Immunogenic composition

Abstract

The present invention relates to an immunogenic composition comprising two or more polypeptides. The 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.

Claims

1. A composition comprising 2, 3, 4 or 5 polypeptides, all of which are different, wherein the amino acid sequence of each polypeptide comprises a first region, wherein the first regions of two or more of the polypeptides each independently comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-17.

2. A composition comprising one or more: (i) hetero-trimers comprising three polypeptides, wherein said polypeptides are all different; or (ii) homotrimers comprising three polypeptides, wherein the polypeptides are all the same; wherein the amino acid sequence of each polypeptide comprises a first region, wherein the first region of each polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-17.

3. A virus-like particle comprising two or more polypeptides, wherein the amino acid sequence of each polypeptide comprises a first region, wherein the first region of two or more of the polypeptides each independently comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 13-17.

4. A method of: (i) treating influenza infection in a subject; or (ii) inducing a T-cell or B-cell response to an influenza antigen in a subject, the method comprising administering a therapeutically effective amount of the composition of claim 1 to a subject in need thereof.

5. The composition of claim 1, wherein the polypeptides are all 280-300 amino acids in length.

6. A method of: (i) treating influenza infection in a subject; or (ii) inducing a T-cell or B-cell response to an influenza antigen in a subject, the method comprising administering a therapeutically effective amount of the composition of claim 2 to a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1: A multi-locus representation of epitopes on a monomer of haemagglutinin (HA). Each influenza strain is assumed to contain specific epitopes of high variability as well as epitopes of low variability shared with other strains.

(2) FIG. 2: Cyclical replacement of dominant antigenic types. The dynamics shown is for a 3-epitope system, each containing 3 possible variants as indicated in the cartoon.

(3) FIG. 3: “Heat map” of plasma from children showing cyclical cross-reactivity with a number of influenza strains. The year on the left hand side of the heat map relates to the strain from which the HA1 domain was taken. Individuals are aligned from 12 to 17 months left to right. Percentage reactivity is stated to the right hand side of the heat map.

(4) FIG. 4: “Heat map” of plasma from children showing cross-reactivity with a number of historical influenza strains. The year on the left hand side of the heat map relates to the strain from which the HA1 domain was taken. Individuals are aligned from 6 to 11 months left to right. Percentage reactivity is stated to the right hand side of the heat map.

(5) FIG. 5: Microneutralisation assays. The x-axis refers to the ratio of the IC50s of the pseudotype viruses to produce a fold-change.

(6) (A) Microneutralisation assay using wild-type (WT) and −147K mutant A/Solomon Islands/3/2006 pseudotyped viruses.

(7) (B) Microneutralisation assay using wild-type (WT) and −147K mutant A/Puerto Rico/8/1934 pseudotyped viruses.

(8) (C) Microneutralisation assay using wild-type (WT) and −147K mutant A/WSN/1933 pseudotyped viruses.

(9) FIG. 6: Sequential vaccination using chimeric HA constructs.

(10) (A) Five groups of mice were sequentially vaccinated with the sequences outlined in (B), substituted into H6, H5 and H11 HAs. Two further groups were sequentially vaccinated with H6, H5 and H11 constructs without any sequence substituted into them and named ‘grey’ and ‘purple’. A further two groups were mock vaccinated and named ‘white’ and ‘black’. The first two vaccinations were administered as a 100 μg intra muscular injection of DNA, whilst the final vaccination was administered as an intra-muscular injection of 8 HI units of lentivirus displaying a chimeric HA (i.e. H11 with or without substitution) with an Alum adjuvant.
(B)-(F) Pseudotype microneutralisation assays using 0.5 μl of sera from the bleed at 21 weeks. Broad neutralising activity occurs against lentiviruses displaying H1 HAs from influenza viruses circulating in 1933, 1934, 1977, 2006 and 2009.
(G)-(J) Influenza challenge of vaccinated mice with either A/PR/8/1934 or A/California/4/2009. The graphs denote daily weight loss and percentage survival of the mice during the challenge.

(11) FIG. 7: Sites of limited variability are present in the head of H1 HA.

(12) (A) Antibody binding sites were mapped to the A/Puerto Rico/8/1934 crystal structure and the variability within those site determined by referring to an alignment of 2,756 H1 sequences. Only parts of A/Puerto Rico/8/1934 crystal structure accessible to antibody binding were considered. In A. an antibody binding site of 800 A.sup.2 was used to determining the variability for three accessibility parameters: amino acids with >30%, >10% or >1% accessibility.
(B) In B. a dataset of amino acids with >10% accessibility was used to determine the variability for three binding site sizes: 600, 800 or 1000 A.sup.2. Both approaches identified the same regions within the head of H1 HA which are of limited variability. One of these regions contains our epitope of limited variability centring of position 156/158. Linear numbering of HA is used for the x-axis.
(C)-(J) Mapping of predicted antibody-binding sites onto the crystal structures of HA domains from specified influenza strains.

(13) FIG. 8: The disrupted peptide sequence corresponding to the site surrounding amino acids 156/158. (A) shows the crystal structures of A/Brevig Mission/1/1918 and (B) shows the crystal structure of A/Puerto Rico/8/1934 from the side and above. The disrupted peptide sequence is mapped on the H1 structures. Amino acid position 147 is highlighted (in white and with an arrow) and is present in A/Brevig Mission/1/1918 but not A/Puerto Rico/8/1934.

(14) FIG. 9: Cyclical activity of the disrupted peptide sequence of a site of limited variability. Disrupted peptide sequences taken from yearly consensus sequences can be groups according to their chemical properties. If arranged with time, the cyclical nature of the disrupted peptide sequence becomes apparent. Other ways of arranging the sequences based on their chemical properties are possible and this is simply one possible incarnation and representative of the cyclical nature of this epitope region.

(15) FIG. 10: Amino acid changes at position 147 cycle between four possibilities. A. The identity of amino acid at position 147 cycles between lysine, arginine, isoleucine and is absent for five periods between 1918-1957 and 1977-2015.

EXAMPLES

(16) The present invention is further illustrated by the following Examples, in which parts and percentages are by weight and degrees are Celsius, unless otherwise stated. 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 can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, various modifications of the invention in addition to those shown and described herein will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims.

Example 1: Antigenic Thrift Model

(17) The existence of protective epitopes of low variability is consistent with the population dynamics of influenza A under the “antigenic thrift model”. This model is based on a multi-locus representation of the virus with each locus corresponding to an epitope region and presents an alternative to the more widely accepted “antigenic drift” model in having the potential to contain protective epitopes of limited variability as well as those of high variability. FIG. 1 shows how these may locate to the known antigenic sites on a monomer of haemagglutinin (HA). The epidemic behaviour of influenza can be readily explained within the antigenic thrift framework by assuming that most influenza strains are in competition with each other because they share epitopes in regions of low variability (Recker et al. 2007; Wikramaratna et al. 2013). Thus although new strains may be generated constantly through mutation, most of these cannot expand in the host population due to pre-existing immune responses against their less variable epitopes. This leads to cyclical dominance of antigenic types (FIG. 2). By contrast with the “antigenic drift” model, antigenic distance between epidemic strains does not necessarily accumulate with time; instead it periodically expands and contracts.

(18) Carter et al. (2013) provides evidence for the antigenic thrift model. Ferrets were infected with one of several historical influenza viruses. Serum antibodies were measured at day 14 and 81 by haemagglutin inhibition (HAI) assays (FIG. 5) and showed a periodic cross-reactivity of antibodies to viruses of historic strains, as predicted by the antigenic thrift hypothesis.

(19) The observed periodic cross-reactivity in Carter et al. (2013) is predicted, in part, by the current structural bioinformatics analysis. For example, infection of ferrets with the 1957 strain is predicted to induce cross-reactive antibodies to 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 on infection, but not the A/Bris/59/2007 strain.

Example 2: Cyclical Cross-Reactivity of Infant Plasma Against Chronologically-Dispersed H1 Influenza Strains

(20) Standardised enzyme-linked immunosorbant assays (ELISAs) were performed using plasma from children aged 12 to 17 months, collected in 2012. The HA1 domains from influenza strains A/California/4/2009, A/USSR/90/1977, A/Brevig Mission/1/1918, A/Solomon Islands/3/2006, A/New Caledonia/20/1999, A/Puerto Rico/8/34 and A/WSN/33 were bought from Sino Biological. Standards used sera from adults based on their date of birth. Two negative controls were run on each plate consisting of a caesin only control and a non-reactive human plasma or sera control.

(21) The results are shown in FIG. 3. Plasma from 81 children aged 12 to 17 months, collected in 2012, cross-reacted with HA1 domains (the head domain and part of the stem domain of HA H1) from influenza strains A/California/4/2009, A/USSR/90/1977 and A/Brevig Mission/1/1918 but not 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 normalised to non-reactive human plasma. ELISA results were accepted or declined based on the criteria set out in Miura et al. (2008).

(22) The fact that this plasma reacted with a panel of historical H1N1 strains in a cyclical manner lead us to infer that epitopes of limited variability are present in the head domain of H1 HA and that they cycle through a limited number of conformations as host population immunity changes.

Example 3: Identification of Epitopes of Limited Variability

(23) To identify epitopes of limited variability, antibody binding sites were mapped to the A/Puerto Rico/8/1934 crystal structure and the variability within those site determined by referring to an alignment of 2,756 H1 sequences (FIG. 7). Only parts of A/Puerto Rico/8/1934 crystal structure accessible to antibody binding were considered. This was determined by aligning the crystal structures of A/Puerto Rico/8/1934, A/Brevig Mission/1/1918 and A/California/04/2009 to determine which residues were present on the surface of the protein and the accessibility of those residues. Typically, the accessibility of the positions were the same in all crystal structures but when a position was more accessible in one crystal structure than another crystal structure, the position was allocated as being more accessible to prevent the false identification of sites of limited variability. Parts of the HA contained within the virion were also not considered for analysis.

(24) In silico analysis was used to determine how the accessibility and binding site area contributed to the variability of hypothetical antibody binding sites. An antibody binding site of 800 A.sup.2 was used to determining the variability for three accessibility parameters: amino acids with >30%, >10% or >1% accessibility. A dataset of positions with >10% accessibility was used to determine the variability for three binding site sizes: 600 A.sup.2, 800 A.sup.2 or 1000 A.sup.2. Both approaches identified the same regions of limited variability within the head of H1 HA (FIG. 7).

(25) Analysis of the sites of limited variability predicted to exist 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 Swiss-pdb viewer. By mapping the predicted sites to the crystal structures, sites that were likely to be epitopes could be identified. One site close to the receptor binding site (RBS), in a region which is known to be under strong immune selection but thought to be highly variable, centred on a 800 A.sup.2 region surrounding this positions 156/158 (FIG. 8; Caton et al. 1982).

Example 4: Cycling of Epitopes

(26) Yearly consensus sequences were generated by dividing the 12,480 curated H1 HA sequences into separate fasta files based on the year that the sequence was collected. The R package ‘seqinr’ was then used to generate consensus sequences.

(27) Analysis of the predicted binding site surrounding positions 156/158 indicated that there were a number of positions in which charged residues could be found, in addition to positions with non-charged residues, which were either conserved or changed between similar residue types. It is generally accepted that antibodies preferentially bind to charged residues and so the possible epitope permutations were defined based on the cycling of charged amino acids in positions 147, 156, 157, 158 and 159 (Kringelum et al. 2013).

(28) 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. Consequently the site was divided up based on this pattern into three groups.

(29) Phylogenetic analysis of position 147 also reveals that strains were identified in which no amino acid was present at position 147 five times during the evolution of H1 influenza in humans between 1918-1957 and 1977-2015 (FIG. 10). It was also found to cycle between lysine, arginine, isoleucine and no amino acid. Consequently, the importance to having a vaccine containing both arginine and lysine as positively charged amino acids were highlighted. This also indicated that the site is structurally limited and cycling between a small number of conformations.

(30) The 147 positive group was then further divided based on the presence of a positively charged amino acid at position 158 or 159, 158 and either a positive charged amino acid at position 156 or 157.

(31) The space filling capacity of non-charged amino acid was then considered, which allowed an addition group to be produced from 147-positive/158 or 157 positive group based on whether alanine or asparagine was present at position 156 (FIG. 9).

Example 5: Loss of Neutralisation Upon Site-Directed Mutagenesis

(32) Sera from children aged 6 to 11 years, taken in late 2006/early 2007, cross-reacted extensively with HA1 domains from historical influenza strains (FIG. 4). Cross-reactivity peaks towards to the HA1 domain from A/WSN/33 in addition to the A/New Caledonia/20/1999 HA1 domain which is closely related to the A/Solomon Islands/3/2006 HA1 domain. Cross-reactivity was also observed towards A/California/4/2009, A/USSR/90/1977, A/Albany/12/1951, A/Puerto Rico/8/34 and A/Brevig Mission/1/1918.

(33) Using a microneutralisation assay (FIG. 5), up to a 32-fold loss of neutralisation to A/Solomon Islands/3/2006 pseudotyped lentivirus was observed when a lysine was inserted at position 147 (p-value: 0.0005). Up to a 18.75-fold loss of neutralisation of A/WSN/1933 pseudotyped lentivirus was also observed with the insertion of a lysine at position 147 (p-value of 0.0056). While only a 11 serum samples from the UK cohort showed cross-reactivity with A/PR/8/1934 pseudotyped lentivirus, the insertion of a lysine at position 147 caused total loss of neutralisation in 8 samples and a reduction in 3 samples. This indicates that the bulk of cross-reactivity between these strains is mediated through an epitope which contains a deletion in position 147.

(34) This data emphasises the importance of amino acid position 147. It should be noted that in the A/Solomon Islands/3/2006, A/PR/8/1934 and A/WSN/1933 strains no amino acid is contained at position 147. Instead monomers of these viruses consist of 565 instead of 566 amino acids.

Example 6: Synthesis of Polypeptides

(35) Invitrogen® GeneArt Strings were used to synthesise the chimeric HA molecules consisting of the epitope of limited variability substituted into the HA1 domain of H5, H6, or H11. Three conformations of the site were initially used.

(36) The chimeric HA1 domain sequences were then cloned into DNA expression constructs and lentiviral glycoprotein expression vectors. The DNA expression were grown up in E. coli and purified using a Qiagen Giga Prep Kit. Lentiviruses were produced displaying the chimeric HAs via the protocol outlined in Carnell et al., (2015) before being purified by sucrose cushion centrifugation. The conformations substituted into the H6, H5 and H11 HA1 domains are provided below (amino acid position is denoted in brackets):

(37) Blue:

(38) N (146), K (147), G (148), V (149), A (151), P (154), H (155), A (156), G (157), A (158), K (159), K (163)

(39) AAC (146) AAG (147) GGC (148) GTG (149) GCC (151) CCC (154) CAC (155) GCC (156) GGC (157) GCC (158) AAG (159) AAG (163)

(40) Hazel:

(41) N (146), I (147), G (148), V (149), A (151), S (154), H (155), A (156), G (157), K (158), S (159), K (163)

(42) AAC (146) ATC (147) GGC (148) GTG (149) GCC (151) AGC (154) CAC (155) GCC (156) GGC (157) AAG (158) AGC (159) AAG (163)

(43) Green:

(44) T (146), R (147), G (148), V (149), A (151), S (154), H (155), K (156), G (157), K (158), S (159), R (163)

(45) ACC (146) AGG (147) GGC (148) GTG (149) GCC (151) AGC (154) CAC (155) AAG (156) GGC (157) AAG (158) AGC (159) AGG (163)

(46) Orange:

(47) T (146), K (147), G (148), V (149), A (151), S (154), H (155), N (156), G (157), K (158), S (159), R (163)

(48) ACC (146) AAG (147) GGC (148) GTG (149) GCC (151) AGC (154) CAC (155) AAC (156) GGC (157) AAG (158) AGC (159) AGG (163)

(49) Red:

(50) T (146), Absent (147), G (148), V (149), A (151), S (154), H (155), N (156), G (157), K (158), S (159), R (163)

(51) ACC (146) Absent (147) GGC (148) GTG (149) GCC (151) AGC (154) CAC (155) AAC (156) GGC (157) AAG (158) AGC (159) AGG (163)

(52) These sequences correspond to those cloned into the vaccine constructs and so any cross-reactivity can be directly attributed to them.

Example 7: Mouse Challenges

(53) Mouse influenza challenges are performed with influenza strains:

(54) (i) A/California/4/2009 at a concentration of 1*10.sup.5 Pfu and (ii) A/PR/8/1934 at a concentration of 1*10.sup.3 Pfu. Weight changes were monitored on a daily basis. The optimisation of challenge experiments enables the protection induced by the vaccine in mice to be quantified in the vaccination studies.

(55) The basic vaccination protocol is shown in FIG. 6A.

(56) Mice were sequentially vaccinated with the sequences outlined below:

(57) TABLE-US-00001 Position Name 146 147 148 149 151 154 155 156 157 158 159 163 Blue N K G V A P H A G A K K Hazel N I G V A S H A G K S K Green T R G V A S H K G K S R Orange T K G V A S H N G K S R Red T Absent G V A S H N G K S R

(58) Five groups of six mice (named blue, red, hazel, orange and green) were vaccinated via intramuscular injection of 100 μg of DNA each with a different conformation of the epitope substituted into H6 HA at 10 weeks. At 13 weeks of age, the same groups were vaccinated via intramuscular injection of 100 μg of DNA with the same conformation substituted into H5 HA. At 18 weeks of age, the same groups were vaccinated via intramuscular with the same conformation substituted into H11 HA displayed on a lentivirus and mixed with Alum adjuvant (Alhydrogel, Invivogen). Two control groups (purple and grey) were vaccinated in the same manner as the aforementioned mice with the HAs without the epitope conformations substituted into them. Finally, two further control groups (black and white) were mock vaccinated at 18 weeks with PBS and Alum (Alhydrogel, Invivogen). At 11 weeks, 14 weeks, 20 weeks and 21 weeks all groups were bled. At 22 weeks, the blue, orange, hazel and purple groups were challenged with mouse adapted A/California/4/2009 virus and weighed daily. At 22 weeks, the red, green, grey and white groups were challenged with mouse adapted A/PR/8/1934 virus and weighed daily. The results are shown in FIG. 6.

Example 8: Vaccination Against the H3 Influenza Subtype

(59) The method of identifying sites of limited variability and subsequent epitopes of limited variability is applied to the H3 subtype of influenza A. As H3 subtype influenza A virus evolves in a similar way to the H1 subtype influenza A virus, 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 limited variability, mapping said regions to H3 structures to identify potential epitopes and then analysing consensus sequence data to identify epitopes behaving in a cyclical manner predicted by antigenic thrift model

(60) Epitope conformations of this type are placed in the HA head domains of H4, H7 H10, H14 and H15 and expressed using VLPs or viral vectors. The vaccine combination is administered as a prime-boost-boost.

Example 9: Cross-Reactivity Produced by the Vaccines

(61) ELISA assays were performed against the HA1 domain 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 a known positive sample reaching an OD of 1.0 in each assay.

(62) TABLE-US-00002 HA1 domains (REU) Vaccine groups A/PR/8/ A/Albany/ A/Memphis/ (pooled sera samples) 1934 A/Bel/1942 14/1951 3/1987 Blue — — — — Hazel — — — 329 Green 205 — — 565 Orange 231 224 317 735 Red 770 — — 307 H5 + H6 + H11 control — — — — Unvaccinated control — — — —

REFERENCES

(63) 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 February; 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 104:7711 Taubenberger, 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.

(64) TABLE-US-00003 SEQUENCES H1 head domain-amino acid SEQ ID NO: 1 CKLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSSSDNGTCYPGDFIDYEE LREQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSY PKLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGTSRYSKKFKPEIAIR PKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDC H6 head domain-amino acid SEQ ID NO: 2 CKILNKAPLDLRGCTIEGWILGNPQCDLLLGDQSWSYIVERPTAQNGICYPGTLNEVEE LKALIGSGERVERFEMFPKSTWAGVDTNSGVTSACPYNSGSSFYRNLLWIIKTKSAAY PVIKGTYNNTGNQPILYFWGVHHPPDTNEQNTLYGSGDRYVRMGTESMNFAKSPEIA ARPAVNGQRGRIDYYWSVLKPGETLNVESNGNLIAPVVYAYKFVSTNNKGAVFKSNLPI ENC H5 head domain-amino acid SEQ ID NO: 3 KRSYNNTNQEDLLVLWGIHHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKV H11 head domain-amino acid SEQ ID NO: 4 CSIDGKAPISLGDCSFAGWILGNPMCDDLIGKTSWSYIVEKPNPTNGICYPGTLENEEE LRLKFSGVLEFSKFEAFTSNGWGAVNSGAGVTAACKFGSSNSFFRNMVWLIHQSGTY PVIRRTFNNTKGRDVLMVWGVHHPATLKEHQDLYKKDSSYVAVGSESYNRRFTPEIST RPKVNGQAGRMTFYVVTIVKPGEAITFESNGAFLAPRYAFELVSLGNGKLFRSDLNIESC H1 head domain-nucleotide SEQ ID NO: 5 TGCAAGCTGAGGGGCGTGGCCCCCCTGCACCTGGGCAAGTGCAACATCGCCGGCTGGATC CTGGGCAACCCCGAGTGCGAGAGCCTGAGCACCGCCAGCAGCTGGAGCTACATCGTGGAG ACCAGCAGCAGCGACAACGGCACCTGCTACCCCGGCGACTTCATCGACTACGAGGAGCTG AGGGAGCAGCTGAGCAGCGTGAGCAGCTTCGAGAGGTTCGAGATCTTCCCCAAGACCAGC AGCTGGCCCAACCACGACAGCAACAAGGGCGTGACCGCCGCCTGCCCCCACGCCGGCGCC AAGAGCTTCTACAAGAACCTGATCTGGCTGGTGAAGAAGGGCAACAGCTACCCCAAGCTG AGCAAGAGCTACATCAACGACAAGGGCAAGGAGGTGCTGGTGCTGTGGGGCATCCACCAC CCCAGCACCAGCGCCGACCAGCAGAGCCTGTACCAGAACGCCGACGCCTACGTGTTCGTG GGCACCAGCAGGTACAGCAAGAAGTTCAAGCCCGAGATCGCCATCAGGCCCAAGGTGAGG GACCAGGAGGGCAGGATGAACTACTACTGGACCCTGGTGGAGCCCGGCGACAAGATCACC TTCGAGGCCACCGGCAACCTGGTGGTGCCCAGGTACGCCTTCGCCATGGAGAGGAACGCC GGCAGCGGCATCATCATCAGCGACACCCCCGTGCACGACTGC H6 head domain-nucleotide SEQ ID NO: 6 TGCAAGATCCTGAACAAGGCCCCCCTGGACCTGAGGGGCTGCACCATCGAGGGCTGGATC CTGGGCAACCCCCAGTGCGACCTGCTGCTGGGCGACCAGAGCTGGAGCTACATCGTGGAG AGGCCCACCGCCCAGAACGGCATCTGCTACCCCGGCACCCTGAACGAGGTGGAGGAGCTG AAGGCCCTGATCGGCAGCGGCGAGAGGGTGGAGAGGTTCGAGATGTTCCCCAAGAGCACC TGGGCCGGCGTGGACACCAACAGCGGCGTGACCAGCGCCTGCCCCTACAACAGCGGCAGC AGCTTCTACAGGAACCTGCTGTGGATCATCAAGACCAAGAGCGCCGCCTACCCCGTGATC AAGGGCACCTACAACAACACCGGCAACCAGCCCATCCTGTACTTCTGGGGCGTGCACCAC CCCCCCGACACCAACGAGCAGAACACCCTGTACGGCAGCGGCGACAGGTACGTGAGGATG GGCACCGAGAGCATGAACTTCGCCAAGAGCCCCGAGATCGCCGCCAGGCCCGCCGTGAAC GGCCAGAGGGGCAGGATCGACTACTACTGGAGCGTGCTGAAGCCCGGCGAGACCCTGAAC GTGGAGAGCAACGGCAACCTGATCGCCCCCTGGTACGCCTACAAGTTCGTGAGCACCAAC AACAAGGGCGCCGTGTTCAAGAGCAACCTGCCCATCGAGAACTGC H5 head domain-nucleotide SEQ ID NO: 7 TGCGACCTGGACGGCGTGAAGCCCCTGATCCTGAGGGACTGCAGCGTGGCCGGCTGGCTG CTGGGCAACCCCATGTGCGACGAGTTCCTGAACGTGCCCGAGTGGAGCTACATCGTGGAG AAGGCCAACCCCGCCAACGACCTGTGCTACCCCGGCAACTTCAACGACTACGAGGAGCTG AAGCACCTGCTGAGCAGGATCAACCACTTCGAGAAGATCCAGATCATCCCCAAGAGCAGC TGGAGCGACCACGAGGCCAGCAGCGGCGTGAGCAGCGCCTGCCCCTACCAGGGCAGGAGC AGCTTCTTCAGGAACGTGGTGTGGCTGATCAAGAAGAACAACGCCTACCCCACCATCAAG AGGAGCTACAACAACACCAACCAGGAGGACCTGCTGGTGCTGTGGGGCATCCACCACCCC AACGACGCCGCCGAGCAGACCAAGCTGTACCAGAACCCCACCACCTACATCAGCGTGGGC ACCAGCACCCTGAACCAGAGGCTGGTGCCCAAGATCGCCACCAGGAGCAAGGTGAACGGC CAGAGCGGCAGGATGGAGTTCTTCTGGACCATCCTGAAGCCCAACGACGCCATCAACTTC GAGAGCAACGGCAACTTCATCGCCCCCGAGTACGCCTACAAGATCGTGAAGAAGGGCGAC AGCACCATCATGAAGAGCGAGCTGGAGTACGGCAACTGC H11 head domain-nucleotide SEQ ID NO: 8 TGCAGCATCGACGGCAAGGCCCCCATCAGCCTGGGCGACTGCAGCTTCGCCGGCTGGATC CTGGGCAACCCCATGTGCGACGACCTGATCGGCAAGACCAGCTGGAGCTACATCGTGGAG AAGCCCAACCCCACCAACGGCATCTGCTACCCCGGCACCCTGGAGAACGAGGAGGAGCTG AGGCTGAAGTTCAGCGGCGTGCTGGAGTTCAGCAAGTTCGAGGCCTTCACCAGCAACGGC TGGGGCGCCGTGAACAGCGGCGCCGGCGTGACCGCCGCCTGCAAGTTCGGCAGCAGCAAC AGCTTCTTCAGGAACATGGTGTGGCTGATCCACCAGAGCGGCACCTACCCCGTGATCAGG AGGACCTTCAACAACACCAAGGGCAGGGACGTGCTGATGGTGTGGGGCGTGCACCACCCC GCCACCCTGAAGGAGCACCAGGACCTGTACAAGAAGGACAGCAGCTACGTGGCCGTGGGC AGCGAGAGCTACAACAGGAGGTTCACCCCCGAGATCAGCACCAGGCCCAAGGTGAACGGC CAGGCCGGCAGGATGACCTTCTACTGGACCATCGTGAAGCCCGGCGAGGCCATCACCTTC GAGAGCAACGGCGCCTTCCTGGCCCCCAGGTACGCCTTCGAGCTGGTGAGCCTGGGCAAC GGCAAGCTGTTCAGGAGCGACCTGAACATCGAGAGCTGC H1 haemagglutinin-amino acid SEQ ID NO: 9 MKAILVVLLYTFATANADTLCIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDKHNGKLC KLRGVAPLHLGKCNIAGWILGNPECESLSTASSWSYIVETSSSDNGTCYPGDFIDYEEL REQLSSVSSFERFEIFPKTSSWPNHDSNKGVTAACPHAGAKSFYKNLIWLVKKGNSYP KLSKSYINDKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGTSRYSKKFKPEIAIR PKVRDQEGRMNYYWTLVEPGDKITFEATGNLVVPRYAFAMERNAGSGIIISDTPVHDC NTTCQTPKGAINTSLPFQNIHPITIGKCPKYVKSTKLRLATGLRNVPSIQSRGLFGAIAGF IEGGVVTGMVDGVVYGYHHQNEQGSGYAADLKSTQNAIDEITNKVNSVIEKMNTQFTAV GKEFNHLEKRIENLNKKVDDGFLDIVVTYNAELLVLLENERTLDYHDSNVKNLYEKVRS QLKNNAKEIGNGCFEFYHKCDNTCMESVKNGTYDYPKYSEEAKLNREEIDGVKLESTR IYQILAIYSTVASSLVLVVSLGAISFWMCSNGSLQCRICI H6 haemagglutinin-amino acid SEQ ID NO: 10 MIAIIVIAILAATGKSDKICIGYHANNSTTQVDTILEKNVTVTHSVELLENQKEERFCKILNK APLDLRGCTIEGWILGNPQCDLLLGDQSWSYIVERPTAQNGICYPGTLNEVEELKALIG SGERVERFEMFPKSTWAGVDTNSGVTSACPYNSGSSFYRNLLWIIKTKSAAYPVIKGT YNNTGNQPILYFWGVHHPPDTNEQNTLYGSGDRYVRMGTESMNFAKSPEIAARPAVN GQRGRIDYYWSVLKPGETLNVESNGNLIAPVVYAYKFVSTNNKGAVFKSNLPIENCDAT CQTIAGVLRTNKTFQNVSPLWIGECPKYVKSESLRLATGLRNVPQIETRGLFGAIAGFIE GGVVTGMIDGVVYGYHHENSQGSGYAADRESTQKAIDGITNKVNSIIDKMNTQFEAVDH EFSNLERRIDNLNKRMEDGFLDVVVTYNAELLVLLENERTLDLHDANVKNLYEKVKSQL RDNANDLGNGCFEFWHKCDNECIESVKNGTYDYPKYQDESKLNRQEIESV KLENLGVYQILAIYSTVSSSLVLVGLIIAMGLWMCSNGSMQCRICI H5 haemagglutinin-amino acid SEQ ID NO: 11 YNNTNQEDLLVLWGIEIHPNDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQ GAINSSMPFHNIEIPLTIGECPKYVKSNRLVLATGLRNSPQRKKRGLFGAIAGFIEGGWQG MVDGWYGYEIHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREENNL ERRIENLNKKMEDGELDVWTYNAELLVLMENERTLDETIDSNVKNLYDKVRLQLRDNA KELGNGCFEEYEIRCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIY STVASSLALAIMVAGLSLWMCSNGSLQCRICI H11 haemagglutinin-amino acid SEQ ID NO: 12 MKKTLLFAAIIICIQADEICIGYLSNNSTEKVDTIIESNVTVTSSVELVENEHTGSFCSIDGK APISLGDCSFAGWILGNPMCDDLIGKTSWSYIVEKPNPTNGICYPGTLENEEELRLKFS GVLEFSKFEAFTSNGWGAVNSGAGVTAACKFGSSNSFFRNMVWLIHQSGTYPVIRRT FNNTKGRDVLMVWGVHHPATLKEHQDLYKKDSSYVAVGSESYNRRFTPEISTRPKVN GQAGRMTFYVVTIVKPGEAITFESNGAFLAPRYAFELVSLGNGKLFRSDLNIESCSTKCQ SEIGGINTNRSFHNVHRNTIGDCPKYVNVKSLKLATGLRNVPAIATRGLFGAIAGFIEGG WPGLINGVVYGFQHRNEEGTGIAADKESTQKAIDQITSKVNNIVDRMNTNFESVQHEFS EIEERINQLSKHVDDSVIDIWSYNAQLLVLLENEKTLDLHDSNVRNLHEKVRRMLKDNA KDEGNGCFTFYHKCDNECIEKVRNGTYDHKEFEEESKLNRQEIEGVKLDSNGNVYKIL SIYSCIASSLVLAAIIMGFILWACSNGSCRCTICI Head domain (blue)-amino acid BLUE SEQUENCE PLACED INTO THE H11 HEAD SEQ ID NO: 13 SFFKNMVWLIHQSGTYPVIRRTFNNTKGRDVLMVWGVHHPATLKEHQDLYKKDSSYV AVGSESYNRRFTPEISTRPKVNGQAGRMTFYVVTIVKPGEAITFESNGAFLAPRYAFELV 0 Head domain (hazel)-amino acid HAZEL SEQUENCE PLACED INTO THE H6 HEAD DOMAIN SEQ ID NO: 14 LKALIGSGERVERFEMFPKSTWAGVDT NIGV TAAC SHAGKS SFYKNLLWIIKTKSAAYPVIKGTYNNTGNQPILYFWGVHHPPDTNEQNTLYGSGDRYVR MGTESMNFAKSPEIAARPAVNGQRGRIDYYWSVLKPGETLNVESNGNLIAPVVYAYKF Head domain (green)-amino acid GREEN SEQUENCE PLACED INTO H6 HEAD DOMAIN. SEQ ID NO: 15 CKILNKAPLDLRGCTIEGWILGNPECELLLGDQSWSYIVERPTAQNGICYPGTLNEVEE LKALIGSGERVERFEMFPKSTWAGVDTTRGVTAACSH KGKSSFYKNLLWIIKTKSAAYP VIKGTYNNTGNQPILYFWGVHHPPDTNEQNTLYGSGDRYVRMGTESMNFAKSPEIAA RPAVNGQRGRIDYYWSVLKPGETLNVESNGNLIAPVVYAYKFVSTNNKGAVFKSNLPIE NC Head domain (orange)-amino acid SEQ ID NO: 16 AVGSESYNRRFTPEISTRPKVNGQAGRMTFYVVTIVKPGEAITFESNGAFLAPRYAFELV Head domain (red)-amino acid SEQ ID NO: 17 NDAAEQTKLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPN Head domain (blue)-nucleotide SEQ ID NO: 18 TGCAGCATCGACGGCAAGGCCCCCATCAGCCTGGGCGACTGCAGCTTCGCCGGCTGGATC CTGGGCAACCCCGAGTGCGAGGACCTGATCGGCAAGACCAGCTGGAGCTACATCGTGGAG AAGCCCAACCCCACCAACGGCATCTGCTACCCCGGCACCCTGGAGAACGAGGAGGAGCTG AGGCTGAAGTTCAGCGGCGTGCTGGAGTTCAGCAAGTTCGAGGCCTTCACCAGCAACGGC TGGGGCGCCGTGAACAGCAACAGGGGCGTGACCGCCGCCTGCCCCCACGCCGGCGCCAAG AGCTTCTTCAAGAACATGGTGTGGCTGATCCACCAGAGCGGCACCTACCCCGTGATCAGG AGGACCTTCAACAACACCAAGGGCAGGGACGTGCTGATGGTGTGGGGCGTGCACCACCCC GCCACCCTGAAGGAGCACCAGGACCTGTACAAGAAGGACAGCAGCTACGTGGCCGTGGGC AGCGAGAGCTACAACAGGAGGTTCACCCCCGAGATCAGCACCAGGCCCAAGGTGAACGGC CAGGCCGGCAGGATGACCTTCTACTGGACCATCGTGAAGCCCGGCGAGGCCATCACCTTC GAGAGCAACGGCGCCTTCCTGGCCCCCAGGTACGCCTTCGAGCTGGTGAGCCTGGGCAAC GGCAAGCTGTTCAGGAGCGACCTGAACATCGAGAGCTGC Head domain (hazel)-nucleotide SEQ ID NO: 19 TGCAAGATCCTGAACAAGGCCCCCCTGGACCTGAGGGGCTGCACCATCGAGGGCTGGATC CTGGGCAACCCCGAGTGCGAGCTGCTGCTGGGCGACCAGAGCTGGAGCTACATCGTGGAG AGGCCCACCGCCCAGAACGGCATCTGCTACCCCGGCACCCTGAACGAGGTGGAGGAGCTG AAGGCCCTGATCGGCAGCGGCGAGAGGGTGGAGAGGTTCGAGATGTTCCCCAAGAGCACC TGGGCCGGCGTGGACACCAACATCGGCGTGACCGCCGCCTGCAGCCACGCCGGCAAGAGC AGCTTCTACAAGAACCTGCTGTGGATCATCAAGACCAAGAGCGCCGCCTACCCCGTGATC AAGGGCACCTACAACAACACCGGCAACCAGCCCATCCTGTACTTCTGGGGCGTGCACCAC CCCCCCGACACCAACGAGCAGAACACCCTGTACGGCAGCGGCGACAGGTACGTGAGGATG GGCACCGAGAGCATGAACTTCGCCAAGAGCCCCGAGATCGCCGCCAGGCCCGCCGTGAAC GGCCAGAGGGGCAGGATCGACTACTACTGGAGCGTGCTGAAGCCCGGCGAGACCCTGAAC GTGGAGAGCAACGGCAACCTGATCGCCCCCTGGTACGCCTACAAGTTCGTGAGCACCAAC AACAAGGGCGCCGTGTTCAAGAGCAACCTGCCCATCGAGAACTGC Head domain (green)-nucleotide SEQ ID NO: 20 TGCAAGATCCTGAACAAGGCCCCCCTGGACCTGAGGGGCTGCACCATCGAGGGCTGGATC CTGGGCAACCCCGAGTGCGAGCTGCTGCTGGGCGACCAGAGCTGGAGCTACATCGTGGAG AGGCCCACCGCCCAGAACGGCATCTGCTACCCCGGCACCCTGAACGAGGTGGAGGAGCTG AAGGCCCTGATCGGCAGCGGCGAGAGGGTGGAGAGGTTCGAGATGTTCCCCAAGAGCACC TGGGCCGGCGTGGACACCACCAGGGGCGTGACCGCCGCCTGCAGCCACAAGGGCAAGAGC AAGAGCTTCTACAAGAACCTGCTGTGGATCATCAAGACCAAGAGCGCCGCCTACCCCGTG ATCAAGGGCACCTACAACAACACCGGCAACCAGCCCATCCTGTACTTCTGGGGCGTGCAC CACCCCCCCGACACCAACGAGCAGAACACCCTGTACGGCAGCGGCGACAGGTACGTGAGG ATGGGCACCGAGAGCATGAACTTCGCCAAGAGCCCCGAGATCGCCGCCAGGCCCGCCGTG AACGGCCAGAGGGGCAGGATCGACTACTACTGGAGCGTGCTGAAGCCCGGCGAGACCCTG AACGTGGAGAGCAACGGCAACCTGATCGCCCCCTGGTACGCCTACAAGTTCGTGAGCACC AACAACAAGGGCGCCGTGTTCAAGAGCAACCTGCCCATCGAGAACTGC Head domain (orange)-nucleotide SEQ ID NO: 21 TGCAGCATCGACGGCAAGGCCCCCATCAGCCTGGGCGACTGCAGCTTCGCCGGCTGGATC CTGGGCAACCCCGAGTGCGAGGACCTGATCGGCAAGACCAGCTGGAGCTACATCGTGGAG AAGCCCAACCCCACCAACGGCATCTGCTACCCCGGCACCCTGGAGAACGAGGAGGAGCTG AGGCTGAAGTTCAGCGGCGTGCTGGAGTTCAGCAAGTTCGAGGCCTTCACCAGCAACGGC TGGGGCGCCGTGAACAGCACCAAGGGCGTGACCGCCGCCTGCAGCCACAACGGCAAGAGC AGCTTCTTCAGGAACATGGTGTGGCTGATCCACCAGAGCGGCACCTACCCCGTGATCAGG AGGACCTTCAACAACACCAAGGGCAGGGACGTGCTGATGGTGTGGGGCGTGCACCACCCC GCCACCCTGAAGGAGCACCAGGACCTGTACAAGAAGGACAGCAGCTACGTGGCCGTGGGC AGCGAGAGCTACAACAGGAGGTTCACCCCCGAGATCAGCACCAGGCCCAAGGTGAACGGC CAGGCCGGCAGGATGACCTTCTACTGGACCATCGTGAAGCCCGGCGAGGCCATCACCTTC GAGAGCAACGGCGCCTTCCTGGCCCCCAGGTACGCCTTCGAGCTGGTGAGCCTGGGCAAC GGCAAGCTGTTCAGGAGCGACCTGAACATCGAGAGCTGC Head domain (red)-nucleotide SEQ ID NO: 22 TGCGACCTGGACGGCGTGAAGCCCCTGATCCTGAGGGACTGCAGCGTGGCCGGCTGGCTG CTGGGCAACCCCGAGTGCGAGGAGTTCCTGAACGTGCCCGAGTGGAGCTACATCGTGGAG AAGGCCAACCCCGCCAACGACCTGTGCTACCCCGGCAACTTCAACGACTACGAGGAGCTG AAGCACCTGCTGAGCAGGATCAACCACTTCGAGAAGATCCAGATCATCCCCAAGAGCAGC TGGAGCGACCACGAGACCGGCGGCGTGAGCGCCGCCTGCGCCAGCCACAACGGCAAGAGC AGCTTCTTCAGGAACGTGGTGTGGCTGATCAAGAAGAACAACGCCTACCCCACCATCAAG AGGAGCTACAACAACACCAACCAGGAGGACCTGCTGGTGCTGTGGGGCATCCACCACCCC AACGACGCCGCCGAGCAGACCAAGCTGTACCAGAACCCCACCACCTACATCAGCGTGGGC ACCAGCACCCTGAACCAGAGGCTGGTGCCCAAGATCGCCACCAGGAGCAAGGTGAACGGC CAGAGCGGCAGGATGGAGTTCTTCTGGACCATCCTGAAGCCCAACGACGCCATCAACTTC GAGAGCAACGGCAACTTCATCGCCCCCGAGTACGCCTACAAGATCGTGAAGAAGGGCGAC AGCACCATCATGAAGAGCGAGCTGGAGTACGGCAACTGC H9 haemagglutinin-amino acid SEQ ID NO: 23 METVSLITILLVVTVSNADKICIGYQSTNSTETVDTLTENNVPVTHAKELLHTEHNGML CATSLGHPLILDTCTIEGLIYGNPSCDLLLGGREWSYIVERPSAVNGLCYPGNVENLEEL RSLFSSARSYQRIQIFPDTIWNVSYSGTSKACSDSFYRSMRWLTQKNNAYPIQDAQYT NNQGKNILFMWGINHPPTDTAQTNLYTRTDTTTSVATEEINRTFKPLIGPRPLVNGLQG RIDYYWSVLKPGQTLRIRSNGNLIAPVVYGHILSGESHGRILKTDLKRGSCTVQCQTEKG GLNTTLPFQNVSKYAFGNCSKYIGIKSLKLAVGLRNVPSRSSRGLFGAIAGFIEGGWSG LVAGVVYGFQHSNDQGVGMAADRDSTQKAIDKITSKVNNIVDKMNKQYEIIDHEFSEVE TRLNMINNKIDDQIQDIWAYNAELLVLLENQKTLDEHDANVNNLYNKVKRALGSNAVED GKGCFELYHKCDDQCMETIRNGTYNRRKYQEESKLERQKIEGVKLESEGTYKILTIYST VASSLVIAMGFAAFLFWAMSNGSCRCNICI