Vaccine composition comprising m2e nanoparticles and ha1 protein

Abstract

The present invenon relates to a vaccine against influenza A viruses, said vaccine comprising HA protein and nanostructures bearing M2e, and its use for vaccinaon against avian influenza, especially in the veterinary field.

Description

[0001] VACCINE COMPOSITION COMPRISING M2E NANOPARTICLES AND HA1 PROTEIN

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to the field of medicine, especially to the vaccine composition, more particularly vaccine composition against infection by influenza A viruses.

[0004] BACKGROUND OF THE INVENTION

[0005] Influenza A viruses (IAV), whose natural reservoirs are wild aquatic birds, infect a wide range of species including domestic birds and mammals such as pigs and humans. Based on genetic and antigenic characterization of the two surface glycoproteins, namely the hemagglutinin (HA) and neuraminidase (NA), avian IAV are categorized into 16 HA and 9 NA subtypes. Generally, avian IAV are classified into low pathogenicity avian influenza (LPAI) or high pathogenic avian influenza (HPAI) viruses on the basis of their pathobiological effects in gallinaceous species. In chickens, infections with LPAI viruses are usually asymptomatic or cause low to mild clinical signs associated with limited viral replication in the respiratory, digestive, and / or reproductive systems. HPAI viruses replicate in multiple tissues causing systemic infections manifested by a complex set of clinical diseases such as central nervous system as well as respiratory involvements, leading to significant mortality. HPAI H5 viruses derived from the Gs / GD lineage, which has evolved worldwide into diverse clades, subclades and reassortants ("H5Nx viruses"), cause not only important economic problems to the poultry industry, but also serious threat to public health worldwide through their zoonotic and pandemic potential.

[0006] Vaccination, when implemented with good farm biosecurity measures, can be an effective approach to control IAV infections in poultry and reduce the economic impacts. Although vaccination of poultry against IAV varies between countries, licensed vaccines include whole inactivated viruses (WIV) emulsified in oil adjuvants and live recombinant viral vectors with HA-encoding gene inserts. These vaccines generate variable levels of neutralizing antibodies directed towards the globular head domain of HA (HA1), which can block viral entry into permissive host cells and subsequent virus replication. While antigenic mismatch between vaccine strains and circulating IAV reduces vaccine efficacy, antigenically updated vaccines can provide improved protection against the clinical disease and virus shedding. However, antigenic drifts that result from mutations, mostly in the HA gene and virus reassortment events occurring periodically in circulating IAV, impact vaccine efficacy in the field.

[0007] Thus, current poultry vaccines against influenza A viruses (IAV) generate immunity directed mainly towards the globular head region of the hemagglutinin (HA1) protein of the virus. These vaccines have demonstrated efficacy against homologous IAV strains, but provided only limited protection against antigenically divergent virus strains. In addition, these vaccines are not suitable to discriminate between vaccinated animals and infected animals.

[0008] Owing to the high genetic and antigenic variability of IAV, current research focuses on developing subunit vaccines which contain "universal" viral antigens, including a conserved sequence located in the ectodomain of the matrix protein (M2), termed M2e. M2 protein is sparsely expressed at the surface of the virus, but is abundant on the surface of IAV infected cells. The protection conferred by M2e-based vaccines is associated with the production of M2e-specific antibodies that can eliminate infected cells mainly through Fc-mediated effector mechanisms, although M2e-specific T cell response may also contribute to the elimination of virus-infected cells. While vaccination trials in mouse or ferret models demonstrated that M2e was a promising antigen candidate for inducing broad protection against a range of IAV, results in chickens were inconsistent (Kolpe et al. Expert Rev Vaccines (2017) 16(2):123-36. Epub 2016 / 09 / 23. doi: 10.1080 / 14760584.2017.1240041 ; Zhang et al. Virol J (2010) 7:89. Epub 2010 / 05 / 13. doi: 10.1186 / 1743-422X-7-89; Hajam et al. Vet Immunol Immunopathol (2019) 209:78-83. Epub 2019 / 03 / 20. doi: 10.1016 / j.vetimm.2019.02.007; Babapoor et al. Influenza Res Treat (2011) 2011:126794. Epub 2011 / 01 / 01. doi: 10.1155 / 2011 / 126794; Dabaghian et al. Vet Microbiol (2014) 174(l-2):116-26. Epub 2014 / 10 / 09. doi: 10.1016 / j.vetmic.2014.09.009 ; Elaish et al. Vaccine (2015) 33(38):4901-9. Epub 2015 / 08 / 02. doi: 10.1016 / j.vaccine.2015.07.049; Elaish et al. PLoS One (2017) 12(2):e0171174. Epub 2017 / 02 / 06. doi: 10.1371 / journal.pone.0171174; Elaish et al. Vaccine (2019) 37(43):6454-62. Epub 2019 / 09 / 12. doi: 10.1016 / j.vaccine.2019.08.082; Hajam et al. Vet Res (2020) 51(1):37. Epub 2020 / 03 / 08. doi: 10.1186 / S13567-020-00762-4 ; Tang et al. Avian Pathol (2013) 42(3):260-7. Epub 2013 / 04 / 24. doi: 10.1080 / 03079457.2013.782096 ; Hajam et al. Vet Res (2018) 49(1):99. Epub 2018 / 10 / 05. doi: 10.1186 / sl3567-018-0593-z ; Kim et al. Vet Res (2018) 49(1):12. Epub 2018 / 02 / 03. doi: 10.1186 / sl3567-018-0509-y ; Layton et al. Poult Sci (2009) 88(ll):2244-52. Epub 2009 / 10 / 17. doi: 10.3382 / ps.2009-00251 ; Yang et al. Appl Microbiol Biotechnol (2018) 102(12):5077-88. Epub 2018 / 04 / 21. doi: 10.1007 / s00253-018-8924-6 ; Hajam et al. Avian Pathol (2020) 49(5):486-95. Epub 2020 / 06 / 03. doi: 10.1080 / 03079457.2020.1775782 ; Kalaiyarasu et al. Front Vet Sci (2020) 7:571999. Epub 2021 / 02 / 23. doi: 10.3389 / fvets.2020.571999 ; Li et al. J Virol Antivir Res (2018) 7:1. doi: 10.4172 / 2324-8955.1000179 ; Reese et al. Vet Med Int (2013) 2013:316926. Epub 2013 / 06 / 15. doi: 10.1155 / 2013 / 316926 ; Song et al. Vaccine (2016) 34(5):678-86. Epub 2015 / 12 / 23. doi: 10.1016 / j.vaccine.2015.11.074 ; Zhang Xet al. Intervirology (2011) 54(5):290-9. Epub 2011 / 01 / 14. doi: 10.1159 / 000319440). Vaccine formulations incorporating single or multiple M2e antigens alone or combined with other conserved epitopes, such as those located in the stalk domain of HA (HA2), were unable to protect chickens against mortality associated with HPAI viruses, nor to prevent virus shedding upon experimental infections with LPAI or HPAI viruses. While subunit vaccines based solely on M2e did not provide complete protection against IAV, two recent studies indicated that the supplementation of H5N1 WIV vaccines with M2e conferred complete clinical protection and totally impeded virus shedding after heterologous HPAI H5 virus infection in chickens (Song et al. Vaccine (2016) 34(5):678-86. Epub 2015 / 12 / 23. doi: 10.1016 / j.vaccine.2015.11.074 ; Zhang et al. Vet Microbiol (2017) 203:143-8. Epub 2017 / 06 / 18. doi: 10.1016 / j.vetmic.2017.03.004). Nonetheless, the WIV and challenge virus strain used in these studies presented an HA gene homology of greater than 90% and WIV vaccines production is complex.

[0009] The inventors previously developed two innovative nanostructures with inherent adjuvant qualities, namely nanorings (NR) and nanofilaments (NF), and used them as vaccine delivery platforms for M2e antigens in mice. NR are composed of recombinant nucleoprotein of the respiratory syncytial virus (RSV), which self-assembles into homogenous rings of about 15 nm diameter when expressed in Escherichia coli (Herve et al. J Virol (2014) 88(l):325-38. Epub 2013 / 10 / 25. doi: 10.1128 / JVI.01141-13; Tawar et al. Science (2009) 326(5957):1279-83. Epub 2009 / 12 / 08. doi: 10.1126 / science.1177634 ; Tran et al. J Gen Virol (2007) 88(Pt l):196-206. Epub 2006 / 12 / 16. doi: 10.1099 / vir.0.82282-0). NF are composed of recombinant curli-specific gene A (CsgA) protein, a major extracellular matrix component contributing to biofilm formation in numerous enteric bacteria. Recombinant CsgA monomers spontaneously self-assemble in vitro into cross-p-sheet quaternary structures forming long and unbranched fibrils with similar structural and mechanical properties to the in vivo assemblies (Wang et al. J Biol Chem (2008) 283(31):21530-9. Epub 2008 / 05 / 30. doi: 10.1074 / jbc.M800466200; Wang et al. J Biol Chem (2007) 282(6):3713-9. Epub 2006 / 12 / 14. doi: 10.1074 / jbc.lVI609228200). Recombinant chimeric NR bearing M2e antigens ("NR- 3M2e") were produced by genetic fusion of three tandem repeats of M2e peptide at the C-terminal end of the nucleoprotein sequence of RSV. Conversely, NF-3M2e were produced by genetically linking the N-terminal domain of CsgA of E. coli with 3M2e. Mice immunized with NR-3M2e or NF- 3M2e developed potent M2e-specific humoral and cellular adaptive immune responses and survived a homologous H1N1 IAV challenge (Herve et al. J Virol (2014) 88(l):325-38. Epub 2013 / 10 / 25. doi: 10.1128 / JVI.01141-13; Calzas et al. Front Immunol (2021) 12:772550. Epub 2021 / 12 / 07. doi: 10.3389 / fimmu.2021.772550; Lamontagne et al. Adv Healthc Mater (2023):e2300224. Epub 2023 / 04 / 09. doi: 10.1002 / adhm.202300224). Nonetheless, the efficacy of NR-3M2e and NF-3M2e in anti-lAV vaccination in chickens has not been demonstrated yet.

[0010] Combination of NR-3M2e and NR-LAH (long alpha helix of the stalk domain H2; Calzas et al. Front Immunol (2021) 12:772550. Epub 2021 / 12 / 07. doi: 10.3389 / fimmu.2021.772550) triggers strong humoral responses in mice against H1N1 and increases mice survival, but this combination in vaccinated chicken failed to confer in protection against a challenge with LPAI H5N8 strain.

[0011] Although experimental subunit IAV vaccines based on the ectodomain of the matrix protein 2 (M2e), a conserved antigen among IAV strains, could trigger antibody-mediated immune responses with cross-reactive properties in vitro, they did not completely abrogate virus shedding after challenge with low pathogenicity avian influenza (LPAI) viruses, and were ineffective against high pathogenicity avian influenza (HPAI) viruses.

[0012] Therefore, there is a strong unmet need for a vaccine composition against IAV that would provide full protection against heterologous virus by a cross-protective potential and prevent virus shedding in the environment. In addition, this vaccine composition must be easy to modulate (exchange of immunogenic epitopes and possibility to combine multiple epitopes on a single particle), in order to allow the adaptation of the vaccine in case of major virus antigenic drifts or of emergence of reassortant virus with a pandemic potential. A vaccine formulation of interest would have the advantage of not being a GMO (no risk of environmental release), to provide a suitable DIVA approach (Differentiating infected from vaccinated animals), and to be produced at lower cost.

[0013] SUMMARY OF THE INVENTION

[0014] The present invention provides a new vaccine or immunogenic composition comprising a recombinant HA1 protein and a protein carrier bearing M2e domain of influenza A virus or a repeat thereof, the protein carrier preferably forming a nanoparticle displaying M2e domain or repeat thereof.

[0015] This vaccine composition induces the production of antibodies specific for M2e and HA1, similarly to a composition comprising either only HA1 protein or only nanoparticle bearing repeats of M2e. Surprisingly, the vaccine composition allows a complete protection against the clinical disease, mortality and histopathological lesions associated with HPAI virus infection, even against antigenically divergent virus strains. Advantageously, the vaccine composition is able to prevent oropharyngeal or cloacal virus shedding in vaccinated subjects, whereas a composition comprising either only HA1 protein or only nanoparticle bearing repeats of M2e is devoid of this capacity. In addition, no lesion or viral antigen was detected in cardiac, lung or brain tissue of infected animals. This effect has been demonstrated with a virus sharing only 81% amino acid sequence identity s with the HA1 sequence of challenge virus (which is a very large difference compared to the virus evolution). In addition, this vaccine composition is inexpensive (contrary to traditional WIV vaccines), easy to administer, and presents a broad-spectrum of protection against HPAI. Finally, an additional advantage of the present vaccine composition is that it can differentiate vaccinated subjects from infected subjects (suitable for DIVA approach). Indeed, the subjects producing antibodies against M2e are vaccinated subjects because the infected subjects are not able to product such antibodies. Therefore, a DIVA assay can be easily developed by testing the presence M2e antibodies.

[0016] Accordingly, the vaccine or immunogenic composition as disclosed herein provides a full cross protection against HPAI virus, prevents virus shedding in the environment, and is suitable for DIVA approach.

[0017] Accordingly, the present invention relates to a vaccine composition comprising:

[0018] - a recombinant hemagglutinin HA1 from an influenza A virus; and

[0019] - a protein carrier fused with an ectodomain of a matrix protein 2 (M2e domain) or a tandem repeat thereof, optionally through a peptide linker, wherein the M2e domain comprises a sequence

[0020] (M / abs ) S L L T E V E T ( P / L ) ( T / I ) ( K / R) ( T / N / K / S ) ( E / G ) W ( E / G )

[0021] ( C / S ) ( R / N / K ) ( C / S ) ( S / N ) ( D / V / G ) S S D (SEQ ID NO: 12).

[0022] In a first aspect, the protein carrier is selected from the group consisting of CRM197 (nontoxic variant of diphtheria toxin isolated from Corynebacterium diphtheriae C7 (P197), tetanus toxoid (TT), a Pseudomonas aeruginosa protein, human serum albumin (HSA), bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), diphtheria toxin fragment B (DTFB), DTFB C8, diphtheria toxoid (DT), fragment C of TT, pertussis toxoid, cholera toxoid, E. coli heat-labile toxin, E. coli heatstable toxin, meningococcal outer membrane protein complex (OMPC), Haemophilus influenzae protein D (HiD), and exotoxin A from Pseudomonas aeruginosa.

[0023] In a second aspect, the protein carrier is a self-assembling protein able to form a nanoparticle, optionally selected from the group consisting of a nucleoprotein N of respiratory syncytial virus (RSV), CsgA (Curli-specific gene A), an amyloid protein such as CspA (E. coli cold shock protein A), ferritin, Soc (Small Outer Capsid protein) of phage T4, hepatitis B virus core antigen (HBc), bacteriophage AP205 coat protein, coat protein of bacteriophage QP, a dodecameric DNA binding protein from starved cells (Dps) of bacteria or archaea such as Dsp of E. coli, Brucella outer membrane protein BP26, capsid protein of PCV2 (porcine circovirus type 2), protrusion domain of the capsid protein of norovirus, a self-assembling an amyloid protein p-peptide such as a ten-mer self-assembling sequence (110) derived from an amyloidogenic peptide (SNNFGAILSS, SEQ ID NO: 36), an antibody such as anti-Clec9A monoclonal antibody, CTA1-DD (ADP-ribosylating Al subunit of cholera toxin CT linked to a dimer of the D-fragment of Staphylococcus aureus protein A), bacterial flagellin such as flagellin of Bacillus subtilis or Salmonella serovar enterica typhimurium (S. typhimurium), Qll peptide (QQKFQFQFEQQ, SEQ ID NO: 37), lumazine synthase, especially Aguifex aelocus (A. aelocus) lumazine synthase, encapsulin, especially from T maritime / , and small Heat shock protein (sHsp) 16.5, especially from Methanocaldoccus jannaschii, a superfolder green fluorescent protein (sfGFP), bacteriophage P22, E2p, especially from Bacillus stearothermophilus (B. stearothermophilus , 13-01 derived from Thermotoga maritima's 2-keto-3-deoxy-6- phosphogluconate (KDPG) aldolase, especially from T. maritima, 153-50, IMX313, SAP, E2, Coil29, FliC, latency-associated nuclear antigen (LANA) from the Kaposi's sarcoma-associated herpesvirus (KSHV), coat of a plant virus such as nepovirus coat virus, in particular GFLV (Grapevine fanleaf virus), hydrophobin, bacteriophage T7 endonuclease, A beta 1-40, Sup35p of Saccharomyces cerevisiae, human Pmell7, Agfa Tafi protein, a Chaplin protein, a peptide derived from islet amyloid polypeptide (IAPP), in particular SNNFGAILSS (SEQ ID NO: 36), and a combination thereof. .

[0024] In a specific aspect, the protein carrier is a N protein of Respiratory syncytial virus (RSV) and the M2e domain or a tandem repeat thereof is fused to the C-terminal end of the N protein. Optionally, the protein carrier is fused to a tandem repeat of 2 to 4 M2e domains, especially of 3 M2e domains. Accordingly, a tandem repeat of 2 to 4 M2e domains, preferably of 3 M2e domains, can be fused to the C-terminal end of the N protein.

[0025] In another specific aspect, the protein carrier is curli specific gene A (CsgA) or a fragment thereof and the M2e domain or a tandem repeat thereof is fused to the N-terminal end of CsgA. Optionally, the protein carrier is fused to a tandem repeat of 2 to 4 M2e domains, especially of 3 M2e domains. Accordingly, a tandem repeat of 2 to 4 M2e domains, preferably of 3 M2e domains, is fused to the N-terminal end of CsgA. Optionally, the protein carrier is CsgA. Optionally, the protein carrier is a fragment of CsgA comprising R2, R3, R4 and R5 repeat units, or R3, R4 and R5 repeat units, or R4 and R5 repeat units, or R5 repeat unit.

[0026] Optionally, the recombinant hemagglutinin HA1 is from an influenza A virus selected from any of the following groups

[0027] - H1N1, H1N2, H1N3, H1N4, H1N5, H1N6, H1N7, H1N8, H1N9, H2N1, H2N2, H2N3, H2N4,

[0028] H2N5, H2N6, H2N7, H2N8, H2N9, H3N1, H3N2, H3N3, H3N4, H3N5, H3N6, H3N7, H3N8,

[0029] H3N9, H4N1, H4N2, H4N3, H4N4, H4N5, H4N6, H4N7, H4N8, H4N9, H5N1, H5N2, H5N3,

[0030] H5N4, H5N5, H5N6, H5N7, H5N8, H5N9, H6N1, H6N2, H6N3, H6N4, H6N5, H6N6, H6N7,

[0031] H6N8, H6N9, H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, H7N9, H8N1, H8N2,

[0032] H8N3, H8N4, H8N6, H8N7, H8N8, H9N1, H9N2, H9N3, H9N4, H9N5, H9N6, H9N7, H9N8,

[0033] H9N9, H10N1, H10N2, H10N3, H10N4, H10N5, H10N6, H10N7, H10N8, H10N9, H11N1, H11N2, H11N3, H11N4, H11N5, H11N6, H11N6, H11N7, H11N8, H11N9, H12N1, H12N2, H12N3, H12N4, H12N5, H12N6, H12N7, H12N8, H12N9, H13N1, H13N2, H13N3, H13N4, H13N6, H13N7, H13N8, H13N9, H14N2, H14N3, H14N4, H14N5, H14N6, H14N7, H14N8, H15N2, H15N4, H15N5, H15N6, H15N7, H15N8, H15N9, H16N3, H16N8, and H16N9; or

[0034] - H1N1, H1N2, H2N2, H2N3, H3N2, H3N8, H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N6, H7N7, H7N9, H9N2, H9N6, H9N7, H10N3, and H10N7; or

[0035] - H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, H5N9, H6N1, H6N2, H6N3, H6N4, H6N5, H6N6, H6N7, H6N8, H6N9, H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, H7N9, H9N1, H9N2, H9N3, H9N4, H9N5, H9N6, H9N7, H9N8, H9N9, H10N1, H10N2, H10N3, H10N4, H10N5, H10N6, H10N7, H10N8, and H10N9; or

[0036] - H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, H7N9, H9N1, H9N2, H9N3, H9N4, H9N5, H9N6, H9N7, H9N8, and H9N9;

[0037] - H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, and H7N9;

[0038] - H5N1, H5N6, H6N1, H7N2, H7N6, H7N9, H9N2, H10N3, H10N7, and H10N8; or

[0039] - H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8 and H5N9, preferably H5N1, H5N6, and H5N8; or

[0040] - H5N1, H5N2, H5N6, H5N8, H5N9, H7N1, H7N3, H7N4, H7N7, and H7N9.

[0041] Preferably, the recombinant hemagglutinin HA1 is from an avian influenza A virus selected from the group consisting of H5N1, H5N2, H5N6, H5N8, H5N9, H7N1, H7N3, H7N4, H7N6, H7N7,and H7N9. More preferably, the recombinant hemagglutinin HA1 is from a H5N1 influenza A virus.

[0042] In a specific aspect, the recombinant hemagglutinin HA1 comprises a sequence selected in the group consisting of:

[0043] SEQ ID NO: 3 or 38 or a sequence having at least 80, 85, 90, or 95 % of identity with SEQ ID NO: 3 or 38;

[0044] SEQ ID NO: 39 or 40 or a sequence having at least 80, 85, 90, or 95 % of identity with SEQ ID NO: 39 or 40; and

[0045] SEQ ID NO: 41 or a sequence having at least 80, 85, 90, or 95 % of identity with SEQ ID NO: 41.

[0046] In a more specific aspect, the recombinant hemagglutinin HA1 comprises a sequence of SEQ ID NO:

[0047] 3 or 38 or a sequence having at least 80, 85, 90, or 95% identity with SEQ ID NO: 3 or 38.

[0048] The M2e domain can comprise a sequence selected in the group consisting of: (M / abs) S L L T E V E T (P / L) T (K / R) (T / N / K / S) (E / G) W (E / G)

[0049] (C / S) (R / N / K) (C / S) (S / N) (D / V / G) S S D (SEQIDN0:13)

[0050] (M / abs) S L L T E V E T (P / L) T (K / R) (T / N) (E / G) W (E / G) (C / S) (R / N / K) (C / S) (S / N) (D / V / G) S S D (SEQ ID NO: 14).

[0051] (M / abs) S L L T E V E T P T R N (E / G) W (E / G) (C / S) (R / K) (C / S) (S / N) D S S D (SEQ ID NO: 15);

[0052] (M / abs) S L L T E V E T P T R N (E / G) W (E / G) (C / S) (R / K) (C / S) S

[0053] D S S D (SEQ ID NO: 16); (M / abs) S L L T E V E T P T R (N / T) (E / G) W (E / G) (C / S) R (C / S)

[0054] S D S S D (SEQ ID NO: 17); (M / abs) S L L T E V E T P T R N (E / G) W E (C / S) (K / R) (C / S) S D S S D (SEQ ID NO: 18); (M / abs) S L L T E V E T P T R N (E / G) W E (C / S) K (C / S) S D S S D (SEQ ID NO: 19);

[0055] (M / abs) S L L T E V E T P T R T E W E C R C S D S S D (SEQ ID NO: 20);

[0056] (M / abs) S L L T E V E T P T R T E W E S R C S D S S D (SEQ ID NO: 21);

[0057] (M / abs) S L L T E V E T P T R T E W E C R S S D S S D (SEQ ID NO: 22);

[0058] (M / abs) S L L T E V E T P T R T E W E S R S S D S S D (SEQ ID NO: 23);

[0059] (M / abs) S L L T E V E T P T R N E W E C R C S D S S D (SEQ ID NO: 24);

[0060] (M / abs) S L L T E V E T P T R N E W E S R C S D S S D (SEQ ID NO: 25);

[0061] (M / abs) S L L T E V E T P T R N E W E C R S S D S S D (SEQ ID NO: 26);

[0062] (M / abs) S L L T E V E T P T R N E W E S R S S D S S D (SEQ ID NO: 27);

[0063] (M / abs) S L L T E V E T P T R T G W E C R C S D S S D (SEQ ID NO: 28);

[0064] (M / abs) S L L T E V E T P T R T G W E S R C S D S S D (SEQ ID NO: 29);

[0065] (M / abs) S L L T E V E T P T R T G W E C R S S D S S D (SEQ ID NO: 30);

[0066] (M / abs) S L L T E V E T P T R T G W E S R S S D S S D (SEQ ID NO: 31);

[0067] (M / abs) S L L T E V E T P T R N G W E C K C S D S S D (SEQ ID NO: 32);

[0068] (M / abs) S L L T E V E T P T R N G W E S K C S D S S D (SEQ ID NO: 33);

[0069] (M / abs) S L L T E V E T P T R N G W E C K S S D S S D (SEQ ID NO: 34); and

[0070] (M / abs) S L L T E V E T P T R N G W E S K S S D S S D (SEQ ID NO: 35).

[0071] Preferably, the M2e domain comprises a sequence (M / abs ) S L L T E V E T P T R T

[0072] E W E S R S S D S S D (SEQ ID NO: 23).

[0073] In a very specific aspect, the vaccine composition comprises: - a recombinant hemagglutinin HA1 comprising a sequence of SEQ ID NO: 3 or 38 or a sequence having at least 80, 85, 90, or 95% identity with SEQ ID NO: 3 or 38; and

[0074] - a nanoparticle made of a carrier protein selected from the group consisting of nucleocapsid protein (N) of Respiratory syncytial virus (RSV) and curli specific gene A (CsgA) or a fragment thereof, the self-assembling protein being fused with a tandem repeat of 3 M2e domains, wherein the M2e domain comprises a sequence (M / abs ) S L L T E V E T P T R T E W E S R S S D S S D (SEQ ID NO: 23).

[0075] Optionally, the vaccine composition is in a form suitable for parenteral administration, especially subcutaneous or intramuscular, for in ovo administration or for mucosal administration, especially by oral or nasal route.

[0076] Optionally, the composition further comprises an adjuvant.

[0077] The present invention further relates to a vaccine composition as disclosed herein for use against influenza A infection, especially by reducing or preventing the effects of an infection by influenza A virus, the effects being selected in the group consisting of mortality, clinical disease associated with infection by influenza A virus, histopathological lesions associated with infection by influenza A, and influenza A virus shedding. It also relates to the use of a vaccine composition as disclosed herein for the manufacture of a vaccine against influenza A infection, especially by reducing or preventing the effects of an infection by influenza A virus, the effects being selected in the group consisting of mortality, clinical disease associated with infection by influenza A virus, histopathological lesions associated with infection by influenza A, and influenza A virus shedding. It further relates to a method for preventing an infection by influenza A virus in a subject, comprising administering to the subject a therapeutically effective amount of a vaccine composition as disclosed herein, thereby reducing or preventing the effects of an infection by influenza A virus, the effects being selected in the group consisting of mortality, clinical disease associated with infection by influenza A virus, histopathological lesions associated with infection by influenza A, and influenza A virus shedding.

[0078] In particular, the subject is a poultry and the influenza A virus is selected from the group consisting of H5N1, H5N2, H5N6, H5N8, H5N9, H7N1, H7N3, H7N4, H7N6, H7N7, and H7N9.

[0079] In particular, the subject is a horse and the influenza A virus is H3N8 or H7N7, preferably H3N8.

[0080] BRIEF DESCRIPTION OF THE FIGURES

[0081] Figure 1. Biochemical and biophysical properties of NR-3M2e and NF-3M2e.

[0082] Recombinant NR-3M2e were produced and purified as described in "Materials and Methods", and then analyzed by (A) SDS-PAGE followed by Coomassie blue staining, (B) Western blot assays with (B, left panel) N-specific polyclonal antibodies and (B, right panel) M2-specific monoclonal antibody, (C) transmission electron microscopy after negative staining.

[0083] Recombinant NF-3M2e were produced and purified as described in "Materials and Methods", and then analyzed by (D) SDS-PAGE followed by Coomassie blue staining, and (E) transmission electron microscopy after negative staining.

[0084] Figure 2. Serum hemagglutination inhibition (HI) titers of chickens immunized with HA1 protein administered alone or in combination with NR-3M2e or NF-3M2e. Chickens received at 2-week intervals three intramuscular administrations of HA1 protein in combination with NR-3M2e or NF- 3M2e, or each single component alone. Chickens receiving PBS were included as a negative control group. All preparations were admixed with Emulsigen®-P. Serum HI titers were determined on days 14, 28, and 42 post-primary vaccination using four HA units of the A / turkey / Ontario / 6213 / 1966 virus. Data are presented as arithmetic means with SEM. *, statistically significant difference (P < 0.05) in comparison to PBS group (at the same day post-primary vaccination). 8, statistically significant difference (P < 0.05) between the indicated groups.

[0085] Figure 3. Serum HAl-specific antibody response of chickens immunized with HA1 protein administered alone or in combination with NR-3M2e or NF-3M2e analyzed by competitive ELISA. Chickens received at 2-week intervals three intramuscular administrations of HA1 protein alone or in combination with NR-3M2e or NF-3M2e. All preparations were admixed with Emulsigen®-P. The serum HAl-specific antibody response was analyzed by competitive ELISA on days 14, 28, and 42 post-primary vaccination (ppv) using HA1 protein from A / turkey / Ontario / 6213 / 1966 as coating antigen. Data are presented as arithmetic means with SEM. The dotted bar represents the positivity threshold (40%).

[0086] Figure 4. Serum M2e-specific antibody response of chickens immunized with NR-3M2e or NF- 3M2e administered alone or in combination with HA1 protein. Chickens received at 2-week intervals three intramuscular administrations of NR-3M2e or NF-3M2e alone or in combination with recombinant HA1 protein. Chickens receiving HA1 or PBS were included as control groups. All preparations were admixed with Emulsigen®-P. Serum M2e-specific antibody response was analyzed by indirect ELISA on day 42 post-primary vaccination. Data are presented as arithmetic means with SEM. *, statistically significant difference (P < 0.05) in comparison to PBS and HA1 groups.

[0087] Figure 5. Clinical protection in chickens immunized with NR-3M2e or NF-3M2e supplemented with HA1, or each component alone, after infection with heterologous HPAI H5N1 virus infection.

[0088] Chickens received at 2-week intervals three intramuscular administrations of HA1 protein in combination with NF-3M2e or NR-3M2e, or each single component alone. Chickens receiving PBS were included as a control group. All preparations were admixed with Emulsigen®-P. Two weeks after the third immunization, all chickens were challenged with 2.3 x 102PFU of heterologous HPAI H5N1 virus and monitored daily for mortality. The survival curves of infected chickens are expressed as the percentages of surviving chickens. The log-rank (Mantel-Cox) test was used to compare survival curves. *, statistically significant difference (P < 0.05) in comparison to PBS group. 8, statistically significant difference (P < 0.05) between the indicated groups.

[0089] Figure 6. Histological and immunohistochemical analyses of lung tissues. Chickens received at 2- week intervals three intramuscular administrations of NR-3M2e or NF-3M2e in combination with HA1 protein, or each single component alone. Chickens receiving PBS were included as a control group. All preparations were admixed with Emulsigen®-P. Two weeks after the third immunization, all chickens were challenged with 2.3 x 102PFU of heterologous HPAI H5N1 virus. Lungs were collected at selected days post-challenge (dpc) and sections were stained with hematoxylin and eosin (HE) before histological analyis (left panels), or stained with anti-NP antibody before immunohistochemical (IHC) analysis (right panels). (A) Histological and (B) immunohistochemical analyses of the lung tissue of the PBS group at 3 dpc. The arrow in panel A indicates areas of necrosis. (C) Histological and (D) immunohistochemical analyses of the lung tissue of the NR-3M2e group at 3 dpc. (E) Histological and (F) immunohistochemical analyses of the lung tissue of the NF- 3M2e group at 3 dpc. (G) Histological and (H) immunohistochemical analyses of the lung tissue of the HA1 group at 19 dpc. (I) Histological and (J) immunohistochemical analyses of the lung tissue of the [NR-3M2e + HA1] group at 21 dpc. (K) Histological and (L) immunohistochemical analyses of the lung tissue of the [NF-3M2e + HA1] group at 21 dpc.

[0090] Figure 7. Histological and immunohistochemical analyses of brain tissues. Chickens received at 2- week intervals three intramuscular administrations of NR-3M2e or NF-3M2e in combination with HA1 protein, or each single component alone. Chickens receiving PBS were included as a control group. All preparations were admixed with Emulsigen®-P. Two weeks after the third immunization, all chickens were challenged with 2.3 x 102PFU of heterologous HPAI H5N1 virus. Brains were collected at selected days post challenge (dpc) and sections were stained with hematoxylin and eosin (HE) before histological analyis (left panels) or stained with anti-NP antibody before immunohistochemical (IHC) analysis (right panels). (A) Histological and (B) immunohistochemical analyses of the brain tissue of the PBS group at 3 dpc. The arrows in panel A indicate areas of necrosis. (C) Histological and (D) immunohistochemical analyses of the brain tissue of the NR-3M2e group at 3 dpc. The arrow and inset in panel C indicate multifocal necrosis gliosis. The arrow and inset in panel D indicate viral antigen. (E) Histological and (F) immunohistochemical analyses of the brain tissue of the NF-3M2e group at 3 dpc. Arrow and inset in panel E indicate multifocal necrosis gliosis. Inset in panel F indicates viral antigen. (G) Histological and (H) immunohistochemical analyses of the brain tissue of the HA1 group at 19 dpc. Arrow in panel G indicates prominent perivascular cuffing of mononuclear inflammatory cells and scattered necrotic cells throughout the neurophil. (I) Histological and (J) immunohistochemical analyses of the brain tissue of the [NR-3M2e + HA1] group at 21 dpc. The brain of the [NF-3M2e + HA1] group was not collected during necropsy.

[0091] Figure 8. Histological and immunohistochemical analyses of heart tissues. Chickens received at 2- week intervals three intramuscular administrations of NR-3M2e or NF-3M2e in combination with HA1 protein, or each single component alone. Chickens receiving PBS were included as a control group. All preparations were admixed with Emulsigen®-P. Two weeks after the third immunization, all chickens were challenged with 2.3 x 102PFU of heterologous HPAI H5N1 virus. Hearts were collected at selected days post challenge (dpc) and sections were stained with hematoxylin and eosin (HE) before histological analyis (left panels) or stained with anti-NP antibody before immunohistochemical (IHC) analysis (right panels). (A) Histological and (B) immunohistochemical analyses of the heart tissue of the PBS group at 3 dpc. (C) Histological and (D) immunohistochemical analyses of the heart tissue of the NR-3M2e group at 3 dpc. (E) Histological and (F) immunohistochemical analyses of the heart tissue of the NF-3M2e group at 3 dpc. (G) Histological and (H) immunohistochemical analyses of the heart tissue of the HA1 group at 19 dpc. (I) Histological and (J) immunohistochemical analyses of the heart tissue of the [NR-3M2e + HA1] group at 21 dpc. (K) Histological and (L) immunohistochemical analyses of the heart tissue of the [NF- 3M2e + HA1] group at 21 dpc.

[0092] DETAILED DESCRIPTION OF THE INVENTION

[0093] In the present invention, the inventors evaluated the benefits of supplementing nanoparticles bearing three tandem repeats of M2e, e.g., in the forms of nanorings (NR-3M2e) or nanofilaments (NF-3M2e), with an HA1 subunit vaccine, in the protection against a heterologous HPAI H5N1 virus challenge in specific pathogen-free chickens. Birds immunized with NR-3M2e or NF-3M2e admixed with HA1 protein developed M2e and HAl-specific antibodies, at levels which were similar to chickens given HA1 protein alone or each of the M2e-containing nanoparticles.

[0094] Surprisingly and in contrast with chickens immunized with HA1, NR-3M2e, or NF-3M2e, all chickens vaccinated with HA1 combined with the M2e-containing nanoparticles were completely protected from clinical disease, mortality, and histopathological lesions associated with HPAI virus infection. In addition, chickens vaccinated with the combined vaccine formulations did not show oropharyngeal or cloacal virus shedding through the 14-days experimental period, whereas chickens vaccinated with either of HA1, NR-3M2e, or NF-3M2e readily shed the virus during that period. The results unequivocally demonstrated that a vaccine formulation containing NR-3M2e or NF-

[0095] 3M2e nanoparticles and HA1 protein provided full cross-protection against HPAI H5N1 heterologous virus and prevented virus shedding in the environment.

[0096] Accordingly, the present invention relates to a vaccine or immunogenic composition comprising: a recombinant hemagglutinin HA1 from an influenza A virus; and a protein carrier fused with an ectodomain of a matrix protein 2 (M2e domain) from an influenza A virus or a tandem repeat thereof, optionally through a peptide linker.

[0097] Preferably, the influenza A virus is a pathogenic influenza A virus. Optionally, the influenza A virus is a highly contagious influenza A virus.

[0098] Optionally, the influenza A virus is any subtype selected from the group consisting of H1N1, H1N2, H1N3, H1N4, H1N5, H1N6, H1N7, H1N8, H1N9, H2N1, H2N2, H2N3, H2N4, H2N5, H2N6, H2N7,

[0099] H2N8, H2N9, H3N1, H3N2, H3N3, H3N4, H3N5, H3N6, H3N7, H3N8, H3N9, H4N1, H4N2, H4N3,

[0100] H4N4, H4N5, H4N6, H4N7, H4N8, H4N9, H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8,

[0101] H5N9, H6N1, H6N2, H6N3, H6N4, H6N5, H6N6, H6N7, H6N8, H6N9, H7N1, H7N2, H7N3, H7N4,

[0102] H7N5, H7N6, H7N7, H7N8, H7N9, H8N1, H8N2, H8N3, H8N4, H8N6, H8N7, H8N8, H9N1, H9N2,

[0103] H9N3, H9N4, H9N5, H9N6, H9N7, H9N8, H9N9, H10N1, H10N2, H10N3, H10N4, H10N5, H10N6,

[0104] H10N7, H10N8, H10N9, H11N1, H11N2, H11N3, H11N4, H11N5, H11N6, H11N6, H11N7, H11N8,

[0105] H11N9, H12N1, H12N2, H12N3, H12N4, H12N5, H12N6, H12N7, H12N8, H12N9, H13N1, H13N2,

[0106] H13N3, H13N4, H13N6, H13N7, H13N8, H13N9, H14N2, H14N3, H14N4, H14N5, H14N6, H14N7,

[0107] H14N8, H15N2, H15N4, H15N5, H15N6, H15N7, H15N8, H15N9, H16N3, H16N8, and H16N9.

[0108] Optionally, the influenza A virus is any subtype selected from the group consisting of H1N1, H1N2, H2N2, H2N3, H3N2, H3N8, H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N6, H7N7, H7N9, H9N2, H9N6, H9N7, H10N3, and H10N7.

[0109] In one particular aspect, the influenza A virus is an avian influenza A virus. Optionally, it is a highly pathogenic avian influenza A virus (HPAI). Especially, the avian influenza A virus is a virus subtype A(H5), A(H6), A(H7), A(H9), or A(H10), especially A(H5) virus subtype, A(H7) virus subtype, or A(H9) virus subtype. Optionally, the avian influenza A virus is any subtype selected from the group consisting of H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, H5N9, H6N1, H6N2, H6N3, H6N4, H6N5, H6N6, H6N7, H6N8, H6N9, H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, H7N9, H9N1, H9N2, H9N3, H9N4, H9N5, H9N6, H9N7, H9N8, H9N9, H10N1, H10N2, H10N3, H10N4, H10N5, H10N6, H10N7, H10N8, and H10N9. Optionally, the avian influenza A virus is any subtype selected from the group consisting of H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, H7N9, H9N1, H9N2, H9N3, H9N4, H9N5, H9N6, H9N7, H9N8, and H9N9. Optionally, the avian influenza A virus is any subtype selected from the group consisting of H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, and H7N9. Optionally, the avian influenza A virus is any subtype selected from the group consisting of H5N1, H5N6, H6N1, H7N2, H7N6, H7N9, H9N2, H10N3, H10N7, and H10N8. Optionally, the avian influenza A virus is any subtype selected from the group consisting of H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, and H5N9, preferably H5N1, H5N6, and H5N8. More particularly, the highly pathogenic influenza A virus is an avian highly pathogenic influenza A virus selected from the group consisting of H5N1, H5N2, H5N6, H5N8, H5N9, H7N1, H7N3, H7N4, H7N7, and H7N9.

[0110] In an alternative aspect, the influenza A virus can be a mammal influenza A virus. Optionally, it is an equine influenza A virus. It can also be a canine, swine, or human influenza A virus.

[0111] Optionally, the influenza A virus is an equine influenza A virus such as H3N8 and H7N7, preferably H3N8. Optionally, the influenza A virus is a swine influenza A virus such as H1N1, H1N2, H2N3, H3N1, H3N2, and H7N9. Optionally, the influenza A virus is a canine influenza A virus such as H3N2. Optionally, the influenza A virus is a human influenza A virus such as H1N1 and H3N2.

[0112] In a very specific aspect, the highly pathogenic influenza A virus is a H5 virus, especially H5N1.

[0113] Alternatively, the influenza A virus is a highly contagious equine influenza A virus, for instance H3N8 or H7N7, more particularly H3N8.

[0114] In a particular aspect, the vaccine or immunogenic composition does not comprise any additional influenza protein or part thereof, for instance Ml (matrix protein 1) or a fragment thereof, full- length M2 (matrix protein 2), neuraminidase NA, or a fragment thereof, full-length hemagglutinin HA or hemagglutinin HA2 region or a fragment thereof. The vaccine or immunogenic composition does not comprise live-attenuated influenza A virus, inactivated influenza A virus such as WIV or split virus.

[0115] Hemagglutinin HA1

[0116] The vaccine or immunogenic composition comprises a recombinant hemagglutinin HA1 from a influenza A virus, especially a pathogenic or contagious influenza A virus.

[0117] The recombinant HA1 can be a HA1 from any influenza A virus as detailed above.

[0118] In a first aspect, the recombinant hemagglutinin HA1 is from a virus selected from the group consisting of H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, and H5N9, preferably H5N1, H5N2, H5N6, H5N8, and H5N9. In a very particular aspect, the recombinant hemagglutinin HA1 is from a H5N1 virus. Optional ly, the recombinant hemagglutinin HA1 from H5N1 virus comprises, essentially consists of or consists of a sequence of SEQ ID NO: 38 or a sequence having at least 80, 85, 90, 95, 96, 97, 98, or 99% of identity with SEQ ID NO: 38. This sequence is from the virus A / duck / France / 22P025647 / 2022 (H5N1). Alternatively, the recombinant hemagglutinin HA1 comprises, essentially consists of or consists of a sequence of SEQ ID NO: 3 or a sequence having at least 80, 85, 90, 95, 96, 97, 98, or 99% of identity with SEQ ID NO: 3. This sequence is from the virus A / Turkey / Ontario / 6213 / 1966 (H5N1).

[0119] In a second aspect, the recombinant hemagglutinin HA1 is from H7 subtype, in particular a virus selected from the group consisting of H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, and H7N9, preferably H7N2 or H7N9. The hemagglutinin HA1 comprises, essentially consists of or consists of a sequence of SEQ ID NO: 41 or a sequence having at least 80, 85, 90, 95, 96, 97, 98, or 99% of identity with SEQ ID NO: 41. This sequence is from the virus A / Anhui / 01 / 2014 (H7N9).

[0120] In a third aspect, the recombinant hemagglutinin HA1 is from H9 subtype, in particular a virus selected from the group consisting of H9N1, H9N2, H9N3, H9N4, H9N5, H9N6, H9N7, H9N8, and H9N9, preferably H9N2, H9N6, H9N6, and H9N7.

[0121] In a fourth aspect, the recombinant hemagglutinin HA1 is from H10 subtype, in particular a virus selected from the group consisting of H10N1, H10N2, H10N3, H10N4, H10N5, H10N6, H10N7, H10N8, and H10N9, preferably H10N3, H10N7, and H10N8.

[0122] In a fifth aspect, the recombinant hemagglutinin HA1 is from H3 subtype, in particular an equine H3N8 virus. Optionally, recombinant hemagglutinin HA1 comprises, essentially consists of or consists of a sequence of SEQ ID NO: 39 or 40 or a sequence having at least 80, 85, 90, 95, 96, 97, 98, or 99% of identity with SEQ ID NO: 39 or 40. These sequences are respectively from the viruses A / Equine / Ohio / 1 / 2003 (H3N8) and A / Equine / Richmond / 1 / 2007 (H3N8).

[0123] The vaccine or immunogenic composition comprises a recombinant HA1 which is not fused to another protein, especially another influenza protein or part thereof such as NA, Ml, M2, M2e, or HA2. In addition, the recombinant HA1 of the vaccine or immunogenic composition is not fused to a VLP or included into a viral-like particle or a viral particle. The recombinant HA1 is not fused to a self-assembling protein.

[0124] Optionally, the vaccine or immunogenic composition may comprise different recombinant HA1 proteins, for instance a recombinant HA1 of a H5 subtype of viruses, and / or a recombinant HA1 of a H7 subtype of viruses, and / or a recombinant HA1 of a H9 subtype of viruses, and / or a recombinant HA1 of a H10 subtype of viruses, especially a recombinant HA1 proteins from a virus selected from the group consisting of H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, and H5N9, preferably H5N1, H5N2, H5N6, H5N8, and H5N9, and / or a recombinant HA1 proteins from a virus selected from the group consisting of H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, and H7N9, preferably H7N2 and H7N9, and / or a recombinant HA1 proteins from a virus selected from the group consisting of H9N1, H9N2, H9N3, H9N4, H9N5, H9N6, H9N7, H9N8, and H9N9, preferably H9N2, H9N6, H9N6, and H9N7, and / or a recombinant HA1 proteins from a virus selected from the group consisting of H10N1, H10N2, H10N3, H10N4, H10N5, H10N6, H10N7, H10N8, and H10N9, preferably H10N3, H10N7, and H10N8.

[0125] Despite the vaccine or immunogenic composition presents the advantage of a cross-protection among antigenically divergent virus strains, the vaccine or immunogenic composition may comprise different recombinant HA1 proteins belonging to the same H virus. For instance, it could comprise several recombinant HA1 proteins of a H3 virus subtype, several recombinant HA1 proteins of a H5 virus subtype, and / or several recombinant HA1 proteins of a H7 virus subtype. For instance, the vaccine or immunogenic composition may comprise a recombinant HA1 proteins from one or several viruses selected from the group consisting of H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, and H5N9, preferably H5N1, H5N2, H5N6, H5N8, and H5N9; and / or a recombinant HA1 proteins from one or several viruses selected from the group consisting of H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, and H7N9, preferably H7N2 and H7N9; and / or a recombinant HA1 proteins from one or several viruses selected from the group consisting of H9N1, H9N2, H9N3, H9N4, H9N5, H9N6, H9N7, H9N8, and H9N9, preferably H9N2, H9N6, H9N6, and H9N7, and / or a recombinant HA1 proteins from one or several viruses selected from the group consisting of H10N1, H10N2, H10N3, H10N4, H10N5, H10N6, H10N7, H10N8, and H10N9, preferably H10N3, H10N7, and H10N8.

[0126] In a particular aspect, the sequence of the recombinant HA1 has more than 80% of identity with the sequence HA1 of the target virus for the vaccine or immunogenic composition, preferably at least 85, 90, 95, 96, 97, 98, or 99% with it.

[0127] Protein carriers

[0128] The vaccine or immunogenic composition comprises a protein carrier fused to a M2e or a tandem repeat of M2e.

[0129] Optionally, the protein carrier is not a protein from influenza A virus, such as nucleoprotein (NP) or hemagglutinin (HA) or a part thereof such as a truncated NP or HA1 or HA2.

[0130] In a first aspect, the carrier protein can be any protein suitable to boost the host immune system so as to induce an immune reaction against M2e or a tandem repeat of M2e. For instance, the carrier protein can be selected from the group consisting of CRM197 (nontoxic variant of diphtheria toxin isolated from Corynebacterium diphtheriae C7 ( 197), tetanus toxoid (TT), a Pseudomonas aeruginosa protein, human serum albumin (HSA), bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), diphtheria toxin fragment B (DTFB), DTFB C8, diphtheria toxoid (DT), fragment C of TT, pertussis toxoid, cholera toxoid, E. coli heat-labile toxin, E. coli heat-stable toxin, meningococcal outer membrane protein complex (OMPC), H. influenzae protein D (HiD), and exotoxin A from Pseudomonas aeruginosa.

[0131] In a second aspect, the protein carrier is a self-assembling protein or a combination of selfassembling proteins, so as to obtain virus-like particle (VLP) or self-assembled protein nanoparticles (SAPN), said VLP or SAPN displaying M2e or tandem repeat thereof.

[0132] According to this aspect, the vaccine or immunogenic composition comprises a nanoparticle bearing M2e or a tandem repeat of M2e. The nanoparticle is made of a self-assembling protein which is fused to M2e or a tandem repeat of M2e, optionally through a peptide linker, thereby the nanoparticle bears the tandem repeat of M2e at its surface.

[0133] Protein carriers that self-assemble in nanoparticles are well-known in the art (see non-exhaustively, Lamontagne et al, Vaccines 2022, 10(11), 1920; https: / / doi.org / 10.3390 / vaccinesl0111920; Zottig et al, Nanomaterials 2020, 10(5), 1008; https: / / doi.org / 10.3390 / nanol0051008; W02020220044, WO2015104352).

[0134] The self-assembling protein refers to individual units or building blocks made of amino acids capable of spontaneous association into highly arranged / ordered structures / patterns. Such highly arranged / ordered structures / patterns are nanoparticles. Virus-like particles (VLPs) are nanoparticles made in vitro by the capsid structural proteins of a virus, especially a non-enveloped which are able to self-assemble into symmetric supramolecular architectures. The nanoparticle can non-exhaustively be a nanoring, an icosahedral nanoparticle, an octahedral cage, a dodecahedral cage, fibrils, nanorods, or nanofilaments. In a particular aspect, the nanoparticle is a nanoring or a nanofilament. The size of VLP or SAPN generally ranges between 5 and 900 nm.

[0135] The protein carrier can be selected in the non-exhaustive list comprising a nucleoprotein N of respiratory syncytial virus (RSV) (Herve et al, J Virol. (2014) 88:325-38. doi: 10.1128 / JVI.01141-13), CsgA (Curli-specific gene A) (WO2008 / 124646), an amyloid protein such as CspA (E. coli cold shock protein A), ferritin, Soc (Small Outer Capsid protein) of phage T4, hepatitis B virus core antigen (HBc) (De Filette et al. Vaccine. (2006) 24:544-51. doi: 10.1016 / j.vaccine.2005.08.061; Chen et al. Biomed Res Int. (2015) 2015:901817. doi: 10.1155 / 2015 / 901817; Zheng et al. Vaccine. (2016) 34:6464-71. doi: 10.1016 / j.vaccine.2016.11.026), bacteriophage AP205 coat protein (Kirsteina et al, Vaccines 2020, 8(2), 197; doi.org / 10.3390 / vaccines8020197), coat protein of bacteriophage Q (Bessa et al. Eur J Immunol. (2008) 38:114-26. doi: 10.1002 / eji.200636959), a dodecameric DNA binding protein from starved cells (Dps) of bacteria or archaea such as Dsp of E. coli, Brucella outer membrane protein BP26, capsid protein of PCV2 (porcine circovirus type 2), protrusion domain of the capsid protein of norovirus (see WO2018 / 182983; Xia et al. Vaccine. (2011) 29:7670-7, doi: 10.1016 / j.vaccine.2011.07.139; Elaish et al. Vaccine. (2015) 33:4901-9. doi:

[0136] 10.1016 / j.vaccine.2015.07.049), a self-assembling an amyloid protein p-peptide, such as a ten-mer self-assembling sequence (110) derived from an amyloidogenic peptide (SNNFGAILSS, SEQ ID NO: 36) (Al Halifa et al, Nanomaterials 2020, 10(10), 1981; https: / / doi.org / 10.3390 / nanol0101981), an antibody such as anti-Clec9A monoclonal antibody, CTA1-DD (ADP-ribosylating Al subunit of cholera toxin CT linked to a dimer of the D-fragment of Staphylococcus aureus protein A), bacterial flagel lin such as flagellin of Bacillus subtilis or Salmonella serovar enterica typhimurium (S. typhimurium), Qll peptide (QQKFQFQFEQQ, SEQ ID NO: 37), lumazine synthase, especially Aquifex aelocus (A. aelocus) lumazine synthase (Ladenstein et al, Biotechnol Rep (Amst). 2020 Sep; 27: e00494), encapsulin, especially from T maritima, and small Heat shock protein (sHsp) 16.5, especially from Methanocaldoccus jannaschii, a superfolder green fluorescent protein (sfGFP) (Wong et al, Vaccines 2023, 11(2), 426; https: / / doi.org / 10.3390 / vaccinesll020426), bacteriophage P22, E2p, especially from Bacillus stearothermophilus (B. stearothermophilusj, 13-01 derived from Thermotoga maritima's 2-Keto-3-deoxy-6-phosphogluconate (KDPG) aldolase, especially from T maritima, 153- 50, IMX313, SAP, E2, Coil29, FliC, latency-associated nuclear antigen (LANA) from the Kaposi's sarcoma-associated herpesvirus (KSHV) (WQ2023170068), coat of a plant virus such as nepovirus coat virus, in particular GFLV (Grapevine fanleaf virus) (WO 2017 / 042367), hydrophobin, bacteriophage T7 endonuclease, A beta 1-40, Sup35p of S. cerevisiae, human Pmell7, Agfa Tafi protein, a Chaplin protein, a peptide derived from islet amyloid polypeptide (IAPP), in particular SNNFGAILSS (SEQ ID NO: 36) (WQ2022000066), an amphiphilic peptide as disclosed in WO2012 / 162637, WQ2013 / 004716 or WQ2023 / 019141, and a combination thereof such as a combination of ferritin, MsDps2 and Encapsulin, a combination of E2p and 13-01. WQ2011 / 063264 and WQ2020 / 223730 discloses alternative self-assembling peptides.

[0137] For instance, the protein carrier can be ferritin, especially heavy chain ferritin, for instance human heavy chain ferritin or Helicobacter pylori ferritin. M2e or tandem repeat thereof are displayed on ferritin nanoparticles (Pan et al, ACS Nano 2023, 17, 14, 13474-13487). The M2e or tandem repeat thereof is fused to the N terminal end of heavy chain ferritin, optionally through a peptide linker.

[0138] In another example, the protein carrier can be phage T4 Soc. The Soc proteins self-assemble into a nanoparticle (Li et al, 2023, https: / / doi.Org / 10.1016 / j.antiviral.2023.105688). The M2e or tandem repeat thereof is fused the Soc protein. The protein carrier can also be a combination of proteins. For instance, the combination can be a combination of ferritin, MsDps2 and Encapsulin (Kar et al, Front. Immunol., 26 May 2022, https: / / doi.org / 10.3389 / fimmu.2022.890622). The M2e or tandem repeat thereof can be fused to C terminal end of MsDps2, and / or to the N-terminal end of ferritin and / or to the C-terminal end of encapsulin, optionally through a peptide linker.

[0139] The carrier protein can also be a Dps of bacteria or archaea such as Dsp of E. coll. The M2e or tandem repeat thereof can be fused to the N-terminal end of Dsp and / or to the C-terminal end of Dsp, optionally through a peptide linker. The Dsp self-assemble as a nanoparticle displaying M2e or tandem repeat thereof (Ni et al, Vaccine. 2017 Dec 15; 35(50): 7026-7032).

[0140] The carrier protein can be Brucella outer membrane protein BP26, which self-assemble to form a nanobarrel displaying M2e or tandem repeat thereof (Kang et al, ACS Nano 2021, 15, 6, 10722- 10732). The M2e or tandem repeat thereof can be fused to BP26, preferably to the N-terminal end of BP26, optionally through a linker.

[0141] The carrier protein can be a capsid protein of PCV2, which self-assemble to form a VLP displaying M2e or tandem repeat thereof (Ding et al, Int J Biol Macromol. 2021 Mar 15;173:244-250. doi: 10.1016 / j.ijbiomac.2021.01.132). The M2e or tandem repeat thereof can be fused to the C-terminal end of Cap of PCV2, optionally through a linker.

[0142] The carrier protein can be a self-assembling p-peptide such 110, forming cross-p fibrils displaying The M2e or tandem repeat thereof. The M2e or tandem repeat thereof can be fused to the N- terminal end of the peptide, preferably through a peptide linker. In a particular aspect, carrier proteins are not fused or covalently linked to any other influenza protein or part thereof, for instance NP, NA, HA1, HA2, or Ml. In addition, the carrier protein is not fused or covalently linked to full- length M2.

[0143] Optionally, the self-assembling protein is a N protein of a virus of the Paramyxoviridae family, such as the mumps virus, measles virus, human metapneumovirus, parainfluenza virus, and respiratory syncytial virus (see W02007 / 119011). Optionally, the N protein can be modified, for instance in the region defined by the last 25 C-terminal amino acids. The N proteins are fused at their C-terminal end to the M2e domain or a tandem repeat thereof, optionally through a peptide linked.

[0144] In a particular aspect, the self-assembling protein is a nucleoprotein N from respiratory syncytial virus. As detailed in WO2017 / 134202 and WO2017 / 134202, the N proteins of RSV are able to self- assemble and form a ring-like structure containing 10 to 11 molecules of N protein (i.e., nanoring). The N proteins are fused at their C-terminal end to the M2e domain or a tandem repeat thereof, optionally through a peptide linked. The ring-like structures induce a strong immune response, even in the absence of adjuvant. Optionally, the N protein is from a human RSV or a bovine RSV. Optionally, the N protein has a sequence of SEQ ID NO: 5 or a variant thereof comprising substitutions within the 25 C-terminal residues or deletion of some or all 25 C-terminal residues.

[0145] In another particular aspect, the self-assembling protein is CsgA (Curli-specific gene A) from E. coli or a fragment thereof (Lamontagne et al, 2023, Adv Healthcare Mater, 2300224, D0l:10.1002 / adhm.2300224). The CsgA or a fragment thereof forms nanofilaments. The M2e domain or a tandem repeat of M2e domains is fused to the N-terminal end of CsgA or a fragment thereof, optionally through a peptide linker. In a particular aspect, the CsgA comprises or consists of the sequence of SEQ ID NO: 11. Optionally, up to 20 amino acid residues of the peptide signal could be deleted and the CsgA comprises or consists of the sequence of SEQ ID NO: 7. CsgA includes 5 repeat units, namely R1 (residues 43-65 of SEQ ID NO: 11), R2 (residues 66-87 of SEQ ID NO: 11), R3 (residues 88-110 of SEQ ID NO: 11), R4 (residues 111-132 of SEQ ID NO: 11), and R5 (residues 133-151 of SEQ ID NO: 11) from N to C terminal end. Rl, R3, and R5 are repeat units capable of assembling into amyloid fibers (Wang et al, J Biol Chem, 2007, 282, 3713-3719. Accordingly, truncated forms of CsgA can be used instead of the full length protein. In particular aspect, the selfassembling protein can be selected from the following truncated forms: a fragment of CsgA comprising R2, R3, R4, and R5 repeat units; a fragment of CsgA comprising R3, R4, and R5 repeat units; a fragment of CsgA comprising R4 and R5 repeat units; and a fragment of CsgA comprising R5 repeat unit.

[0146] M2e domain and repeat thereof

[0147] The carrier protein, in particular the self-assembling protein, is fused to an ectodomain of the matrix protein 2 (M2e domain) or a tandem repeat thereof.

[0148] In a particular aspect, the carrier protein, in particular the self-assembling protein, is fused to one M2e domain. In an alternative aspect, the carrier protein, in particular the self-assembling protein, is fused to a repeat of 2 to 10 M2e sequences, for instance 2, 3, 4, 5, 6, 7, 8, 9, or 10 M2e sequences.

[0149] In a more particular aspect, the tandem repeat of M2e domains comprises from 2 to 5 M2e sequences or from 2 to 4 M2e sequences. For instance, the tandem repeat comprises 2, 3, 4, or 5 M2e sequences. In a preferred aspect, the tandem repeat has 3 M2e sequences.

[0150] The sequence of M2e domain is highly conserved among the influenza A viruses. Accordingly, it is possible to define consensus sequences for these viruses, especially the HPAI viruses. In one aspect, the sequence of M2e domain comprises, essentially consists of or consists of a sequence of SEQ ID NO: 12:

[0151] (M / abs) S L L T E V E T (P / L) (T / I) (K / R) (T / N / K / S) (E / G) W (E / G)

[0152] (C / S) (R / N / K) (C / S) (S / N) (D / V / G) S S D (SEQ ID NO: 12).

[0153] In another aspect, the sequence of M2e domain comprises, essentially consists of or consists of a sequence of SEQ ID NO: 13:

[0154] (M / abs) S L L T E V E T (P / L) T (K / R) (T / N / K / S) (E / G) W (E / G) (C / S)

[0155] (R / N / K) (C / S) (S / N) (D / V / G) S S D (SEQ ID NO: 13).

[0156] In a particular aspect, the sequence of M2e domain comprises, essentially consists of or consists of a sequence selected in the group consisting of:

[0157] (M / abs) S L L T E V E T (P / L) T (K / R) (T / N) (E / G) W (E / G) (C / S) (R / N / K) (C / S) (S / N) (D / V / G) S S D (SEQ ID NO: 14).

[0158] (M / abs) S L L T E V E T P T R N (E / G) W (E / G) (C / S) (R / K) (C / S) (S / N) D S S D (SEQID NO: 15);

[0159] (M / abs) S L L T E V E T P T R N (E / G) W (E / G) (C / S) (R / K) (C / S)

[0160] S D S S D (SEQID NO: 16);

[0161] (M / abs) S L L T E V E T P T R (N / T) (E / G) W (E / G) (C / S) R (C / S)

[0162] S D S S D (SEQID NO: 17);

[0163] (M / abs) S L L T E V E T P T R N (E / G) W E (C / S) (K / R) (C / S) S D S S D (SEQ ID NO: 18); and

[0164] (M / abs) S L L T E V E T P T R N (E / G) W E (C / S) K (C / S) S D S S D (SEQID NO: 19).

[0165] M / abs means a methionine or no amino acid. P / L means proline or leucine. K / R means lysine or arginine. T / N / K / S means threonine, asparagine, lysine, or serine. T / N means threonine or asparagine. E / G means glutamic acid or glycine. C / S means cysteine or serine. R / N / K means arginine, asparagine, or lysine. K / R means lysine or arginine. S / N means serine or asparagine. D / V / G means aspartic acid, valine, or glycine.

[0166] The M2e sequence of SEQ ID NO: 12 is a consensus sequence of M2e of influenza A viruses. The M2e sequence of SEQ ID NOs: 13 and 14-16 are consensus sequences from influenza viruses of H3, H5, and H7 subtypes. The M2e sequence of SEQID NO: 17 is more specific to H5 subtype of influenza virus. The M2e sequence of SEQ ID NO: 18 is more specific to H7 subtype of influenza virus. The M2e sequence of SEQ ID NO: 19 is more specific to H3 subtype of influenza virus. In a very particular aspect, the M2e sequence comprises the M2e sequence comprises, essentially consists of or consists of a sequence selected in the group consisting of:

[0167] (M / abs) S L L T E V E T P T R T E W E C R C S D S S D(SEQIDNO

[0168] 20);

[0169] (M / abs) S L L T E V E T P T R T E W E S R C S D S S D(SEQIDNO

[0170] 21);

[0171] (M / abs) S L L T E V E T P T R T E W E C R S S D S S D(SEQIDNO

[0172] 22);

[0173] (M / abs) S L L T E V E T P T R T E W E S R S S D S S D(SEQIDNO

[0174] 23);

[0175] (M / abs) S L L T E V E T P T R N E W E C R C S D S S D(SEQIDNO

[0176] 24);

[0177] (M / abs) S L L T E V E T P T R N E W E S R C S D S S D(SEQIDNO

[0178] 25);

[0179] (M / abs) S L L T E V E T P T R N E W E C R S S D S S D(SEQIDNO

[0180] 26);

[0181] (M / abs) S L L T E V E T P T R N E W E S R S S D S S D(SEQIDNO

[0182] 27);

[0183] (M / abs) S L L T E V E T P T R T G W E C R C S D S S D(SEQIDNO

[0184] 28);

[0185] (M / abs) S L L T E V E T P T R T G W E S R C S D S S D(SEQIDNO

[0186] 29);

[0187] (M / abs) S L L T E V E T P T R T G W E C R S S D S S D(SEQIDNO

[0188] 30);

[0189] (M / abs) S L L T E V E T P T R T G W E S R S S D S S D(SEQIDNO

[0190] 31);

[0191] (M / abs) S L L T E V E T P T R N G W E C R C S D S S D(SEQIDNO

[0192] 32);

[0193] (M / abs) S L L T E V E T P T R N G W E S R C S D S S D(SEQIDNO

[0194] 33);

[0195] (M / abs) S L L T E V E T P T R N G W E C R S S D S S D(SEQIDNO

[0196] 34); and

[0197] (M / abs) S L L T E V E T P T R N G W E S R S S D S S D(SEQIDNO

[0198] 35). In a very particular aspect, the tandem repeat includes 3 M2e sequences and the M2e sequence is (M / abs ) S L L T E V E T P T R T E W E S R S S D S S D (SEQ ID NO: 23).

[0199] Optionally, the M2e sequence can be shorter and may have a deletion of 1, 2, or 3 amino acid(s) at the C-terminal end. Optionally, the M2e sequence can be longer and include 1, 2, or 3 additional amino acid(s) at the C-terminal end, for instance the addition of P, PV, or PVL.

[0200] Optionally, the M2e sequences are the same in the tandem repeat. Alternatively, the M2e sequences in the tandem repeat can be different. For instance, the tandem repeat may comprise a M2e sequence from influenza A H5 subtype, a M2e sequence from influenza A H7 subtype, a M2e sequence from influenza A H9 subtype, and a M2e sequence from influenza A H10 subtype, or any combination of 2, 3 or 4 different subtypes.

[0201] Optionally, the first M2e sequence includes a M residue at the N terminal end whereas the other M2e sequences are devoid of the N-terminal M residue. Optionally, the M2e sequences do not include a M residue at the N-terminal end of the M2e sequence. Optionally, the M2e sequences include a M residue at the N-terminal end of the M2e sequence.

[0202] Optionally, the M2e sequences in the tandem repeat are connected directly or through a peptide linker. The peptide linker can include 1-10 amino acid residues, for instance 1, 2, 3, 4, 5, or 6 amino acid residues. In a particular aspect, the M2e sequences in the tandem repeat are connected through 2, 3, or 4 amino acid residues, preferably 2 amino acid residues. The linker can include any amino acid. Optionally, the amino acids are G, T, E, or F. Optionally, the amino acids are G or S. Optionally, the linker can be selected from the group consisting of GT, EF, and GG.

[0203] In a very particular aspect of the disclosure, the vaccine or immunogenic composition comprises:

[0204] - a recombinant hemagglutinin HA1 from H5 virus comprising a sequence of SEQ ID NO: 3 or 38 or a sequence having at least 80, 85, 90, or 95% identity with SEQ ID NO: 3 or SEQ ID NO: 38; and

[0205] - a nanoparticle made of a self-assembling protein being selected from the group consisting of nucleocapsid protein (N) of respiratory syncytial virus (RSV) and curli specific gene A (CsgA) or a fragment thereof, the self-assembling protein being fused with a tandem repeat of 3 M2e sequences and the M2e sequence comprises, essentially consists of or consists of a sequence (M / abs ) S L L T E V E T P T R T E W E S R S S D S S D (SEQ ID NO: 23).

[0206] More specifically, the self-assembling protein is N protein of SEQ ID NO: 5 and the tandem repeat of M2e is fused to the C-terminal end of the N protein. Optionally, the N protein of RSV fused with a tandem repeat of 3 M2e sequences comprises or consists in the sequence as shown in SEQ ID NO: 6 and the coding sequence can be for instance the nucleic acid sequence of SEQ ID NO: 9.

[0207] Alternatively, the self-assembling protein is CsgA protein of SEQ ID NO: 7 or a fragment thereof and the tandem repeat of M2e is fused to the N-terminal end of the CsgA protein of the fragment thereof. Optionally, the CsgA fused with a tandem repeat of 3 M2e sequences comprises or consists in the sequence as shown in SEQ ID NO: 8 and the coding sequence can be for instance the nucleic acid sequence of SEQ ID NO: 10.

[0208] This vaccine or immunogenic composition is appropriate for vaccination against viruses of H5 subtype, especially a virus selected from the group consisting of H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, and H5N9, preferably a virus selected from the group consisting of H5N1, H5N2, H5N6, H5N8, and H5N9, more specifically the H5N1 virus.

[0209] In another very specific aspect, the vaccine or immunogenic composition comprises:

[0210] - a recombinant hemagglutinin HA1 from H3N8 virus comprising a sequence of SEQ ID NO: 39 or 40 or a sequence having at least 80, 85, 90, or 95% identity with SEQ ID NO: 39 or 40; and

[0211] - a nanoparticle made of a carrier protein being selected from the group consisting of nucleocapsid protein (N) of respiratory syncytial virus (RSV) and curli specific gene A (CsgA) or a fragment thereof, the carrier protein being fused with a tandem repeat of 3 M2e sequences and the M2e sequence comprises, essentially consists of or consists of a sequence (M / abs) S L L T E V E T P T R N (E / G) W E (C / S) K (C / S) S D S S D (SEQ ID NO: 19), preferably (M / abs) S L L T E V E T P T R N G W E C K C S D S S D (SEQ ID NO: 32) or (M / abs) S L L T E V E T P T R N G W E S K S S D S S D (SEQ ID NO: 35).

[0212] More specifically, the self-assembling protein is N protein of SEQ ID NO: 5 and the tandem repeat of M2e is fused to the C-terminal end of the N protein.

[0213] Alternatively, the self-assembling protein is CsgA protein of SEQ ID NO: 7 or a fragment thereof and the tandem repeat of M2e is fused to the N-terminal end of the CsgA protein of the fragment thereof.

[0214] This vaccine or immunogenic composition is suitable for vaccination against H3N8, especially for horse vaccination.

[0215] Adjuvant The vaccine or immunogenic composition may further comprise an adjuvant. The adjuvant is optional in the composition because the nanoparticle already provides an adjuvant effect. Accordingly, in one aspect of the present disclosure, the vaccine or immunogenic composition is devoid of adjuvant.

[0216] Optionally, the adjuvant can be selected in the group consisting of bacterial enterotoxins and derivatives thereof such as cholera toxin and E. coli heat-labile toxin, flagell in, proteosome-based adjuvants, bacterial-like particles, lipid-based adjuvants, organic polymers such as chitosan, polyamine polymers and polyesters, inorganic nanoparticles, nucleotide-based adjuvants, and cytokines / chemokines. See Calzas and Chevalier, Frontiers in Immunology, 2019, 10, article 1605.

[0217] Optionally, the vaccine composition may further comprise a lipid-based adjuvant, in particular so as to obtain a water-in-oil emulsion, an oil in water emulsion or a water in oil in water emulsion, preferably an oil in water emulsion.

[0218] The lipid-based adjuvant can be an Emulsigen® P-based adjuvant. Alternatively, suitable W / O or O / W emulsifiers may be selected from mannide mono-oleate and sorbitan mono-oleate, for instance. Examples of appropriate oily adjuvants are a mixture of 5-20% mannide mono-oleate with 80-95% mineral oil (Montanide® ISA 51 sold by SEPPIC) or squalene (Montanide® ISA 720 sold by SEPPIC) and similar mixtures. In a specific aspect, the adjuvant is a mixture of mineral oil and mannide mono-oleate, especially Montanide® ISA. The ratio can be from 50 to 70% in weight of adjuvant. The adjuvant can also be suitable for prepared micro-emulsion. For instance, the adjuvant can be Montanide® IMS. The ratio can be from 15 to 50% in weight of adjuvant.

[0219] The adjuvant may, alternatively or in addition to the above oily adjuvants, be selected from micro- and nanoparticles, such as liposomes and microspheres, of PLG (poly-a-L-glutamine), PLA (poly D,L- lactic acid), PLGA (poly (D,L-lactic-co-glycolic acid) or other natural polymers such as gelatin, collagen, and chitosan. Other adjuvants may comprise TLR ligands, Toll-like receptor ligands (such as TLR3, TLR4, TLR7, and TLR9), stimulators of IFN genes (STING) agonists, cytokines such as GM- CSF and IL2, carbohydrates, bacterial derivatives, mineral salts, and immune stimulating complexes (ISCOM).

[0220] Exemplary adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant. Other adjuvants comprise GMCSP, BCG, aluminum hydroxide, MDP compounds, such as thur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A (MPL). RIBI, which contains three components extracted from bacteria, MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2 percent squalene / Tween 80 emulsion also is contemplated. MF-59, Novasomes(R), and MHC antigens may also be used.

[0221] Optionally, the adjuvant can be a mucoadhesive adjuvant. A mucoadhesive adjuvant is a hydrophilic polymer (e.g. having hydroxyl, carboxyl, amine, or sulphate groups), having a molecular weight of at least 2,000 (two thousand) Da, preferably at least 5,000 (five thousand) Da, which is to be used as delivery system and carrier of vaccine antigens to improve the adhesion to mucosal membranes enhancing local mucosal residence time and controlled local release of the antigen. Preferably the polymer has a pK value that differs from the pH of the target mucosa of the host animal to be vaccinated, such that ionization of the polymer upon contact with the mucosal surface is induced. The pH of poultry mucosal surfaces of the oral cavity is typically near neutral, between 6.5 and 7.5, for healthy animals. Preferably the mucosal adjuvant is a biodegradable polymer, i.e. a polymer that is capable of being decomposed by the target organism. Depending on the host animal and target mucosal surface, the following substances are typically used as mucoadhesive adjuvants: natural polymers (or derivates thereof) such as albumin, pectin, cellulose, gelatin, alginate (a series of polymers of various combi's of G-C blocks; final weight varies from 60-600 kDa), chitosan (a series of chitosan polymers ranging from 110-360 kDa), hyaluronic acid; synthetic polymers such as poloxamer (polyalkylene oxide block copolymer; commercially available as Synperonic F127 from Croda Health Care, a 12 kDa ethylene oxide / propylene oxide block copolymer), PEG (polyethylene glycol), PAA (polyacrylic acid, carbomer, Carbopol[R[, 70-4000 kDa), PLGA, PLA, PVP (polyvinylpyrrolidone), and PVA (polyvinylalcohol).

[0222] Alternatively, the mucosal can be selected in the group consisting in CARBIGEN™; maleic anhydrides, such as ethylene maleic anhydride or polyethylene maleic anhydride; aluminum salts, such as aluminum hydroxide or aluminum phosphate; EMULSIGEN®-based adjuvants including EMULSIGEN®, EMULSIGEN®-D (containing dimethyldioctadecylammonium bromide [DDA]), EMULSIGEN®-BCL (containing a block copolymer immunostimulant), and EMULSIGEN®-P (containing with a proprietary immunostimulant) (Phibro Animal Health Corporation, Omaha, NE, USA); chitosan, hydroxymethyl chitosan, water-in-oil emulsions; oil-in-water emulsions; saponins including VetSap, QS21 (Antigenics, Framingham, MA.) and derivatives of saponins; polyacrylic acids; copolymers, including low molecular weight copolymers such as Polygen™ (available from Phibro Animal Health Corporation, Omaha, NE, USA); Carbopol-based adjuvants; synthetic polynucleotides such as oligonucleotides containing a CpG motif (e.g., U.S. Patent no. 6,207,646); TRIGEN™; Poly-IC; Poly-ICLC; MONTANIDES; or combinations thereof.

[0223] The vaccine or immunogenic composition may further comprise a pharmaceutically acceptable carrier, vehicle, or excipient. Some examples of materials which can serve as pharmaceutically 1 acceptable carriers, vehicles, or excipients include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other nontoxic compatible substances employed in pharmaceutical formulations.

[0224] Optionally, the vaccine or immunogenic composition is in bulk or in unit dosage form. The unit dosage form is any of a variety of forms, including, for example, a capsule, a tablet, or a vial. The quantity of active ingredient in a unit dose of composition is an effective amount and is varied according to the particular treatment involved.

[0225] Optionally, the vaccine or immunogenic composition can be administered or in a form suitable to be administered by parenteral route, especially subcutaneous or intramuscular, by in ovo administration or by mucosal route, for instance by oral or nasal route.

[0226] Optionally, the vaccine or immunogenic composition can be administered on a schedule, for instance with an initial administration with subsequent booster administrations. For example, a second dose may be administered anywhere from 2 weeks to 6 months after the initial administration, optionally from 2 to 8 weeks. A third dose may be administered after the second dose and from about 2 weeks to about one year after the initial administration, optionally from 2 to 8 weeks. The third dose may be optionally administered when no or low levels of specific immunoglobulins are detected in a subject sample after the second dose.

[0227] Optionally, when the subject is a poultry, the administration can be a mass administration. The delivery of the compositions can be via drinking water, spray, or feed.

[0228] The present invention relates to a vaccine or immunogenic composition as disclosed herein for use against influenza A infection; or to the use of a vaccine or immunogenic composition as disclosed herein for the manufacture of a vaccine against influenza A infection; or to a method for preventing or treating an infection by influenza A in a subject comprising administering an effective amount of a vaccine or immunogenic composition as disclosed herein to said subject.

[0229] The present invention relates to a vaccine or immunogenic composition as disclosed herein for use in immunizing a subject against influenza A infection, or to the use of a vaccine or immunogenic composition as disclosed herein for the manufacture of a vaccine for immunizing a subject against influenza A infection, or to a method for immunizing a subject against influenza A infection comprising administering an effective amount of a vaccine or immunogenic composition as disclosed herein to said subject.

[0230] The present invention relates to a vaccine or immunogenic composition as disclosed herein for use in reducing or preventing the effects of an infection by influenza A in a subject; or to the use of a vaccine or immunogenic composition as disclosed herein for the manufacture of a vaccine for reducing or preventing the effects of an infection by influenza A in a subject; or to a method for reducing or preventing the effects of an infection by influenza A in a subject comprising administering an effective amount of a vaccine or immunogenic composition as disclosed herein to said subject. In a particular aspect, the effects can be selected in the group consisting of mortality, clinical disease associated with infection by influenza A virus, histopathological lesions associated with infection by influenza A, and influenza A virus shedding, especially oropharyngeal or cloacal virus shedding. In a very specific aspect, the effects are influenza A virus shedding, especially oropharyngeal or cloacal virus shedding.

[0231] The influenza A virus can be any of the influenza A viruses disclosed herein and the subject can be any animal or human susceptible to be infected by the influenza A virus. For instance, the subject can be a human. Alternatively, the subject can be a mammal such as a horse, a swine, a dog or a cat.

[0232] In a particular aspect, the vaccine or immunogenic composition as disclosed herein is for use for immunizing birds against avian influenza A. Especially, birds are poultry, in particular chickens, ducks, geese, turkeys, bantams, quails, pheasants, and pidgeons, more specifically chickens. Optionally, the poultry is a layer hen. Optionally, the poultry is used for breeding. Optionally, the influenza A virus is a virus subtype A(H5), A(H6), A(H7), A(H9), or A(H10), especially A(H5) virus subtype, A(H7) virus subtype or A(H9) virus subtype. Optionally, the avian influenza A virus is any subtype selected from the group consisting of H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, H5N9, H6N1, H6N2, H6N3, H6N4, H6N5, H6N6, H6N7, H6N8, H6N9, H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, H7N9, H9N1, H9N2, H9N3, H9N4, H9N5, H9N6, H9N7, H9N8, H9N9, H10N1, H10N2, H10N3, H10N4, H10N5, H10N6, H10N7, H10N8, and H10N9. Optionally, the avian influenza A virus is any subtype selected from the group consisting of H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, H7N9, H9N1, H9N2, H9N3, H9N4, H9N5, H9N6, H9N7, H9N8, and H9N9. Optionally, the avian influenza A virus is any subtype selected from the group consisting of H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, and H7N9. Optionally, the avian influenza A virus is any subtype selected from the group consisting of H5N1, H5N6, H6N1, H7N2, H7N9, H9N2, H10N3, H10N7, and H10N8. Optionally, the influenza A virus is selected from the group consisting of H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N8, H5N9, H7N1, H7N3, H7N4, H7N7, H7N9, H9N2, H9N6, H9N6, and H9N7. Preferably, the influenza A virus is an avian highly pathogenic influenza A virus, such as H5N1, H5N2, H5N6, H5N8, H5N9, H7N1, H7N3, H7N4, H7N7, and H7N9. More specifically, the influenza A virus is H5N1 virus.

[0233] In this context, the vaccine or immunogenic composition comprises a recombinant HA1 from a virus subtype A(H5), A(H6), A(H7), A(H9), or A(H10), especially A(H5) virus subtype, A(H7) virus subtype or A(H9) virus subtype and a self-assembling protein being fused with a tandem repeat of M2e sequences from a virus subtype A(H5), A(H6), A(H7), A(H9), or A(H10), especially A(H5) virus subtype, A(H7) virus subtype, A(H9) virus subtype, or a consensus sequence thereof. In a particular aspect, the vaccine or immunogenic composition comprises a recombinant HA1 from a virus subtype A(H5) and a self-assembling protein being fused with a tandem repeat of M2e sequences from a virus subtype A(H5) or a consensus sequence thereof. In another particular aspect, the vaccine or immunogenic composition comprises a recombinant HA1 from a A(H7) virus subtype and a self-assembling protein being fused with a tandem repeat of M2e sequences from a A(H7) virus subtype or a consensus sequence thereof. In an additional particular aspect, the vaccine or immunogenic composition comprises a recombinant HA1 from a A(H9) virus subtype and a selfassembling protein being fused with a tandem repeat of M2e sequences from a A(H9) virus subtype or a consensus sequence thereof. The recombinant HA1 and the tandem repeat of M2e sequences can be as detailed above.

[0234] In another particular aspect, the vaccine or immunogenic composition as disclosed herein is for use for immunizing horse against an equine influenza A, especially H3N8. In this context, the vaccine or immunogenic composition comprises a recombinant HA1 from an equine H3N8 virus and a selfassembling protein being fused with a tandem repeat of M2e sequences from a H3N8 virus or a consensus sequence thereof, in particular as detailed above.

[0235] In a particular aspect, the vaccine or immunogenic composition against an influenza virus is selected for having a recombinant HA1 having at least 80, 85, 90, or 95% identity with the sequence of the HA1 domain of the target virus.

[0236] The effect amount of the vaccine or immunogenic composition can be within the range of 0.001 to 1000 mg per dose or of 0.01 to 500 mg per dose or of 0.01 to 100 mg per dose, optionally within a range of 0.01 to 1, 5 or 10 mg per dose.

[0237] Definitions "consists of'," "consists essentially of" or "substantially comprises": a peptide or protein described herein as comprising a particular sequence should be understood as also describing a peptide or protein consisting of that sequence, unless otherwise stated or clearly contradicted by context. By "consists essentially of" is intended that the peptide or protein consists of that sequence, but it may also include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 substitutions, additions or deletions. In particular, by "essentially consist in", it may be intended that the peptide may include 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional amino acids at the N- or C-terminal end and 1, 2, or 3 substitution(s), deletion(s), or addition(s).

[0238] The use of "sequence identity" means that the same nucleotides or amino acid residues are found within the variant sequence and a reference sequence when a specified, contiguous segment of the nucleotide sequence, or amino acid sequence of the variant is aligned and compared to the nucleotide sequence or amino acid sequence of the reference sequence. Methods for sequence alignment and for determining identity between sequences are well known by those skilled in the art. With respect to optimal alignment of two nucleotide sequences, the contiguous segment of the variant nucleotide sequence may have additional nucleotides or deleted nucleotides with respect to the reference nucleotide sequence. Likewise, for purposes of optimal alignment of two amino acid sequences, the contiguous segment of the variant amino acid sequence may have additional amino acid residues or deleted amino acid residues with respect to the reference amino acid sequence. The contiguous segment used for comparison to the reference nucleotide sequence or reference amino acid sequence will comprise at least 20 contiguous nucleotides, or amino acid residues, and may be 30, 40, 50, 100, or more nucleotides or amino acid residues. Corrections for increased sequence identity associated with inclusion of gaps in the variant's nucleotide sequence or amino acid sequence can be made by assigning gap penalties. Methods of sequence alignment are well known in the art. The determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, percent identity of an amino acid sequence can be determined using the Smith-Waterman homology search algorithm using an affine 6 gap search with a gap open penalty of 12 and a gap extension penalty of 2, BLOSUM matrix 62. Alternatively, percent identity of a nucleotide sequence is determined using the Smith-Waterman homology search algorithm using a gap open penalty of 25 and a gap extension penalty of 5. Such a determination of sequence identity can be performed using, for example, the DeCypher Hardware Accelerator from TimeLogic Version G. The Smith-Waterman homology search algorithm is taught in Smith and Waterman (1981) Adv. Appl. Math 2:482-489. Alternatively, the alignment program GCG Gap (Wisconsin Genetic Computing Group, Suite Version 10.1) using the default parameters may be used. The GCG Gap program applies the Needleman and Wunch algorithm and for the alignment of nucleotide sequences with an open gap penalty of 3 and an extend gap penalty of 1 may be used. Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul (1990) Proc. Natl. Acad. Sci. USA 87:2264, modified as in Karlin and Altschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990) J. Mol. Biol. 2 / 5:403. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength 12, to obtain nucleotide sequences having sufficient sequence identity. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences having sufficient sequence identity. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSLBIast can be used to perform an iterated search that detects distant relationships between molecules. See Altschul et al. (1997) supra. When utilizing BLAST, Gapped BLAST, and PSLBIast programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov. Another non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller (1988) CABIOS 4: 11-17. Such an algorithm is incorporated into the ALIGN program (version 2.0), which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.

[0239] "Recombinant" refers to a nucleic acid construct, a vector and a protein produced by genetic engineering.

[0240] Amino acids: The amino acid sequences defined herein are represented by a one-letter symbol as shown below: A, Ala, (alanine); R, Arg, (arginine); N, Asn, (asparagine); D, Asp, (aspartic acid); C, Cys, (cysteine); Q, Gin, (glutamine); E, Glu, (glutamic acid); G, Gly, (glycine); H, His, (histidine); I, He, (isoleucine); L, Leu, (leucine); K, Lys, (lysine); M, Met, (methionine); F, Phe, (phenylalanine); P, Pro, (proline); S, Ser, (serine); T, Thr, (threonine); W, Trp, (tryptophan); Y, Tyr, (tyrosine); and V, Vai, (valine).

[0241] By "adjuvant", it is meant a compound or a mixture of compounds which is / are able to increase the immune response generated by the peptides and direct this immune response, for instance towards a CTL (Cytotoxic T lymphocyte) response. They may act either directly on the immune system, or indirectly, by enabling the suitable delivery of the peptides to the immune system.

[0242] The term "administering in ovo" or "in ovo administration," as used herein, unless otherwise indicated, means administering a vaccine or immunogenic composition to a bird egg containing a live, developing embryo by any means of penetrating the shell of the egg and introducing the vaccine or immunogenic composition. The terms "poultry" and "poultry subjects" as used herein, are intended to include males and females of any avian or bird species, especially, domesticated species, and in particular are intended to encompass poultry which are commercially raised for eggs, meat or as pets. Accordingly, the terms "poultry" and "poultry subject" encompass chickens, turkeys, ducks, geeses, quails, pheasants, parakeets, parrots, cockatoos, cockatiels, ostriches, emus and the like. Commercial poultry includes broilers and layers, which are raised for meat and egg production, respectively.

[0243] The term "effective amount" as used herein refers to an amount effective to achieve an intended purpose, especially to induce an immunological response. The term "immunogenically effective amount," as used herein, unless otherwise indicated, means an amount or dose of a composition sufficient to induce an immune response in the treated subjects that is greater than the inherent immunity of non-inoculated subjects. An immunogenically effective amount in any particular context can be routinely determined using methods known in the art.

[0244] The term "immunogenic" refers to the ability of a polypeptide to elicit an immunological response in a subject.

[0245] An "immunological response" includes but is not limited to one or more of the following effects: the production or activation of antibodies, B cells, helper T cells, suppressor T cells, and / or cytotoxic T cells and / or gamma-delta T cells, directed specifically to an antigen or antigens included in the immunogenic composition of the invention. Preferably, the host will display either a protective immunological response or a therapeutically response.

[0246] The disclosure of any or all references cited in the present application is incorporated herein by reference.

[0247] EXAMPLES

[0248] Results

[0249] Biochemical and biophysical properties of NR-3M2e and NF-3M2e

[0250] The inventors evaluated the effect of the supplementation of nanoparticles incorporating M2e antigens with an HA1 subunit vaccine in the cross-protection against HPAI H5N1 virus challenge in chickens. To this end, chimeric NR-3M2e and NF-3M2e were produced and their physicochemical characteristics were evaluated using previous protocols (Herve et al. J Virol (2014) 88(l):325-38. Epub 2013 / 10 / 25. doi: 10.1128 / JVI.01141-13; Tran et al. J Gen Virol (2007) 88(Pt l):196-206. Epub 2006 / 12 / 16. doi: 10.1099 / vir.0.82282-0; Calzas et al. Front Immunol (2021) 12:772550. Epub 2021 / 12 / 07. doi: 10.3389 / fimmu.2021.772550 ; Lamontagne et al. Adv Healthc Mater (2023):e2300224. Epub 2023 / 04 / 09. doi: 10.1002 / adhm.202300224). The purified NR-3M2e and NF-3M2e preparations appeared as a single band at the predicted molecular weight by SDS-PAGE and Coomassie blue staining (Figures 1A and ID). Concerning NR-3M2e, Western blotting using specific antibodies validated the identity and antigenicity of preparation (Figure IB). NR-3M2e exhibited ring-like morphological features by TEM (Figure 1C), and DLS analysis indicated that the preparation was mainly composed of a homogenous population with a hydrodynamic radius of around 20 nm.

[0251] The self-assembly and morphology of the purified NF-3M2e were analyzed by CD spectroscopy, ThT and ANS fluorescence spectroscopy, AFM, and TEM. CD spectroscopy indicated NF-3M2e being largely unstructured immediately after its purification (0 h spectrum). However, after sufficient assembly time (24 h and 48 h), NF-3M2e adopted structural conformations and displayed a characteristic minimum at ~218 nm and maximum at ~197 nm, corresponding to a p-sheet-rich secondary structure. The NF-3M2e structural morphology was also probed by measuring the fluorescence intensity of ThT, a dye commonly used to probe cross- -sheet quaternary structure (Groenning et al. J Chem Biol (2010) 3(1):1-18. Epub 2009 / 08 / 21. doi: 10.1007 / sl2154-009-0027- 5). The ThT fluorescence emission increased sharply after a self-assembly period of 24h, suggesting the polymerization of freshly purified NF-3M2e from a soluble to an assembled state. Moreover, the ANS dye, whose fluorescence quantum yield increases upon binding to protein hydrophobic domains, revealed the formation of accessible hydrophobic pockets upon nanofilament formation (at 24 h), which were absent in the monomeric / pre-fibrillar NF-3M2e solution (0 h). TEM (Figure IE) analyses of NF-3M2e revealed the formation of a dense network of fibrilla assemblies at 24 h of incubation. The long and interwind NF-3M2e fibrils had widths in the order of 2.5-5 nm. These observations supported the typical morphological heterogeneities of CsgA, in accordance with previous studies (Wang et al. J Biol Chem (2007) 282(6):3713-9. Epub 2006 / 12 / 14. doi: 10.1074 / jbc.M609228200 ; Nguyen et al. Common Biol (2021) 4(1):939. Epub 2021 / 08 / 07. doi: 10.1038 / s42003-021-02466-7; Zottig et al. Small (2019) 15(33):el901806. Epub 2019 / 07 / 04. doi: 10.1002 / smll.201901806 ; Dueholm et al. Biochemistry (2011) 50(39):8281-90. Epub 2011 / 09 / 01. doi: 10.1021 / bi200967c).

[0252] The co-administration of NR-3M2e or NF-3M2e with HA1 protein triggered M2e-and HAl-specific antibody responses in chickens

[0253] Firstly, the inventors evaluated the immunogenicity of the vaccine formulations. Chickens received three intramuscular administrations, at 2-week intervals, of NR-3M2e or NF-3M2e admixed with HA1 protein, or each single component alone. Vaccine antigens were emulsified with Emulsigen’-P, an oil-in-water adjuvant used in veterinary vaccination. The HAl-specific antibody responses were assessed by an HI assay (Figure 2) and competitive ELISA (Figure 3). Chickens immunized with HA1 protein alone or in combination with NF-3M2e exhibited significant HI titers two weeks after the first boost, whereas chickens immunized with HA1 admixed with NR-3M2e generated significant HI titers after the second boost. The mean HI titers of chickens immunized with HA1 protein or [NF- 3M2e + HA1] reached ~5 Iog2, whereas the co-administration of NR-3M2e with HA1 protein slightly reduced the magnitude of the response (mean HI titers of 4 logz) (Figure 2). The competitive ELISA confirmed that chickens immunized with HA1, [NR-3M2e + HA1] or [NF-3M2e + HA1] developed significant HAl-specific antibody responses at day 42 post-primary vaccination (Figure 3). Finally, chickens immunized with NR-3M2e or NF-3M2e administered alone or in combination with HA1 developed significant M2e-specific IgG at day 42 post-primary vaccination, and there was no difference in the magnitude of the response whether HA1 was incorporated in the formulation or not (Figure 4). In conclusion, chickens immunized with HA1 protein in combination with NR-3M2e or NF-3M2e developed significant HA1- and M2e-specific antibody responses, and the coadministration of the two IAV antigens did not notably hinder the induction of the response directed against each of the antigens.

[0254] The supplementation of NR-3M2e or NF-3M2e with HA1 protein enhanced the clinical and virological protection of chickens against heterologous HPAI H5N1 virus challenge

[0255] Two weeks after the third immunization, chickens were experimentally infected with a heterologous HPAI H5N1 virus and monitored daily for mortality and clinical signs for up to 21 days after challenge (Figure 5). The challenge virus belonged to clade 2.3.4.4c, whose HA gene was derived from the Eurasian Gs / GD-like H5N8 lineage (Berhane et al. Sci Rep (2016) 6:23380. Epub 2016 / 03 / 19. doi: 10.1038 / srep23380). The HA1 vaccine protein was derived from a North American lineage LPAI H5N1 virus and shared only 81% amino acid sequence identity s with the HA1 sequence of the challenge virus, allowing to evaluate the broad-protective potential of our vaccine formulations. Unvaccinated chickens (PBS group) and chickens immunized with NR-3M2e or NF-3M2e started to die from 2 to 3 days post-infection, with 100% mortality at day 7 post-infection. Forty percent of chickens immunized with HA1 protein survived the challenge, and no clinical signs were observed in the surviving chickens. Finally, all chickens immunized with HA1 protein in combination with NR- 3M2e or NF-3M2e survived the infection, with no protracted clinical signs. The virus was detected in the oropharyngeal and cloacal swabs of all unvaccinated chickens and in chickens immunized with NR-3M2e or NF-3M2e (Table 2). Viral RNA was detected in the swabs of all dead chickens and all surviving chickens with the exception of three surviving chickens in the HA1 group. By severe contrast, no viral RNA was detected in both cloacal and oropharyngeal swabs of birds immunized with [NR-3M2e + HA1] or [NF-3M2e + HA1] during the course of the experiment (Table 2). Thus, the supplementation of NR-3M2e or NF-3M2e with HA1 protein was essential to completely prevent chickens from clinical disease, mortality, and virus shedding after a challenge with heterologous

[0256] H5N1 virus.

[0257] The supplementation of NR-3M2e or NF-3M2e with HA1 protein promoted protection against histological lesions after heterologous HPAI H5N1 virus challenge in chickens

[0258] The protective potential of the vaccine formulations against lung, brain, and heart tissue lesions associated with H5N1 virus challenge was evaluated by histological and immunohistochemical analyses 3 days post-challenge from surviving but euthanized chickens which received PBS, NR- 3M2e, or NF-3M2e preparations. Infections with HPAI H5N1 viruses usually result in moderate pulmonary lesions in chickens (Y. K. Kwon, D. E. Swayne (2010). Avian Diseases, Vol. 54, No. 4 (December 2010), pp. 1260-1269. https: / / www.jstor.org / stable / 409830581. However, in this study, chickens in the PBS group exhibited obvious lung lesions, consisting of necrosis and hypercellularity (Figure 6A). Mild increase in cellularity due to inflammatory cell infiltration was observed in the NR- 3M2e (Figure 6C) and NF-3M2e (Figure 6E) groups. By contrast, no lung lesions were noticeable in the HA1 (Figure 6G), [NR-3M2e + HA1] (Figure 61), or [NF-3M2e + HA1] (Figure 6K) groups at 19- and 21-days post-challenge respectively. The virus antigen load was scanty detected in PBS and HA1 groups (Figures 6B and 6H), whereas it presented an unusual extensive spread in the lungs of chickens vaccinated with NR-3M2e or NF-3M2e (Figures 6D and 6F). No virus antigen was visible in the lungs of chickens immunized with the supplemented vaccine formulations (Figures 6J and 6L). Histological lesions in the brain of the PBS group (Figure 7A) were similar to that in the NR-3M2e (Figure 7C), NF-3M2e (Figure 7E), or HA1 (Figure 7G) groups, and included areas of multifocal to focal necrotizing encephalitis with perivascular cuffing of mononuclear inflammatory cell infiltrates. By contrast, no traceable microscopic brain lesions were observed in the group immunized with [NR-3M2e + HA1] (Figure 71) at 21 days post-challenge. Although the extent of the virus antigen load varied between the groups, the virus was detected in the brain of the PBS (Figure 7B), NR- 3M2e (Figure 7D), NF-3M2e (Figure 7F), and HA1 (Figure 7H) groups. No viral antigen was detected in the [NR-3M2e + HA1] group (Figure 7J). Finally, whereas necrotic foci were observed in the heart tissue of chickens immunized with HA1 (Figure 8G), no cardiac lesions were detected in the PBS group (Figure 8A) or in chickens immunized with the M2e-based nanoparticles administered alone (Figures 8C and 8E) or in combination with HA1 (Figures 81 and 8K). Remarkable amounts of virus antigen were detected in the cardiac fibers in all groups of chickens (Figure 8B, 8D, 8F and 8H) with the exception of chickens immunized with the supplemented vaccine formulations, which did not harbor any traces of virus antigen in the cardiac tissue (Figures 8J and 8L). To conclude, the supplementation of NR-3M2e or NF-3M2e with HA1 protein prevented virus replication and subsequent pathological alterations in the lung, brain, and heart tissues after infection of chickens with a heterologous HPAI H5N1 virus. Table 1: Experimental groups and vaccine formulations

[0259] Groups Vaccine formulations Synonymous

[0260] 1 PBS + Emulsigen’-P PBS

[0261] 2 HA1 + Emulsigen’-P HA1

[0262] 3 NF-3M2e + HA1 + Emulsigen’-P [NF-3M2e + HA1]

[0263] 4 NR-3M2e + HA1 + Emulsigen’-P [NR-3M2e + HA1]

[0264] 5 NF-3M2e + Emulsigen’-P NF-3M2e

[0265] 6 NR-3M2e + Emulsigen’-P NR-3M2e

[0266] Table 2. Frequency of lAV-positive swabs in vaccinated and unvaccinated chickens after heterologous HPAI H5N1 virus challenge dpc, days post-challenge; (-), all animals died. Materials and Methods

[0267] Viruses and vaccine antigens

[0268] A / chicken / BC / FAV-002 / 2015 HPAI H5N1 (2.3.4.4c) virus isolated from a backyard layer chicken flock near Chilliwack, British Columbia (National Centre for Foreign Animal Disease [NCFAD], Winnipeg, Canada) was used in the heterologous challenge experiment (Berhane et al. Sci Rep (2016) 6:23380. Epub 2016 / 03 / 19. doi: 10.1038 / srep23380). The virus was inoculated via the allantoic route in 9- day-old specific pathogen free (SPF) embryonated chicken eggs (Canadian Food Inspection Agency [CFIA] Fallowfield Laboratory, Ottawa, Canada) and incubated at 37°C. The allantoic fluid washarvested and the virus titer was determined in Madin-Darby Canine Kidney (MDCK) cells and expressed as PFU / mL. The M2e sequence MSLLTEVETPTRTEWECRCSDSSD (SEQ ID NO: 1) was derived from A / chicken / BC / FAV-002 / 2015 HPAI H5N1 virus. The two cysteine residues of the M2e sequence (amino acids 17 and 19) were substituted to two serine residues (SEQ ID NO: 2) to avoid formation of a disulfide bond that could impair the assembly of the nanoparticles. The recombinant HA1 protein (SEQ ID NO: 3) was produced from North American lineage A / turkey / Ontario / 6213 / 1966 H5N1 LPAI virus.

[0269] Production of HA1 protein

[0270] The expression and purification of soluble recombinant HA1 protein was performed as described (Yang et al. J Virol Methods (2009) 162(l-2):194-202. Epub 2009 / 08 / 25. doi: 10.1016 / j.jviromet.2009.08.006). Briefly, the codon-optimized HA1 gene (SEQ ID NO: 4) for expression in insect cells was cloned into a pAB-bee™-FH vector (AB Vector, San Diego, CA) through GenScript services (GenScript USA Inc, Piscataway, NJ). The vector was purified and co-transfected with linearized baculovirus vector DNA and ProFold™-ERl (AB Vector) into Spodoptera frugiperda (Sf9) insect cells to generate recombinant baculovirus containing the HA1 gene. The baculovirus was plaque-purified and sequenced for HA1 gene validation. Sf9 cells were infected with the baculovirus and incubated for 72 h at 27°C under agitation. Cells were pelleted and lysed with l-PER insect cell protein extraction reagent (Pierce Biotechnology, Rockford, IL). The supernatant was purified using Ni-NTA bead resin and the expression of the recombinant protein was confirmed via Western blotting using a 6x histidine-specific monoclonal antibody (Novagen, Billerica, MA).

[0271] Production of NR-3M2e

[0272] The pET-N-Sac plasmid was obtained from the pET-N plasmid, which contained the full-length coding sequence of the N protein derived from RSV Long strain (ATCC VR-26, GenBank accession number AY911262.1), by introducing a Sad restriction enzyme site in frame and at the C-terminal end of the N sequence (Herve et al. J Virol (2014) 88(l):325-38. Epub 2013 / 10 / 25. doi: 10.1128 / JVI.01141-13). To obtain the pET-N-3M2e plasmid containing three repetitions of the M2e sequence, the synthesized pUC57-3M2e plasmid (ProteoGenix, Schiltigheim, France) was used as a template to amplify the 3M2e sequence by PCR using the Phusion high-fidelity DNA polymerase (Thermo Fisher Scientific, Waltham, MA, USA) with gene-specific primers flanked with Sacl / Sacl (forward primer / reverse primer) sites. The PCR-amplified 3M2e sequence was then digested by Sad and inserted into the pET-N-Sac plasmid using the same restriction site. NR-3M2e were expressed and purified as previously described (Herve et al. J Virol (2014) 88(l):325-38. Epub 2013 / 10 / 25. doi: 10.1128 / JVI.01141-13; Tran et al. J Gen Virol (2007) 88(Pt l):196-206. Epub 2006 / 12 / 16. doi: 10.1099 / vir.0.82282-0; Calzas et al. Front Immunol (2021) 12:772550. Epub 2021 / 12 / 07. doi: 10.3389 / fimmu.2021.772550). The identity and antigenicity of NR-3M2e were validated by SDS- PAGE followed by Western blot assay with N- and M2e-specific antibodies. The structural integrity of the NR was confirmed by dynamic light scattering and transmission electron microscopy (TEM), as described previously (Calzas et al. Front Immunol (2021) 12:772550. Epub 2021 / 12 / 07. doi: 10.3389 / fimmu.2021.772550).

[0273] Amino acid sequence of NR-3M2e: SEQ ID NO: 6

[0274] Coding sequence of NR-3M2e: SEQ ID NO: 9

[0275] Production of NF-3M2e

[0276] The pET-29a (+) plasmid containing three repetitions of the M2e sequence fused in frame at the N- terminal end of the E. coll CsgA sequence (GenBank accession number WP124055573) was provided by GenScript (Piscataway, NJ). To express NF-3M2e, E. coll NiCo21(DE3) competent cells (New England Biolabs Inc., Ipswich, MA, USA) transformed with the pET-3M2e-CsgA plasmid were grown at 37°C under agitation (225 rpm) in LB medium containing 50 pg / mL of kanamycin. Protein expression was induced by adding 1 pM IPTG to mid-log-phase cells (OD60onm ~1) and subsequent incubation for 1 h at 37°C. Cells were then harvested by centrifugation at 5,000 x g for 15 min and stored at -80°C. All purification steps were performed under denaturing conditions with 8 M guanidine hydrochloride (GdnHCI) to prevent uncontrolled aggregation, until the assembly was precisely initiated by switching from 8 M GdnHCI to PBS buffer. Thawed cell pellet was resuspended in 8 M GdnHCI, PBS pH 7.2 and sonicated on ice using six cycles of 20 s. Clarification of lysate was achieved by centrifugation at 30,000 x g for 30 min at 4°C. The protein of interest was purified over a Ni-charged Profinity™ metal ion affinity chromatography (IMAC) resin (Bio-Rad Laboratories, Inc., Hercules, CA, USA) in a gravity flow column equilibrated in 8 M GdnHCI, PBS pH 7.2. The column was washed with 8 M GdnHCI, PBS pH 7.2 supplemented with 12.5 mM imidazole, and protein was eluted using 250 mM imidazole in PBS pH 7.4. A final desalting step was performed using a Sephadex G-25 fine desalting column in PBS pH 7.4. NF-3M2e was analyzed by SDS-PAGE, and the (supra)structural characterization of the NF was performed by circular dichroism (CD) spectroscopy, fluorescence spectroscopy, atomic force microscopy (AFM) and TEM, as described below.

[0277] Amino acid sequence of NF-3M2e: SEQ ID NO: 8

[0278] Coding sequence of NF-3M2e: SEQ ID NO: 10

[0279] Fluorescence spectroscopy

[0280] Thioflavin T (ThT; 1 mM) was added to NF-3M2e sample to reach a final ThT concentration of 40 pM, and fluorescence emission was recorded by a PTI QuantaMaster™ (Horiba Ltd., Kyoto, Japan) spectrofluorometer from 450 nm to 550 nm at an excitation wavelength of 440 nm. For 8-anilino-l- naphthalenesulfonic acid (ANS), the final concentration of ANS was 450 pM and the fluorescence emission was measured between 385 and 550 nm with a constant excitation at 370 nm. Fluorescence was measured in ultramicro 10 mm length quartz cells. Data are expressed as arithmetic means with the standard error of the mean (± SEM) of at least three individual experiments performed in triplicate.

[0281] Atomic force microscopy

[0282] NF-3M2e samples at 0, 24, and 48 h of self-assembly were diluted in 1% acetic acid and immediately applied to freshly cleaved mica. The mica was washed twice with deionized water and air-dried for 24 h. Samples were analyzed using a Veeco / Bruker Multimode AFM as described elsewhere (Nguyen et al. Commun Biol (2021) 4(1):939. Epub 2021 / 08 / 07. doi: 10.1038 / s42003-021-02466-7; Zottig et al. Small (2019) 15(33):el901806. Epub 2019 / 07 / 04. doi: 10.1002 / smll.201901806). The scanner had an upper limit of 10 pm x 10 pm. The images were recorded at 1 line / s for a total resolution of 512 lines / scan. The probe was a silicon nitride (SizNa) cantilevered tip with an apex measuring 2-12 nm in diameter.

[0283] Transmission electron microscopy

[0284] NF-3M2e samples at 0, 24, and 48 h of self-assembly were diluted in 20 mM Tris-HCI buffer (pH 7.4) before being applied to glow-discharged carbon films on 400 mesh copper grids. After adsorption, samples were negatively stained with 1.5% uranyl formate for 1 min and air-dried for 15 min. Images were recorded using a FEI Tecnai 12 BioTwin microscope as described elsewhere (Nguyen et al. Commun Biol (2021) 4(1):939. Epub 2021 / 08 / 07. doi: 10.1038 / s42003-021-02466-7; Zottig et al. Small (2019) 15(33):el901806. Epub 2019 / 07 / 04. doi: 10.1002 / smll.201901806). Electron micrographs of NR-3M2e were acquired using a CM12 TEM (Royal Philips Electronics, Amsterdam, Netherlands) at 80 kV excitation voltage. Samples at 0.05-1 mg / mL were applied onto an airglow- discharged carbon-coated 200-mesh copper grid and stained with a 2% uranyl acetate aqueous solution. Animal experiments

[0285] All animal experiments were conducted after approval by the Canadian Science Centre for Human and Animal Health institutional guidelines for animal care. One-day-old SPF white Leghorn layer chickens (CFIA, Ottawa, Canada) were housed in heated biosafety level 3 (BSL-3) plus animal cubicles at the NCFAD on a floor system. Feed and water were provided ad libitum and the animal cubicles were enriched with cupboards and porches. When chickens were 21 days old, they were randomly assigned to six experimental groups (n = 14-15 / group) as indicated in Table 1 and kept in a separate cubicle. The vaccine formulations consisted of HA1 protein (15 pg) admixed with NF-3M2e (50 pg) or with NR-3M2e (50 pg), or each single component administered alone. The last group served as unvaccinated PBS group. Before administration, vaccine antigens were mixed with Emulsigen’-P (MVP Adjuvants, Phibro Animal Health, NJ) for 2 h under gentle agitation. Three vaccinations were applied via the intramuscular route. The two booster vaccinations were applied two weeks apart. On days 14, 28, and 42 post-primary vaccination, sera were collected from each bird. Chickens were challenged at day 42 post-primary vaccination with 2.3 x 102PFU of heterologous A / chicken / BC / FAV-002 / 2015 virus in 100 pL endotoxin-free PBS via the intranasal route (50 pL / nostril). They were observed daily for clinical signs and scored as follows: 1) clinically normal - chickens fed, drank and moved well; 2) mild clinical disease - chickens showed either one or a combination of the following clinical signs: depression, mouth breathing, diarrhea, mild neurological signs, wing drooping, facial oedema - but moved freely, ate and drank; 3) severely sick - chickens showed any of the above clinical signs, were unable to move and drink- chickens euthanized and recorded as dead birds; 4) dead - chickens died acutely with no overt clinical signs. Moreover, oropharyngeal and cloacal swabs were collected on days 3, 5, 7, 10, and 14 after challenge. At day 3 (groups 1, 5, and 6), day 19 (group 2) and day 21 (groups 3 and 4) post-challenge, chickens (n = 1) were euthanized and tissue samples from brain, lung, and heart were collected and fixed in 10% neural-buffered formalin for histopathological / immunohistochemistry assessments.

[0286] The HI assay was performed according to OIE manual standardized protocol. Briefly, two-fold serially diluted serum samples from each chicken were incubated with four HA units of the A / Turkey / Ontario / 6213 / 1966 (H5N1) virus in a V-bottom plates (Costar, Cole-Parmer Canada, Quebec, Canada) and incubated for 30 min. Subsequently, 0.5% chicken red blood cells were added and incubated for another 30 min. The hemagglutination inhibition (HI) titers were scored as the reciprocal of the highest serum dilution resulting in 100% inhibition of chicken red blood cells from hemagglutination. Enzyme-linked immunosorbent assay (ELISA)

[0287] HAl-specific antibody response in serum samples was evaluated by competitive ELISA as described previously (Yang et al. J Virol Methods (2009) 162(l-2):194-202. Epub 2009 / 08 / 25. doi: 10.1016 / j.jviromet.2009.08.006) with modifications. Briefly, 96-well flat-bottom polystyrene plates (Nunc MaxiSorp™; Nunc, Roskilde, Denmark) were coated with recombinant HA1 protein in carbonate-bicarbonate buffer 0.1 M (pH 9.6). After blocking, diluted sera (1:10) were added to the plates right before the addition of F73-H5-3 monoclonal antibody (1:300) and subsequently incubated for 1 h at 37°C with agitation. Each serum was assayed in duplicate. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (1:2000) (Jackson) was added and incubated at 37°C for 1 h with agitation. Tetramethylbenzidine (TMB) substrate was added and plates were further incubated for 15 min at room temperature with shaking in the dark. Enzyme reaction was stopped with 2 M H2SO4, and absorbance was read at 405 nm with an ELISA plate reader. Results are expressed as percent inhibition (PI) [l-(ODChicken serum+Mab / ODMab)*100], Values above 40% inhibition are considered positive.

[0288] The presence of M2e-specific antibodies in serum samples was evaluated by indirect ELISA in 96- well flat-botom microtiter plates (Nunc MaxiSorp™). The plates were coated with synthetic M2e peptide (200 ng / well in 0.1 M carbonate-bicarbonate buffer [pH 9.6]) overnight at 4°C. Plates were washed five times with PBS containing 0.05% Tween 20 (PBS-T), blocked in 0.5% BSA for 1 h at room temperature, and diluted sera (1:3200) were added and incubated at 37°C for 1 h. Negative control wells only contained PBS-T. Plates were washed and then incubated with HRP-conjugated donkey anti-chicken IgG (1:5000 in PBS-T) at 37°C for 1 h under agitation. TMB substrate was then added and plates were further incubated for 10 min at room temperature in the dark with gentle agitation. The enzymatic reaction was stopped with 1% 2M H2SO4. The plates were read with an ELISA plate reader at an absorbance of 450 nm. The results were expressed as OD values whose background (twice the OD45onm of negative wells) was subtracted.

[0289] Determination of viral RNA real-time

[0290] Total RNA was extracted from the oropharyngeal and cloacal swabs using the MagMAX 1835 Nucleic Acid Isolation Kit (ThermoFisher Scientific, Waltham, MA, USA). The spiked Enteroviral armoured RNA (ARM-ENTERO; Asuragen) was used as an exogenous extraction and reaction control. Quantitative real-time polymerase chain reaction (qRT-PCR) targeting the matrix protein 1 gene of influenza A virus was used to evaluate viral RNA shedding frequency as a measure of challenge virus replication as described previously (Spackman et al. J Clin Microbiol (2002) 40(9):3256-60. Epub 2002 / 08 / 31. doi: 10.1128 / JCM.40.9.3256-3260.2002). Histopathology and immunohistochemistry

[0291] Tissues fixed in 10% neutral phosphate buffered formalin were sectioned at 5 pm and stained with haematoxylin and eosin (HE) for histopathologic analysis. For immunohistochemistry (IHC), paraffin tissue sections were quenched for 10 min in aqueous 3% H2O2and then pretreated with proteinase K for 15 min for epitope retrieval. A mouse monoclonal antibody specific for influenza A nucleoprotein (F26NP9) (43) was applied on the sections at a dilution of 1:10,000 for 1 h. The microscopic sections were then visualized using a horseradish peroxidase labelled polymer, Envision®+system (anti-mouse) (Dako, USA), and reacted with the chromogen diaminobenzidine (DAB). The sections were then counter stained with Gill's hematoxylin. The virus antigen load and distribution in the tissue section were visualized under the microscope and the intensity of coloration (brown) in the tissues of the different groups were qualitatively evaluated.

[0292] Statistical data analysis

[0293] The log-rank (Mantel-Cox) test was used to compare the survival rates between groups. Otherwise, differences between the experimental groups were analyzed for significance using the Mann- Whitney rank sum test. All analyses were done using the Sigma Plot system vll.O (Systat Software, San Jose, CA, USA). A P value < 0.05 was considered statistically significant.

Claims

CLAIMS1- A vaccine composition comprising- a recombinant hemagglutinin HA1 from an influenza A virus; and- a protein carrierfused with an ectodomain of a matrix protein 2 (M2e domain) or a tandem repeat thereof, optionally through a peptide linker, wherein the M2e domain comprises a sequence(M / abs ) S L L T E V E T ( P / L ) ( T / I ) ( K / R ) ( T / N / K / S ) ( E / G ) W( E / G ) ( C / S ) ( R / N / K ) ( C / S ) ( S / N ) ( D / V / G ) S S D (SEQ ID NO: 12).2- The vaccine composition according to claim 1, wherein the protein carrier is selected from the group consisting of CRM197 (nontoxic variant of diphtheria toxin isolated from Corynebacterium diphtheriae C7 (P197), tetanus toxoid (TT), a Pseudomonas aeruginosa protein, human serum albumin (HSA), bovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), diphtheria toxin fragment B (DTFB), DTFB C8, diphtheria toxoid (DT), fragment C of TT, pertussis toxoid, cholera toxoid, E. coli heat-labile toxin, E. coli heat-stable toxin, meningococcal outer membrane protein complex (OMPC), Haemophilus influenzae protein D (HiD), and exotoxin A from Pseudomonas aeruginosa.3- The vaccine composition according to claim 1, wherein the protein carrier is a self-assembling protein able to form a nanoparticle, optionally selected from the group consisting of a nucleoprotein N of respiratory syncytial virus (RSV), CsgA (Curli-specific gene A), an amyloid protein such as CspA (E coli cold shock protein A), ferritin, Soc (Small Outer Capsid protein) of phage T4, hepatitis B virus core antigen (HBc), bacteriophage AP205 coat protein, coat protein of bacteriophage QP, a dodecameric DNA binding protein from starved cells (Dps) of bacteria or archaea such as Dsp of E. coli, Brucella outer membrane protein BP26, capsid protein of PCV2 (porcine circovirus type 2), protrusion domain of the capsid protein of norovirus, a self-assembling an amyloid protein p- peptide such as a ten-mer self-assembling sequence (110) derived from an amyloidogenic peptide (SNNFGAILSS, SEQ ID NO: 36), an antibody such as anti-Clec9A monoclonal antibody, CTA1-DD (ADP- ribosylating Al subunit of cholera toxin CT linked to a dimer of the D-fragment of Staphylococcus aureus protein A), bacterial flagellin such as flagellin of Bacillus subtilis or Salmonella serovar enterica typhimurium (S. typhimurium), Qll peptide (QQKFQFQFEQQ, SEQ ID NO: 37), lumazinesynthase, especially Aquifex aelocus (A. aelocus) lumazine synthase, encapsulin, especially from T maritime / , and small Heat shock protein (sHsp) 16.5, especially from Methanocaldoccus jannaschii, a superfolder green fluorescent protein (sfGFP), bacteriophage P22, E2p, especially from Bacillus stearothermophilus (B. stearothermophilus , 13-01 derived from Thermotoga maritima's 2-keto-3- deoxy-6-phosphogluconate (KDPG) aldolase, especially from T. maritima, 153-50, IMX313, SAP, E2, Coil29, FliC, latency-associated nuclear antigen (LANA) from the Kaposi's sarcoma-associated herpesvirus (KSHV), coat of a plant virus such as nepovirus coat virus, in particular GFLV (Grapevine fanleaf virus), hydrophobin, bacteriophage T7 endonuclease, A beta 1-40, Sup35p of S. cerevisiae, human Pmell7, Agfa Tafi protein, a Chaplin protein, a peptide derived from islet amyloid polypeptide (IAPP), in particular SNNFGAILSS (SEQ. ID NO: 36), and a combination thereof. .4- The vaccine composition according to claim 3, wherein the protein carrier is a N protein of Respiratory syncytial virus (RSV) and the M2e domain or a tandem repeat thereof is fused to the C- terminal end of the N protein.5- The vaccine composition according to claim 4, wherein a tandem repeat of 2 to 4 M2e domains is fused to the C-terminal end of the N protein.6- The vaccine composition according to claim 5, wherein a tandem repeat of 3 M2e domains is fused to the C-terminal end of the N protein.7- The vaccine composition according to claim 3, wherein the protein carrier is curli specific gene A (CsgA) or a fragment thereof and the M2e domain or a tandem repeat thereof is fused to the N- terminal end of CsgA.8- The vaccine composition according to claim 7, wherein a tandem repeat of 2 to 4 M2e domains is fused to the N-terminal end of CsgA.9- The vaccine composition according to claim 7, wherein a tandem repeat of 3 M2e domains is fused to the N-terminal end of CsgA.10- The vaccine composition according to any one of claims 7-9, wherein the protein carrier is CsgA.11- The vaccine composition according to any one of claims 7-9, wherein the protein carrier is a fragment of CsgA comprising R2, R3, R4 and R5 repeat units, or R3, R4 and R5 repeat units, or R4 and R5 repeat units, or R5 repeat unit.12- The vaccine composition according to any one of claims 1-11, wherein the recombinant hemagglutinin HA1 is from an influenza A virus selected from any of the following groups- H1N1, H1N2, H1N3, H1N4, H1N5, H1N6, H1N7, H1N8, H1N9, H2N1, H2N2, H2N3, H2N4,H2N5, H2N6, H2N7, H2N8, H2N9, H3N1, H3N2, H3N3, H3N4, H3N5, H3N6, H3N7, H3N8,H3N9, H4N1, H4N2, H4N3, H4N4, H4N5, H4N6, H4N7, H4N8, H4N9, H5N1, H5N2, H5N3,H5N4, H5N5, H5N6, H5N7, H5N8, H5N9, H6N1, H6N2, H6N3, H6N4, H6N5, H6N6, H6N7,H6N8, H6N9, H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, H7N9, H8N1, H8N2,H8N3, H8N4, H8N6, H8N7, H8N8, H9N1, H9N2, H9N3, H9N4, H9N5, H9N6, H9N7, H9N8,H9N9, H10N1, H10N2, H10N3, H10N4, H10N5, H10N6, H10N7, H10N8, H10N9, H11N1,H11N2, H11N3, H11N4, H11N5, H11N6, H11N6, H11N7, H11N8, H11N9, H12N1, H12N2,H12N3, H12N4, H12N5, H12N6, H12N7, H12N8, H12N9, H13N1, H13N2, H13N3, H13N4,H13N6, H13N7, H13N8, H13N9, H14N2, H14N3, H14N4, H14N5, H14N6, H14N7, H14N8,H15N2, H15N4, H15N5, H15N6, H15N7, H15N8, H15N9, H16N3, H16N8, and H16N9; or- H1N1, H1N2, H2N2, H2N3, H3N2, H3N8, H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N6, H7N7, H7N9, H9N2, H9N6, H9N7, H10N3, and H10N7; or- H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, H5N9, H6N1, H6N2, H6N3, H6N4, H6N5, H6N6, H6N7, H6N8, H6N9, H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, H7N9, H9N1, H9N2, H9N3, H9N4, H9N5, H9N6, H9N7, H9N8, H9N9, H10N1, H10N2, H10N3, H10N4, H10N5, H10N6, H10N7, H10N8, and H10N9; or- H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, H7N9, H9N1, H9N2, H9N3, H9N4, H9N5, H9N6, H9N7, H9N8, and H9N9;- H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, H5N9, H7N1, H7N2, H7N3, H7N4, H7N5, H7N6, H7N7, H7N8, and H7N9;- H5N1, H5N6, H6N1, H7N2, H7N6, H7N9, H9N2, H10N3, H10N7, and H10N8; or- H5N1, H5N2, H5N3, H5N4, H5N5, H5N6, H5N7, H5N8, and H5N9, preferably H5N1, H5N6 and H5N8; or- H5N1, H5N2, H5N6, H5N8, H5N9, H7N1, H7N3, H7N4, H7N7, and H7N9.13- The vaccine composition according to any one of claims 1-12, wherein the recombinant hemagglutinin HA1 is from an avian influenza A virus selected from the group consisting of H5N1, H5N2, H5N6, H5N8, H5N9, H7N1, H7N3, H7N4, H7N6, H7N7, and H7N9.14- The vaccine composition according to claim 13, wherein the recombinant hemagglutinin HA1 is from a H5N1 influenza A virus.15- The vaccine composition according to any one of claims 1-12, wherein the recombinant hemagglutinin HA1 comprises a sequence selected in the group consisting of:SEQ ID NO: 3 or 38 or a sequence having at least 80, 85, 90, or 95 % of identity with SEQ ID NO: 3 or 38;SEQ ID NO: 39 or 40 or a sequence having at least 80, 85, 90, or 95 % of identity with SEQ ID NO: 39 or 40; andSEQ ID NO: 41 or a sequence having at least 80, 85, 90, or 95 % of identity with SEQ ID NO: 41.16- The vaccine composition according to claim 15, wherein the recombinant hemagglutinin HA1 comprises a sequence of SEQ ID NO: 3 or 38 or a sequence having at least 80, 85, 90, or 95% identity with SEQ ID NO: 3 or 38.17- The vaccine composition according to any one of claims 1-16, wherein the M2e domain comprises a sequence selected in the group consisting of:(M / abs) S L L T E V E T (P / L) T (K / R) (T / N / K / S) (E / G) W (E / G)(C / S) (R / N / K) (C / S) (S / N) (D / V / G) S S D (SEQIDNO: 13)(M / abs) S L L T E V E T (P / L) T (K / R) (T / N) (E / G) W (E / G) (C / S)(R / N / K) (C / S) (S / N) (D / V / G) S S D (SEQ ID NO: 14).(M / abs) S L L T E V E T P T R N (E / G) W (E / G) (C / S) (R / K) (C / S)(S / N) D S S D (SEQ ID NO: 15);(M / abs) S L L T E V E T P T R N (E / G) W (E / G) (C / S) (R / K) (C / S) S D S S D (SEQ ID NO: 16);(M / abs) S L L T E V E T P T R (N / T) (E / G) W (E / G) (C / S) R (C / S)S D S S D (SEQ ID NO: 17);(M / abs) S L L T E V E T P T R N (E / G) W E (C / S) (K / R) (C / S) SD S S D (SEQ ID NO: 18);(M / abs) S L L T E V E T P T R N (E / G) W E (C / S) K (C / S) S D SS D (SEQ ID NO: 19);(M / abs) S L L T E V E T P T R T E W E C R C S D S S D (SEQ ID NO:20);(M / abs) S L L T E V E T P T R T E W E S R C S D S S D (SEQ ID NO:21);(M / abs) S L L T E V E T P T R T E W E C R S S D S S D (SEQ ID NO:22);(M / abs) S L L T E V E T P T R T E W E S R S S D S S D (SEQ ID NO:23);(M / abs) S L L T E V E T P T R N E W E C R C S D S S D (SEQ ID NO:24);(M / abs) S L L T E V E T P T R N E W E S R C S D S S D (SEQ ID NO:25);(M / abs) S L L T E V E T P T R N E W E C R S S D S S D (SEQ ID NO:26);(M / abs) S L L T E V E T P T R N E W E S R S S D S S D (SEQ ID NO:27);(M / abs) S L L T E V E T P T R T G W E C R C S D S S D (SEQ ID NO:28);(M / abs) S L L T E V E T P T R T G W E S R C S D S S D (SEQ ID NO:29);(M / abs) S L L T E V E T P T R T G W E C R S S D S S D (SEQ ID NO:30);(M / abs) S L L T E V E T P T R T G W E S R S S D S S D (SEQ ID NO:31);(M / abs) S L L T E V E T P T R N G W E C K C S D S S D (SEQ ID NO:32);(M / abs) S L L T E V E T P T R N G W E S K C S D S S D (SEQ ID NO:33);(M / abs) S L L T E V E T P T R N G W E C K S S D S S D (SEQ ID NO:34); and(M / abs) S L L T E V E T P T R N G W E S K S S D S S D (SEQ ID NO:35).18- The vaccine composition according to any one of claims 1-16, wherein the M2e domain comprises a sequence (M / abs) S L L T E V E T P T R T E W E S R S S D S S D (SEQ ID NO: 23).19- The vaccine composition according to claim 1, wherein the vaccine composition comprises:- a recombinant hemagglutinin HA1 comprising a sequence of SEQ ID NO: 3 or 38 or a sequence having at least 80, 85, 90, or 95% identity with SEQ ID NO: 3 or 38; and- a nanoparticle made of a carrier protein selected from the group consisting of nucleocapsid protein (N) of respiratory syncytial virus (RSV) and curli specific gene A (CsgA), the self-assembling protein being fused with a tandem repeat of 3 M2e domains, wherein the M2e domain comprises a sequence (M / abs) S L L T E V E T P T R T E W E S R S S D S S D (SEQ ID NO: 23).20- The vaccine composition according to any one of claims 1-19, wherein the vaccine composition is in a form suitable for parenteral administration, especially subcutaneous or intramuscular, for in ovo administration or for mucosal administration, especially by oral or nasal route.21- The vaccine composition according to any one of claims 1-20, wherein the composition further comprises an adjuvant.22- The vaccine composition according to any one of claims 1-21 for use against influenza A infection, especially by reducing or preventing the effects of an infection by influenza A virus, the effects being selected in the group consisting of mortality, clinical disease associated with infection by influenza A virus, histopathological lesions associated with infection by influenza A, and influenza A virus shedding.23- The vaccine composition for use according to claim 22 for use against avian influenza A infection in poultry, in particular infection by an influenza A virus selected from the group consisting of H5N1, H5N2, H5N6, H5N8, H5N9, H7N1, H7N3, H7N4, H7N6, H7N7, and H7N9.24- The vaccine composition for use according to claim 22 for use against avian H5N1 influenza A infection in poultry .25- The vaccine composition for use according to claim 22 for use against influenza A infection in horse, in particular infection by H3N8 or H7N7, preferably H3N8.26- Use of the vaccine composition according to any one of claims 1-21 for the manufacture of a vaccine against influenza A infection, especially by reducing or preventing the effects of infection by influenza A virus, the effects being selected in the group consisting of mortality, clinical disease associated with infection by influenza A virus, histopathological lesions associated with infection by influenza A, and influenza A virus shedding.27- The use of claim 26, wherein the vaccine is a veterinary vaccine against avian influenza A infection in poultry, in particular infection by an influenza A virus selected from the group consisting of H5N1, H5N2, H5N6, H5N8, H5N9, H7N1, H7N3, H7N4, H7N6, H7N7, and H7N9.28- The use of claim 26, wherein the vaccine is a veterinary vaccine against avian H5N1 influenza A infection in poultry.29- A method for preventing or treating an infection by influenza A virus in a subject comprising administering an effective amount of a vaccine composition according to any one of claims 1-21 to said subject. 30- The method according to claim 29, wherein the subject is a poultry and / or the influenza A virus is H5N1.

Images