Immunogenic components

Abstract

Immunogenic compositions for treating or preventing influenza A infection are provided. [Solution] An immunogenic composition is provided which comprises at least five different virus-like particles (VLPs), optionally together with one or more pharmaceutically acceptable carriers, adjuvants, excipients or diluents, as a combined preparation suitable for simultaneous, separate or sequential use to treat or prevent influenza A infection, wherein each VLP independently comprises one or more homotrimers composed of polypeptides of specific sequences.

Description

[Technical Field]

[0001] The present invention relates to immunogenic compositions comprising two or more polypeptides. The present invention also provides nucleic acid molecules and vectors encoding the polypeptides, and methods of using the compositions, nucleic acid molecules, and vectors for the prevention or treatment of influenza. [Background technology]

[0002] Seasonal influenza is a major public health problem that causes severe illness and death. Globally, seasonal influenza is estimated to cause 3 to 5 million cases of serious illness and 250,000 to 500,000 deaths (Non-Patent Document 1). Demographics at highest risk for complications include children under 2 years of age, adults over 65 years of age, pregnant women, and people of all ages with certain medical conditions, such as diabetes or a weakened immune system (Non-Patent Document 2). It is estimated that the majority of child deaths in developing countries are influenza-related. Seasonal influenza also causes high levels of worker absenteeism and reduced productivity.

[0003] Influenza pandemics occur sporadically when a unique influenza strain originating from a reservoir animal begins to circulate widely in the human population. The most recent influenza pandemic occurred in 2009, resulting in an increase in severe influenza illness and hospitalizations among individuals under the age of 35 (Non-Patent Documents 3 and 4). The 1918 influenza pandemic was the most serious pandemic in history, causing 50 to 100 million deaths. There is concern about the emergence of new pandemic influenza strains.

[0004] Vaccination is the most effective method for preventing illness resulting from influenza infection. Currently, vaccination against influenza requires a trivalent or quatrivalent vaccine consisting of the most recent circulating strains of influenza A subtypes H1N1 and H3N2, and also includes one or two influenza B strains (Non-Patent Document 5). Rapid antigenic evolution of influenza requires constant vaccine updates, and time constraints often result in the wrong vaccine strain being selected for the next influenza season. For these reasons, conventional trivalent vaccines are estimated to be 10-60% effective, and at-risk groups are vaccinated annually (Non-Patent Documents 6 and 7).

[0005] As a result, there are clear societal and economic benefits to improving current influenza vaccines. This has been recognized by pharmaceutical companies such as GSK and Pfizer, who are developing their own new influenza vaccines. These approaches generally target epitopes that are weakly immune selected and therefore "immunorecessive."

[0006] Influenza viruses are currently conceptualized as containing (i) highly variable, highly immunogenic (and protective) epitopes, and (ii) less immunogenic, invariant epitopes. These together form the backbone of the theory of "antigenic drift," whereby virus populations gradually acquire changes in highly variable epitope regions, necessitating vaccines directed against these sites that are constantly updated. The only alternative is seen as artificial boosting of the naturally less potent invariant epitopes.

[0007] In contrast, we propose that influenza viruses also contain less variable, highly immunogenic epitopes, and that identifying these protective epitopes will allow the construction of a universal vaccine. This idea is supported by an alternative theory of influenza evolution known as "antigenic thrift," in which viral dynamics are driven by pre-existing immunity to common epitopes, but the existence of such epitopes remains questionable, and their use in vaccination has not previously been discussed.

[0008] Using a combination of bioinformatics, structural analysis, and serological analysis, one epitope of limited variability that is under strong immune selection in the major influenza antigen, hemagglutinin (HA), has now been identified and characterized.

[0009] HA is the major surface antigen of influenza viruses. It binds to sialic acid, triggers membrane fusion, and induces endocytosis. It is a trimeric protein, generally 565 / 566 amino acids long. Each monomer consists of a head domain and a stem domain.

[0010] The epitope identified here is under strong immune selection and is therefore "immunodominant," enabling the design of a new "universal" influenza vaccine that protects against the majority of H1N1 influenza strains by targeting this epitope with limited variability.

[0011] As a result, the vaccine of the present invention has several advantages over conventional trivalent vaccines and other influenza vaccines in development.

[0012] These advantages include: (i) Infection with circulating influenza strains potentially undermines but enhances vaccine protection. (ii) The vaccine should be more immunogenic, have a lower threshold of protection, and provide longer-lasting protection than other "universal" vaccines in development. (iii) it should require only 1 to 3 administrations (i.e., a prime and one boost, or two primes and boosts); (iv) The theoretical and experimental framework from which the vaccine is derived suggests that H1N1 influenza is unlikely to escape the protection conferred by the proposed vaccine. [Prior art documents] [Non-patent literature]

[0013] [Non-Patent Document 1] Lozano, R. et al., 2012. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010 : a systematic analysis for the Global Burden of Disease Study 2010. Lancet, 380, pp. 2095-2128. [Non-patent document 2] Mertz, D., Hyong, T. & Johnstone, J., 2013. Populations at risk for severe or complicated influenza illness : systematic review and meta-analysis. British Medical Journal, 5061(August), pp.1-15. [Non-patent document 3] Presanis, AM et al., 2011. Changes in severity of 2009 pandemic A / H1N1 influenza in England : a Bayesian evidence synthesis. British Medical Journal, (343), pp.1-14. [Non-patent document 4] Manicassamy, B. et al., 2010. Protection of mice against lethal challenge with 2009 H1N1 influenza A virus by 1918-like and classical swine H1N1 based vaccines. PLoS Pathogens, 6(1). [Non-patent document 5] WHO 2016. Recommended composition of influenza virus vaccine for use in the 2016-2017 northern hemisphere influenza season. [Non-patent document 6] Treanor, JJ et al., 2012. Effectiveness of Seasonal Influenza Vaccines in the United States During a Season With Circulation of All Three Vaccine Strains., pp.1-9. [Non-Patent Document 7] Belongia, EA et al., 2009. Effectiveness of Inactivated Influenza Vaccines Varied Substantially with Antigenic Match from the 2004-2005 Season to the 2006-2007 Season Linked references are available on JSTOR for this article : Effectiveness of Inactivated Influenza Vaccines Varied. The Journal of Infectious Disease, 199(2), pp.159-167. Summary of the Invention [Means for solving the problem]

[0014] It is therefore an object of the present invention to provide an influenza vaccine composition capable of conferring protection against one or more influenza A subtypes, preferably against the H1N1 subtype.

[0015] In one embodiment, the present invention therefore provides an immunogenic composition comprising two or more polypeptides, optionally together with one or more pharmaceutically acceptable carriers, adjuvants, excipients or diluents, each polypeptide independently comprising a first region of contiguous amino acids: (a) the amino acid sequence of the first region has at least 80% sequence identity to an influenza A hemagglutinin head domain; (b) the first region has one or more amino acid substitutions at positions corresponding to the following positions in SEQ ID NO:9: Position 83 is E Position 85 is a negatively charged amino acid Position 146 is T, N, I, or A Position 147 is a positively charged amino acid, I, or is absent Position 148 is G Position 149 is V Position 151 is A Position 154 is S or P Position 155 is H Position 156 is a positively charged amino acid or A or G or N or E Position 157 is a positively charged amino acid or A or G Position 158 is a positively charged amino acid or A or S or N or C or E Position 159 is K or A or S or N or C Position 163 is a positively charged amino acid; Immunogenic compositions are provided in which two or more polypeptides differ in amino acid sequence, and the compositions are capable of inducing antibodies in a subject against influenza A virus.

[0016] The present invention relates to a polypeptide comprising an amino acid sequence comprising a first region, (a) the amino acid sequence of the first region has at least 80% sequence identity to the hemagglutinin head domain of influenza A subtype H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, or H18, preferably to the hemagglutinin head domain of influenza A subtype H1, H5, H6, H9, or H11; (b) the first region has one or more amino acid substitutions at positions corresponding to the following positions in SEQ ID NO:9: Position 83 is E Position 85 is a negatively charged amino acid Position 146 is T, N, I, or A Position 147 is a positively charged amino acid, I, or is absent Position 148 is G Position 149 is V Position 151 is A Position 154 is S or P Position 155 is H Position 156 is a positively charged amino acid or A or G or N or E Position 157 is a positively charged amino acid or A or G Position 158 is a positively charged amino acid or A or S or N or C or E Position 159 is K or A or S or N or C Also provided is a polypeptide wherein position 163 is a positively charged amino acid.

[0017] The present invention also provides compositions comprising such polypeptides, optionally together with one or more pharmaceutically acceptable carriers, adjuvants, excipients or diluents (preferably immunogenic compositions, wherein the compositions are capable of inducing antibodies in a subject against influenza A virus).

[0018] The present invention also provides nucleic acid molecules (preferably DNA molecules) encoding such polypeptides, which preferably are vectors or plasmids.

[0019] In a preferred embodiment, (b) the first region has one or more amino acid substitutions at positions corresponding to the following positions in SEQ ID NO:9: Position 83 is E Position 85 is E or D Position 146 is T or N Position 147 is R, K, I, or absent Position 148 is G Position 149 is V Position 151 is A Position 154 is S or P Position 155 is H Position 156 is A or G or K or N or E Position 157 is A or G Position 158 is A or K Position 159 is A, K, C, N, or S Position 163 is K or R.

[0020] In another preferred embodiment, (b) the first region has one or more amino acid substitutions at positions corresponding to the following positions in SEQ ID NO:9: Position 83 is E Position 85 is a negatively charged amino acid Position 146 is T, N, I, or A Position 147 is a positively charged amino acid; Position 148 is G Position 149 is V Position 151 is A Position 154 is S or P Position 155 is H Position 156 is A Position 157 is G Position 158 is K or A or S or N or C Position 159 is K or A or S or N or C Position 163 is a positively charged amino acid.

[0021] In another preferred embodiment, (b) the first region has one or more amino acid substitutions at positions corresponding to the following positions in SEQ ID NO:9: Position 83 is E Position 85 is E Position 146 is N Position 147 is R Position 148 is G Position 149 is V Position 151 is A Position 154 is P Position 155 is H Position 156 is A Position 157 is G Position 158 is A Position 159 is K Position 163 is K.

[0022] Preferably, the first region of one polypeptide comprises or consists of the amino acid sequence given in SEQ ID NO:13.

[0023] In another preferred embodiment, (b) the first region has one or more amino acid substitutions at positions corresponding to the following positions in SEQ ID NO:9: Position 83 is E Position 85 is a negatively charged amino acid Position 146 is N Position 147 is I Position 148 is G Position 149 is V Position 151 is A Position 154 is S Position 155 is H Position 156 is K or A Position 157 is G Position 158 is A or K Position 159 is K or S Position 163 is a positively charged amino acid.

[0024] In another preferred embodiment, (b) the first region has one or more amino acid substitutions at positions corresponding to the following positions in SEQ ID NO:9: Position 83 is E Position 85 is E Position 146 is N Position 147 is I Position 148 is G Position 149 is V Position 151 is A Position 154 is S Position 155 is H Position 156 is A Position 157 is G Position 158 is K Position 159 is S Position 163 is K.

[0025] Preferably, the first region of one of the polypeptides comprises or consists of the amino acid sequence given in SEQ ID NO:14.

[0026] In another preferred embodiment, (b) the first region has one or more amino acid substitutions at positions corresponding to the following positions in SEQ ID NO:9: Position 83 is E Position 85 is a negatively charged amino acid T at position 146 Position 147 is a positively charged amino acid Position 148 is G Position 149 is V Position 151 is A Position 154 is S Position 155 is H Position 156 is a positively charged amino acid or A or G Position 157 is a positively charged amino acid or A or G Position 158 is K Position 159 is S or C Position 163 is a positively charged amino acid.

[0027] In another preferred embodiment, (b) the first region has one or more amino acid substitutions at positions corresponding to the following positions in SEQ ID NO:9: Position 83 is E Position 85 is E T at position 146 Position 147 is R Position 148 is G Position 149 is V Position 151 is A Position 154 is S Position 155 is H Position 156 is K Position 157 is G Position 158 is K Position 159 is S Position 163 is K.

[0028] Preferably, the first region of one polypeptide comprises or consists of the amino acid sequence given in SEQ ID NO:15.

[0029] In another preferred embodiment, (b) the first region has one or more amino acid substitutions at positions corresponding to the following positions in SEQ ID NO:9: Position 83 is E Position 85 is a negatively charged amino acid T at position 146 Position 147 is a positively charged amino acid Position 148 is G Position 149 is V Position 151 is A Position 154 is S Position 155 is H Position 156 is N Position 157 is G Position 158 is a positively charged amino acid Position 159 is S Position 163 is a positively charged amino acid.

[0030] In another preferred embodiment, (b) the first region has one or more amino acid substitutions at positions corresponding to the following positions in SEQ ID NO:9: Position 83 is E Position 85 is E T at position 146 Position 147 is K Position 148 is G Position 149 is V Position 151 is A Position 154 is S Position 155 is H Position 156 is N Position 157 is G Position 158 is K Position 159 is S Position 163 is R.

[0031] Preferably, the first region of one polypeptide comprises or consists of the amino acid sequence given in SEQ ID NO:16.

[0032] In another preferred embodiment, (b) the first region has one or more amino acid substitutions at positions corresponding to the following positions in SEQ ID NO:9: Position 83 is E Position 85 is a negatively charged amino acid Position 146 is T or N Position 147 does not exist Position 148 is G Position 149 is V Position 151 is A Position 154 is S or P Position 155 is H Position 156 is N or E Position 157 is G Position 158 is K or E Position 159 is S Position 163 is a positively charged amino acid.

[0033] In another preferred embodiment, (b) the first region has one or more amino acid substitutions at positions corresponding to the following positions in SEQ ID NO:9: Position 83 is E Position 85 is E T at position 146 Position 147 does not exist Position 148 is G Position 149 is V Position 151 is A Position 154 is S Position 155 is H Position 156 is N Position 157 is G Position 158 is K Position 159 is S Position 163 is R.

[0034] Preferably, the first region of one polypeptide comprises or consists of the amino acid sequence given in SEQ ID NO:17.

[0035] In one embodiment, the present invention relates to an immunogenic composition.

[0036] As used herein, the term "immunogenicity" refers to the ability to induce a specific immune response against an influenza A subtype. This response may be, for example, when one composition of the invention is administered at an appropriate dose and in an appropriate formulation, which may include / require an appropriate adjuvant. Booster doses, including doses similar to or smaller than the initial dose, may be required to obtain the required immunogenic response.

[0037] In particular, the immunogenic compositions of the present invention are capable of inducing antibodies (preferably neutralizing antibodies) in a subject against influenza A virus.

[0038] Preferably, the immunogenic compositions of the invention are capable of providing protection in a subject against influenza A virus.

[0039] More preferably, the immunogenic compositions of the present invention are capable of inducing antibodies (preferably neutralizing antibodies) in a subject against the H1N1 influenza A subtype.

[0040] The ability of a composition of the invention to induce neutralizing antibodies in a subject (eg, a human subject) can be analyzed by purifying serum from the blood of a subject to which the composition has been administered.

[0041] Antibodies can be measured by ELISA or pseudotype micro-neutralization assay (pMN). ELISA is the most sensitive of the two assays and quantifies all antibodies, whereas pMN is less sensitive but quantifies neutralizing antibodies.

[0042] The term "influenza" as used herein relates to influenza viruses, preferably influenza A viruses, more preferably influenza A viruses of the subtype H1, and most preferably influenza A viruses of the subtype H1N1.

[0043] An immunogenic composition may comprise one, two or more polypeptides, the amino acid sequences of which preferably differ.

[0044] The composition can include, for example, 2, 3, 4, 5, 6, 7, 8, 9 or 10 different polypeptides.

[0045] Preferably, the composition comprises 2, 3, 4 or 5 different polypeptides, more preferably 3 different polypeptides.

[0046] The sequences of the present invention are derived from or based on the head domain of the influenza A hemagglutinin protein.

[0047] The naturally occurring hemagglutinin protein is a homotrimer of three polypeptides.

[0048] In one preferred composition of the invention, the composition comprises three polypeptides as defined herein that form a homotrimer. The composition may comprise more than one (e.g., 2, 3, 4, or 5) different homotrimers of a polypeptide as defined herein.

[0049] In another preferred embodiment, three polypeptides of the invention form a heterotrimer in the composition. The composition may comprise more than one (e.g., 2, 3, 4, or 5) different heterotrimers of the polypeptides defined herein.

[0050] Each polypeptide independently comprises a first region of contiguous amino acids, which is a contiguous series of covalently linked amino acids.

[0051] In one embodiment, the amino acid sequence of the first region has at least 80% sequence homology to an influenza A hemagglutinin (HA) head domain.

[0052] The intention is for this first region to adopt the conformation of an influenza A hemagglutinin head domain.

[0053] The hemagglutinin head domain can be, for example, from any influenza A subtype, such as H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, or H18.

[0054] Preferably, the hemagglutinin head domain is derived from an influenza A H1, H5, H6, H9 or H11 subtype.

[0055] In one embodiment, the hemagglutinin head domain is derived from influenza A H1 subtype.

[0056] In one embodiment, the hemagglutinin head domain is derived from influenza A H5 subtype.

[0057] In one embodiment, the hemagglutinin head domain is derived from influenza A H6 subtype.

[0058] In one embodiment, the hemagglutinin head domain is derived from influenza A H9 subtype.

[0059] In one embodiment, the hemagglutinin head domain is derived from influenza A H11 subtype.

[0060] Preferably, the influenza AN subtype is N1.

[0061] The consensus amino acid sequences of the H1, H5, H6 and H11 hemagglutinin polypeptides are set forth herein as SEQ ID NOs: 9 to 12, respectively. The consensus amino acid sequence of the H9 hemagglutinin polypeptide is set forth herein as SEQ ID NO: 23.

[0062] The H9 sequence may be used herein in place of the H1, H5, H6 or H9 embodiments disclosed herein, mutatis mutandis.

[0063] The amino acid sequence numbers used herein are based on the numbering given to the influenza A H1 hemagglutinin head domain as set forth in SEQ ID NO:9.

[0064] Note that there are three ways to number the influenza A H1 hemagglutinin polypeptides, and linear numbering is used throughout this specification, with Met=1.

[0065] The HA polypeptide contains two regions, the HA1 region and the HA2 region, which are separated by a potential cleavage site.

[0066] The H1 cleavage site consensus sequence is PSIQSR / GLF (sequence number 24), the H5 cleavage site consensus sequence is PQRKKR / GLF (sequence number 25), the H6 cleavage site consensus sequence is PQIETR / GLF (sequence number 26), the H9 cleavage consensus sequence is PSRSSR / GLF (sequence number 27), and the H11 cleavage site consensus sequence is PAIATR / GLF (sequence number 28).

[0067] Cleavage of HA0 into HA1 and HA2 occurs between the R and GLF and is carried out by proteases. All of the cleavage sites are described as monobasic. In some H5 viruses, polybasic cleavage sites exist, which differ from monobasic sites by having multiple arginine (R) and / or lysine (K) residues at key base positions.

[0068] Further details of the cleavage site can be found in Sun et al., Journal of Virology, September 2010, Vol. 84, No. 17, pp. 8683-8690.

[0069] The HA1 region contains 1-60 amino acids of the stalk, followed by the head domain and then additional amino acids of the stalk, whereas the HA2 region contains only amino acids of the stalk.

[0070] The head domain of hemagglutinin is defined as occurring between two cysteines within the HA1 region: the first cysteine ​​is generally at position 58, 59, or 60, and the second cysteine ​​is generally at position 290, 291, or 292.

[0071] In influenza A H1 hemagglutinins, these cysteines are at positions 59 and 291 / 292 due to the absence of an amino acid at position 147 in some H1 hemagglutinins.

[0072] In the H6 hemagglutinin, the head domain corresponds to the sequence between positions 58 and 292.

[0073] However, in H5 and H11 hemagglutinins, the head domain corresponds to the region between positions 58 and 290, respectively.

[0074] In the H9 hemagglutinin, the head domain corresponds to the region between positions 60 and 290.

[0075] The consensus amino acid sequences of the H1, H5, H6 and H11 hemagglutinin head domains are set forth herein as SEQ ID NOs: 1 to 4, respectively.

[0076] In some embodiments, the amino acid sequence of the first region has at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:1.

[0077] In some embodiments, the amino acid sequence of the first region has at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:2.

[0078] In some embodiments, the amino acid sequence of the first region has at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:3.

[0079] In some embodiments, the amino acid sequence of the first region has at least 80%, 85%, 90% or 95% sequence identity to SEQ ID NO:4.

[0080] In some embodiments, the amino acid sequence of the first region has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the influenza A hemagglutinin head domain at positions other than those corresponding to positions 83, 85, 146-149, 151, 154-159 and 163 of SEQ ID NO:9.

[0081] In some embodiments, the amino acid sequence of the first region has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the influenza A H1 hemagglutinin head domain (preferably of SEQ ID NO: 1) at positions other than those corresponding to positions 83, 85, 146-149, 151, 154-159 and 163 of SEQ ID NO: 9.

[0082] In some embodiments, the amino acid sequence of the first region has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the influenza A H6 hemagglutinin head domain (preferably of SEQ ID NO: 2) at positions other than those corresponding to positions 83, 85, 146-149, 151, 154-159 and 163 of SEQ ID NO: 9.

[0083] In some embodiments, the amino acid sequence of the first region has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to an influenza A H5 hemagglutinin head domain (preferably of SEQ ID NO: 3) at positions other than those corresponding to positions 83, 85, 146-149, 151, 154-159 and 163 of SEQ ID NO: 9.

[0084] In some embodiments, the amino acid sequence of the first region has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to the influenza A H11 hemagglutinin head domain (preferably of SEQ ID NO: 4) at positions other than those corresponding to positions 83, 85, 146-149, 151, 154-159 and 163 of SEQ ID NO: 9.

[0085] In some embodiments, each polypeptide independently further comprises one or more amino acids adjacent to and linked to the first region at the N- and / or C-terminus.

[0086] The additional N-terminal amino acids are preferably a series of adjacent amino acids derived from the hemagglutinin N-terminal stalk region, preferably derived from the hemagglutinin N-terminal stalk region of an influenza AH subtype, most preferably an H1, H5, H6, H9 or H11 subtype.

[0087] Preferably, 58 to 60 amino acids of the influenza A subtype hemagglutinin N-terminal stalk region are contiguous to the N-terminus of the first region in one or more of the polypeptides.

[0088] In some embodiments, the amino acid sequence of the stalk region is: (i) amino acids 1 to 59 of SEQ ID NO: 9; (ii) amino acids 1 to 58 of SEQ ID NO: 10; (iii) amino acids 1 to 58 of SEQ ID NO: 11, or (iv) has at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to amino acids 1 to 58 of SEQ ID NO: 12.

[0089] The additional C-terminal amino acids, if present, are preferably a series of contiguous amino acids (e.g., 1 to 300, 1 to 200, 1 to 100, 1 to 50 or 1 to 10 amino acids) from the hemagglutinin C-terminal stalk region of an influenza A subtype, preferably H1, H5, H6, H9 or H11.

[0090] Preferably, the stretch of contiguous amino acids is derived from the hemagglutinin stalk region of the same influenza A subtype from which the head region is derived.

[0091] In some embodiments, the polypeptide does not include an influenza A subtype HA2 region.

[0092] In some embodiments, the polypeptide does not include the HA2 region of SEQ ID NOs: 9-12.

[0093] In some preferred embodiments, one or more or all of the one, two or more polypeptides independently comprise an influenza A HA1 domain comprising a first region as defined herein, most preferably an influenza A H1, H5, H6 or H11 subtype HA1 domain comprising a first region as defined herein.

[0094] Preferably, the polypeptides are independently less than 600, more preferably less than 400, and most preferably less than 300 amino acids in length.

[0095] Preferably, the polypeptides are independently 250 to 350, more preferably 280 to 300, and most preferably 290 to 292 amino acids in length.

[0096] The first region of the polypeptide has one or more amino acid substitutions at designated positions corresponding to positions in SEQ ID NO:1.

[0097] For example, a first region of the polypeptide can have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 of the specified amino acid substitutions.

[0098] Preferably, the first region of the polypeptide has all 14 of the designated amino acid substitutions.

[0099] As used herein, the term "positively charged amino acid" includes lysine, arginine, and histidine. As used herein, the term "negatively charged amino acid" includes aspartic acid and glutamic acid.

[0100] In some preferred embodiments, the amino acid sequences of the polypeptides independently comprise or consist of the amino acid sequences of SEQ ID NOs: 13-17.

[0101] The polypeptides of the present invention can be produced by recombinant methods, for example, as described in "Molecular Cloning: A Laboratory Manual" (4th Edition) by Michael R. Green and Joseph Sambrook.

[0102] Alternatively, a nucleotide sequence encoding the polypeptide can be produced by chemical synthesis. Such a nucleotide sequence can then be ligated into an appropriate vector for transformation or introduction into a host cell. The polypeptide can then be expressed in such a host cell.

[0103] For modification of existing HA genes, use the CRISPR-Cas:A Laboratory CRISPR-based techniques such as those described in "The Genetic Manual" (2016), edited by Jennifer Doudna (University of California, Berkeley) and Prashant Mali (University of California, San Diego), can also be used. TALEN-based techniques can also be used.

[0104] Alternatively, the polypeptides of the invention can be synthesized by standard chemical peptide synthesis techniques, for example, solid phase synthesis of peptides can be used in which the C-terminal amino acid of the sequence is attached to an insoluble support, followed by sequential addition of the remaining amino acids.

[0105] In a further embodiment, the invention provides a nucleic acid molecule encoding one or more polypeptides of the invention. Preferably, the nucleic acid molecule encodes one, two or more, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10, of the polypeptides of the invention.

[0106] Preferred nucleotide sequences include those comprising SEQ ID NOs: 18-22, which encode polypeptides capable of inducing antibodies in a subject against influenza A virus, and nucleotide sequences having at least 80%, 85%, 90%, or 95% sequence identity thereto.

[0107] Nucleic acid molecules encoding the polypeptides of SEQ ID NOs: 13-17 are also preferred.

[0108] The terms "nucleic acid sequence," "nucleic acid molecule," and "polynucleotide" are used interchangeably herein and do not imply any length restriction. They include DNA (including cDNA) and RNA sequences.

[0109] Nucleic acid molecules of the present invention include isolated nucleic acid molecules removed from their natural environment, recombinant or cloned DNA isolates, and chemically synthesized analogs or biologically synthesized analogs in heterologous systems.

[0110] The nucleic acid molecules of the present invention can be prepared by any means known in the art. For example, large amounts of polynucleotides can be generated by replication in suitable host cells. Natural or synthetic DNA fragments encoding the desired fragments can be incorporated into recombinant nucleic acid constructs, generally DNA constructs, that can be introduced into and replicated in prokaryotic or eukaryotic cells. Typically, DNA constructs are suitable for autonomous replication in unicellular hosts such as yeast or bacteria, but may also be intended for introduction and integration into the genomes of cultured insects, mammals, plants, or other eukaryotic cell systems.

[0111] The nucleic acid molecules of the present invention can also be produced by chemical synthesis, for example, by the phosphoramidite or triester method, which can be performed on commercially available automated oligonucleotide synthesizers. Double-stranded fragments can be obtained from the single-stranded product of chemical synthesis by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by using a DNA polymerase to add an appropriate primer sequence to the complementary strand.

[0112] The initial (eg, wild-type) codon in a nucleic acid molecule can be optimized for expression in a desired cell line using online tools such as those available at, for example, http: / / genomes.urv.es / OPTIMIZER / .

[0113] In one embodiment of the present invention, the nucleic acid molecule is therefore codon-optimized for expression in a host cell, preferably a human cell.

[0114] As used herein, the term "product of the invention" refers inter alia to a polypeptide of the invention, a nucleic acid of the invention, a vector of the invention, a particle of the invention, and a composition of the invention.

[0115] The present invention also provides a vector or plasmid comprising a nucleic acid molecule of the invention. Preferably, the vector is an expression vector.

[0116] Vectors and / or plasmids can include one or more control sequences, such as one or more enhancers, promoters, and / or transcription termination sequences, operably linked to the polypeptide-encoding sequence.

[0117] In a particularly preferred embodiment, there is provided an immunogenic composition comprising one, two or more vectors encoding the polypeptides of SEQ ID NOs: 13-17, optionally together with one or more pharmaceutically acceptable carriers, adjuvants, excipients or diluents, as a combined preparation suitable for simultaneous, separate or sequential use to treat or prevent influenza A infection.

[0118] Preferably, an initial immunization is administered to the subject with a vector(s) encoding SEQ ID NOs: 14 and 15, then a first booster immunization is administered with a vector(s) encoding SEQ ID NOs: 13 and 16, and then a final booster immunization is administered with a vector encoding SEQ ID NO: 17.

[0119] In some embodiments, the vector is a viral vector, such as a poxvirus vector.

[0120] In another embodiment, the vector is an adenovirus vector or a Modified Vaccinia Ankara (MVA) virus vector.

[0121] Preferably, the vector is a non-replicating vector.

[0122] Non-replicating poxviruses and adenoviruses are groups of viruses that can be used as vectors to deliver genetic material to target cells. Viral vectors act as antigen delivery vehicles and also have the ability to activate the innate immune system through the binding of viral elements to cell surface molecules. Recombinant viral vectors can be generated that carry nucleic acids encoding a given antigen. The viral vector can then be used to deliver the nucleic acid to target cells, where the encoded antigen is produced by the target cell's own molecular machinery. As a "non-self," the produced antigen generates an immune response in the target subject.

[0123] Without being bound to any particular theory, the inventors believe that antigen delivery using the vectors of the present invention stimulates T cell responses, among other responses, in a subject. Thus, the inventors believe that one way in which the present invention confers protection against influenza infection is by stimulating T cell responses and the cellular immune system. Additionally, humoral (antibody) protection may also be obtained.

[0124] The vector of the present invention can be a non-replicating poxvirus vector. As used herein, a non-replicating (or replication-deficient) viral vector is a viral vector that lacks the ability to productively replicate after infecting a target cell. Therefore, a non-replicating viral vector cannot generate copies of itself after infecting a target cell. Therefore, a non-replicating viral vector can advantageously have an improved safety profile compared to a replication-competent viral vector.

[0125] In one embodiment, the non-replicating poxvirus vector is selected from a modified vaccinia virus Ankara (MVA) vector, a NYVAC vaccinia virus vector, a canarypox (ALVAC) vector, and a fowlpox (FPV) vector. Both MVA and NYVAC are attenuated derivatives of vaccinia virus. Compared to vaccinia virus, MVA lacks approximately 26 of the approximately 200 open reading frames.

[0126] In one embodiment, the non-replicating poxvirus vector is an MVA vector.

[0127] The vector of the present invention can be an adenoviral vector. In one embodiment, the adenoviral vector is a non-replicating adenoviral vector (non-replicating is defined above). An adenovirus can be rendered non-replicating by deleting the El or both the El and E3 gene regions. Alternatively, an adenovirus can be rendered non-replicating by modifying the El or El and E3 gene regions to render them non-functional. For example, a non-replicating adenovirus can lack a functional El region or functional El and E3 gene regions. In this way, the adenovirus is replication-incompetent in most mammalian cell lines and does not replicate in immunized mammals. Most preferably, deletions of both the El and E3 gene regions are present in the adenovirus, thereby allowing for the insertion of larger transgenes. This is particularly important for expressing larger antigens, or when multiple antigens are expressed in a single vector, or when using large promoter sequences such as the CMV promoter. Deletions of the E3 as well as the El region are particularly preferred for recombinant Ad5 vectors. Optionally, the E4 region can also be manipulated.

[0128] In one embodiment, the adenovirus vector is selected from a human adenovirus vector, a simian adenovirus vector, a group B adenovirus vector, a group C adenovirus vector, a group E adenovirus vector, an adenovirus 6 vector, a PanAd3 vector, an adenovirus C3 vector, a ChAdY25 vector, an AdC68 vector, and an Ad5 vector.

[0129] As described above, the viral vectors of the present invention can be used to deliver a single antigen to a target cell. Advantageously, the viral vectors of the present invention can also be used to deliver multiple (different) antigens to a target cell.

[0130] In one embodiment, a vector of the invention further comprises a nucleic acid sequence encoding an adjuvant (eg, cholera toxin, E. coli lethal toxin, or flagellin).

[0131] Nucleic acid sequences encoding a vector (as described above) can be produced using any technique known in the art for manipulating and producing recombinant nucleic acids. In one aspect, the invention provides a method of making a vector (as described above) comprising providing nucleic acid comprising a nucleic acid molecule encoding a vector of the invention, introducing the nucleic acid molecule into a host cell, culturing the host cell under conditions suitable for propagation of the vector, and obtaining the vector from the host cell.

[0132] As used herein, "introduction" can refer to any non-viral method of introducing a nucleic acid molecule into a cell. The nucleic acid molecule can be any nucleic acid molecule suitable for introduction into a host cell. Thus, in one embodiment, the nucleic acid molecule is a plasmid. The host cell can be any cell in which a vector (i.e., a non-replicating poxvirus vector or an adenovirus vector, as described above) can propagate. As used herein, "culturing a host cell under conditions suitable for propagation of the vector" refers to the use of any cell culture conditions and techniques known in the art that are suitable for the selected host cell and that can produce the vector in the host cell. As used herein, "obtaining the vector" refers to the use of any technique known in the art that is suitable for separating the vector from the host cell. Thus, the host cell can be lysed to release the vector. The vector can then be isolated and purified using any suitable method or methods known in the art.

[0133] The present invention also provides a host cell comprising the nucleic acid molecule, vector or plasmid of the present invention. Preferably, the host cell is a eukaryotic host cell. Examples of eukaryotic host cells include yeast and mammalian cells.

[0134] The host cell is preferably a cell in which the vector (e.g., a non-replicating poxvirus vector or adenovirus vector, as described above) can grow or propagate. The host cell may be selected from 293 cells (also known as HEK, i.e., human embryonic kidney cells), CHO cells (Chinese hamster ovary), CCL81.1 cells, Vero cells, HeLa cells, Per.C6 cells, BHK cells (baby hamster kidney), primary CEF cells (chicken embryo fibroblasts), duck embryo fibroblasts, or DF-1 cells.

[0135] In another embodiment, the host cell is a human cell (eg, an isolated human cell).

[0136] In a further embodiment, there is provided a virus-like particle (VLP) comprising one, two or more (e.g., 3, 4, 5, 6, 7, 8, 9 or 10) polypeptides of the invention. The particle is preferably immunogenic.

[0137] Virus-like particles resemble viruses but do not contain viral genetic material and are therefore non-infectious. The particles can also be described as multimeric lipoprotein particles.

[0138] When expressed in an appropriate system, these VLPs are able to spontaneously assemble into lipoprotein structures / particles composed of one or more monomers of the polypeptide.

[0139] The invention also provides VLPs having one, two, or more (e.g., 3, 4, 5, 6, 7, 8, 9, or 10) polypeptides of the invention (preferably, different polypeptides) covalently attached to the VLP. For example, polypeptides of the invention can be covalently attached to the VLP using chemical cross-linkers, reactive unnatural amino acids, or SpyTag / SpyCatcher reactions.

[0140] In a particularly preferred embodiment, there is provided an immunogenic composition comprising at least five different virus-like particles (VLPs), optionally together with one or more pharmaceutically acceptable carriers, adjuvants, excipients or diluents, as a combined formulation suitable for simultaneous, separate or sequential use to treat or prevent influenza A infection, wherein each VLP independently comprises one or more homotrimers consisting of or consisting of a polypeptide of SEQ ID NO: 13 to 17.

[0141] Preferably, the initial immunization is administered to the subject using the homotrimer of SEQ ID NO: 14 and 15, the first booster immunization is administered using the homotrimer of SEQ ID NO: 13 and 16, and the final booster immunization is administered using the homotrimer of SEQ ID NO: 17.

[0142] The present invention also provides compositions comprising one, two or more polypeptides of the invention, one or more nucleic acid molecules of the invention, one or more vectors of the invention, or VLPs of the invention, optionally together with one or more pharmaceutically acceptable carriers, excipients or diluents.

[0143] The composition is preferably an immunogenic composition.

[0144] Suitable materials for use as pharmaceutically acceptable carriers are known in the art. Non-limiting examples of pharmaceutically acceptable carriers include water, saline, and phosphate-buffered saline. However, in some embodiments, the composition is in lyophilized form, which may include a stabilizer such as bovine serum albumin (BSA). In some embodiments, it may be desirable to formulate the composition with a preservative such as thiomersal or sodium azide to facilitate long-term storage. Examples of buffers include, but are not limited to, sodium succinate (pH 6.5) and phosphate-buffered saline (PBS: pH 7.4).

[0145] In addition to pharmaceutically acceptable carriers, the compositions of the present invention may be further combined with one or more salts, excipients, diluents, adjuvants, immunomodulators, and / or antimicrobial compounds.

[0146] In one embodiment, a product of the invention may comprise 5% to 95% active ingredient (i.e., polypeptide, nucleic acid, vector or VLP), such as at least 10% or 25% active ingredient, or at least 40% active ingredient, or at least 50%, 55%, 60%, 70% or 75% active ingredient.

[0147] The products of the present invention can be administered in a manner compatible with the dosage formulation, and in such amount as will be prophylactically and / or therapeutically effective.

[0148] Administration of the products of the invention will generally be by conventional routes, for example intravenous, subcutaneous, intraperitoneal or mucosal. Administration can also be parenteral, for example by subcutaneous or intramuscular injection.

[0149] Thus, the products of the present invention can be prepared for injection as liquid solutions or suspensions. Alternatively, solid dosage forms suitable for solution or suspension in liquid prior to injection can be prepared. The preparation can also be emulsified, or the peptides can be encapsulated in liposomes or microcapsules. The active ingredient may be mixed with excipients that are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. In addition, if desired, the products of the present invention can contain minor amounts of auxiliary substances, such as wetting agents, emulsifying agents, and / or pH buffering agents.

[0150] Additional formulations suitable for alternative modes of administration include oral formulations or formulations suitable for dispersal as an aerosol. Oral formulations contain commonly employed excipients such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained-release formulations, or powders.

[0151] It may also be desirable to target the products of the invention to the respiratory system of a subject (as discussed above). Effective delivery of therapeutic / prophylactic compositions or medicaments to the site of infection in the lungs can be by oral or intranasal administration.

[0152] Formulations for intranasal administration can be in the form of nasal drops or nasal sprays. Intranasal formulations can comprise droplets having an approximate diameter in the range of 100 to 5000 μm, such as 500 to 4000 μm, 1000 to 3000 μm, or 100 to 1000 μm. Alternatively, the volume of the droplets can be in the range of about 0.001 to 100 μl, such as 0.1 to 50 μl, 1.0 to 25 μl, or 0.001 to 1 μl.

[0153] Alternatively, the therapeutic / prophylactic formulation or pharmaceutical product can be an aerosol formulation. The aerosol formulation can be in the form of a powder, suspension, or solution. The size of the aerosol particles is related to the delivery capacity of the aerosol. Smaller particles can travel further down the respiratory tract toward the alveoli than larger particles. In one embodiment, the aerosol particles have a diameter distribution that promotes delivery along the entire length of the bronchi, bronchioles, and alveoli. Alternatively, the particle size distribution can be selected to target specific portions of the respiratory tract, such as the alveoli. For aerosol delivery of pharmaceutical products, the particles can range in diameter from approximately 0.1 to 50 μm, preferably 1 to 25 μm, and more preferably 1 to 5 μm.

[0154] The aerosol particles can be for delivery by nebulizer (e.g., oral) or nasal spray. The aerosol formulation can optionally include a propellant and / or surfactant.

[0155] Preferably, the composition of the invention is a vaccine composition, eg suitable for parenteral administration, optionally with one or more adjuvants.

[0156] As used herein, a vaccine is a formulation that, when administered to an animal subject, such as a mammal (e.g., a human, bovine, porcine, ovine, caprine, equine, cervid, canine, or feline subject, particularly a human subject), stimulates a protective immune response against infectious disease. The immune response can be a humoral and / or cellular immune response. Thus, a vaccine can stimulate B cells and / or T cells.

[0157] Examples of suitable adjuvants include those selected from the group consisting of: -metal salts such as aluminum hydroxide and aluminum phosphate, -oil-in-water emulsion, - Toll-like receptor agonists (Toll-like receptor 2 agonists, Toll-like receptor 3 agonists, Toll-like receptor 4 agonists, Toll-like receptor 7 agonists, Toll-like receptor 8 agonists, Toll-like receptor 9 agonists, etc.), saponins, such as Quil A and its derivatives such as QS7 and / or QS21; CpG-containing oligonucleotides, -3D-MPL, -(2-deoxy-6-o-[2-deoxy-2-[(R)-3-dodecanoyloxytetradecanoylamino]-4-o-phosphono-β-D-glucopyranosyl]]-2-[(R)-3-hydroxytetradecanoylamino]-α-D-glucopyranosyl dihydrogen phosphate), -DP(3S,9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9(R)-[(R)-3-hydroxytetradecanoylamino]decane-1,10-diol, 1,10-bis(dihydrogen phosphate), and -MP-AcDP(3S-,9R)-3-[(R)-dodecanoyloxytetradecanoylamino]-4-oxo-5-aza-9-[(R)-3-hydroxytetradecanoylamino]decane-1,10-diol, 1-dihydrogen phosphate 10-(6-aminohexanoate), or a combination thereof.

[0158] Preferably, the adjuvant is selected from the group comprising: saponin associated with a metal salt such as aluminium hydroxide or aluminium phosphate, 3D-MPL, QS21 and CpG oligonucleotides, for example as an oil-in-water formulation; the saponin in liposomal form further comprises a sterol, such as, for example, QS21, a sterol, and -ISCOM.

[0159] In some particularly preferred embodiments, the adjuvant comprises a saponin, which is a steroid or triterpenoid glycoside present in many plant species.

[0160] Saponin-based adjuvants act in part by stimulating the influx of antigen-presenting cells to the injection site and enhancing antigen presentation in local lymph nodes.

[0161] Preferably, the adjuvant comprises saponin, cholesterol and phospholipids, for example ISCOM Matrix-M™ (Isconova, Novavax).

[0162] In Matrix-M, a purified saponin fraction is mixed with synthetic cholesterol and phospholipids to form stable particles that can be easily formulated with a variety of vaccine antigens. Matrix-M™ induces both cell- and antibody-mediated immune responses.

[0163] In some alternative preferred embodiments, the adjuvant comprises a squalene oil-in-water nanoemulsion, such as AddaVax™ (InvivoGen).

[0164] Squalene is an oil that is more easily metabolized than the paraffin oil used in Freund's adjuvants. Squalene oil-in-water emulsions are known to induce both cellular (Th1) and humoral (Th2) immune responses. This class of adjuvants is thought to act by recruiting and activating APCs and stimulating cytokine and chemokine production by macrophages and granulocytes.

[0165] The composition may further comprise a surfactant. Examples of suitable surfactants include Tween (such as Tween 20), briji and polyethylene glycol.

[0166] Vaccine formulations are listed in the "New Trends and Developments in Vaccines," edited by Voller et al., University Park Press, Baltimore, Maryland, USA, 1978. Encapsulation within liposomes is described, for example, in Fullerton, US Pat. No. 4,235,877.

[0167] The amount of polypeptide, nucleic acid molecule, vector, or particle of the present invention present in each vaccine dose is selected to induce a protective immune response without significant adverse side effects associated with typical vaccines. Such amounts will vary depending on the specific immunogen employed and whether the vaccine contains an adjuvant. Generally, each dose is expected to contain 1-1000 μg of protein, e.g., 1-200 μg, e.g., 10-100 μg, more specifically, 10-40 μg. The optimal amount for a particular vaccine can be ascertained through standard studies involving observation of antibody titers and other responses in subjects. Following the initial vaccination, subjects preferably receive a booster immunization approximately four weeks later, followed by repeat boosters every six months for as long as the risk of infection remains. The immune response to the products of the present invention can be enhanced through the use of adjuvants and / or immunostimulants.

[0168] The amount of saponin used in the adjuvant of the present invention can be 1 to 1000 μg / dose, generally 1 to 500 μg / dose, such as 1 to 250 μg / dose, and more specifically 1 to 100 μg / dose (for example, 10, 20, 30, 40, 50, 60, 70, 80, or 90 μg / dose).

[0169] The present invention also provides a combined preparation comprising two or more components selected from two or more polypeptides of the present invention, two or more particles of the present invention, two or more nucleic acids of the present invention, two or more vectors of the present invention and two or more compositions of the present invention, preferably as a combined preparation suitable for simultaneous, separate or sequential use for treating or preventing influenza A infection.

[0170] In yet another aspect, the present invention provides an antibody against a polypeptide of the present invention.

[0171] In a further embodiment, the present invention provides a polypeptide of the invention, a particle of the invention, a nucleic acid of the invention, a vector of the invention or a composition of the invention for use in therapy or for use as a medicament.

[0172] In a further aspect, the present invention provides a polypeptide of the invention, a particle of the invention, a nucleic acid of the invention, a vector of the invention, or a composition of the invention for use in a method of preventing or treating influenza infection in a subject.

[0173] In a further aspect, the present invention provides a polypeptide of the invention, a particle of the invention, a nucleic acid of the invention, a vector of the invention or a composition of the invention for use in a method of inducing a T cell or B cell response to an influenza antigen in a subject.

[0174] In particular, the non-replicating poxvirus vectors of the present invention can be used to stimulate a protective immune response via the cellular immune system. In one embodiment, the T cells are helper T cells (T h In one embodiment, the T cells are T h There are 17 cells.

[0175] In a further embodiment, the present invention provides the use of a polypeptide of the invention, a particle of the invention, a nucleic acid of the invention, a vector of the invention, or a composition of the invention in the manufacture of a medicament for use in a method of preventing or treating influenza infection in a subject.

[0176] In a further embodiment, the present invention provides the use of a polypeptide of the invention, a particle of the invention, a nucleic acid of the invention, a vector of the invention or a composition of the invention in the manufacture of a medicament for use in a method of inducing a T cell or B cell response to an influenza antigen in a subject.

[0177] The present invention also provides a method for treating a subject susceptible to influenza infection, comprising administering to the subject an effective amount of a polypeptide of the present invention, a particle of the present invention, a nucleic acid of the present invention, a vector of the present invention, or a composition of the present invention.

[0178] The present invention also provides a method for inducing a T cell or B cell response against an influenza antigen in a subject, comprising administering to the subject an effective amount of a polypeptide of the present invention, a particle of the present invention, a nucleic acid of the present invention, a vector of the present invention, or a composition of the present invention.

[0179] A polypeptide of the invention, a particle of the invention, a nucleic acid of the invention, a vector of the invention, or a composition of the invention can also be used in similar uses and methods to produce neutralizing antibodies in vivo against influenza antigens.

[0180] Preferably, the influenza antigen is the hemagglutinin protein, more preferably the HA1 or head domain of the hemagglutinin protein.

[0181] Preferably, the influenza is influenza A.

[0182] The effectiveness of the uses and methods for treating / preventing influenza infection can be determined by demonstrating the presence or absence of neutralizing antibodies to influenza virus in the subject's blood (eg, by ELISA).

[0183] Also provided is an immunogenic composition comprising two or more polypeptides, two or more nucleic acid molecules, or two or more vectors or plasmids as defined herein as a combined preparation suitable for simultaneous, separate or sequential use for the treatment or prevention of influenza, preferably influenza A, or for inducing a T cell or B cell response in a subject against influenza virus, preferably influenza A virus.

[0184] The subject is preferably a mammal, more preferably a human.

[0185] As used herein, the term "prevention" includes preventing the onset of influenza infection and / or reducing the severity of the intensity of influenza infection. Thus, "prevention" encompasses vaccination.

[0186] As used herein, the term "treatment" encompasses treatment and preventative / prophylactic treatment (including post-exposure prophylaxis), including post-infection treatment and recovery from influenza infection. Each of the above methods and uses may comprise the step of administering to a subject an effective, such as a therapeutically effective, amount of the polypeptide of the present invention, the particle of the present invention, the nucleic acid of the present invention, the vector of the present invention, or the composition of the present invention.

[0187] As used herein, an effective amount is a dosage or amount sufficient to achieve a desired biological result. As used herein, a therapeutically effective amount is an amount that, when administered in single or multiple doses to a subject (such as a mammalian subject, particularly a human subject), is effective to treat, prevent, cure, delay, reduce the severity of, or ameliorate at least one symptom of a disorder or recurring disorder, or to prolong the survival of the subject beyond that expected in the absence of such treatment.

[0188] Thus, the amount of active ingredient administered will depend on the subject being treated, the capacity of the subject's immune system to mount a protective immune response, and the degree of protection desired. The precise amount of active ingredient required for administration will depend on the judgment of the practitioner and may be particular to each subject. Administering to a subject can include administering to the subject a polypeptide of the invention, a particle of the invention, a nucleic acid of the invention, a vector of the invention, or a composition of the invention (i.e., a product of the invention), wherein the product of the invention is administered multiple times sequentially (e.g., the composition is administered two, three, or four times). Thus, in one embodiment, a polypeptide of the invention, a particle of the invention, a nucleic acid of the invention, a vector of the invention, or a composition of the invention is administered to the subject, followed by the administration of the same product of the invention (or a substantially similar product) again at a different time.

[0189] In one embodiment, administering to a subject comprises administering a polypeptide of the invention, a particle of the invention, a nucleic acid of the invention, a vector of the invention, or a composition of the invention to a subject, wherein said product of the invention is administered substantially before, simultaneously with, or subsequent to another immunogenic composition.

[0190] The present invention also extends to prime-boost regimens.

[0191] For example, priming and / or boosting can be carried out with one or more products of the invention. The products can be administered to a subject sequentially, simultaneously, or separately.

[0192] A preferred prime-boost strategy of the present invention provides a method of preventing or treating influenza infection in a subject or inducing a T cell or B cell response to an influenza antigen in a subject, the method comprising: (i) administering to a subject in need thereof effective amounts of one, two, three, four, five or more different polypeptides simultaneously, separately or sequentially; each polypeptide independently comprising a first region of contiguous amino acids; (a) the amino acid sequence of the first region has at least 80% sequence identity to an influenza A hemagglutinin head domain; (b) the first region has one or more amino acid substitutions at positions corresponding to the following positions in SEQ ID NO:9: Position 83 is E Position 85 is a negatively charged amino acid Position 146 is T, N, I, or A Position 147 is a positively charged amino acid, I, or is absent Position 148 is G Position 149 is V Position 151 is A Position 154 is S or P Position 155 is H Position 156 is a positively charged amino acid or A or G or N or E Position 157 is a positively charged amino acid or A or G Position 158 is a positively charged amino acid or A or S or N or C or E Position 159 is K or A or S or N or C Position 163 is a positively charged amino acid.

[0193] Preferred influenza A hemagglutinin head domain sequences and first region substitutions are disclosed herein, mutatis mutandis.

[0194] The polypeptide may be in the form of a pharmaceutical composition, preferably a vaccine composition, optionally together with one or more pharmaceutically acceptable carriers, diluents, excipients and adjuvants.

[0195] Preferably, one or more of the polypeptides (as defined above) are in the form of one or more trimers. In some embodiments, the trimers are homotrimers. In other embodiments, the trimers are heterotrimers.

[0196] Preferably, the method comprises: (ii) the additional step of administering to said subject a booster dose of a second polypeptide, and optionally, (iii) administering to said subject a booster dose of a third polypeptide; The second and third polypeptides (defined above) are preferably different from each other, and preferably different from the first polypeptide.

[0197] Preferably, the method comprises: (ii) the additional step of administering a second trimer boost to the subject, and optionally, (iii) administering a third trimer booster to the subject; The second and third trimers are preferably different from each other and preferably different from the first trimer.

[0198] Preferably, the first, second and third polypeptides are independently selected from the group consisting of polypeptides comprising or consisting of SEQ ID NOs: 13-17.

[0199] Preferably, the first, second and third trimers independently consist of polypeptides comprising or consisting of SEQ ID NOs: 13-17.

[0200] In a preferred embodiment, the polypeptides or homotrimers of SEQ ID NOs: 14 and 15 are administered to the subject first, the polypeptides or homotrimers of SEQ ID NOs: 13 and 16 are then administered to the subject, and the polypeptide or homotrimer of SEQ ID NO: 17 is then administered to the subject.

[0201] In another preferred embodiment, the polypeptide or trimer is administered in the form of a VLP, ie a VLP comprising polypeptide(s) trimer(s) is administered.

[0202] In another preferred embodiment, a nucleic acid molecule (preferably a vector) is administered to the subject, the nucleic acid molecule encoding one or more of the polypeptides defined above. Preferred vectors are described herein.

[0203] In one embodiment, the first and second products are administered as part of a prime-boost administration protocol, such that a first product can be administered to a subject as a "prime," followed by administration of a second product to the same subject as a "boost."

[0204] In one embodiment, the first product is a prime with an adenoviral vector of the invention and the second product is a boost with a non-replicating poxvirus vector of the invention.

[0205] In one embodiment, each of the above methods further comprises the step of administering to the subject a product of the invention.

[0206] In one embodiment, a polypeptide of the invention is administered separately from the administration of a viral vector of the invention. Preferably, the polypeptide and viral vector are administered sequentially in any order. Thus, in one embodiment, the viral vector ("V") and polypeptide ("P") can be administered in the order of VP or PV.

[0207] In certain embodiments, the method further comprises administering to the subject an adjuvant, which can be administered with any of the products of the invention.

[0208] The products of the invention can be administered in a single dose schedule (i.e., the entire dose is administered in approximately one dose), or alternatively, the products of the invention can be administered in a multiple dose schedule.

[0209] A multiple dose schedule can be one in which the main treatment course (e.g., vaccination) is one to six separate doses, followed by another dose at subsequent time intervals as needed to maintain and / or enhance the immune response, for example (for human subjects) a second dose at 1 to 4 months, and then, if necessary, a subsequent dose(s) after a further 1 to 4 months.

[0210] The dosage regimen will, at least in part, be determined by the need of the individual and be dependent on the judgment of the practitioner (eg, physician or veterinarian).

[0211] Simultaneous administration means administration at (approximately) the same time.

[0212] Sequential administration of two or more products according to the invention means that the products are administered one after the other at (approximately) different times.

[0213] For example, sequential administration can include administration of two or more products of the invention at different times, where the different times are separated by a number of days (e.g., 1, 2, 5, 10, 15, 20, 30, 60, 90, 100, 150, or 200 days).

[0214] For example, in one embodiment, the vaccines of the present invention may be administered as part of a "prime-boost" vaccination regimen.

[0215] In one embodiment, the products of the invention can be administered to a subject, such as a mammal (e.g., a human, bovine, porcine, ovine, caprine, equine, cervid, canine, or feline subject), in combination (simultaneously or sequentially) with one or more immunomodulatory agents selected from, for example, immunoglobulins, antibiotics, interleukins (e.g., IL-2, IL-12), and / or cytokines (e.g., IFN-γ).

[0216] In a further embodiment, the present invention provides a process for producing one or more of the polypeptides of the invention, the process comprising expressing in a suitable host one or more nucleic acid molecules encoding one, two or more of the polypeptides, and recovering the polypeptide product(s).

[0217] Preferably, the host is a human cell.

[0218] There are many established algorithms available for aligning two amino acid sequences. Generally, one sequence serves as a reference sequence to which a test sequence can be compared. The sequence comparison algorithm calculates the percentage of sequence homology of the test sequence(s) to the reference sequence based on predetermined program parameters. The alignment of amino acid sequences for comparison can be performed, for example, by computer-implemented algorithms (e.g., GAP, BESTFIT, FASTA, or TFASTA), or by BLAST and BLAST 2.0 algorithms.

[0219] The percentage of amino acid sequence homology and nucleotide sequence homology can be obtained by the BLAST method of alignment (Altschul et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs," Nucleic Acids Res. 25:3389-3402 and http: / / www.ncbi.nlm.nih.gov / BLAST). Preferably, standard or default alignment parameters are used.

[0220] Standard protein-protein BLAST (blastp) can be used to find similar sequences in protein databases. Like other BLAST programs, blastp is designed to find local regions of similarity. When sequence similarity spans the entire sequence, blastp also reports a global alignment, which is the preferred result for protein identification purposes. Preferably, standard or default alignment parameters are used. In some cases, the "low complexity filter" can be turned off.

[0221] BLAST protein searches can also be performed with the BLASTX program, score = 50, word length = 3. To obtain gapped alignments for comparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) can be used to perform an iterated search that detects distant relationships between molecules. (See Altschul et al. (1997) supra.) When using BLAST, Gapped BLAST, or PSI-BLAST, the default parameters of the respective programs can be used.

[0222] For nucleotide sequence comparison, MEGABLAST, discontinuous megablast, and blastn can be used to achieve this purpose. Preferably, standard or default alignment parameters are used. MEGABLAST is specifically designed to efficiently find long alignments between highly similar sequences. Discontinuous MEGABLAST can be used to find nucleotide sequences that are similar but not identical to the nucleic acids of the present invention.

[0223] The BLAST nucleotide algorithm finds similar sequences by breaking down a query into short subsequences called words. The program first identifies exact matches (word hits) to the query word. The BLAST program then extends these word hits in multiple steps to generate a final gapped alignment. In some embodiments, BLAST nucleotide searches can be performed using the BLASTN program, score=100, word length=12.

[0224] One of the important parameters that governs the sensitivity of a BLAST search is the word size. The most important reason why blastn is more sensitive than MEGABLAST is that it uses a shorter default word size (11). This makes blastn better than MEGABLAST at finding alignments with related nucleotide sequences from different organisms. The word size is adjustable in blastn and can be reduced from the default value to a minimum of 7 to increase search sensitivity.

[0225] More sensitive searches can be performed using the newly introduced non-contiguous megablast page (www.ncbi.nlm.nih.gov / Web / Newsltr / FallWinter02 / blastlab.html). This page uses an algorithm similar to that reported by Ma et al. (Bioinformatics. 2002 Mar;18(3):440-5). Non-contiguous megablast uses non-contiguous words within a longer template window as seeds for alignment extension, rather than requiring exact word matches. In coding mode, it accounts for third-base wobble by focusing on finding matches at the first and second codon positions while ignoring mismatches at third positions. Searches in non-contiguous MEGABLAST using the same word size are more sensitive and efficient than standard blastn searches using the same word size. Parameters unique to non-contiguous megablast are word size 11 or 12, template: 16, 18, or 21, and template type: coding (0), non-coding (1), or both (2).

[0226] In some embodiments, the BLASTP 2.5.0+ algorithm can be used with default parameters (such as those available from NCBI).

[0227] In another embodiment, the BLAST Global Alignment program can be used using a Needleman-Wunsch alignment of two protein sequences (such as those available from NCBI) with gap costs: Existence 11 and Extension 1.

[0228] The methods disclosed herein for identifying defined variable sites and subsequent defined variable epitopes can be applied to all subtypes of influenza A. In particular, because H3 subtype influenza A viruses evolve similarly to H1 subtype influenza A viruses, the disclosed techniques for identifying epitopes are particularly applicable to the H3 subtype of influenza A.

[0229] Thus, in a further embodiment, the present invention provides a method for identifying epitopes on the hemagglutinin head domain of an influenza virus of a defined subtype, comprising: The method comprises: (i) identifying potential antibody binding sites on the hemagglutinin head domain polypeptide of an influenza virus of a defined subtype; (ii) identifying one or more contiguous or non-contiguous sequences of head domain polypeptides within a potential antibody binding site; and (iii) comparing these sets of amino acid sequences to amino acid sequences of hemagglutinin head domains from multiple influenza strains of a defined subtype to identify defined regions of variability in these sets of amino acid sequences; Thereby, a set of positions within the hemagglutinin head domain of influenza viruses of a defined subtype that have limited variability in their amino acid composition and form epitopes are identified.

[0230] Epitopes identified in this way are under strong immune selection and are periodically repeated in a limited number of forms throughout the evolution of influenza viruses.

[0231] The methods and processes can be applied to any influenza virus. Preferably, the influenza virus is an influenza A virus.

[0232] The influenza virus can be of any subtype, for example, H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, or H18. Preferably, the influenza virus is of the H1 or H3 subtype.

[0233] Step (i) involves identifying potential antibody binding sites on the hemagglutinin head domain polypeptide of an influenza virus of a defined subtype.

[0234] This can be done by analyzing the crystal structure of a hemagglutinin head domain polypeptide. Crystal structures of two or more head domains from influenza viruses of a given subtype can be aligned to determine the residues present on the polypeptide surface and the accessibility of those residues. Generally, the accessibility of a position is the same in all crystal structures, but if a position in one crystal structure is more accessible than another, that position is designated as more accessible to prevent erroneous identification of limited variability sites.

[0235] In silico analysis can be used to determine how accessibility and binding site regions contribute to the variability of a hypothetical antibody binding site. 2 and 1000A 2 The antibody binding site between can be used to determine the variability of the accessibility parameters for amino acids with accessibility >30% to >1%.

[0236] Step (ii) entails identifying one or more contiguous or non-contiguous stretches of head domain polypeptides within a potential antibody binding site, which stretches can be contacted by the antibody.

[0237] Once a potential antibody binding site has been identified, one or more contiguous or non-contiguous stretches of head domain polypeptides within the potential antibody binding site can be identified, for example using the Swiss-pdb viewer.

[0238] Step (iii) involves comparing a series of contiguous or non-contiguous amino acid sequences with the amino acid sequences of the hemagglutinin head domain from multiple influenza strains of a defined subtype to identify regions of limited variability within these series of amino acid sequences.

[0239] For example, multiple influenza strain sequences can be derived from consensus sequences of the hemagglutinin head domain of influenza strains of a defined subtype per year. Consensus sequences per year can be generated by dividing curated hemagglutinin sequences into separate datasets based on the year the sequences were collected. Consensus sequences can then be generated using the R package "seqinr" or another consensus sequence generation program.

[0240] As used herein, the term "limited sequence variability" refers to amino acids or sequences of amino acids that have a limited number of different epitope conformations that they can form.

[0241] In another embodiment, the term "limited sequence variability" refers to amino acid positions where 0, 1, 2, 3, or 4 (preferably 0 or 1) different amino acids are found during comparison of the amino acid sequences.

[0242] In this way, it is possible to identify a conserved stretch of amino acids within the hemagglutinin head domain of influenza viruses of a defined subtype that forms an epitope.

[0243] The epitope may be one that is bound by an antibody. Preferably, the epitope is an epitope of limited variability.

[0244] The present invention provides a process for producing a polypeptide, comprising the steps of: (i) identifying a set of amino acids with limited variability within the hemagglutinin head domain of an influenza virus of a defined subtype using the methods for identifying epitopes defined herein; (ii) generating a polypeptide comprising a first region of contiguous amino acids; (a) the amino acid sequence of the first region has at least 80% sequence identity to an influenza A hemagglutinin head domain; (b) the first region has one or more amino acid substitutions at positions corresponding to amino acid positions with limited variability, where the substitutions introduce a conserved amino acid at that position; Also provided is a process whereby the polypeptide is capable of inducing antibodies in a subject against influenza A virus.

[0245] The present invention provides a process for producing an immunogenic composition, comprising the steps of: (i) identifying a set of amino acid positions of limited variability within the hemagglutinin head domain of an influenza virus of a defined subtype using the method for identifying epitopes defined herein; (ii) generating a polypeptide comprising a first region of contiguous amino acids, (a) the amino acid sequence of the first region has at least 80% sequence identity to an influenza A hemagglutinin head domain; (b) the first region has one or more amino acid substitutions at positions corresponding to conserved amino acid positions, where the substitutions introduce the amino acid that is conserved at that position; the polypeptide is capable of inducing antibodies in a subject against influenza A virus; and (iii) formulating one or more of the polypeptides into an immunogenic composition, wherein the composition optionally comprises one or more pharmaceutically acceptable carriers, adjuvants, excipients or diluents.

[0246] The composition is capable of inducing antibodies in a subject against influenza A virus.

[0247] For epitopes of influenza virus subtype H3, the amino acid sequence of the first region preferably has at least 80%, 90%, 95% or 100% sequence identity to the influenza A subtype H4, H7, H10, H14 or H15 hemagglutinin head domain.

[0248] The immunogenic composition is preferably a vaccine composition, which may be administered as a prime-boost-boost regime, for example, according to the compositions and dosing regimens described herein.

[0249] The disclosure of each reference cited herein is specifically incorporated herein by reference in its entirety. [Brief explanation of the drawings]

[0250] [Figure 1] Figure 1. Multilocus map of epitopes on the hemagglutinin (HA) monomer. Each influenza strain is thought to contain highly variable specific epitopes and less variable epitopes that are shared with other strains. [Figure 2] Figure 1 shows the cyclical replacement of major antigen types. The change pattern shown is for a three-epitope system, each with three possible variants shown in the diagram. [Figure 3] "Heat maps" of children's plasma showing periodic cross-reactivity with several influenza strains. The years on the left side of the heat map refer to the strains from which the HA1 domains were collected. Individuals are aligned left to right from 12 to 17 months of age. Percent reactivity is shown to the right of the heat map. [Figure 4] "Heatmap" of children's plasma showing cross-reactivity with several historical influenza strains. The year on the left of the heatmap refers to the strain from which the HA1 domain was obtained. Individuals are aligned left to right from 6 to 11 months of age. Percent reactivity is shown to the right of the heatmap. [Figure 5A]Figure 1 shows microneutralization assay. The x-axis shows the ratio of IC50 of pseudotyped viruses resulting in fold change. Microneutralization assay with wild-type (WT) and -147K mutant A / Solomon Islands / 3 / 2006 pseudotyped viruses. [Figure 5B] Figure 1 shows microneutralization assay. The x-axis shows the ratio of IC50 of pseudotyped viruses resulting in fold change. Microneutralization assay with wild-type (WT) and -147K mutant A / Puerto Rico / 8 / 1934 pseudotyped viruses. [Figure 5C] Figure 1 shows microneutralization assay. The x-axis shows the ratio of IC50 of pseudotyped viruses resulting in fold change. Microneutralization assay with wild-type (WT) and -147K mutant A / WSN / 1933 pseudotyped viruses. [Figure 6A] Figure 1. Sequential vaccination with chimeric HA constructs. Five groups of mice were sequentially vaccinated with the sequences outlined in (B) substituted into the H6, H5, and H11 HAs. Two additional groups were sequentially vaccinated with the H6, H5, and H11 constructs without the substituted sequences; these groups were designated "gray" and "purple." Two additional groups were mock-vaccinated; these groups were designated "white" and "black." The first two vaccinations were administered as 100 μg intramuscular injections of DNA, and the final vaccination was administered as an intramuscular injection of 8 HI units of lentivirus displaying the chimeric HA (i.e., H11 with or without substitutions) with Alum adjuvant. [Figure 6B] Pseudotype microneutralization assay using 0.5 μl of serum from a bleed at week 21. Broad neutralizing activity is generated against a lentivirus displaying an H1 HA from the 2009 influenza virus. [Figure 6C] Pseudotype microneutralization assay using 0.5 μl of serum from a bleed at week 21. Broad neutralizing activity is generated against a lentivirus displaying an H1 HA from the 1933 influenza virus. [Figure 6D] Pseudotype microneutralization assay using 0.5 μl of serum from a bleed at week 21. Broad neutralizing activity is generated against a lentivirus displaying an H1 HA from the 1934 influenza virus. [Figure 6E] Pseudotype microneutralization assay using 0.5 μl of serum from a bleed at week 21. Broad neutralizing activity is generated against a lentivirus displaying an H1 HA from the 1977 influenza virus. [Figure 6F] Pseudotype microneutralization assay using 0.5 μl of serum from a bleed at week 21. Broad neutralizing activity is generated against a lentivirus displaying the H1 HA from the 2006 influenza virus. [Figure 6G]

[0023] Figure 1 shows influenza challenge of vaccinated mice with A / California / 4 / 2009. The graph depicts the daily weight loss of mice during challenge. [Figure 6H]

[0023] Figure 1 shows influenza challenge of vaccinated mice with A / California / 4 / 2009. The graph represents daily survival of mice during challenge. [Figure 6I] Figure 1 shows influenza challenge of vaccinated mice with A / PR / 8 / 1934. The graph depicts the daily weight loss of mice during challenge. [Figure 6J] Figure 1 shows influenza challenge of vaccinated mice with A / PR / 8 / 1934. The graph represents daily survival of mice during challenge. [Figure 7A]Figure 1 shows that sites of limited variability are present in the head of H1 HA. Antibody binding sites were mapped to the A / Puerto Rico / 8 / 1934 crystal structure, and variability within those sites was determined by reference to an alignment of 2,756 H1 sequences. Only the portion of the A / Puerto Rico / 8 / 1934 crystal structure accessible for antibody binding was considered. In A, the antibody binding site of 800A2 was used to determine variability for three accessibility parameters: amino acids with accessibility >30%, >10%, or >1%. [Figure 7B] Figure 1 shows that sites of limited variability are present in the head of H1 HA. A dataset of amino acids with >10% accessibility was used to determine variability for three binding site sizes: 600, 800, or 1000 A2. Both approaches identified the same regions of limited variability within the head of H1 HA. One of these regions contains our epitope with limited variability centrally located at positions 156 / 158. The linear numbering of HA is used for the x-axis. [Figure 7C] Figure 1 shows that sites of limited variability are present in the head of H1 HA. Mapping predicted antibody binding sites onto crystal structures of HA domains from specific influenza strains. [Figure 7D] Figure 1 shows that sites of limited variability are present in the head of H1 HA. Mapping predicted antibody binding sites onto crystal structures of HA domains from specific influenza strains. [Figure 7E] Figure 1 shows that sites of limited variability are present in the head of H1 HA. Mapping predicted antibody binding sites onto crystal structures of HA domains from specific influenza strains. [Figure 7F] Figure 1 shows that sites of limited variability are present in the head of H1 HA. Mapping predicted antibody binding sites onto crystal structures of HA domains from specific influenza strains. [Figure 7G] Figure 1 shows that sites of limited variability are present in the head of H1 HA. Mapping predicted antibody binding sites onto crystal structures of HA domains from specific influenza strains. [Figure 7H] Figure 1 shows that sites of limited variability are present in the head of H1 HA. Mapping predicted antibody binding sites onto crystal structures of HA domains from specific influenza strains. [Figure 7I] Figure 1 shows that sites of limited variability are present in the head of H1 HA. Mapping predicted antibody binding sites onto crystal structures of HA domains from specific influenza strains. [Figure 7J] Figure 1 shows that sites of limited variability are present in the head of H1 HA. Mapping predicted antibody binding sites onto crystal structures of HA domains from specific influenza strains. [Figure 8A] Figure 1 shows the disordered peptide sequence corresponding to the region around amino acids 156 / 158. Crystal structures of A / BrevigMission / 1 / 1918 from the side and top are shown. The disordered peptide sequence is located on the H1 structure. Amino acid position 147 is highlighted (white and arrowed) and is present in A / BrevigMission / 1 / 1918 but absent from A / PuertoRico / 8 / 1934. [Figure 8B] Figure 1 shows the disordered peptide sequence corresponding to the region around amino acids 156 / 158. The crystal structures of A / Puerto Rico / 8 / 1934 are shown from the side and from above. The disordered peptide sequence is located on the H1 structure. Amino acid position 147 is highlighted (white and arrowed) and is present in A / Brevig Mission / 1 / 1918 but absent from A / Puerto Rico / 8 / 1934. [Figure 9A]

[0023] Figure 1 shows the cyclical activity of disordered peptide sequences of limited variability sites. Disordered peptide sequences taken from consensus sequences by year can be grouped according to their chemical properties. When aligned by time, the cyclical nature of the disordered peptide sequences becomes apparent. Other alignments of the sequences based on their chemical properties are possible; this is simply one possible embodiment and representation of the cyclical nature of this epitope region. [Figure 9B]

[0023] Figure 1 shows the cyclical activity of disordered peptide sequences of limited variability sites. Disordered peptide sequences taken from consensus sequences by year can be grouped according to their chemical properties. When aligned by time, the cyclical nature of the disordered peptide sequences becomes apparent. Other alignments of the sequences based on their chemical properties are possible; this is simply one possible embodiment and representation of the cyclical nature of this epitope region. [Figure 9C]

[0023] Figure 1 shows the cyclical activity of disordered peptide sequences of limited variability sites. Disordered peptide sequences taken from consensus sequences by year can be grouped according to their chemical properties. When aligned by time, the cyclical nature of the disordered peptide sequences becomes apparent. Other alignments of the sequences based on their chemical properties are possible; this is simply one possible embodiment and representation of the cyclical nature of this epitope region. [Figure 9D]

[0023] Figure 1 shows the cyclical activity of disordered peptide sequences of limited variability sites. Disordered peptide sequences taken from consensus sequences by year can be grouped according to their chemical properties. When aligned by time, the cyclical nature of the disordered peptide sequences becomes apparent. Other alignments of the sequences based on their chemical properties are possible; this is simply one possible embodiment and representation of the cyclical nature of this epitope region. [Figure 10] Figure 1 shows the amino acid change at position 147 cycling among four possibilities: A. Amino acid homology at position 147 cycles among lysine, arginine, isoleucine, and absence over five periods: 1918-1957 and 1977-2015. [Example]

[0251] The present invention is further illustrated by the following examples. Unless otherwise specified, parts and percentages are by weight and degrees are in degrees Celsius. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these examples, one skilled in the art will be able to ascertain the essential features of the present invention and can make various changes and modifications to adapt the present invention to various uses and conditions without departing from the spirit and scope thereof. Thus, various modifications of the present invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.

[0252] Example 1: Antigenic variation model The existence of less variable protective epitopes is consistent with influenza A population dynamics under the "antigenic variation model." This model offers an alternative to the more widely accepted "antigenic variation" model, based on a multilocus map of the virus, where each locus corresponds to an epitope region and potentially includes both limited and highly variable protective epitopes. Figure 1 shows how these may be located at known antigenic sites on the hemagglutinin (HA) monomer. The epidemic behavior of influenza can be easily explained within an antigenic variation framework, given that most influenza strains compete with each other because they share epitopes in less variable regions (Recker et al., 2007; Wikramaratna et al., 2013). Thus, although new strains can constantly be generated through mutation, the majority of these are unable to expand in the host population due to pre-existing immune responses to their less variable epitopes. This results in the cyclical dominance of antigenic types (Figure 2). In contrast to the "antigenic variation" model, antigenic distance between circulating strains does not necessarily accumulate over time, but instead expands and contracts periodically.

[0253] Carter et al. (2013) provide evidence for the antigenic drift model. Ferrets were infected with one of several historical influenza viruses. Serum antibodies were measured by hemagglutinin inhibition (HAI) assay on days 14 and 81 (Figure 5). Serum antibodies showed periodic cross-reactivity of antibodies to historical strains of virus, as predicted by the antigenic drift hypothesis.

[0254] The periodic cross-reactivity observed in Carter et al. (2013) is partially predicted by current structural bioinformatics analysis. For example, infection of ferrets with the 1957 strain is predicted to induce cross-reactive antibodies against the A / R / 8 / 1934, A / Den / 1 / 1957, A / NC / 20 / 1999, and A / Bris / 59 / 2007 strains. Cross-reactivity is observed between the A / R / 8 / 1934, A / Den / 1 / 1957, and A / NC / 20 / 1999 strains, but not the A / Bris / 59 / 2007 strain, depending on the infection.

[0255] Example 2: Cyclic cross-reactivity of infant plasma to time-dispersing H1 influenza strains A standardized enzyme-linked immunosorbent assay (ELISA) was performed using plasma samples from 12- to 17-month-old children collected in 2012. The HA1 domains from influenza strains A / California / 4 / 2009, A / USSR / 90 / 1977, A / Brevigmission / 1 / 1918, A / Solomon Islands / 3 / 2006, A / New Caledonia / 20 / 1999, A / Puerto Rico / 8 / 34, and A / WSN / 33 were purchased from Sino Biological. Adult serum was used as a standard based on their birth date. Two negative controls were run on each plate: a casein-only control and a nonreactive human plasma or VI serum control.

[0256] The results are shown in Figure 3. Plasma from 81 children aged 12 to 17 months collected in 2012 cross-reacted with the HA1 domain (the head and part of the stalk domain of HA H1) from influenza strains A / California / 4 / 2009, A / USSR / 90 / 1977, and A / BrevigMission / 1 / 1918, but not with A / Solomon Islands / 3 / 2006, A / New Caledonia / 20 / 1999, A / Puerto Rico / 8 / 34, or A / WSN / 33. All ELISA results were completed in triplicate and normalized to nonreactive human plasma. ELISA results were accepted or rejected based on the criteria described in Miura et al. (2008).

[0257] The fact that this plasma periodically reacted with a panel of historical H1N1 strains leads us to speculate that epitopes of limited variability reside in the head domain of H1 HA and circulate through a limited number of conformations as immune responses change in the host population.

[0258] Example 3: Identification of epitopes with limited variability To identify epitopes with limited variability, antibody binding sites were mapped to the A / Puerto Rico / 8 / 1934 crystal structure, and variability within those sites was determined by reference to an alignment of 2,756 H1 sequences (Figure 7). Only the portions of the A / Puerto Rico / 8 / 1934 crystal structure accessible to antibody binding were considered. This was followed by alignment of the crystal structures of A / Puerto Rico / 8 / 1934, A / Brevig Mission / 1 / 1918, and A / California / 04 / 2009 to determine the residues present on the protein surface and their accessibility. Generally, the accessibility of positions was the same across all crystal structures; however, when a position in one crystal structure was more accessible than another, that position was designated as more accessible to prevent false identification of sites with limited variability. Portions of HA contained within the virion were also not considered in the analysis.

[0259] In silico analysis was used to determine how accessibility and binding site regions contribute to the variability of a hypothetical antibody binding site. 2 The antibody binding sites were used to determine variability for three accessibility parameters: amino acids with accessibility >30%, >10%, or >1%. The dataset of positions with accessibility >10% was used to determine the variability for three binding site sizes: 600A. 2 , 800A 2 or 1000A 2 Both approaches identified the same region of limited variability within the H1 HA head (Fig. 7).

[0260] Analysis of the restricted variability sites predicted from the in silico analysis was performed by mapping the predicted epitopes to the A / Puerto Rico / 8 / 1934, A / Brevig Mission / 1 / 1918, and A / California / 04 / 2009 crystal structures using the Swiss-pdb viewer. By mapping the predicted sites to the crystal structures, potential epitopes could be identified. One site near the receptor binding site (RBS) was found within the 800A region surrounding positions 156 / 158, in a region known to be under strong immune selection but thought to be highly variable. 2 was centrally located above the area (Fig. 8; Caton et al., 1982).

[0261] Example 4: Circulating epitopes Consensus sequences for each year were generated by splitting the 12,480 curated H1 HA sequences into separate fasta files based on the year of sequence collection, and then using the R package "seqinr" to generate consensus sequences.

[0262] Analysis of the predicted binding site around positions 156 / 158 revealed that in addition to positions with uncharged residues, there were several positions where charged residues could be found, either conserved or varying between similar residue types. It is generally accepted that antibodies bind preferentially to charged residues, and therefore possible epitope permutations were defined based on the cycling of charged amino acids at positions 147, 156, 157, 158, and 159 (Kringelum et al., 2013).

[0263] At position 147, the amino acid alternated between a positively charged amino acid, lysine or arginine, a neutral amino acid, isoleucine, and no amino acid, and the site was divided into three groups based on this pattern.

[0264] Phylogenetic analysis of position 147 also identified five strains in which the amino acid was absent at position 147 during the evolution of H1 influenza in humans between 1918 and 1957 and between 1977 and 2015 (Figure 10). Cycling between lysine, arginine, isoleucine, and no amino acid was also found. The results emphasize the importance of having a vaccine that contains both arginine and lysine as positively charged amino acids. This also indicated that the site is structurally restricted and cyclable among a small number of conformations.

[0265] The 147 positive groups were then further divided based on the presence of a positively charged amino acid at positions 158 or 159, 158, and 156 or 157.

[0266] We then considered the space-filling ability of uncharged amino acids, which allows additional groups to be generated from the 147-positive / 158- or 157-positive groups based on whether an alanine or asparagine is present at position 156 (Figure 9).

[0267] Example 5: Reduction of neutralization by site-directed mutagenesis Sera from children aged 6–11 years collected in late 2006 / early 2007 broadly cross-reacted with HA1 domains from historical influenza strains (Figure 4). Cross-reactivity was greatest with the HA1 domain from A / WSN / 33, as well as with the closely related A / Solomon Islands / 3 / 2006 HA1 domain and the A / New Caledonia / 20 / 1999 HA1 domain. Cross-reactivity was also observed with A / California / 4 / 2009, A / USSR / 90 / 1977, A / Albany / 12 / 1951, A / Puerto Rico / 8 / 34, and A / Brevig Mission / 1 / 1918.

[0268] Using microneutralization assays (Figure 5), we observed that the insertion of a lysine at position 147 resulted in a decrease in neutralization of up to 32-fold against A / Solomon Islands / 3 / 2006 pseudotyped lentivirus (p-value 0.0005). We also observed a decrease in neutralization of A / WSN / 1933 pseudotyped lentivirus (p-value 0.0056) with the insertion of a lysine at position 147. Only 11 serum samples from the UK cohort showed cross-reactivity with A / PR / 8 / 1934 pseudotyped lentivirus, but the insertion of a lysine at position 147 completely abolished neutralization in eight samples and reduced it in three samples. This indicates that the majority of cross-reactivity between these strains is mediated by the epitope containing the deletion at position 147.

[0269] This data highlights the importance of amino acid position 147. Note that in the A / Solomon Islands / 3 / 2006, A / PR / 8 / 1934, and A / WSN / 1933 strains, no amino acid is included at position 147. Instead, the monomers of these viruses consist of 565 instead of 566 amino acids.

[0270] Example 6: Synthesis of Polypeptides Using Invitrogen® GeneArt Strings, chimeric HA molecules were synthesized consisting of limited variability epitopes substituted into the HA1 domain of H5, H6, or H11. Three conformations of the site were initially used.

[0271] The chimeric HA1 domain sequence was then inserted into a DNA expression construct and a lentiviral glycoprotein expression vector. DNA expression was carried out in Escherichia coli (E. coli) and purified using a Qiagen Giga Prep Kit. Lentiviruses displaying the chimeric HA were produced according to the procedure outlined in Carnell et al. (2015) and then purified by sucrose cushion centrifugation. The conformations substituted into the H6, H5, and H11 HA1 domains are shown below (amino acid positions are indicated in parentheses): blue: N(146), K(147), G(148), V(149), A(151), P(154), H(155), A(156), G(157), A(158), K(159), K(163) AAC(146)AAG(147)GGC(148)GTG(149)GCC(151)CCC(154)CAC(155)GCC(156)GGC(157)GCC(158)AAG(159)AAG(163) Hazel: N(146), I(147), G(148), V(149), A(151), S(154), H(155), A(156), G(157), K(158), S(159), K(163) AAC(146)ATC(147)GGC(148)GTG(149)GCC(151)AGC(154)CAC(155)GCC(156)GGC(157)AAG(158)AGC(159)AAG(163) green: T(146), R(147), G(148), V(149), A(151), S(154), H(155), K(156), G(157), K(158), S(159), R(163) ACC(146)AGG(147)GGC(148)GTG(149)GCC(151)AGC(154)CAC(155)AAG(156)GGC(157)AAG(158)AGC(159)AGG(163) orange: T(146), K(147), G(148), V(149), A(151), S(154), H(155), N(156), G(157), K(158), S(159), R(163) ACC(146)AAG(147)GGC(148)GTG(149)GCC(151)AGC(154)CAC(155)AAC(156)GGC(157)AAG(158)AGC(159)AGG(163) Red: T(146), None(147),), G(148), V(149), A(151), S(154), H(155), N(156), G(157), K(158), S(159), R(163) ACC(146) None(147) GGC(148) GTG(149) GCC(151) AGC(154) CAC(155) AAC(156) GGC(157) AAG(158) AGC(159) AGG(163)

[0272] These sequences correspond to those inserted into the vaccine construct and therefore any cross-reactivity can be directly attributed to them.

[0273] Example 7: Mouse sensitization Mice were immunized with influenza using the following influenza strains: (i) Concentration 1×10 5 Pfu A / California / 4 / 2009 and (ii) a concentration of 1 × 10 3 Pfu A / PR / 8 / 1934. Weight changes were monitored daily. Optimization of the sensitization experiments allows the vaccine-induced protection in mice to be quantified in vaccination studies.

[0274] The basic vaccination procedure is shown in Figure 6A.

[0275] Mice were vaccinated sequentially with the sequences outlined below.

[0276] [Table 1]

[0277] Five groups of six mice (designated Blue, Red, Hazel, Orange, and Green) were each vaccinated at week 10 by intramuscular injection of 100 μg of DNA with a different conformation of the epitope substituted into the H6 HA. At 13 weeks of age, the same groups were vaccinated intramuscularly with 100 μg of DNA with the same conformation substituted into the H5 HA. At 18 weeks of age, the same groups were vaccinated intramuscularly with the same conformation substituted into the H11 HA displayed on a lentivirus and mixed with Alum adjuvant (Alhydrogel, Invivogen). Two control groups (purple and gray) were vaccinated in the same way as the above mice, but with HA without the epitope conformation substituted into them. Finally, two additional control groups (black and white) were vaccinated at week 18 with a mock vaccine of PBS and Alum (Alhydrogel, Invivogen). Blood was collected from all groups at weeks 11, 14, 20, and 21. At week 22, the blue, orange, hazel, and purple groups were challenged with the mouse-modified A / California / 4 / 2009 virus and weighed daily. At week 22, the red, green, gray, and white groups were challenged with the mouse-modified A / PR / 8 / 1934 virus and weighed daily. The results are shown in Figure 6.

[0278] Example 8: Vaccination against H3 influenza subtypes The method for identifying sites of restricted variability and subsequent epitopes of restricted variability is applied to the H3 subtype of influenza A. Because H3 subtype influenza A viruses evolve similarly to H1 subtype influenza A viruses, this approach to identifying epitopes is equally applicable to H3 subtype influenza A. Consequently, epitopes can be identified by mapping the variability of H3 strains to the head of H3 influenza, identifying regions of restricted variability, mapping the regions to the H3 structure to identify potential epitopes, and then analyzing the consensus sequence data to identify epitopes that behave periodically as predicted by antigenic variation models.

[0279] This type of epitope conformation is located in the HAhead domains of H4, H7, H10, H14 and H15 and is expressed using VLPs or viral vectors. The vaccine combination is administered as prime-boost-boost.

[0280] Example 9: Vaccine-induced cross-reactivity ELISA assays were performed against the HA1 domains of A / PR / 8 / 1934, A / Bel / 1942, A / Albany / 14 / 1951, and A / Memphis / 3 / 1987. Relative ELISA units (REU) were calculated based on known positive samples that reached an OD of 1.0 in each assay.

[0281] [Table 2]

[0282] References Belongia, E.A. et al., 2009. Effectiveness of Inactivated Influenza Vaccines Varied Substantially with Antigenic Match from the 2004-2005 Season to the 2006-2007 Season Linked references are available on JSTOR for this article : Effectiveness of Inactivated Influenza Vaccines Varied. The Journal of Infectious Disease, 199(2), pp.159-167.Carnell et al., (2015) Pseudotype-based neutralization assays for influenza: a systematic analysis. Front Immunol. 2015 Apr 29;6:161. doi: 10.3389 / fimmu.2015.00161. eCollection 2015.Carter et al., (2013) Sequential seasonal H1N1 influenza virus infections protect ferrets against novel 2009 H1N1 influenza virus. J Virol. 2013 Feb;87(3):1400-10.Caton et al., 1982. The antigenic structure of the influenza virus A / PR / 8 / 34 hemagglutinin (H1 subtype). Cell, 31(2 Pt 1), pp.417-427.Gupta S. 2016 Immune Driven Pathogen Evolution, Encyclopaedia of Immunology (Ed. Kaye, P.) Elsevier.Krammer, F. et al., 2013. Broadly Protective Stalk-Specific Antibodies., 87(12), pp.6542-6550.Li, Y. et al., 2013. Immune history shapes specificity of pandemic H1N1 influenza antibody responses. 210(8), pp.1493-1500.Lozano, R. et al., 2012. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010 : a systematic analysis for the Global Burden of Disease Study 2010. Lancet, 380, pp. 2095-2128.Manicassamy, B. et al., 2010. Protection of mice against lethal challenge with 2009 H1N1 influenza A virus by 1918-like and classical swine H1N1 based vaccines. PLoS Pathogens, 6(1).Matsuzaki, Y. et al., 2014. Epitope Mapping of the Hemagglutinin Molecule of A / ( H1N1 ) pdm09 Influenza Virus by Using Monoclonal Antibody Escape Mutants. Journal of Virology, 88(21), pp.12364-12373.Mertz, D., Hyong, T. & Johnstone, J., 2013. Populations at risk for severe or complicated influenza illness : systematic review and meta-analysis. British Medical Journal, 5061(August), pp.1-15.Miura et al. 2008 Vaccine 26:193.Presanis, A.M. et al., 2011.Changes in severity of 2009 pandemic A / H1N1 influenza in England : a Bayesian evidence synthesis. British Medical Journal, (343), pp.1-14.Recker, M. et al., 2007. The generation of influenza outbreaks by a network of host immune responses against a limited set of antigenic types. PNAS 104:7711Taubenberger, J.K. & Morens, D.M., 2006. 1918 Influenza : the Mother of All Pandemics. Lancet, 12(1), pp.15-22.Treanor, J.J. et al., 2012. Effectiveness of Seasonal Influenza Vaccines in the United States During a Season With Circulation of All Three Vaccine Strains., pp.1-9.WHO 2016. Recommended composition of influenza virus vaccines for use in the 2016- 2017 northern hemisphere influenza season.Wikramaratna, P.S. et al., 2013. The antigenic evolution of influenza: drift or thrift? Philosophical transactions of the Royal Society of London. Series B, Biological sciences, 368(1614), p.20120200. Available at: http: / / www.pubmedcentral.nih.gov / articlerender.fcgi?artid=3678325&tool=pmcentrez&rendertype=abstract. [Sequence List Free Text]

[0283] SEQ ID NO: 1: H1 head domain - amino acid SEQ ID NO: 2: H6 head domain - amino acid SEQ ID NO: 3: H5 head domain - amino acid SEQ ID NO: 4: H11 head domain - amino acid SEQ ID NO: 5: H1 head domain - nucleotides SEQ ID NO: 6: H6 head domain - nucleotides SEQ ID NO: 7: H5 head domain - nucleotides SEQ ID NO: 8: H11 head domain - nucleotides SEQ ID NO: 9: H1 hemagglutinin-amino acid SEQ ID NO: 10: H6 hemagglutinin-amino acid SEQ ID NO: 11: H5 hemagglutinin-amino acid SEQ ID NO: 12: H11 hemagglutinin-amino acid SEQ ID NO: 13: Head domain (Blue) - Amino acid sequence Blue located in H11head SEQ ID NO: 14: Head domain (Hazel) - Amino acid Hazel sequence located in the H6 head domain SEQ ID NO: 15: head domain (green) - Green sequence of amino acids located in the H6 head domain SEQ ID NO: 16: head domain (orange) - amino acid SEQ ID NO: 17: head domain (red) - amino acids SEQ ID NO: 18: head domain (blue) - nucleotides SEQ ID NO: 19: head domain (Hazel) - nucleotides SEQ ID NO: 20: head domain (green) - nucleotides SEQ ID NO: 21: head domain (orange) - nucleotides SEQ ID NO: 22: head domain (red) - nucleotides SEQ ID NO: 23: H9 hemagglutinin-amino acid SEQ ID NO: 24: H1 cleavage site consensus sequence SEQ ID NO: 25: H5 cleavage site consensus sequence SEQ ID NO: 26: H6 cleavage site consensus sequence SEQ ID NO: 27: H9 cleavage consensus sequence SEQ ID NO: 28: H11 cleavage site consensus sequence

Claims

1. optionally together with one or more pharmaceutically acceptable carriers, adjuvants, excipients or diluents, An immunogenic composition comprising two or more polypeptides, each polypeptide independently comprising a first region of contiguous amino acids; (a) the amino acid sequence of the first region has at least 95% sequence identity to the hemagglutinin head domain of influenza A subtype H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, or H18; (b) the first region has amino acid substitutions at positions corresponding to the following positions of SEQ ID NO:9: Position 83 is E Position 85 is a negatively charged amino acid Position 146 is T, N, I or A Position 147 is a positively charged amino acid or I, or is absent Position 148 is G Position 149 is V Position 151 is A Position 154 is S or P Position 155 is H Position 156 is a positively charged amino acid or A or G or N or E Position 157 is a positively charged amino acid or A or G Position 158 is a positively charged amino acid or A or S or N or C or E Position 159 is K or A or S or N or C, and Position 163 is a positively charged amino acid; An immunogenic composition for treating influenza A, wherein the amino acid sequences of the two or more polypeptides are different, and the composition is capable of inducing antibodies in a subject against influenza A virus.

2. A composition comprising a polypeptide, wherein the amino acid sequence of the polypeptide comprises a first region; (a) the amino acid sequence of the first region has at least 95% sequence identity to the hemagglutinin head domain of influenza A subtype H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, H16, H17, or H18; (b) the first region has amino acid substitutions at positions corresponding to the following positions of SEQ ID NO:9: Position 83 is E Position 85 is a negatively charged amino acid Position 146 is T, N, I or A Position 147 is a positively charged amino acid or I, or is absent Position 148 is G Position 149 is V Position 151 is A Position 154 is S or P Position 155 is H Position 156 is a positively charged amino acid or A or G or N or E Position 157 is a positively charged amino acid or A or G Position 158 is a positively charged amino acid or A or S or N or C or E Position 159 is K or A or S or N or C, and Position 163 is a positively charged amino acid, A composition for treating influenza A, wherein the composition is capable of inducing antibodies in a subject against influenza A virus.

3. The composition of claim 1 or 2, wherein the amino acid sequence of the influenza A hemagglutinin head domain is selected from the group consisting of SEQ ID NOs: 1 to 4.

4. (A) the first region has an amino acid substitution at a position corresponding to the following position in SEQ ID NO:9: Position 83 is E Position 85 is E or D Position 146 is T or N Position 147 is R, K, I, or absent Position 148 is G Position 149 is V Position 151 is A Position 154 is S or P Position 155 is H Position 156 is A or G or K or N or E Position 157 is A or G Position 158 is A, K, or E Position 159 is A, K, C, N or S, and Position 163 is K or R; Or, (B) the first region has an amino acid substitution at a position corresponding to the following position in SEQ ID NO:9: Position 83 is E Position 85 is a negatively charged amino acid Position 146 is T, N, I or A Position 147 is a positively charged amino acid; Position 148 is G Position 149 is V Position 151 is A Position 154 is S or P Position 155 is H Position 156 is A Position 157 is G Position 158 is K or A or S or N or C Position 159 is K or A or S or N or C, and Position 163 is a positively charged amino acid; Or, (C) the first region has an amino acid substitution at a position corresponding to the following position in SEQ ID NO:9: Position 83 is E Position 85 is a negatively charged amino acid Position 146 is N Position 147 is I Position 148 is G Position 149 is V Position 151 is A Position 154 is S Position 155 is H Position 156 is K or A Position 157 is G Position 158 is A or K Position 159 is K or S, and Position 163 is a positively charged amino acid; Or, (D) the first region has an amino acid substitution at a position corresponding to the following position in SEQ ID NO:9: Position 83 is E Position 85 is a negatively charged amino acid Position 146 is T Position 147 is a positively charged amino acid Position 148 is G Position 149 is V Position 151 is A Position 154 is S Position 155 is H Position 156 is a positively charged amino acid or A or G Position 157 is a positively charged amino acid or A or G Position 158 is K Position 159 is S or C, and Position 163 is a positively charged amino acid; Or, (E) the first region has an amino acid substitution at a position corresponding to the following position in SEQ ID NO:9: Position 83 is E Position 85 is a negatively charged amino acid Position 146 is T Position 147 is a positively charged amino acid Position 148 is G Position 149 is V Position 151 is A Position 154 is S Position 155 is H Position 156 is N Position 157 is G Position 158 is a positively charged amino acid Position 159 is S, and Position 163 is a positively charged amino acid; or (F) the first region has an amino acid substitution at a position corresponding to the following position in SEQ ID NO:9: Position 83 is E Position 85 is a negatively charged amino acid Position 146 is T or N Position 147 does not exist Position 148 is G Position 149 is V Position 151 is A Position 154 is S or P Position 155 is H Position 156 is N Position 157 is G Position 158 is K Position 159 is S, and 4. The composition of claim 1, wherein position 163 is a positively charged amino acid.

5. 2. The composition of claim 1, wherein the first regions of the two or more polypeptides independently comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-17.

6. 3. The composition of claim 2, wherein the first region of the polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-17.

7. (A) one or more of the polypeptides further comprises a stretch of contiguous amino acids derived from the hemagglutinin N-terminal stalk region; or (B) the polypeptides are all 280 to 300 amino acids in length; or (C) the composition comprises two, three, four, or five of the polypeptides, all of which are different; or (D) The composition of any one of claims 1 to 6, wherein the composition comprises one or more heterotrimers of three different polypeptides or homotrimers of the same polypeptide.

8. A virus-like particle for treating influenza A, comprising two or more polypeptides according to any one of claims 1 to 7.

9. 10. A composition for treating influenza A comprising the virus-like particle of claim 8, optionally together with one or more pharmaceutically acceptable carriers, adjuvants, excipients or diluents.

10. A vaccine composition for treating influenza A, comprising a composition according to any one of claims 1 to 7 and an adjuvant.

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