A broadly protective mosaic vaccine against highly pathogenic avian influenza viruses

Abstract

Disclosed herein are mosaic avian influenza (e.g., H5N1) proteins or fragments thereof. Also disclosed herein are nucleic acid constructs, avian influenza vaccine vectors, avian influenza vaccines, host cells, pharmaceutical compositions, and kits comprising the mosaic avian influenza (e.g., H5N1) proteins or fragments thereof. Also disclosed herein are methods of eliciting an immune response in a subject and methods of preventing, reducing the incidence of, attenuating, or treating avian influenza infection in a subject in need thereof.

Description

A BROADLY PROTECTIVE MOSAIC VACCINE AGAINST HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUSESCROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application incorporates by reference in their entirety U.S. Provisional Application No. 63 / 673,019, filed July 18, 2024, and U.S. Provisional Application No. 63 / 756,643, filed February 10, 2025.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The content of the electronically submitted sequence listing in xml file (Name:4997_006PC02_Seqlisting_ST26.xml; Size: 25,866 bytes; and Date of Creation: July 9, 2025) filed with the application is incorporated herein by reference in its entirety.FIELD

[0003] The present disclosure provides vaccine compositions and methods for eliciting an immune response in an avian subject against one or more avian influenza antigens and / or for preventing, reducing the incidence of, attenuating, or treating avian influenza in an avian subject.BACKGROUND OF THE DISCLOSURE

[0004] Influenza A viruses are widely distributed in nature and their ability to undergo constant changes through antigenic drift and shift raises one of the most important public health concerns. Since its first detection in 1996, the highly pathogenic avian influenza (HP Al) H5N1 lineage has evolved into major genetic groups, leading to the high prevalent 2.3.4.4.b clade responsible of the largest avian influenza outbreak in the US.

[0005] Influenza A virus is a member of the Orthomyxoviridae family of segmented, negative-sense, single-stranded RNA viruses. Virions are enveloped and pleomorphic; they are generally spherical or elliptical in shape, ranging from approximately 80-120 nm in diameter, but are occasionally filamentous, reaching more than 20 pm in length, or irregular. The genome ranges from 10.0 to 14.6 kb in length and comprises 6 to 8 single-stranded, negative-sense RNA segments: polymerase basic protein 2 (PB2), polymerase basic protein 1 (PB1), polymerase acidic (PA), hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix (M), and non-structural (NS). These segments code for 10 structural and 9 regulatory proteins. See Public Health Agency of Canada, Influenza A virus subtypes H5, H7, and H9: Infectious substances pathogen safety data sheet, March 2023, Canada. ca / en / public-health / services / laboratory -biosafety -biosecurity / pathogen- safety-data-sheets-ri sk-assessment / influenza-a-virus-subtypes-h5 -h7 -h9. html ; see also ICTV 9thReport, Orthomyxoviridae, 2011, i ctv . gl ob al / report_9th / RN Aneg / Orthomyxoviridae .

[0006] Influenza A viruses (lAVs) are divided into subtypes based on the antigenicity of their membrane-bound surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Eighteen distinct HA subtypes (H1-H18) and eleven NA subtypes (Nl-Nl 1) have been identified that circulate in wild bird populations. There are three primary HA subtypes (Hl, H2, H3) and two NA subtypes (N1 and N2) that have established stable lineages in the human population. Humans may be sporadically infected by lAVs of the H5, H7, and H9 subtypes. lAVs are further divided into highly pathogenic avian influenza (HP Al) and low pathogenic avian influenza (LPAI), which refers to their ability to cause disease in domestic poultry (e.g., Chickens). All HP Al viruses identified to date belong to the H5 and H7 subtypes. Id.

[0007] The HA, PB2, NS1 and PB1-F2 proteins are known determinants of host-range restriction and pathogenicity, along with other contributing viral proteins. In avian lAVs, HA is a critical determinant of pathogenicity. The HA proteins of HP Al viruses contain multiple basic amino acids at the cleavage site, forming a polybasic motif that is recognized by ubiquitous proteases; the presence of a polybasic HA cleaving site greatly expands IAV tropism in poultry, thereby leading to systemic infection in poultry. Conversely, HA proteins of LPAI viruses and non-avian lAVs contain a single arginine residue at the HA cleavage site and are only cleaved in a few organs. Reassortment, the swapping of gene segments between alphainfluenza viruses during co-infection, occurs frequently and generates viruses with significantly altered antigenicity and pathogenicity. lAVs also undergo antigenic drift, the gradual change in the HA and / or NA proteins as a result of accumulation of point mutations in the antigenic epitopes, further contributing to virus evolution. Id.

[0008] The influenza virus infection cycle starts with the attachment of virus through the HA glycoprotein to sialic acid-containing glycan receptors on the host cell surface. Avian lAVs preferentially bind the avian a2,3-galactose linked sialic acid receptors, whereas mammalian lAVs preferentially bind the mammalian a2,6-galactose linked sialic acid receptors. The virus is then internalized and, after fusion of the viral envelope to the endosomal membrane, viral RNAs are translocated into the cell nucleus, transcribed, and replicated to direct the production of new viral components. The NA is critically important during the final stages of infection, where it cleaves off sialic acid from glycans on the target host cell, as well as from newly formed budding virions, thus preventing virus aggregation and facilitating progeny release from the host cell. Host specificity among lAVs is due to the type of sialic acid receptor on the cell surface. Id.

[0009] HA is a type 1 transmembrane protein that is assembled as a homotrimer in the endoplasmic reticulum and transported to the plasma membrane via the secretory pathway. HA is further cleaved into HA1 and HA2 by a protease provided by the host system. The two subunits remain linked by a disulphide bridge. Structurally, each subunit consists of a membrane-proximal helix-rich stem structure primarily composed of HA2 with some HA1 residues, and a membrane-distal receptor-binding globular domain (RBD) comprised of HA1. In addition to the RBD, HA1 also comprises a Vestigial Esterase domain (VE) and a fusion peptide (F). See Zheng Z., et al., The Vestigial Esterase Domain of Haemagglutinin of H5N1 Avian Influenza A Virus: Antigenicity and Contribution to Viral Pathogenesis, Vaccines (Basel) 6(3): 53 (2018).

[0010] Currently, several veterinary vaccine candidates are at different development stages to protect birds against avian influenza, however, the efficacy studies have revealed limitations regarding the administration route and the evolving dynamic circulation of the virus.

[0011] Therefore, a need remains for preventative strategies for avian influenza and for broadly protective vaccines that will protect against both known highly pathogenic avian influenza strains (e.g., H5N1), but also against new viruses that emerge via zoonotic transfer.BRIEF SUMMARY

[0012] In some aspects, provided herein is a nucleic acid construct comprising a nucleic acid sequence encoding a mosaic H5 protein or fragment thereof.

[0013] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0014] In some aspects, the nucleic acid sequence has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 6.

[0015] In some aspects, provided herein is a nucleic acid construct comprising one or more nucleic acid sequences selected from the group consisting of (i) a first nucleic acid sequence encoding a first mosaic H5 protein or fragment thereof; (ii) a first nucleic acid sequence encoding a first mosaic H5 protein or antigenic fragment thereof and a second nucleic acid sequence encoding a second mosaic H5 protein or fragment thereof; and (iii) combinations thereof.

[0016] In some aspects, the first mosaic H5 protein or fragment thereof encodes an H5 hemagglutinin (HA) protein or fragment thereof.

[0017] In some aspects, the first mosaic H5 HA protein or fragment thereof is a fragment of the Hl HA protein. In some aspects, the fragment comprises an HA1 region and / or an HA2 region.

[0018] In some aspects, the second mosaic H5 protein or fragment thereof encodes an H5 hemagglutinin (HA) protein or fragment thereof.

[0019] In some aspects, the second mosaic H5 HA protein or fragment thereof is a fragment of the H5 HA protein. In some aspects, the fragment comprises an HA1 region and / or an HA2 region.

[0020] In some aspects, the first mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3 or 9.

[0021] In some aspects, the second mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0022] In some aspects, the first nucleic acid sequence has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 6 or 13.

[0023] In some aspects, the second nucleic acid sequence has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 14.

[0024] In some aspects, the nucleic acid construct further comprises a promoter. In some aspects, the promoter is an S E / L promoter.

[0025] In some aspects, provided herein is a mosaic H5 protein or fragment thereof, wherein the mosaic H5 protein or fragment thereof comprises an H5 hemagglutinin (HA) protein or fragment thereof.

[0026] In some aspects, the H5 HA protein or fragment thereof is a fragment of the H5 HA protein. In some aspects, the fragment comprises an HA1 region and / or an HA2 region.

[0027] In some aspects, the fragment comprises an HA1 region.

[0028] In some aspects, the fragment comprises an HA2 region.

[0029] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0030] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9.

[0031] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0032] In some aspects, provided herein is a pharmaceutical composition comprising i) any of the nucleic acid constructs described herein or any of the mosaic H5 proteins or fragments thereof described herein; and ii) one or more pharmaceutically acceptable excipients or carriers.

[0033] In some aspects, provided herein is an isolated host cell comprising any of the nucleic acid constructs described herein or any of the mosaic H5 proteins or fragments thereof described herein.

[0034] In some aspects, the host cell is a eukaryotic host cell.

[0035] In some aspects, the host cell is an avian host cell.

[0036] In some aspects, provided herein is an avian influenza vaccine vector comprising any of the nucleic acid constructs described herein.

[0037] In some aspects, provided herein is an avian influenza vaccine vector comprising a polynucleotide encoding a mosaic H5 protein or fragment thereof.

[0038] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0039] In some aspects, wherein the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9.

[0040] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0041] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 6.

[0042] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 13.

[0043] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 14.

[0044] In some aspects, the avian influenza vaccine vector further comprises a promoter. In some aspects, the promoter is an S E / L promoter.

[0045] In some aspects, the avian influenza vaccine vector is a viral vector.

[0046] In some aspects, the viral vector is a poxvirus vector.

[0047] In some aspects, the poxvirus vector is a vaccinia virus vector.

[0048] In some aspects, the vaccinia virus vector is a modified vaccinia Ankara (MV A) vector.

[0049] In some aspects, provided herein is an avian influenza vaccine comprising a polynucleotide encoding a mosaic H5 protein or fragment thereof and one or more carriers.

[0050] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0051] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 sequence identity to the amino acid sequence of SEQ ID NO: 9.

[0052] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0053] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 6.

[0054] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 13.

[0055] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 14.

[0056] In some aspects, the carrier is a poxvirus.

[0057] In some aspects, the poxvirus is a vaccinia virus.

[0058] In some aspects, the vaccinia virus is a MVA virus.

[0059] In some aspects, provided herein is a kit comprising any of the nucleic acid constructs described herein, any of the mosaic H5 proteins or fragments thereof described herein, any of the pharmaceutical compositions described herein, any of the avianinfluenza vaccine vectors described herein, or any of the avian influenza vaccines described herein.

[0060] In some aspects, provided herein is a method of eliciting an immune response in an avian subject against one or more avian influenza antigens, the method comprising administering one or more doses of any of the pharmaceutical compositions described herein, any of the avian influenza vaccine vectors described herein, or any of the avian influenza vaccines described herein to the avian subject.

[0061] In some aspects, the method elicits a neutralizing antibody response against avian influenza in the avian subject.

[0062] In some aspects, the avian influenza is an H5N1 strain.

[0063] In some aspects, the H5N1 strain is a member of a clade selected from the group consisting of clade 2.2, clade 2.3.4.4 (including sub lineages 2.3.4.4a, 2.3.4.4b, 2.3.4.4c, 2.3.4.4d, 2.3.4.4e, or 2.3.4.4f), and clade 2.5.

[0064] In some aspects, provided herein is a method of preventing, reducing the incidence of, attenuating, or treating avian influenza infection in an avian subject in need thereof, the method comprising administering one or more doses of any of the pharmaceutical compositions described herein, any of the avian influenza vaccine vectors described herein, or any of the avian influenza vaccines described herein to the avian subject.

[0065] In some aspects, the avian infection is caused by an H5N1 strain.

[0066] In some aspects, the H5N1 strain is a member of a clade selected from the group consisting of clade 2.2, clade 2.3.4.4 (including sub lineages 2.3.4.4a, 2.3.4.4b, 2.3.4.4c, 2.3.4.4d, 2.3.4.4e, or 2.3.4.4f), and clade 2.5.

[0067] In some aspects, the one or more doses of the pharmaceutical composition, the avian influenza vaccine vector, or avian influenza vaccine are administered to the avian subject by mucosal route of administration.

[0068] In some aspects, the avian subject is a domestic poultry.

[0069] In some aspects, the domestic poultry is selected from the group consisting of chickens, ducks, turkeys, quails, geese, or guinea fowls.

[0070] In some aspects, provided herein is a method of eliciting an immune response in a mammalian subject against one or more avian influenza antigens, the method comprising administering one or more doses of any of the pharmaceutical compositions, any of theavian influenza vaccine vectors, or any of the avian influenza vaccines described herein to the mammalian subject.

[0071] In some aspects, the method elicits a neutralizing antibody response against avian influenza in the mammalian subject.

[0072] In some aspects, the avian influenza is an H5N1 strain.

[0073] In some aspects, the H5N1 strain is a member of a clade selected from the group consisting of clade 2.2, clade 2.3.4.4 (including sub lineages 2.3.4.4a, 2.3.4.4b, 2.3.4.4c, 2.3.4.4d, 2.3.4.4e, or 2.3.4.4f), and clade 2.5.

[0074] In some aspects, provided herein is a method of preventing, reducing the incidence of, attenuating, or treating avian influenza infection in a mammalian subject in need thereof, the method comprising administering one or more doses of any of the pharmaceutical compositions, any of the avian influenza vaccine vectors, or any of the avian influenza vaccines described herein to the mammalian subject.

[0075] In some aspects, the avian infection is caused by an H5N1 strain.

[0076] In some aspects, the H5N1 strain is a member of a clade selected from the group consisting of clade 2.2, clade 2.3.4.4 (including sub lineages 2.3.4.4a, 2.3.4.4b, 2.3.4.4c, 2.3.4.4d, 2.3.4.4e, or 2.3.4.4f), and clade 2.5.

[0077] In some aspects, the one or more doses of the pharmaceutical composition, the avian influenza vaccine vector, or avian influenza vaccine are administered to the mammalian subject by an oral, intranasal, subcutaneous, or intramuscular route of administration.

[0078] In some aspects, the mammalian subject is a human subject.

[0079] In some aspects, provided herein is a composition comprising a lipid nanoparticle and a messenger RNA (mRNA) comprising an open reading frame (ORF) that comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14; wherein the nucleotide sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0080] In some aspects, the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

[0081] In some aspects, the lipid nanoparticle comprises a PEG-modified lipid, a noncationic lipid, a sterol, an ionizable cationic lipid, or any combination thereof.

[0082] In some aspects, the mRNA comprises a 5' untranslated region (UTR) and a 3' UTR.

[0083] In some aspects, the mRNA comprises a chemical modification.BRIEF DESCRIPTION OF THE DRAWINGS

[0084] Figure 1 (FIG. 1) shows a comparison of the hemagglutinin protein of H5N1 outbreak strains and designed mosaics. Amino acid positions are denoted above the mosaic protein map (e.g., position 3, 8, 10, 11, 51, 52 ...). Dashes indicate deletions.

[0085] Figures 2A-2C (FIGs. 2A-2C) shows Van der Waals surfaces, computed from the HA 4th protein structures for: the proposed second-generation (H5M2) mosaic (FIG. 2A), the MVA-H5M vaccine (FIG. 2B) relative to the recently emerged H5N1 virus (overlap indicated by the darker shading), and the proposed second-generation (H5M3) mosaic (FIG. 3 A). The mosaics are aligned with the circulating strains to qualify and quantify coverage, identity and molecular similarities.

[0086] Figure 3 (FIG. 3) shows a phylogenetic tree of H5N1 sequences to classify clades of H5M (clade 2.5), H5M2 (clade 2.2), and H5M3 (clade 2.3.4.4b).

[0087] Figure 4 (FIG. 4) shows survival rate of White Leghorn SPF chickens after administration with 1) the MVA-H5M3 vaccine, 2) formulation #1 of the self-amplifying mRNA (saRNA)-H5M3 vaccine at low dose (0.1 pg), medium dose (0.5 pg), or high dose (1 pg), 3) formulation #2 of the sa-RNA-H5M3 vaccine, or 4) an inactivated H5N1 virus. The chickens received an initial administration 4 week prior to infection and a boost 2 weeks prior to infection. After the prime and boost with the vaccine, the chickens were infected with H5N1 and survival rates were tracked for two weeks. Unvaccinated chickens were used as a control.

[0088] Figures 5A-5B (FIGs. 5A-5B) show clinical signs of White Leghorn SPF chickens after administration with 1) the MVA-H5M3 vaccine, 2) formulation #1 of the self-amplifying mRNA (saRNA)-H5M3 vaccine at low dose (0.1 pg), medium dose (0.5 pg), or high dose (1 pg), 3) formulation #2 of the sa-RNA-H5M3 vaccine, or 4) an inactivated H5N1 virus. The chickens received an initial administration 6 weeks prior to infection and a boost 2 weeks prior to infection. After the prime and boost with thevaccine, the chickens were infected with H5N1 and survival rates were tracked for two weeks. Both vaccinated (FIG. 5A) and unvaccinated (FIG. 5B) were observed.

[0089] Figures 6A-6B (FIGs. 6A-6B) show viral shedding in the oropharynx (FIG. 6A) and cloaca (FIG. 6B) of White Leghorn SPF chickens after administration with 1) the MVA-H5M3 vaccine, 2) formulation #1 of the self-amplifying mRNA (saRNA)-H5M3 vaccine at low dose (0.1 pg), medium dose (0.5 pg), or high dose (1 pg), 3) formulation #2 of the sa-RNA-H5M3 vaccine, or 4) an inactivated H5N1 virus. The chickens received an initial administration 6 weeks prior to infection and a boost 2 weeks prior to infection. After the prime and boost with the vaccine, the chickens were infected with H5N1 and survival rates were tracked for two weeks.

[0090] Figure 7 (FIG. 7) shows hemagglutination inhibiting (HI) antibody titer of White Leghorn SPF chickens after administration with 1) the MVA-H5M3 vaccine, 2) formulation #1 of the self-amplifying mRNA (saRNA)-H5M3 vaccine at low dose (0.1 pg), medium dose (0.5 pg), or high dose (1 pg), 3) formulation #2 of the sa-RNA-H5M3 vaccine, or 4) an inactivated H5N1 virus. The chickens received an initial administration 6 weeks prior to infection and a boost 2 weeks prior to infection. After the prime and boost with the vaccine, the chickens were infected with H5N 1 and survival rates were tracked for two weeks.

[0091] Figure 8 (FIG. 8) shows H5-specific antibodies and nucleoprotein (NP) specific antibodies of White Leghorn SPF chickens after administration with 1) the MVA-H5M3 vaccine, 2) formulation #1 of the self-amplifying mRNA (saRNA)-H5M3 vaccine at low dose (0.1 pg), medium dose (0.5 pg), or high dose (1 pg), 3) formulation #2 of the sa- RNA-H5M3 vaccine, or 4) an inactivated H5N1 virus. The chickens received an initial administration 6 weeks prior to infection and a boost 2 weeks prior to infection. After the prime and boost with the vaccine, the chickens were infected with H5N1 and survival rates were tracked for two weeks.DETAILED DESCRIPTION OF THE DISCLOSURE

[0092] In some aspects, provided herein is a nucleic acid construct comprising a nucleic acid sequence encoding a mosaic H5 protein or fragment thereof.

[0093] In some aspects, provided herein is a nucleic acid construct comprising one or more nucleic acid sequences selected from the group consisting of: (i) a first nucleic acidsequence encoding a first mosaic H5 protein or fragment thereof; (ii) a first nucleic acid sequence encoding a first mosaic H5 protein or antigenic fragment thereof and a second nucleic acid sequence encoding a second mosaic H5 protein or fragment thereof; and (iii) combinations thereof.

[0094] In some aspects, provided herein is a mosaic H5 protein or fragment thereof. In some aspects, the mosaic H5 protein or fragment thereof comprises an H5 hemagglutinin (HA) protein or fragment thereof.

[0095] In some aspects, provided herein is an avian influenza vaccine vector comprising a polynucleotide encoding a mosaic H5 protein or fragment thereof.

[0096] In some aspects, provided herein is an avian influenza vaccine comprising a polynucleotide encoding a mosaic H5 protein or fragment thereof and one or more carriers.I. Definitions

[0097] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0098] The term "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0099] It is understood that wherever aspects are described herein with the language "comprising," otherwise analogous aspects described in terms of "consisting of and / or "consisting essentially of' are also provided.

[0100] As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value and within a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwiseevident from the context (except where such number would exceed 100% of a possible value). When the term "approximately" or "about" is applied herein to a particular value, the value without the term "approximately" or "about" is also disclosed herein.

[0101] As described herein, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.

[0102] Units, prefixes, and symbols are denoted in their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range. The headings provided herein are not limitations of the various aspects of the disclosure, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification in its entirety.

[0103] As used herein, the term "isolated" refers to in vitro preparation and / or isolation of a nucleic acid molecule (e.g., a polynucleotide, vector, or plasmid), peptide, or polypeptide (protein), or virus of the disclosure so that it is not associated with in vivo substances, or is substantially purified from in vitro substances. An isolated virus preparation is generally obtained by in vitro culture and propagation and is substantially free from other infectious agents.

[0104] A "recombinant" virus is one which has been manipulated in vitro, e.g., using recombinant DNA techniques, to introduce changes to the viral genome.

[0105] As used herein, the term "construct" refers to a nucleic acid molecule of any length (e.g., single-stranded or double-stranded DNA, single-stranded or double-stranded RNA, double-stranded DNA-RNA, polynucleotides comprising one or more phosphoester analogs, mRNA, siRNA, RNAi), a plasmid, a non-viral vector (e.g., a bacterial vector, a yeast vector, a cosmid, an artificial chromosome), a viral vector (e.g., a recombinant virus, such as recombinant MVA virus), a virus like particle, a host cell, or a tissue. The constructs of the present disclosure may include any of a number of suitable transcription or translation elements, including, but not limited to, constitutive or inducible promoters, transcription enhancer elements, and transcription terminators.

[0106] The terms "nucleic acids," "nucleic acid molecules, "nucleotides," "nucleotide(s) sequence," and "polynucleotide" can be used interchangeably and refer to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNAmolecules", including mRNA) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules"), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Single stranded nucleic acid sequences refer to single-stranded DNA (ssDNA) or single-stranded RNA (ssRNA). Double stranded DNA- DNA, DNA-RNA and RNA-RNA helices are possible. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, supercoiled DNA and chromosomes. In discussing the structure of particular double-stranded DNA molecules, sequences can be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the non-transcribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA). A "recombinant DNA molecule" is a DNA molecule that has undergone a molecular biological manipulation. DNA includes, but is not limited to, cDNA, genomic DNA, DNA plasmid, synthetic DNA, and semi-synthetic DNA. A "nucleic acid composition" or "nucleic acid construct" of the disclosure comprises one or more nucleic acids as described herein.

[0107] RNA can be obtained by transcription of a DNA-sequence, e.g., inside a cell. In eukaryotic cells, transcription is typically performed inside the nucleus or the mitochondria. In vivo, transcription of DNA usually results in premature RNA, which has to be processed into messenger RNA (mRNA). Processing of the premature RNA, e.g., in eukaryotic organisms, comprises a variety of different posttranscriptional-modifications such as splicing, 5'-capping, polyadenylation, export from the nucleus or the mitochondria and the like. The sum of these processes is also called maturation of RNA. The mature mRNA usually provides the nucleotide sequence that can be translated into an amino acid sequence of a particular peptide, protein, or protein antigen. Typically a mature mRNA comprises a 5' cap, optionally a 5 -UTR, an open reading frame, optionally a 3'-UTR, and a poly(A) sequence.

[0108] The term "5'-cap," as used herein, refers to an entity, typically a modified nucleotide entity, which generally "caps" the 5'-end of a mature mRNA. A 5'-cap can typically be formed by a modified nucleotide, particularly by a derivative of a guanine nucleotide. In some aspects, the 5'-cap is linked to the 5'-terminus via a 5'-5'-triphosphatelinkage. A 5'-cap can be methylated, e.g., m7GpppN, wherein N is the terminal 5' nucleotide of the nucleic acid carrying the 5'-cap, typically the 5'-end of an RNA. The naturally occurring 5'-cap is m7GpppN.

[0109] As used herein, a "poly(A) sequence," also called "poly(A) tail" or "3'-poly(A) tail," is typically understood to be a sequence of adenine nucleotides, e.g., of up to about 400 adenine nucleotides. A poly(A) sequence can be located at the 3' end of an mRNA. In some aspects, a poly(A) sequence can also be located within an mRNA or any other nucleic acid molecule, such as, e.g., in a vector, for example, in a vector serving as template for the generation of an RNA, preferably an mRNA, e.g., by transcription of the vector. In some aspects, a poly(A) sequence is present in the 3'-UTR of the mRNA as defined herein.

[0110] In some aspects, a 3'-UTR sequence is part of an mRNA, which is located between the protein coding region (i.e. the open reading frame) and the 3' terminus of the mRNA molecule. If a 3'-terminal poly(A) sequence ('poly(A) tail') was added to the RNA (e.g. by polyadenylation), then the term 3'-UTR can refer to that part of the molecule, which is located between the protein coding region and the 3'-terminal poly(A) sequence. In some aspects, a 3'-UTR can also comprise a poly(A) sequence (e.g., a poly(A) sequence which is not located at the very 3' terminus of the RNA molecule). A 3'-UTR of the mRNA is not translated into an amino acid sequence. The 3'-UTR sequence is generally encoded by the gene, which is transcribed into the respective mRNA during the gene expression process. The genomic sequence is first transcribed into pre-mature mRNA, which comprises optional introns. The pre-mature mRNA is then further processed into mature mRNA in a maturation process. This maturation process comprises the steps of 5' capping, splicing the pre-mature mRNA to excise optional introns and modifications of the 3 '-end, such as polyadenylation of the 3 '-end of the pre-mature mRNA and optional endo- / or exonuclease cleavages etc. In some aspects, a 3'-UTR corresponds to the sequence of a mature mRNA, which is located 3' to the stop codon of the protein coding region (e.g., immediately 3' to the stop codon of the protein coding region), and which extends to the 3' terminus of the RNA molecule or to the 5'-side of a 3' terminal poly(A) sequence (e.g., to the nucleotide immediately 5' to the 3' terminus or immediately 5' to the 3' terminal poly(A) sequence). The term "corresponds to" means that the 3'-UTR sequence can be an RNA sequence, such as in the mRNA sequence used for defining the 3'-UTR sequence, or a DNA sequence, which corresponds to such RNAsequence. In some aspects, the term "a 3'-UTR of a gene", such as "3'-UTR of alpha or beta globin", is the sequence, which corresponds to the 3'-UTR of the mature mRNA derived from this gene, i.e. the mRNA obtained by transcription of the gene and maturation of the pre-mature mRNA. The term "3'-UTR of a gene" encompasses the DNA sequence and the RNA sequence of the 3'-UTR. In some aspects, the 3'-UTR is derived from a gene that relates to an mRNA with an enhanced half-like (i.e., that provides a stable mRNA), for example a 3'-UTR of a gene selected from the group consisting of: albumin gene, an a-globin gene, a P-globin gene, a tyrosine hydroxylase gene, a lipoxygenase gene, and a collagen alpha gene, such as a collagen alpha 1(1) gene [OHl] A 5'-UTR is typically understood to be a particular section of messenger RNA (mRNA). It is located 5' of the open reading frame of the mRNA. In some aspect, the 5'- UTR starts with the transcriptional start site and ends one nucleotide before the start codon of the open reading frame. The 5'-UTR can comprise elements for controlling gene expression, also called regulatory elements. Such regulatory elements can be, for example, ribosomal binding sites or a 5'-Terminal Oligopyrimidine Tract. The 5'-UTR can be posttranscriptionally modified, for example by addition of a 5'-cap. In some aspects, a 5'-UTR corresponds to the sequence of a mature mRNA which is located between the 5' cap and the start codon. In some aspects, the 5'-UTR corresponds to the sequence which extends from a nucleotide located 3' to the 5'-cap (e.g., from the nucleotide located immediately 3' to the 5' cap) to a nucleotide located 5' to the start codon of the protein coding region (e.g., to the nucleotide located immediately 5' to the start codon of the protein coding region). The nucleotide located immediately 3' to the 5' cap of a mature mRNA typically corresponds to the transcriptional start site. The term "corresponds to" means that the 5'-UTR sequence can be an RNA sequence, such as in the mRNA sequence used for defining the 5'-UTR sequence, or a DNA sequence which corresponds to such RNA sequence. In some aspects, the term "a 5'-UTR of a gene", is the sequence, which corresponds to the 5'-UTR of the mature mRNA derived from this gene.

[0112] As used herein, the term "transfecting" or "transfection" refers to the transport of nucleic acids from the environment external to a cell to the internal cellular environment, with particular reference to the cytoplasm and / or cell nucleus. Without being bound by any particular theory, it is to be understood that nucleic acids can be delivered to cells either after being encapsulated within or adhering to one or more cationicpolymer / nucleic acid complexes or being entrained therewith. Particular transfecting instances deliver a nucleic acid to a cell nucleus. Nucleic acids include DNA and RNA as well as synthetic congeners thereof. Such nucleic acids include missense, antisense, nonsense, as well as protein producing nucleotides, on and off and rate regulatory nucleotides that control protein, peptide, and nucleic acid production. In particular, but not limited to, they can be genomic DNA, cDNA, mRNA, tRNA, rRNA, hybrid sequences or synthetic or semi-synthetic sequences, and of natural or artificial origin. In addition, the nucleic acid can be variable in size, ranging from oligonucleotides to chromosomes. These nucleic acids can be of human, animal, vegetable, bacterial, viral, or synthetic origin. They can be obtained by any technique known to a person skilled in the art.

[0113] As used herein, a "transformed" cell is a cell into which has been introduced a nucleic acid molecule by molecular biology techniques. The term encompasses all techniques by which a nucleic acid molecule might be introduced into such a cell, including transfection with viral vectors, transformation with plasmid vectors, and introduction of naked DNA by electroporation, lipofection, and particle gun acceleration.

[0114] As used herein, "peptide" means peptides of any length and includes proteins. The terms "polypeptide" and "oligopeptide" are used herein without any particular intended size limitation, unless a particular size is otherwise stated.

[0115] "Administering" and similar terms refer to the physical introduction of a therapeutic agent (e.g., the polynucleotides, avian influenza vaccine vectors, avian influenza vaccines, mosaic H5 proteins or antigenic fragments thereof, pharmaceutical compositions described herein) to an avian subject, using any of the various methods and delivery systems known to those skilled in the art. Exemplary routes of administration include a buccal, epidermal, epidural, intraarterial, intraarticular, intracapsular, intracardiac, intracoronary, intradermal, intralesional, intralymphatic, intramuscular, intranasal, intraorbital, intraperitoneal, intraspinal, intrastema, intrathecal, intravenous, mucosal, oral, rectal, subarachnoid, subcapsular, subcutaneous, subcuticular, sublingual, topical, transtracheal, or vaginal route of administration or any combination thereof. Administering can also be performed, for example, once, a plurality of times, and / or over one or more extended periods.

[0116] Treatment" or "therapy" of an avian subject refers to any type of intervention or process performed on, or the administration of an active agent to, an avian subject withthe objective of reversing, alleviating, ameliorating, inhibiting, slowing down, or preventing the onset, progression, development, severity, or recurrence of a symptom, complication, condition, or biochemical indicia associated with a disease

[0117] A "therapeutically effective amount," "effective amount," "therapeutic dose," "effective dose," or "effective dosage," as used herein, means an amount or a dose that achieves a therapeutic goal, as described herein. One of ordinary skill in the art will further understand that a therapeutically effective amount etc. can be administered in a single dose, or can be achieved by administration of multiple doses (i.e., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses). The ability of a therapeutic agent to promote disease regression or inhibit the development or recurrence of the disease can be evaluated using a variety of methods known to the skilled practitioner, such as by assaying the activity of the agent in in vitro assays.

[0118] As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment," and the like, refer to reducing the probability of developing a disease or condition in an avian subject, who does not have, but is at risk of or susceptible to developing a disease or condition.

[0119] As used herein, the term "adjuvant" refers to any component which improves the body's response to a vaccine.

[0120] As used herein, the term "vaccine" or "vaccine composition" refers to an immunogenically active composition for the prophylaxis and / or treatment of diseases. Accordingly, in some aspects, vaccines are medicaments which comprise or deliver antigens and are intended to be used in animals (e.g., avians) for generating specific defense and protective substance by vaccination.

[0121] A used herein, the terms "inducing immunity," "eliciting an immune response," or "immunogenically active" refers to the ability to stimulate an immune response, i.e., to stimulate the production of antibodies, particularly humoral antibodies, or to stimulate a cell-mediated response. For example, the ability to stimulate the production of circulating or secretory antibodies or the production of a cell-mediated response in local mucosal regions, peripheral blood, cerebral spinal fluid or the like. In some aspects, the effective immunizing amount of the immunogenically active component(s) of this disclosure can vary and can be any amount sufficient to evoke an immune response and provide a protective immune response against avian influenza infection (e.g., an avian influenza virus infection). A dosage unit comprising a composition (e.g., a polynucleotide, nucleicacid construct, mosaic polypeptide, avian influenza vaccine vector, avian influenza vaccine, or pharmaceutical composition) of the disclosure is contemplated. At least one dosage unit per patient is contemplated herein as a vaccination regimen. In some embodiments, two or more dosage units can be useful. The skilled artisan will quickly recognize that a particular quantity of vaccine composition per dosage unit, as well as the total number of dosage units per vaccination regimen, can be optimized, so long as an effective immunizing amount of the virus or a component thereof is ultimately delivered to the avian subject.

[0122] An "immunological response" or "immune response" to a substance such as a composition or vaccine is the development in the subject of a cellular and / or antibody- mediated immune response to a composition or vaccine of interest. Usually, an "immunological response" includes but is not limited to one or more of the following effects: the production of antibodies, B cells, helper T cells, and / or cytotoxic T cells, directed specifically to an antigen or antigens included in the composition or vaccine of interest. In some aspects, the subject can display either a therapeutic or protective immunological response so resistance to new infection is enhanced and / or the clinical severity of the disease reduced. In some aspects, such protection can be demonstrated by either a reduction or lack of symptoms normally displayed by an infected subject, a quicker recovery time and / or a lowered viral titer in the infected subject

[0123] It is recognized that the antigenic polypeptides of the disclosure can be full length polypeptides or active fragments or variants thereof. In some aspects, the term "active fragments" or "active variants" or "antigenic fragments" refers to fragments or variants that retain all or some of the antigenic nature of the polypeptide. Thus, in some aspects, the present disclosure encompasses any mosaic avian influenza protein, antigen, epitope or immunogen that elicits, induces, or stimulates an immunogenic response in an avian subject.

[0124] The term "epitope" refers to the site on an antigen or hapten to which specific B cells and / or T cells respond. The term is also used interchangeably with "antigenic determinant" or "antigenic determinant site." Antibodies that recognize the same epitope can be identified in a simple immunoassay showing the ability of one antibody to block the binding of another antibody to a target antigen.

[0125] As used herein, the term "pharmaceutical agent," "pharmaceutical composition," or "drug" or any other similar term means any chemical or biological material orcompound suitable for administration by the methods previously known in the art and / or by the methods taught in the present disclosure, which induce a desired biological or pharmacological effect, which can include but are not limited to (1) having a prophylactic effect on the organism and preventing an undesired biological effect such as preventing an infection, (2) alleviating a condition caused by a disease, for example, alleviating pain or inflammation caused as a result of disease, and / or (3) either alleviating, reducing, or completely eliminating a disease from the organism. The effect can be local or it can be systemic.

[0126] A "pharmaceutically acceptable carrier" or "carrier that renders the composition suitable for pharmaceutical use" refers to a carrier that can be administered to an avian subject, together with an agent, and which does not destroy the pharmacological activity thereof and is nontoxic when administered in doses sufficient to deliver a therapeutic amount of the agent. In certain aspects, the pharmaceutically acceptable carrier is an aqueous solvent, z.e., a solvent comprising water, optionally with additional co-solvents. Exemplary pharmaceutically acceptable carriers include water, buffer solutions in water (such as phosphate-buffered saline (PBS), and 5% dextrose in water (D5W). In certain embodiments, the aqueous solvent further comprises dimethyl sulfoxide (DMSO), e.g., in an amount of about 1-4%, or 1-3%. In certain aspects, the pharmaceutically acceptable carrier is isotonic (i.e., has substantially the same osmotic pressure as a body fluid such as plasma).

[0127] A "subject" includes any non-human animals. The term "nonhuman animal" includes, but is not limited to, vertebrates such as nonhuman primates, sheep, dogs, avians (e.g., chickens) and rodents such as mice, rats, and guinea pigs. In some aspects, the subject is a chicken. The terms "subject" and "patient" are used interchangeably herein.

[0128] The term "expression" as used herein refers to a process by which a polynucleotide produces a gene product, for example, a mosaic H5N 1 protein or antigenic fragment thereof. In some aspects, it includes, without limitation, transcription of the polynucleotide into messenger RNA (mRNA) and the translation of an mRNA into a polypeptide. Expression produces a "gene product." As used herein, a gene product can be either a nucleic acid, e.g., a messenger RNA produced by transcription of a gene, or a polypeptide which is translated from a transcript. Gene products described herein can further include nucleic acids with post transcriptional modifications, e.g., polyadenylation or splicing, or polypeptides with post translational modifications, e.g., methylation,glycosylation, the addition of lipids, association with other protein subunits, or proteolytic cleavage. In some aspects, the avian influenza proteins or antigenic fragments thereof disclosed herein are expressed in yeast, such as in a Pichia pastoris strain, and isolated (e.g., isolated using pressurized mechanical lysis).

[0129] As used herein, the term "5"' or "5 prime" refers to the 5' end of a nucleic acid or nucleic acid sequence, and the term "3"' or "3 prime" refer to the 3' end of nucleic acid or nucleic acid sequence.

[0130] The terms "identical," percent "identity," or percent "sequence identity" in the context of two or more nucleic acids refer to two or more sequences that are the same or have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned (introducing gaps, if necessary) for maximum correspondence, not considering any conservative amino acid substitutions as part of the sequence identity. The percent identity can be measured using sequence comparison software or algorithms or by visual inspection. Various algorithms and software are known in the art that can be used to obtain alignments of amino acid or nucleotide sequences.

[0131] As used herein, the term "promoter" refers to DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In some aspects, a coding sequence is located 3' to a promoter sequence. Promoters can be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. For example, suitable promoters may be selected from the Eukaryotic Promoter Database (EPDB). It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters." Promoters that cause a gene to be expressed in a specific cell type are commonly referred to as "cell-specific promoters" or "tissue-specific promoters." Promoters that cause a gene to be expressed at a specific stage of development or cell differentiation are commonly referred to as "developmentally-specific promoters" or "cell differentiation-specific promoters." Promoters that are induced and cause a gene to be expressed following exposure or treatment of the cell with an agent, biological molecule, chemical, ligand, light, or the like that induces the promoter are commonly referred to as "inducible promoters" or "regulatable promoters." It is further recognized that since inmost cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity.

[0132] The term "operably linked" refers to genetic elements that are joined together in a manner that enables them to carry out their normal functions. For example, a gene is operably linked to a promoter when its transcription is under the control of the promoter and this transcription results in the production of the product encoded by the gene.

[0133] The term "antibody" includes molecules or active fragments (z.e., antigen binding fragments) of molecules that bind to antigens. These active fragments can be derived from an antibody of the present disclosure by a number of techniques. For further description of general techniques for the isolation of active fragments of antibodies, see for example, Khaw, B. A. et al. J. Nucl. Med. 23: 1011-1019 (1982). The term "antibody" also includes bispecific and chimeric antibodies and antibodies in nonmammalian species.

[0134] The term "vector," as used herein, is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked; or an entity comprising such a nucleic acid molecule capable of transporting another nucleic acid. In some aspects, the vector is a non-viral vector (e.g., a DNA plasmid, a bacterial vector, a cosmid, or artificial chromosome). In some aspects, the non-viral vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. In some aspects, the vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. In some aspects, such viral vectors include, but are not limited to: a poxvirus vector (e.g., a vaccinia vector or a modified vaccinia Ankara (MV A) vector). In some aspects, the viral vector is a replication defective viral vector. In some aspects, the viral vector is a virus-like particle. Certain vectors, or polynucleotides that are part of vectors, are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication, and episomal mammalian vectors). Other vectors (e.g., non- episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked.

[0135] Various additional aspects of the disclosure are described, disclosed or illustrated in further detail in the following subsections.II. Exemplary Compositions and Methods

[0136] The present disclosure relates to compositions and methods which employ polynucleotides (e.g., nucleic acid constructs, isolated polynucleotides, and recombinant nucleic acid molecules), vaccine vectors, mosaic H5 proteins, or antigenic fragments thereof (e.g., viral vaccine vectors, non-viral vaccine vectors, and recombinant host cells which encode or express one or more avian influenza proteins or antigenic fragments thereof, or extracts of recombinant host cells). In some aspects, the mosaic H5 protein or antigenic fragment thereof is obtained from a recombinant host cell, such as a recombinant prokaryotic cell, a recombinant eukaryotic cell, or a eukaryotic cell line.

[0137] In some aspects, the compositions and methods of the present disclosure are useful for eliciting an immune response in a subject against one or more avian influenza antigens. In some aspects, the compositions and methods of the present disclosure are useful for preventing, reducing the incidence of, attenuating, or treating avian influenza infection in an avian subject in need thereof. The compositions of the present disclosure, for example a single dose thereof, are broad spectrum immunotherapeutics and provide for prophylactic and / or therapeutic activity against a variety of avian influenza strains.

[0138] In some aspects, the compositions of the present disclosure further comprise a pharmaceutically acceptable carrier. In some aspects, the compositions are administered orally, for instance, in a formulation suitable to deliver protein(s). In some aspects, the compositions are administered through a various other acceptable delivery routes, for example, through parenteral injection, intranasally, or via an intramuscular injection. In some aspects, the compositions are administered to the avian subject one or more times, at times including but not limited to 1 to 7 days, 1 to 3 weeks, or about 1, 2, 3, 4, or more moths (e.g., up to about 6 months) before the avian subject is exposed to an avian influenza. In some aspects, the compositions are administered to the avian subject one or more times after exposure to an avian influenza, e.g., at 1 hour, 6 hours, 12 hours, 1 day, 2 days, 4 days or more (e.g., up to about 2 weeks) after exposure.III. Polynucleotides and Nucleic Acid Constructs

[0139] The polynucleotides of the present disclosure (e.g., a nucleic acid construct) can include DNA and / or RNA sequences for use in the pharmaceutical compositions and vaccines described or exemplified herein.

[0140] The present disclosure also features nucleic acid constructs (e.g., isolated polynucleotides or recombinant nucleic acid molecules) comprising one or more nucleic acid sequences encoding one or more mosaic H5 proteins or antigenic fragments thereof described or exemplified herein that are immunogenic for antigens derived from two or more avian influenza strains (e.g., derived from H5N1 sequences reported between 1996 and 2023).

[0141] In some aspects, the nucleic acid construct is a DNA polynucleotide. In some aspects, the DNA polynucleotide is a single-stranded DNA (ssDNA) polynucleotide. In some aspects, the DNA polynucleotide is a double-stranded DNA polynucleotide. In some aspects, the nucleic acid construct is an RNA polynucleotide. In some aspects, the RNA polynucleotide is a single-stranded RNA (ssRNA). In some aspects, the RNA polynucleotide is a double-stranded RNA. In some aspects, the nucleic acid construct is a double stranded DNA-RNA polynucleotide. In some aspects, the nucleic acid construct is an mRNA. In some aspects, the mRNA comprises a 5' cap. In some aspects, the mRNA comprises a 5 -UTR. In some aspects, the mRNA comprises a 3 -UTR. In some aspects, the mRNA comprises a poly(A). In some aspects, the mRNA comprises a 5' cap and a poly(A). In some aspects, the mRNA comprises a 5' cap, a 5'-UTR, and a poly(A). In some aspects, the mRNA comprises a 5' cap, a 3'-UTR, and a poly(A). In some aspects, the mRNA comprises a 5' cap, a 5'-UTR, a 3'-UTR, and a poly(A).

[0142] In some aspects, provided herein is a nucleic acid construct comprising a nucleic acid sequence encoding a mosaic H5 protein or fragment thereof.

[0143] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1-3.

[0144] In some aspects, the nucleic acid sequence has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 4-6.

[0145] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 1-3.

[0146] In some aspects, the nucleic acid sequence has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, atleast about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 4-6.

[0147] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence corresponding to any one of SEQ ID NO: 1-3.

[0148] In some aspects, the nucleic acid sequence corresponds to any one of SEQ ID NO: 4-6.

[0149] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 7-10.

[0150] In some aspects, the nucleic acid sequence has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 11-14.

[0151] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 7-10.

[0152] In some aspects, the nucleic acid sequence has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 11-14.

[0153] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence corresponding to any one of SEQ ID NO: 7-10.

[0154] In some aspects, the nucleic acid sequence corresponds to any one of SEQ ID NO: 11-14.

[0155] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence disclosed in Table 1.

[0156] In some aspects, the nucleic acid sequence corresponds to any of the sequences disclosed in Table 2.Table 1. Mosaic Protein Amino Acid SequencesTable 2. Mosaic Protein Nucleic Acid Sequences

[0157] In some aspects, provided herein is a nucleic acid construct comprising one or more nucleic acid sequences selected from the group consisting of: (i) a first nucleic acid sequence encoding a first mosaic H5 protein or fragment thereof; (ii) a first nucleic acid sequence encoding a first mosaic H5 protein or antigenic fragment thereof and a second nucleic acid sequence encoding a second mosaic H5 protein or fragment thereof; and (iii) combinations thereof.

[0158] In some aspects, the first mosaic H5 protein or fragment thereof encodes an H5 hemagglutinin (HA) protein or fragment thereof.

[0159] In some aspects, the first mosaic H5 HA protein or fragment thereof is a fragment of the H5 HA protein. In some aspects, the fragment comprises an HA1 region and / or an HA2 region.

[0160] In some aspects, the second mosaic H5 protein or fragment thereof encodes an H5 hemagglutinin (HA) protein or fragment thereof.

[0161] In some aspects, the second mosaic H5 HA protein or fragment thereof is a fragment of the H5 HA protein. In some aspects, the fragment comprises an HA1 region and / or an HA2 region.

[0162] In some aspects, the first mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 7-9.

[0163] In some aspects, the second mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0164] In some aspects, the first nucleic acid sequence has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: I lls.

[0165] In some aspects, the second nucleic acid sequence has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 14.

[0166] In some aspects, the first mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 7-9.

[0167] In some aspects, the second mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0168] In some aspects, the first nucleic acid sequence has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 11-13.

[0169] In some aspects, the second nucleic acid sequence has at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity to the nucleic acid sequence of SEQ ID NO: 14.

[0170] In some aspects, the first mosaic H5 protein or fragment thereof comprises an amino acid sequence corresponding to any one of SEQ ID NO: 7-9.

[0171] In some aspects, the second mosaic H5 protein or fragment thereof comprises an amino acid sequence corresponding to any one of SEQ ID NO: 10.

[0172] In some aspects, the first nucleic acid sequence corresponds to any one of SEQ ID NO: 11-13.

[0173] In some aspects, the second nucleic acid sequence corresponds to any one of SEQ ID NO: 14.

[0174] In some aspects, the nucleic acid construct further comprises a promoter. In some aspects, the promoter is an S E / L promoter. In some aspects, the S E / L promoter comprises a nucleic acid sequence corresponding to aaaaattgaaattttattttttttttttggaatataaataag (SEQ ID NO: 15).

[0175] In some aspects, the first nucleic acid sequence or the second nucleic acid sequence is codon optimized. In some aspects, the first nucleic acid sequence and / or the second nucleic acid sequence has a reduced number of RNA secondary structures relative to an unmodified nucleic acid sequence. In some aspects, the first nucleic acid sequence and / or the second nucleic acid sequence has a reduced number of RNA destabilization sequence relative to an unmodified nucleic acid sequence. In some aspects, the first nucleic acid sequence and / or the second nucleic acid sequence has a reduced number of or no transcription termination sequences relative to an unmodified nucleic acid sequence.

[0176] In some aspects, the first nucleic acid sequence and / or the second nucleic acid sequence is optimized for expression in at least one selected host. Optimized sequences include sequences which are codon optimized, i.e., codons which are employed more frequently in one organism relative to another organism, e.g., a distantly related organism, or balance the usage of codons so that the most frequently used codon is not used to exhaustion. Other modifications can include addition or modification of Kozak sequences and / or introns, and / or to remove undesirable sequences, for instance, potential transcription factor binding sites.

[0177] In some aspects, the first nucleic acid sequence and / or the second nucleic acid sequence is optimized for expression in a mammalian host cell. In some aspects, an optimized nucleic acid sequence no longer hybridizes to a corresponding non-optimized (e.g., wild-type) sequence, e.g., does not hybridize to the non-optimized sequence under medium or high stringency conditions. The term "stringency" is used in reference to the conditions of temperature, ionic strength, and the presence of other compounds, under which nucleic acid hybridizations are conducted. With "high stringency" conditions, nucleic acid base pairing will occur only between nucleic acid fragments that have a high frequency of complementary base sequences. Thus, conditions of "medium" or "low"stringency are often required when it is desired that nucleic acids that are not completely complementary to one another be hybridized or annealed together. Numerous equivalent conditions known in the art can be employed to comprise medium or low stringency conditions. Exemplary "high stringency conditions" when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42° C in a solution comprising 5x SSPE (43.8 g / 1 NaCl, 6.9 g / 1 NaHzPCUHzO and 1.85 g / 1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5x Denhardt' s reagent and 100 pg / ml denatured salmon sperm DNA followed by washing in a solution comprising 0. lx SSPE, 1.0% SDS at 42° C when a probe of about 500 nucleotides in length is employed. Exemplary "medium stringency conditions" when used in reference to nucleic acid hybridization comprise conditions equivalent to binding or hybridization at 42° C in a solution consisting of 5x SSPE (43.8 g / 1 NaCl, 6.9 g / 1 NaftPCU^O and 1.85 g / 1 EDTA, pH adjusted to 7.4 with NaOH), 0.5% SDS, 5x Denhardt' s reagent and 100 pg / ml denatured salmon sperm DNA followed by washing in a solution comprising l.Ox SSPE, 1.0% SDS at 42° C when a probe of about 500 nucleotides in length is employed.

[0178] In some aspects, the optimized nucleic acid sequences have less than 90% (e.g., less than 80%) nucleic acid sequence identity to a corresponding non-optimized (e.g., wild-type) sequence. Avian influenza vaccine vectors comprising the optimized nucleic acid sequences are also provided.

[0179] In some aspects, a nucleic acid sequence encoding a mosaic H5 (e.g., H5M2 and H5M3) protein or antigenic fragment thereof is optimized by replacing codons, e.g., at least 25% of the codons, in a non-optimized sequence (e.g., a wild type sequence) with codons which are preferentially employed in a particular (selected) cell. Preferred codons have a relatively high codon usage frequency in a selected cell, and their introduction results in the introduction of relatively few undesirable structural attributes. Thus, the optimized nucleic acid product may have an improved level of expression due to improved codon usage frequency, and a reduced number of undesirable transcription regulatory sequences.

[0180] An optimized nucleic acid sequence may have a codon composition that differs from that of the corresponding non-optimized nuclei acid sequence (e.g., a wild-type nucleic acid sequence) at more than 30%, 35%, 40% or more than 45% (e.g., 50%, 55%, 60% or more) of the codons. Exemplary codons for use in the disclosure are those which are employed more frequently than at least one other codon for the same amino acid in aparticular organism and, in some aspects, are also not low-usage codons in that organism and are not low-usage codons in the organism used to clone or screen for the expression of the nucleic acid molecule. Moreover, codons for certain amino acids (i.e., those amino acids that have three or more codons), may include two or more codons that are employed more frequently than the other (non-preferred) codon(s). The presence of codons in the nucleic acid molecule that are employed more frequently in one organism than in another organism results in a nucleic acid molecule which, when introduced into the cells of the organism that employs those codons more frequently, is expressed in those cells at a level that is greater than the expression of the wild type or parent nucleic acid sequence in those cells.

[0181] In some aspects, the codons that are different are those employed more frequently in an avian (e.g., a chicken). Codons for different organisms are known to the art, e.g., see kazusa.or.jp. / codon / .

[0182] The nucleic acid constructs described or exemplified herein can further comprise one or more post-transcriptional regulatory elements. In some aspects, the post- translational regulatory element is positioned 3' to a coding region of the polynucleotide. Non-limiting examples of post-transcriptional regulatory elements that are useful for the present disclosure include a Woodchuck Hepatitis virus post-transcriptional regulatory element (WPRE), a Hepatitis B virus post-transcriptional regulatory element (HPRE), polyadenylation signal sequences, intron / exon junctions / splicing signals, synthetic elements, or any combination thereof.

[0183] The nucleic acid constructs can also comprise one or more polyadenylation (poly(a)) signals, which can be downstream of any protein coding sequence. Examples of polyadenylation signals include but are not limited to a SV40 poly(a) tail, a LTR poly(a) tail, a bovine growth hormone (bGH) poly(a) tail, a human growth hormone (hGH) poly(a) tail, or a human P-globin poly(a) tail. In some aspects, the nucleic acid constructs described or exemplified herein further comprise at least one 3' UTR poly(a) tail sequence operably linked to the first nucleic acid sequence, the second nucleic acid sequence, the third nucleic acid sequence, or any combination thereof. In some aspects, the 3' UTR poly(a) tail sequence is a 3' UTR SV40 poly(a) tail sequence, a 3' UTR bovine growth hormone (bGH) poly(A) sequence, a 3' UTR actin poly(A) tail sequence, a 3' UTR hemoglobin poly(A) sequence, or any combination thereof.

[0184] The nucleic acid constructs described or exemplified herein can further comprise at least one enhancer sequence upstream of any protein coding sequence. In some aspects, the enhancer sequence is a viral enhancer sequence. In some aspects, the enhancer sequence is a non-viral enhancer sequence. Examples of viral and non-viral enhancer sequences include but are not limited to a SV40 enhancer sequence, a polyoma virus enhancer sequence, a cytomegalovirus enhancer sequence, an HIV enhancer sequence, an immunoglobulin enhancer sequence, an interferon enhancer sequence, a chymotrypsin enhancer sequence, an insulin enhancer sequence, a metallothionein enhancer sequence, a beta-actin enhancer sequence, and a synthetic enhancer sequence.

[0185] The nucleic acid constructs described or exemplified herein may be employed alone or with one or more immunogenic agents, such as other virus in a vaccine, to raise virus-specific antisera, in gene therapy, and / or in diagnostics.

[0186] The nucleic acid constructs described or exemplified herein may be employed in a vector to express one or more Mosaic H5 proteins or antigenic fragments thereof, e.g., for recombinant protein vaccine production or to raise antisera, as a nucleic acid vaccine, for use in diagnostics, for viral RNA (vRNA) production, to prepare chimeric genes, e.g., with other viral genes including other avian influenza strains, and / or to prepare recombinant virus.

[0187] In some aspects, the mosaic H5 protein or fragment thereof comprises an H5 hemagglutinin (HA) protein or fragment thereof.

[0188] In some aspects, the H5 HA protein or fragment thereof is a fragment of the H5 HA protein.

[0189] In some aspects, the fragment comprises an HA1 region and / or an HA2 region. In some aspects, the fragment comprises an HA1 region. In some aspects, the fragment comprises an HA2 region.

[0190] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0191] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0192] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0193] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7.

[0194] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0195] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9.

[0196] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0197] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0198] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0199] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0200] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7.

[0201] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at leastabout 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0202] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9.

[0203] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10.IV. Avian Influenza Vaccine Vectors

[0204] The present disclosure also features avian influenza vaccine vectors comprising any nucleic acid construct (e.g., an isolated polynucleotide or a recombinant nucleic acid molecule) described or exemplified herein.

[0205] In some aspects, provided herein is an avian influenza vaccine vector comprising any of the nucleic acid constructs described herein.

[0206] In some aspects, provided herein is an avian influenza vaccine vector comprising a polynucleotide encoding a mosaic H5 protein or fragment thereof.

[0207] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0208] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0209] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0210] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7.

[0211] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0212] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9.

[0213] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0214] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 4.

[0215] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 5.

[0216] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 6.

[0217] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 11.

[0218] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 12.

[0219] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 13.

[0220] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 14.

[0221] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0222] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0223] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0224] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 7.

[0225] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0226] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9.

[0227] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0228] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:4.

[0229] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:5.

[0230] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:6.

[0231] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 11.

[0232] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:12.

[0233] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:13.

[0234] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, atleast about 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 14.

[0235] In some aspects, the avian influenza vaccine vector further comprises a promoter. In some aspects, the promoter is an S E / L promoter.

[0236] In some aspects, the avian influenza vaccine vector is a viral vector.

[0237] In some aspects, the viral vector is a poxvirus vector.

[0238] In some aspects, the poxvirus vector is a vaccinia virus vector.

[0239] In some aspects, the vaccinia virus vector is a modified vaccinia Ankara (MV A) vector.

[0240] In some aspects, the avian influenza vaccine vector is a viral vector, a non-viral vector, or a combination thereof. In some aspects, the viral vector is a poxvirus vector (e.g., a vaccinia vector or a modified vaccinia Ankara (MV A) vector). In some aspects, the viral vector is a replication defective viral vector. In some aspects, the viral vector is a poxvirus vector. In some aspects, the poxvirus vector is a vaccinia virus vector. In some aspects, the vaccinia virus vector is a modified vaccinia Ankara (MV A) vector. In some aspects, the viral vector is a virus-like particle. In some aspects, the non-viral vector is a DNA plasmid, a cosmid, a bacterial vector, an artificial chromosome, or any combination thereof.

[0241] In some aspects, the viral vector or non-viral vector exhibits an adjuvant property. Not to be bound by any theory, in some aspects, the adjuvant property of the viral vector or the non-viral vector promotes mobilization of antigen presenting cells to the site of vaccine delivery and antigen expression, thereby augmenting the uptake of the polynucleotide and the expressed antigens into professional antigen presenting cells to elicit antigen presentation.V. Avian Influenza Vaccines

[0242] The present disclosure also features Avian Influenza vaccines comprising any mosaic H5 protein or antigenic fragment thereof described or exemplified herein (e.g., H5M2 and H5M3) and one or more carriers.

[0243] In some aspects, provided herein is an avian influenza vaccine comprising a polynucleotide encoding a mosaic H5 protein or fragment thereof and one or more carriers.

[0244] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0245] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0246] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0247] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 sequence identity to the amino acid sequence of SEQ ID NO: 7.

[0248] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0249] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 sequence identity to the amino acid sequence of SEQ ID NO: 9.

[0250] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0251] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 4.

[0252] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 5.

[0253] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 6.

[0254] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 11.

[0255] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 12.

[0256] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 13.

[0257] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 14.

[0258] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 1.

[0259] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100 sequence identity to the amino acid sequence of SEQ ID NO: 2.

[0260] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100 sequence identity to the amino acid sequence of SEQ ID NO: 3.

[0261] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100 sequence identity to the amino acid sequence of SEQ ID NO: 7.

[0262] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100 sequence identity to the amino acid sequence of SEQ ID NO: 8.

[0263] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100 sequence identity to the amino acid sequence of SEQ ID NO: 9.

[0264] In some aspects, the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100 sequence identity to the amino acid sequence of SEQ ID NO: 10.

[0265] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:4.

[0266] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:5.

[0267] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:6.

[0268] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 11.

[0269] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:12.

[0270] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:13.

[0271] In some aspects, the polynucleotide comprises a nucleic acid sequence having at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO:14.

[0272] In some aspects, the carrier is a poxvirus.

[0273] In some aspects, the poxvirus is a vaccinia virus.

[0274] In some aspects, the vaccinia virus is a MVA virus.

[0275] In some aspects, the carrier is a virus (e.g., a recombinant virus). In some aspects, the carrier is a vaccinia virus (e.g., a poxvirus such as MVA). In some aspects, the carrier is a poxvirus. In some aspects, the carrier is a vaccinia virus. In some aspects, the carrier is a modified vaccinia Ankara (MVA) virus. In some aspects, the carrier is a virus-like particle. In some aspects, the carrier is a non-viral carrier. In some aspects, the carrier comprises two or more different types of viruses and / or virus-like particles. In some aspects, the carrier is a pharmaceutically acceptable carrier (e.g., suitable for a buccal, epidermal, epidural, intraarterial, intraarticular, intracap sul ar, intracardiac, intracoronary, intradermal, intralesional, intralymphatic, intramuscular, intranasal, intraorbital, intraperitoneal, intraspinal, intrasterna, intrathecal, intravenous, mucosal, oral, rectal, subarachnoid, subcapsular, subcutaneous, subcuticular, sublingual, topical, transtracheal, or vaginal route of administration or any combination thereof). In some aspects, the carrier is a pharmaceutically acceptable carrier suitable for mucosal delivery.

[0276] In some aspects, the carrier exhibits an adjuvant property. Not to be bound by any theory, in some aspects, the adjuvant property of the delivery component promotesmobilization of antigen presenting cells to the site of vaccine delivery and antigen expression, thereby augmenting the uptake of the polynucleotide and the expressed antigens into professional antigen presenting cells to elicit antigen presentation.

[0277] In some aspects, the avian influenza vaccine is in freeze-dried form.

[0278] In some aspects, the avian influenza vaccine further comprises an adjuvant.

[0279] In some aspects, the avian influenza vaccine described herein is an mRNA vaccine.

[0280] In some aspects, the mRNA vaccine comprises a single-stranded RNA that may be translated into the respective protein upon entering cells of a recipient. In addition to wildtype or codon-optimized sequences encoding the antigen sequence, the RNA may contain one or more structural elements optimized for maximal efficacy of the RNA with respect to stability and translational efficiency (5' cap, 5' UTR, Togaviridae replicase (Venezuelan equine encephalitis virus non- structural proteins from TC83 strain), promoter, 3' UTR, poly(A)-tail). In some aspects, the RNA contains all of these elements. In some aspects, the RNA comprises a chemical modification.

[0281] In some aspects, the avian influenza vaccine described herein is a self-amplifying mRNA vaccine (saRNA) or a self-replicating RNA molecule. Self-replicating RNA molecules are well known in the art and can be produced by using replication elements derived from, e.g., alphaviruses, and substituting the structural viral proteins with a nucleotide sequence encoding a protein of interest. A self-replicating RNA molecule is typically a +-strand molecule which can be directly translated after delivery to a cell, and this translation provides a RNA-dependent RNA polymerase which then produces both antisense and sense transcripts from the delivered RNA. Thus the delivered RNA leads to the production of multiple daughter RNAs. These daughter RNAs, as well as collinear subgenomic transcripts, may be translated themselves to provide in situ expression of an encoded antigen (i.e. a mosaic H5 antigen), or may be transcribed to provide further transcripts with the same sense as the delivered RNA which are translated to provide in situ expression of the antigen. The overall result of this sequence of transcriptions is a huge amplification in the number of the introduced replicon RNAs and so the encoded antigen becomes a major polypeptide product of the cells.

[0282] One suitable system for achieving self-replication in this manner is to use an alphavirus-based replicon. These replicons are +-stranded (positive sense-stranded) RNAs which lead to translation of a replicase (or replicase-transcriptase) after delivery to a cell.The replicase is translated as a polyprotein which auto-cleaves to provide a replication complex which creates genomic- strand copies of the +-strand delivered RNA. These negative sense (- -strand) transcripts can themselves be transcribed to give further copies of the +-stranded parent RNA and also to give a subgenomic transcript which encodes the antigen. Translation of the subgenomic transcript thus leads to in situ expression of the antigen by the infected cell. Suitable alphavirus replicons can use a replicase from a Sindbis virus, a Semliki forest virus, an eastern equine encephalitis virus, a Venezuelan equine encephalitis virus, etc. Mutant or wild- type virus sequences can be used e.g. the attenuated TC83 mutant of VEEV has been used in replicons, see the following reference: W02005 / 113782, the context of which is incorporated by reference.

[0283] In some aspects, the self-replicating RNA molecule described herein encodes (i) a RNA-dependent RNA polymerase which can transcribe RNA from the self-replicating RNA molecule and (ii) a mosaic H5 protein antigen. The polymerase can be an alphavirus replicase e.g. comprising one or more of alphavirus proteins nsPl, nsP2, nsP3 and nsP4.

[0284] Whereas natural alphavirus genomes encode structural virion proteins in addition to the non-structural replicase polyprotein, in certain embodiments, the self- replicating RNA molecules do not encode alphavirus structural proteins. Thus, the self- replicating RNA can lead to the production of genomic RNA copies of itself in a cell, but not to the production of RNA-containing virions. The inability to produce these virions means that, unlike a wild-type alphavirus, the self-replicating RNA molecule cannot perpetuate itself in infectious form. The alphavirus structural proteins which are necessary for perpetuation in wild- type viruses are absent from self-replicating RNAs of the present disclosure and their place is taken by gene(s) encoding the immunogen of interest, such that the subgenomic transcript encodes the immunogen rather than the structural alphavirus virion proteins. Thus a self-replicating RNA molecule useful with the invention may have two open reading frames. The first (5') open reading frame encodes a replicase; the second (3') open reading frame encodes an antigen. In some embodiments the RNA may have additional (e.g. downstream) open reading frames e.g. to encode further antigens or to encode accessory polypeptides.

[0285] Furthermore, a secretory signal peptide may be fused to the antigen-encoding regions preferably in a way that the secretory signal peptide is translated as N terminal tag. Sequences coding for short linker peptides predominantly consisting of the aminoacids glycine (G) and serine (S), as commonly used for fusion proteins may be used as GS / Linkers.

[0286] The mRNA vaccine may be complexed with proteins and / or lipids, preferably lipids, to generate RNA-particles for administration. If a combination of different RNAs is used, the RNAs may be complexed together or complexed separately with proteins and / or lipids to generate RNA-particles for administration.

[0287] In some aspects, the mRNA vaccine is administered in the form of a lipid nanoparticle. The lipid nanoparticle may comprise any lipid capable of forming a particle to which the one or more nucleic acid molecules are attached, or in which the one or more nucleic acid molecules are encapsulated.

[0288] In some aspects, the lipid nanoparticle comprises one or more cationic lipids, and one or more stabilizing lipids. In some aspects, the lipid nanoparticle comprises a PEG- modified lipid, a non-cationic lipid, a sterol, an ionizable cationic lipid, or any combination thereof.

[0289] In some embodiments, the lipid nanoparticle comprises a cationic lipid, a neutral lipid, a steroid, a polymer conjugated lipid; and the RNA, encapsulated within or associated with the lipid nanoparticle.

[0290] In some aspects, the mRNA vaccine comprises a mRNA encoding the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, or combinations thereof. In some aspects, the mRNA vaccine comprises a mRNA corresponding to SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, or combinations thereof.

[0291] In some aspects, provided herein is a composition comprising a lipid nanoparticle and a messenger RNA (mRNA) comprising an open reading frame (ORF) that comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14; wherein the first nucleotide sequence encodes a first polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

[0292] In some aspects, the lipid nanoparticle comprises a PEG-modified lipid, a noncationic lipid, a sterol, an ionizable cationic lipid, or any combination thereof. In someaspects, the mRNA comprises a 5' untranslated region (UTR) and a 3' UTR. In some aspects, the mRNA comprises a chemical modification.

[0293] Lipids and lipid nanoparticles comprising nucleic acids and their method of preparation are disclosed in, e.g., U.S. Patent No. 8,569,256, U.S. Patent No. 5,965,542 and U.S. Patent Publication Nos. 2016 / 0199485, 2016 / 0009637, 2015 / 0273068, 2015 / 0265708, 2015 / 0203446, 2015 / 0005363, 2014 / 0308304, 2014 / 0200257, 2013 / 0338210, 2013 / 0323269, 2013 / 0245107, 2013 / 0195920, 2013 / 0123338, 2013 / 0022649, 2013 / 0017223, 2012 / 0295832, 2012 / 0183581, 2012 / 0172411, 2012 / 0027803, 2012 / 0058188, 2011 / 0311583, 2011 / 0311582, 2011 / 0262527, 2011 / 0216622, 2011 / 0117125, 2011 / 0091525, 2011 / 0076335, 2011 / 0060032, 2010 / 0130588, 2007 / 0042031, 2006 / 0240093, 2006 / 0083780, 2006 / 0008910, 2005 / 0175682, 2005 / 0118253, 2005 / 0064595, 2004 / 0142025, 2007 / 0042031, and PCT Pub. Nos. WO 99 / 39741, WO 2018 / 081480, WO 2018 / 078053, WO 2017 / 004143, WO 2017 / 075531, WO 2015 / 199952, WO 2014 / 008334, WO 2013 / 086373, WO 2013 / 086322, WO 2013 / 016058, WO 2013 / 086373, WO 2011 / 141705, and WO 1999 / 009076, the full disclosures of which are herein incorporated by reference in their entirety for all purposes.

[0294] Other exemplary lipids and lipid nanoparticles and their manufacture are described in the art, for example in U.S. Patent Application Publication No. U.S. 2012 / 0276209, Semple et al., 2010, Nat Biotechnol., 28(2): 172-176; Akinc et al., 2010, Mol Ther., 18(7): 1357-1364; Basha et al., 2011, Mol Ther, 19(12): 2186-2200; Leung et al., 2012, J Phys Chem C Nanomater Interfaces, 116(34): 18440-18450; Lee et al., 2012, Int J Cancer., 131(5): E781-90; Belliveau et al., 2012, Mol Ther nucleic Acids, 1 : e37; Jayaraman et al., 2012, Angew Chem Int Ed Engl., 51(34): 8529-8533; Mui et al., 2013, Mol Ther Nucleic Acids.2, el39; Maier et al., 2013, Mol Ther., 21(8): 1570-1578; and Tam et al., 2013, Nanomedicine, 9(5): 665-74, each of which are incorporated by reference in their entirety. Lipids and their manufacture can be found, for example, in U.S. Pub. No. 2015 / 0376115 and 2016 / 0376224, both of which are incorporated herein by reference.VI. Pharmaceutical Formulations

[0295] The compositions (e.g., the polynucleotides, the avian influenza vaccine vectors, the avian influenza vaccines, the mosaic H5 proteins or antigenic fragment thereof, or anycombination thereof) described or exemplified herein may be formulated with conventional carriers and excipients, which is selected in accord with ordinary practice. Aqueous formulations are prepared in sterile form, and when intended for delivery by other than oral administration, will generally be isotonic. All formulations will optionally contain excipients such as those set forth in the Handbook of Pharmaceutical Excipients, Sheskey, P. J. et al., eds., 9thEd., American Pharmacists Association, Washington, DC, and Pharmaceutical Press, Grayslake, IL (2020). Excipients include ascorbic acid and other antioxidants, chelating agents such as EDTA, carbohydrates such as dextrin, hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and the like. The pH of the formulations ranges from about 3 to about 11, but is ordinarily about 7 to 10 or about 8 to 9, e.g., for poxviruses.

[0296] While it is possible for the active ingredients to be administered alone, they may be present as pharmaceutical formulations. The formulations, e.g., for veterinary use, of the disclosure comprise at least one active ingredient, together with one or more acceptable carriers, and optionally other therapeutic ingredients. The formulations include those suitable for the foregoing administration routes. The formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods known in the art of pharmacy. Techniques and formulations generally are found in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Company, Easton, Pa. (1990). Such methods include the step of bringing into association the active ingredient with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

[0297] Formulations of the disclosure suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient may also be administered as a bolus, electuary or paste.

[0298] Pharmaceutical formulations according to the present invention may include one or more pharmaceutically acceptable carriers or excipients and optionally other therapeutic agents. Pharmaceutical formulations containing the active ingredient may bein any form suitable for the intended method of administration. When used for oral use for example, tablets, troches, lozenges, aqueous or oil suspensions, dispersible powders or granules, emulsions, hard or soft capsules, syrups or elixirs may be prepared. Compositions intended for oral use may be prepared according to any method known to the art for the manufacture of pharmaceutical compositions and such compositions may contain one or more agents including sweetening agents, flavoring agents, coloring agents and preserving agents, in order to provide a palatable preparation.

[0299] Formulations for oral use may be also presented as hard gelatin capsules where the active ingredient is mixed with an inert solid diluent, for example calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.

[0300] Aqueous suspensions of the invention contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia, and dispersing or wetting agents such as a naturally occurring phosphatide (e.g., lecithin), a condensation product of an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (e.g., heptadecaethyleneoxy cetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxy -benzoate, one or more coloring agents, one or more flavoring agents and one or more sweetening agents, such as sucrose or saccharin.

[0301] Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, such as arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oral suspensions may contain a thickening agent, such as beeswax, hard paraffin or cetyl alcohol. Sweetening agents, such as those set forth above, and flavoring agents may be added to provide a palatable oral preparation. These compositions may be preserved by the addition of an antioxidant such as ascorbic acid.

[0302] The amount of active ingredient that may be combined with the carrier material to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. The pharmaceutical composition can be prepared to provideeasily measurable amounts for administration. For example, an aqueous solution intended for intravenous infusion may contain from about 3 to 500 pg of the active ingredient per milliliter of solution in order that infusion of a suitable volume at a rate of about 30 mL / hour can occur.

[0303] Formulations suitable for intrapulmonary or nasal administration may have a particle size for example in the range of 0.1 to 500 microns (including particle sizes in a range between 0.1 and 500 microns in increments microns such as 0.5, 1, 30 microns, 35 microns, etc.), which is administered by rapid inhalation through the nasal passage or by inhalation through the mouth so as to reach the alveolar sacs. Suitable formulations include aqueous or oily solutions of the active ingredient. Formulations suitable for aerosol or dry powder administration may be prepared according to conventional methods and may be delivered with other therapeutic agents such as compounds heretofore used in the treatment or prophylaxis of a given condition.

[0304] Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.

[0305] The formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injection, immediately prior to use. Extemporaneous injection solutions and suspensions are prepared from sterile powders, granules and tablets of the kind previously described. Exemplary unit dosage formulations are those containing a daily dose or unit daily subdose, as herein above recited, or an appropriate fraction thereof, of the active ingredient.

[0306] It should be understood that in addition to the ingredients particularly mentioned above the formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavoring agents.

[0307] The invention further provides veterinary compositions comprising at least one active ingredient as above defined together with a veterinary carrier therefor. Veterinary carriers are materials useful for the purpose of administering the composition and may be solid, liquid or gaseous materials which are otherwise inert or acceptable in the veterinaryart and are compatible with the active ingredient. These veterinary compositions may be administered orally, parenterally or by any other desired route.VII. Pharmaceutical Compositions

[0308] The present disclosure also features pharmaceutical compositions comprising any of the polynucleotides, nucleic acid constructs, avian influenza vaccine vectors, avian influenza vaccines, mosaic H5 proteins or fragments thereof described herein, or any combination thereof.

[0309] In some aspects, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients or carriers.

[0310] In some aspects, the pharmaceutical compositions are suitable for inoculation, e.g., nasal, ocular, parenteral or oral administration. In some aspects, the pharmaceutical compositions further comprise sterile aqueous or non-aqueous solutions, suspensions, or emulsions. In some aspects, the pharmaceutical compositions further comprise auxiliary agents or excipients, as known in the art. In some aspects, the pharmaceutical compositions are presented in the form of individual doses (unit doses).

[0311] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and / or emulsions, which may contain auxiliary agents or excipients known in the art. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyloleate. Carriers or occlusive dressings can be used to increase skin permeability and enhance antigen absorption. Liquid dosage forms for oral administration may generally comprise a liposome solution containing the liquid dosage form. Suitable forms for suspending liposomes include emulsions, suspensions, solutions, syrups, and elixirs containing inert diluents commonly used in the art, such as purified water. Besides the inert diluents, such compositions can also include adjuvants, wetting agents, emulsifying and suspending agents, or sweetening, flavoring, or perfuming agents.

[0312] As apparent to one skilled in the art, the optimal concentration of the active agent in a pharmaceutical composition of the disclosure will necessarily depend upon the specific agent(s) used, the characteristics of the mammal, the type and amount of adjuvant, if any, and / or the nature of the infection. These factors can be determined by those of skill in the medical and pharmaceutical arts in view of the present disclosure.

[0313] Specific dosages may be adjusted depending on conditions of disease, the age, body weight, ethnic background, general health conditions, sex, diet, lifestyle and / or current therapeutic regimen of the mammal, as well as for intended dose intervals, administration routes, excretion rate, and combinations of drugs.

[0314] In addition to the polynucleotides, avian influenza vaccine vectors, avian influenza vaccines, mosaic H5 proteins or antigenic fragments thereof described or exemplified herein, the pharmaceutical composition may further comprise one or more pharmaceutically acceptable carriers. As used herein, the term "pharmaceutically acceptable carrier" refers to an acceptable vehicle for administering a composition to mammals comprising one or more non-toxic excipients which do not react with or reduce the effectiveness of the pharmacologically active agents contained therein. The proportion and type of pharmaceutically acceptable carrier in the pharmaceutical composition may vary, depending on the chosen route of administration. Suitable pharmaceutically acceptable carriers for the compositions of the present disclosure are described in the standard pharmaceutical texts. See, e.g., "Remington's Pharmaceutical Sciences", 18th Ed., Mack Publishing Company, Easton, Pa. (1990). Specific non-limiting examples of suitable pharmaceutically acceptable carriers include water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.

[0315] In some aspects, the pharmaceutical composition further comprises minor amounts of auxiliary substances such as agents that enhance the effectiveness of the preparation, stabilizers, preservatives, and the like.

[0316] In some aspects, the pharmaceutical composition further comprises a bile acid or a derivative thereof, in particular in the form of a salt. These include derivatives of cholic acid and salts thereof, in particular sodium salts of cholic acid or cholic acid derivatives. Examples of bile acids and derivatives thereof include cholic acid, deoxy cholic acid, chenodeoxycholic acid, lithocholic acid, ursodeoxycholic acid, hyodeoxycholic acid and derivatives such as glyco-, tauro-, amidopropyl- 1 -propanesulfonic-, and amidopropyl-2- hydroxy-l-propanesulfonic-derivatives of the aforementioned bile acids, or N, N- bis(3Dgluconoamidopropyl) deoxycholamide. A particular example is sodium deoxycholate (NaDOC).

[0317] Examples of suitable stabilizers include protease inhibitors, sugars such as sucrose and glycerol, encapsulating polymers, chelating agents such as ethylene-diaminetetraceticacid (EDTA), proteins and polypeptides such as gelatin and polyglycine and combinations thereof.

[0318] In some aspects, the pharmaceutical composition further comprises one or more adjuvants. Suitable adjuvants for inclusion in the pharmaceutical compositions of the present disclosure include, but are not limited to, those that are well known in the art, such as complete Freund's adjuvant (CFA), incomplete Freund's adjuvant (IF A), squalene, squalane, alum, and various oils, all of which are commercially from several sources, such as Novartis (e.g., Novartis' MF59 adjuvant).

[0319] Depending on the route of administration, the compositions (e.g., any the polynucleotides, avian influenza vaccine vectors, avian influenza vaccines, mosaic H5 proteins or antigenic fragments thereof, pharmaceutical compositions described or exemplified herein) may take the form of a solution, suspension, emulsion, or the like. A composition can be administered by a buccal, epidermal, epidural, intraarterial, intraarticular, intracapsular, intracardiac, intracoronary, intradermal, intralesional, intralymphatic, intramuscular, intranasal, intraorbital, intraperitoneal, intraspinal, intrasterna, intrathecal, intravenous, mucosal, oral, rectal, subarachnoid, subcapsular, subcutaneous, subcuticular, sublingual, topical, transtracheal, or vaginal route of administration or any combination thereof to an avian (e.g., a chicken). Compositions may be formulated for a particular route of delivery, e.g., formulated for oral delivery, nasal delivery, or intravenous delivery.

[0320] In some aspects, the compositions are formulated for a mucosal delivery.

[0321] For parenteral administration (e.g., intravenous, subcutaneous, intramuscular, intraperitoneal, or intradermal injection), the compositions may further comprise pharmaceutically accepted carriers. For administration by injection, the composition may be in a solution in a sterile aqueous vehicle which may also contain other solutes such as buffers or preservatives as well as sufficient quantities of pharmaceutically acceptable salts or of glucose to make the solution isotonic.

[0322] The compositions may be delivered to the respiratory system, for example to the nose, sinus cavities, sinus membranes or lungs, in any suitable manner, such as by inhalation via the mouth or intranasally. The composition may be dispensed as a powdered or liquid nasal spray, suspension, nose drops, a gel or ointment, through a tube or catheter, by syringe, by packtail, by pledget, or by submucosal infusion. The composition may be conveniently delivered in the form of an aerosol spray using apressurized pack or a nebulizer and a suitable propellant, e.g., without limitation, dichlorodifluoromethane, trichlorofluoromethane, di chlorotetrafluoroethane, or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be controlled by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the composition and a suitable powder base such as lactose or starch. Examples of intranasal formulations and methods of administration can be found in PCT publications WO 01 / 41782, WO 00 / 33813, and U.S. Pat. Nos. 6,180,603; 6,313,093; and 5,624,898, all of which are incorporated herein by reference and for all purposes. A propellant for an aerosol formulation may include compressed air, nitrogen, carbon dioxide, or a hydrocarbon based low boiling solvent. The composition may be conveniently delivered in the form of an aerosol spray presentation from a nebulizer or the like. In some aspects, the active ingredients are suitably micronized so as to permit inhalation of substantially all of the active ingredients into the lungs upon administration of the dry powder formulation, thus the active ingredients will have a particle size of less than 100 microns (e.g., less than 20 microns, 1 to 10 microns, or 0.2 to 0.4 microns). In some aspects, the composition is packaged into a device that can deliver a predetermined, and generally effective, amount of the pharmaceutical composition via inhalation, for example a nasal spray or inhaler.

[0323] In some aspects, the composition is administered prophylactically. For instance, administration of the composition may be commenced before or at the time of infection. In some aspects, the composition is administered up to about 1 month or more (e.g., up to about 4 months or more) before the mammal is exposed to an avian influenza virus. In some aspects, the composition is administered as soon as 1 week before infection (e.g., 1 to 5 days before infection).

[0324] In some aspects, the composition is presented in a single dose or as divided doses administered at appropriate intervals, for example as two, three, four or more sub-doses per day. In some aspects, a dose of the composition is administered on one day, followed by one or more booster doses spaced as desired thereafter. In some aspects, an initial vaccination is given, followed by a boost of the same vaccine at about one week up to about two months (e.g., about two weeks, about three weeks, about four weeks, about five weeks, about six weeks, about seven weeks, or about eight weeks) after the initial vaccination.

[0325] In some aspects, therapeutically effective and optimal dosage ranges for the compositions can be determined using methods known in the art. For example, subjects or test animals can be inoculated with varying dosages at scheduled intervals and test blood samples can be evaluated for levels of antibody and / or avian influenza neutralizing activity present in the blood, for example, by Western blot analysis. Such results can be used to refine an optimized immunization dosage and schedule for effective immunization of avian, specifically chicken, subjects.VIII. Host Cells

[0326] The present disclosure also features host cells (e.g., isolated host cells) comprising one or more of the polynucleotides, nucleic acid constructs, avian influenza vaccine vectors, avian influenza vaccines, mosaic H5 proteins or antigenic fragments thereof described herein or any combination thereof. Host cells include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, and higher eukaryotic cells such as avian cells.

[0327] In some aspects, the host cell is a eukaryotic host cell.

[0328] In some aspects, the host cell is an avian host cell.

[0329] In some aspects, the host cells are prepared by introducing polynucleotides, avian influenza vaccine vectors, avian influenza vaccines, mosaic H5 proteins or antigenic fragments thereof into the cells by techniques readily available to the person of ordinary skill in the art. These include, but are not limited to, calcium phosphate transfection, DEAE-dextran-mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection, lipofection, and other techniques such as those found in Sambrook et al. (Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)).IX. Kits and Containers

[0330] The present disclosure also features kits comprising any of the compositions (e.g., any polynucleotide, nucleic acid construct, avian influenza vaccine vector, avian influenza vaccine, mosaic H5 protein or antigenic fragment thereof, pharmaceutical composition described herein, or any combination thereof). The kits can be used to supplypolynucleotides, vectors, vaccines, pharmaceutical compositions, and other agents for use in diagnostic, basic research, or therapeutic methods, among others.

[0331] In some aspects, the kit further comprises a glass vial. In some aspects, the kit further comprises instructions for using the composition in a method for eliciting an immune response in an avian subject against one or more avian influenza antigens. In some aspects, the kit further comprises instructions for using the in a method for preventing, reducing the incidence of, attenuating, or treating avian influenza infection in an avian subject in need thereof.

[0332] The present disclosure also features containers comprising any composition (e.g., any polynucleotide, nucleic acid construct, avian influenza vaccine vector, avian influenza vaccine, mosaic H5 protein or antigenic fragment thereof, pharmaceutical composition described herein, or any combination thereof).

[0333] In some aspects, the container is a glass vial.X. Methods

[0334] The present disclosure also features methods of eliciting an immune response in an mammalian subject (e.g., a human, primate, or ferret) against one or more avian influenza antigens, the method comprising administering one or more doses (e.g., an effective amount) of any polynucleotide, nucleic acid construct, avian influenza vaccine vector, avian influenza vaccine, mosaic H5 protein or fragment thereof, pharmaceutical composition described herein, or any combination thereof, to the mammalian subject.

[0335] In some aspects, provided herein is a method of preventing, reducing the incidence of, attenuating, or treating avian influenza infection in a mammalian subject in need thereof, the method comprising administering one or more doses of any of the pharmaceutical compositions, any of the avian influenza vaccine vectors, or any of the avian influenza vaccines described herein to the mammalian subject.

[0336] In some aspects, the one or more doses of the pharmaceutical composition, the avian influenza vaccine vector, or avian influenza vaccine are administered to the mammalian subject by an oral, intranasal, subcutaneous, or intramuscular route of administration.

[0337] In some aspects, the mammalian subject is a human, a non-human primate, a ferret, a dog, a cat, a rodent, a porcine, or a bovine. In some aspects, the mammalian subject is a human.

[0338] In some aspects, the method elicits a neutralizing antibody response against avian influenza in the mammalian subject.

[0339] In some aspects, the mammalian subject is a human subject.

[0340] The present disclosure also features methods of eliciting an immune response in an avian subject (e.g., a chicken) against one or more avian influenza antigens, the method comprising administering one or more doses (e.g., an effective amount) of any polynucleotide, nucleic acid construct, avian influenza vaccine vector, avian influenza vaccine, mosaic H5 protein or fragment thereof, pharmaceutical composition described herein, or any combination thereof, to the avian subject.

[0341] In some aspects, the method elicits a neutralizing antibody response against avian influenza in the avian subject.

[0342] In some aspects, the method comprises administering i) a first dose (i.e., a priming dose) of any polynucleotide, nucleic acid construct, avian influenza vaccine vector, avian influenza vaccine, mosaic H5 protein or fragment thereof, pharmaceutical composition described herein, or any combination thereof, to the subject (e.g., an avian subject or a mammalian subject), and ii) a second dose (i.e., a boost dose) of any polynucleotide, nucleic acid construct, avian influenza vaccine vector, avian influenza vaccine, mosaic H5 protein or fragment thereof, pharmaceutical composition described herein, or any combination thereof, to the subject (e.g., an avian subject or a mammalian subject).

[0343] In some aspects, the second dose is about one week, about two weeks, about three weeks, about four weeks, about five weeks, about six weeks, about seven weeks, or about eight weeks after the first dose.

[0344] In some aspects, the first dose and the second dose comprise the same amount of any polynucleotide, nucleic acid construct, avian influenza vaccine vector, avian influenza vaccine, mosaic H5 protein or fragment thereof, pharmaceutical composition described herein, or any combination thereof.

[0345] In some aspects, the first dose and the second dose comprise a different amount of any polynucleotide, nucleic acid construct, avian influenza vaccine vector, avian influenza vaccine, mosaic H5 protein or antigenic fragment thereof, pharmaceutical composition described herein, or any combination thereof.

[0346] In some aspects, the first dose comprises an Avian Influenza vaccine comprising any mosaic H5 protein or antigenic fragment thereof described or exemplified herein (e.g., MVA-H5M2 and MVA-H5M3) and a modified vaccinia Ankara (MV A) virus, andthe second dose comprises an Avian Influenza self-amplifying mRNA vaccine comprising any mosaic H5 protein or antigenic fragment thereof described or exemplified herein (e.g., saRNA-H5M2 and saRNA-H5M3).

[0347] In some aspects, the first dose comprises an Avian Influenza self-amplifying mRNA vaccine comprising any mosaic H5 protein or antigenic fragment thereof described or exemplified herein (e.g., saRNA-H5M2 and saRNA-H5M3), and the second dose comprises an Avian Influenza vaccine comprising any mosaic H5 protein or antigenic fragment thereof described or exemplified herein (e.g., MVA-H5M2 and MVA- H5M3) and a modified vaccinia Ankara (MV A) virus.

[0348] In some aspects, the first dose and the second dose comprise Avian Influenza vaccine comprising any mosaic H5 protein or antigenic fragment thereof described or exemplified herein (e.g., H5M2 and H5M3) and a modified vaccinia Ankara (MV A) virus.

[0349] In some aspects, the first dose and the second dose comprise an Avian Influenza self-amplifying mRNA vaccine comprising any mosaic H5 protein or antigenic fragment thereof described or exemplified herein (e.g., saRNA-H5M2 and saRNA-H5M3).

[0350] In some aspects, the avian influenza is an H5N1 strain.

[0351] In some aspects, the H5N1 strain is a member of a clade selected from the group consisting of clade 2.2, clade 2.3.4.4 (e.g., sub lineages 2.3.4.4a, 2.3.4.4b, 2.3.4.4c, 2.3.4.4d, 2.3.4.4e, or 2.3.4.4f), and clade 2.5.

[0352] In some aspects, the H5N1 strain is a member of clade 2.2.

[0353] In some aspects, the H5N1 strain is a member of clade 2.3.4.4 (e.g., sub lineages2.3.4.4a, 2.3.4.4b, 2.3.4.4c, 2.3.4.4d, 2.3.4.4e, or 2.3.4.4f).

[0354] In some aspects, the H5N1 strain is a member of clade 2.5.

[0355] In some aspects, the avian influenza comprises a H5 hemagglutinin and a neuraminidase selected from the group consisting of Nl, N2, N3, N4, N5, N6, N7, N8, N9, N10, and Nl 1. In some aspects, the avian influenza comprises a H5 hemagglutinin and a Nl neuraminidase. In some aspects, the avian influenza comprises a H5 hemagglutinin and a N6 neuraminidase. In some aspects, the avian influenza comprises a H5 hemagglutinin and a N8 neuraminidase. In some aspects, the avian influenza comprises a H5 hemagglutinin and a N9 neuraminidase.

[0356] In some aspects, the one or more doses of the polynucleotides, nucleic acid constructs, avian influenza vaccine vectors, avian influenza vaccines, mosaic H5 proteinsor antigenic fragments thereof, pharmaceutical compositions, or any combination thereof are effective to elicit an immune response in the avian subject to the one or more avian influenza antigens. In some aspects, the one or more doses of the polynucleotides, nucleic acid constructs, avian influenza vaccine vectors, avian influenza vaccines, mosaic H5 proteins or antigenic fragments thereof, pharmaceutical compositions, or any combination thereof are effective to elicit an immune response in the avian subject to avian influenza antigens from at least two different avian influenza strains. In some aspects, the one or more doses of the polynucleotides, nucleic acid constructs, avian influenza vaccine vectors, avian influenza vaccines, mosaic H5 proteins or antigenic fragments thereof, pharmaceutical compositions, or any combination thereof are effective to elicit an immune response in the avian subject to avian influenza antigens from at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, or at least eleven different avian influenza strains. In some aspects, the one or more doses of the polynucleotides, nucleic acid constructs, avian influenza vaccine vectors, avian influenza vaccines, mosaic H5 proteins or antigenic fragments thereof, pharmaceutical compositions, or any combination thereof are effective to elicit an immune response in the avian subject to one or more avian influenza antigens from a newly emergent avian influenza (e.g., a highly pathogenic avian influenza).

[0357] In some aspects, the one or more doses of the polynucleotides, nucleic acid constructs, avian influenza vaccine vectors, avian influenza vaccines, mosaic H5 proteins or antigenic fragments thereof, pharmaceutical compositions, or any combination thereof are effective to induce an adaptive immune response in the avian subject to at least one avian influenza antigen. In some aspects, the adaptive immune response is a cellular T cell response to the at least one avian influenza antigen. In some aspects, the adaptive immune response is a humoral antibody response to the at least one avian influenza antigen. In some aspects, the adaptive immune response is a cellular T cell response to the at least one avian influenza antigen and a humoral antibody response to the at least one avian influenza antigen.

[0358] An adaptive immunological response to a composition or vaccine is the development in the host organism of a cellular and / or humoral (e.g., antibody-mediated) immune response to a viral polypeptide, e.g., an administered viral preparation, polypeptide or one encoded by an administered nucleic acid molecule, which can prevent or inhibit infection to closely structurally related viruses as well as more distantly relatedviruses. Usually, such a response involves the avian subject producing antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells directed specifically to an antigen or antigens included in the composition or vaccine of interest.

[0359] In some aspects, the one or more doses of the polynucleotides, nucleic acid constructs, avian influenza vaccine vectors, avian influenza vaccines, mosaic H5 proteins or antigenic fragments thereof, pharmaceutical compositions, or any combination thereof are administered to the avian subject by a buccal, epidermal, epidural, intraarterial, intraarticular, intracapsular, intracardiac, intracoronary, intradermal, intralesional, intralymphatic, intramuscular, intranasal, intraorbital, intraperitoneal, intraspinal, intrasterna, intrathecal, intravenous, mucosal, oral, rectal, subarachnoid, subcapsular, subcutaneous, subcuticular, sublingual, topical, transtracheal, or vaginal route of administration or any combination thereof.

[0360] In some aspects, the one or more doses of the polynucleotides, nucleic acid constructs, avian influenza vaccine vectors, avian influenza vaccines, mosaic H5 proteins or antigenic fragments thereof, pharmaceutical compositions, or any combination thereof are administered to the avian subject by a mucosal route.

[0361] In some aspects, the subject is an avian. In some aspects, the avian is a chicken.

[0362] The present disclosure also features methods of preventing, reducing the incidence of, attenuating, or treating an avian influenza infection in an avian subject in need thereof, the method comprising administering one or more doses (e.g., an effective amount) of any polynucleotide, nucleic acid construct, avian influenza vaccine vector, avian influenza vaccine, mosaic H5 protein or fragment thereof, pharmaceutical composition described herein, or any combination thereof to the avian subject.

[0363] In some aspects, the avian infection is caused by an H5N1 strain.

[0364] In some aspects, the H5N1 strain is a member of a clade selected from the group consisting of clade 2.2, clade 2.3.4.4 (e.g., sub lineages 2.3.4.4a, 2.3.4.4b, 2.3.4.4c, 2.3.4.4d, 2.3.4.4e, or 2.3.4.4f), and clade 2.5.

[0365] In some aspects, the avian infection is caused by a newly emergent avian influenza (e.g., a highly pathogenic avian influenza).

[0366] In some aspects, the one or more doses of the polynucleotides, nucleic acid constructs, avian influenza vaccine vectors, avian influenza vaccines, mosaic H5 proteins or antigenic fragments thereof, pharmaceutical compositions, or any combination thereof prevent, reduce the incidence of, attenuate or treat infection with at least two, at leastthree, at least four, or at least five different strains of avian influenza. In some aspects, the one or more doses of the polynucleotides, nucleic acid constructs, avian influenza vaccine vectors, avian influenza vaccines, mosaic H5 proteins or antigenic fragments thereof, pharmaceutical compositions, or any combination thereof prevent, reduce the incidence of, attenuate or treat infection with a newly emergent avian influenza and at least two, at least three, at least four, or at least five different strains of avian influenza.

[0367] In some aspects, the one or more doses of the polynucleotides, nucleic acid constructs, avian influenza vaccine vectors, avian influenza vaccines, mosaic H5 proteins or antigenic fragments thereof, pharmaceutical compositions, or any combination thereof are effective to induce an adaptive immune response in the subject to at least one avian influenza antigen. In some aspects, the adaptive immune response is a cellular T cell response to the at least one avian influenza antigen. In some aspects, the adaptive immune response is a humoral antibody response to the at least one avian influenza antigen. In some aspects, the adaptive immune response is a cellular T cell response to the at least one avian influenza antigen and a humoral antibody response to the at least one avian influenza antigen.

[0368] In some aspects, the one or more doses of the polynucleotides, nucleic acid constructs, avian influenza vaccine vectors, avian influenza vaccines, mosaic H5 proteins or antigenic fragments thereof, pharmaceutical compositions, or any combination thereof are administered to the avian subject by a buccal, epidermal, epidural, intraarterial, intraarticular, intracapsular, intracardiac, intracoronary, intradermal, intralesional, intralymphatic, intramuscular, intranasal, intraorbital, intraperitoneal, intraspinal, intrasterna, intrathecal, intravenous, mucosal, oral, rectal, subarachnoid, subcapsular, subcutaneous, subcuticular, sublingual, topical, transtracheal, or vaginal route of administration or any combination thereof.

[0369] In some aspects, the one or more doses of the polynucleotides, nucleic acid constructs, avian influenza vaccine vectors, avian influenza vaccines, mosaic H5 proteins or antigenic fragments thereof, pharmaceutical compositions, or any combination thereof are administered to the avian subject by a mucosal route of administration.

[0370] Administration can be accomplished by any means appropriate for the therapeutic agent, for example, by parenteral, mucosal, pulmonary, topical, catheter-based, or oral means of delivery. Parenteral delivery can include for example, subcutaneous, intravenous, intramuscular, intra-arterial, intraperitoneal, intralymphatic, and injectioninto the tissue of an organ. Mucosal delivery can include, for example, intranasal delivery, preferably administered into the airways of a patient, i.e., nose, sinus, throat, lung, for example, as nose drops, by nebulization, vaporization, or other methods known in the art. Oral or intranasal delivery can include the administration of a propellant. Pulmonary delivery can include inhalation of the agent. Catheter-based delivery can include delivery by iontophoretic catheter-based delivery. Oral delivery can include delivery of a coated pill, or administration of a liquid by mouth. Administration can generally also include delivery with a pharmaceutically acceptable carrier, such as, for example, a buffer, a polypeptide, a peptide, a polysaccharide conjugate, a liposome, and / or a lipid, according to methods known in the art.

[0371] In some aspects, the avian subject is a domestic poultry.

[0372] In some aspects, the domestic poultry is selected from the group consisting of chickens, ducks, turkeys, quails, geese, or guinea fowls.

[0373] In some aspects, the effective amount of a viral vector (e.g., a poxvirus such asMV A) or virus-like particle may be from IxlO7to IxlO8plaque-forming units (PFU) orTCIDso, which may be administered as a single dose or in two or more doses or each dose may include from IxlO7to IxlO8PFU, e.g., from IxlO7to IxlO8PFU. For instance, each dose may have the same number of PFU, or the booster dose(s) may have higher or lower amounts relative to the initial dose. The initial booster may be administered from 1 to 8 weeks after the priming dose, for instance 3 to 4 weeks after the priming dose. The priming dose and / or booster dose(s) may include an adjuvant.

[0374] In some aspects, the dose of the viral vector is between about IxlO6to about IxlO9PFU or TCID50, between about IxlO7to about IxlO9PFU or TCID50, between about IxlO8to about IxlO9PFU or TCID50, between about IxlO6to about IxlO8PFU or TCID50, or between about IxlO6to about IxlO7PFU or TCID50.

[0375] In some aspects, the dose of the viral vector is about IxlO6, about IxlO7, about IxlO8, or about IxlO9PFU or TCID50.

[0376] The practice of the present disclosure will employ, unless otherwise indicated, conventional techniques of cell biology, cell culture, molecular biology, transgenic biology, microbiology, recombinant DNA, and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. See, for example, Sambrook et al., ed. (1989) Molecular Cloning A Laboratory Manual (2nd ed.; Cold Spring Harbor Laboratory Press); Sambrook et al., ed. (1992) Molecular Cloning: A Laboratory Manual,(Cold Springs Harbor Laboratory, NY); D. N. Glover ed., (1985) DNA Cloning, Volumes I and II; Gait, ed. (1984) Oligonucleotide Synthesis; Mullis et al. U.S. Pat. No. 4,683,195; Hames and Higgins, eds. (1984) Nucleic Acid Hybridization; Hames and Higgins, eds.(1984) Transcription And Translation; Freshney (1987) Culture Of Animal Cells (Alan R. Liss, Inc.); Immobilized Cells And Enzymes (IRL Press) (1986); Perbal (1984) A Practical Guide To Molecular Cloning; the treatise, Methods In Enzymology (Academic Press, Inc., N.Y.); Miller and Calos eds. (1987) Gene Transfer Vectors For Mammalian Cells, (Cold Spring Harbor Laboratory); Wu et al., eds., Methods In Enzymology, Vols.154 and 155; Mayer and Walker, eds. (1987) Immunochemical Methods In Cell And Molecular Biology (Academic Press, London); Weir and Blackwell, eds., (1986) Handbook Of Experimental Immunology, Volumes I-FV; Manipulating the Mouse Embryo, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1986);Crooks, Antisense drug Technology: Principles, strategies and applications, 2ndEd. CRC Press (2007) and in Ausubel et al. (1989) Current Protocols in Molecular Biology (John Wiley and Sons, Baltimore, Md.).

[0377] All of the references cited above, as well as all references cited herein and the amino acid or nucleotide sequences (e.g., GenBank numbers and / or Uniprot numbers), are incorporated herein by reference in their entireties.

[0378] The following examples are offered by way of illustration and not by way of limitation.EXAMPLESExample 1 - MVA Based Recombinant Vaccine against Highly Pathogenic Avian InfluenzaStrain H5N1

[0379] A MVA platform is ideal for broad-protective vaccine designs and mucosal delivery approaches, widely used to express vaccine antigens against a variety of pathogens including influenza and SARS-CoV-2. In prior work, a MVA-based recombinant vaccine against HP Al H5N1 was developed under an in-silico mosaic approach (MVA-H5M) that showed protection in mice when challenged with multiple viral clades.

[0380] Here, an updated version of MVA-H5M (MVA-H5M2) was developed. The sequence will be analyzed using a self-amplifying mRNA, and it is predicted that thesevaccines will provide protection against recently emerged H5N1 viruses. A mosaic protein was constructed using H5N1 sequences reported between 1996 and 2023 that virtually covers the entire diversity of the virus to date. These sequences were downloaded automatically from The National Center for Biotechnology Information (NCBI) and GISAID databases, using a Selenium based web scrapping tool, and screened by bash preprocessing and map-reduce algorithms to exclude incomplete, low-coverage, ambiguous nucleotide and redundant sequences. Subsequently, the nucleotide sequences were translated to protein sequences to design the mosaic according to the final sequences using the platform Mosaic Vaccine Designer tool webpage. The obtained H5 mosaic (H5M2 and H5M3) were back translated, and codon optimized for chickens.

[0381] To generate the H5M, H5M2, and H5M3 protein sequences, a “genetic algorithm” (see the epigraph vaccine design tool developed by Los Alamos National Labs for HIV work and available at hiv.lanl.gov / content / sequence / EPIGRAPH / epigraph.html; see also Theiler, J., et al., Epigraph: A Vaccine Design Tool Applied to an HIV Therapeutic Vaccine and a Pan-Filovirus Vaccine, Sci Rep 6, 33987 (2016)) was used to generate, select, and “recombine” (in 811100 potential T cell epitopes (12 amino acids in length) into “mosaic” proteins, which can provide greater coverage of avian influenza A strains, and thus optimized immunogenicity, than any single wild-type avian influenza A protein. In the parameter options, the epitope length parameter was set to an amino acid length of 12-mer. The number of trials parameter was set to 1 with 0 iterative refinement steps. The random seed parameter was set to 0, and epitopes with 2 counts or less were removed.

[0382] The mosaic sequence accounts for the complete or full-length sequence of the protein and / or regions of interest, as well as the full diversity of the “core” sequences provided. The use of a mosaic sequence for an HIV-1 vaccine, which recombined potential T cell epitopes into Gag, Pol, and Env proteins, has been reported (Fischer et al., Nature Medicine 13: 100 (2007)). The combined antigenic coverage of the Ml, M2, and M3 mosaic proteins is 85%, which is similar to results of previously described and effective mosaic proteins (Callaway, E., Nature 579(7800):481 (2020)).

[0383] Additionally, T cell and B cell linear epitope identification against different AIV strains, and a discontinuous B cell prediction and functional analysis of the antigenic protein was analyzed. A genetic and proteomic diversity analysis with the final nucleotide and amino acids sequences was performed, which allowed the construction of a phylogenetic tree to identify the most crucial clades based on the recent outbreaks of Alover the years. In this analysis, the H5M mosaic vaccine developed before and the new mosaic vaccines (H5M2 and H5M3) was included to analyze the evolution and coverage of the existing strains. Comparing H5M, H5M2, and H5M3 with the HA sequence of other outbreaks causing H5N 1 s, it was found that H5M3 can comprehensively cover most of the representative viral clades and had important conserved T and B cell epitopes derived from H5N1 isolates (Tables 3-5). The H5M HA1 region (receptor binding site) does not show any evolution changes compared with the new emergence strains (Figure 1). In contrast, the HA1 region from H5M2 and H5M3 have some critical changes compared with the new emerging viruses, such as mutations that may promote a better binding site between the virus and the host cell (Figure 2). The HA2 region is highly conserved and plays an important role for broadly neutralizing antibodies against all Al subtypes within the mosaics. Interestingly, H5M2 and H5M3 contain a deletion of a single amino acid at position 344 which is also present in all recent HP Al viruses (Figure 1).Table 3. Percent of identity, positives, and gaps between HA protein of H5N1 viruses and the MVA-H5M Mosaic (first-generation vaccine)Table 4. Percent of identity, positives, and gaps between HA protein of H5N1 viruses and the H5M2 Mosaic (second-generation vaccine candidate)Table 5. Percent of identity, positives, and gaps between HA protein of H5N1 viruses and the H5M3 Mosaic (third-generation vaccine candidate)

[0384] A preliminary phylogenetic analysis was performed to assess the clade classification and mutations identification on the mosaic sequences throughout the evolution of the virus between 1980 and 2023. Based on this analysis, H5M mosaic is classified into clade 2.5, H5M2 into clade 2.2, and H5M3 into clade 2.3.4.4b (Figure 3). Crucial mutations were identified that were reported in the literature (FluSurver by GISAID) for H5M, H5M2, and H5M3 related to host specificity shift, antigenic drift, and escape mutant (Table 6). Additionally, four mutations in H5M were identified that associated with structural interactions that promote antibody recognition, viral oligomerization interfaces, and binding of small ligands (K52T, V102T, R22K, and 1529). In contrast, seven of these mutations were identified in H5M2 (1190 V, S197P, D243E, A279T, V281M, V285L, and L338Q) and fourteen mutations in H5M3 (K3N, G16S, R88N, R98K, Li l IF, Q185R, V194I, A201E, N252D, E284G, M285V, I298V, K492E, and I547M).Table 6. Mutations related to antigenic shift and escape mutant reported in FluSurver of GISAID database for H5M and H5M2 mosaics.Example 2 - Evaluation of the immunogenicity and efficacy of an H5 mosaic sa-mRNA vaccine in mice and chickens against recently emerged H5N1 viruses. (Prophetic)

[0385] The MVA-H5M vaccine provided homosubtypic and heterosubtypic protection in mice against H5Nls and HlNls. This vaccine candidate was designed and tested with the potential to be used for poultry immunizations in the context of future H5N1 outbreaks. Under that premise, is imperative to test if the second-generation of the H5 mosaic protein (H5M2) can elicit a protective response against recently emerged AIVs as an sa- mRNA vaccine.

[0386] Evaluate the efficacy of sa-mRNA-H5M2 against recently emerged HP Al H5N1 viruses in mice.

[0387] Experimental Approach. To test the hypothesis that sa-mRNA-H5M2 can confer protective immune response against recently emerged HP Al H5N1 viruses, groups of 5- week-old BALB / c mice (8 animals per group) will be vaccinated with 1 pg or 0.01 pg of the selected sa-mRNA-H5M2 vaccine, but additionally will receive 1 x 107PFU (Plaque Forming Unit) of A / H5N1 influenza vaccine (inactivated whole virion vaccine as positive control), or PBS (negative controls) via the intramuscular (IM) injection in the rear quadriceps. Four weeks after the initial immunization, some of the groups will receive a boost inoculation with the corresponding vaccine or control. At two weeks post boost inoculation, mice will be challenged intranasally with 100 LD50s of A / Turkey / Indiana / 2022 (H5N1 clade 2.3.4.4.b) virus contained in 20 pL of PBS. On days 28 (pre-boost) and 42 (pre-challenge) post prime immunization, blood samples will be collected for serological analysis by hemagglutination inhibition (HI) assay. Four mice from each group will be euthanized on day 4 post challenge for lung tissue collection that will be used for subsequent histopathology analysis, viral load determination by qRT- PCR and viral titration by plaque assay on MDCK cells. The remaining animals within each group (4 mice per group) will be monitored daily for 14 days for survival, bodyweight changes and scoring of clinical parameters. Mice showing greater than or equal to a 20% body weight loss will be humanely euthanized. See Table 7.Table 7. Immunogenicity and Efficacy of MVA-H5M2 in mice (5-week-old BALB / c mice)Evaluate if sa-mRNA-H5M2 can elicit a humoral neutralizing response in chickens to drive protection against recently emerged HPAI H5N1 viruses.

[0388] Experimental Approach. The capacity of sa-mRNA-H5M2 to elicit a humoral immune response in chickens against viral lethal challenges through a passive transfer assay to a murine model will be assessed. For this, groups of 2-week-old SPF chickens (8 animals per group) will receive two inoculations four weeks apart with 1 pg or 0.01 pg ofthe selected sa-mRNA-H5M2 vaccine, but additionally will receive 1 x 107PFU (Plaque Forming Unit) of A / H5N1 influenza vaccine (inactivated whole virion vaccine as positive control), or PBS (negative controls) via the intramuscular (IM) injection. Blood samples will be collected on days 28 (pre-boost) and 42 (two weeks post-boost) post-priming for serological analysis. On day 42, sera from each group will be pooled to obtain six pools. See Table 8.

[0389] Groups of naive 5-week-old BALB / c mice (8 animals per group) will receive pooled sera from the corresponding chicken groups by intraperitoneal (IP) administration. At 24 hours post-transfer, mice will be challenged IN with 100 LDsos of A / Turkey / Indiana / 2022 (H5N1 clade 2.3.4.4.b) virus contained in 20 pL of PBS. On days 7 and 14 post challenge, blood samples will be collected for serological analysis by HI assay. Four mice from each group will be euthanized on day 4 post-challenge for lung tissue collection, for subsequent histopathology analysis, viral load determination by qRT-PCR and viral titration by plaque assay on MDCK cells. The remaining animals within each group (4 mice per group) will be monitored daily for 14 days for survival, body weight changes and scoring of clinical parameters. Mice showing greater than or equal to a 20% body weight loss will be humanely euthanized. See Table 9.Table 8. Evaluation of MVA-H5M2 capacity to induce humoral protective response (Vaccination in 2 week-old SFP chickens)Table 9. Evaluation of MVA-H5M2 capacity to induce humoral protective response (Passive Transfer in 5-week-old BALB / c mice)Example 3 - Evaluation of the immunogenicity and efficacy of MVA-H5M3 and sa-mRNA H5M3 vaccine in chickens against recently emerged H5N1 viruses.

[0390] The three H5M, H5M2, and H5M3 mosaic sequences generated in example 1 above were used to generate self-amplifying mRNA vaccine vectors (saRNA-H5M, saRNA-H5M2, and saRNA-H5M3).

[0391] These saRNA vaccine vectors were loaded into two different lipid nanoparticle delivery systems developed to effectively and safely deliver therapeutic nucleic acids into the cytosol of various cell types after local administration in vivo (e.g., formulation #1 and formulation #2).

[0392] Next, the MVA-H5M3 vaccine, formulation #1 of the self-amplifying mRNA (saRNA)-H5M3 vaccine at low dose (0.1 pg), medium dose (0.5 pg), or high dose (1 pg), and formulation #2 of the sa-RNA-H5M3 vaccine, were analyzed, with an inactivated H5N1 virus used as a positive control.

[0393] Experimental Approach. White Leghorn SPF chickens (10 per group) received a prime dose and a boost dose four weeks apart, with a H5N1 challenge 2 weeks after the boost dose. The SPF chickens received IxlO8PFU of MVA-H5M3 vaccine, a low dose(0.1 pg), medium dose (0.5 pg), or high dose (1 pg) of formulation #1 of the selfamplifying mRNA (saRNA)-H5M3 vaccine, a medium dose (0.5 pg) of formulation #2 of the self-amplifying mRNA (saRNA)-H5M3 vaccine, or 512 hemagglutinating units (HAU) whole inactivated H5N1 virion. Unvaccinated SPF chickens were used as a negative control.

[0394] Results. After the prime and boost with the vaccine, the chickens were infected with an H5N1 virus and survival rates were tracked for two weeks. Figure 4 shows survival rate of White Leghorn SPF chickens after administration with vaccines or control. While 100% of the vaccinated animals survived, only 20% of the unvaccinated animals survived by the end of the two week period.

[0395] Furthermore, no clinical signs of H5N1 infection were seen in vaccinated animals over the two week period (Figure 5A), while unvaccinated animals began showing mild symptoms, depression, or death shortly after infection with the H5N1 virus (Figure 5B).

[0396] Viral shedding was not seen either the oropharynx or cloaca of vaccinated animals over the two week period, while H5N1 viral detection in unvaccinated animals was seen 2 days post infection in both the oropharynx and cloaca (Figures 6A and 6B).

[0397] Hemagglutination inhibiting (HI) antibody titer was measured in each group at the following time points: 1) 3 weeks after the prime dose, 2) 4 weeks after the prime dose, 3) 6 weeks after the prime dose (i.e., after the boost dose, but before the challenge with the virus), and 4) 8 weeks after the prime does (i.e., two weeks after the challenge). The MVA-H5M3 vaccine showed 10% seropositivity before boost, 100% seropositivity with boost, and slightly lower HI titers compared to saRNA-H5M3. Formulation #1 of the saRNA-H5M3 vaccine showed 20% seropositivity before boost, 100% seropositivity with boost, and a dose-response pattern on HI titers. Formulation #2 of the saRNA-H5M3 vaccine showed 50% seropositivity before boost, 100% seropositivity after boost, and higher HI titers than formulation #1. The whole inactivated H5N1 virion positive control showed 100% seropositivity before boost and a high increase in HI titers with the boost. Finally, unvaccinated control did not show any detectable HI titers. Taken together, both the MVA-H5M3 vaccine and saRNA-H5M3 vaccine formulations showed titers above the recommended HI titer to protect from mortality, thus showing their effectiveness. See Figure 7.

[0398] Additionally, both H5-specific Antibodies and nucleoprotein (NP) specific antibodies were measured at the following time points: 1) 3 weeks after the prime dose, 2)4 weeks after the prime dose, 3) 6 weeks after the prime dose (i.e., after the boost dose, but before the challenge with the virus), and 4) 8 weeks after the prime does (i.e., two weeks after the challenge). The MVA-H5M3 vaccine showed 20% seropositivity for the H5-specific antibodies before the boost and 100% seropositivity with the boost. Formulation #1 of the saRNA-H5M3 vaccine showed 20%-70% seropositivity for the H5-specific antibodies before boost (depending on dose), and 100% seropositivity after the boost for all doses. Formulation #2 of the saRNA-H5M3 vaccine showed 100% seropositivity for the H5-specific antibodies before boost and 100% seropositivity after the boost. The MVA-H5M3 and saRNA-H5M3 vaccines did not show seropositivity for the NP-specific antibodies before the challenge with the H5N1 virus. However, one animal in the MVA-H5M3 group showed NP-specific antibodies at the 8 week time point. The whole inactivated H5N1 virion positive control showed 100% seropositivity for both H5-specific antibodies and NP-specific antibodies at all measured time points. The unvaccinated controls only had 1 animal be seropositive for both H5-specific and NP- specific antibodies, and the data point was 1 week after the challenge with the H5N1 virus. See Figure 8.

[0399] The data in this experiment demonstrates that both the MVA-H5M3 vaccine and the saRNA-H5M3 vaccines (both formulations) are effective tools for combating H5N1 influenza.Example 4 - Evaluation of the immunogenicity of an H5 mosaic sa-mRNA vaccine and an H5 mosaic MVA vaccine in Balb / c mice against recently emerged H5N1 viruses. (Prophetic)

[0400] Experimental Approach. Groups of 5-week-old BALB / c mice (10 animals per group) will be vaccinated with 1) the MVA-H5M3 vaccine for both prime and boost dosages, 2) the saRNA-H5M3 vaccine for both prime and boost dosages, 3) the saRNA- H5M3 vaccine for the prime dose and the MVA-H5M3 vaccine for the boost dose, 4) the MVA-H5M3 vaccine for the prime dose and the saRNA-H5M3 vaccine for the boost dose, 5) whole inactivated H5N1 virion vaccine for prime and boost dosages, or 6) native MVA vector for both prime and boost dosages as a negative control. Prime and boost vaccination will be performed four weeks apart from each other.

[0401] At two weeks post boost inoculation, mice will be challenged intranasally with 100 LD50s of H5N1 (clade 2.3.4.4.b) virus. On days 28 (pre-boost) and 42 (prechallenge) post prime immunization, blood samples will be collected for serological analysis by hemagglutination inhibition (HI) assay, H5-specific antibody titermeasurements and cross-neutralization assay for other avian (H7N9 and H10N8) and seasonal (H1N1 and H3N2) influenza viruses. Four mice from each group will be euthanized on day 4 post challenge for lung tissue collection that will be used for subsequent histopathology analysis, viral load determination by RT-qPCR and viral titration by plaque assay on MDCK cells. The remaining animals within each group (6 mice per group) will be monitored daily for 14 days for survival, body weight changes and scoring of clinical parameters.

[0402] After testing the magnitude of humoral response and protection against the H5N 1 virus, one vaccination regime will be selected for further studies.Example 5 - Evaluation of the immunogenicity of an H5 mosaic sa-mRNA vaccine and an H5 mosaic MVA vaccine in primates against recently emerged H5N1 viruses. (Prophetic)

[0403] Experimental Approach. To test the hypothesis that the saRNA-H5M3 vaccine and the MVA-H5M3 vaccine can confer protective immune response against recently emerged HP Al H5N 1 viruses, groups of non-human primates (NHPs) will be administered the vaccination regime selected from Example 4 (e.g., 1) the MVA-H5M3 vaccine for both prime and boost dosages, 2) the saRNA-H5M3 vaccine for both prime and boost dosages, 3) the saRNA-H5M3 vaccine for the prime dose and the MVA-H5M3 vaccine for the boost dose, or 4) the MVA-H5M3 vaccine for the prime dose and the saRNA-H5M3 vaccine for the boost dose). If the saRNA-H5M3 vaccine is selected, then both 10 pg and 50 pg dosages will be tested. Prime and boost vaccination will be performed four weeks apart from each other.

[0404] The magnitude of humoral response will be tested via blood samples collected on days 28 (before boost vaccination), 42 (two weeks after boost vaccination) and 56 (four weeks after boost vaccination) post-prime vaccination for serological analysis by hemagglutination inhibition (HI) assay, as well as measurement of H5-specific antibody titer.Example 6 - Evaluation of the immunogenicity and efficacy of an H5 mosaic sa-mRNA vaccine and an H5-MVA vaccine in ferrets against recently emerged H5N1 viruses. (Prophetic)

[0405] Experimental Approach. To test the hypothesis that the saRNA-H5M3 vaccine and the MVA-H5M3 vaccine can confer protective immune response against recently emerged HP Al H5N1 viruses, groups of ferrets will be administered the vaccination regime selected from Example 4 (e.g., 1) the MVA-H5M3 vaccine for both prime andboost dosages, 2) the saRNA-H5M3 vaccine for both prime and boost dosages, 3) the saRNA-H5M3 vaccine for the prime dose and the MVA-H5M3 vaccine for the boost dose, or 4) the MVA-H5M3 vaccine for the prime dose and the saRNA-H5M3 vaccine for the boost dose). If the saRNA-H5M3 vaccine is selected, then both 10 pg and 50 pg dosages will be tested. Prime and boost vaccination will be performed four weeks apart from each other.

[0406] The ferrets will be challenged intranasally with an H5N1 virus from the 2.3.4.4b clade two weeks after boost vaccination and followed for two weeks after infection to monitor clinical signs and survival. The magnitude of humoral response will be tested via blood samples collected on days 27 (before boost vaccination) and 41 (before challenge) post-prime vaccination for serological analysis by hemagglutination inhibition (HI) assay, as well as measurement of H5-specific antibody titer. The magnitude of the cellular response will be tested via peripheral blood mononuclear cells (PBMC) separated from blood samples collected on day 35 (one week after boost vaccination) post-prime vaccination for virus-specific T lymphocytes analysis by flow cytometry. Nasal swabs and lung tissue post-infection will be collected to test for viral load determination by RT- qPCR and viral titration by plaque assay on MDCK cells.Example 7 - Evaluation of the cross protection of an H5 mosaic sa-mRNA vaccine and an H5 mosaic MVA vaccine in Balb / c mice against other avian and seasonal influenza viruses. (Prophetic)

[0407] Experimental Approach. Groups of 5-week-old BALB / c mice (10 animals per group) will be administered the vaccination regime selected from Example 4 (e.g., 1) the MVA-H5M3 vaccine for both prime and boost dosages, 2) the saRNA-H5M3 vaccine for both prime and boost dosages, 3) the saRNA-H5M3 vaccine for the prime dose and the MVA-H5M3 vaccine for the boost dose, or 4) the MVA-H5M3 vaccine for the prime dose and the saRNA-H5M3 vaccine for the boost dose).

[0408] At two weeks post boost inoculation, mice will be challenged intranasally with both avian influenza viruses (e.g., H7N9 or H10N8) and seasonal influenza viruses (H1N1 or H3N2). On days 28 (pre-boost) and 42 (pre-challenge) post prime immunization, blood samples will be collected for serological analysis by hemagglutination inhibition (HI) assay. Four mice from each group will be euthanized on day 4 post challenge for lung tissue collection that will be used for subsequent histopathology analysis, viral load determination by qRT-PCR and viral titration byplaque assay on MDCK cells. The remaining animals within each group (6 mice per group) will be monitored daily for 14 days for survival, body weight changes and scoring of clinical parameters.Example 8 - Evaluation of the immunogenicity of an H5 mosaic sa-mRNA vaccine and an H5-MVA vaccine in humans against recently emerged H5N1 viruses. (Prophetic)

[0409] Healthy adult subjects (n=40) with receive two doses of the saRNA-H5M3 vaccine (Group 1, n=10), 2 doses of the MVA-H5M3 vaccine (Group 2, n=10), 1 dose of each the saRNA-H5M3 vaccine and the MVA-H5M3 vaccine (Group 3, n=10), or placebo (Control group, n=10). Specific dose levels and vaccination sequence will be chosen selected on the experimental data in Examples 4-7.

[0410] Two participants in each group will receive vaccinations 48 hours apart. 7 days after the sentinel vaccination, additional participants will be randomized. Blood will be drawn at day 0 (1 day before prime dose of the vaccine), day 28, day 56, and day 180. Immunogenicity, hemagglutination inhibition, and microneutralization (MN) assays will be analyzed on each blood sample drawn after vaccination. Safety and reactogenicity will be analyzed for each group, along with any adverse effects.* * *

[0411] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary aspects or embodiments of the present invention as contemplated by the inventor(s), and thus, are not intended to limit the present invention and the appended claims in any way.

[0412] The present invention has been described above with the aid of functional building blocks illustrating the implementation of specified functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed.

[0413] The foregoing description of the specific aspects or embodiments will so fully reveal the general nature of the invention that others can, by applying knowledge within the skill of the art, readily modify and / or adapt for various applications such specific aspects or embodiments, without undue experimentation, without departing from the general concept of the present invention. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects orembodiments, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0414] The breadth and scope of the present invention should not be limited by any of the above-described exemplary aspects or embodiments, but should be defined only in accordance with the following claims and their equivalents.

Claims

WHAT IS CLAIMED IS:

1. A nucleic acid construct comprising a nucleic acid sequence encoding a mosaic H5 protein or fragment thereof, wherein the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2 or 3.

2. The nucleic acid construct of claim 1, wherein the nucleic acid sequence has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 5 or 6.

3. The nucleic acid construct of claim 1, wherein the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

4. The nucleic acid construct of claim 1, wherein the nucleic acid sequence has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 6.

5. The nucleic acid construct of claim 1, wherein the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2.

6. The nucleic acid construct of claim 1, wherein the nucleic acid sequence has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 5.

7. A nucleic acid construct comprising one or more nucleic acid sequences selected from the group consisting of:(i) a first nucleic acid sequence encoding a first mosaic H5 protein or fragment thereof;(ii) a first nucleic acid sequence encoding a first mosaic H5 protein or antigenic fragment thereof and a second nucleic acid sequence encoding a second mosaic H5 protein or fragment thereof; and(iii) combinations thereof; wherein the first mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NOs: 2, 3, 8, or 9.

8. The nucleic acid construct of claim 7, wherein the first mosaic H5 protein or fragment thereof encodes an H5 hemagglutinin (HA) protein or fragment thereof.

9. The nucleic acid construct of claim 8, wherein the first mosaic H5 HA protein or fragment thereof is a fragment of the Hl HA protein, wherein the fragment comprises an HA1 region and / or an HA2 region.

10. The nucleic acid construct of any one of claims 7-9, wherein the second mosaic H5 protein or fragment thereof encodes an H5 hemagglutinin (HA) protein or fragment thereof.

11. The nucleic acid construct of claim 10, wherein the second mosaic H5 HA protein or fragment thereof is a fragment of the H5 HA protein, wherein the fragment comprises an HA1 region and / or an HA2 region.

12. The nucleic acid construct of any one of claims 7-11, wherein the first mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3 or 9.

13. The nucleic acid construct of any one of claims 7-12, wherein the second mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10.

14. The nucleic acid construct of any one of claims 7-13, wherein the first nucleic acid sequence has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 6 or 13.

15. The nucleic acid construct of any one of claims 7-14, wherein the second nucleic acid sequence has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 14.

16. The nucleic acid construct of any one of claims 1-15, wherein the nucleic acid construct further comprises a promoter, optionally wherein the promoter is a S E / L Promoter.

17. A mosaic H5 protein or fragment thereof, wherein the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 2, 3, 8 or 9.

18. The mosaic H5 protein or fragment thereof of claim 17, wherein the H5 HA protein or fragment thereof is a fragment of the H5 HA protein, wherein the fragment comprises an HA1 region.

19. The mosaic H5 protein or fragment thereof of claim 18, wherein the fragment further comprises an HA2 region.

20. The mosaic H5 protein or fragment thereof of claim 17, wherein the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 3.

21. The mosaic H5 protein or fragment thereof of claim 17, wherein the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 9.

22. The mosaic H5 protein or fragment thereof of claim 19, wherein the HA2 region comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10.

23. A pharmaceutical composition comprising i) the nucleic acid construct of any one of claims 1- 16 or the mosaic H5 protein or fragment thereof of any one of claims 17-22; and ii) one or more pharmaceutically acceptable excipients or carriers.

24. An isolated host cell comprising the nucleic acid construct of any one of claims 1-16 or the mosaic H5 protein or fragment thereof of any one of claims 17-22.

25. The isolated host cell of claim 24, wherein the host cell is a eukaryotic host cell.

26. The isolated host cell of claim 25, wherein the host cell is an avian host cell.

27. An avian influenza vaccine vector comprising the nucleic acid construct of any one of claims 1-16.

28. The avian influenza vaccine vector of claim 27, wherein the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity sequence identity to the amino acid sequence of SEQ ID NO: 2 or 3.

29. The avian influenza vaccine vector of claim 27, wherein the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 8 or 9.

30. The avian influenza vaccine vector of claim 27, wherein the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the amino acid sequence of SEQ ID NO: 10.

31. The avian influenza vaccine vector of claim 27, wherein the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 5 or 6.

32. The avian influenza vaccine vector of claim 27, wherein the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 12 or 13.

33. The avian influenza vaccine vector of claim 27, wherein the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 14.

34. The avian influenza vaccine vector of any one of claims 27-33, wherein the avian influenza vaccine vector further comprises a promoter, optionally wherein the promoter is a S E / L promoter.

35. The avian influenza vaccine vector of any one of claims 27-34, wherein the avian influenza vaccine vector is a viral vector.

36. The avian influenza vaccine vector of claim 35, wherein the viral vector is a poxvirus vector.

37. The avian influenza vaccine vector of claim 36, wherein the poxvirus vector is a vaccinia virus vector.

38. The avian influenza vaccine vector of claim 37, wherein the vaccinia virus vector is a modified vaccinia Ankara (MV A) vector.

39. An avian influenza vaccine comprising a polynucleotide encoding a mosaic H5 protein or fragment thereof and one or more carriers, wherein the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity sequence identity to the amino acid sequence of SEQ ID NO: 2 3, 8 or 9.

40. The avian influenza vaccine of claim 39, wherein the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identity sequence identity to the amino acid sequence of SEQ ID NO: 3.

41. The avian influenza vaccine of claim 39, wherein the mosaic H5 protein or fragment thereof comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 sequence identity to the amino acid sequence of SEQ ID NO: 9.

42. The avian influenza vaccine of claim 39 or 41, wherein the mosaic H5 protein or fragment thereof further comprises an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100 sequence identity to the amino acid sequence of SEQ ID NO: 10.

43. The avian influenza vaccine of claim 39, wherein the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 5 or 6.

44. The avian influenza vaccine of claim 39, wherein the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 12 or 13.

45. The avian influenza vaccine of claim 42, wherein the polynucleotide comprises a nucleic acid sequence having at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to the nucleic acid sequence of SEQ ID NO: 14.

46. The avian influenza vaccine of any one of claims 39-45, wherein the carrier is a poxvirus.

47. The avian influenza vaccine of claim 46, wherein the poxvirus is a vaccinia virus.

48. The avian influenza vaccine of claim 47, wherein the vaccinia virus is a MVA virus.

49. A kit comprising the nucleic acid construct of any one of claims 1-16, the mosaic H5 protein or fragment thereof of any one of claims 17-22, the pharmaceutical composition of claim 23, the avian influenza vaccine vector of any one of claims 27-38, or the avian influenza vaccine of any one of claims 39-48.

50. A method of eliciting an immune response in an avian subject against one or more avian influenza antigens, the method comprising administering one or more doses of the pharmaceutical composition of claim 23, the avian influenza vaccine vector of any one of claims 27-38, or the avian influenza vaccine of any one of claims 39-48 to the avian subject.

51. The method of claim 50, wherein the method elicits a neutralizing antibody response against avian influenza in the avian subject.

52. The method of claim 51, wherein the avian influenza is an H5N1 strain.

53. The method of claim 52, wherein the H5N1 strain is a member of a clade selected from the group consisting of clade 2.2, clade 2.3.4.4 (including sub lineages 2.3.4.4a, 2.3.4.4b, 2.3.4.4c, 2.3.4.4d, 2.3.4.4e, or 2.3.4.4f), and clade 2.5.

54. A method of preventing, reducing the incidence of, attenuating, or treating avian influenza infection in an avian subject in need thereof, the method comprising administering one or more doses of the pharmaceutical composition of claim 23, the avian influenza vaccine vector of any one of claims 27-38, or the avian influenza vaccine of any one of claims 39-48 to the avian subject.

55. The method of claim 54, wherein the avian infection is caused by an H5N1 strain.

56. The method of claim 55, wherein the H5N1 strain is a member of a clade selected from the group consisting of clade 2.2, clade 2.3.4.4 (including sub lineages 2.3.4.4a, 2.3.4.4b, 2.3.4.4c, 2.3.4.4d, 2.3.4.4e, or 2.3.4.4f), and clade 2.5.

57. The method of any one of claims 50-56, wherein the one or more doses of the pharmaceutical composition, the avian influenza vaccine vector, or avian influenza vaccine are administered to the avian subject by mucosal route of administration.

58. The method of any one of claims 50-57, wherein the avian subject is a domestic poultry.

59. The method of claim 58, wherein the domestic poultry is selected from the group consisting of chickens, ducks, turkeys, quails, geese, or guinea fowls.

60. A method of eliciting an immune response in a mammalian subject against one or more avian influenza antigens, the method comprising administering one or more doses of the pharmaceutical composition of claim 23, the avian influenza vaccine vector of any one of claims 27-38, or the avian influenza vaccine of any one of claims 39-48 to the mammalian subject.

61. The method of claim 60, wherein the method elicits a neutralizing antibody response against avian influenza in the mammalian subject.

62. The method of claim 61, wherein the avian influenza is an H5N1 strain.

63. The method of claim 62, wherein the H5N1 strain is a member of a clade selected from the group consisting of clade 2.2, clade 2.3.4.4 (including sub lineages 2.3.4.4a, 2.3.4.4b, 2.3.4.4c, 2.3.4.4d, 2.3.4.4e, or 2.3.4.4f), and clade 2.5.

64. A method of preventing, reducing the incidence of, attenuating, or treating avian influenza infection in a mammalian subject in need thereof, the method comprising administering one or more doses of the pharmaceutical composition of claim 23, the avian influenza vaccine vector of any one of claims 27-38, or the avian influenza vaccine of any one of claims 39-48 to the mammalian subject.

65. The method of claim 64, wherein the avian infection is caused by an H5N1 strain.

66. The method of claim 65, wherein the H5N1 strain is a member of a clade selected from the group consisting of clade 2.2, clade 2.3.4.4 (including sub lineages 2.3.4.4a, 2.3.4.4b, 2.3.4.4c, 2.3.4.4d, 2.3.4.4e, or 2.3.4.4f), and clade 2.5.

67. The method of any one of claims 60-66, wherein the one or more doses of the pharmaceutical composition, the avian influenza vaccine vector, or avian influenza vaccine are administered to the mammalian subject by an oral, intranasal, subcutaneous, or intramuscular route of administration.

68. The method of any one of claims 60-67, wherein the mammalian subject is a human subject.

69. A composition comprising a lipid nanoparticle and a messenger RNA (mRNA) comprising an open reading frame (ORF) that comprises a nucleotide sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence, or 100% identity to the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14; wherein the nucleotide sequence encodes a polypeptide comprising the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, or SEQ ID NO: 10.

70. The composition of claim 69, wherein the nucleotide sequence comprises the nucleotide sequence of SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, or SEQ ID NO: 14.

71. The composition of claim 69 or 70, wherein the lipid nanoparticle comprises a PEG-modified lipid, a non-cationic lipid, a sterol, an ionizable cationic lipid, or any combination thereof.

72. The composition of any one of claims 69-71, wherein the mRNA comprises a 5' untranslated region (UTR) and a 3' UTR.

73. The composition of any one of claims 69-72, wherein the mRNA comprises a chemical modification.

Images