response in a subject.
  • an immunogenic composition e.g., vaccine formulation, comprising one or more PVPs of the instant invention
  • the amount produces an enhancement in both cellular and humoral immune responses in the subject.
  • an effective dosage form may be in the form of a transdermal patch (e.g., 3M 1 ml patch) delivering lml of phage product (lxlO 10 pfu).
  • a transdermal patch e.g., 3M 1 ml patch
  • lml of phage product lxlO 10 pfu
  • patients may receive either a single dose, a 2-dose series (14-days apart), or a 3-dose series (14-days apart). Dosages may be with or without added adjuvant.
  • immunogenic compositions of the instant invention may comprise equal or varying amounts of the different expression products; the exact proportions of different antigenic peptides may be adjusted by one of skill in the art.
  • compositions and vaccines disclosed herein may be administered to a subject alone or in combination with other vaccines, and/or in combination with one or more other APIs.
  • Such other vaccines and APIs include, e.g., other active therapeutic or immunoregulatory agents which can enhance a subject’s immune response to a coronavims, or other virus from the family Coronaviridae.
  • additional vaccines and active agents may be administered to a subject in any manner, e.g., before, after, or concurrently with one or more immunogenic compositions of the instant invention comprising PVPs, and '' or nucleic acid encoding targeted antigenic coronavims polypeptides.
  • the immunogenic compositions of the instant invention may be co-administered with a DNA vaccine.
  • DNA vaccines are familiar to one of skill in the art and comprise administering genetic material encoding a particular antigen of interest, e.g., in the form of plasmid DNA comprising nucleic acid encoding the antigen, into a subject. Expression of the genetic material in the subject can then trigger an immune response against the expressed antigen in the subject.
  • one or more viral antigens for use in the methods and compositions of the instant invention may be administered to a subject to induce an immune response, not only in the form of a recombinant protein displayed on the surface of a PVP, but also in the form of nucleic acid encoding the antigen, e.g., as a DNA plasmid, or otherwise conveyed by phage for expression in the subject.
  • phages not only expressing the immunizing antigen on its surface (e.g., fusion protein) in the phage display format, but also incorporating foreign antigen genes to be transduced into the host cell (e.g., APCs) for expression may be administered [00112]
  • DNA vaccines can comprise an optimized gene sequence of interest cloned into a bacterial plasmid.
  • the amount of antigen produced in vivo after DNA inoculation is in the picogram to nanogram range. Since small amounts of protein are synthesized, an effective amount for humans may be determined through experimentation and dosing trials without undue experimentation.
  • nucleic acid vaccines may be administered between 1 mg and 5 mg per dose.
  • an immunogenic composition of the instant invention may be administered to a subject according to a variety of conventional methods. These methods include but not limited to, parenteral (e.g., by intracistemal injection and infusion techniques), intradermal, transmembranal, transdermal (including topical), intramuscular, intraperitoneal, intravenous, intra-arterial, intralesional, subcutaneous, oral, and intranasal (e.g., inhalation) routes of administration. Administration can also be by continuous infusion or bolus injection.
  • the immunogenic compositions of the instant invention can be administered in a variety of dosage forms. These include, e.g., liquid preparations and suspensions, including preparations for parenteral, subcutaneous, intradermal, intramuscular, intraperitoneal or intravenous administration (e.g., injectable administration), such as sterile isotonic aqueous solutions, suspensions, emulsions or viscous compositions that may be buffered to a selected pH.
  • injectable administration such as sterile isotonic aqueous solutions, suspensions, emulsions or viscous compositions that may be buffered to a selected pH.
  • the immunogenic compositions of the instant invention are administered to a subject as an injectable, including but not limited to injectable compositions for delivery by intramuscular, intravenous, subcutaneous, or transdermal injection.
  • intradermal delivery using microneedles, e.g., engineered in the form of small patches is also contemplated herein.
  • compositions of the instant invention may be administered orally.
  • Oral formulations for administration according to the methods of the present invention may include a variety of dosage forms, e.g., solutions, powders, suspensions, tablets, pills, capsules, caplets, sustained release formulations, or preparations which are time-released or which have a liquid filling, e.g., gelatin covered liquid, whereby the gelatin is dissolved in the stomach for delivery to the gut.
  • Such formulations may include a variety of pharmaceutically acceptable excipients described herein, including but not limited to mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate.
  • a composition for oral administration may be a liquid formulation.
  • Such formulations may comprise a pharmaceutically acceptable thickening agent which can create a composition with enhanced viscosity which facilitates mucosal delivery of the immunogen, e.g., by providing extended contact with the lining of the stomach.
  • Such viscous compositions may be made by one of skill in the art employing conventional methods and employing pharmaceutical excipients and reagents, e.g., methylcellulose, xanthan gum, carboxymethyl cellulose, hydroxypropyl cellulose, and carbomer.
  • immunogenic compositions of the instant invention may also be lyophilized and may be delivered to a subject with or without rehydration using conventional methods.
  • the viral vaccines and other pharmaceutical compositions disclosed herein may be formulated by one of skill in the art using a variety of pharmaceutical excipients, carriers, diluents, etc. familiar to one of skill in the art using art recognized methods.
  • Such vaccines and compositions may be administered to a subject alone, e.g., as individual dosage forms, or administered in combination in the form of an immunogenic composition comprising a coronavirus (e.g., SARS-CoV-2) vaccine formulation(s) comprising PVPs displaying one or more antigen polypeptides and/or comprising nucleic acid, e.g., for use as a DNA vaccine.
  • a coronavirus e.g., SARS-CoV-2
  • PVPs displaying one or more antigen polypeptides and/or comprising nucleic acid, e.g., for use as a DNA vaccine.
  • the methods of the instant invention comprise administering the immunogenic compositions of the instant invention to a subject according to various regimens familiar to one of skill in the art, i.e., in an amount and in a manner and for a time sufficient to provide a clinically meaningful benefit to the subject.
  • a subject i.e., in an amount and in a manner and for a time sufficient to provide a clinically meaningful benefit to the subject.
  • different variables e.g., choice of antigen, delivery route, dose, adjuvant, and boosting regimen may affect the degree of immune response achieved in a subject.
  • Suitable administration regimens for use with the instant invention may be determined by one of skill in the art according to conventional methods.
  • an effective amount of an immunogenic composition of the instant invention may be administered to a subject as a single dose, a series of multiple doses administered over a period of days, or using a “prime boost” schedule comprising an initial single “priming” dose followed by a “boosting” dose thereafter, e.g., several weeks or even years later.
  • a “prime boost” schedule comprising an initial single “priming” dose followed by a “boosting” dose thereafter, e.g., several weeks or even years later.
  • Various types of “prime boost” schedules are familiar to one of skill in the art and may comprise administering the same antigen, e.g., using a “homologous prime-boost strategy” or using a “heterologous prime-boost” strategy using different antigens (different vaccines). See Kardani, K et al. Vaccine 2016 Jan 20; 34(4):413-423.
  • dose refers to physically discrete units suitable for administration to a subject, each dosage containing a predetermined quantity of a viral vaccine comprising PVPs displaying antigen polypeptide and/or nucleic acid sequences as active pharmaceutical ingredients calculated to produce a desired response on the immune system of the subject.
  • An immunogenic composition of the instant invention may be administered to a subject as contemplated herein prior to exposure to infection, or after infection.
  • the administrative regimen e.g., the quantity to be administered, the number of treatments, and effective amount per unit dose, etc. will depend on the judgment of the practitioner and are peculiar to each subject. Factors to be considered in this regard include physical and clinical state of the subject, route of administration, intended goal of treatment, as well as the potency, stability, and toxicity of the particular construct or composition.
  • a “boosting dose” may comprise the same dosage amount as the initial dosage, or a different dosage amount. Indeed, when a series of immunizations is administered in order to produce a desired immune response in the subject, one of skill in the art will appreciate that in that case, an “effective amount” may encompass more than one administered dosage amount.
  • the immunogenic compositions of the instant invention are preferably sterile and contain an amount of the viral vaccine formulation in a unit of weight or volume suitable for administration to a subject.
  • the volume of the composition administered to a subject will depend on the method of administration and is discernible by one of skill in the art.
  • the pharmaceutical compositions of the instant invention comprise an amount of phage (PVPs) in a unit of weight or volume suitable for administration to a subject.
  • PVPs phage
  • the volume administered typically may be between 0.1 and 1.0 ml, e.g., approximately 0.5 ml. In another embodiment, up to 10 ml may be delivered in conjunction with a saline IV.
  • kits comprising one or more compositions or reagents disclosed herein which may be provided to a user, e.g., a clinician treating a subject with a coronavirus infection.
  • a user e.g., a clinician treating a subject with a coronavirus infection.
  • kits of the instant invention may enhance the rate of immunizations against COVID-19 by facilitating clinical access, e.g., by bringing the vaccine to subjects in need of vaccination, including subjects in remote geographic locations.
  • kits of the instant invention may comprise, e.g., coronavirus vaccine formulations, e.g., a SARS-CoV-2 vaccine formulation comprising one or more PVPs of the instant invention displaying an antigenic viral peptide or fusion protein thereof, and/or comprising nucleic acid encoding antigenic viral peptides and/or fragments, of the instant invention, as well as isolated, purified, and/or synthetic or recombinant fusion proteins of the instant invention.
  • coronavirus vaccine formulations e.g., a SARS-CoV-2 vaccine formulation comprising one or more PVPs of the instant invention displaying an antigenic viral peptide or fusion protein thereof, and/or comprising nucleic acid encoding antigenic viral peptides and/or fragments, of the instant invention, as well as isolated, purified, and/or synthetic or recombinant fusion proteins of the instant invention.
  • the kit can optionally also contain one or more other therapeutic or immunoregulatory agents and/or one or more additional reagents or items for use therewith, e.g., buffers, diluents, etc. as well as instructions or other information describing and/or facilitating the administration of the kit contents.
  • additional reagents or items for use therewith e.g., buffers, diluents, etc. as well as instructions or other information describing and/or facilitating the administration of the kit contents.
  • kits of the instant invention may comprise various articles or medical devices made from a variety of pharmaceutically acceptable materials or reagents for facilitating treatment of a subject.
  • the kits may contain suitable delivery devices, e.g., syringes, inhalation devices, and the like, along with instructions for administering the compositions.
  • kits can contain separate doses of the immunogenic compositions and vaccine formulations for serial or sequential administration, or compositions comprising the active pharmaceutical ingredients in combination.
  • the kits can optionally contain instructions for storage, reconstitution (if applicable), and administration of any or all therapeutic agents included.
  • the kits can include a plurality of containers reflecting the number of administrations to be given to a subject. If the kit contains a first and second container, then a plurality of these can be present. Kits comprising pharmaceuticals or other agents or items for clinical use are familiar to one of skill in the art and typically comprise one or more packaging containers designed to safeguard the integrity and viability of the contents during transit and/or storage.
  • a kit may comprise reagents for performing one or more diagnostic methods disclosed hereinbelow.
  • a kit may comprise reagents for detecting antibodies that specifically bind a fusion protein expressed by Construct 1 and/or Construct 2.
  • the kit may comprise, in separate containers, one or more fusion proteins disclosed herein, and one or more detection reagents, e.g., a reagent that detects an antibody that specifically binds the fusion protein.
  • the fusion protein may be various forms, e.g., in an isolated, purified form and/or as displayed on the surface of a recombinant phage as a chimeric capsid protein (PVP).
  • nucleic acid sequences, constructs, recombinant bacteriophage (PVPs) and compositions thereof disclosed herein can also be used in various assays, e.g., as reagents for immune diagnostics.
  • PVPs recombinant bacteriophage
  • SARS-CoV-2 epitopes on their surface of the instant invention are not infectious to humans, they may be used as surrogates for SARS-CoV-2 in certain assays, thus allowing some research to be performed at Biosafety Level 2 (BSL-2), rather than BSL-3.
  • the invention relates to additional laboratory and pre-clinical uses of the constructs, fusion proteins, PVPs, and compositions of the instant invention.
  • Such uses include but are not limited to, use as diagnostic reagents for testing a biological sample, e.g., a sample of sera from humans or animals, for antibodies to SARS-CoV-2 spike (e.g. serving as antigen to which the antibodies bind).
  • a biological sample e.g., a sample of sera from humans or animals
  • SARS-CoV-2 spike e.g. serving as antigen to which the antibodies bind
  • the present invention also relates to methods of detecting antibodies that specifically bind the fusion proteins of the present invention.
  • kits form are useful in, for instance, detecting whether a subject has antibodies that specifically binds fusion proteins and/or a targeted antigenic sequence of the present invention, and diagnosing whether a subject may have an infection caused by a coronavirus, e.g., SARS-CoV-2.
  • a coronavirus e.g., SARS-CoV-2.
  • such diagnostic systems are in kit form.
  • the diagnostic methods contemplated herein may comprise contacting an antibody with a preparation that includes at least one fusion protein or targeted antigenic sequence of the present invention to result in a mixture.
  • the antibody is present in a biological sample, e.g., a blood (serum) sample.
  • the method further includes incubating the mixture under conditions to allow the antibody to specifically bind the fusion protein or targeted antigenic sequence to form a protein.antibody complex.
  • protein: antibody complex refers to the complex that results when an antibody specifically binds to a protein.
  • the preparation that includes the proteins present in a composition of the present invention may also include reagents, for instance a buffer, that provide conditions appropriate for the formation of the protein.antibody complex.
  • reagents for instance a buffer
  • the protein.antibody complex is then detected.
  • suitable incubation protocols may be discerned by one of skill in the art without undue experimentation.
  • the detection of antibodies is also known in the art and can include, for instance, immunofluorescence and peroxidase.
  • the methods for detecting the presence of antibodies that specifically bind to polypeptides of the present invention can be used in various conventional formats that have been used to detect antibodies, including radioimmunoassay and enzyme-linked immunosorbent assays familiar to one of skill in the art. Additional methods used in immunodiagnostic tests include, e.g., fluorescent or chemiluminescent immunoassays. Such assays are described, e.g., in US Pub. 20200408749, and reviewed in Darwish, I. A., Int. J Biomed Sci. 2006 Sep; 2(3): 217 — 235, the entire contents of which are incorporated by reference herein.
  • Such antibody assays may take various forms familiar to one of skill in the art. These include, e.g., assays in a plate format, on a blot, or in a liquid, bead-based or other “pull-down” type assay.
  • a COVID-19 diagnostic test may comprise culture plates coated with PVPs and/or expression products of Construct 1 and/or Construct 2 of the instant invention. Such coated culture plates could be used, e.g., in an ELISA-based diagnostic assay for the identification of COVID-19 antibodies from human convalescent serum.
  • such assay could comprise dispensing and incubating serial dilutions of human convalescent sera in coated 96 well microtiter plates, followed by incubating, washing, and detecting steps familiar to one of skill in the art.
  • such assay could comprise a two -hour incubation at room temperature, followed by subsequent washes with mild detergent, e.g., Triton X, for removing unbound serum antibodies. Bound COVID-19 specific antibodies can be detected, e.g., using HRP conjugated rabbit sera against human IgG.
  • Additional diagnostic assays contemplated herein include a phage neutralization assay such as provided in Example 12 below.
  • PVPs expressing a gpD-SARS-CoV-2 chimeric capsid protein may be able to pull antibodies against the specific SARS-CoV-2 antigen from the serum, and such binding may be detected by measuring inhibitory or reduced effects on phage infectivity in vitro.
  • Additional contemplated diagnostic uses of the constructs, fusion proteins, PVPs, and/or compositions of the instant invention include use as reagents in ACE-2 or other receptor binding assays, e.g., to identify viral entry inhibitors. Methods of performing such assays are familiar to one of skill in the art and materials for performing such methods are available from a variety of commercial vendors. In a particular embodiment, it is contemplated herein that such studies may comprise focusing on fusion proteins in SARS-CoV-2 that initiate “class I fusion” to host cell membranes in order to identify potential fusion-inhibitory peptides or other agents.
  • such studies may focus on viral and host cell membrane fusion by examining protein-protein binding and/or conformational changes that are mediated via protein binding in order to facilitate coronavirus-host membrane fusion, and screening for potential reagents for inhibiting fusion.
  • such studies may include, e.g., binding assays that comprise the use of fusion proteins of the instant invention and/or PVPs expressing NVlOl-COVID-19 and/or NV102-COVID-19 constructs as substrates to which various parts of viral receptor and/or co-receptor(s) could be tested for binding.
  • Additional diagnostic uses include use of the constructs, fusion proteins, PVPs, and/or compositions of the instant invention as positive controls for novel virus detection assays aimed at viral spike (S) protein.
  • the gpD-spike fusion proteins expressed by Construct 1 and Construct 2 could serve as positive controls for binding in an assay where spike is the analyte intended to be bound to either a column, or beads, or a blot.
  • the detection method could make use of an antibody that binds to spike RBD or HR2, or a construct based on either full or partial ACE-2 protein, or various other proteins or matrices that may bind spike protein in either human or environmental samples (e.g. waste water).
  • Such viral detection assays employing the constructs, fusion proteins, PVPs, and/or compositions of the instant invention can be performed using commercially available materials by one of skill in the art without undue experimentation. See also, e.g., methods reviewed in Sridhar, S. et al J Mol Diagn 2015, 17:230-241, the contents of which are incorporated by reference in their entirety herein. [00138] Similarly, it is also contemplated herein that the constructs, fusion proteins, PVPs, and compositions of the instant invention may be used as positive controls in diagnostic western immunoblots to identify spike protein-specific COVID-19 IgG from human convalescent serum.
  • the PVPs and/or peptides expressed by Construct 1 and Construct 2 may be used as marker antigens for COVID-19 positive serum.
  • diagnostic western immunoblots which employ PVPs and fusion polypeptides of the instant invention as reagents may be performed according to a protocol as provided in the below examples.
  • Example 1 Phage displayed, multi-subunit vaccine targeting two parts of the spike glycoprotein: the receptor binding domain (RBD) in SI and the heptad repeat 2 (HR2) domain within the stalk region of S2
  • RBD receptor binding domain
  • HR2 heptad repeat 2
  • NV101 -COVID-19 encodes a fusion protein comprising a 210 amino acid sequence of the receptor binding domain (RBD) in the SI subunit
  • NV102-COVID-19 encodes a fusion protein comprising a 163 amino acid sequence of the heptad repeat 2 (HR2) domain within the stalk region of the S2 subunit.
  • Lambda phage viral particles (PVPs) expressing one or both constructs may be administered to a subject to elicit antibodies to the head (RBD) and/or stalk of spike protein of SARS-CoV-2.
  • RBD head
  • SARS-CoV-2 spike protein of SARS-CoV-2.
  • a single subunit vaccine comprising either of the constructs may be administered, the focus of this example is on a multi-subunit vaccine. Indeed, a multi- subunit vaccine expressing these two different constructs would be expected to elicit protective antibodies to different antigens and thus block virus entry into host cells at two different stages of the infection process.
  • the antibodies might block host receptor binding and also prevent conformational changes that facilitate viral entry into host cells, while simultaneously promoting opsonization of virus particles.
  • SARS-CoV-2 utilizes class I fusion, as does Influenza virus.
  • Knowledge of the fusion process reveals functional targets for antibodies, akin to the use of peptide inhibitors (Xia et al Cell Research (2020) 30:343-355).
  • NVl0l-COVID-19 contains a 210 amino acid (AA) sequence chosen from the RBD of SARS-CoV-2 based on our nucleotide alignments and protein modeling results using PHYRE2. (Kelley LA et al. Nat Protoc. 2015 June; 10(6)845-848.) This is the region predicted to interact directly with ACE2, making antibodies to this region potentially very potent viral inhibitors because they would compete with ACE2 for binding to virus.
  • AA 210 amino acid
  • NV102-COVID-19 contains a 163 amino acid domain chosen from the stalk domain in S2. This region is the HR2 domain, which, upon binding to ACE2 by the RBD, and associated conformational changes in spike, would interact with HR1 to form a classical six-helix bundle, further refolding the spike and pulling viral and host cell membranes together for fusion and entry.
  • the nucleic acid (codon optimized cDNA) and amino acid sequences for this targeted viral antigen are provided below:
  • the selected domains are functionally relevant (e.g. blocking their function would negatively affect the virus life cycle) and immunogenic.
  • the RBD domain we have utilized to make construct NV101-COVID-19 (AA383-592) overlaps with domains 587-628 identified as a domain of interest via immunogenicity prediction using IEDB’s Immunobrowser tool with Response Factor score >0.3 (Grifoni A et al Cell Host & Microbe 2020; 27 671-680DOT. 10.1016/j.chom.2020.03.002).
  • IEDB Immunobrowser tool with Response Factor score >0.3
  • the constructs disclosed herein were designed based on the strategy to reduce the chances of immimopathology by reducing the number of S protein epitopes displayed to the human immune system.
  • the odds of a negative event are reduced.
  • the odds of vaccine escape arising due to genetic drift are reduced.
  • Construct 1 and Construct 2 employs the start codon from gpD gene and then, rather than a stop codon at the end of gpD, transcription continues right through to a stop codon at the end of the vaccine antigen target. Therefore, it is critical this gene fusion is in frame.
  • the resulting fusion protein expressed from the constructs of the instant invention and displayed on lambda phage is gpD with part of a SARS-CoV-2 protein attached to it.
  • SARS-CoV-2 spike protein specific antigenic peptides were selected for synthesis of two separate phage vaccine constructs.
  • the nucleic acid sequence were codon optimized for optimal expression in the phage bacterial host, E.coli strain W3110 using OPTIMIZER, as discussed above.
  • DNA sequences encoding the targeted antigenic SARS-CoV-2 sequences were synthesized (gBlock synthesis platform. Integrated DNA Technologies, Redwood City, CA.) The synthesized DNA fragments were PCR amplified to create restriction sites for in-frame cloning with the gpD sequence. Specifically, the oligo NT sequence of each PCR primer was modified slightly to produce Nhel and BssHII restriction sites at the 5 ’ and 3 ends of the amplified DNA segments.
  • Recombinant plasmid DNA of these clones were harvested using GeneJET Plasmid Miniprep Kit (Thermo Fisher Scientific, Waltham, MA) and subsequently restriction digested with appropriate enzymes. Restriction digested recombinant plasmid DNA were electrophoresed on 1% agar gel at constant volt of 80. After electrophoresis, gels were stained with ethidium bromide and visualized under ultraviolet (UV) light.
  • UV ultraviolet
  • Nhel and BssHII restriction digestion analysis of recombinant plasmid DNA extracted from NV101-COVID-19 clones indicate that six colonies had recombinant donor plasmid harboring the correct size insert (630bp) (data not shown.)
  • Recombinant plasmid DNA extracted from NV102-COVID-19 clones were analyzed by restriction digestion of EcoRl and HinDIII enzymes. Result indicate that four clones had recombinant donor plasmid containing the correct size insert (909bp) (data not shown.)
  • Cre-expressing cells E. coli
  • Cre-expressing cells were transformed with these recombinant donor plasmids and subsequently infected with a recipient lambda phage that carries a stuffer DNA segment flanked by loxPwt and loxP511 sites.
  • Lambda phage infected Cre- expressing E. coli were grown in Luria Bartani (LB) Ampicillin (100 ug/ml) at 37° C for four hours in presence of 0.2% maltose and 0.1M CaCl2 Recombination occurred in vivo at the lox sites and Amp 1 cointegrates were formed, which spontaneously lysed the E. coli thus releasing the phage into the culture media (data not shown.)
  • the cloning strategy is depicted in Figure 6.
  • Lambda cointegrates were used to produce lambda lysogens and were selected on LB ampicillin agar (15% agar) plates. Briefly, cointegrates from spontaneously lysed E. coli culture were used to infect Cre-ve, suppressor-ve E. coli cells and spread on LB ampicillin agar plates. Plates were incubated at 30° C for 48 hours to obtain Amp r colonies which were actually recombinant lambda lysogens and carry a recombinant lambda phage genome in their chromosome.
  • This cell free supernatant was used to infect E. coli cells and were plated on solid LB agar plate to obtain phage plaques.
  • the resulting phage plaques were harvested from the plate and single plaques were purified three times on E. coli by the standard procedures described by Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 4 th Edition (2001). Approximately 220 copies of COVID specific peptides are believed displayed on a single phage head, but the level of expression was not experimentally quantitated.
  • lambda cointegrates of NV101-COVID-19 and NV102-COVID- 19 were amplified using XbaI-5' and XbaI-3 primers to confirm the proper integration of recombinant donor plasmid in lambda genome. Integration of recombinant donor plasmid in lambda produces an insertion of new DNA segment in Xbal site of lambda genome.
  • PCR amplified products (2m1) were elecirophoresed on 1% agar gel at constant volt of SO. After electrophoresis gels were stained with ethidium bromide and visualized under ultraviolet (UV) light.
  • Phage DNA containing cointegrates were also subjected to complete genome sequencing and subsequent bioinformatics analysis using conventional methods to confirm the proper orientation of the SARS-CoV-2 antigen sequence in lambda genome as gpD fusion. Specifically, sequencing results verifed that there is one complete copy of the SARS-CoV-2 insert fused 5’ to 3’ direction directly downstream of the gpD and prior to stop codon (data not shown).
  • FIG. 7 Depicted in Figure 7 is a whole genome characterization of new recombinant virus NVlll-COVID-19, "SARS-CoV-2 Spike protein receptor binding domain fused to lambda gpD” illustrating the RBD insert was successfully integrated into lambda backbone.
  • Figure 8 depicted in Figure 8 is a whole genome characterization of new recombinant virus NV102-COVID-19, "HR2 domain fused to lambda gpD” illustrating the HR2 insert was successfully integrated into lambda backbone.
  • each recombinant lambda virus has the indicated antigen from SARS-CoV-2 fused to the lambda gpD protein, by nature of an in frame gene fusion. Specifically, all the genes of lambda will be expressed as full length gene products, whereas the sequences cloned in from SARS-CoV-2 will be expressed as partial protein sequences fused to full length lambda gpD protein.
  • recombinant virus expressing the NVl0l-COVID-19 construct has the receptor binding domain of SARS-CoV-2 fused to gpD
  • recombinant virus expressing NV102-COVID-19 construct has the HR2 domain of SARS-CoV-2 fused to gpD.
  • each construct also has two AAs “AR” at the 3’ end, which is generated via the Bsshll restriction enzyme site during cloning.
  • Genetic analysis has also detected an Arg -> His mutation in the S1 subunit (depicted as the enlarged histidine residue in Figure 5(A)).
  • lysate preparation procedure may be employed as an integral step for GMP grade phage vaccine preparation, including large- scale production and purification of COVID-19 phage vaccines disclosed herein.
  • the materials and equipment listed below are familiar to one of skill in the art and may be obtained from a variety of commercial vendors.
  • An enhanced PVP purification process comprising the use of TFF is provided herein and may be used to reduce the major contaminants in bacterial lysates such as media components, host cell protein, and nucleic acids as well as endotoxin (LPS). It is contemplated herein that the disclosed method can be used to create PVP preparations from bacterial culture lysates of sufficient purity and activity for pre-clinical studies. It is also contemplated that this method can be scaled up and used to create materials for clinical and commercial use. A prophetic example is provided below. The materials and equipment listed below are familiar to one of skill in the art and may be obtained from a variety of commercial vendors. As described below, a Millipore Cognet Ml TFF device is used for this filtration (Millipore Sigma, Burlinton, MA).
  • the pressure is controlled on input and output of the chamber.
  • Recirculation rate is set to 400 m1/min, feed pressure to 10 psig and permeate pressure to 5 psig (maintain TMP 2-4 psig).
  • a total 2L of lysate can be concentrated to 200ml following the above settings (retentate & permeate valves were adjusted to maintain TMP 2-4 psig).
  • the TFF procedure detailed above is based on screening different molecular weight cut-off membranes and chemistries and operational parameters to maximize contaminant removal and PVP recovery. If deemed necessary, further scale up and optimization of this process may be performed to meet clinical and commercial needs.
  • the purification process may comprise the following stages: (i) an initial clarification step, (ii) a tangential flow filtration (TFF) step, and (iii) a final formulation step to place the PVPs in the desired formulation buffer.
  • the process maintains the physico-chemical characteristics, quality, and potency of the PVP -based vaccines while yielding a preparation of sufficient purity.
  • Tangential flow filtered lysates can be achieved by concentrating 5 L of phage lysate down to 100 ml total volume. After concentration, media is replaced by dia-filtration against 4L of 0.5M Phosphate Buffered Saline (PBS).
  • PBS Phosphate Buffered Saline
  • An immimoblot assay was carried out to analyze the expression of the antigenic peptide sequence encoded by NV101-COVID-19 on the phage capsid.
  • phage were separately plated on LB agar plates to obtain 100 to 150 plaques on each plate. The plates were incubated at 37° C. for 18 hours, until the plaques were about one mm in size.
  • a 137 mm colony/plaque screen membrane (NEN® Research products) was soaked in distilled water and blotted dry on a filter paper. This membrane was carefully placed on the top agar and incubation was continued at 37° C for another 15 minutes.
  • the membrane was peeled from the agar, and washed three times with Tris saline to remove the debris and bacteria. The plates were then stored at 4° C. and the washed membranes were then blocked with 2% fat free dried milk-powder suspended in Tris saline solution for 1 hour.
  • the membranes were incubated in a dried milk-powder solution (milk powder suspended in Tris saline with 0.05% TRITOO-X 100) containing 1 :500 dilution of commercially available polyclonal antibodies raised against SARS spike protein (SARS-S protein polyclonal, Invitrogen (Carlsbad, CA); SARS-S protein polyclonal, Bioss Antibodies Inc (Woburn, MA)). After incubation at room temperature for two hours, the membranes were washed twice in Tris saline with 0.05% TRITON-X 100 and once in Tris saline for 15 minutes each.
  • a dried milk-powder solution milk powder suspended in Tris saline with 0.05% TRITOO-X 100
  • the polyclonal treated membranes were incubated with 1:000 dilutions of horseradish peroxidase labeled rabbit anti-goat IgG secondary antibody (Kirkegaard & Perry Laboratories, Inc. Gaithersburg, MD) for one hour at room temperature. The membranes were consecutively washed three times in the same way as before, followed by a final wash with 0.9% NaC1. Finally, the membranes were treated with TMB One Component HRP Membrane Substrate solution (Sigma, St. Louis, MO) for approximately 5 minutes and the reaction was then stopped by washing the membrane in distilled water. Purple immunoreactive spots that formed correspond to the recombinants expressing SARS-CoV-2 specific peptides (data not shown.)
  • results indicate that the SARS spike protein—specific polyclonal antibodies obtained from Bioss Antibodies Inc. did not bind with recombinant lambda plaques of NV1Ol-COVID-19 and non-recombinant lambda plaques, whereas Invitrogen polyclonal antibodies bind to both non-recombinant lambda plaques and recombinant lambda plaques of NV1Ol-COVID-19. These results indirectly indicate that Invitrogen polyclonal antibodies probably contribute nonspecific binding with some of the lambda phage specific protein.
  • Construct 1 encoding the fusion protein RBD fragment-gpD and Construct 2 encoding the fusion protein HR2 fragment-gpD were created as detailed in Example 1.
  • the predicted molecular weight of the fusion protein encoded by Construct 1 is 36.23 kDa (24.66 kDa viral RBD antigen peptide + 11.57 kDa of gpD).
  • the predicted molecular weight of the fusion protein encoded by Construct 2 is 30.90 kDa (19.33 kDa viral HR2 antigen peptide + 11.57 kDa of gpD).
  • Primary antibodies Commercial rabbit antibodies (Sino Biological, Wayne, PA; Cat # 40591-T62 and 40590-T62).
  • Towbin transfer buffer (BioRad, Hercules, CA)
  • TBS Tris-buffered saline
  • TBS-T TBS + 0.05% Triton X-100
  • TMB substrate (SeraCare KPL, ref# 5420-0028)
  • BioRad PowerPac High-Current Electrophoresis Power Supply BioRad, Hercules, CA
  • Boil samples at 95°C for 10m followed by centrifugation at 13k xg for Irn Place XCell SureLock Mini-Gel apparatus into a plastic pan of ice. Cut open packaging for 4-20% tris-glycine gel and rinse gel cassette and packaging with deionized water. Place gel into apparatus- if only running 1 gel, put the plastic spacer in the other gel slot and clamp apparatus closed. Add SDS running buffer to fill the center chamber and 1/3 volume of the outer chamber in the gel apparatus. Gently remove the comb from the wells- gently pipette up and down in each well to wash them. Load 10ul of PageRuler prestained ladder into 1 well of the gel. Load the other samples into the gel.
  • Towbin Transfer Buffer pH 8.3:25mM tris base, 192mM glycine, 20% methanol.
  • TBS Tris-buffered saline
  • TBS- Triton TBS with 0.05% Triton X- 100.
  • Example 8 Prophetic Mouse Assay to Assess Safety, Reactogenicity, and Immunogenicity of SARS-CoV-2 Vaccine Formulations:
  • compositions comprising PVPs of the instant invention may be assessed for safety, reactogenicity, and immunogenicity in various animal models.
  • a prophetic mouse assay is provided below. The materials and equipment listed below are familiar to one of skill in the art and may be obtained from a variety of commercial vendors.
  • group A three separate groups (group A, group B, and group C), of male BALB/c mice (body wt.18 to 25 g), will be immunized via intramuscular route (i/m) with PVPs expressing a SARS-CoV-2 vaccine construct disclosed herein, e.g., NV101- COVID-19 and/or NV102-COVID-19.
  • Their serum, lungs, spleens and lymph nodes will be collected for additional immunological and histopathological analysis.
  • 50ul of blood will be collected at 7 hours post challenge from each group of mice. The blood will be clotted for collection of serum.
  • mice sera will be pooled and analyzed to determine the level of various cytokines.
  • TNF-a, IL-la, IL-6, and IFN-g in mouse sera may be measured using a commercial available sandwich ELISA using cytokine-specific antibodies according to the manufacturer’s instructions.
  • Chimeric protein specific and lambda capsid gpD specific antibody titer of mouse sera before and after vaccinations will be monitored by ELISA and western blot using conventional methods to evaluate the humoral immune response against various components of the PVP construct. Histopathology of various organs may also be analyzed to determine the extent of inflammatory tissue damage. This will be especially important to evaluate any cytotoxic effect of phage vaccines which will be vaccinated but not challenged. See Figure 9 for a depiction of this study.
  • Example 9 Analysis of immune response in a live host upon immunization using the lambda phage-display vaccine candidate
  • PVPs expressing the capsid protein comprising the 163 amino acid sequence of the HR2 domain fused to gpD encoded by Construct 2 (NV 102-C OVID- 19) compared to the respective lambda control.
  • mice Female C57BL/6 mice of 6-8-week old were immunized intramuscularly and immunized intranasally (6 mice each). Physical observations were conducted daily, and body weights were measured before administration, 2 days after administration, and then once a week until study termination. Animals underwent blood collection for serum processing at 4 timepoints- pre- immunization (i.e.
  • Materials for administration included two formulations of PVPs expressing Construct 2, inactivated by radiation: 1x10 8 /100ul and lxl0 8 /25ul (dose/volume); and two formulations of inactivated control lambda phage, Ixl0 8 /100ul and lxl0 8 /25ul (dose/volume).
  • Female mice species Mas muscahis strain C57BL/6, 6-8 weeks old w'ere chosen for study.
  • Test articles were stored at -80°C until dosing. Frozen TAs were opaque in color and solid in form. TAs were thawed prior to dispensing and dosing by placing TA vials into a decontaminated biological safety cabinet to thaw' at room temperature for no longer than 30 minutes. Thawed TA w'ere completely transparent and contained no visible solids. Once the TA was thawed, the vial was vortexed for 10 seconds (but not shaken) prior to dispensing.
  • mice were housed in individually ventilated cages.
  • Female C57BL/6 mice of 6-8-week old upon study initiation were selected for use in the study.
  • a control groups was also present in the study.
  • the mice were immunized intramuscularly (IM) and immunized intranasally (IN). With regard to the latter, 25ul is considered the optimal dose to be administered 12.5ul per nostril (25ul total).
  • test formulations were thawed fully prior to administration as described above.
  • the formulations were dosed at 1x10 8 particles on Study Days 0 and 28 by intramuscular (IM) lOOuL injection or intranasal administration (IN) 12.5ul per nostril (25ul total).
  • IM intramuscular
  • IN intranasal administration
  • Serum was obtained from the whole blood placed in the tubes by centrifugation and stored in cryovials at -20°C prior to shipment on dry ice for further analysis by VisMederi (Siena, Italy). On day 38 spleens were collected and processed to splenocytes and stored at -80°C for two days after which they were stored in liquid nitrogen.
  • a goal of this study is to assess the presence of neutralizing antibodies and Thl/Th2 immunogenicity profile of C57BL/6 mice immunized with SARS-CoV-2 bacteriophage vaccines in order to address the FDA's concern about Th2 skewing that may lead to VAERD.
  • T cell cytokine profile (IL-4, IL- 6, IFN-y and TNF-a) will also be evaluated by ELISpot and Intracellular staining (ICS) assays according to VisMederi SOP: "ELISPOT ASSAY USING IMMUNOSPOT COMMERCIAL KIT (SOP-ELD)" and “CELL STIMULATION AND STAINING FOR FACS ANALYSIS OF SURFACE AND INTRACELLULAR MOLECULES (SOP-CSSFA)", respectively.
  • ICS Intracellular staining
  • Immune endpoints will be (i) neutralizing antibodies measured by virus neutralization assays, (ii) IgM, IgG 1 and IgG2 measured by ELISA assay, (iii) IFN-y and IL-4 production by splenocytes measured by ELISpot assay and (iv) IFN-y, TNF-a, IL-6 and IL-4 cytokine production in CD4 and CD8 T cells by intracellular cytokine staining (ICS) and flow cytometry.
  • ICS intracellular cytokine staining
  • Vis Mederi will use proprietary SOPs to test murine sera for the presence of neutralizing antibodies in a neutralization assay.
  • murine sera will be assessed for the presence of IgM and IgG 1 and IgG2 recognition of SARS-CoV-2 Spike Protein and Nuclear Protein following VisMederi SOPs for ELISAs with isotyping.
  • ELISpot assays will also be performed by VisMederi to detect frequencies of IFN-y- and IL-4-producing splenocytes stimulated by vaccine- matched SARS-CoV-2 antigens (peptides/proteins) following VisMederi's SOP.
  • Intracellular cytokine staining (ICS)/flow cytometry Thl and Th2 cell frequencies will be determined by ICS and flow cytometry following stimulation of splenocytes with vaccine-matched SARS-CoV-2 antigens.
  • Cells will be stained for surface lineage markers (CD3, CD4 andCD8) and intracellular cytokines (IFN-gamma, TNF-a, IL-4, and IL-6).
  • the data will be analyzed and the amount of CD4+ or CD8+ cells positive for each cytokine investigated gated on live/singlet/ lymphocyte/CD3+ events will be enumerated. Till cells will be defined as IFN-gamma-producing CD4 T cells and Th2 as IL-4-producing CD4 T cells.
  • the number ofCD4+ and CD8+ cells positive for each marker will be evaluated versus the "Live" cell population. See below
  • the formulations to be tested may comprise PVPs expressing either Construct 1 or Construct 2 or PVPs expressing both, administered in combination or administered in a heterologous prime-boost strategy.
  • the volume of the dose administered may change, depending on the species, size and/or weight of the animals to be tested.
  • Example 10 Evaluation of Expression of SARS-CoV-2 antigens in Bacteriophages by Liquid Chromatography - Tandem Mass Spectrometry (LC/MS/MS) analysis
  • Mass spectrometry system Q-Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer, Dionex Ultimate 3000 RSLCnano system, Proteome Discoverer 1.4 software (ThermoFisher Scientific, Waltham, MA)
  • PicoFrit Column 360 um OD/75 um ID, 15um tip ID, packed with 5um ProteoPep II C18 300A, 20 cm (New' Objective, Inc. Woburn, MA)
  • Nanospray LC/MS/MS analysis and database search The LC/MS/MS analysis was carried out using a Thermo Scientific Q-Exactive hybrid Quadrupole- Orbitrap Mass Spectrometer and a Thermo Dionex UltiMate 3000 RSLCnano System. TWO 12 m ⁇ of each digestion (analyzed in duplicates) was loaded onto a peptide trap cartridge at a flow' rate of 5 pL/min. The trapped peptides w'ere eluted onto a reversed- phase 20 cm Cl 8 PicoFrit column (New' Objective, Woburn, MA) using a linear gradient of acetonitrile (3-36%) in 0.1% formic acid.
  • the elution duration was 110 min at a flow rate of 0.3 ⁇ L/min.
  • Eluted peptides from the PicoFrit column w'as ionized and sprayed into the mass spectrometer, using a Nanospray Flex Ion Source ES071 (Thermo) under the following settings: spray voltage, 1.8 kV, Capillary temperature, 250°C.
  • the Q Exactive instrument was operated in the data dependent mode to automatically switch between full scan MS and MS/MS acquisition.
  • Survey full scan MS spectra (m/z 300-1800) was acquired in the Orbitrap with 70,000 resolutions (m/z 200) after accumulation of ions to a 1 x 106 target value based on predictive automatic gain control (AGC).
  • AGC predictive automatic gain control
  • Dynamic exclusion was set to 20 s.
  • the 15 most intense multiply charged ions (z > 2) were sequentially isolated and fragmented in the octopole collision cell by higher-energy collisional dissociation (HCD) using normalized HCD collision energy 28% with an AGC target 1x105 and a maxima injection time of 100 ms at 17,500 resolutions.
  • HCD collisional dissociation
  • MS Raw data files were searched against a protein sequences database including all reviewed protein sequences obtained from UniprotKB website (https://wwvMiniprot.org) and Construct 1 and Construct 2 sequences using the Proteome Discoverer 2.4 software (Thermo, San Jose, CA) based on the SEQUEST and percolator algorithms.
  • the false positive discovery rates (FDR) was set on 1%.
  • the resulting Proteome Discoverer Report contains all assembled proteins with peptides sequences and peptide spectrum match counts (PSM#). The relative abundance of proteins identified was estimated based on MS1 peak area. The calculation and data analysis used Microsoft Excel functions.
  • Detection of Expression of SARS-CoV-2 antigens A set of 6 samples were analyzed in this assay. First, the protein content was measured by using Pierce BCA kit. Results are summarized in Table 3. The samples (180 ⁇ 1) were then processed for trypsin digestion followed by LC/MS/MS analysis. The results are summarized in Table 3. Three gpD-SARS-CoV-2 antigen fusion proteins were detected in three groups of six samples respectively. The gpD-CoV-2 antigens are estimated to be 2.5% -3.1% of total phage proteins in the sample tested. The expression of phage lambda capsid decoration protein D only was not detected in all six samples.
  • Fusion protein expression on PVPs of the instant invention may be examined using immunoelectron microscopy.
  • a prophetic example to fix high concentration bacteriophage samples e.g., TFF purified as per Example 3 with paraformaldehyde and glutaraldehyde for subsequent immuno-transmission electron microscopy is provided below.
  • the materials and equipment listed below are familiar to one of skill in the art and may be obtained from a variety of commercial vendors.
  • PBS Phosphate Buffered Saline
  • SARS-CoV-2 (2019 nCoV) Spike, rabbit pAb (SinoBiological, cat# 40591-T62, Chesterbrook, PA)
  • SARS-CoV-2 (2019-nCoV) Spike S2, rabbit pAb (SinoBiological, cat# 40590-T62, Chesterbrook, PA)
  • Bovine Serum Albumin (Fraction V) 1.00 gram
  • Samples are floated on a drop (sample side down) of primary antiserum that has been diluted in BTT (for a minimum of 30 minutes). Samples are floated on drops of BTT (sample side down) to wash away unbound antiserum (5 minutes each for two drops). Samples are floated on a drop (sample side down) of nano gold-labelled secondary antibody that has been diluted in BTT (for a minimum of 30 minutes). 24. Samples are floated on drops of BTT (sample side down) to wash away unbound antiserum (5 minutes each for twO drops).
  • Samples are floated on a drop of purified water (sample side down) for 30 seconds.
  • a sample of plasma e.g., convalescent sera
  • PVPs expressing a gpD-SARS-CoV-2 chimeric capsid protein may pull antibodies against the specific SARS-CoV-2 antigen from the serum, and such binding will be detected by measuring effects on phage infectivity in vitro.
  • the materials and equipment listed below are familiar to one of skill in the art and may be obtained from a variety of commercial vendors.
  • Serum samples to be tested e.g., COVID-19 convalescent serum
  • phage Bacteriophage (phage) stock above 10 s pfu/ml Host bacteria strain Tryptic soy broth (TSB)
  • PBS Phosphate buffered saline
  • the Investigational Product is contemplated as a transdermal patch (3M, Flemington, New Jersey) comprising at least 1 ml of phage at a concentration of lxl0 10 pfu.
  • Study Design This is a Phase I open-label dose ranging study to evaluate the safety, tolerability, and immunogenicity of PVPs expressing Construct 1.
  • This phage display based vaccine utilizes the lambda phage in which upon codon optimization of the targeted antigenic sequence of the RBD within the E coli vector was cloned into the lambda phage, and expressed as a chimeric capsid protein fused with the gpD protein on the lambda phage capsid.
  • Our hypothesis presumes that antibody binding to the aforementioned viral epitope will block host receptor binding, prevent conformational changes for entry and promote opsonization.
  • Immunogenicity assessment Blood will be obtained for antibody and T-cell responses at baseline and then at weeks 1 and 2, 4, 8, and 12 weeks following each vaccination; and at 3, 6, and 12 months (as applicable for each cohort) following the final vaccination. Serum will be separated and analyzed for humoral responses (neutralizing and binding antibody titers. Whole blood will be processed to obtain peripheral blood mononuclear cells (PBMCs) for determination of cell mediated immune responses (CD4 and CD8 T-cell responsiveness to the RBD peptide of SARS-CoV-2).
  • PBMCs peripheral blood mononuclear cells
  • Inclusion Criteria a. Age 18-50 years; military, civilian, male and female; b. Able to provide consent to participate and having signed an Informed Consent Form (IGF); c. Able and willing to comply w ith all study procedures; d. Women of childbearing potential agree to either remain sexually abstinent, use medically effective contraception (oral contraception, barrier methods, spermicide, etc.) or have a partner who is sterile from enrollment to 3 months following the last administration, or have a partner who is unable to induce pregnancy; e.
  • IGF Informed Consent Form
  • Sexually active men who are considered sexually fertile must agree to use either a barrier method of contraception during the study, and agree to continue the use for at least 3 months following the last administration, or have a partner who is permanently sterile or unable to become pregnant. All information about the volunteer’s medical history and that of his or her sexual partner wall be based on self-report. Only the female volunteer’s current pregnancy status will be verified with either a urine or serum pregnancy test; f. Normal screening ECG or screening ECG with no clinically significant findings; g. Screening laboratory must be within normal limits or have Grade 0-1 findings; h. No history of clinically significant immunosuppressive or autoimmune disease; i.
  • immunosuppressive agents excluding inhaled, topical skin and/or eye drop-containing corticosteroids, low-dose methotrexate, or corticosteroids at a dose less than 20 mg/day
  • j Willing to allow storage and future use of samples for 2019nCoV related research.
  • Exclusion Criteria a. Administration of an investigational compound either currently or within 30 days of first dose; b. Previous receipt of an investigational product for the treatment or prevention of COVID-19 except if participant is verified to have received placebo; c. Previous infection with COVID-19; as assessed by self-report and solicited exposure history; d. Positive results on the “current” optimized COVID-19 diagnostic (currently under the EUA as a SARS-CoV-2 Real-Time RT-PCR Diagnostic Panel); e. Administration of any vaccine within 4 weeks of first dose; f. A BMI greater than or equal to 35; g. Administration of any monoclonal or polyclonal antibody product w ithin 4 weeks of the first dose; h.
  • Immunosuppressive illness including hematologic malignancy, history of solid organ or bone marrow transplantation; p. Current or anticipated concomitant immunosuppressive therapy (excluding inhaled, topical skin and/or eye drop-containing corticosteroids, low-dose methotrexate, or corticosteroids at a dose less than 20 mg/day); q. Current or anticipated treatment with TNF-a inhibitors, e.g. infliximab, adalimumab, etanercept; r. Prior major surgery or any radiation therapy within 4 weeks of group assigmnent; s. Any pre-excitation syndromes, e.g., Wolff-Parkinson- White syndrome; t.
  • TNF-a inhibitors e.g. infliximab, adalimumab, etanercept
  • Presence of a cardiac pacemaker or automatic implantable cardioverter defibrillator (AICD); u. Current prisoner or participants who are compulsorily detained (involuntary incarceration) for treatment of either a physical or psychiatric illness; v. Active drug or alcohol use or dependence that, in the opinion of the investigator, would interfere with adherence to study requirements or assessment of immunologic endpoints; or w. Any illness or condition that in the opinion of the investigator may affect the safety of the participant or the evaluation of any study endpoint. x. Extensive tattoos preventing interpretation of adverse reactogenicity.
  • Example 14 Prophetic Clinical Trial Protocol Using a Multi-Subunit Vaccine
  • contemplated is a Phase I open-label dose ranging study to evaluate the safety, tolerability, and immunogenic ity of a multisubunit vaccine comprising an immunogenic composition of the instant invention comprising PVPs displaying SARS-CoV-2 antigens encoded by Construct 1 and encoded by Construct 2. Additional details of this prophetic study are found in co-pending priority application, U.S. provisional application, Ser. No. 63/008,412 filed April 10, 2020, the disclosure of which is hereby incorporated herein by reference.
  • the Investigational Product is contemplated as a transdermal patch (3M, Flemington, New Jersey) comprising at least 1 ml of phage at a concentration of 1x10 10 pfu.
  • Study Design This is a Phase I open-label dose ranging study to evaluate the safety, tolerability, and immunogenicity of a multisubunit vaccine consisting of two coadministered phage displayed constructs called NVlOl-COVID-19 and NV102- COVID-19.
  • This phage display based vaccine utilizes the lambda phage, into which codon optimized nucleic acid sequences encoding two domains of the SARS-CoV-2 spike protein (210 and 163 amino acids in length, respectively) have been cloned and expressed as fusion proteins comprising gpD protein on the lambda phage capsid.
  • Our hypothesis presumes that antibody binding to the aforementioned viral epitopes would block host receptor binding and prevent conformational changes that facilitate viral entry into host cells, while simultaneously promoting opsonization of vims particles.
  • ICF informed consent form
  • the contemplated objectives of this clinical trial include, e.g. evaluating the cellular and humoral response of antigens when delivered by TDM administration as well as evaluating the response for cellular and humoral reactivity of antigens when delivered TDM administration after 1, 2 or 3 doses.
  • PBMCs peripheral blood mononuclear cells
  • the study will explore humoral and cell mediated immune responses in blood samples collected at the following times: Enrollment (pre 1 st dose of vaccine); Week 1 thru Week 5 post 1 st vaccination (in all groups); Week 8, 12 and 52 post 1st vaccination (in all groups).
  • Enrollment pre 1 st dose of vaccine
  • Week 1 thru Week 5 post 1 st vaccination in all groups
  • Week 8, 12 and 52 post 1st vaccination in all groups.
  • the study design will expedite clarity and characterization of the immune response post a single immunization as well as durability (across receipt of 1, 2 and 3 immunizations).
  • secondary immuno logic endpoints that might be determined include using conventional methods for determining quantitative binding antibody titers to the full length SARS-CoV-2 antigens as well as determining the qualitative and quantitative levels of neutralizing antibodies against SARS-CoV-2.
  • Antigen- specific cellular immune responses to SARS-CoV-2 may also be determined using conventional methods to perform interferon-gamma (IFN-g) ELISpot assays and/or intracellular cytokine staining (ICS) (cytotoxic T lymphocyte phenotype, lytic granule loading, granzyme B killing of target cells).
  • IFN-g interferon-gamma
  • ICS intracellular cytokine staining
  • ELISA A standardized binding ELISA may be performed to measure the binding of generated antibodies to vaccine antigens. Briefly, in a typical assay, 96-well enzyme immunoassay plates will be coated with RBD and HR2 from the SARS-CoV-2 virus. Samples will be scored as positive if the average OD is greater than 0.15 absorbance units and greater than the average OD before immunization plus 2.5 times the standard deviation (SD) of the OD before immunization at the same dilution. Results will be presented as end-point titer, i.e. the last dilution where the OD value of a sample meets the above criteria.
  • SD standard deviation
  • the neutralizing activity of generated antibodies against the SARS-CoV-2 virus may be assayed, e.g., by performing a median tissue culture infectious dose (TCID5o) assay to determine the number of infectious virus particles, i.e., the amount of virus required to kill 50% of infected hosts or to produce a cytopathic effect in 50% of inoculated tissue culture cells. Briefly, in a typical assay, host tissue cells are cultured, and then serial dilutions of the virus are added to the wells. After incubation, the percentage of infected cells is observed for each dilution, and the results are used to calculate the TCID5o value.
  • TCID5o median tissue culture infectious dose
  • sera from immunized participants may be mixed with 100 infectious particles of SARS-CoV-2 and overlaid onto a monolayer of Vero cells.
  • the titer of neutralizing antibody is reported as the reciprocal of the highest dilution for which less than 50% of the cells show cytopathic effects. Pseudovirus assays will be used to determine the breadth of response.
  • ELISpot enzyme- linked immune absorbent spot
  • ELISpot enzyme- linked immune absorbent spot
  • conventional methods may be used to determine the number of antigen-specific IFN-g secreting spot forming units (SFU) using an IFN-g ELISpot assay in response to stimulation with SARS-CoV-2 peptides, e.g., using 15-mer peptides overlapping by 11 amino acids spanning the entire regions of RBD and HR2 that were targeted (GenScript, Piscataway, NJ).
  • SFU antigen-specific IFN-g secreting spot forming units
  • PBMCs will be thawed and plated at 200,000 cells/well.
  • SARS-CoV-2 peptides will be incubated with SARS-CoV-2 peptides, incubated overnight, and IFN-g release detected using standard procedures.
  • the average number of SFU counted in media control wells will be subtracted from the average in individual SARS-CoV-2 peptide wells and then adjusted to lx10 6 PBMCs for each SARS-CoV-2 peptide pool.
  • ICS and flow' cytometric assays may be performed to assay the influence of vaccination on the ability of participant T cells to exhibit phenotypic markers associated with cytolytic potential after short-term stimulation by SARS-CoV-2 (“CTL Phenotyping”), the ability of participant T cells to remain active in the presence of long- term antigen exposure and efficiently synthesize proteins used in lytic activity (“Lytic Granule Loading”), and/or the ability of participant T cells to effectively employ Granzyme B for the purposes of lytic degranulation and killing of target cells expressing the RBD and/or HR2 (“Killing”).
  • CTL Phenotyping phenotypic markers associated with cytolytic potential after short-term stimulation by SARS-CoV-2
  • Lytic Granule Loading the ability of participant T cells to remain active in the presence of long- term antigen exposure and efficiently synthesize proteins used in lytic activity
  • Killing the ability of participant T cells to effectively employ Granzyme B for the purposes of lytic degranulation and
  • the assay for CTL Phenotyping will employ a 6-hour in vitro stimulation of imfractionated participant PBMCs using peptides spanning the S glycoprotein as described above along with a positive control (Staphylococcal enterotoxin B or a combination of phorbol 12-myristate 13-acetate and Ionomycin).
  • the CTL Phenotyping assay will examine the following external cellular markers: CD3, CD4, CD 8 (T-cell identification): CD45RO, CCR7 (memory subset identification); CDI 07a (marker of lytic degranulation).
  • the CTL Phenotyping assay may also analyze the following intracellular markers: interferon gamma (IFN-g, Thl biasing cytokine), tumor necrosis factor alpha (TNF-a), Granzyme A, Granzyme B, and perforin (proteins involved in lytic degranulation and cytotoxic potential).
  • IFN-g interferon gamma
  • TNF-a tumor necrosis factor alpha
  • Granzyme A proteins involved in lytic degranulation and cytotoxic potential
  • perforin proteins involved in lytic degranulation and cytotoxic potential
  • a prophetic Lytic Granule Loading assay may employ a 120-hour in vitro stimulation of unfractionated participant PMBCs using peptides spanning the SARS- CoV-2 RBD and/or HR2, an irrelevant peptide control (OVA), and a positive control (concanavalin A).
  • the Lytic Granule Loading assay will examine the following external cellular markers: CD3, CD4, CD8 (T cell identification); and CD137 (also known as 41 BB, marker of T cell activation).
  • the Lytic Granule Loading assay will additionally analyze the following intracellular markers: Granzyme A, Granzyme B, Granulysin, and Perforin (proteins involved in cytotoxic potential). The markers evaluated in this assay may change as new relevant data become available.
  • a prophetic Killing assay may employ 120-hour in vitro stimulation of unfractionated PBMC using peptides spanning the SARS-CoV-2 RBD and/or HR2. Stimulated whole PBMC or CDS T-cells isolated after 120-hours will be co-incubated for 60 minutes with target cells that have received a stain that differentiates them from participant PBMC/CD8 + T-cells and have additionally been pulsed with a reagent which is activated upon cleavage of terminal caspases. Target cells are identified using a stain and analyzed for the presence of cleaved caspase activity in targets as a measure of functional cytolytic degranulation and killing. Employment of this assay may change pending readouts from the Lytic Granule Loading assay. The markers evaluated in this assay may change as new relevant data become available.
  • B lymphocytes specific for epitopes on the SARS-CoV-2 RBD and/or HR2 may be sorted by flow cytometry.
  • Neutralizing and non-neutralizing antibodies may be screened and expressed. Resolution of antibody structures in complex with the RBD and/or HR2 may be resolved.
  • Non-neutralizing antibody functional assays may be performed using conventional methods including antibody-dependent cell- mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), antibody-dependent cell-mediated viral inhibition (ADCVI), and viral capture.
  • ADCC antibody-dependent cell- mediated cytotoxicity
  • ADCP antibody-dependent cell-mediated phagocytosis
  • ADCVI antibody-dependent cell-mediated viral inhibition
  • host immune- genotyping may be performed as resources are made available, e.g., investigators may perform targeted analyses of genetic polymorphisms within host genes that may influence immune response to vaccination.
  • targeted genes involved in humoral and cell-mediated immune responses may be assayed, including, e.g., Fey receptors, capable of binding diverse immunoglobulin isotypes; and the alleles comprising Class I HLA-A, -B, and -C loci, respectively.
  • HLA typing procedures that might be used in this study have yet to be validated for clinical use but represent essential research tools for the analyses of adaptive immune responses to vaccination. HLA typing data will be unlinked from personal identifiers, and reported in aggregate for the populations studied.
  • Additional studies contemplated herein include exploring whether end point antibody titers to SARS-CoV-2 antigens are related to frequency of dosing (e.g., after 1, 2, or 3 doses 14 days apart); exploring the time to onset of antibody production and longevity of serologic response; exploring the time to onset of T cell responsiveness and longevity of cell mediated immunity; evaluating host genetics as a potential predictor of vaccine immune response; evaluating additional cellular immune responses; neutralizing and non-neutralizing antibody repertoire analysis; exploring innate immune responses to the SARS-CoV-2 antigen(s), and assessing cross-neutralization activity against other human coronaviruses.
  • Contemplated exploratory endpoints include, e.g., comparing S binding antibody and SARS-CoV-2 neutralizing antibody titers; analyzing the kinetics and durability of S binding antibody and SARS-CoV-2 neutralizing antibody titers; comparing IFN-y ELISpot, and ICS responses across different frequency of dosing regimens; comparing kinetics and durability of IFN-y ELISpot, and ICS responses across different frequency of dosing regimens; analyzing host immune-genotyping; epitope mapping of CD4+ and CD8+ T lymphocyte responses; immunophenotyping and functional characterization of cellular subsets of interest, including natural killer (NK) cells; isolating, expressing and characterizing monoclonal antibodies against the SARS- CoV-2 antigens and assessing their neutralizing and non-neutralizing functional activity.
  • Molecular characterization of study vaccine-elicited antibodies may include, e.g., performing structural biology analysis, Fc analysis, isotype analysis, and epitop

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  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biotechnology (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Plant Pathology (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Mycology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne un procédé de prévention, d'amélioration ou de traitement d'une maladie provoquée par le coronavirus chez un sujet en ayant besoin, comprenant l'administration au sujet d'une composition immunogène comprenant une quantité thérapeutiquement efficace d'un ou de plusieurs bactériophages affichant sur sa capside un ou plusieurs peptides de coronavirus antigéniques fusionnés avec une ou plusieurs protéines de capside de bactériophage (PVP).
PCT/US2021/023002 2020-03-19 2021-03-18 Constructions de vaccin et compositions et procédés d'utilisation de celles-ci Ceased WO2021188818A1 (fr)

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US202062992053P 2020-03-19 2020-03-19
US62/992,053 2020-03-19
US202063008412P 2020-04-10 2020-04-10
US63/008,412 2020-04-10

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113908264A (zh) * 2021-10-21 2022-01-11 中国人民解放军空军军医大学 基于噬菌体载体的新型冠状病毒疫苗及其制备方法
WO2023114727A1 (fr) * 2021-12-13 2023-06-22 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Système de vaccin du bactériophage lambda

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US20100284967A1 (en) * 2006-11-21 2010-11-11 National Institutes of Health (NIH), U.S. Dept. of Health and Human Services (DHHS) U.S. Govt. Modified phage for displaying post-translationally modified proteins and uses thereof
US7915218B2 (en) * 2004-07-23 2011-03-29 Novartis Ag Polypeptides for oligomeric assembly of antigens
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US20150337014A1 (en) * 2013-01-18 2015-11-26 University Of Washington Through Its Center For Commercialization Theragnostic Particles

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US8080642B2 (en) * 2003-05-16 2011-12-20 Vical Incorporated Severe acute respiratory syndrome DNA compositions and methods of use
US7915218B2 (en) * 2004-07-23 2011-03-29 Novartis Ag Polypeptides for oligomeric assembly of antigens
US20100284967A1 (en) * 2006-11-21 2010-11-11 National Institutes of Health (NIH), U.S. Dept. of Health and Human Services (DHHS) U.S. Govt. Modified phage for displaying post-translationally modified proteins and uses thereof
US20150337014A1 (en) * 2013-01-18 2015-11-26 University Of Washington Through Its Center For Commercialization Theragnostic Particles

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113908264A (zh) * 2021-10-21 2022-01-11 中国人民解放军空军军医大学 基于噬菌体载体的新型冠状病毒疫苗及其制备方法
CN113908264B (zh) * 2021-10-21 2023-06-23 中国人民解放军空军军医大学 基于噬菌体载体的新型冠状病毒疫苗及其制备方法
WO2023114727A1 (fr) * 2021-12-13 2023-06-22 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Système de vaccin du bactériophage lambda

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