CN120435313A - Vaccines containing new nanoparticle scaffolds - Google Patents

Abstract

The present invention provides novel engineered nanoparticle scaffold sequences derived from the I3-01 protein. Compared to known I3-01 proteins or variants thereof, the novel I3-01-derived scaffold sequences of the present invention comprise an extended N-terminal helix. Also provided are vaccine constructs comprising a variety of immunogenic proteins displayed on the novel nanoparticle scaffold sequences described herein. For example, the vaccine constructs of the present invention include nanoparticles displaying tandem repeats of the influenza M2e protein or the HCV E2 core protein.

Description

Vaccines comprising novel nanoparticle scaffolds

Cross Reference to Related Applications

This patent application claims priority from U.S. provisional patent application Ser. No. 63/385,224 (filed on day 29 at 11 at 2022; presently pending). The entire disclosure of this priority application is incorporated herein by reference in its entirety for all purposes.

Sequence listing

The present application includes a sequence table 2189_1PC_Sequence Listing in XML format, which was created at 2023, 11, 9 and contains 37KB of content.

Background

Influenza viruses (influenza virus, IAV) belong to the orthomyxoviridae family (Orthomyxoviridae family) and can be divided into four types, a, b, c and t. All influenza viruses are negative-sense single-stranded RNA viruses with envelopes of segmented genomes, wherein influenza a and b viruses (IAV and IBV) comprise 8 gene segments encoding at least 17 proteins. The most abundant surface glycoprotein hemagglutinin (hemagglutinin, HA) allows the virus to bind to host cell receptors and mediate cell entry. Neuraminidase (NA) aids in the release of the viral particles by cleaving residues on the surface of the host cell. Matrix-1protein (m 1) contributes to the budding of the virus from the plasma membrane of the infected cell, and matrix-2protein (m 2) contributes to the maintenance of pH during viral entry into and replication in the host cell. IAVs can be further categorized by subtype based on the antigenic properties of the two surface glycoproteins HA (HA 1-18) and NA (NA 1-11). IAV can infect many hosts, whereas IBV is limited to humans and differentiates into two lineages (Victoria and Yamagata) by intra-host evolution. Avian-derived influenza viruses recognize the alpha-2, 3 sialic acid receptor, whereas human influenza viruses preferentially bind to the alpha-2, 6 sialic acid receptor in the upper respiratory tract. Influenza viruses use two mechanisms to evade the immune system. Antigen drift (ANTIGENIC DRIFT) consists of small changes introduced into HA and NA under immune pressure and is responsible for the annual prevalence of human influenza. Antigen conversion occurs when IAV undergoes complete alteration of HA and/or NA genes due to its large animal reservoir (reservoir). Antigen shift results in increased spread of new IAV strains in humans and is a major cause of pandemic.

Seasonal influenza vaccines have been used as efficient and cost effective tools to minimize influenza epidemics and improve public health since the 40 s of the 20 th century. Current vaccines use inactivated or attenuated live strains. The most common type of inactivated viral vaccine is known as split vaccines, in which detergents or chemicals are used to destroy the viral particles. Live attenuated vaccines use cold-adapted live viruses that do not replicate at human temperatures and are typically administered intranasally to induce strong local immunity. Subunit vaccines utilize viral HA or NA proteins that are partially purified after cleavage by chemicals or detergents. Viral strains selected for tetravalent vaccines (comprising H1N1, H3N2 and two Flu B strains) were produced in chicken eggs. However, due to mutations in influenza virus and the propensity for immune evasion, current influenza vaccines must be updated annually to include predicted strains. Strain mismatches generally result in low potency, which underscores the need for better vaccines.

There is a strong unmet need in the medical field for more reliable and effective influenza vaccines. The present invention is directed to this and other unmet needs in the art.

Disclosure of Invention

In one aspect, the invention provides an N-terminally elongated I3-01 nanoparticle scaffold sequence. These scaffold sequences comprise an extended N-terminal helix compared to the N-terminal helix in the original or wild-type I3-01 scaffold sequence. In some embodiments, the novel I3-01-derived NP scaffold sequences of the present invention comprise a heterologous helical motif of about 6 to about 12 amino acid residues fused to the N-terminus of the I3-01 scaffold sequence shown in SEQ ID NO 27 (I3-01 v 9). In some of these embodiments, the inserted heterologous helical motif comprises AKLAEELQK (SEQ ID NO: 25), conservatively modified variants thereof, or essentially the same sequence. Some of the N-terminally elongated I3-01 nanoparticle scaffold sequences of the invention comprise SEQ ID NO. 4, conservatively modified variants thereof or essentially identical sequences. In a related aspect, the present invention provides self-assembled nanoparticles formed from the novel N-terminally elongated I3-01 nanoparticle scaffold sequences of the present invention.

In another aspect, the invention provides nanoparticle vaccine constructs comprising an immunogenic protein or polypeptide immunogen fused to an N-terminal elongated I3-01 nanoparticle scaffold sequence. The N-terminal extended I3-01 nanoparticle scaffold sequences in these NP vaccine constructs comprise an extended N-terminal helix compared to the N-terminal helix in the original or wild-type I3-01 scaffold sequence. In some of these vaccine constructs, the N-terminal elongated I3-01 nanoparticle scaffold sequence comprises a heterologous helical motif of about 6 to about 12 amino acid residues fused to the N-terminal of the I3-01 scaffold sequence shown in SEQ ID NO. 27 (I3-01 v 9). In some embodiments, the extended heterologous helical motif comprises AKLAEELQK (SEQ ID NO: 25), conservatively modified variants thereof, or essentially the same sequence. In some vaccine constructs, the N-terminal elongated I3-01 nanoparticle scaffold sequence comprises SEQ ID NO. 4, conservatively modified variants thereof, or essentially the same sequence. Typically, the immunogenic protein displayed in the vaccine construct is fused to the N-terminus of the N-terminally elongated I3-01 nanoparticle scaffold sequence by a linker at its C-terminus. In some embodiments, the linker used comprises GGGGS (SEQ ID NO: 3).

In some vaccine constructs of the invention, the displayed polypeptide immunogen is an influenza fusion polypeptide comprising 2 or more tandem repeats of the extracellular domain (M2 e) of the influenza M2 protein. In some further constructs, the displayed polypeptide immunogen is an HCV immunogenic protein. In some embodiments, displayed tandem influenza M2e repeats are separated by a peptide spacer (e.g., GGGG (SEQ ID NO: 9)). In some influenza vaccine constructs, at least one displayed M2e tandem repeat comprises a missense mutation at conserved Cys17 and Cys19 residues. In some of these embodiments, the missense mutation comprises replacing each of the two Cys residues with an amino acid residue having an uncharged polar side chain. In various embodiments, each of the Cys residues is independently replaced with an amino acid residue selected from serine, glycine, asparagine, glutamine, threonine and tyrosine. In some preferred embodiments, both Cys residues in the same M2e sequence are replaced with Ser.

In some influenza vaccines of the invention, the displayed immunogenic protein comprises 3 tandem M2e sequences. In some of these embodiments, the 3 tandem M2e sequences are independently human M2e sequences, avian/porcine consensus M2e sequences, or human/porcine consensus M2e sequences, except for missense mutations at residues Cys17 and Cys19 in at least 2 of the 3 tandem M2e sequences. In some of these embodiments, the displayed influenza fusion polypeptide comprises, in any order, human M2e (SEQ ID NO: 2), an avian/porcine consensus M2e sequence (SEQ ID NO: 30) in which Cys17 and Cys19 are each replaced with a Ser residue, and a human/porcine consensus M2e sequence (SEQ ID NO: 31) in which Cys17 and Cys19 are each replaced with a Ser residue. In some embodiments, the influenza M2e tandem repeat fusion polypeptide displayed comprises, in any order, human M2e in which Cys17 and Cys19 are each replaced by a Ser residue (SEQ ID NO: 29), an avian/porcine consensus M2e sequence in which Cys17 and Cys19 are each replaced by a Ser residue (SEQ ID NO: 30), and a human/porcine consensus M2e sequence in which Cys17 and Cys19 are each replaced by a Ser residue (SEQ ID NO: 31). In some embodiments, the displayed influenza M2e tandem fusion polypeptide comprises SEQ ID NO. 23, SEQ ID NO. 24, conservatively modified variants thereof or essentially identical sequences. Some of these influenza vaccine constructs comprise the subunit or scaffold sequences shown in SEQ ID NO. 11, SEQ ID NO. 12, conservatively modified variants thereof or essentially identical sequences.

In addition to the immunogenic protein sequences, the novel I3-01 scaffold-based vaccine constructs of the invention may additionally comprise T cell epitopes and locking domains at the C-terminus. For example, they may comprise at the C-terminus the locking domain shown in SEQ ID NO. 5 and the T cell epitope shown in SEQ ID NO. 6. Some of these vaccine constructs comprise the subunit or scaffold sequences shown in SEQ ID NO. 14, SEQ ID NO. 15, conservatively modified variants thereof or essentially identical sequences. Some vaccine constructs of the invention may additionally comprise an N-terminal leader sequence. Some of these vaccine constructs comprise the subunit or scaffold sequences shown in SEQ ID NO. 17, SEQ ID NO. 18, conservatively modified variants thereof or essentially identical sequences. Some vaccine constructs of the invention may additionally comprise an N-terminal leader sequence, as well as T cell epitopes and locking domains at the C-terminus. Some of these vaccine constructs comprise the subunit or scaffold sequences shown in SEQ ID NO. 20, SEQ ID NO. 21, conservatively modified variants thereof or essentially identical sequences.

In some vaccine constructs of the invention, the immunogenic protein displayed is an HCV immunogen, e.g., an E2 core or E1E2 dimer protein. In some of these embodiments, the displayed protein comprises an HCV E2 core as shown in any one of SEQ ID NOs 32 to 35. In some embodiments, the displayed HCV immunogenic protein comprises 2 tandem copies of the E2 core sequence. In some of these embodiments, the 2E 2 core sequences are from different HCV isolates. For example, 2 tandem E2 core sequences may comprise SEQ ID NOS 32 and 33 or SEQ ID NOS 34 and 35, respectively. Some HCV vaccine constructs of the present invention additionally comprise a T cell epitope at the C-terminus and a locking domain. For example, the vaccine construct may comprise the locking domain shown in SEQ ID NO. 5 and/or the T cell epitope shown in SEQ ID NO. 6. Some of these HCV vaccines comprise a subunit or scaffold sequence having different structural motifs from the N-terminus to the C-terminus shown in (a) SEQ ID NO:4, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:5 and SEQ ID NO:6, or (b) SEQ ID NO:4, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:5 and SEQ ID NO:6, respectively. Some additional constructs comprise subunits or scaffold sequences that are conservatively modified variants or essentially identical sequences of one of the exemplified scaffold sequences.

In another aspect, the invention provides a polynucleotide sequence encoding a subunit or scaffold sequence of one of the nanoparticle vaccine constructs described herein. The invention also encompasses vectors or expression constructs comprising one or more of these polynucleotide sequences. Also provided in the invention are pharmaceutical compositions or kits comprising the nanoparticle vaccine constructs or encoding polynucleotide sequences described herein.

A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and the claims.

Drawings

Figure 1. Rational design of the best display of I3-01v9a of monomeric antigen on Nanoparticle (NP) surface was achieved. The complete amino acid sequence of the I3-01v9a scaffold is shown (SEQ ID NO: 4).

FIG. 2 (A) construct design of HCV E2 core I3-01v9a nanoparticle. Restriction sites and linkers between the E2 core and the nanoparticle scaffold are shown (SEQ ID NO: 36). (B) EM images of three HCV E2 core I3-01v9a nanoparticles. (C) Construct design of HCV tandem E2 core I3-01v9a nanoparticles. The junction sequence between 2E 2 cores (SEQ ID NO: 3) and the junction sequence between the second E2 core and the nanoparticle scaffold (SEQ ID NO: 36) are shown. (D) SEC and EM analysis of two tandem E2 core I3-01v9a nanoparticles. SEC spectra of two expression volumes in ExpiCHO cells 50ml vs 200ml.

FIG. 3. (A) M2 e-based vaccine construct design. Left side hM2e structure and sequence (SEQ ID NO: 2). The model of hM2E-5GS-1TD0 trimer, and the models of hM2E-5GS-FR, hM2E-5GS-E2p-LD4-PADRE and hM2E-5GS-I3-01v9a-LD7-PADRE 1 c-SApNP. Schematic representation of (B) 1c-SApNP expression and purification. (C) SEC spectra of hM2e trimer and 1C-SApNP. (D) Micrographs of Fab148 purified hM2e 1c-SApNP by negative staining of EM.

FIG. 4 (A) mice immunization/challenge protocol. (B) Survival and weight loss following A/Puerto Rico/8/1934 (PR 8) H1N1 virus challenge. (C) Survival and weight loss following A/Hong Kong/1/1968 (HK 68) H3N2 virus challenge. (D) ELISA of hM2 e-specific antibody responses in mouse serum against hM2e probes.

FIG. 5. (A) negative staining EM image of tandem M2e 1 c-SApNP. The M2ex3-5GS-FR sample, which was maintained at 70℃for 10 minutes, showed no visible change in structure, and it bound to the anti-M2 e antibody with almost the same affinity. (B) Survival and weight loss of alum adjuvant group following A/Puerto Rico/8/1934 (PR 8) H1N1 virus challenge. (C) Survival and weight loss of the AddaVax adjuvant group following A/Puerto Rico/8/1934 (PR 8) H1N1 virus challenge.

Detailed Description

I overview

A variety of antigens and vaccine strategies have been explored to develop universal influenza vaccines. The most common antigen is HA, and recent vaccine design efforts have focused on conserved epitopes within the conserved stem or head domains. These methods include the use of headless HA (headless HA), chimeric HA, and mosaic HA (mosaicha). NA is also an attractive target for broadly neutralizing antibodies (broadly neutralizing antibody, bNAb) to seasonal and pandemic strains. The extracellular domain of the M2 protein (M2 e) is a highly conserved target for universal IAV vaccines. Although M2e is small (about 23 aa) and non-immunogenic, it can be linked to large vectors to elicit cross-protection and reduce viral replication. Endoproteases (e.g., nucleoprotein and M1) have been explored for T cell targeting. The use of adjuvants (e.g., MF59 and AS 03) has been shown to significantly improve the efficacy of influenza vaccines. Thus, in developing a universal influenza vaccine targeting the subdominant (subdominant) HA stems and M2e, the adjuvant effect must be carefully examined.

M2 e-based vaccines provide protection through mechanisms such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), which eliminate virus-infected cells. M2e was linked to various vectors for vaccine development. One of the early M2e vaccines used hepatitis b core protein (HEPATITIS B CORE PROTEIN, HBc) as a carrier. Tobacco mosaic virus (tobacco mosaic virus, TMV) coat protein, keyhole limpet hemocyanin (keyhole limpet hemocyanin, KLH), rotavirus NSP4, GCN4, bacterial flagellin, and liposomes have been tested in M2e vaccine development. As the search for better vectors continues, many vaccine candidates advance to human trials, providing important feedback for future M2e vaccine development. In phase I trials (clinicaltrias. Gov: NCT 00819013), adjuvanted M2e-HBc fusion proteins induced anti-M2 e antibodies at 90% of the time and were well tolerated. However, vaccine-induced anti-M2 e antibody responses were rapidly declining. In phase I trials (clinicaltrias. Gov NCT 00921206), M2 e-flagellin fusion vaccines were highly immunogenic but caused undesirable side effects such as fever, diarrhea, fatigue, headache and muscle pain at higher doses. A vaccine combining M2e with multiple cytotoxic T lymphocyte (cytotoxic Tlymphocyte, CTL) epitopes can stimulate intense cellular immunity in humans (clinicaltrias. Gov: NCT 01181336), but the T cell response range is narrow and slow, making the vaccine unsuitable for the newly emerging pandemic situation. Thus, the correct balance between carrier, adjuvant and antibody and T cell responses is a major challenge facing M2e vaccine development.

The present inventors designed a multi-layered single-component self-assembled protein nanoparticle (1 c-SApNP) as a carrier for foreign antigens for vaccine development based on E2p and I3-01 (two bacterial proteins self-assembled into a 60-mer of 22 to 25 nm). These 1c-SApNP have been successfully applied to HIV-1, HCV, ebola virus (EBOV) and SARS-CoV-2 to produce nanoparticle vaccines. In the embodiments described herein, the invention encompasses novel I3-01 derived NP platforms that have been shown to have activity in presenting immunogenic proteins (e.g., influenza M2e protein and HCV immunogen). The invention also encompasses a broad protective vaccine comprising immunogenic proteins (e.g., HCV E2 core protein and influenza M2E protein) displayed on a novel NP scaffold. Details of making and using the compositions and methods encompassed by the present invention are described below.

The nanoparticle scaffolds and vaccine constructs may have a variety of applications in a clinical setting. For example, the novel I3-01 derived NP scaffolds described herein (e.g., I3-01v9a (SEQ ID NO: 4)) may be used to present a variety of other monomeric antigens in addition to the HCV and influenza immunogenic proteins exemplified herein. The vaccine thus constructed (e.g. HCV vaccine or tandem hM2e vaccine as exemplified herein) can be used as a broad protective vaccine. Influenza vaccines are used as an example, which can be added as "performance enhancers" to seasonal vaccines (e.g., HCV or influenza vaccines) to improve protection against endemic (human strains) and pandemic (swine and avian strains) influenza viruses. In other applications, a vaccine based on the new I3-01 scaffold (e.g., a vaccine displaying M2 e) may be combined with other vaccine forms (e.g., a Hemagglutinin (HA) stem-based vaccine) to produce a truly universal influenza vaccine.

The vaccines of the present invention also have a number of advantageous properties relative to related vaccines known in the art. Using influenza vaccine for illustration, the uniform distribution of antigen anchor sites on the surface and (just-above-sea-level) exposure slightly above the basal plane makes the novel I3-01 scaffold described herein an ideal nanoparticle platform for presentation of monomeric antigen. Icosahedral symmetry and dense surface display make 1c-SApNP an ideal carrier for multivalent display of suitable antigens (e.g., influenza M2 e). As a single segment or tandem construct design, it can be optimally displayed on the nanoparticle surface and can produce high quality antibody responses. In addition, the combination of gene fusion and self-assembly will result in robust production of 1c-SApNP in laboratory and industrial environments. As demonstrated herein, after immunoaffinity (Fab 148) purification, the vaccine can be produced in ExpiCHO cells in reasonable yield and extremely high purity. Since CHO is one of the major mammalian cell lines used in the industrial manufacture of protein therapeutics and vaccines, and ExpiCHO is a transient form of this CHO cell line, vaccines obtained from ExpiCHO cells (e.g., influenza M2e vaccine) are expected to have the same characteristics as those from industrial CHO cell lines. This would enable GMP manufacture of 1c-SApNP vaccine for human use. Furthermore, the multilayer structure will ensure the thermal stability of 1c-SApNP (e.g., influenza M2e 1 c-SApNP) and allow for multiple delivery routes and use in combination with other related vaccines. As exemplified herein, a high temperature of 10 minutes at 70 ℃ did not cause any structural change, and ELISA showed nearly the same binding as M2 e-specific antibodies (e.g., fab65 and Fab 148). The excellent thermal stability of 1c-SApNP (e.g., influenza M2e 1 c-SApNP) of the present invention will allow them to be used in demanding conditions, such as embedding in autolytic microneedles for transdermal immunization.

Unless otherwise indicated herein, the various compositions and methods of the present invention may be produced or performed according to the procedures exemplified herein or according to conventional practice methods well known in the art. See for example,

Methods in Enzymology, volume 289, solid-PHASE PEPTIDE SYNTHESIS, J.N.ABELSON, M.I.SIMON, G.B.FIELDS (editions), ACADEMIC PRESS, version 1 (1997) (ISBN-13:978-0121821906), U.S. Pat. Nos. 4,965,343 and 5,849,954;Sambrook et al, molecular Cloning: A Laboratory Manual, cold Spring Harbor Press, N.Y. (3 rd edition, 2000), brent et al, current Protocolsin Molecular Biology, john Wiley & Sons, inc. (ringbou editions ,2003);Davis et al.,Basic Methodsin Molecular Biology,Elsevier Science Publishing,Inc.,New York,USA(1986); or Methods in Enzymology: guide to Molecular Cloning Techniques, S.L Berger and A.R.Kimmerl editions ,Academic Press Inc.,San Diego,USA(1987);Current Protocols in Protein Science(CPPS)(John E.Coligan,et.al., john Wiley and Sons, inc.), current Protocolsin Cell Biology (CPCB) (Juan S.Bonifacino et al editions, john Wiley and Sons, inc.), and R.Ian Freshney's cult ANIMAL CELLS: A Manual of Basic Technique, publishers: wiley-Lists, 5 th edition (2005), ANIMAL CELL culure Methods (Methodsin Cell Biology, volume 57, rn57 P.heat and David Baes, 1998 edition, matpp.1, 75)

. The following sections provide additional guidance for practicing the compositions and methods of the present invention.

II. Definition of

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide the skilled artisan with a general definition of many of the terms used in the present invention:

ACADEMIC PRESS Dictionary of SCIENCE AND Technology, morris (edit), ACADEMIC PRESS (1 st edition, 1992), oxford Dictionary of Biochemistry and Molecular Biology, smith et al (edit), oxford University Press (edit ,2000);Encyclopaedic Dictionary of Chemistry,Kumar (Ed.),Anmol Publications Pvt.Ltd.(2002);Dictionary of Microbiology and Molecular Biology,Singleton et al.(), john Wiley & Sons (3 rd edition, 2002), dictionary of Chemistry, hunt (edit), routledge (1 st edition, 1999), dictionary of Pharmaceutical Medicine, nahler (edit), springer-Verlag Telos (1994): dictionary of Organic Chemistry, kumar and Anandand (edit), anmol Publications Pvt.Ltd. (2002), and A Dictionary of Biologgy (Oxford Paperback Reference), martin and fine (edit), oxford University Press (4 th edition, 2000)

. Further description of some of these terms as applied specifically to the present invention is provided herein.

As used herein, a noun without a quantitative word modification means one or more than one, unless the context clearly indicates otherwise. For example, "Env-derived trimer" may refer to both single or multiple Env-derived trimer molecules, and may be considered equivalent to the phrase "at least one Env-derived trimer".

The term "antigen" or "immunogen" as used herein is used interchangeably to refer to a substance, typically a protein, capable of inducing an immune response in a subject. The term also refers to a protein having an immunological activity in the sense that it is capable of eliciting an immune response against a humoral and/or cellular type of the protein upon administration to a subject (either directly or by administering to the subject a nucleotide sequence or vector encoding the protein). The term "vaccine immunogen" is used interchangeably with "protein antigen" or "immunogenic polypeptide" unless otherwise indicated.

The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acids do not encode an amino acid sequence, essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For polypeptide sequences, "conservatively modified variants" refers to variants having conservative amino acid substitutions (amino acid residues are replaced by other amino acid residues having similarly charged side chains). The art has defined families of amino acid residues with similarly charged side chains. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

Epitope refers to an antigenic determinant. These are specific chemical groups or peptide sequences on molecules that are antigenic such that they elicit a specific immune response, e.g., an epitope is an antigenic region of a B and/or T cell response. Epitopes can be formed by both contiguous amino acids or non-contiguous amino acids juxtaposed by tertiary folding of the protein.

The vaccine or other agent is in an effective amount sufficient to produce a desired response, such as reducing or eliminating signs or symptoms of a disorder or disease (e.g., seasonal influenza). For example, this may be the amount required to inhibit viral replication or measurably alter the extrinsic symptoms of a viral infection (e.g., an increase in T cell count in the case of influenza infection). Generally, the amount is sufficient to measurably inhibit replication or infectivity of the virus. When administered to a subject, dosages that reach target tissue concentrations (e.g., in lymphocytes) that have been shown to achieve in vitro inhibition of viral replication are typically used. In some embodiments, an "effective amount" is an amount that treats (including prevents) one or more symptoms and/or underlying causes of any disorder or disease (e.g., for treating influenza infection). In some embodiments, the effective amount is a therapeutically effective amount. In some embodiments, an effective amount is an amount that prevents the occurrence of one or more signs or symptoms of a particular disease or disorder (e.g., one or more signs or symptoms associated with the disease).

Fusion proteins as used herein are recombinant proteins comprising amino acid sequences from at least two unrelated proteins that have been linked together by peptide bonds to form a single protein. The unrelated amino acid sequences may be directly linked to each other or they may be linked using a linker sequence. A protein used herein is irrelevant if the amino acid sequences of the protein used herein are generally found not linked together by peptide bonds in their natural environment (e.g., in a cell). For example, the amino acid sequence of the enzyme of the anaerobic bacterium Thermotoga maritima (Thermotoga maritima) from which the I3-01 NP scaffold is derived is not naturally linked by peptide bonds to the amino acid sequence of the influenza M2E or HCV E2 core.

An immunogen is a protein or portion thereof capable of inducing an immune response in a mammal (e.g., a mammal infected by or at risk of being infected by a pathogen). Administration of the immunogen may result in protective and/or active immunity against the pathogen of interest.

An immune response refers to a response of cells of the immune system (e.g., B cells, T cells, or monocytes) to a stimulus. In some embodiments, the response is specific for a particular antigen ("antigen-specific response"). In some embodiments, the immune response is a T cell response, such as a cd4+ response or a cd8+ response. In some further embodiments, the response is a B cell response and results in the production of specific antibodies.

An immunogenic composition refers to a composition comprising an immunogenic polypeptide that induces a measurable CTL response against a virus expressing the immunogenic polypeptide, or induces a measurable B cell response (e.g., antibody production) against the immunogenic polypeptide.

Amino acid numbering or amino acid numbering system as used herein refers to the numbering or linear position of amino acid residues in an immunogenic protein or polypeptide (e.g., influenza M2 e) from a prototype strain or species. By standardized sequence alignment, sequences of different orthologs of the same immunogenic protein (e.g., M2 e) from other strains or species, or engineered versions of the same protein described herein, are allowed to be compared to sequences of the prototype sequence. Using such standard or standardized amino acid numbering, conserved amino acid residues in immunogenic proteins from various viral strains or engineered proteins can be readily identified and named. For example, unless otherwise indicated herein, the amino acid numbering of the M2e proteins may be based on the consensus sequence of the human influenza M2e proteins. According to this numbering, the conserved Cys residues to be mutated are referred to as residues Cys17 and Cys19 for all influenza strains.

Sequence identity or similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed as identity or similarity between the sequences. Sequence identity can be measured in terms of percent identity, with higher percentages being the more identical the sequences. Two sequences are "substantially identical" if they have a specified percentage of identical amino acid residues or nucleotides (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95% or 99% identity over the entire sequence, either in the specified region or when not specified) when compared and aligned for maximum correspondence over a comparison window or specified region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, identity exists over a region of at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region of 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.

Homologs or orthologs of nucleic acid or amino acid sequences have a relatively high degree of sequence identity/similarity when aligned using standard methods. Sequence alignment methods for comparison are well known in the art. Various procedures and alignment algorithms are described below:

Smith&Waterman,Adv.Appl.Math.2:482,1981;Needleman&Wunsch,J.Mol.Biol.48:443,1970;Pearson&Lipman,Proc.Natl.Acad.Sci.USA 85:2444,1988;Higgins&Sharp,Gene,73:237-44,1988;Higgins&Sharp,CABIOS 5:151-3,1989;Corpet et al.,Nuc.Acids Res.16:10881-90,1988;Huang et al.Computer Appls.in the Biosciences 8,155-65,1992; And Pearson et al, meth.mol.Bio.24:307-31,1994.Altschul et al, J.mol.biol.215:403-10,1990

Detailed considerations of sequence alignment methods and homology calculations are shown.

The term "subject" refers to any animal classified as a mammal, such as a human and a non-human mammal. Some examples of non-human animals include dogs, cats, cows, horses, sheep, pigs, goats, rabbits, and the like. The terms "patient" or "subject" are used interchangeably herein unless otherwise indicated. Preferably, the subject is a human.

The term "treating" or "alleviating" includes administering a compound or agent to a subject to prevent or delay the onset of symptoms, complications, or biochemical indicators of a disease (e.g., influenza infection), alleviate symptoms, or prevent or inhibit further development of the disease, condition, or disorder. Subjects in need of treatment include those already with the disease or disorder as well as those at risk of developing the disorder. Treatment may be prophylactic (preventing or delaying the onset of a disease, or preventing the manifestation of clinical or subclinical symptoms thereof), or therapeutic inhibition or alleviation of symptoms after manifestation of a disease.

A vaccine refers to a pharmaceutical composition that elicits a prophylactic or therapeutic immune response in a subject. In some cases, the immune response is a protective immune response. Generally, vaccines elicit antigen-specific immune responses against antigens of pathogens (e.g., viral pathogens) or against cellular components associated with pathological conditions. A vaccine may include a polynucleotide (e.g., a nucleic acid encoding a disclosed antigen), a peptide or polypeptide (e.g., a disclosed antigen), a virus, a cell, or one or more cellular components. In some embodiments of the invention, the vaccine or vaccine immunogen or vaccine composition is expressed from a fusion construct and self-assembled into nanoparticles displaying the immunogenic polypeptide or protein on the surface.

A vaccine (e.g., influenza or HCV vaccine) refers to an immunogenic composition capable of stimulating an immune response that is administered to prevent, ameliorate or treat a disease or infection (e.g., an influenza virus infection). Vaccines may include, for example, attenuated or killed (e.g., split) pathogens (e.g., viruses), virus-LIKE PARTICLE, VLPs, and/or antigenic polypeptides or DNA derived therefrom, or any recombinant form of such immunogenic material.

A virus-like particle (VLP) refers to a non-replicating viral capsid derived from any one of several viruses. VLPs are typically composed of one or more viral proteins, such as, but not limited to, those proteins known as capsid proteins, coat proteins, capsid proteins, surface proteins and/or envelope proteins, or particle-forming polypeptides derived from these proteins. VLPs may spontaneously form upon recombinant expression of the protein in a suitable expression system. Methods for producing specific VLPs are known in the art. The presence of VLPs following recombinant expression of viral proteins can be detected using conventional techniques known in the art (e.g., by electron microscopy, biophysical characterization, etc.). See, for example, baker et al (1991) Biophys.J.60:1445-1456, and HAGENSEE ET al (1994) J.Virol.68:4503-4505. For example, VLPs may be isolated by density gradient centrifugation and/or identified by characteristic density bands. Alternatively, a vitrified aqueous sample of the VLP formulation in question may be subjected to freeze electron microscopy and the image recorded under appropriate exposure conditions.

Self-assembled nanoparticles refer to spherical protein shells with diameters of tens of nanometers and well-defined surface geometry, formed from identical copies of non-viral proteins that can be automatically assembled into nanoparticles with a similar appearance to VLPs. Some examples that are known include ferritin (ferritin, FR) which is conserved among species and forms a 24-mer, and bacillus stearothermophilus (b.stearothermophilus) dihydrolipoyl acyltransferase (E2 p), liquid-borne fungus (Aquifex aeolicus) dioxytetrahydropteridine synthase (lumazine synthase, LS), variants of I3-01 origin, and Thermotoga maritima encapulin, all forming 60-mers. Self-assembled nanoparticles can spontaneously form after recombinant expression of the protein in a suitable expression system. The nanoparticle generation, detection and characterization methods can be performed using the same techniques developed for VLPs.

NP scaffolds with improved activity

The present invention provides novel nanoparticle scaffold sequences suitable for presentation of a variety of viral immunogenic proteins to elicit potent neutralizing antibody responses. These rational design and functional test scaffold sequences are based on the I3-01 protein. I3-01 is an engineered protein (SEQ ID NO: 22) that self-assembles into ultra-stable nanoparticles. Original ("unextended" or "wild-type") I3-01 proteins are described in Hsia et al, nature535,136-139,2016. Several ultra-stable nanoparticle scaffolds derived from I3-01 were previously developed and used to present viral proteins, such as those from HIV-1 and HCV. See, for example, WO21/021603, WO22/035739, U.S. Pat. No. 10,906,944 and WO19/089817. To identify new NP scaffolds with improved activity, the inventors rationally designed and tested for function on known I3-01 variant scaffolds. The original I3-01 proteins and variants known in the art (i.e., the unextended I3-01 scaffold sequences) comprise an N-terminal helical motif KMEELFKKHK (SEQ ID NO: 26). The novel scaffold sequences of the invention are obtained by extending the N-terminal helix of existing I3-01 variant scaffolds (e.g., I3-01v9 (SEQ ID NO: 27)) by grafting heterologous helical motifs, followed by rational design using a set-based protein design program. An example of a new scaffold is I3-01v9a (SEQ ID NO: 4), as exemplified herein. As described in more detail in the examples, the resulting novel variant I3-01 scaffold (e.g., SEQ ID NO: 4) is capable of providing optimal surface display of monomeric protein antigens.

The I3-01 sequence without the first Met residue (SEQ ID NO: 22) (N-terminal helical band underlined):

KMEELFKKHKIVAVLRANSVEEAKKKALAVFLGGVHLIEITFTVPDADTVIKELSFLKEMGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTPVEVAEKAKAFVEKIRGCTE

In various embodiments, the novel I3-01-derived NP scaffolds of the present invention comprise an I3-01 variant sequence (e.g., SEQ ID NO: 27), except that a helical motif of about 6 to about 12 amino acid residues is added at the N-terminus. This inserted helical motif results in an elongation of the original N-terminal helix KMEELFKKHK (SEQ ID NO: 26) in the I3-01 protein. In some embodiments, the inserted helical motif comprises AKLAEELQK (SEQ ID NO: 25), conservatively modified variants thereof, or essentially the same sequence.

I3-01v9 (SEQ ID NO: 27) (N-terminal helical band underlined):

KMEELFKKHKIVAVLRANSVEEAKMKALAVFVGGVHLIEITFTVPDADTVIKELSFLKELGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTIAEVAAKAAAFVEKIRGCTE

i3-01v9a (SEQ ID NO: 4) (extension of the N-terminal helix underlined):

AKLAEELQKKMEELFKKHKIVAVLRANSVEEAKMKALAVFVGGVHLIEITFTVPDADTVIKELSFLKELGAIIGAGTVTSVEQCRKAVESGAEFIVSPHLDEEISQFCKEKGVFYMPGVMTPTELVKAMKLGHTILKLFPGEVVGPQFVKAMKGPFPNVKFVPTGGVNLDNVCEWFKAGVLAVGVGSALVKGTIAEVAAKAAAFVEKIRGCTE

In various embodiments, the novel I3-01-derived NP scaffolds of the present invention comprise SEQ ID NO 4, conservatively modified variants thereof, or essentially identical sequences (e.g., at least 90%, 95%, 96%, 97%, 98%, or 99% identical). In some of these embodiments, the helical motif inserted at the N-terminus is identical to SEQ ID NO. 25, while the remainder of the scaffold sequence is a conservatively modified variant or essentially identical sequence of SEQ ID NO. 27. In some further embodiments, the entire extended N-terminal helix of the new I3-01 variant scaffold is identical to the N-terminal helix in I3-01v9a (i.e., AKLAEELQKKMEELFKKHK (SEQ ID NO: 28)), while the remaining sequences are conservatively modified variants of or essentially identical to the corresponding sequences of SEQ ID NO:27 (i.e., SEQ ID NO:27 minus the N-terminal helix).

Immunogenic polypeptides or proteins for the production of vaccine compositions

The novel I3-01-derived nanoparticle scaffolds described herein can be used to construct vaccines that present a number of different immunogenic proteins, including monomeric and multimeric polypeptides. These include any protein or polypeptide from a pathogen against which an immune response is desired to be elicited. Thus, the vaccine compositions of the present invention may utilize immunogenic polypeptides derived from any virus, bacteria or other pathogenic organism. Suitable immunogenic polypeptides for use in the present invention may also be derived from non-pathogenic species, including human proteins, against which the eliciting immune response may have a therapeutic effect, alleviating symptoms of a disease or improving overall health. In general, an immunogenic polypeptide can be any structural or functional polypeptide or peptide comprising at least about 10 amino acid residues. In some embodiments, the immunogenic polypeptide is about 10 to about 10,000 amino acid residues in length. In some embodiments, the immunogenic polypeptide is about 25 to about 2,000 amino acid residues in length. In some embodiments, the immunogenic polypeptide is about 50 to about 500 amino acid residues in length. Thus, an immunogenic polypeptide or protein suitable for the present invention may have a molecular weight of about 1kDa to about 1,000kDa, and preferably about 2.5kDa to about 250kDa. In some more preferred embodiments, the immunogenic polypeptides employed have a molecular weight of about 5kDa to about 25kDa or 50kDa.

In some embodiments, the immunogenic polypeptides or proteins used in the vaccine compositions of the invention may be derived from viral surface or core proteins (target polypeptides). There are many known viral proteins that are important for viral infection of host cells. Examples include, but are not limited to, the glycoprotein (or surface antigen such as GP120 and GP 41) and capsid protein (or structural protein such as P24 protein), the surface antigen or core protein of hepatitis A, B, C, D or E virus (such as small hepatitis B virus surface antigen (S-HBsAg) and hepatitis C virus core proteins NS3, NS4 and NS5 antigen), the glycoprotein GP350/220 of EB virus (Epstein-Barr virus, EBV), the glycoprotein (G protein) or fusion protein (F protein) of respiratory syncytial virus (respiratory syncytial virus, RSV), the surface and core proteins of herpes simplex virus HSV-1 and HSV-2 (such as glycoprotein D from HSV-2), the surface proteins of polio virus (such as gB, gC, gD, gH and gL), the envelope glycoprotein hemagglutinin (H) and fusion protein (F) of measles virus (measles virus, MV), the fibrous protein G of lymphocytic choriomeningitis virus (lymphocytic choriomeningitis virus, LCM, V), the fibrous protein G of adenovirus and the pentravus (34), and the envelope virus (E), the viral envelope-free virus (e.g.g. the viral capsid protein of the human adenovirus (J) of the human virus, and the human virus (human virus).

In some preferred embodiments, the immunogen or immunogenic protein displayed on the novel I3-01 NP scaffold is a monomeric protein. Some examples of such proteins include, for example, influenza M2 extracellular domain (M2 e) proteins exemplified herein. As detailed below, some embodiments of influenza vaccines of the present invention encompass NP vaccines comprising a novel I3-01 scaffold (e.g., SEQ ID NO: 4) that displays tandem repeats (e.g., 2,3, 4 or more copies) of the M2e protein. In some of these embodiments, one or more of the tandem M2e copies comprises substitutions at conserved Cys17 and Cys19 residues to prevent random disulfide bond formation.

Some additional embodiments of the invention relate to HCV vaccines comprising a novel I3-01 NP scaffold (e.g., SEQ ID NO: 4) that displays HCV immunogenic proteins. In general, the HCV immunogenic proteins to be displayed on the NP scaffold are derived from HCV glycoproteins E1 and E2, which form heterodimers on the HCV envelope that mediate viral entry into the host hepatocytes. In some embodiments, the displayed HCV protein comprises an E2 core. The E2 core as known in the art refers to a portion of E2 that forms a three-dimensional structure recognized by the broadly neutralizing antibody AR3C Fab (Law et al, nat. Med.2008;14:25, 2008). As exemplified herein, the I3-01 variant scaffolds of the invention may be used to display single copies of E2 core protein or tandem E2 core fusion proteins. One specific HCV E2 core protein that can be used in the HCV vaccine constructs of the present invention is a redesigned E2mc3 protein as described in U.S. patent No. 11,008,368. E2mc3 sequences derived from a variety of HCV subtypes or isolates, including HCV H77, J6, ED43 and UKN3A1.28c isolates (SEQ ID NOS: 32 to 35, respectively) as exemplified herein, may be used. Conservatively modified variants or essentially identical sequences of these exemplified E2 core sequences may also be used in the HCV vaccine constructs of the present invention.

In some embodiments, the displayed HCV immunogenic protein is a tandem E2 core fusion protein comprising SEQ ID NO. 32 and SEQ ID NO. 33 in any order. In some further embodiments, the HCV immunogenic proteins displayed are tandem E2 core fusion proteins comprising SEQ ID NO. 34 and SEQ ID NO. 35 in any order. In some further embodiments, the HCV immunogenic protein displayed by the NP scaffold comprises an E1E2 heterodimer, e.g., a rationally redesigned HCV E1E2 dimer. In addition to the HCV-derived immunogenic proteins, the novel I3-01 scaffold-displayed HCV vaccine may additionally comprise a locking domain and/or a T cell epitope. For example, the vaccine construct may have an LD7 motif (SEQ ID NO: 5) and a PADRE epitope (SEQ ID NO: 6) at the C-terminus, as exemplified herein.

In the construction of the HCV vaccines of the present invention, any E2 core protein sequence, tandem E2 core fusion molecule, and E1E2 dimer known in the art or that can be easily engineered are suitable. Detailed guidance for obtaining such HCV immunogenic proteins and constructing NP vaccines comprising such HCV immunogenic proteins is provided below, for example

WO21/021603;McGregor et al.,J.Virol.96：e01675-21;Lin et al.,Front Immunol.2022:13：831285;Wang et al.,Proc.Natl.Acad.Sci.USA 119:e2112008119,2022:Clarke et al.,Plant Biotechnol.J.15:1611-21,2017; And Sepulveda-Crespo et al J.biomed.Sci 27,78,2020

The various immunogenic proteins or polypeptides (e.g., the tandem influenza M2E fusion protein or the tandem HCV E2 core fusion protein) displayed on the novel I3-01NP scaffold of the invention can be obtained or produced according to the protocols illustrated herein or by methods well known in the art. See, e.g., sambrook et al, molecular Cloning: A Laboratory Manual, cold Spring Harbor Press, n.y. (3 rd edition, 2000), and brunt et al, current Protocols in Molecular Biology, john Wiley & Sons, inc. (ringbou edit, 2003).

V NP vaccine comprising novel I3-01NP scaffold

The present invention provides nanoparticle vaccines with the novel I3-01NP scaffolds disclosed herein. As described above, some vaccine constructs display HCV immunogenic proteins such as tandem E2 core proteins. Some additional vaccine constructs of the invention display a single copy of the influenza M2 protein extracellular domain (M2 e). Several examples of such influenza NP vaccines are exemplified herein. In yet other embodiments, fusion polypeptides comprising tandem repeats of influenza M2e protein are displayed on novel I3-01 derived nanoparticle scaffolds. In these embodiments, the displayed immunogenic protein displayed on the novel I3-01 scaffold sequence is a fusion polypeptide comprising 2 or more influenza M2e sequence tandem repeats. In various embodiments, at least one M2e tandem repeat comprises missense mutations at conserved Cys17 and Cys19 residues to prevent the formation of random disulfide bonds. In some of these influenza NP vaccine constructs, the engineered missense mutation is the replacement of each of the Cys residues with an amino acid residue comprising an uncharged polar side chain. For example, each of the two CY residues in one or more tandem M2e repeats may be independently replaced with serine, glycine, asparagine, glutamine, threonine, or tyrosine. In some embodiments, one or both of the Cys residues is replaced with Ser.

Typically, the tandem M2e fusion polypeptide sequence is fused to the N-terminus of the novel I3-01 scaffold sequence, e.g., via a linker motif such as GGGGS (SEQ ID NO: 3) as exemplified herein. Preferably, tandem M2e repeats in the displayed fusion polypeptide are separated by a short linker or spacer. For example, GGGG (SEQ ID NO: 9) spacers herein may be used to separate different M2e sequences, as exemplified herein. Some influenza NP vaccines of the invention comprise 3 tandem M2e repeats. In various embodiments, the tandem M2e repeats displayed on the novel I3-01 scaffold of the invention may be the same or different. Orthologous M2e sequences from many species and modified versions thereof are known in the art. See, e.g., mezhenskaya et al, j.biomed.sci.26,76,2019. Thus, for example, each M2e tandem repeat may independently be a human M2e sequence (SEQ ID NO: 2), an avian/porcine consensus M2e sequence (SEQ ID NO: 7), or a human/porcine consensus M2e sequence (SEQ ID NO: 8). When M2e sequences from different sources are used for tandem M2e molecules, the different M2e motifs may be linked in any order. As one example, the tandem M2e repeat sequence in the influenza vaccine of the invention may comprise a human M2e sequence, an avian/porcine consensus M2e sequence, and a human/porcine consensus M2e sequence. In these embodiments, 3 different Me2 sequences can be linked to the scaffold sequence in any of 6 possible sequence orders.

In some embodiments, at least 2 of the 3 tandem M2e repeats comprise substitutions at residues Cys17 and Cys 19. For example, the human M2e sequence may retain unmutated residues at Cysl and Cys19, while the avian/porcine consensus M2e sequence and the human/porcine consensus M2e sequence comprise substituted residues (e.g., all substituted with Ser) at Cys17 and Cys 19. In some of these embodiments, the mutated Cys residues are all replaced with Ser residues. Thus, the displayed M2e fusion polypeptide may comprise, in any order, the unmutated human M2e (SEQ ID NO: 2), the mutated avian/porcine consensus M2e sequence SLLTEVETPTRNGWESKSSDSSD (SEQ ID NO: 30), and the mutated human/porcine consensus M2e sequence SLLTEVETPTRSEWESRSSGSSD (SEQ ID NO: 31). In some embodiments, all 3 tandem M2e repeats contain substitutions at residues Cys17 and Cys 19. Thus, the displayed M2e fusion polypeptide may comprise, in any order, mutated human M2ESLLTEVETPIRNEWGSRSNDSSD (SEQ ID NO: 29), mutated avian/porcine consensus M2e sequence SLLTEVETPTRNGWESKSSDSSD (SEQ ID NO: 30), and mutated human/porcine consensus M2e sequence SLLTEVETPTRSEWESRSSGSSD (SEQ ID NO: 31). As a specific example, a fusion M2e polypeptide may comprise the sequence shown in SEQ ID NO. 23 or SEQ ID NO. 24, conservatively modified variants thereof or essentially identical sequences.

In some embodiments, NP vaccines (e.g., influenza M2E vaccine in tandem or HCV E2 vaccine) comprising the novel I3-01 variant scaffolds of the invention can optionally comprise a trimerization motif, e.g., SHP or foldon. Some nanoparticle vaccine compositions may additionally comprise other structural components whose function is to further enhance the stability and antigenicity of the displayed immunogens. In some embodiments, the locked protein domain (LD) may be inserted into the nanoparticle construct, for example, by covalent fusion with the C-terminus of the nanoparticle subunit. The locking domain may be any dimeric protein capable of forming an interface through specific interactions, such as hydrophobic (van der waals (VAN DER WAALS)) contacts, hydrogen bonds, and/or salt bridges. One example of a locking domain that can be used in the vaccine of the invention is LD7 (SEQ ID NO: 5) as exemplified herein. General guidelines for selecting a locking domain and various other examples (e.g., LD 4) are described in the art, for example, in WO19/241483, U.S. Pat. No. 10,906,944, and U.S. Pat. No. 11,305,004.

In some embodiments, the stented influenza vaccines of the present invention may further comprise T cell epitopes to promote a robust T cell response and direct B cell development to bNAb. T cell epitopes can be located anywhere relative to other structural components as long as they do not affect the presentation of the engineered HA protein on the nanoparticle surface. Any T cell epitope sequence or peptide known in the art may be used in the practice of the invention. It includes any polypeptide sequence that comprises an MHC class II epitope and that is effective to activate cd4+ and cd8+ T cells after immunization, e.g., a T-helper epitope that activates cd4+ T helper cells. See, e.g., ,Alexander et al.,Immunity 1,751-761,1994;Ahlers et al.,J.Clin.Invest.108:1677-1685,2001;Fraser et al.,Vaccine 32,2896-2903,2014;De Groot et al.,Immunol.Cell Biol.8:255-269,2002; and Gene Ther.21:225-232,2014. In some embodiments, the T cell epitope inserted into the nanoparticle vaccine construct is the universal pan DR epitope peptide (pan DR epitope peptide, PADRE) AKFVAAWTLKAAA (SEQ ID NO: 6), as exemplified herein for influenza and HCV vaccines. More detailed information on T cell epitopes suitable for the present invention is described, for example, in Hung et al, mobile. Ther.15:1211-19,2007; wu et al, J.biomed. Sci.17:88,2010, and Bissati et al, npj Vaccines 2:24,2017. Other examples of suitable T cell epitopes are also described in the art, for example, the D and TpD epitopes (Fraser et al, vaccine 32,2896-2903,2014).

The novel I3-01 scaffold based nanoparticle vaccines of the present invention can be constructed according to standard recombinant techniques and other methods described in the art, such as ,He et al.,Nat.Comm.7,12041,2016;Kong et al.,Nat.Comm.7,12040,2016;He et al.,Sci Adv.4(11):eaau6769,2018; and PCT publications WO2017/192434, WO2019/089817 and WO19/241483. In various embodiments, a novel I3-01 scaffold-based nanoparticle vaccine can be constructed by fusing an immunogenic protein of interest (e.g., a tandem HCV E2 core or a tandem influenza M2E polypeptide) to an I3-01 scaffold subunit. Preferably, the C-terminus of the immunogenic protein sequence is fused to the N-terminus of the nanoparticle subunit sequence. In some embodiments, a short peptide linker or spacer (e.g., SEQ ID NOS: 3 and 9) may be inserted between the sequence of the immunogenic protein and the nanoparticle subunit sequence or between tandem copies of the immunogenic protein.

After recombinant expression (e.g., in ExpiCHO cells as detailed herein), the nanoparticle vaccines of the present invention can be substantially purified by any conventionally practiced procedure. See, e.g., guide to Protein Purification, ed.Deutscher, meth.Enzymol.185, academic Press, san Diego,1990, and scope, protein Purification: PRINCIPLES AND PRACTICE, SPRINGER VERLAG, new York,1982. Substantially purified means purified from other proteins or cellular components. The substantially purified protein is at least 60%, 70%, 80%, 90%, 95% or 98% pure. Once purified, the antigenicity and other properties of the vaccine can also be readily detected by standard methods, such as antigen profiling using known bNAb and non-Nab, differential Scanning Calorimetry (DSC), electron microscopy, binding analysis by ELISA, biol-cal layer interferometry (Biolayer Interferometry, BLI), surface plasmon resonance (Surface Plasmon Resonance, SPR), and eutectic analysis. Some of these assays are exemplified herein for analysis of novel I3-01 stented HCV vaccines or influenza vaccines.

V. Polynucleotide and expression constructs

The novel I3-01 scaffolds and vaccines based thereon of the present invention are typically produced by first generating an expression construct (i.e., an expression vector) comprising the coding sequences of the various structural components described herein in operable linkage. In some embodiments, the vaccine compositions of the invention are polynucleotide-based (e.g., mRNA-based vaccines). Thus, in some related aspects, the invention provides polynucleotides (e.g., DNA or RNA) encoding subunit sequences of a novel I3-01 scaffold or nanoparticle vaccine based on the scaffold, expression vectors comprising such polynucleotides, and host cells (e.g., expiCHO cells as exemplified herein) for the production of novel NP scaffolds and vaccines. The invention also encompasses fusion polypeptides encoded by the polynucleotides or expressed by the vectors.

Polynucleotides and related vectors can be readily produced using standard molecular biology techniques or protocols exemplified herein. For example, general protocols for cloning, transfection, transient gene expression and obtaining stably transfected cell lines are described in the art, such as Sambrook et al, molecular Cloning: ALaboratory Manual, cold Spring Harbor Press, n.y. (3 rd edition, 2000), and brunt et al, current Protocols in Molecular Biology, john Wiley & Sons, inc (ringbou edition, 2003). The introduction of mutations into polynucleotide sequences by PCR can be performed as described, for example, in PCR Technology: PRINCIPLES AND Applications for DNA Amplification, H.A.erlich (eds.), FREEMAN PRESS, NY, NY,1992;PCR PROTOCOLS:A GUIDE TO METHODS AND APPLICATIONS,INNIS ET AL (eds.), ACADEMIC PRESS, san Diego, calif., 1990;Mattila et al, nucleic Acids Res.19:967,1991, and Eckert et al, PCR Methods and Applications 1:17,1991.

The choice of the particular vector will depend on the intended use of the fusion polypeptide. For example, the vector selected must be capable of driving expression of the fusion polypeptide in the desired cell type, whether the cell type is prokaryotic or eukaryotic. Many vectors contain sequences that allow both eukaryotic expression of operably linked gene sequences and replication of prokaryotic vectors. Vectors useful in the present invention may autonomously replicate, i.e., the vector is present extrachromosomally, and its replication is not necessarily directly linked to replication of the host cell's genome. Or replication of the vector may be linked to replication of the chromosomal DNA of the host, e.g. the vector may be integrated into the chromosome of the host cell, as is achieved by retroviral vectors and in stably transfected cell lines. Both viral-based and non-viral expression vectors can be used to produce immunogens in mammalian host cells. Non-viral vectors and systems include plasmids, episomal vectors (typically with an expression cassette for expression of a protein or RNA) and human chromosomes (see, e.g., harrington et al, nat. Genet.15:345,1997). Useful viral vectors include those based on lentiviruses or other retroviruses, adenoviruses, adeno-associated viruses, cytomegaloviruses, herpes viruses, SV40, papillomaviruses, HBP EB viruses, vaccinia virus vectors, and Semliki forest viruses (Semliki Forest virus, SFV). See Brent et al, supra, smith, annu. Rev. Microbiol.49:807,1995, and Rosenfeld et al, cell 68:143,1992.

Depending on the particular vector used to express the fusion polypeptide, a variety of known cells or cell lines may be used in the practice of the invention. The host cell may be any cell into which a recombinant vector carrying a fusion of the invention may be introduced, and wherein a vector that allows for driving expression of the fusion polypeptide may be used in the invention. It may be prokaryotic, for example, any of a number of bacterial strains, or eukaryotic, for example, yeast or other fungal cells, insect or amphibian cells, or mammalian cells, including, for example, rodent, simian, or human cells. The cells expressing the fusion polypeptides of the invention may be primary cultured cells or may be established cell lines. Thus, in addition to the cell lines exemplified herein (e.g., CHO cells), many other host cell lines that can be known in the art can also be used in the practice of the present invention. These include, for example, a variety of Cos cell lines, heLa cells, HEK293, atT20, BV2 and N18 cells, myeloma cell lines, transformed B cells and hybridomas.

The use of mammalian tissue cell cultures to express polypeptides is generally discussed in, for example, winnacker, from Genes to Clones, VCH Publishers, n.y., 1987. The vector expressing the fusion polypeptide may be introduced into the selected host cell by any of a number of suitable methods known to those skilled in the art. For introducing a vector encoding a fusion polypeptide into a mammalian cell, the method used will depend on the form of the vector. For plasmid vectors, DNA encoding the fusion polypeptide sequence may be introduced by any of a number of transfection methods including, for example, lipid-mediated transfection ("lipofection"), DEAE-dextran-mediated transfection, electroporation, or calcium phosphate precipitation. These methods are described in detail, for example, in Brent et al, supra. Lipid transfection reagents and methods suitable for transient transfection of a wide variety of transformed and untransformed or primary cells are widely available, making lipid transfection an attractive method for introducing constructs into eukaryotic and, in particular, cultured mammalian cells. For example, lipofectAMINE ^TM (Life Technologies) or LipoTaxi ^TM (Stratagene) kits are available. Other companies that provide reagents and methods for lipid transfection include Bio-Rad Laboratories、Clontech、Glen Research、Life Technologies、JBL Scientific、MBI Fermentas、PanVera、Promega、Quantum Biotechnologies、Sigma-Aldrich and Wako Chemicals USA.

For long-term high-yield production of recombinant fusion polypeptides, stable expression is preferred. Instead of using an expression vector comprising a viral origin of replication, the host cell may be transformed with a fusion polypeptide coding sequence and a selectable marker controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.). Selectable markers in recombinant vectors confer resistance to selection and allow the cells to stably integrate the vector into their chromosomes. Common selectable markers include neo (Colberre-Garapin, et al, J.mol.biol.,150:1, 1981) which confers resistance to the aminoglycoside G-418, and hygro (SANTERRE ET al, gene,30:147, 1984) which confers resistance to hygromycin. By appropriate selection, the transfected cells may comprise an integrated copy of the fusion polypeptide coding sequence.

Pharmaceutical composition and therapeutic use

The present invention provides pharmaceutical or immunogenic compositions and related methods of treatment using novel I3-01 scaffold-based vaccines (e.g., influenza or HCV vaccines). In some embodiments, the vaccine composition can be used to prevent and treat a disease or infection (e.g., HCV infection or influenza). In some embodiments, the nanoparticle displaying an immunogenic protein (e.g., tandem M2E or HCV E2 core protein) is included in a pharmaceutical composition. The pharmaceutical composition may be a therapeutic or prophylactic formulation. Generally, the composition additionally comprises one or more pharmaceutically acceptable carriers, and optionally other therapeutic ingredients (e.g., antibiotics or antiviral drugs). Various pharmaceutically acceptable additives may also be used in the composition.

Some pharmaceutical compositions of the invention are vaccines. For vaccine compositions, a suitable adjuvant may additionally be included. Some examples of suitable adjuvants include, for example, aluminum hydroxide, lecithin, freund's adjuvant, MPL ^TM, and IL-12. In some embodiments, the novel I3-01 scaffold-based vaccines of the present invention can be formulated as controlled-release or timed-release (time-release) formulations. This may be achieved in a composition comprising a slow release polymer or by a microencapsulated delivery system or bioadhesive gel. A variety of pharmaceutical compositions may be prepared according to standard procedures well known in the art. See, e.g., ,Remington's Pharmaceutical Sciences,19.sup.th Ed.,Mack Publishing Company,Easton,Pa.,1995;Sustained and Controlled Release Drug Delivery Systems,J.R.Robinson editions, MARCEL DEKKER, inc., new York, 1978), U.S. Pat. nos. 4,652,441 and 4,917,893, U.S. Pat. nos. 4,677,191 and 4,728,721, and U.S. Pat. No. 4,675,189.

The methods of treatment of the invention involve administering a suitable vaccine of the invention (e.g., an influenza vaccine or an HCV vaccine) to a subject suffering from or at risk of developing a disease or infection (e.g., an influenza or HCV infection). Using influenza vaccines as an example, the immunogenic compositions of the invention are typically administered in an amount sufficient to induce an immune response against an influenza virus or group of viruses. For prophylactic use, the immunogenic composition is provided prior to any symptoms (e.g., prior to infection). The prophylactic administration of the immunogenic composition serves to prevent or ameliorate any subsequent infection. Thus, in some embodiments, the subject to be treated is a subject suffering from or at risk of developing an influenza virus infection, e.g., due to exposure or possible exposure to a virus. After administration of a therapeutically effective amount of the disclosed therapeutic compositions, the subject can be monitored for viral infection, symptoms associated with viral infection, or both. For therapeutic use, the immunogenic composition is provided at or after the onset of symptoms of the disease or infection (e.g., after onset of influenza symptoms, or after diagnosis of viral infection). Thus, the immunogenic composition may be provided prior to the intended exposure to the virus to attenuate the severity, duration, or extent of the intended infection and/or associated disease symptoms, after exposure to the virus or suspected exposure to the virus, or after the actual infection has begun. The appropriate amount of vaccine may be determined based on the particular disease or condition to be treated or prevented, the severity, the age of the subject, and other personal attributes of the particular subject (e.g., the overall state of the subject's health and the robustness of the subject's immune system). Determination of an effective dose is also guided by animal model studies followed by human clinical trials and by administration regimens that significantly reduce the occurrence or severity of the targeted disease symptoms or conditions in the subject.

The pharmaceutical compositions of the present invention may be combined with other agents known in the art for treating or preventing diseases or infections (e.g., influenza virus infection). The administration of the pharmaceutical composition and the known antiviral agent may be performed simultaneously or sequentially. Pharmaceutical compositions comprising suitable vaccines of the present invention may be provided as a component of a kit. Optionally, such kits comprise additional components, including packaging, instructions, and a variety of other reagents, such as buffers, substrates, antibodies or ligands (e.g., control antibodies or ligands), and detection reagents. Optional instructions may additionally be provided in the kit.

Examples

The following examples are provided to illustrate, but not to limit, the present invention.

Example 1 rational design of novel I3-01v9a nanoparticle scaffolds

The I3-01v9 nanoparticle scaffold was rationally optimized to achieve optimal surface display of monomeric protein antigen (fig. 1). The N-terminus of I3-01v9 forms a broad triangle that is desirable for displaying monomeric antigen (FIG. 1A). However, the first residue (antigen anchor site) is located below the nanoparticle surface and therefore long linkers must be used to link the antigen to the I3-01v 9N-terminus, which will increase structural instability. The goal is to lengthen the I3-01v 9N-terminal helix so that its first residue is at the same level as the nanoparticle surface. To achieve this goal, a helical backbone from residues 953 to 982 of the c-MYC transcription factor protein (PDB ID:6G 6L) was selected and structure fitted using residues E2 and E3 of I3-01v9, grafted onto the I3-01v9 subunit (SEQ ID NO: 27) (FIG. 1B). The extended N-terminal helix is then truncated to 11 residues such that its first residue is just above the nanoparticle surface (fig. 1C). Next, protein structure sampling program CONCOORD was used to generate 1000 slightly interfering conformations for the modified I3-01v9 subunit (fig. 1D). Thereafter, using the aggregate-based protein design program previously used to optimize HIV gp140 and HCV E2 antigens, the first 9 residues of amino acids in the 11 residue segment were predicted using a RAPDF scoring function based on ca and ca (fig. 1E). By combining the data from predictions using both energy functions, the final design I3-01v9a (SEQ ID NO: 4) was selected (FIG. 1F).

EXAMPLE 2 HCV display of antigen on I3-01v9a nanoparticle scaffold

This example describes multivalent display of HCV E2 cores and tandem E2 cores on an I3-01v9a nanoparticle scaffold. A newly designed I3-01v9a nanoparticle scaffold (SEQ ID NO: 4) has been used to present monomeric HCV E2 cores of different genotypes (FIG. 2A). E2mc3I3-01v9a-LD7-PADRE nanoparticles designed for H77 (genotype 1 a), HCV1 (genotype 1B) and ED43 (genotype 4) showed good nanoparticle formation in negatively stained EM (FIG. 2B). The sequences of the different E2mc3 proteins are shown in SEQ ID NOS 32 to 35, respectively. Based on this outcome, a tandem E2 core antigen was designed in which two HCV E2 cores of different genotypes were connected in tandem with a 5GS linker, and this tandem E2 core antigen was connected to I3-01v9a with an enzymatic restriction site "AS" (for facilitating molecular cloning) and another 5GS linker (fig. 2C). Based on this design strategy, the H77 (genotype 1) and J6 (genotype 2) E2 cores were displayed together on the I3-01v9a-LD7-PADRE nanoparticle, and the ED43 (genotype 4) and UKN3A1.28c (genotype 3) E2 cores were displayed together on the I3-01v9a-LD7-PADRE nanoparticle. Both nanoparticles were expressed in 50ml or 200ml ExpiCHO cells and subsequently characterized by size exclusion chromatography (size-exclusion chromatography, SEC) on a Superose 6 column and by negative staining of EM (fig. 2D). In summary, I3-01v9a has been successfully used to display monomeric antigens for vaccine development.

E2mc3 of H77 isolate (SEQ ID NO: 32):

QLINTNGSWHINSTALNCNESLNTGWLAGLFYQHKFDSSGCPERASGHYPRPCGIVPAKSVCGPVYCFTPSPVVVGTTDRSGAPTYSWGANDTDVFVLNNTGNWFGCTWMNSTGFTKVCGAPPGGPTDGGSGPWITPRCMVDYPYRLWHYPCTINYTIFKVRMYVGGVEHRLEAACN

E2mc3 of J6 isolate (SEQ ID NO:):

QLVNTNGSWHINRTALNCNDSLHTGFIASLFYTHSFNS SGCPERASGHYPRQCGVVSAKTVCGPVYCFTPSPVVVGTTDRLGAPTYTWGENETDVFLLNSTGSWFGCTWMNSSGYTKTCGAPPGGPTDGGSGPWLTPRCLIDYPYRLWHYPCTVNYTIFKIRMYVGGVEHRLTAACN

e2mc3 of ED43 isolate (SEQ ID NO: 34):

QLINSNGSWHINRTALNCNDSLNTGFLASLFYTHKFNSSGCSERASGHYARPCGIVPASSVCGPVYCFTPSPVVVGTTDHVGVPTYTWGENETDVFLLNSTGAWFGCVWMNSTGFTKTCGAPPGGPTDGGSGPWITPRCLIDYPYRLWHFPCTANFSVFNIRTFVGGIEHRMQAACN

E2mc3 of UKN3A1.28c isolate (SEQ ID NO: 35):

QLVNTNGSWHINRTALNCNESINTGFIAGLFYYHKFNSTGCPQRASGHYARPCESVPASKVCGPVYCFTPSPVVVGTTDAKGVPTYTWGANETDVFLLNSLGRWFGCTWMNSTGFTKTCGAPPGGPTDGGAGPWLTPRCMVDYPYRLWHYPCTVNFTLFQVRMFVGGFEHRFTAACN

example 3 design and characterization of a Single hM2e (1 c-SApNP) vaccine

The crystal structure of human M2e (hM 2 e) complexed with monoclonal antibodies Fab65 and Fab148 is available. Fab 65-bound hM2E is folded into a compact conformation comprising the beta-turn (T5-E8) and the 3 ₁₀ helices (I11-W15). Fab 148-bound hM2e adopts a hook conformation with an N-terminal β -turn (S2-T5). A trimer scaffold (PDB ID:1TD 0) was used to present hM2e (S2-D24) with a 5GS spacer (fig. 3A) because 1TD0 was used as a C-terminal motif in previous studies by the inventors to stabilize EBOV GP trimers. Structural modeling showed that two hM2e peptides on the 1TD0 scaffold were measured at P10 across 9.1nm.The hM2E peptide was fused to 24-mer Ferritin (FR) and two "multilayers" 1c-SApNP of E2P-LD4-PADRE (alternatively referred to as E2P-L4P) and I3-01v9a-LD7-PADRE (also referred to as I3-01v9 a-L7P) yielding 20.9nm, 29.1nm and 32.4nm vaccine particles, respectively (FIG. 3A). Four hM2e immunogens (one trimer and three 1 c-SApNP) were transiently expressed in 25ml ExpiCHO cells and purified by immunoaffinity chromatography (immunoaffinity chromatography, IAC) using antibodies Fab65 and Fab148 (fig. 3B). Size Exclusion Chromatography (SEC) spectra of the hM2e scaffold and three 1C-SApNP were obtained on Superdex 75 and Superose 6 columns, respectively (FIG. 3C). Although multiple peaks were observed in SEC, IAC purified 1c-SApNP showed high purity in negatively stained EM images collected at SCRIPPS EM Core, indicating good NP formation (fig. 3D).

EXAMPLE 4 immunization with Single hM2e vaccine and influenza Virus challenge

Immunogenicity and protective efficacy of M2 e-based vaccines were evaluated in a comprehensive mouse study. Briefly, 10 mice/group were immunized by intradermal injection into the footpad with a total of 10 μg (2.5 μg/footpad) of hM2E-5GS-1TD0, hM2E-5GS-FR, hM2E-5GS-E2p-LD4-PADRE, or hM2E-5GS-I3-01v9a-LD7-PADRE mixed with aluminum phosphate. Mice were immunized twice, 3 weeks apart, and blood was collected 2 weeks after each injection. In this study, one group of naive mice was included as a negative control and a second group of mice was immunized with beta-propiolactone (beta-propiolactone, BPL) inactivated PR 8H 1N1 virus (also known as an inactivated H1N1 vaccine) to serve as a positive control. Three weeks after the second immunization, mice were challenged intranasally (i.n.) with 10×ld ₅₀ (semi-lethal dose (determined in previous studies)) of vaccine matched a/Puerto Rico/8/1934 (PR 8) H1N1 virus. Mice were weighed daily and monitored for visible symptoms of infection (including fur pucker, humpback posture and/or reduced activity) 14 days after their infection (dpi). Mice that were significantly in distress or lost 75% of their original body weight were euthanized. After the surviving mice returned to their baseline after immunization, prior to challenge, heterologous IAV challenge was performed with a/Hong Kong/1/1968 (HK 68) H3N2 virus of 10×ld ₅₀ and monitored for 14dpi. Immunization/challenge study protocol is shown in fig. 4A.

After the first challenge (fig. 4B), 8 out of all primary mice as well as 10 mice in the 1TD0 trimer group died within 8 dpi. In contrast, the survival rate of all three groups 1c-SApNP and groups receiving the inactivated PR8H1N1 vaccine was 100%. Mice receiving strain matched PR8H1N1 vaccine lost minimal body weight, began to regain body weight by 6dpi, and regained their original body weight by 14 dpi. Mice receiving hM2e-5GS-FR and hM2e-5GS-I3-01v9a-L7P lost more weight and began to recover at 8dpi, but recovered to their original weights by 14 dpi. Mice receiving the hM2e-5GS-1TD0 trimer lost significantly more body weight than those in group 1 c-SApNP. Two surviving mice began to regain body weight to 9dpi and did not regain their original body weight until 14 dpi. After the second challenge (fig. 4C), 4 of the 9 mice in the group receiving the inactivated PR8H1N1 vaccine died, while all hM2e immunized mice survived the H3N2 challenge. Consistently, mice receiving inactivated H1N1 virus vaccine lost significantly more weight after H3N2 challenge than the hM2e trimer and 1c-SApNP vaccine groups, and began to regain their weight and at a slower rate after one day (at dpi 6). The challenge data thus underscores the effectiveness and broad protection of the M2e1c-SApNP vaccine. To determine if protection of mice vaccinated with hM2e correlated with hM2 e-specific antibody responses, enzyme-linked immunosorbent assay (enzyme-linked immunosorbent assay, ELISA) was performed on mouse serum at w5 (one week prior to the first challenge) using hM2e-5GS-foldon antigen probe (fig. 4D). Foldon (PDB ID:4 NCU) was used in this antigen probe to avoid detection of 1TD 0-specific antibodies in the hM2e-5GS-1TD0 trimer group. As expected, the naive mice did not exhibit an hM2 e-specific response. Notably, mice receiving the inactivated H1N1 virus vaccine also showed no signal, consistent with the low abundance of M2 in virions 32. Although all groups 1c-SApNP exhibited high hM2 e-specific antibody titers, two surviving mice in the 1TD0 trimer group produced a detectable hM2e antibody response. In summary, the results indicate that 1c-SApNP displaying M2e can effectively protect mice from lethal challenge with two different IAVs, and that this protection is closely related to M2 e-specific antibodies.

EXAMPLE 5 sequences of some single hM2e vaccine constructs

The sequences of some of the hM2e vaccines described herein were determined. The complete sequence of the construct based on I3-01v9a is shown below. In this sequence, the underlined sequence indicates the leader sequence (SEQ ID NO: 1). The italic sequence represents 23 residues of hM2e (SEQ ID NO: 2). Two conserved Cys residues in the mutated hM2e sequence in some tandem M2e constructs discussed below are also underlined. It should be noted that the first Met residue is removed from the hM2e sequence inserted into the NP constructs described herein. Thus, while referred to herein (and also in the literature) as Cys17 and Cys19, respectively, based on the original complete hM2e sequence, they are actually residues 16 and 18 in the hM2e sequence present in the vaccine construct. Two bolded and underlined residues represent the restriction sites for PCR. The double underlined sequence indicates the I3-01v9a NP scaffold subunit sequence (SEQ ID NO: 4). The construct may optionally further comprise a locking domain and/or a T cell epitope. As exemplified in the constructs herein, the locking domain used may be LD7 (SEQ ID NO: 5) (shown in double underlined and bold), and the T cell epitope may be a PADRE epitope (SEQ ID NO: 6) (shown in double underlined and italic). The linkers or spacers separating the different structural motifs of the nanoparticle constructs are shown in italics and underlined residues in the construct sequences herein, e.g., GS, GGGGGGGG spacers (SEQ ID NO: 9) and 5GS linkers (SEQ ID NO: 3).

HM2e-5GS-I3-01v9a-LD7-PADRE construct without N-terminal leader and LD/PADRE motif (SEQ ID NO: 10)

HM2e-5GS-I3-01v9a-LD7-PADRE construct comprising the C-terminal LD/PADRE motif (SEQ ID NO: 13)

HM2e-5GS-I3-01v9a-LD7-PADRE construct comprising an N-terminal leader (SEQ ID NO: 16)

HM2e-5GS-I3-01v9a-LD7-PADRE construct (SEQ ID NO: 19) comprising an N-terminal leader and a C-terminal LD/PADRE motif

EXAMPLE 6 design, characterization and challenge Studies of tandem M2e vaccines

Phylogenetic analysis avian, porcine or human split into several lineages according to the original host species of IAV. Although M2e is highly conserved, there are small but important sequence differences between IAVs from different species, which have been demonstrated to limit cross-protection. Thus, in addition to seasonal endemic strains, a universal influenza vaccine based on M2e must provide protection against pandemic strains typically derived from avian or porcine IAVs. The use of combinations of M2e sequences from multiple species has been previously reported. Here, tandem M2eX3 constructs comprising human, avian/porcine and human/porcine M2e sequences were designed with GGGG (SEQ ID NO: 9) spacers between consecutive M2e segments. Notably, cys17 and Cys19 in the second (avian/porcine) and third (human/porcine) repeats were mutated to serine to avoid random disulfide bonds. Optionally, cys17 and Cys19 in all three repeats may be mutated to serine, as exemplified herein. The M2 eX3 antigen is fused to lTD0 and three 1c-SApNP through a 5GS spacer, yielding a nucleic acid sequence known as

Four constructs of M2eX3-5 GS-1TD0, M2eX3-5 GS-FR, M2eX3-5 GS-E2P-LD4-PADRE (or M2eX3-5 GS-E2P-L4P), and M2eX3-5 GS-I3-01v9a-LD7-PADRE (or M2eX3-5 GS-I3-01v9 a-L7P). These four tandem M2e immunogens were transiently expressed in ExpiCHO cells and purified by IAC using Fab148 antibody columns. The Fab148 purified 1c-SApNP samples were analyzed using negative staining EM at SCRIPPS EM Core. Consistent with hM2e 1c-SApNP, all of the tandem M2e 1c-SApNP showed good NP formation (FIG. 5A).

The immunogenicity and protective efficacy of the tandem M2e vaccine was evaluated in a mouse study following a protocol similar to the study of the hM2e immunogen (fig. 4A). Two adjuvants, aluminum hydroxide (aluminum hydroxide, AH) and oil-in-water emulsion AddaVax were tested in this study. For the AH adjuvant group (fig. 5B), 4 out of all naive mice as well as 8 mice in the 1TD0 trimer group died within 9dpi after PR 8H 1N1 challenge. In contrast, the survival rate of FR 1c-SApNP was 88% and the survival rate of the two large multi-layered 1c-SApNP groups and the group receiving the inactivated PR 8H 1N1 vaccine was 100%. In terms of weight loss, AH formulated E2p 1c-SApNP showed closest approach to inactivated vaccine and was more effective than other tandem M2E immunogens. For the AddaVax adjuvant group (fig. 5C), after PR 8H 1N1 challenge, 3 out of all naive mice as well as 8 mice in the 1TD0 trimer group died within 9 dpi. In contrast, the survival rate of all groups 1c-SApNP and groups receiving the inactivated PR 8H 1N1 vaccine was 100%. In terms of weight loss, addaVax formulated I3-01v9a 1c-SApNP was shown to be closest to the inactivated vaccine and more effective than the other tandem M2e immunogens. The formulation also outperformed the E2p/AH formulation in preventing weight loss (fig. 5B). Overall, tandem M2e immunogens showed broad protection, with tandem M2e-5GS-1TD0 trimer significantly better than its hM2e counterpart, and when paired with AddaVax, I3-013 a 1c-SApNP was the best performing for all immunogens.

EXAMPLE 7 sequences of several tandem M2eX3 immunogen constructs

The amino acid sequences (SEQ ID NOS: 11 and 12) of two exemplary tandem M2E vaccine constructs (M2eX3-5 GS-I3-01v9a-LD7-PADRE; also known as M2EX3-5 GS-I3-01v9 a-L7P) are shown below. Each of these two constructs contained a rationally designed I3-01v9a variant scaffold (SEQ ID NO: 4) and a tandem M2e molecule with 3M 2e sequences (SEQ ID NO:23 or SEQ ID NO: 24). These 3M 2e sequences are respectively human M2e sequence SLLTEVETPIRNEWGCRCNDSSD (SEQ ID NO:2; underlined Cys17 and Cys 19), avian/porcine consensus M2e sequence SLLTEVETPTRNGWECKCSDSSD (SEQ ID NO:7; underlined Cys17 and Cys 19) and human/porcine consensus M2e sequence SLLTEVETPTRSEWECRCSGSSD (SEQ ID NO:8; underlined Cys17 and Cys 19). In addition, both constructs have conserved Cys17 and Cys19 residues replaced by Ser residues in 2 or all 3 of the M2e tandem repeats, respectively. In addition to the NP scaffold sequence and the displayed tandem M2e molecule, the construct also comprises a locking domain LD7 (SEQ ID NO: 5) and a universal PADRE T cell epitope (SEQ ID NO: 6). Finally, each of the constructs may additionally have a leader sequence at the N-terminus. The leader sequence may comprise MGILPSPGMPALLSLVSLLSVLLMGCVAE (SEQ ID NO: 1) as exemplified herein.

Tandem M2e polypeptide having a conserved CYs residue mutated in repeats 2 and 3 (SEQ ID NO: 23):

tandem M2e polypeptide with all 3 mutated conserved CYs residues in the repeat (SEQ ID NO: 24):

In each of the two exemplified tandem M2e NP construct sequences shown below, each of the 3 tandem M2e sequences is italicized. Conserved Cys 17 and Cys19 residues or substituted Ser residues in the M2e sequence are also underlined. Two bolded and underlined residues represent the restriction sites for PCR. The linkers or spacers linking the different motifs of the M2e sequences and/or constructs are italicized and underlined. These include the GS linker GGGGS (SEQ ID NO: 3) and GGGGGG (SEQ ID NO: 9) spacers separating the 5aa tandem M2e sequences. The double underlined sequence indicates that the I3-01v9a scaffold sequence is displayed. The sequence of the locking domain (LD 7) is double underlined and bolded. Finally, PATRE T cell epitopes are shown in double underlined and italics.

Tandem M2eNP construct with Cys17/CYs19 mutated to serine in M2e repeats #2 and #3, without N-terminal leader and C-terminal LD/PADRE motif (SEQ ID NO: 11):

A tandem M2eNP construct with Cys17/Cys19 mutated to serine in M2e repeats #2 and #3 comprising a C-terminal LD/PADRE motif (SEQ ID NO: 14):

a tandem M2e NP construct with Cys17/Cys19 mutated in 2M 2e repeats, comprising an N-terminal leader sequence (SEQ ID NO: 17):

A tandem M2e NP construct with Cysl/CYsl 9 mutated in 2M 2e repeats comprising an N-terminal leader sequence and a C-terminal LD/PADRE motif (SEQ ID NO: 20):

Tandem M2eNP construct with Cys17/CYs19 mutated to serine in all 3M 2e repeats, without N-terminal leader and C-terminal LD/PADRE motif (SEQ ID NO: 12):

A tandem M2eNP construct with Cys17/Cys19 mutated to serine in all 3M 2e repeats, comprising a C-terminal LD/PADRE motif (SEQ ID NO: 15):

A tandem M2eNP construct with Cys17/Cys19 mutated to serine in all 3M 2e repeats, comprising an N-terminal leader sequence (SEQ ID NO: 18):

A tandem M2eNP construct with Cys17/Cys19 mutated to serine in all 3M 2e repeats, comprising an N-terminal leader sequence and a C-terminal LD/PADRE motif (SEQ ID NO: 21):

***

accordingly, the present invention has been widely disclosed and illustrated with reference to the above-described representative embodiments. It will be understood that various modifications may be made without departing from the spirit and scope of the invention.

It should also be noted that all publications, serial numbers, patents and patent applications cited herein are hereby incorporated by reference in their entirety and for all purposes as if each were individually and so indicated. To the extent that conflicts with definitions in this disclosure, the definitions contained in the text incorporated by reference are excluded.

Claims (42)

1. An N-terminally extended I3-01 nanoparticle scaffold sequence comprising an N-terminal helix extended compared to the N-terminal helix in the original I3-01 scaffold sequence. 2. The N-terminally extended I3-01 nanoparticle scaffold sequence of claim 1, comprising a heterologous helical motif of about 6 to about 12 amino acid residues, which is fused to the N-terminus of the I3-01 scaffold sequence shown in SEQ ID NO: 27. 3. The N-terminally extended I3-01 nanoparticle scaffold sequence of claim 2, wherein the heterologous helical motif comprises AKLAEELQK (SEQ ID NO: 25), a conservatively modified variant thereof, or a substantially identical sequence. 4. The N-terminally extended I3-01 nanoparticle scaffold sequence of claim 2, comprising SEQ ID NO: 4, a conservatively modified variant thereof, or a substantially identical sequence. 5. Self-assembling nanoparticles, which are formed by the N-terminally extended I3-01 nanoparticle scaffold sequence according to claim 1. 6. A nanoparticle vaccine construct comprising a polypeptide immunogen fused to an N-terminally extended I3-01 nanoparticle scaffold sequence, wherein the N-terminally extended I3-01 nanoparticle scaffold sequence comprises an extended N-terminal helix compared to the N-terminal helix in the original I3-01 scaffold sequence. 7. The nanoparticle vaccine construct of claim 6, wherein the N-terminally extended I3-01 nanoparticle scaffold sequence comprises a heterologous helical motif of about 6 to about 12 amino acid residues, which is fused to the N-terminus of the I3-01 scaffold sequence shown in SEQ ID NO: 27. 8. The nanoparticle vaccine construct of claim 6, wherein the heterologous helical motif comprises AKLAEELQK (SEQ ID NO: 25), a conservatively modified variant thereof, or a substantially identical sequence. 9. The nanoparticle vaccine construct of claim 6, wherein the N-terminally extended I3-01 nanoparticle scaffold sequence comprises SEQ ID NO: 4, a conservatively modified variant thereof, or a substantially identical sequence. 10. The nanoparticle vaccine construct of claim 6, wherein the polypeptide immunogen is fused to the N-terminus of the N-terminally extended I3-01 nanoparticle scaffold sequence via a linker at its C-terminus. 11. The nanoparticle vaccine construct of claim 10, wherein the linker comprises GGGGS (SEQ ID NO: 3). 12. The nanoparticle vaccine construct of claim 6, wherein the polypeptide immunogen comprises (a) an influenza fusion polypeptide comprising two or more tandem repeats of the influenza M2 protein extracellular domain (M2e) or (b) an HCV immunogenic protein. 13. The nanoparticle vaccine construct of claim 12, wherein the tandem M2e repeats are separated by a peptide spacer. 14. The nanoparticle-displayed immunogenic protein of claim 13, wherein the peptide spacer comprises GGGG (SEQ ID NO: 9). 15. The nanoparticle vaccine construct of claim 12, wherein at least one M2e tandem repeat comprises a missense mutation at the conserved Cys17 and Cys19 residues. 16. The nanoparticle vaccine construct of claim 15, wherein the missense mutation comprises replacing each of two Cys residues with an amino acid residue having an uncharged polar side chain. 17. The nanoparticle vaccine construct of claim 15, wherein each of the Cys residues is independently replaced with an amino acid residue selected from the group consisting of serine, glycine, asparagine, glutamine, threonine, and tyrosine. 18. The nanoparticle vaccine construct of claim 15, wherein both of the Cys residues are replaced by Ser. 19. The nanoparticle vaccine construct of claim 12, wherein the influenza fusion polypeptide comprises three tandem M2e sequences. 20. The nanoparticle vaccine construct of claim 19, wherein the three tandem M2e sequences are independently human M2e sequences, avian/porcine consensus M2e sequences, or human/porcine consensus M2e sequences, except for missense mutations at residues Cys17 and Cys19 in at least two of the three tandem M2e sequences. 21. The nanoparticle vaccine construct of claim 20, wherein the influenza fusion polypeptide comprises, in any order, human M2e (SEQ ID NO: 2), an avian/porcine consensus M2e sequence in which Cys17 and Cys19 are each replaced by a Ser residue (SEQ ID NO: 30), and a human/porcine consensus M2e sequence in which Cys17 and Cys19 are each replaced by a Ser residue (SEQ ID NO: 31). 22. The nanoparticle vaccine construct of claim 20, wherein the influenza fusion polypeptide comprises, in any order, human M2e in which Cys17 and Cys19 are each replaced with a Ser residue (SEQ ID NO: 29), an avian/porcine consensus M2e sequence in which Cys17 and Cys19 are each replaced with a Ser residue (SEQ ID NO: 30), and a human/porcine consensus M2e sequence in which Cys17 and Cys19 are each replaced with a Ser residue (SEQ ID NO: 31). 23. The nanoparticle vaccine construct of claim 20, wherein the influenza fusion polypeptide comprises SEQ ID NO: 23, SEQ ID NO: 24, conservatively modified variants thereof, or substantially identical sequences. 24. The nanoparticle vaccine construct of claim 20, comprising the sequence set forth in SEQ ID NO: 11, SEQ ID NO: 12, conservatively modified variants thereof, or substantially identical sequences. 25. The nanoparticle vaccine construct of claim 20, further comprising a T cell epitope and a locking domain at the C-terminus. 26. The nanoparticle vaccine construct of claim 25, wherein the locking domain comprises SEQ ID NO: 5 and the T cell epitope comprises SEQ ID NO: 6. 27. The nanoparticle vaccine construct of claim 25, comprising SEQ ID NO: 14, SEQ ID NO: 15, conservatively modified variants thereof, or substantially identical sequences. 28. The nanoparticle vaccine construct of claim 20, further comprising an N-terminal leader sequence. 29. The nanoparticle vaccine construct of claim 28, comprising SEQ ID NO: 17, SEQ ID NO: 18, conservatively modified variants thereof, or substantially identical sequences. 30. The nanoparticle vaccine construct of claim 20, further comprising an N-terminal leader sequence and a T cell epitope and locking domain at the C-terminus. 31. The nanoparticle vaccine construct of claim 30, comprising SEQ ID NO: 20, SEQ ID NO: 21, conservatively modified variants thereof, or substantially identical sequences. 32. The nanoparticle vaccine construct of claim 12, wherein the HCV immunogenic protein comprises E2 core or E1E2 dimer. 33. The nanoparticle vaccine construct of claim 32, wherein the HCV E2 core comprises any one of SEQ ID NOs: 32 to 35. 34. The nanoparticle vaccine construct of claim 32, wherein the HCV immunogenic protein comprises two tandem copies of the E2 core sequence. 35. The nanoparticle vaccine construct of claim 34, wherein the two E2 core sequences are from different HCV isolates. 36. The nanoparticle vaccine construct of claim 34, wherein the two E2 core sequences comprise SEQ ID NOs: 32 and 33 or SEQ ID NOs: 34 and 35, respectively. 37. The nanoparticle vaccine construct of claim 34, further comprising a T cell epitope and a locking domain at the C-terminus. 38. The nanoparticle vaccine construct of claim 37, wherein the locking domain comprises SEQ ID NO: 5 and the T cell epitope comprises SEQ ID NO: 6. 39. The nanoparticle vaccine construct of claim 37, comprising (1) a subunit sequence comprising, from N-terminus to C-terminus, (a) SEQ ID NO: 4, SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 5, and SEQ ID NO: 6 or (b) SEQ ID NO: 4, SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 5, and SEQ ID NO: 6; or (2) a conservatively modified variant of the subunit sequence or a substantially identical sequence. 40. A polynucleotide sequence encoding a subunit sequence of the nanoparticle vaccine construct of claim 6. 41. A vector comprising the polynucleotide sequence of claim 40. 42. A pharmaceutical composition comprising the nanoparticle vaccine construct of claim 6 or the polynucleotide sequence of claim 40.