Methods for producing broadly protective vaccine compositions comprising neuraminidase

Abstract

The present disclosure relates to a cluster-based consensus method for producing recombinant neuraminidase (NA) polypeptides. The present disclosure further relates to influenza vaccine compositions comprising the recombinant NA polypeptides.

Description

[0001] This application claims the benefit of priority to U.S. Provisional Application No. 62 / 649,002, filed on March 28, 2018, and U.S. Provisional Application No. 62 / 718,527, filed on August 14, 2018, the contents of both of which are incorporated by reference in their entirety.

[0002] This application contains a sequence listing that has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy created on March 27, 2019 is named 01121-0037-00PCT_SL.txt and is 69,015 bytes in size.

[0003] Influenza is a highly contagious respiratory disease caused by one or more influenza viruses. Currently, vaccines provide the most effective defense against influenza. The World Health Organization updates the vaccine composition every year to adapt to changes in circulating influenza strains. However, vaccine mismatches due to incorrect predictions can lead to high morbidity and mortality even in vaccinated populations.

[0004] Influenza viruses contain two structural glycoproteins on the surface of the viral membrane, namely hemagglutinin (HA) and neuraminidase (NA). HA binds sialic acid and is responsible for viral entry, while NA is responsible for releasing the virus from infected cells by removing sialic acid. Current influenza vaccines are generally based on inducing an immune response to HA. However, the variability of HA may cause such vaccines to be effective only for a small number of related circulating strains. NA is also generally included in influenza vaccine compositions. However, in current vaccine formulations, the content and activity of NA have not yet been standardized.

[0005] There remains a need for effective influenza vaccines that can provide broad, long-term (eg, multi-season) protection against influenza viruses, including mismatched strains. Summary of the invention

[0006] The present invention provides, inter alia, a cluster-based consensus (CBC) method for generating NA polypeptides capable of eliciting a broadly reactive and protective immune response against a variety of influenza strains. In various embodiments, the method comprises:

[0007] Selecting more than one influenza NA polypeptide sequence and aligning the sequences;

[0008] Compute pairwise similarity / dissimilarity matrix;

[0009] Identifying and creating clusters of similar sequences from the pairwise similarity / dissimilarity matrix;

[0010] In each cluster, determining the presence or absence of a consensus amino acid at each position in the sequence alignment using a pairwise alignment method, wherein if the amino acid frequency at a given position is 50% or greater, the amino acid is designated as a consensus amino acid, and if the amino acid frequency at a given position is less than 50%, the amino acid is designated as a variable amino acid;

[0011] Generate a first sequence including consensus amino acids and variable amino acids for each cluster;

[0012] Optionally, if more than one cluster is analyzed, the first sequence generated in one cluster is compared to sequences generated in another cluster or clusters by:

[0013] o Align the resulting sequences for each cluster;

[0014] o determining the presence or absence of a consensus amino acid at each position in the sequence alignment using a pairwise alignment method, wherein if the amino acid frequency at a given position is 50% or greater, the amino acid is designated as a consensus amino acid, and if the amino acid frequency at a given position is less than 50%, the amino acid is designated as a variable amino acid; and

[0015] o generating a second sequence comprising consensus amino acids and variable amino acids;

[0016] In either the generated first sequence or the generated second sequence, the consensus amino acid for each variable amino acid position is determined by:

[0017] o generating a set of test sequences based on the first sequence or the second sequence, wherein the test amino acid is located at the variable amino acid position;

[0018] o perform molecular modeling on each of said test sequences;

[0019] o determining a consensus amino acid for each variable amino acid position by selecting one or more amino acids that result in a polypeptide having a negative total energy value; and

[0020] - Producing an influenza NA polypeptide comprising said consensus amino acids.

[0021] In various embodiments, the present invention provides NA polypeptides produced using the methods of the present invention. In an exemplary embodiment, the NA polypeptide comprises an amino acid sequence of any one of SEQ ID NOs: 1-3 or a fragment thereof. In some embodiments, the present invention further relates to a tetrameric NA protein comprising one or more NA polypeptides described herein.

[0022] In various embodiments, the NA polypeptides and / or tetrameric NA proteins of the present invention are used as vaccine antigens. In some embodiments, the NA polypeptides and tetrameric NA proteins provide a broad protective immune response against multiple influenza strains, types or subtypes. Without wishing to be bound by theory, it is believed that the NA polypeptides and tetrameric NA proteins can elicit neutralizing antibody responses against multiple epitopes (e.g., conserved epitopes) within influenza viruses.

[0023] Further embodiments of the present application are as follows:

[0024] Embodiment A1. A method of producing a recombinant influenza neuraminidase (NA) polypeptide comprising a consensus amino acid, wherein the method comprises:

[0025] a. selecting more than one influenza NA polypeptide sequence and aligning the sequences;

[0026] b. Calculate the pairwise similarity / dissimilarity matrix;

[0027] c. identifying and creating clusters of similar sequences from the pairwise similarity / dissimilarity matrix;

[0028] d. In each cluster, determining whether a common amino acid exists at each position in the sequence alignment using a pairwise alignment method, wherein if the frequency of the amino acid at a given position is 50% or greater, the amino acid is designated as a common amino acid, and if the frequency of the amino acid at a given position is less than 50%, the amino acid is designated as a variable amino acid;

[0029] e. generating a first sequence including common amino acids and variable amino acids for each cluster;

[0030] f. Optionally, if multiple clusters are analyzed, the first sequence generated in one cluster in step (e) is compared to the first sequence generated in another cluster or multiple clusters by:

[0031] i. aligning the sequences generated in step (e) for each cluster;

[0032] ii. determining the presence or absence of a consensus amino acid at each position in the sequence alignment using a pairwise alignment method, wherein if the amino acid frequency at a given position is 50% or greater, the amino acid is designated as a consensus amino acid, and if the amino acid frequency at a given position is less than 50%, the amino acid is designated as a variable amino acid; and

[0033] iii. generating a second sequence comprising a consensus amino acid and a variable amino acid;

[0034] g. Determine the consensus amino acid for each variable amino acid position in the first sequence generated in step (e) or the second sequence generated in step (f)(iii) by:

[0035] iv. generating a set of test sequences based on the first sequence or the second sequence, wherein the test amino acid is located at the variable amino acid position;

[0036] v. performing molecular modeling on each of the test sequences;

[0037] vi. determining a consensus amino acid for each variable amino acid position by selecting one or more amino acids that result in a polypeptide having a negative total energy value; and

[0038] h. Producing a recombinant influenza NA polypeptide comprising the consensus amino acid.

[0039] Embodiment A 2. The method according to embodiment A1, wherein aligning the sequences comprises using MAFFT, MUSCLE, CLUSTAL OMEGA, FASTA, a combination thereof, or any other multiple sequence alignment software package.

[0040] Embodiment A 3. The method according to embodiment A1 or A 2, wherein calculating the pairwise similarity / dissimilarity matrix includes using BLOSUM, PAM, IDENTITY substitution matrix or a combination thereof.

[0041] Embodiment A 4. A method according to any one of embodiments A1-3, wherein identifying and creating clusters of similar sequences from the pairwise similarity / dissimilarity matrix includes using K-means clustering, minimax clustering, principal component analysis (PCA), multidimensional scaling (MDS) or a combination thereof.

[0042] Embodiment A 5. The method of any one of Embodiments A 1-4, wherein the molecular modeling comprises comparison with a crystal structure of an influenza NA polypeptide or protein.

[0043] Embodiment A 6. The method according to any one of embodiments A 1-5, wherein molecular modeling comprises using Rosetta or any other molecular modeling software.

[0044] Embodiment A 7. The method according to any one of Embodiments A 1-6, wherein the test amino acid comprises any natural or unnatural amino acid found in proteins.

[0045] Embodiment A 8. A recombinant influenza NA polypeptide produced using the method according to any one of Embodiments A 1-7.

[0046] Embodiment A 9. A recombinant influenza NA polypeptide according to Embodiment A 8, wherein the polypeptide comprises an amino acid sequence selected from SEQ ID NO: 1, 2 or 3, or a fragment thereof, or an amino acid sequence having at least 85%, 90%, 95%, 96%, 97%, 98% or 99% identity with SEQ ID NO: 1, 2 or 3, or a fragment thereof.

[0047] Embodiment A 10. The recombinant NA polypeptide according to embodiment A 9, wherein the polypeptide comprises amino acids 75-469 of SEQ ID NO: 1, 2 or 3.

[0048] Embodiment A 11. A recombinant tetrameric NA protein comprising one or more of the recombinant NA polypeptides according to any one of Embodiments A 8-10.

[0049] Embodiment A 12. An isolated nucleic acid encoding the recombinant HA polypeptide according to any one of Embodiments A 8-10 or the recombinant tetrameric NA protein according to Embodiment A 11.

[0050] Embodiment A 13. A vector comprising the nucleic acid according to embodiment A 12.

[0051] Embodiment A 14. An isolated cell comprising the vector according to embodiment A 13.

[0052] Embodiment A 15. The isolated cell according to embodiment A 14, wherein the cell is a mammalian cell.

[0053] Embodiment A 16. The isolated cell according to embodiment A 15, wherein the isolated cell is a HEK293T cell or a CHO cell.

[0054] Embodiment A 17. The isolated cell of embodiment A 14, wherein the isolated cell is an insect cell.

[0055] Embodiment A 18. A fusion protein comprising the recombinant NA polypeptide according to any one of embodiments A 8-10 or the recombinant tetrameric NA protein according to embodiment A 11.

[0056] Embodiment A 19. An influenza virus-like particle (VLP) comprising the recombinant NA polypeptide according to any one of Embodiments A 8-10 or the recombinant tetrameric NA protein according to Embodiment A 11.

[0057] Embodiment A 20. The influenza VLP according to embodiment A 19, further comprising one or more of influenza hemagglutinin (HA) protein, influenza matrix (M1) protein, human immunodeficiency virus (HIV) gag protein, or a combination thereof.

[0058] Embodiment A 21. A pharmaceutical composition comprising a recombinant NA polypeptide according to any one of Embodiments A 8-10, a recombinant tetrameric NA protein according to Embodiment A 11, a fusion protein according to Embodiment A 18, or an influenza VLP according to Embodiment A 19 or A 20, and a pharmaceutically acceptable carrier, excipient, or adjuvant.

[0059] Embodiment A 22. The pharmaceutical composition of embodiment A 21, wherein the composition elicits an immune response against one or more influenza strains, types and / or subtypes.

[0060] Embodiment A 23. A method for immunizing a subject against influenza virus, comprising administering to the subject an effective amount of a recombinant NA polypeptide according to any one of embodiments A 8-10, a recombinant tetrameric NA protein according to embodiment A 11, a fusion protein according to embodiment A 18, an influenza VLP according to embodiment A 19 or A 20, or a pharmaceutical composition according to embodiment A 21 or A 22.

[0061] Embodiment A 24. A method of inducing an immune response against influenza virus in a subject, comprising administering to the subject an effective amount of a recombinant NA polypeptide according to any one of embodiments A 8-10, a recombinant tetrameric NA protein according to embodiment A 11, a fusion protein according to embodiment A 18, an influenza VLP according to embodiment A 19 or A20, or a pharmaceutical composition according to embodiment A 21 or A 22.

[0062] Embodiment A 25. The method according to embodiment A 23 or A 24, wherein the influenza virus is a seasonal or pandemic influenza virus.

[0063] Embodiment A 26. The method of embodiment A 24 or A 25, wherein the immune response comprises the production of antibodies against one or more influenza virus strains, types or subtypes.

[0064] Embodiment A 27. The method according to any one of Embodiments A 23-26, wherein the subject is a mammal.

[0065] Embodiment A 28. The method according to embodiment A 27, wherein the subject is human.

[0066] Embodiment A 29. The method according to any one of embodiments A 23-28, wherein the administration is performed by intramuscular, intranasal, intradermal, subcutaneous, oral or intravenous route.

[0067] Other purposes and advantages will be set forth in part in the following description and in part will be obvious from the description or may be learned by practice. These purposes and advantages will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims.

[0068] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the claims.

[0069] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate one (several) embodiments and, together with the description, serve to explain the principles described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0070] Figure 1A-1B An exemplary cluster-based consensus (CBC) method for designing NA polypeptides is shown. Figure 1A Shown are three NA polypeptide candidates, NA5200, NA7900 and NA9100, generated using the methods described herein. Figure 1B Shown are the positions of the three NA polypeptides in sequence space relative to other known H1N1 subtype sequences, as determined by multidimensional calibration.

[0071] Figure 2A-2B Schematic representation and activities of NA constructs are shown. Figure 2A Schematic representations of monomeric and tetrameric NA constructs are provided. Figure 2B Relative fluorescence units (RFU, mean increase / minute) of various NA constructs when expressed in HEK293T cells from different expression plasmids (i.e., pCAXL, pcDNA3, or pEF) are shown. TB Pan represents the soluble tetrabrachion pandemic A / Belgium / 1 / 2009 (= A / California / 07 / 2009) construct. cNA1 represents NA5200, cNA2 represents NA7900, and cNA3 represents NA9100.

[0072] Figures 3A-3F Results from an ELISA assay measuring the binding activity of sera from mice immunized with DNA encoding different NA constructs are presented. Binding activity to different influenza viruses is depicted: A / Puerto Rico / 8 / 1934 (PR8; Figure 3A ); A / USSR / 90 / 1977 (USSR / 77; Figure 3B); A / Singapore / 6 / 1986(Sing / 86; Figure 3C ); A / New Caledonia / 20 / 1999 (N.Cal / 99; Figure 3D ); A / Brisbane / 59 / 2007 (Bris / 07; Figure 3E ); and A / Belgium / 1 / 2009 (Pdm / 09; Figure 3F ). In the figure, the X-axis represents serum dilution, and the Y-axis represents OD.

[0073] Figures 4A-4E Results of an enzyme-linked lectin (ELLA) assay measuring the NA inhibition (NAI) activity of sera from mice immunized with DNA encoding different NA constructs are provided. The NAI activity is depicted as: A / Puerto Rico / 8 / 1934 (PR8; Figure 4A ); Empty vector ( Figure 4B );NA5200( Figure 4C );NA7900( Figure 4D ); and NA9100( Figure 4E ).

[0074] Figures 5A-5F Shown are the percentage survival and percent weight loss of mice vaccinated twice with DNA encoding different NA constructs and subsequently challenged with 1 LD50 of pandemic 2009 virus. Figure 5A , 5C and 5E show the percentage of survival. Figure 5B , 5D and 5F show the percent weight loss. Figure 5A-5B Results related to NA5200 are provided. Figure 5C-5D Results related to NA7900 are provided. Figure 5E-5F Results related to NA9100 are provided. As shown, * indicates statistical significance compared to empty vector.

[0075] Figures 6A-6F Shown are the percentage survival and percent weight loss of mice vaccinated twice with DNA encoding different NA constructs and subsequently challenged with 1 LD50 of PR8 virus. Fig. 6A , 6C and 6E show the percentage of survival. Figure 6B , 6D and 6F show the percent weight loss. Figure 6A-6B Results related to NA5200 are provided. Figure 6C-6D Results related to NA7900 are provided. Figure 6E-6F Results related to NA9100 are provided. As shown in the figure, * indicates statistical significance compared with the empty vector group, while Statistical significance compared to the pdm09 group is indicated.

[0076] Figure 7A-7B Purification curves and specific enzyme activities of recombinant tetrameric NA proteins are provided. Specifically, soluble tetrameric proteins including NA5200, NA7900 or NA9100 NA polypeptides were produced in HEK293T cells. The supernatant was collected and purified by affinity chromatography followed by size exclusion chromatography. Fig. 7A Shown is the UV absorption curve of tetrameric soluble NA protein after size exclusion chromatography. The vertical lines above the graph represent standards of known protein size. Figure 7B Results from a MUNAA assay are shown, measuring the enzymatic activity of recombinant tetrameric NA protein as well as wild-type A / Belgium / 2009 (Bel / 09) NA. Specific activity was calculated from a standard curve of 4-methylumbelliferone.

[0077] Fig. 8A and 8B It was shown that antisera raised against tetrameric NA proteins including NA5200, NA7900 or NA9100 exhibited NA inhibition (NAI) against a wider range of influenza A viruses (IAVs) than WT NA antisera. Mice were primed and boosted subcutaneously (three weeks apart) with 1 μg of tetrameric NA proteins (including A / Puerto Rico / 8 / 1934 (PR8 / 34) NA, A / New Caledonia / 1999 (NC / 99) NA, A / Belgium 1 / 2009 (Bel / 09; as described in www.ncbi.nlm.nih.gov / nuccore / ?term=txid1502382) NA, NA5200, NA7900, NA9100 or PBS alone) in the presence of the Sigma adjuvant system (SAS). Three weeks after the boost, antisera were collected and tested for their ability to inhibit NA activity of the N1 virus panel. Increasing serial dilutions of heat-inactivated sera raised against tetrameric NA protein were mixed with the H1N1 virus panel. NA activity was determined by enzyme-linked lectin (ELLA) assay on fetuin-coated plates 18 h post infection. Data show 1:IC determined by nonlinear regression analysis 50 Values ​​and plotted as log 2 Value. Fig. 8A In , data are reported as means (± 1SD) of experiments performed in triplicate. The dashed line on the y-axis represents the highest concentration of serum tested, 4.3 (i.e., 1:20). Values ​​below this line were extrapolated from the titration curve using nonlinear regression analysis, with 2.3 (i.e., 1:5) set as a true positive titer. Figure 8B In the data display, 1:IC 50Heatmap of values, next to which is a phylogenetic tree of NAs clustered by the amino acid sequence of the NA head domain.

[0078] Figures 9A-9L Vaccination with tetrameric NA proteins including NA5200, NA7900 or NA9100 protects mice from lethal infection with influenza A virus (IAV). Mice were primed and boosted subcutaneously (3 weeks later) with 1 μg of tetrameric proteins including NA5200 (black circles), NA7900 (white squares) or NA9100 (white circles) in the Sigma adjuvant system (SAS), or mock vaccinated with PBS (white triangles). After vaccination, 5LD 50 A / Puerto Rico / 8 / 1934 (PR8 / 34; Figures 9A-9B )、A / USSR / 90 / 1977(USSR / 77; Figure 9C-9D ), A / New Caledonia / 20 / 1999 (NC / 99; Figures 9E-9F ), A / Belgium / 1 / 2009 (Bel / 09; Figure 9G-9H ), A / Sw / Belgium / 1 / 1998(Sw / Bel / 98; Figures 9I-9J ) or H5N1 strain NIBRG-14(H5N1)( Figure 9K-9L ) were intranasally infected with , and weight loss (left panel) or mortality (right panel) were monitored. When mice lost ≥25% of their original body weight, the animals were sacrificed. Weight loss data are reported as the mean percentage (± SEM) of original body weight over time ( Fig.9A , 9C , 9E, 9G, 9I and 9K, n=8-10). Survival data are shown as percentage of survival over time ( Fig. 9B , 9D , 9F, 9H, 9J and 9L, n=8-10). Weight loss over time was analyzed by two-way ANOVA to assess main column effects. Survival was assessed using a two-tailed log-rank (Mantel Cox) test. *p<0.05, **p<0.01 compared to mock-vaccinated mice.

[0079] Figures 10A-10F It was shown that protection mediated by tetrameric NA proteins including NA5200, NA7900 or NA9100 resulted in a reduction in lung virus titers and inflammation in the lung environment. Mice were primed and boosted subcutaneously (3 weeks later) with 1 μg of tetrameric proteins including NA5200, NA7900 or NA9100 in the Sigma adjuvant system (SAS), or mock vaccinated with PBS. After vaccination, 5LD 50 A / Puerto Rico / 8 / 1934 (PR8 / 34; Figures 10A-10B ), A / New Caledonia / 20 / 1999 (NC / 99; Figures 10C-10D ) or A / Belgium / 1 / 2009 (Bel / 09; Figures 10E-10F ) were intranasally infected with 100 μg / mL of 4% paraformaldehyde. On the 3rd or 7th day after infection, BAL fluid and lungs were collected and analyzed by TCID 50 Evaluation of viral titers in lung homogenates ( Fig. 10A , 10C and 10E). The total protein levels of cell-free BAL fluid at day 7 were investigated ( Fig. 10B , 10D and 10F). The dashed lines in the viral titer graphs indicate TCID 50 The cut-off limit of the assay was (0.7). For protein levels; bars represent mean viral titers ± SD. Data were pooled from two independent experiments. Significance was assessed using one-way ANOVA. *p < 0.05, **p < 0.01 compared to mock-vaccinated mice.

[0080] Figures 11A-11D It shows that after three rounds of DNA immunization, Pdm09 ( Fig.11A )、NA5200( Fig. 11B )、NA7900( Fig. 11C ) or NA9100( Fig.11D ) induced a broad range of NAI activity. Antiserum was used for Figure 12-14F Passive transfer experiments shown.

[0081] Fig.12 The experimental schedule for the passive transfer experiment described in Example 6 is shown. Mice were treated intraperitoneally with 100 μl of heat-inactivated antiserum raised against NA5200, NA7900, NA9100, preimmune serum (negative; -ve controls) or the corresponding positive (+ve) controls (immunized with tetNA+SAS; Bel09 or NewCal99). Mice were challenged 24 hours later.

[0082] Figures 13A-13H Shown is the weight loss protection and survival of mice after 1 LD50 A / Belgium / 1 / 2009 (Be109) challenge (5 animals per group). 24 hours before challenge, mice were immunized with DNA against NA5200 ( Fig.13A and 13C )、NA7900( Fig. 13B and 13D )、NA9100( Fig.13E and 13G ) or Bel / 09( Fig.13F and 13H ) were used to passively immunize mice. Fig.13A , 13B, 13E and 13F show body weight data. Fig. 13C , 13D , 13G, and 13H show the percent survival data.

[0083] Figures 14A-14F A / Belgium / 1 / 2009 (Bel09, Fig.14A and 14B ), A / New Caledonia / 20 / 1999(NewCal99, Fig. 14C and 14D ) or A / Puerto Rico / 8 / 1934 (PR8, Fig.14E and 14F ) challenge, weight loss protection and survival rate of mice (7-8 animals per group). Fig.14A , 14C and 14E show body weight data. Fig. 14B , 14D Survival data are shown in Figures 14 and 14F. Mice were passively immunized by DNA immunization with antisera against NA5200, NA7900 or NA9100 24 hours prior to challenge.

[0084] Figures 15A-15F Antibodies were demonstrated to play a major role in the protection afforded by protein vaccination with tetrameric NA proteins including NA5200, NA7900, or NA9100. Antisera raised against CBC NA, wild-type PR8 / 34 rNA, wild-type NC / 99 rNA, or wild-type Bel / 09 rNA were evaluated. Figures 8A-8B The ability of the same to control influenza virus infection by passive transfer was tested. Mice were treated intranasally with 20 μl of heat-inactivated antiserum raised against tetrameric NA proteins including NA5200, NA7900, NA9100, PBS (negative; -ve control) or the corresponding homologous rNA positive (+ve) control. After 24 hours, 2LD 50 A / Puerto Rico / 8 / 1934 (PR8 / 34; Figures 15A-15B ), A / New Caledonia / 20 / 1999 (NC / 99; Figures 15C-15D ) or A / Belgium / 1 / 2009 (Bel / 09; Figures 15E-15F ) mice were challenged intranasally and weight loss was observed over 14 days ( Fig.15A , 15C and 15E) and survival rate ( Fig. 15B , 15Dand 15F). Weight loss data are reported as the mean percentage (± SEM) of original body weight over time (n=8), while survival data are shown as the percentage of survival over time (n=8). Data were combined from two independent experiments. Two-way ANOVA was used to analyze weight loss over time, assessing the main column effect, while survival was assessed using a two-tailed log-rank (Mantel Cox) test. *p<0.05 compared to negative control mice.

[0085] Figures 16A-16L The results showed that CBC NA provided broader protection than wild-type NA and provided increased breadth when added to monovalent inactivated vaccines. In the presence of SAS adjuvant, 1 μg of wild-type Bel / 09 rNA, wild-type NC / 99 rNA, CBC NAs NA5200 or NA9100 ( Figures 16A-16C and 16G-16I), or with 0.1 μg monovalent pdm09 vaccine alone, or in combination with 1 μg wild-type Bel / 09 rNA, wild-type NC / 99 rNA, CBC NAs NA5200 or NA9100 ( Figures 16D-16F and 16J-16L), the mice were primed and boosted subcutaneously (3 weeks apart). A group of mice were also mock vaccinated. Three weeks after the last booster immunization, 5LD 50 The mice were challenged intranasally with Bel / 09 or NC / 99. The weight loss of the mice was monitored over 14 days ( Fig.16A , 16D , 16G) and survival rate ( Fig. 16B , 16E , 16F). Mice were sacrificed if they lost ≥25% of their original body weight. Lung homogenates were obtained on day 7 of Bel / 09 infection and day 6 of NC / 99 infection and analyzed by TCID 50 Viral load was assessed. Weight loss data are presented as mean percentage of original body weight over time (± SEM), while survival data are presented as percentage of survival over time (n=9-10, pooled from 2 independent homologous rNA vaccination experiments) ( Figures 16A-16C and 16G-16I); n = 5 (from one experiment with monovalent single-vaccinated mice) ( Figures 16D-16F and 16J-16L). Weight loss over time was analyzed by two-way ANOVA, examining for main column effects, and survival was assessed using a two-tailed log-rank (Mantel Cox) test. Viral titer significance was assessed using a one-way ANOVA. Figures 16A-16C and 16G-16I), or compared with mice vaccinated with monovalent vaccines alone ( Figures 16D-16FCompared with 16J-16L), *p<0.05, **p<0.01.

[0086] Figures 17A-17C Antisera raised against tetrameric NA proteins including NA5200 and NA9100 were shown to mediate NAI against A(H1N1)pdm09 HA drift strains (HA drift was identified by the CDC due to increased infection rates in middle-aged individuals) ( Figures 17A-17B ), and the percentage of identity shared by CBC NA and A(H1N1)pdm09 viruses ( Fig. 17C ). Mice were primed and boosted (3 weeks apart) with tetrameric NA proteins containing either NA5200 or NA9100 in the presence of SAS. Dilutions of heat-inactivated sera raised against A / Belgium / 1 / 2009 (Bel / 09) and recombinant consensus NA were tested for their ability to mediate NAI against A / Belgium / 1 / 2009 (Bel / 09) and A / Singapore / GP1908 / 2015 (Sing / 15) in the ELLA assay. Fig.17A In the figure, the dashed line at 1:4.3 represents the highest concentration of serum tested. Values ​​below this line are extrapolated from the curve determined by the nonlinear regression equation, in which 1:2.3 is set as a true positive signal. Data are reported as means (±1SD) of experiments performed in triplicate. *p<0.05, one-way ANOVA. Fig. 17C The percentage of identity shared by CBCNA and A(H1N1)pdm09 virus as determined using BlastP suite-2 sequence software is shown. Bold numbers indicate 1:IC values ​​> 2.3 (i.e. 1:5) were observed in the NAI assay. 50 .

[0087] Sequence Description

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[0092] DETAILED DESCRIPTION

[0093] definition

[0094] Adjuvant: As used herein, an adjuvant refers to a substance or vehicle that non-specifically enhances the immune response to an antigen. An adjuvant may include, but is not limited to, a suspension of a mineral (e.g., alum, aluminum hydroxide, or phosphate) on which an antigen is adsorbed; an oil-in-water or water-in-oil emulsion, in which an antigen solution is emulsified in mineral oil or water (e.g., Freund's incomplete adjuvant). Killed mycobacteria (e.g., Freund's complete adjuvant) are sometimes included to further enhance antigenicity. Immunostimulatory oligonucleotides (e.g., CpG motifs) may also be used as adjuvants (e.g., see U.S. Patent Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068; 6,406,705; and 6,429,199). Adjuvants may also include biomolecules, such as Toll-like receptor (TLR) agonists and co-stimulatory molecules. Exemplary biological adjuvants include, but are not limited to, IL-2, RANTES, GM-CSF, TNF-a, IFN-γ, G-CSF, LFA-3, CD72, B7-1, B7-2, OX-40L, 41BBL, or a combination thereof.

[0095] Antibody: As used herein, an antibody refers to a protein or polypeptide that includes sufficient canonical immunoglobulin sequence elements to confer specific binding to a particular antigen. In some embodiments, the antibody is a classical antibody that includes two heavy chains and two light chains. Each heavy chain includes a variable region (e.g., V H ) and at least three constant regions (e.g., CH 1 , CH 2 and CH 3 ), and each light chain includes a variable region (V L ) and a constant region (C L). The variable region determines the specificity of the antibody. Each variable region includes three hypervariable regions, also called complementarity determining regions (CDRs), which are flanked by four relatively conserved framework regions (FRs). The three CDRs, called CDR1, CDR2, and CDR3, contribute to the antibody binding specificity. In some embodiments, antibodies also refer to "antibody fragments (antibody fragment or antibody fragments)", which include a portion of a complete antibody, such as, for example, the antigen binding region or variable region of an antibody. Examples of "antibody fragments" include Fab, Fab', F(ab')2, Fv fragments, tri-antibodies, tetra-antibodies, linear antibodies, single-chain antibody molecules, and CDR-containing portions included in multispecific antibodies. In certain embodiments, any polypeptide or polypeptide complex including sufficient immunoglobulin domain sequences found in natural antibodies can be referred to and / or used as an "antibody", whether such polypeptide is naturally produced (e.g., produced by an organism in response to an antigen), or produced by recombinant engineering, chemical synthesis, or other artificial systems or methods. In some embodiments, antibodies are monoclonal; in some embodiments, antibodies are polyclonal. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is humanized.

[0096] Antigen: As used herein, an antigen refers to an agent that triggers an immune response when exposed to or administered to an organism, and / or a factor that binds to a T cell receptor (e.g., when presented by an MHC molecule) or a factor that binds to an antibody (e.g., produced by a B cell). In some embodiments, an antigen triggers a humoral response in an organism (e.g., including the production of antigen-specific antibodies). Alternatively or additionally, in some embodiments, an antigen triggers a cellular response in an organism (e.g., involving T cells whose receptors specifically interact with the antigen). Those skilled in the art will recognize that a particular antigen may trigger an immune response in one or several members of a target organism (e.g., mouse, rabbit, primate, human), but not in all members of the target organism species. In some embodiments, the antigen triggers an immune response in at least about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% of the members of the target organism species. In some embodiments, the antigen binds to antibodies and / or T cell receptors and may or may not induce a specific physiological response in the organism. In some embodiments, for example, the antigen can bind to antibodies and / or T cell receptors in vitro, regardless of whether this interaction occurs in vivo. In some embodiments, the antigen reacts with the product of a specific humoral or cellular immunity, including those induced by a heterologous immunogen. In some embodiments, the influenza HA polypeptide or its immunogenic fragment is an antigen.

[0097] Antigenic drift: As used herein, antigenic drift refers to relatively frequent mutations in the HA or NA antigens. Antigenic drift can allow influenza viruses to evade immune recognition and may reduce vaccine efficacy.

[0098] Antigenic shift: As used herein, antigenic shift refers to a major change in the HA or NA antigens that results from the reassortment of genetic material between different influenza strains.

[0099] Approximately: As used herein, the term "approximately" or "about" as applied to one or more target values ​​refers to a value similar to the stated reference value. In certain embodiments, unless otherwise stated or the context has a clear meaning, the term "approximately" or "approximately" refers to a range of values ​​(except where this number exceeds 100% of the possible value) falling in either direction (greater than or less than) of the stated reference value of 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less.

[0100] Epitope: As used herein, refers to any part that is specifically recognized in whole or in part by an immunoglobulin (e.g., an antibody or receptor) binding component. In some embodiments, an epitope consists of multiple chemical atoms or groups on an antigen. In some embodiments, such chemical atoms or groups are surface exposed when the antigen adopts a related three-dimensional conformation. In some embodiments, such chemical atoms or groups are physically close to each other in space when the antigen adopts this conformation. In some embodiments, when the antigen adopts an alternative conformation (e.g., linearized), at least some of the chemical atoms or groups are physically separated from each other.

[0101] Host: As used herein, the term "host" refers to a system (e.g., cell, organism, etc.) in which the polypeptide of interest is present. In some embodiments, the host is a system susceptible to infection by a specific infectious agent. In some embodiments, the host is a system that expresses a specific polypeptide or protein of interest.

[0102] Host cell: As used herein, "host cell" refers to a cell into which exogenous DNA (recombinant or otherwise) has been introduced. For example, host cells can be used to produce the influenza NA polypeptides described herein by standard recombinant techniques. It is understood by those skilled in the art that such terms refer not only to a particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, such progeny may in fact be different from the parent cell, but are still included within the scope of the term "host cell" as used herein. In some embodiments, host cells include any prokaryotic and eukaryotic cells suitable for expressing exogenous DNA (e.g., a recombinant nucleic acid sequence). Exemplary cells include prokaryotic or eukaryotic cells (single or multicellular), bacterial cells (e.g., strains of E. coli, Bacillus species, Streptomyces species, etc.), mycobacterial cells, fungal cells, yeast cells (e.g., S. cerevisiae, S. pombe, P. pastoris, P. methanolica, etc.), plant cells, insect cells (e.g., SF-9, SF-21, baculovirus-infected insect cells, Trichoplusia ni, etc.), non-human animal cells, human cells, or cell fusions (e.g., hybridomas or quadruple hybridomas). In some embodiments, the cell is a human, monkey, ape, hamster, rat, or mouse cell. In some embodiments, the cell is a eukaryotic cell and is selected from, but is not limited to, CHO (e.g., CHO K1, DXB-11 CHO, Veggie-CHO), COS (e.g., COS-7), retinal cells, Vero, CV1, kidney cells (e.g., HEK293, 293EBNA, MSR 293, MDCK, HaK, BHK), HeLa, HepG2, WI38, MRC 5, Colo205, HB 8065, HL-60 (e.g., BHK21), Jurkat, Daudi, A431 (epidermal), CV-1, U937, 3T3, L cells, C127 cells, SP2 / 0, NS-0, MMT060562, Sertoli cells, BRL 3A cells, HT1080 cells, myeloma cells, tumor cells, and cell lines derived from the above cells. In some embodiments, the cell comprises one or more viral genes, e.g., a retinal cell expressing a viral gene (e.g., PER.C6 TM cell).

[0103] Immune response: As used herein, the term "immune response" refers to the response of cells of the immune system (such as B cells, T cells, dendritic cells, macrophages or polymorphonuclear cells) to stimuli (such as antigens or vaccines). The immune response can include any cell of the body involved in the host defense response, including, for example, epithelial cells that secrete interferons or cytokines. The immune response includes, but is not limited to, innate and / or adaptive immune responses. As used herein, a protective immune response refers to an immune response that protects a subject from infection (e.g., preventing infection or preventing the development of a disease associated with infection). Methods for measuring immune responses are well known in the art and include, for example, by measuring the proliferation and / or activity of lymphocytes (such as B or T cells), the secretion of cytokines or chemokines, inflammation, antibody production, and the like.

[0104] Immunogen: As used herein, the term "immunogen" refers to a compound, composition or substance capable of stimulating an immune response (such as antibody production or T cell response) in an animal under appropriate conditions, including compositions injected or absorbed into an animal. As used herein, an "immunogenic composition" is a composition comprising an immunogen (such as an HA polypeptide or tetrameric HA protein). As used herein, "immunization" refers to protecting a subject from an infectious disease, such as by vaccination.

[0105] Influenza Virus: as used herein refers to a segmented negative-strand RNA virus belonging to the family Orthomyxoviridae.

[0106] Flu vaccine: As used herein, refers to an immunogenic composition capable of stimulating an immune response, which is administered to prevent, improve or treat influenza virus infection. Flu vaccines may include, for example, attenuated or killed (e.g., lysed) influenza viruses, virus-like particles (VLPs) and / or antigenic polypeptides or proteins (e.g., NA polypeptides or tetrameric HA proteins described herein) or DNA derived therefrom, or any recombinant form of such immunogenic materials. Flu vaccines also include vaccines based on DNA and viral vectors. Vaccines contemplated herein may optionally include one or more adjuvants.

[0107] Isolated: as used herein, refers to an agent or entity that is (i) separated from at least some of the components with which it is associated when originally produced (whether in nature or in an experimental setting); or (ii) produced artificially. An isolated agent or entity can be separated from at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% or more of the other components with which it was originally associated. In some embodiments, the purity of the isolated agent is greater than 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. As used herein, a substance is "pure" if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the art, a substance may still be considered "isolated" or even "pure" after being combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffers, solvents, water, etc.); in such embodiments, the percent isolation or purity of the substance is calculated excluding such carriers or excipients. In exemplary embodiments, a biopolymer such as a naturally occurring polypeptide or polynucleotide is considered "isolated" when: a) it is not associated with some or all of the components that accompany it in its native state in nature due to its source or source of derivation; b) it is substantially free of other polypeptides or nucleic acids from the same species as the species in which it is produced in nature; c) it is expressed by a cell or other expression system of a species other than the species in which it is produced in nature or is associated with components from a cell or other expression system of a species other than the species in which it is produced in nature. Thus, for example, in some embodiments, a polypeptide that is chemically synthesized or synthesized in a cell system different from the cell system in which it is produced in nature is considered an "isolated" polypeptide. Alternatively or additionally, in some embodiments, a polypeptide that has been subjected to one or more purification techniques may be considered an "isolated" polypeptide to the extent that it has been separated from other components a) with which it is associated in nature and / or b) with which it was originally produced.

[0108] Neuraminidase or NA protein: As used herein, the neuraminidase of the NA protein refers to a structural glycoprotein on the surface of the influenza virus membrane. NA is responsible for releasing influenza virus from infected cells by removing sialic acid from cell surface proteins. Currently, there are 11 known NA subtypes (i.e., N1, N2, N3, N4, N5, N6, N7, N8, N9, N10 and N11), which are defined by interactions with antibodies. All variants of a given NA subtype can be neutralized by a similar set of antibodies. NA inhibition (NAI) assays can be used to identify NA glycoprotein subtypes in specific influenza viruses, and thus classify the viruses. Humans are usually infected with N1 or N2 subtype viruses. In some embodiments, the NA protein can be monomeric and include a single NA polypeptide. In other embodiments, the NA protein is a tetramer and includes four NA polypeptides. Figure 2A Embodiments of monomeric and tetrameric NA proteins described herein are described. As used herein, "neuraminidase polypeptide" or "NA polypeptide" refers to a polypeptide whose amino acid sequence includes at least one characteristic sequence of NA. NA polypeptides may include full-length influenza NA polypeptide sequences and fragments thereof. One of ordinary skill in the art can easily identify the following sequences, which are generally characteristic of NA polypeptides and / or specific NA polypeptides (e.g., N1 or N2 polypeptides) or NA that mediates infection in specific hosts (e.g., birds, camels, dogs, cats, civets, horses, humans, leopards, minks, mice, seals, stone martins, pigs, tigers, whales, etc.). The National Center for Biotechnology Information (NCBI) maintains a database of NA polypeptide sequences.

[0109] Outbreak: As used herein, an influenza virus "outbreak" refers to a collection of virus isolates from a single country in a given year.

[0110] Pandemic influenza, seasonal influenza and swine flu strains: As used herein, "pandemic" influenza strains are influenza strains that have caused pandemic infection in the population or have the ability to cause pandemic infection in the population. In some embodiments, pandemic influenza strains have caused pandemic infection. In some embodiments, this pandemic infection involves a pandemic infection across multiple regions. In some embodiments, pandemic infection involves infection across regions separated from each other (e.g., separated by mountains, waters, as part of different continents, etc., so that infection is usually not spread between them). In some embodiments, pandemic influenza strains include those derived from the reassignment (antigenic shift occurs approximately every 20-30 years) between human influenza virus and avian influenza virus or swine influenza virus, and the reassignment results in a virus with a novel HA or NA of avian or pig origin, and humans lack immunity to the virus. In other words, due to previous vaccination or previous exposure, the population is considered to be initial, with no or only little resistance. Pandemic influenza strains and seasonal influenza strains are antigenically distinct, and also very different in sequence. In general, seasonal influenza strains can be defined as circulating strains (including non-pandemic 2009 sequences) of a particular season or a particular year (e.g., 1986 to 2009) and other strains (i.e., similar in antigen sequence space) with substantially similar coding antigenic regional genetic sequences. Swine influenza strains refer to any influenza strains relevant to viruses prevalent in pigs. Exemplary pandemic influenza strains include, but are not limited to, A / California / 07 / 2009, A / California / 04 / 2009, A / Belgium / 145 / 2009, A / South Carolina / 01 / 1918, and A / New Jersey / 1976. Pandemic subtypes particularly include H5N1, H2N2, H9N2, H7N7, H7N3, H7N9, and H10N7 subtypes. Exemplary seasonal influenza strains include, but are not limited to, A / Puerto Rico / 8 / 1934, A / Ford / Monmouth / 1 / 1947, A / Chile / 1 / 1983, A / Texas / 36 / 1991, A / Singapore / 6 / 1986, A / Beijing / 32 / 1992, A / New Caledonia / 20 / 1999, A / Solomon Islands / 03 / 2006, and A / Brisbane / 59 / 2007. Exemplary swine flu strains include, but are not limited to, A / New Jersey / 1976 isolates and A / California / 07 / 2009. Other pandemic influenza, seasonal influenza, and / or swine flu strains are known in the art.

[0111] Prevention: As used herein, refers to preventing, avoiding disease manifestation, delaying onset, and / or reducing the frequency and / or severity of one or more symptoms of a particular disease, disorder, or condition (e.g., an infection, such as an influenza virus infection). In some embodiments, prevention is assessed on a population basis, such that if a statistically significant reduction in the development, frequency, and / or intensity of one or more symptoms of the disease, disorder, or condition is observed in a population susceptible to the disease, disorder, or condition, then the agent is considered to have "prevented" the particular disease, disorder, or condition.

[0112] Recombinant: As used herein, the term "recombinant" refers to a polypeptide or protein (e.g., a NA polypeptide or tetrameric NA protein described herein) that is designed, engineered, prepared, expressed, created, or isolated by recombinant means, such as a polypeptide expressed using a recombinant expression vector transfected into a host cell, a polypeptide isolated from a recombinant combinatorial polypeptide library, or a polypeptide prepared, expressed, created, or isolated by any other means involving splicing selected sequence elements to each other. In some embodiments, one or more such selected sequence elements are found in nature. In some embodiments, one or more such selected sequence elements are computer designed. In some embodiments, one or more such selected sequence elements are generated by mutagenesis (e.g., in vivo or in vitro) of known sequence elements of, for example, natural or synthetic origin. In some embodiments, one or more such selected sequence elements are generated by a combination of multiple (e.g., two or more) known sequence elements that are not naturally present in the same polypeptide (e.g., two epitopes from two independent NA polypeptides).

[0113] Signal sequence, secretion signal, or secretion signal peptide: As used herein, the terms refer to a peptide sequence that signals secretion from a cell. A secretion signal can result in the secretion of a polypeptide or protein that would not otherwise be secreted.

[0114] Tetramerization domain: As used herein, the term refers to an amino acid sequence encoding a domain that results in tetrameric assembly of a polypeptide or protein. A tetramerization domain that is not native to a particular protein may be referred to as an artificial or heterologous tetramerization domain. Exemplary tetramerization domains include, but are not limited to, sequences from four arms, a GCN4 leucine zipper, or vasodilator-stimulated phosphoprotein (VASP).

[0115] Sequence identity: Similarity between amino acid sequences or nucleic acid sequences is expressed as similarity and / or identity between sequences. Sequence similarity can include elements with sequence identity and sequences that are closely related in homology. Sequence similarity is often measured as a percentage similarity (or identity or homology); the higher the percentage, the more similar the two sequences are. When aligned using standard methods, homologs or variants of a given gene or protein will have a relatively high degree of sequence identity. Sequence alignment methods for comparison are well known in the art. Various programs and alignment algorithms are described in the prior art: Smith and Waterman, Adv. Appl. Math. 2:482, 1981; Needleman and Wunsch, J. Mol. Biol. 48:443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988; Higgins and Sharp, Gene 73:237-244, 1988; Higgins and Sharp, CABIOS 5:151-153, 1989; Corpet et al., Nucleic Acids Research 16:10881-10890, 1988; and Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444, 1988. Altschul et al., Nature Genet. 6: 119-129, 1994. The NCBI Basic Local Alignment Search Tool is available from several sources, including the National Center for Biotechnology Information (NCBI, Bethesda, Maryland). (Altschul et al., J. Mol. Biol. 215:403-410, 1990), for use in conjunction with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx.

[0116] Subject: As used herein, refers to any member of the animal kingdom. In some embodiments, "subject" refers to humans. In some embodiments, "subject" refers to non-human animals. In some embodiments, subjects include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects and / or worms. In certain embodiments, the non-human subject is a mammal (e.g., rodents, mice, rats, rabbits, monkeys, dogs, cats, sheep, cattle, primates and / or pigs). In some embodiments, the subject can be a transgenic animal, a genetically engineered animal and / or a clone. In certain embodiments of the present invention, the subject is an adult, a teenager or an infant. In some embodiments, the term "individual" or "patient" is used and is intended to be interchangeable with "subject". The present invention also contemplates the administration of a pharmaceutical composition and / or the execution of an intrauterine treatment method.

[0117] Vaccination: As used herein, the term "vaccination" or "vaccinating" refers to the administration of a composition intended to generate an immune response, such as an immune response to a disease-causing agent. Vaccination can be administered before, during, and / or after exposure to a disease-causing agent, and / or before the development of one or more symptoms, and in some embodiments, before, during, and / or after exposure to the agent. In some embodiments, vaccination comprises multiple administrations of a vaccine composition appropriately spaced in time.

[0118] Virus-like particles (VLPs): As used herein, the phrase "virus-like particles" or "VLPs" refers to particles that are similar to viruses but lack any viral genetic material and are therefore non-infectious. "Virus-like particles" or "VLPs" can be produced by heterologous expression in various cell culture systems including mammalian cell lines, insect cell lines, yeast, and plant cells. In addition, VLPs can be purified by methods known in the art. In some embodiments, the influenza VLPs described herein comprise HA polypeptides and / or NA polypeptides. In some embodiments, the influenza VLPs described herein comprise HA polypeptides, NA polypeptides, and / or structural polypeptides. In some certain embodiments, the influenza VLPs described herein comprise HA polypeptides, NA polypeptides, and / or influenza M1 polypeptides. In some embodiments, the influenza VLPs described herein comprise HA polypeptides, NA polypeptides, and / or HIV gag polypeptides. It is appreciated by those skilled in the art that other viral structural proteins can be used as substitutes for those illustrated herein. Influenza VLPs can be produced by transfecting host cells (e.g., mammalian cells) with plasmids encoding HA and NA proteins and optionally M1 proteins and / or HIV gag proteins. After the transfected cells are incubated for an appropriate time to allow protein expression, VLP can be separated from the cell culture supernatant. In some embodiments, influenza VLP as described herein is produced by transient transfection in mammalian cells (e.g., human cells). In some embodiments, influenza VLP is analyzed by using one or more assays. For example, influenza VLP particle size can be analyzed by dynamic light scattering, and the hemagglutinin activity and hemagglutinin content of such VLP can also be analyzed by protein staining.

[0119] Wild-type (WT): As understood in the art, the term "wild-type" generally refers to the normal form of a protein or nucleic acid found in nature. For example, wild-type NA polypeptides are found in natural isolates of influenza virus. A variety of different wild-type NA sequences can be found in the NCBI influenza virus sequence database.

[0120] Methods for designing neuraminidase (NA) peptides

[0121] A significant challenge associated with generating a consensus influenza protein sequence involves temporal and geographic sequence biases. Part of the reason for this bias is that sequence records provided in public and / or private sequence databases are often heavily biased toward newer sequences. Further, sequences associated with certain geographic regions (such as the United States) are often over-represented. On the one hand, the present invention provides a novel method for generating influenza NA polypeptides comprising consensus amino acids using a cluster-based consensus method that overcomes such temporal and geographic sequence biases. The method of the present invention is independent of phylogenetic information and depends only on the information contained in the primary amino acid sequence. Therefore, the present method is capable of generating NA polypeptide sequences that reflect overall sequence diversity and are not biased toward temporally or geographically over-represented sequences.

[0122] In various embodiments, the method involves designing NA polypeptide sequences based on computer analysis of sequence variations between multiple influenza NA sequences, applying a consensus sequence-based algorithm to generate clusters of similar sequences, and performing structural analysis on NA polypeptides having a consensus amino acid sequence. In some embodiments, the method generates a pairwise similarity / dissimilarity matrix that can be clustered using tools such as K-means, minimax clustering, and farthest-first clustering. Alternatively or additionally, a ranking technique such as multidimensional scaling (MDS) or principal component analysis (PCA) is used to visualize the pairwise similarity / dissimilarity matrix in a compact representation to define appropriate clusters (e.g., to separate and define the number of clusters). Further, the method utilizes molecular modeling and comparison of crystal structures to resolve variable amino acid positions within the consensus sequence and to rank candidates that are likely to fold correctly and therefore function.

[0123] Without wishing to be bound by theory, it is believed that the methods of the present invention produce NA polypeptides that include epitopes that are conserved across different influenza strains, types, and / or subtypes. Thus, in various embodiments, the methods produce NA polypeptides that are capable of inducing an enhanced cross-reactive immune response against a wide range of influenza strains (e.g., one or more seasonal influenza, pandemic influenza, or swine influenza strains), influenza types (e.g., one or more influenza A, B, or C), and / or influenza subtypes (e.g., one or more influenza subtypes, such as, but not limited to, H1N1, H3N2, or H5N1).

[0124] In some embodiments, the method comprises selecting various influenza NA polypeptide sequences. Various different NA sequences can be found in sequence databases, such as the National Center for Biotechnology Information (NCBI) influenza virus sequence database. In some embodiments, a non-redundant subset of unique sequences is selected for sequence alignment.

[0125] In some embodiments, the method comprises aligning influenza NA polypeptide sequences. Any multiple sequence alignment tool known in the art can be used. See, for example, Katoh and Kuma (2002) NAR, 30: 3059; Katoh and Standley (2013) Mol BioL Evol 30: 772; Edgar, RC (2004) NAR, 32: 1792; Edgar, RC (2004) BMC Bioinf, 113; Sievers et al. (2011) Mol Sys Biol 7: 539; and Pearson and Lipman. (1988) PNAS, 85: 2444). Exemplary sequence alignment tools that can be used in the present invention include, but are not limited to, MAFFT, MUSCLE, CLUSTAL OMEGA, FASTA, or a combination thereof.

[0126] In some embodiments, specific sequence regions may be masked from further analysis. For example, any of the NA signal peptide sequence, transmembrane domain sequence, or any other conserved NA domains may be masked from further analysis.

[0127] In some embodiments, the method includes calculating a pairwise similarity / dissimilarity matrix from the aligned sequences. Any method for calculating the distance between two or more sequences can be used. Exemplary tools for calculating a pairwise similarity / dissimilarity matrix include, but are not limited to, BLOSUM, PAM, IDENTITY substitution matrix or a combination thereof. In one embodiment, an alternative method for calculating a pairwise similarity / dissimilarity matrix can be used, such as FastTree (Price, MN, Dehal, PS, and Arkin, AP (2009) Molecular Biology and Evolution 26: 1641-1650).

[0128] In some embodiments, the method also includes identifying and creating clusters of similar sequences from a pairwise similarity / dissimilarity matrix. Exemplary tools for identifying clusters of similar sequences include, but are not limited to, K-means clustering, minimax clustering, farthest-first clustering, principal component analysis (PCA), multidimensional scaling (MDS) or a combination thereof. In one embodiment, the K-means method for clustering is utilized (see Hartigan, JA et al. (1979) Journal of the Royal Statistical Society, Series C, Applied Statistics, 28 (1): 100-108). In another embodiment, minimax clustering (e.g., minimax-connected hierarchical clustering of a similarity matrix) is utilized (see, Bien, J. et al., (2011) The Journal of the American Statistical Association). In a further embodiment, the farthest-first traversal is used (see Rosenkrantz et al. (1977) SIAM J Comp, 6: 563).

[0129] In some embodiments, sorting techniques can be used to identify and create clusters of similar sequences from a pairwise similarity / dissimilarity matrix. For example, in some embodiments, PCA is used for dimensionality reduction of a pairwise similarity / dissimilarity matrix. PCA can be used to convert a high-dimensional pairwise similarity / dissimilarity matrix into a lower-dimensional subspace to facilitate visualization and identification of clusters of similar sequences (see Pearson, K. (1901) Philosophical Magazine 2 (11): 559-572; and Hotelling, H. (1933) Journal of Educational Psychology, 24, 417-441, and 498-520). In further embodiments, multidimensional scaling (MDS) is used. MDS refers to a set of dimensionality reduction methods that calculate and visualize the levels of similarity and dissimilarity of a multidimensional data set and find the distance between all pairs of a set of points that is reproduced to the greatest extent. In some embodiments, MDS is used to place each object in an N-dimensional space, thereby maintaining the distance between objects. In some embodiments, MDS allows the information contained in the distance matrix to be displayed. In some embodiments, MDS places the NA sequences in a reduced dimensional space, thereby accurately maintaining the relative distances between pairs of viral sequences. In some embodiments, MDS overcomes the shortcomings in phylogenetic methods, because phylogenetic methods may be inconsistent in the presence of reassortment and / or recombination. In some embodiments, MDS filters out random neutral substitutions in influenza viruses. In various embodiments, sorting techniques such as MDS or PCA help convert high-dimensional paired distance matrices into lower dimensional subspaces to facilitate visualization and identification of clusters.

[0130] In some embodiments, the methods described herein create more than one cluster of similar sequences (e.g., seasonal flu-like, pandemic flu-like, or swine flu-like sequences). Exemplary sequence clusters for use in the methods described herein are provided, but are not limited to Figure 1A Those in .

[0131] In some embodiments, within each cluster, a consensus sequence is calculated based on the most frequent amino acid at each position in the multiple sequence alignment. For example, if the amino acid frequency at a given position is 50% or higher (or any other user-defined threshold), the amino acid is designated as a consensus amino acid. Alternatively, if the amino acid frequency at a given position is less than 50% (or any other user-defined threshold), the amino acid is designated as a variable amino acid. In some embodiments, a first sequence is generated for each cluster, which includes a consensus amino acid and a variable amino acid. In some embodiments, the first sequence generated for each cluster is designated as a cluster consensus sequence.

[0132] In some embodiments, consensus sequences are generated for multiple sequence clusters. In such embodiments, the selected cluster consensus sequences for multiple clusters are merged based on the specified result attributes to derive other consensus sequences. For example, the cluster consensus sequences relevant to a specific geographic region, host, or time period can be merged to generate a cross-cluster consensus sequence (e.g., a second sequence).

[0133] In various embodiments, in order to generate a cross-cluster consensus sequence, the intra-cluster consensus sequence (e.g., first sequence) generated from a cluster is compared with the intra-cluster consensus sequence (e.g., first sequence) generated from another cluster or multiple clusters. In some embodiments, the sequences generated are compared with each other. In some embodiments, a pairwise comparison method is utilized to determine whether there is a common amino acid at each position in the comparison. As previously described, if the amino acid frequency at a given position is 50% or higher (or any other user-defined threshold), the amino acid is designated as a common amino acid, and if the amino acid frequency at a given position is less than 50% (or any other user-defined threshold), the amino acid is designated as a variable amino acid. In some embodiments, a cross-cluster consensus sequence (e.g., second sequence) including a common amino acid and a variable amino acid is generated by this multi-cluster analysis. In various embodiments, the process of comparing sequences and determining the common amino acid at each position can be iteratively performed until all target sequence clusters are considered.

[0134] In some embodiments, other steps are performed to determine the consensus sequence within the generated cluster and / or the consensus sequence across the cluster (e.g., the first sequence and / or the second sequence) in each variable amino acid position. In such embodiments, a group of test sequences are generated based on the consensus sequence (e.g., the first sequence and / or the second sequence), wherein the test amino acid is placed at the variable amino acid position. The test amino acid used in the methods described herein covers any natural or non-natural (e.g., non-classical) amino acid found in the protein, including essential and non-essential amino acids. Exemplary amino acids include the amino acids provided in the following table 2 and the amino acids described elsewhere herein.

[0135] Table 2

[0136] Required Nonessential Histidine (H) Alanine (A) Isoleucine (I) Arginine(R) Leucine (L) Aspartic acid (D) Lysine (K) Cysteine ​​(C) Methionine (M) Glutamate (E) Phenylalanine (F) Glutamine (Q) Threonine (T) Glycine (G) Tryptophan (W) Proline (P) Valine (V) Serine (S) Tyrosine (Y) Asparagine (N) Selenocysteine(U) Pyrrolysine(O)

[0137] In various embodiments, the present method contemplates the use of molecular modeling to analyze the test sequence. In some embodiments, molecular modeling is performed on each test sequence. In some embodiments, molecular modeling includes comparison with the crystal structure of the influenza protein (i.e., NA) being analyzed. Such crystal structure information can be easily obtained from, for example, a protein database. In one embodiment, the molecular modeling includes the use of Rosetta (https: / / www.rosettacommons.org / software) or any other similar molecular modeling software (see, for example, Leaver-Fay et al. (2011) Meth. Enzymol. 487: 545-74). For example, in order to resolve the variable amino acid positions in the consensus sequence, the Metropolis-Monte Carlo simulated annealing scheme in Rosetta can be used to sample the substitutions of all possible combinations of amino acid residues present in the identified variant sites. The possible substitutions are then scored according to the energy value.

[0138] In some embodiments, the consensus amino acid for each variable amino acid position is determined by selecting one or more amino acids that result in the NA polypeptide having a calculated total energy value that is similar to or lower than the starting value. In some embodiments, the consensus amino acid for each variable amino acid position is determined by selecting one or more amino acids that result in the NA polypeptide having a negative total energy value. Without wishing to be bound by theory, it is believed that NA polypeptides with negative total energy scores are more likely to fold into stable proteins, while polypeptides with positive energy scores are less likely to fold correctly. In some embodiments, one or more NA polypeptides are generated and ranked according to their negative total energy scores and / or comparison to a reference structure.

[0139] In various embodiments, the present methods generate NA polypeptide sequences that include consensus amino acids at various positions. Table 1 provides exemplary NA polypeptides generated using the methods of the present invention. The sequence listing in Table 1 is identical to the sequence listing in Table 1 of U.S. Provisional Application No. 62 / 649,002.

[0140] Neuraminidase (NA) Peptides and Proteins

[0141] In another aspect, the present invention provides NA polypeptides produced using the methods described herein. In some embodiments, the NA polypeptides include a consensus amino acid sequence and are capable of eliciting an immune response against multiple influenza strains (e.g., one or more pandemic influenza, seasonal influenza, and / or swine influenza strains), types (e.g., one or more influenza A, B, and / or C viruses) and / or subtypes (e.g., one or more of H1N1, H3N2, or H5N1). Therefore, in some embodiments, the NA polypeptides can be incorporated into vaccine compositions as antigens to provide improved protective immunity against influenza.

[0142] In some embodiments, the present invention provides a NA polypeptide comprising an amino acid sequence selected from SEQ ID NO: 1, 2 or 3 or a fragment thereof. For example, in some embodiments, the NA polypeptide comprises amino acids 75-469 of SEQ ID NO: 1, 2 or 3. In some embodiments, the NA polypeptide comprises an amino acid sequence having at least about 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO 1, 2 or 3 or a fragment thereof (e.g., a fragment comprising amino acids 75-469 of SEQ ID NO: 1, 2 or 3).

[0143] In some embodiments, the present invention provides a NA polypeptide comprising an amino acid sequence selected from SEQ ID NO: 7-18 or a fragment thereof. In some embodiments, the NA polypeptide comprises an amino acid sequence or a fragment thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO 7-18.

[0144] In some embodiments, the present invention provides a NA polypeptide (which has one or more amino acid mutations relative to SEQ ID NO: 1, 2 or 3 or any one of SEQ ID NOs: 7-18) or a fragment thereof. For example, the NA polypeptide may include about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51, about 52, about 53, about 54, about 55, about 56, about 57, about 58, about 59, about 60, about 61, about 62, about 63, about 64, about 65, about 66, about 67, about 68, about 69, about 70, about 71 In the present invention, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41, about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, or about 50 amino acid mutations are present.

[0145] In some embodiments, the one or more amino acid mutations may be independently selected from substitutions, insertions, deletions, and truncations. In some embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and / or non-conservative substitutions. In some embodiments, the amino acid mutations are amino acid substitutions, and may include conservative and / or non-conservative substitutions.

[0146] Conservative substitutions can be made, for example, based on similarities in polarity, charge, size, solubility, hydrophobicity, hydrophilicity, and / or amphipathic properties of the amino acid residues involved. For example, the 20 naturally occurring amino acids can be divided into the following six standard amino acid groups: (1) hydrophobic: Met, Ala, Val, Leu, Ile; (2) neutral hydrophilic: Cys, Ser, Thr; Gln, Asn; (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) residues affecting chain orientation: Gly, Pro; and (6) aromatic: Trp, Tyr, Phe. As used herein, "conservative substitution" is defined as the exchange of an amino acid with another amino acid listed in the same group of the six standard amino acid groups shown above. For example, the exchange of Asp by Glu will retain a negative charge in the modified polypeptide. In addition, glycine and proline can be substituted for each other based on their ability to disrupt α-helices. As used herein, a "non-conservative substitution" is defined as the exchange of an amino acid with another amino acid listed in a different group of the six standard amino acid groups shown above.

[0147] In various embodiments, the substitutions can also include non-classical amino acids (e.g., selenocysteine, pyrrolysine, N-formylmethionine, β-alanine, GABA and δ-aminolevulinic acid, 4-aminobenzoic acid (PABA), D-isomers of common amino acids, 2,4-diaminobutyric acid, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteine, tert-butylglycine, tert-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acids, designer amino acids (e.g., β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids), and amino acid analogs in general).

[0148] In various embodiments, the present invention further provides a tetrameric NA protein comprising four NA polypeptides. In some embodiments, at least 1, at least 2, at least 3 or all 4 NA polypeptides within the tetrameric NA protein comprise an amino acid sequence as described herein. For example, at least 1, at least 2, at least 3 or all 4 NA polypeptides of the tetrameric NA protein may comprise an amino acid sequence of SEQ ID NO: 1, 2 or 3 or SEQ ID NO: 7-18 or a fragment thereof. In an exemplary embodiment, the tetrameric NA protein comprises at least 1, at least 2, at least 3 or at least 4 NA polypeptides, which comprise amino acids 75-469 of SEQ ID NO: 1, 2 or 3. In some embodiments, the tetrameric NA protein comprises four identical NA polypeptides. In other embodiments, the tetrameric NA protein comprises two or more different NA proteins having different amino acid sequences.

[0149] In various embodiments, the present invention further provides a fusion protein comprising the NA polypeptide of the present invention or a fragment thereof.

[0150] In various embodiments, the NA polypeptides of the present invention may include a tetramerization sequence or tetramerization domain that promotes the assembly of monomeric NA polypeptides into tetrameric NA proteins. In some embodiments, the NA polypeptides include a tetramerization sequence or tetramerization domain that is present in NA polypeptides found in natural influenza isolates. For example, the NA polypeptides may include a stem region sequence of a NA polypeptide found in a natural influenza isolate, which is known to promote tetramer formation. In other embodiments, the NA polypeptides may include an engineered or heterologous tetramerization sequence or tetramerization domain that is not naturally present in a wild-type NA polypeptide. For example, in one embodiment, the NA polypeptides may be engineered to include a tetramerization domain derived from a tetramer or vasodilator-stimulated phosphoprotein (VASP). In another embodiment, the NA polypeptides may include a GCN4 leucine zipper domain. In an exemplary embodiment, the NA polypeptides of the present invention are engineered to include a tetramerization domain that includes the amino acid sequence of SEQ ID NO: 4. Any sequence or domain that can promote tetramerization can be used in the present invention.

[0151] In some embodiments, the NA polypeptide may include a secretion signal peptide. In one embodiment, the incorporation of the secretion signal peptide allows the NA polypeptide or tetrameric NA protein to be secreted from the host cell. In another embodiment, the incorporation of the secretion signal peptide allows the purification of the NA polypeptide or tetrameric NA protein from the host cell (e.g., the supernatant of the host cell) used for protein expression and production.

[0152] It is contemplated that any secretion signal peptide known in the art may be incorporated into the NA polypeptide of the present invention. In some embodiments, the secretion signal peptide is specific for the host cell used for protein expression and production. Exemplary secretion signal peptides include, but are not limited to, the CD33 signal peptide sequence, the human IgGκ light chain signal peptide (for expression in human cells), the honey bee melittin signal sequence (for expression in insect cells), and the yeast alpha factor signal sequence (for expression in yeast cells). In an exemplary embodiment, the secretion signal sequence is a CD5 secretion signal peptide. In one embodiment, the CD5 secretion signal peptide comprises the amino acid sequence of SEQ ID NO: 5.

[0153] In some embodiments, the NA polypeptide includes other functional sequences, such as linker sequences. In some embodiments, the linker is a polypeptide. In some embodiments, the length of the linker is less than about 100 amino acids. For example, the length of the linker can be less than about 100, about 90, about 80, about 70, about 60, about 50, about 40, about 30, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 12, about 11, about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3 or about 2 amino acids.

[0154] In various embodiments, the linker consists essentially of glycine and / or serine residues (e.g., about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 97% glycine and serine). For example, in some embodiments, the linker is (Gly 4 Ser) n , wherein n is from about 1 to about 8, e.g., 1, 2, 3, 4, 5, 6, 7, or 8 (SEQ ID NO: 21). In one embodiment, the linker sequence is GGSGGSGGGGSGGGGS (SEQ ID NO: 22). Other illustrative linkers include, but are not limited to, those having the sequences LE, GGGGS (SEQ ID NO: 23), (GGGGS) n (n=1-4) (SEQ ID NO: 24), (Gly) 8 (SEQ ID NO: 25), (Gly) 6 (SEQ ID NO: 26), (EAAAK) n (n=1-3) (SEQ ID NO: 27), A (EAAAK) n A (n=2-5) (SEQ ID NO: 28), AEAAAKEAAAKA (SEQ ID NO: 29), A (EAAAK) 4 ALEA(EAAAK) 4 A (SEQ ID NO: 30), PAPAP (SEQ ID NO: 31), KESGSSVSSEQLAQFRSLD (SEQ ID NO: 32), EGKSSGSGSESKST (SEQ ID NO: 33), GSAGSAAGSGEF (SEQ ID NO: 34) and (XP) n (wherein X represents any amino acid, e.g., Ala, Lys, or Glu). In an exemplary embodiment, the linker is GGS or GSG. In another exemplary embodiment, the linker is SA.

[0155] In some embodiments, the NA polypeptide further comprises a functional tag sequence (e.g., to facilitate protein purification). Without limitation, the NA polypeptide can include an affinity tag, such as glutathione-S-transferase (GST), polyhistidine (e.g., 6xHis (SEQ ID NO: 35)), protein A, streptavidin / biotin-based tags, and any other protein tags known in the art.

[0156] In some embodiments, the NA polypeptide further comprises a protease cleavage site, such as a thrombin cleavage site, a trypsin cleavage site, an enterokinase cleavage site, or any other protease cleavage site known in the art.

[0157] In various embodiments, the NA polypeptides (or tetrameric NA proteins) described herein provide improved protective immunity (e.g., a broadly reactive immune response) against a range of influenza viruses with different sequences. In various embodiments, the NA polypeptides (or tetrameric NA proteins) described herein elicit an enhanced immune response against conserved epitopes in multiple influenza strains. In various embodiments, the NA polypeptides (or tetrameric NA proteins) described herein induce an immune response against different influenza viruses that exhibit antigenic shift or antigenic drift.

[0158] In some embodiments, the NA polypeptides or tetrameric NA proteins described herein exhibit greater immunogenicity in different influenza strains / types / subtypes compared to NA polypeptides or tetrameric NA proteins from wild-type or naturally occurring influenza virus strains. In some embodiments, the NA polypeptides or tetrameric NA proteins described herein have greater stability compared to NA polypeptides or tetrameric NA proteins from wild-type or naturally occurring influenza virus strains.

[0159] Nucleic acid construction and expression

[0160] In various embodiments, methods for producing recombinant NA polypeptides are provided. The methods include producing NA polypeptides comprising a consensus amino acid sequence using the cluster-based consensus method described herein, and producing the NA polypeptides by transfecting a host cell with a vector encoding the NA polypeptide. In various embodiments, methods for producing recombinant tetrameric NA proteins are also provided.

[0161] In some embodiments, the NA polypeptides described herein can be produced from nucleic acids using molecular biology methods known in the art. For example, a nucleic acid molecule is inserted into a vector that is capable of expressing the NA polypeptide when introduced into an appropriate host cell. Suitable host cells include, but are not limited to, bacteria, yeast, insect, and mammalian cells, as well as any other cell types described elsewhere herein. Any method known to those skilled in the art for inserting DNA fragments into a vector can be used to construct an expression vector encoding the NA polypeptide under the control of transcription / translation control signals. These methods may include in vitro recombinant DNA and synthetic cloning techniques as well as in vivo recombination techniques (see Sambrook et al. Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory; Current Protocols in Molecular Biology, Ausubel, et al., Greene Publ. Assoc., Wiley-Interscience, New York).

[0162] In some embodiments, the present invention provides nucleic acids encoding NA polypeptides or characteristic or biologically active portions of NA polypeptides. In some embodiments, the present invention provides nucleic acids complementary to nucleic acids encoding NA polypeptides or characteristic or biologically active portions of NA polypeptides. In other embodiments, the present invention further provides nucleic acids encoding tetrameric NA proteins.

[0163] In some embodiments, the invention provides nucleic acid molecules that hybridize to nucleic acids encoding NA polypeptides or characteristic or biologically active portions of NA polypeptides. Such nucleic acids can be used, for example, as primers or probes. In exemplary embodiments, such nucleic acids can be used as primers in polymerase chain reaction (PCR), as probes for hybridization (including in situ hybridization) and / or as primers for reverse transcription PCR (RT-PCR).

[0164] In some embodiments, nucleic acid can be DNA or RNA, and can be single-stranded or double-stranded. In some embodiments, nucleic acid according to the present invention can include one or more non-natural nucleotides. In some embodiments, nucleic acid according to the present invention only includes natural nucleotides.

[0165] The expression of nucleic acid molecules according to the present invention can be regulated by the second nucleic acid sequence so that the molecule is expressed in a host transformed with a recombinant DNA molecule. For example, the expression of nucleic acid molecules of the present invention can be controlled by promoters and / or enhancer elements known in the art.

[0166] In some embodiments, an expression vector containing a nucleic acid molecule is transformed into a suitable host cell to allow production of a NA polypeptide or protein encoded by the nucleic acid construct. The host cell transformed with the expression vector is then grown under conditions that allow production of the NA polypeptide or tetrameric NA protein of the invention, followed by recovery of the polypeptide or protein. Exemplary cell types that can be used in the present invention include, but are not limited to, mammalian cells, insect cells, yeast cells, plant cells, and bacterial cells. Insect cells include, but are not limited to, SF cells, caterpillar cells, butterfly cells, moth cells, SF9 cells, SF21 cells, fruit fly cells, S2 cells, fall armyworm cells, cabbage looper cells, Spodoptera frugiperda cells, and cabbage looper cells. Suitable mammalian cells include, but are not limited to, beagle kidney (MDCK) cells, VERO cells, EBx cells, chicken embryo cells, Chinese hamster ovary (CHO) cells, monkey kidney cells, human embryonic kidney cells, HEK293T cells, NSO cells, myeloma cells, hybridoma cells, primary adenoid cell lines, primary bronchial epithelial cells, transformed human cell lines, and Per.C6 cells. Other useful cells or cell systems include, but are not limited to, plant-based systems (e.g., tobacco plants; see, e.g., Jul-Larsen, A., et al., Hum Vaccin Immunother., 8(5): 653-61, 2012), yeast (see, e.g., Athmaram, TN et al., Virol J., 8: 524, 2011) and fungi (see, e.g., Allgaier, S. et al., Biologicals, 37: 128-32, 2009). The present invention also encompasses bacterial-based expression systems (see, e.g., Davis, AR et al., Gene, 21: 273-284, 1983). The present invention further contemplates the use of baculovirus systems.

[0167] The NA polypeptide or tetrameric NA protein of the present invention can be purified by any technique known in the art. For example, the NA polypeptide or tetrameric NA protein can be recovered from the cells as a soluble fraction or as inclusion bodies, and the NA polypeptide or tetrameric NA protein can be extracted therefrom by, for example, guanidine hydrochloride and dialysis. In order to further purify the NA polypeptide or tetrameric NA protein, conventional ion exchange chromatography, hydrophobic interaction chromatography, reverse phase chromatography, size exclusion chromatography, affinity chromatography, gel filtration or a combination thereof can be used. In some embodiments, the NA polypeptide or tetrameric NA protein can also be recovered from the conditioned medium after secretion from eukaryotic or prokaryotic cells. In such embodiments, a purified recombinant NA polypeptide or tetrameric NA protein is produced by culturing host cells under conditions sufficient for the cells to secrete the polypeptide or protein into the culture supernatant and purifying the polypeptide or protein from the supernatant.

[0168] In some embodiments, the recombinant NA polypeptide is purified from the host cell as a monomer. In other embodiments, the recombinant NA polypeptide is purified from the host cell as a tetramer.

[0169] Evaluation of NA peptides and proteins

[0170] In some embodiments, the present invention contemplates evaluating NA polypeptides or tetrameric NA proteins produced using the methods described herein to determine whether they (i) elicit an immune response against one or more influenza viruses; (ii) provide a protective immune response against one or more influenza viruses, or (iii) produce antibodies against one or more influenza viruses after administration to a subject. Various methods for testing such functions are well known in the art and can be utilized.

[0171] In some embodiments, the desired expression and conformation of the NA polypeptides or tetrameric NA proteins produced according to the methods described herein are evaluated. Screening methods are well known in the art and include cell-free assays, cell-based assays, and animal assays. In vitro assays include solid-state or soluble target molecule detection methods involving the use of detectable markers. In such assays, the NA polypeptides or tetrameric NA proteins can be identified by binding to a target molecule (e.g., an immunoglobulin). In some embodiments, the NA polypeptides or proteins described herein can be selected based on desired expression and conformational characteristics.

[0172] The present invention further provides a method for testing NA polypeptides in an animal host. As used herein, "animal host" includes any animal model suitable for influenza research. For example, animal hosts include mammalian hosts, including but not limited to primates, ferrets, cats, dogs, cows, horses, rabbits and rodents (such as mice, hamsters and rats). In some embodiments, the animal host is inoculated, infected or otherwise exposed to influenza virus before or simultaneously with the administration of NA polypeptide or tetrameric NA protein. Alternatively, a DNA molecule encoding the NA polypeptide or tetrameric NA protein can be administered to the animal host. The animal host can be inoculated, infected or otherwise exposed to influenza virus by any method known in the art, including by an intranasal route.

[0173] In some embodiments, the animal host is naive to viral exposure or infection prior to administration of the NA polypeptide or tetrameric NA protein (optionally, as a component in a composition). Naive and / or inoculated animals can be used in any of a variety of studies. For example, such animal models can be used in viral transmission studies, as is known in the art. For example, airborne transmission of viral influenza from inoculated animals (e.g., ferrets) to naive animals is known (Tumpey et al., 2007, Science 315; 655-59). In exemplary viral transmission studies, NA polypeptides or tetrameric NA proteins can be administered to a suitable animal host to determine the efficacy of the NA polypeptide or protein in eliciting a broad immune response in the animal host. Using this information collected from animal host studies, the efficacy of NA polypeptides or proteins in eliciting an immune response in a human host can be predicted.

[0174] Influenza virus-like particles (VLPs)

[0175] In some embodiments, the invention provides influenza virus-like particles (VLPs) comprising NA polypeptides or tetrameric NA proteins as described herein. In some embodiments, influenza VLPs are generally composed of HA, NA and / or viral structural proteins (e.g., HIV gag, influenza M1 proteins). The production of influenza VLPs is known in the art. For example, influenza VLPs can be produced by transfecting host cells with plasmids encoding HA, NA and / or HIV gag or M1 proteins. In an exemplary embodiment, suitable host cells include human cells (e.g., HEK293T). After the transfected cells are incubated for an appropriate time to allow protein expression (e.g., about 72 hours), VLPs can be separated from cell culture supernatants. In some embodiments, influenza VLPs disclosed herein can be used as influenza vaccines to induce a broad neutralization immune response against one or more influenza viruses.

[0176] Pharmaceutical compositions and administration

[0177] In various embodiments, the present invention provides pharmaceutical compositions comprising NA polypeptides or tetrameric NA proteins and / or related entities as described herein. In some embodiments, the pharmaceutical composition is an immunogenic composition (e.g., a vaccine) capable of eliciting an immune response such as a protective immune response against influenza virus.

[0178] For example, in some embodiments, the pharmaceutical composition may include one or more of the following: (1) a live attenuated influenza virus, e.g., a replication-defective virus, (2) an inactivated virus, (3) a virus-like particle (VLP), (4) a recombinant NA polypeptide or a recombinant tetrameric NA protein of the present invention, or a characteristic or biologically active portion thereof, (5) a nucleic acid encoding a NA polypeptide or a tetrameric NA protein of the present invention, or a characteristic or biologically active portion thereof, (6) a DNA vector encoding a NA polypeptide or a tetrameric NA protein of the present invention, or a characteristic or biologically active portion thereof, and / or (7) an expression system, e.g., a cell expressing a NA polypeptide or a tetrameric NA protein of the present invention.

[0179] In some embodiments, the present invention provides a pharmaceutical composition comprising an antibody or other agent related to the NA polypeptide or tetrameric NA protein of the present invention. In one embodiment, the pharmaceutical composition comprises an antibody that binds and / or competes with the NA polypeptide or tetrameric NA protein described herein. Alternatively, the antibody can recognize a viral particle comprising the NA polypeptide or tetrameric NA protein described herein. In another embodiment, the pharmaceutical composition comprises a small molecule that interacts or competes with the NA polypeptide or tetrameric NA protein described herein. In a further embodiment, the pharmaceutical composition comprises a nucleic acid that can be used for gene silencing, such as a nucleic acid having a sequence complementary to the NA polypeptide sequence.

[0180] In some embodiments, the pharmaceutical compositions described herein are administered alone or in combination with one or more agents to enhance immune response. For example, in some embodiments, the pharmaceutical composition is administered in combination with an adjuvant. The present invention contemplates the use of any known adjuvant. Exemplary adjuvants include, but are not limited to, Freund's incomplete adjuvant or Freund's complete adjuvant. In some embodiments, one or more cytokines (e.g., IL-2, IL-6, IL-12, RANTES, GM-CSF, TNF-α or IFN-γ), one or more growth factors (e.g., GM-CSF or G-CSF), one or more molecules (such as OX-40L or 41BBL) or a combination thereof, may be used as biological adjuvants. In some embodiments, the pharmaceutical composition may include aluminum salts and monophosphoryl lipid A as adjuvants. Alternatively or additionally, adjuvants used in human vaccines, such as MF59 (Chiron Corporation), CPG 7909 (Cooper et al., (2004) Vaccine, 22: 3136) and saponins, such as QS21 (Ghochikyan et al., (2006) Vaccine, 24: 2275) can be used. Other examples of adjuvants include, but are not limited to, poly[di(carboxybenzoyloxy)phosphazene] (PCCP; Payne et al., (1998) Vaccine, 16: 92), block copolymer P1205 (CRL1005; Katz et al., (2000) Vaccine, 18: 2177) and polymethyl methacrylate (PMMA; Kreuter et al., (1981) J. Pharm. Sci., 70: 367). Other adjuvants are described elsewhere herein.

[0181] In some embodiments, the pharmaceutical composition also includes a pharmaceutically acceptable carrier or excipient. As used herein, the term "carrier" refers to a diluent, adjuvant, excipient or vehicle administered with the pharmaceutical composition. In an exemplary embodiment, the carrier may include a sterile liquid, such as, for example, water and oil, including oils of petroleum, animal, plant or synthetic origin, such as, for example, peanut oil, soybean oil, mineral oil, sesame oil, etc. In some embodiments, the carrier is or includes one or more solid components. Pharmaceutically acceptable carriers may also include, but are not limited to, saline, buffered saline, dextrose, glycerol, ethanol and combinations thereof. As used herein, excipients are any non-therapeutic agents that may be included in a pharmaceutical composition, such as to provide or contribute to a desired consistency or stabilization. Suitable pharmaceutical excipients include, but are not limited to, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene, glycol, water, ethanol, etc. In various embodiments, the pharmaceutical composition is sterile.

[0182] In some embodiments, the pharmaceutical composition contains a small amount of wetting agent or emulsifier or pH buffer. In some embodiments, the pharmaceutical composition may include any of a variety of additives, such as stabilizers, buffers or preservatives. In addition, adjuvants, stabilizers, thickeners, lubricants and coloring agents may be included.

[0183] In various embodiments, the pharmaceutical composition can be formulated to be suitable for any desired mode of administration. For example, the pharmaceutical composition can take the form of a solution, suspension, emulsion, drops, tablets, pills, pellets, capsules, capsules containing liquids, gelatin capsules, powders, sustained-release formulations, suppositories, emulsions, aerosols, sprays, suspensions, lyophilized powders, lyophilized suspensions, dry powders, or any other suitable use. General considerations in the preparation and manufacture of medicaments can be found in, for example, Remington's Pharmaceutical Sciences, 19th edition, Mack Publishing Co., Easton, Pennsylvania, 1995 (incorporated herein by reference).

[0184] The pharmaceutical composition can be administered by any route of administration. Routes of administration include, for example, oral, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, mucosal, epidural, sublingual, intranasal, intracerebral, intravaginal, transdermal, rectal, by intratracheal instillation, bronchial instillation, inhalation or topical. Administration can be local or systemic. In some embodiments, administration is oral. In another embodiment, administration is by parenteral injection. In some cases, administration results in the release of the NA polypeptide or tetrameric NA protein described herein into the bloodstream. The mode of administration can be determined at the practitioner's discretion.

[0185] In one embodiment, the pharmaceutical composition is suitable for oral administration. Compositions for oral delivery may be in the form of, for example, tablets, lozenges, aqueous or oily suspensions, granules, powders, emulsions, capsules, syrups or elixirs.

[0186] In another embodiment, the pharmaceutical composition is suitable for parenteral administration (e.g., intravenous, intramuscular, intraperitoneal and subcutaneous). Such compositions can be formulated into, for example, solutions, suspensions, dispersions, emulsions, etc. They can also be manufactured in the form of sterile solid compositions (e.g., freeze-dried compositions, which can be dissolved or suspended in sterile injection media immediately before use). For example, parenteral administration can be achieved by injection. In such embodiments, injections can be prepared in conventional forms, i.e. as liquid solutions or suspensions, solid forms suitable for dissolving or being suspended in liquids before injection, or as emulsions. In some embodiments, injection solutions and suspensions are prepared by sterile powders, freeze-dried powders or granules.

[0187] In further embodiments, the pharmaceutical composition is formulated to be delivered by inhalation (for example, delivered directly to the lungs and respiratory system). For example, the composition can take the form of a nasal spray or any other known aerosol formulation. In some embodiments, the preparation for inhalation or aerosol delivery comprises a plurality of particles. In some embodiments, such preparations can have an average particle size of about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12 or about 13 microns. In some embodiments, the preparation for inhalation or aerosol delivery is formulated into a dry powder. In some embodiments, the preparation for inhalation or aerosol delivery is formulated into a wet powder, for example, by comprising a wetting agent. In some embodiments, the wetting agent is selected from the liquid of water, saline or other physiological pH.

[0188] In some embodiments, the pharmaceutical composition according to the present invention is administered to the nasal or buccal cavity in the form of drops. In some embodiments, the dosage may include multiple drops (e.g., 1-100, 1-50, 1-20, 1-10, 1-5 drops, etc.).

[0189] In some embodiments, the pharmaceutical composition will comprise a NA polypeptide or tetrameric NA protein encapsulated, captured or bound within a lipid vesicle, a bioavailable and / or biocompatible and / or biodegradable matrix or other microparticle. In some embodiments, the pharmaceutical composition comprises nanoparticles displaying the NA polypeptide or tetrameric NA protein. In some embodiments, the nanoparticle is a ferritin nanoparticle (see, e.g., U.S. Patent Publication No. 2014 / 0072958).

[0190] The pharmaceutical composition of the present invention can be administered in any dosage suitable for achieving the desired result. In some embodiments, the desired result is to induce a long-term adaptive immune response to one or more influenza strains. In some embodiments, the desired result is to reduce the intensity, severity, frequency and / or delayed onset of one or more symptoms of influenza infection. In some embodiments, the desired result is to inhibit or prevent influenza virus infection. Depending on the species, age, weight and general condition of the subject, the severity of the infection being prevented or treated, the specific composition used and its mode of administration, the required dosage will vary from subject to subject.

[0191] In some embodiments, the pharmaceutical composition according to the present invention is administered in a single dose or multiple doses. In some embodiments, the pharmaceutical composition is administered in multiple doses on different days (e.g., primary immunity-boosting immunization strategy). In some embodiments, the pharmaceutical composition is administered according to a continuous administration regimen so that the subject does not experience a period less than the therapeutic dose inserted between treatment dose periods. In some embodiments, the pharmaceutical composition is administered according to an intermittent administration regimen so that the subject experiences at least one period less than the therapeutic dose inserted between two treatment dose periods. In some embodiments, the pharmaceutical composition is administered as a part of a booster immunization regimen.

[0192] In various embodiments, the pharmaceutical composition is co-administered with one or more other therapeutic agents. If the timing is such that the pharmacological activity of the other therapeutic agent overlaps in time with that of the NA polypeptide or the tetrameric NA protein, then co-administration does not require simultaneous administration of the therapeutic agents to exert a combined therapeutic effect. Typically, each agent will be administered at a dose and schedule determined for that agent.

[0193] In some embodiments, the pharmaceutical composition is co-administered with other conventional influenza vaccines. For example, the pharmaceutical composition of the present invention can be co-administered with a seasonal influenza vaccine. In some embodiments, the seasonal influenza vaccine can be monovalent, bivalent, trivalent, or quadrivalent.

[0194] In some embodiments, the present invention encompasses the delivery of a pharmaceutical composition in combination with an agent that can improve its bioavailability, reduce or modify its metabolism, inhibit its excretion, and / or modify its distribution in the body. In some embodiments, the pharmaceutical compositions described herein are combined with one or more antiviral agents (e.g., oseltamivir, Zanamivir etc.) in combination.

[0195] Antibodies to NA Peptides

[0196] The present invention provides antibodies against NA polypeptides or tetrameric NA proteins produced according to the present invention. These may be monoclonal or polyclonal and may be prepared by any of a variety of techniques known to those of ordinary skill in the art (e.g., see Harlow and Lane, 1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory; incorporated herein by reference). For example, antibodies may be produced by cell culture techniques, including the production of monoclonal antibodies, or by transfecting antibody genes into suitable bacterial or mammalian host cells to produce antibodies.

[0197] In some embodiments, the antibody may be a classical antibody consisting of two heavy chains and two light chains. In other embodiments, the antibody may be an antibody derivative. For example, in some embodiments, the antibody may be a single domain antibody, a recombinant antibody with only a heavy chain (VHH), a single chain antibody (scFv), a shark antibody with only a heavy chain (VNAR), a microprotein (cysteine ​​knot protein, knottin), a DARPin, a tetranectin, an affinity body, a trans body, an Anticalin, an AdNectin, an Affilin, a microbody, a peptide aptamer, an Ortress, a plastic antibody, a Filobody, a Stardubody, a giant body, an Ivybody, a Phinobody, an Armadillo repeat protein, a Kunitz-type domain, a high affinity multimer, an Atrebody, a Probody, an Immunobody, a Triocumab, a Trojan body; a Papebody; a Vaccinebody, a Monospecific; an Affinity Multimer, a Bispecific, a Fv, a Fab, a Fab′, a F(ab′) 2 , peptide mimetic molecules, or synthetic molecules or any other antibody format known in the art.

[0198] Methods of immunizing and protecting against influenza viruses

[0199] On the other hand, the present invention provides a method for immunizing a subject against one or more influenza viruses in the subject. The present invention further provides a method for eliciting an immune response against one or more influenza viruses in a subject. In some embodiments, the method comprises administering to the subject an effective amount of a pharmaceutical composition as described herein. In some embodiments, the method comprises administering to the subject an effective amount of a recombinant NA polypeptide or tetrameric NA protein as described herein. In some embodiments, the method comprises administering to the subject an effective amount of a VLP comprising a NA polypeptide or tetrameric NA protein as described herein.

[0200] In various embodiments, the immunization methods provided herein cause a wide range of protective immune responses for multiple epitopes in one or more influenza viruses. In various embodiments, the immunization methods provided herein cause a wide range of neutralizing immune responses for one or more influenza viruses. In some embodiments, the immune response includes an antibody response. Therefore, in various embodiments, the pharmaceutical composition described herein can provide a wide range of cross protection for different types of influenza viruses. In some embodiments, the pharmaceutical composition provides cross protection for avian, swine, seasonal and / or pandemic influenza viruses. In some embodiments, the pharmaceutical composition provides cross protection for one or more A, B or C subtype influenzas. In some embodiments, the pharmaceutical composition provides cross protection for multiple strains of influenza A H1 subtype virus (e.g., H1N1), influenza A H3 subtype virus (e.g., H3N2) and / or influenza A H5 subtype virus (e.g., H5N1).

[0201] In some embodiments, the method of the present invention can cause an improved immune response for one or more seasonal influenza strains. Exemplary seasonal influenza strains include but are not limited to A / Puerto Rico / 8 / 1934, A / Ford / Monmouth / 1 / 1947, A / Chile / 1 / 1983, A / Texas / 36 / 1991, A / Singapore / 6 / 1986, A / Beijing / 32 / 1992, A / New Caledonia / 20 / 1999, A / Solomon Islands / 03 / 2006 and A / Brisbane / 59 / 2007. In some embodiments, the method of the present invention can cause an improved immune response for one or more pandemic influenza strains. Exemplary pandemic influenza strains include but are not limited to A / California / 07 / 2009, A / California / 04 / 2009, A / Belgium / 145 / 2009, A / South Carolina / 01 / 1918 and A / New Jersey / 1976. Pandemic subtypes include H5N1, H2N2, H9N2, H7N7, H7N3, H7N9 and H10N7 subtypes. In some embodiments, the method of the present invention can induce an improved immune response to one or more swine flu strains. Exemplary swine flu strains include, but are not limited to, A / New Jersey / 1976 isolates and A / California / 07 / 2009. Other pandemic influenza, seasonal influenza and / or swine flu strains are known in the art.

[0202] In various embodiments, the present method can extend immune protection to a series of influenza strains with different antigens. For example, the method of the present invention can induce an immune response to a new pandemic influenza strain caused by antigenic shift (i.e., so that they cover antigenic strains that are far apart across an extended timeline in genetic sequence space). The present method can also be used to solve the genetic changes that occur in a relatively short period of time, so that the pharmaceutical composition of the present invention continues to be effective by inducing an immune response (e.g., improved seasonal response) to a circulating seasonal influenza strain with antigenic drift. Therefore, in various embodiments, the method of the present invention can be used: (1) to expand the coverage (i.e., the ability to induce a neutralizing immune response) for one or more seasonal influenza strains; (2) to expand the coverage (to solve antigenic drift) for one or more pandemic influenza strains; and (3) to expand the coverage for any other antigenic different influenza strains. It is considered that the present method can provide extensive, long-term (e.g., multi-season) protection against influenza viruses (including mismatched strains).

[0203] In some embodiments, the present invention provides a method for preventing or treating influenza infection by administering a pharmaceutical composition of the present invention to a subject in need thereof. In some embodiments, the subject suffers from or is susceptible to influenza infection. In some embodiments, if the subject exhibits one or more symptoms typically associated with influenza infection, the subject is considered to suffer from influenza infection. In some embodiments, it is known or believed that the subject has been exposed to influenza virus. In some embodiments, if it is known or believed that the subject has been exposed to influenza virus, the subject is considered to be susceptible to influenza infection. In some embodiments, if the subject has been in contact with other individuals known or suspected of having been infected with influenza virus and / or if the subject is now or has appeared in a place known or believed to be infected with influenza, it is known or believed that the subject has been exposed to influenza virus.

[0204] In various embodiments, the pharmaceutical compositions described herein can be administered before or after the occurrence of one or more influenza infection symptoms. In some embodiments, the pharmaceutical compositions are administered as a prophylactic drug. In such embodiments, the methods of the present invention effectively prevent or protect subjects from influenza virus infection. In some embodiments, the pharmaceutical compositions of the present invention are used as components of seasonal and / or pandemic influenza vaccines or as part of an influenza vaccination regimen intended to give long-term (multi-season) protection. In some embodiments, the pharmaceutical compositions of the present invention are used to treat the symptoms of influenza infection.

[0205] In some embodiments, a subject suffering from or susceptible to influenza infection is tested for antibodies to the NA polypeptides or tetrameric NA proteins of the invention before, during, or after administration of a pharmaceutical composition according to the invention. In some embodiments, a subject having such antibodies is not administered a pharmaceutical composition of the invention. In some embodiments, an appropriate dose of a pharmaceutical composition is selected based on the detection of such antibodies (or the lack thereof).

[0206] In various embodiments, the subject is any member of the animal kingdom. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a mammal, avian (e.g., chicken or bird), reptile, amphibian, fish, insect and / or worm. In certain embodiments, the non-human subject is a mammal (e.g., rodent, mouse, rat, rabbit, monkey, dog, cat, sheep, cattle, primate and / or pig). In some embodiments, the subject is a transgenic animal, a genetically engineered animal and / or a clone.

[0207] In some embodiments, the subject is a human. In certain embodiments, the subject is an adult, a teenager, or an infant. In some embodiments, the human subject is less than 6 months old. In some embodiments, the human subject is 6 months old or older, 6 months to 35 months old, 36 months to 8 years old, or 9 years old or older. In some embodiments, the human subject is 55 years old or older, such as 60 years old or older, or 65 years old or older. The present invention also contemplates the administration of pharmaceutical compositions and / or the execution of intrauterine treatment methods.

[0208] This description and exemplary embodiments should not be considered as limiting. For the purpose of this specification and the appended claims, unless otherwise indicated, all numerals and other numerical values ​​expressing quantities, percentages or ratios used in the specification and claims should be understood to be modified by the term "about" in all cases to the extent that it cannot be so modified. Therefore, unless otherwise indicated, the numerical parameters set forth in the following specification and the appended claims are approximate values, which can vary according to the desired characteristics attempted to be obtained. At least and without attempting to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be interpreted as being based on the number of reported significant figures and by applying ordinary rounding techniques.

[0209] Note that, as used in this specification and the appended claims, the singular forms "a," "an," and "the," as well as any singular use of any term, include plural referents unless expressly and unambiguously limited to one referent. As used herein, the term "include" and its grammatical variations are intended to be non-limiting, such that recitation of items in a list does not exclude other similar items that can be substituted or added to the listed items.

[0210] The present invention will be more fully understood by reference to the following examples.All literature citations included herein are incorporated herein by reference.

[0211] Example

[0212] The following examples are provided to illustrate certain disclosed embodiments and should not be construed as limiting the scope of the present disclosure in any way.As used herein, CBC NA refers to a NA polypeptide or protein designed using the cluster-based consensus (CBC) method described herein.

[0213] Example 1. Cluster-based consensus approach for designing recombinant influenza NA polypeptides

[0214] Recombinant influenza NA polypeptides were generated using a cluster-based consensus (CBC) method. As a first step, influenza A neuraminidase (NA) protein sequences (H1N1 subtype) from 1918 to 2011 were downloaded from the influenza virus resources of the National Center for Biotechnology Information (see Bao et al. (2008) J. Virol. 82, 596-601). Specifically, a set of non-redundant 1796 full-length human host NA protein sequences were identified for analysis and generation of consensus amino acid sequences. MAFFT v7 (Katoh, S. (2013) Mol. Biol. Evol. 30, 772-780) was used for non-redundant sequence alignment to generate multiple sequence alignments. Subsequently, python was used to calculate all pairs of all paired identity matrices. Then, classical multidimensional scaling (MDS) was performed on the paired identity matrix to visualize the dimensionality reduction and similarity between sequences, thereby generating clusters of similar sequences. Retaining only the first two dimensions of the MDS, the relationship between each sequence in the high-dimensional identity matrix can be mapped into a two-dimensional scatter plot.

[0215] From the two-dimensional representation of the pairwise identity matrix, a total of five non-overlapping clusters of similar protein sequences were defined ( Figure 1A The five clusters represent seasonal influenza-like sequences (three clusters: 1933-1950; 1948-1997; 1998-2009), swine influenza-like sequences (one cluster: 1976-2008), and pandemic influenza-like sequences (one cluster: 2009-2011).

[0216] To generate consensus sequences between sequence clusters, consensus sequences within each sequence cluster were first generated by majority voting (i.e., the most frequent amino acid at each position). For example, if the frequency of an amino acid at a given position was 50% or higher, the amino acid was designated as a consensus amino acid, and if the frequency of an amino acid at a given position was less than 50%, the amino acid was designated as a variable amino acid. In the case of amino acid variation at a particular position in the alignment (e.g., maximum frequency < 0.5), the determination of the representative amino acid at that position was based on an analysis of the structural model of the consensus sequence generated by the comparison model.

[0217] Specifically, in cases where an unambiguous majority vote cannot define a single amino acid at a particular position in the sequence, multiple consensus sequences are generated (one for each possible amino acid based on the alignment). Positions that cannot be unambiguously determined are coded as 'X' and resolved by molecular modeling from the unique set of possible amino acids that may occur at any particular position. Therefore, an important aspect of the design is to refine it through molecular modeling to resolve potential structural issues and select appropriate amino acids at variable positions, and to select sequences based on low calculated energies. For example, the 3D structures of sequences generated by the consensus method were modeled using the Rosetta molecular modeling software package (Leaver-Fay et al. (2011) Meth. Enzymol. 487: 545-74). Molecules with negative total energy values ​​are predicted to have a high probability of folding into stable and / or functional proteins, while molecules with positive energy values ​​are considered unlikely to fold correctly. Therefore, in cases where residue positions cannot be unambiguously assigned using the consensus generation method, amino acids that result in structures with the lowest or near-lowest calculated potential energy are selected because they are assumed to be more stable and therefore likely to be expressed and function. Using this process, a set of energy-minimized designs including multiple candidate sequences are generated. In some cases, a single representative sequence (intra-cluster prototype sequence) was selected for each of the five clusters for further evaluation.

[0218] To further expand the breadth of antigen coverage, multiple consensus sequences (i.e., intra-cluster sequences) were combined to generate cross-cluster consensus sequences using the same procedures for defining consensus sequences and structural modeling as previously described. Thus, NA polypeptide sequences comprising consensus amino acids were generated by: (i) generating NA polypeptides - NA5200 (SEQ ID NO: 1) of swine flu-like (1976-2008) and pandemic flu-like (2009-2011) sequences, (ii) generating three seasonal flu-like (1933-1950, 1948-1997, 1998-2009) sequence clusters, and iii) generating NA9100 (SEQ ID NO: 3) of all five sequence clusters (see Figure 1A ). Figure 1B The positions of NA5200, NA7900 and NA9100 in sequence space relative to other known H1N1 subtype sequences, as determined by multidimensional calibration, are shown. Other exemplary NA sequences are represented in Table 1 as SEQ ID NOs: 7-18.

[0219] Example 2. Functional characterization of CBC NA polypeptide

[0220] To evaluate the functional activity of CBC NA polypeptides, a MUNANA (4-methylumbelliferone) assay was performed. Specifically, HEK293T cells were transfected with 1 μg of endo-free mega-prep NA plasmid (i.e., full-length NA containing the NA transmembrane and stem domains) or 100 ng of a GFP control plasmid. Results using pCAXL (derived from pCAGG with novel restriction enzyme sites), pcDNA3, and PEF plasmids were compared. Samples were collected three days after transfection.

[0221] NA activity was determined by cleavage of 4-methylumbelliferyl from 2'-(4-methylumbelliferyl)-α-DN-acetylneuraminic acid (1 mM) by CBC NA in 200 mM NaAc (pH 6.5), 2 mM CaCl2, and 1% butanol at 37°C. The release of 4-methylumbelliferyl was measured every 2 minutes for 1 hour (355 nm and 460 nm). One neuraminidase unit was defined as the amount of NA that released 1 nmol of 4-methylumbelliferyl per minute.

[0222] like Figure 2B As shown, the highest NA activity was observed with the pCAXL vector, which corresponds to the highest expression level in this plasmid. When expressed in pCAXL, NA7900 had similar expression levels as NA9100, but had lower enzyme activity. When expressed in pcDNA3, NA7900 was not expressed and had no enzyme activity.

[0223] Next, the immunogenicity of the CBC NA polypeptides was tested by DNA immunization. In one set of experiments, BALB / C mice were primed and boosted (3 weeks apart) with 30 μg of full-length NA5200, NA7900, or NA9100 cloned into the pCAXL vector. An empty plasmid was included as a negative control. Immunization was performed by intramuscular electroporation. Specifically, electroporation was performed around the injection site with a 2-pronged needle with 8 200 V electrical pulses spaced 100 ms apart for 20 ms. Figures 3A-3F Results of an ELISA assay measuring the binding activity of sera to a panel of influenza H1N1 virus antigens (i.e., PR8, USSR / 77, Sing / 86, N.Cal / 99, Bris / 07, or Pdm / 09) are shown. The results indicate that sera from mice immunized with the CBC NA constructs exhibit broad cross-reactivity to a panel of H1N1 virus antigens. In fact, in this ELISA assay, sera from mice immunized with the CBC NA constructs showed evidence of broader binding to H1N1 viruses than sera generated from mice immunized with wild-type PR8 NA.

[0224] To further characterize the CBC NA polypeptides, the NA inhibitory (NAI) activity of sera from mice immunized with 30 μg of full-length NA5200, NA7900, or NA9100 cloned into the pCAXL vector was determined using the ELLA assay ( Figures 4A-4E ELLA assays were performed as described in Couzens, L. et al. (2014) J. Virol. Methods 210, 7-14. Briefly, sera from mice immunized with NA constructs encoding NA5200, NA7900, or NA9100 were prepared. Serial dilutions of heat-inactivated sera (with a starting dilution factor of 1:20) were incubated with influenza A virus (IAV) at a predetermined concentration of virus to provide 90% of maximal NA activity. The sera were cultured at 37°C in a 5% PBS solution supplemented with 10 mg / ml BSA, 1 mM CaCl2, 0.5 mM MgCl 2 and 0.5% Tween20 in PBS for 30 minutes. The dilutions were added to the wells of the plate coated with fetuin (Sigma Aldrich, 5 μg / ml) and incubated at 37°C for 18 hours. HRP-conjugated peanut agglutinin (PNA, SigmaAldrich, 2.5 μg / ml) was used to detect the galactose residues exposed after NA-mediated removal of sialic acid from fetuin. The 50% inhibitory concentration (IC50) was calculated by nonlinear regression analysis (GraphPadPrism). Figures 4A-4E As shown, sera from mice immunized with CBC NA exhibited broadly cross-reactive NAI activity.

[0225] The survival and body weight of mice immunized with CBC NA constructs were analyzed. Specifically, these mice were challenged with 1 LD50 of pdm / 09 (A / Belgium / 1 / 2009) or PR8 virus (respectively Figures 5A-5F The results showed that mice vaccinated with DNA constructs encoding NA5200, NA7900, and NA9100 showed higher survival and less weight loss to viral challenge compared to mice vaccinated with wild-type NA.

[0226] Example 3. Expression and purification of tetrameric CBC NA protein

[0227] Since the NA polypeptides are designed according to criteria different from the natural evolutionary process, it is important to confirm the correct folding and maintenance of epitope integrity. Therefore, NA enzyme activity is used as a surrogate indicator of structural integrity. It has been shown that although NA inhibitory antibodies can be induced by immunization with NA antigen (enzymatically inhibited by the addition of zanamivir) (see Sultana, I., et al. (2011) Vaccine 29, 2601-2606), it is still necessary to maintain NA in its native tetrameric form (see Bucher, DJ and Kilbourne, ED (1972) J. Virol. 10, 60-6 and Deroo, T. et al. (1996) Vaccine 14, 561-569). Therefore, the NA5200, NA7900 and NA9100 polypeptide constructs were modified to allow production and purification as soluble tetrameric proteins in mammalian expression systems. Soluble tetrameric NA derived from the 2009 pandemic influenza A (H1N1pdm) was also produced and used as a naturally occurring control (see Schotsaert et al. 2016).

[0228] Recombinant tetrameric NA proteins (also referred to as rNA) were produced essentially as previously described for A / Belgium / 1 / 2009 rNA (see Schotsaert et al. 2016). Briefly, the stem region of the monomeric NA polypeptides (i.e., NA5200, NA7900, and NA9100) was removed such that the polypeptides included amino acids 75-469 of SEQ ID NO: 1, 2, or 3. The stem region was replaced by a four-arm derived helical peptide (SEQ ID NO: 4) that can drive self-assembly into a tetrameric coiled coil, thereby stabilizing the tertiary and quaternary structures of the NA enzyme domain. Figure 2A A schematic diagram of the tetrameric NA protein structure is provided in .

[0229] An N-terminal CD5-derived secretion signal (SEQ ID NO: 5) promotes secretion of the NA polypeptide and / or NA protein. To facilitate purification of the protein, a Strep-tag (SEQ ID NO: 6) or a HIS tag (e.g., 6X His (SEQ ID NO: 35)) is cloned between the secretion signal and the tetramerization sequence (Schmidt et al. (2011) PLoS One 6, e16284). Further, a linker sequence (SEQ ID No: 19) is also incorporated. It is contemplated that other linker sequences known in the art, such as those comprising a thrombin cleavage site (SEQ ID NO: 20), may also be used.

[0230] In order to purify the strep-tagged NA protein, the NA construct was expressed in Expi293 cells. The culture supernatant was first clarified by low-speed centrifugation (1000Xg), followed by 0.2um filtration to remove insoluble cell debris. The cell supernatant was concentrated by ultrafiltration and tangential flow filtration, and then PBS supplemented with avidin was dialyzed to remove any biotin present in the expression medium. The dialyzed supernatant was loaded onto a Strep-Trap column (GE Healthcare) equilibrated with PBS. The bound protein was eluted with 2.5mM desthiobiotin in PBS. The eluted fractions were combined and concentrated and polished by preparative SEC on a HiLoad 16 / 600 Superdex 200 (GE Healthcare) equilibrated with PBS.

[0231] In order to purify the His-tagged recombinant NA protein, the NA construct was also expressed in Expi293 cells. The culture supernatant was clarified and concentrated as described above. The dialyzed supernatant was captured on a His Trap Excel (GE Healthcare) resin balanced with PBS at pH 7.4. Unbound proteins were washed away with PBS, and bound proteins were eluted with a linear gradient of 20-500mM imidazole in PBS. The eluted proteins were then purified on a HiLoad XK50 / 70Superdex200 preparative column (GE Healthcare) balanced with PBS in size exclusion mode. In some embodiments, the dialyzed sample was loaded onto a HiTrap QHP column (GE Healthcare) balanced with 50mM Tris, pH 8. Any unbound proteins were washed away with 50mM Tris, pH 8, and bound proteins were eluted with a linear gradient of 0-1M NaCl in 50mM Tris, pH 8.0. The pooled fractions were filtered through a 0.2 um filter, dialyzed against PBS and a small aliquot was snap frozen in liquid nitrogen.

[0232] Size exclusion chromatography analysis showed major peaks including proteins of NA5200, NA7900 and NA9100, with retention times corresponding to the predicted molecular weight of soluble tetrameric NA ( Fig. 7A ). For NA5200 and NA9100 tetrameric proteins, a secondary peak was observed that eluted faster from the size exclusion column than the primary peak. These fractions likely corresponded to aggregated forms of NA and were discarded.

[0233] The NA activity of the tetrameric NA protein was determined by the MUNANA assay. Figure 7BAs shown, all three tetrameric CBC NA proteins (including NA5200, NA7900 or NA9100 polypeptides) were enzymatically active as determined by the release of 4-methylumbelliferone from a small molecule sialic acid conjugate precursor. The specific activities of these three tetrameric NAs were similar to or slightly higher than those of the soluble tetrameric NA derived from 2009H1N1pdm ( Figure 7B ).

[0234] Next, the possible N-glycosylation sites in the CBC NA head domain were predicted using the NetNGlyc 1.0 server. The potential N-glycosylation sites were compared with the sites in the related N1. The NA5200, NA7900 and NA9100 polypeptides carry three potential N-glycosylation sites, namely, positions 88, 146 and 235 in almost all N1 NAs (see Table 1) (Sun et al., PLoS One, 6, e22844, 2011).

[0235] Altogether, these data indicate that the tetrameric CBC NA protein exhibits correct folding and maintenance of epitope integrity.

[0236] Example 4. Immunological study of tetrameric CBC Na protein

[0237] It is known that protection by vaccination with NA depends on the induction of antibodies that can mediate neuraminidase inhibition (NAI) (see Wohlbold et al. 2015). Therefore, as a first step to examine the potential breadth of the antibody response to the tetrameric CBC NA protein, a protein immunization study was performed. Specifically, six-week-old mice were primed with the Sigma adjuvant system (SAS) containing the immunostimulants monophosphoryl lipid A and synthetic trehalose dimycolate, and then boosted subcutaneously at three-week intervals with 1 μg of recombinant tetrameric CBC NA protein or control NA or 0.1 μg of monovalent inactivated vaccine HA alone or in combination with NA. Serum samples were collected by tail bleeding three weeks after the primary and booster immunizations. In some cases, terminal bleeding was performed by retro-orbital bleeding three weeks after the booster immunization.

[0238] Heat-inactivated sera from mice immunized with CBC NA proteins were compared with heat-inactivated sera from mice immunized with three wild-type NA proteins for their ability to mediate NAI against a panel of human H1N1 viruses. Specifically, the influenza A viruses (IAVs) used in this study were mouse-adapted H1N1 strains A / USSR / 90 / 1977 (USSR / 77), A / New Caledonia / 20 / 1999 (NC / 99), A / Brisbane / 59 / 2007 (Bris / 07), and A / Belgium / 1 / 2009 (Bel / 09; as described at www.ncbi.nlm.nih.gov / nuccore / ?term=txid1502382). The A / swine / Belgium / 1 / 98 strain was adapted to mice by serial passage in mouse lungs (see Neirynck, S. et al. (1999) Nat. Med. 5, 1157-1163). Also included in the panel were A / Puerto Rico / 8 / 34 (PR8 / 34) and the H5N1 virus NIBRG-14. In particular, the NIBRG-14 strain was included because it represents a potential pandemic-causing virus. NIBRG-14 is a 6:2 reverse genetics-derived reassortant virus that expresses the NA (polybasic cleavage site removed) and HA segments of A / Vietnam / 1194 / 2004 (an avian influenza virus isolated from an infected human) and an additional 6 gene segments from PR8 / 34. In general, these strains were selected to look for cross-reactivity of any induced anti-NA antibodies with NA from these distantly related viruses.

[0239] NAI activity was assessed using the ELLA assay as described previously. As observed in previous studies (Wohlbold et al. 2015), immune sera raised against native NA have some cross-reactivity ( Fig. 8A and 8B ). PR8 / 34 NA antiserum mediated NAI against PR8 / 34 and, to a lesser extent, against USSR / 77 and Sw / Bel / 98. Likewise, antisera induced by vaccination with purified recombinant tetrameric NA derived from NC / 99 reacted with self and Bris / 07. Bel / 09 NA antiserum mediated the strongest NAI titers against self and Sw / Bel / 98, followed by NIBRG-14.

[0240] The results showed that the tetrameric CBC NA protein significantly elicited enhanced cross-protection against multiple strains, including distantly related strains ( Fig. 8A and 8B). Anti-NA5200 antiserum strongly inhibited the NA activity of Bel / 09, Sw / Bel / 98 and NIBRG-14, and to a lesser extent PR8 / 34 and USSR / 77. Anti-NA7900 antiserum mediated NAI against all four seasonal H1N1 viruses (PR8 / 34, USSR / 77, NC / 99 and Bris / 07) and NIBRG-14, thus demonstrating a broader NAI span than antiserum against NC / 99 tetrameric rNA. Finally, antiserum raised against NA9100 showed substantial NAI against all H1N1 viruses tested.

[0241] In addition, the percentage of sequence identity shared between the CBC NA or wild-type (WT) NA used in the ELLA assay and the H1N1 virus was determined. For the evaluation, only amino acids above 75 (relative to SEQ ID No 1, 2 or 3) were considered, as the individual NA polypeptides lack the native NA stem sequence. The identity percentage was determined using the BlastP suite-2 sequence software (see Bao 2008 and Katoh 2013). Bold numbers indicate that a 1:IC50 of >2.3 (i.e. 1:5) was observed in the NAI assay.

[0242] Table 3. Percentage of identity shared by CBC design and WT NA

[0243]

[0244] As shown in Table 3, NA9100 shared the highest percent identity with the H1N1 viruses tested, followed by NA7900 and then NA5200. In contrast, the identity of WT NA to other H1N1 NAs varied widely. In general, higher sequence identity between WT virus and CBC-designed NAs correlated with greater breadth of coverage of NAIs (as shown in bold in Table 3). Although NC / 99 NA shared 92% identity with US / 77, no NAIs were detected, even though most of the shared NAs exhibited NAIs with ≥88% identity.

[0245] Taken together, these data suggest that while wild-type tetrameric NA induces a certain degree of cross-reactive antibody response, tetrameric CBC NA proteins provide significantly enhanced cross-protection against multiple influenza strains. Without wishing to be bound by theory, it is believed that CBC NA is able to elicit a broadly reactive immune response against multiple conserved epitopes within influenza NA, even if native NA is not.

[0246] Example 5. Vaccination challenge study of tetrameric CBC NA protein

[0247] Active vaccination challenge experiments were performed to further investigate the protective ability of tetrameric CBC NA proteins. In these experiments, mice were primed and boosted subcutaneously with tetrameric CBC NA proteins including NA5200, NA7900, or NA9100 polypeptides and adjuvants. Mice mock-vaccinated with buffer and adjuvant alone were used as controls. Three weeks after the booster immunization, mice were challenged with intranasal infection with 5LD50 of PR8 / 34, USSR / 77, NC / 99, Bel / 09, Sw / Bel / 98, or NIBRG-14. The mice were subsequently evaluated for changes in body weight and survival rate over 14 days ( Figures 9A-9L ).

[0248] By day 9, all challenged mice that received adjuvant alone succumbed to infection ( Figures 9A-9L Vaccination with tetrameric CBC NA proteins, including NA5200, significantly protected mice from challenge with all tested H1N1 viruses, except USSR / 77. In contrast, NA5200 only partially protected vaccinated mice from challenge with USSR / 77, as these mice exhibited transient weight loss over time and were only partially protected from mortality ( Figures 9C-9D ). Vaccination with tetrameric CBC NA proteins including NA7900 significantly protected mice from challenge with PR8 / 34, USSR / 77, NC / 99 or NIBRG-14 infection, but not against Bel / 09 and Sw / Bel / 98, where only partial protection was observed. Vaccination with tetrameric CBC NA proteins including NA9100 showed broad protection and significantly protected mice from challenge with PR8 / 34, NC / 99, Bel / 09, Sw / Bel / 98 and NIBRG-14 infection. Mice infected with USSR / 77 and vaccinated with NA9100 did not show significant differences compared to mock-vaccinated mice when considering the main column effect of the two-way ANOVA (p value = 0.08). However, the weight loss of mice vaccinated with NA9100 was significantly different from that of mice vaccinated with mock vaccination from day 6 to day 9 (p < 0.01, two-way ANOVA), and 100% of the mice survived the infection ( Fig. 9C Altogether, these results suggest that the tetrameric CBC NA protein, including the NA5200 and NA9100 polypeptides, provides broad protection against influenza virus infection, likely across a long time frame and corresponding antigenic space.

[0249] Previous studies have shown that when anti-NA immunity is induced, the viral load in the lung is reduced (Bosch, BJ et al. (2010) J. Virol. 84, 10366-74; Schulman, JL, et al. (1968) J. Virol. 2, 778-86; and Webster, RG and Laver, WGJ, (1967) Immunology 99). Therefore, the ability of vaccination with tetrameric CBC NA proteins including NA5200, NA7900 or NA9100 polypeptides to reduce viral lung load was also evaluated. Specifically, three weeks after the booster immunization, mice were primed and boosted with tetrameric CBC NA proteins including NA5200, NA7900 or NA9100 and infected with 5LD50 of PR8 / 34, NC / 99 or Bel / 09. On days 3 and 7 after infection, the lungs were collected and the viral load was examined.

[0250] The mice were monitored for weight loss and survival, and euthanized if they lost ≥25% of their original body weight. In some experiments, mice were killed on days 3, 6, or 7 with an overdose of sodium pentobarbital (final concentration 3 mg / mouse), bronchoalveolar lavage (BAL) was performed, and the lungs were removed. According to Van Hoecke et al. (see Van Hoecke, et al. (2017) J. Vis. Exp. e55398-e55398), BAL was performed and the viral load of the cell-free supernatant was assessed by tissue culture infection dose (TCID50). The total protein level in the cell-free BAL fluid was determined by Bradford protein dye using a standard curve of BSA. Lung homogenate was prepared and clarified as previously described (see De Baets, S. et al. (2015) PLoS One 10), and viral titers were assessed by TCID50 determination.

[0251] The virus titer in the lung homogenate or BAL fluid (BALF) of clarification was assessed using standard TCID50 assay. The confluent monolayer MDCK cells cultivated in the DMEM supplemented with 10% FCS, non-essential amino acids, 2mM L-glutamine, 0.4mM sodium pyruvate in 96-well plates were washed in serum-free medium and incubated with 10 times of sample dilutions in the serum-free DMEM (Sigma) containing the trypsin processed by 1 μg / ml TPCK. After 7 days post-infection, the virus was detected in the wells by agglutinating chicken erythrocytes and calculated by Reed and Muench method (Reed and Muench 1938).

[0252] like Figures 10A-10FAs shown, the viral load of the group vaccinated with CBC NA did not show any significant reduction compared to the mock-treated animals on day 3. However, by day 7 after challenge, the lung viral titers of all mice vaccinated with tetrameric CBC NA proteins were significantly lower than those of mice vaccinated with mock vaccination. Since vascular leakage and pulmonary edema indicate severe influenza infection (see Tate, MD et al. J. Immunol. (2009) 183 and Job, ER et al. J. Immunol. (2014) 192), the potential benefits of NA vaccination were also evaluated based on the total protein content in cell-free BAL fluid. Compared with controls, mice vaccinated with tetrameric CBC NA proteins and infected with PR8 / 34, NC / 99 or Bel / 09 did not show significant differences in protein levels in BAL fluid isolated on day 3 after challenge, but total protein was significantly reduced on day 7 after infection ( Figures 10A-10F ).

[0253] In summary, these data demonstrate that vaccination with tetrameric CBC NA proteins including NA5200, NA7900 or NA9100 exhibits significant antiviral efficacy as measured by a variety of different assay platforms.

[0254] Example 6. Passive transfer experiment

[0255] As mentioned previously, the major correlate of protection by vaccination with NA is the ability of NA to induce inhibitory antibodies. Therefore, passive transfer experiments were performed to determine whether antibodies are the major mediator of protection induced by CBC NA. In such experiments, heat-inactivated antisera were prepared from mice immunized with DNA encoding NA5200, NA7900, NA9100, or buffer alone (PBS). Figures 11A-11D As shown, after three rounds of immunization, the antisera showed a wide range of NAI activity. The antisera were then passively transferred intranasally into six-week-old mice one day before infection with 2 LD50 of PR8 / 34, NC / 99, or Bel / 09. Antisera raised against each wild-type rNA served as homologous positive controls. The antisera were compared with those previously described in Example 4 ( Figures 8A-8B ) were used to assess the expansion of NAI responses in the same sera used in the previous study. In all viral challenges, the positive control antiserum completely protected mice from weight loss and survival after potentially lethal infection with the homologous virus. Fig.12 An exemplary experimental schedule for passive transfer experiments is provided.

[0256] Figures 13A-13H Results of passive experiments showing protection against weight loss and increased survival after 1 LD50 Be109 challenge provided by CBC NA are presented. Figures 14A-14FProtection against weight loss and increased survival after challenge with 1 LD50 Be109, NC / 99 or PR8 / 34 was shown by CBC NA.

[0257] Passive transfer experiments were also performed by immunizing mice with tetrameric CBC NA protein. Figures 15A-15F As shown, although active vaccination with tetrameric CBC NA including NA5200 protected against PR8 / 34, NC / 99 and Bel / 09 infection, passive transfer of NA5200 antiserum only provided significant protection against weight loss and mortality in mice infected with Bel / 09 or PR8 / 34, but had no significant effect on the case of NC / 99 (p < 0.01, two-way ANOVA or log-rank test). Passive transfer of antiserum from mice immunized with tetrameric CBC NA including NA7900 immune serum significantly protected against morbidity and mortality when infected with PR8 / 34 and NC / 99 compared to the negative control group (p < 0.01, two-way ANOVA or log-rank test). However, passive transfer of NA7900 antiserum did not significantly protect mice from challenge with Bel / 09 virus. Finally, passive transfer of NA9100 antiserum provided significant protection against weight loss and survival against all three challenge viruses (p<0.01, two-way ANOVA or log-rank test).

[0258] In all passive transfer experiments, complete protection against mortality correlated with the ability of the antisera to mediate NAI (see Fig. 8A ). Therefore, it can be concluded that antibodies play a major role in the protection induced by tetrameric CBC NA proteins, although in some cases, such as when protection is apparent in the absence of NAI, another mechanism may also contribute to protection.

[0259] Example 7. Evaluation of the scope of protection and combination methods with monovalent vaccines

[0260] Next, experiments were performed to test whether CBC NA (i) could provide broader protection in vivo compared to wild-type recombinant tetrameric NA protein (rNA), and (ii) could increase the protection provided by split inactivated vaccines. Specifically, mice were vaccinated with NA5200 and NA9100 or wild-type soluble recombinant NA derived from Bel / 09 and NC / 99 alone or in combination with a monovalent H1N1pdm09 vaccine. Subsequently, mice were challenged with Bel / 09 or NC / 99.

[0261] Mice vaccinated with NC / 99 rNA were not significantly protected from weight loss and mortality compared to mice vaccinated with Bel / 09 rNA after Bel / 09 challenge. Mice vaccinated with the homologous vaccine exhibited slightly less but significant weight loss compared to mice vaccinated with NA5200 and NA9100; however, 100% of the mice survived infection ( Fig.16A , 16B ). Further, the viral load in the lungs of mice vaccinated with NC / 99 on day 7 was similar on average to that of mice vaccinated with mock vaccination. The viral titers of mice vaccinated with NA5200 and NA9100 on day 7 were significantly reduced compared with those of mice vaccinated with mock vaccination (p < 0.01, one-way ANOVA), but both homologous rNA and NA5200 outperformed NA9100 NA ( Fig. 16C This pattern was also observed for mice challenged with NC / 99. Mice vaccinated with Bel / 09 RNA lost significantly more weight on day 6 post infection, exhibited increased mortality, and had higher viral loads compared to mice vaccinated with NC / 99 ( Figure 16G , 16H , 16I).

[0262] and Fig.9E and 9F Compared to the data shown in , NA5200 was unable to protect NC / 99 challenged mice to the same extent, but NA9100 did not show a significant difference in its ability to protect mice compared to mice vaccinated with the homologous vaccine. In addition, Bel / 09 rNA was also unable to protect mice from morbidity and mortality when infected with PR8 / 34 virus (data not shown). Taken together, these data indicate that NA9100 exhibits a broader range of protection than the wild-type NA tested.

[0263] Mice vaccinated with the monovalent H1N1 pdm09 vaccine (alone or in combination with recombinant tetrameric NA protein) and challenged with Bel / 09 showed minimal signs of weight loss and almost no mortality ( Fig.16D and 16E Furthermore, by day 7 post-infection, there was no evidence of virus in the lungs ( Fig.16F ). These results demonstrate that immunization with a monovalent vaccine, especially in combination with an adjuvant, is potent against homologous viruses.

[0264] When mice were challenged with NC / 99, the adjuvant vaccine alone also provided some degree of protection compared with mock-vaccinated mice ( Figure 16G-16L ). Neither NA5200 nor NA9100 showed additive effects on weight loss and survival compared with monovalent alone ( Fig.16J and 16KHowever, the addition of NC / 99 or NA9100NA to the monovalent vaccine significantly reduced viral titers in the lung compared to either monovalent vaccine alone ( Figure 16L ). Thus, these data demonstrate the ability of NA to contribute to protection in combination with traditional vaccine approaches.

[0265] Example 8. Evaluation of efficacy against HA variants

[0266] For the 2017 southern hemisphere influenza season, the World Health Organization (WHO) recommended replacing the A(H1N1)pdm09-like virus in seasonal influenza vaccines with an A / Michigan / 45 / 2015-like strain. This influenza variant is hypothesized to have changes within the HA that result in increased infection rates in middle-aged individuals (B. Flannery et al. 2018, JID).

[0267] Therefore, experiments were performed to test whether the tetrameric CBC NA protein could mediate NA inhibition against this variant, as its sequence was not included in the original CBC NA design strategy.

[0268] Initially, HA antigenic differences were investigated between Bel / 09 and the A / Michigan / 45 / 2015-like virus A / Singapore / GP1908 / 2015 (Sing / 15) using hemagglutination inhibition (HAI). A two-fold difference in the ability of mouse antisera raised against the monovalent split A(H1N1)pdm09 vaccine to mediate HAI against Bel / 09 and Sing / 15 was observed (1280 HAU vs. 640 HAU). This result is consistent with previous studies in which ferret reference sera did not indicate evidence of significant antigenic drift.

[0269] Next, the ability of antisera raised against tetrameric CBC NA protein or tetrameric Bel / 09 NA to mediate NA inhibition against Bel / 09 and Sing / 15 was tested ( Figures 17A-17C Although Bel / 09 antisera had significantly lower NAI titers against Sing / 15 drift variants compared to Bel / 09 (p<0.05, one-way ANOVA), antisera raised against tetrameric CBC NAs including NA5200 or NA9100 mediated NAI against Sing / 15 and Bel / 09 equally well, albeit at lower levels than wild-type Bel / 09 antisera ( Figures 17A-17B When considering amino acid identity, starting from amino acid 75, Sing / 15 shares 91%, 87%, 89% and 97% identity with NA5200, NA7900, NA9100 and Bel / 09, respectively ( Fig. 17C ). Therefore, these data indicate that CBC NA exhibits the same level of inhibition against strains that were not included in the initial design of the protein.

[0270] Therefore, it is believed that the cluster-based consensus approach can provide NA polypeptides capable of generating anti-NA responses that span strains that undergo HA antigenic drift. Therefore, the present approach can be used to expand immune responses and provide long-term (i.e., multi-season) protection against a variety of influenza strains, types, and subtypes. Sequence Listing <110> SANOFI PASTEUR INC. VIB Research Institute (VIB VZW) Ghent University <120> Methods for producing broadly protective vaccine compositions comprising neuraminidase <130> 01121-0037-00PCT <140> <141> <150> 62 / 718,527 <151> 2018-08-14 <150> 62 / 649,002 <151> 2018-03-28 <160> 35 <170> PatentIn Version 3.5 <210> 1 <211> 469 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 1 Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Cys Met Thr 1 5 10 15 Ile Gly Met Ala Asn Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile 20 25 30 Trp Ile Ser His Ser Ile Gln Leu Gly Asn Gln Asn Gln Ile Glu Thr 35 40 45 Cys Asn Gln Ser Val Ile Thr Tyr Glu Asn Asn Thr Trp Val Asn Gln 50 55 60 Thr Tyr Val Asn Ile Ser Asn Thr Asn Phe Ala Ala Gly Gln Ser Val 65 70 75 80 Val Ser Ile Lys Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Ser Gly 85 90 95 Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly 100 105 110 Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu 115 120 125 Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His 130 135 140 Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser 145 150 155 160 Cys Pro Ile Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser 165 170 175 Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr 180 185 190 Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr 195 200 205 Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Lys Ile Leu 210 215 220 Arg Thr Gln Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr 225 230 235 240 Val Met Thr Asp Gly Pro Ser Asp Gly Gln Ala Ser Tyr Lys Ile Phe 245 250 255 Lys Ile Glu Lys Gly Lys Ile Val Lys Ser Val Glu Met Asp Ala Pro 260 265 270 Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Ser Gly Lys Val 275 280 285 Met Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val 290 295 300 Ser Phe Asp Gln Asn Leu Asp Tyr Gln Ile Gly Tyr Ile Cys Ser Gly 305 310 315 320 Ile Phe Gly Asp Asn Pro Arg Ser Asn Asp Gly Thr Gly Ser Cys Gly 325 330 335 Pro Val Ser Ser Asn Gly Ala Asn Gly Val Lys Gly Phe Ser Phe Arg 340 345 350 Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Ile Ser Ser Arg 355 360 365 Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp 370 375 380 Asn Ser Phe Ser Ile Lys Gln Asp Ile Val Ala Ile Thr Glu Trp Ser 385 390 395 400 Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Met Asn 405 410 415 Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Gln Pro Lys 420 425 430 Glu Asn Thr Ile Trp Thr Ser Gly Ser Ser Ser Ile Ser Phe Cys Gly Val 435 440 445 Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp Gly Ala Asp Leu Pro 450 455 460 Phe Thr Ile Asp Lys 465 <210> 2 <211> 469 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 2 Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Ala 1 5 10 15 Ile Gly Ile Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile 20 25 30 Trp Val Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile 35 40 45 Cys Asn Gln Arg Ile Ile Thr Tyr Glu Asn Ser Thr Trp Val Asn Gln 50 55 60 Thr Tyr Val Asn Ile Asn Asn Thr Asn Val Val Ala Gly Lys Asp Thr 65 70 75 80 Thr Ser Val Ile Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Arg Gly 85 90 95 Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly 100 105 110 Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu 115 120 125 Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His 130 135 140 Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Ala Leu Met Ser 145 150 155 160 Cys Pro Val Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser 165 170 175 Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr 180 185 190 Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr 195 200 205 Asn Gly Ile Ile Thr Glu Thr Ile Lys Ser Trp Arg Lys Asn Ile Leu 210 215 220 Arg Thr Gln Glu Ser Glu Cys Val Cys Val Asn Gly Ser Cys Phe Thr 225 230 235 240 Ile Met Thr Asp Gly Pro Ser Asp Gly Leu Ala Ser Tyr Lys Ile Phe 245 250 255 Lys Ile Glu Lys Gly Lys Val Thr Lys Ser Ile Glu Leu Asp Ala Pro 260 265 270 Asn Ser His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Thr Gly Lys Val 275 280 285 Met Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val 290 295 300 Ser Phe Asn Gln Asn Leu Asp Tyr Gln Ile Gly Tyr Ile Cys Ser Gly 305 310 315 320 Val Phe Gly Asp Asn Pro Arg Pro Lys Asp Gly Thr Gly Ser Cys Gly 325 330 335 Pro Val Thr Val Asp Gly Ala Asn Gly Val Lys Gly Phe Ser Tyr Arg 340 345 350 Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Asn Ser Ser Arg 355 360 365 Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp 370 375 380 Ser Ser Phe Leu Val Lys Gln Asp Val Val Ala Ile Thr Asp Trp Ser 385 390 395 400 Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp 405 410 415 Cys Met Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Leu Pro Arg 420 425 430 Glu Asp Thr Ile Trp Thr Ser Gly Ser Ser Ser Ile Ser Phe Cys Gly Val 435 440 445 Asn Ser Asp Thr Val Asn Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro 450 455 460 Phe Thr Ile Asp Lys 465 <210> 3 <211> 469 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 3 Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Ala 1 5 10 15 Ile Gly Ile Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile 20 25 30 Trp Val Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile 35 40 45 Cys Asn Gln Arg Ile Ile Thr Tyr Glu Asn Ser Thr Trp Val Asn Gln 50 55 60 Thr Tyr Val Asn Ile Asn Asn Thr Asn Val Val Ala Gly Lys Asp Thr 65 70 75 80 Thr Ser Val Ile Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Arg Gly 85 90 95 Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly 100 105 110 Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu 115 120 125 Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His 130 135 140 Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser 145 150 155 160 Cys Pro Val Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser 165 170 175 Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr 180 185 190 Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr 195 200 205 Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu 210 215 220 Arg Thr Gln Glu Ser Glu Cys Val Cys Ile Asn Gly Ser Cys Phe Thr 225 230 235 240 Ile Met Thr Asp Gly Pro Ser Asp Gly Gln Ala Ser Tyr Lys Ile Phe 245 250 255 Lys Ile Glu Lys Gly Lys Val Thr Lys Ser Ile Glu Leu Asp Ala Pro 260 265 270 Asn Ser His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Thr Gly Lys Val 275 280 285 Val Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val 290 295 300 Ser Phe Asp Gln Asn Leu Asp Tyr Gln Ile Gly Tyr Ile Cys Ser Gly 305 310 315 320 Val Phe Gly Asp Asn Pro Arg Pro Asn Asp Gly Thr Gly Ser Cys Gly 325 330 335 Pro Val Thr Ser Asn Gly Ala Asn Gly Val Lys Gly Phe Ser Phe Arg 340 345 350 Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Asn Ser Ser Arg 355 360 365 Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp 370 375 380 Ser Ser Phe Ser Val Lys Gln Asp Ile Val Ala Ile Thr Asp Trp Ser 385 390 395 400 Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp 405 410 415 Cys Met Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Leu Pro Lys 420 425 430 Glu Asn Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val 435 440 445 Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro 450 455 460 Phe Thr Ile Asp Lys 465 <210> 4 <211> 50 <212> PRT <213> unknown <220> <223> Unknown description: Four-armed polypeptide <400> 4 Ile Ile Asn Glu Thr Ala Asp Asp Ile Val Tyr Arg Leu Thr Val Ile 1 5 10 15 Ile Asp Asp Arg Tyr Glu Ser Leu Lys Asn Leu Ile Thr Leu Arg Ala 20 25 30 Asp Arg Leu Glu Met Ile Ile Asn Asp Asn Val Ser Thr Ile Leu Ala 35 40 45 Ser Ile 50 <210> 5 <211> twenty three <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 5 Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly 1 5 10 15 Met Leu Val Ala Ser Val Leu 20 <210> 6 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 6 Trp Ser His Pro Gln Phe Glu Lys 1 5 <210> 7 <211> 469 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 7 Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Cys Met Thr 1 5 10 15 Ile Gly Met Ala Asn Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile 20 25 30 Trp Ile Ser His Ser Ile Gln Leu Gly Asn Gln Asn Gln Ile Glu Thr 35 40 45 Cys Asn Gln Ser Val Ile Thr Tyr Glu Asn Asn Thr Trp Val Asn Gln 50 55 60 Thr Tyr Val Asn Ile Ser Asn Thr Asn Phe Ala Ala Gly Gln Ser Val 65 70 75 80 Val Ser Val Lys Leu Ala Gly Asn Ser Ser Leu Cys Pro Val Ser Gly 85 90 95 Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly 100 105 110 Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser Pro Leu Glu 115 120 125 Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His 130 135 140 Ser Asn Gly Thr Ile Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser 145 150 155 160 Cys Pro Ile Gly Glu Val Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser 165 170 175 Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Ile Asn Trp Leu Thr 180 185 190 Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr 195 200 205 Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu 210 215 220 Arg Thr Gln Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr 225 230 235 240 Val Met Thr Asp Gly Pro Ser Asp Gly Gln Ala Ser Tyr Lys Ile Phe 245 250 255 Arg Ile Glu Lys Gly Lys Ile Val Lys Ser Val Glu Met Asn Ala Pro 260 265 270 Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Ser Ser Glu Ile 275 280 285 Thr Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val 290 295 300 Ser Phe Asn Gln Asn Leu Glu Tyr Gln Ile Gly Tyr Ile Cys Ser Gly 305 310 315 320 Ile Phe Gly Asp Asn Pro Arg Pro Asn Asp Lys Thr Gly Ser Cys Gly 325 330 335 Pro Val Ser Ser Asn Gly Ala Asn Gly Val Lys Gly Phe Ser Phe Lys 340 345 350 Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Ile Ser Ser Arg 355 360 365 Asn Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Gly Thr Asp 370 375 380 Asn Asn Phe Ser Ile Lys Gln Asp Ile Val Gly Ile Asn Glu Trp Ser 385 390 395 400 Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp 405 410 415 Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys 420 425 430 Glu Asn Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val 435 440 445 Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro 450 455 460 Phe Thr Ile Asp Lys 465 <210> 8 <211> 469 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 8 Met Asn Thr Asn Gln Arg Ile Ile Thr Ile Gly Thr Val Cys Leu Ile 1 5 10 15 Val Gly Ile Ile Ser Leu Leu Leu Gln Ile Gly Asn Ile Val Ser Leu 20 25 30 Trp Ile Ser His Ser Ile Gln Thr Gly Glu Lys Asn His Pro Glu Ile 35 40 45 Cys Asn Gln Ser Val Ile Thr Tyr Glu Asn Asn Thr Trp Val Asn Gln 50 55 60 Thr Tyr Val Asn Ile Ser Asn Thr Asn Ile Ala Ala Gly Gln Gly Val 65 70 75 80 Thr Ser Ile Ile Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Ser Gly 85 90 95 Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly 100 105 110 Asp Ile Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu 115 120 125 Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Arg His 130 135 140 Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser 145 150 155 160 Cys Pro Ile Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser 165 170 175 Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr 180 185 190 Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr 195 200 205 Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Lys Ile Leu 210 215 220 Arg Thr Gln Glu Ser Glu Cys Val Cys Ile Asn Gly Ser Cys Phe Thr 225 230 235 240 Ile Met Thr Asp Gly Pro Ser Asn Gly Gln Ala Ser Tyr Lys Ile Phe 245 250 255 Lys Met Glu Lys Gly Lys Ile Ile Lys Ser Ile Glu Leu Asp Ala Pro 260 265 270 Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Thr Gly Lys Val 275 280 285 Val Cys Val Cys Arg Asp Asn Trp His Ala Ser Asn Arg Pro Trp Val 290 295 300 Ser Phe Asp Gln Asn Leu Asp Tyr Gln Ile Gly Tyr Ile Cys Ser Gly 305 310 315 320 Val Phe Gly Asp Asn Pro Arg Ser Asn Asp Gly Lys Gly Asn Cys Gly 325 330 335 Pro Val Leu Ser Asn Gly Ala Asn Gly Val Lys Gly Phe Ser Phe Arg 340 345 350 Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Ile Ser Ser Arg 355 360 365 Ser Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp 370 375 380 Ser Ser Phe Ser Met Lys Gln Asp Ile Ile Ala Leu Thr Asp Trp Ser 385 390 395 400 Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Met Asn 405 410 415 Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Gln Pro Lys 420 425 430 Glu Ser Thr Ile Trp Thr Ser Ser Gly Ser Ser Ser Ile Ser Phe Cys Gly Val 435 440 445 Asp Ser Glu Thr Ala Ser Trp Ser Trp Pro Asp Gly Ala Asp Leu Pro 450 455 460 Phe Thr Ile Asp Lys 465 <210> 9 <211> 469 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 9 Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Ile Ser Ile Ala 1 5 10 15 Ile Gly Ile Ile Ser Leu Met Leu Gln Ile Gly Asn Ile Ile Ser Ile 20 25 30 Trp Ala Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile 35 40 45 Cys Asn Gln Arg Ile Ile Thr Tyr Glu Asn Ser Thr Trp Val Asn His 50 55 60 Thr Tyr Val Asn Ile Asn Asn Thr Asn Val Val Ala Gly Lys Asp Lys 65 70 75 80 Thr Ser Val Thr Leu Ala Gly Asn Ser Ser Leu Cys Ser Ile Ser Gly 85 90 95 Trp Ala Ile Tyr Thr Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly 100 105 110 Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu 115 120 125 Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His 130 135 140 Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Ala Leu Met Ser 145 150 155 160 Cys Pro Leu Gly Glu Ala Pro Ser Pro Tyr Asn Ser Lys Phe Glu Ser 165 170 175 Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr 180 185 190 Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr 195 200 205 Asn Gly Ile Ile Thr Gly Thr Ile Lys Ser Trp Lys Lys Gln Ile Leu 210 215 220 Arg Thr Gln Glu Ser Glu Cys Val Cys Met Asn Gly Ser Cys Phe Thr 225 230 235 240 Ile Met Thr Asp Gly Pro Ser Asn Gly Ala Ala Ser Tyr Lys Ile Phe 245 250 255 Lys Ile Glu Lys Gly Lys Val Thr Lys Ser Ile Glu Leu Asn Ala Pro 260 265 270 Asn Phe His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Thr Gly Lys Val 275 280 285 Met Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val 290 295 300 Ser Phe Asn Gln Asn Leu Asp Tyr Gln Ile Gly Tyr Ile Cys Ser Gly 305 310 315 320 Val Phe Gly Asp Asn Pro Arg Pro Glu Asp Gly Glu Gly Ser Cys Asn 325 330 335 Pro Val Thr Val Asp Gly Ala Asn Gly Val Lys Gly Phe Ser Tyr Lys 340 345 350 Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Asn Arg Leu Arg 355 360 365 Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Asn Thr Asp 370 375 380 Ser Asp Phe Ser Val Lys Gln Asp Val Val Ala Ile Thr Asp Trp Ser 385 390 395 400 Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp 405 410 415 Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Val Arg Gly Leu Pro Arg 420 425 430 Glu Asn Thr Ile Trp Thr Ser Gly Ser Ser Ser Ile Ser Phe Cys Gly Val 435 440 445 Asn Ser Asp Thr Ala Asn Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro 450 455 460 Phe Thr Ile Asp Lys 465 <210> 10 <211> 469 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 10 Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Ala 1 5 10 15 Ile Gly Ile Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile 20 25 30 Trp Val Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile 35 40 45 Cys Asn Gln Arg Ile Ile Thr Tyr Glu Asn Ser Thr Trp Val Asn Gln 50 55 60 Thr Tyr Val Asn Ile Asn Asn Thr Asn Val Val Ala Gly Lys Asp Thr 65 70 75 80 Thr Ser Met Thr Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Arg Gly 85 90 95 Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly 100 105 110 Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu 115 120 125 Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His 130 135 140 Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Ala Leu Met Ser 145 150 155 160 Cys Pro Ile Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser 165 170 175 Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr 180 185 190 Ile Gly Ile Ser Gly Pro Asp Asp Gly Ala Val Ala Val Leu Lys Tyr 195 200 205 Asn Gly Ile Ile Thr Glu Thr Ile Lys Ser Trp Arg Lys Arg Ile Leu 210 215 220 Arg Thr Gln Glu Ser Glu Cys Val Cys Val Asn Gly Ser Cys Phe Thr 225 230 235 240 Ile Met Thr Asp Gly Pro Ser Asp Gly Pro Ala Ser Tyr Arg Ile Phe 245 250 255 Lys Ile Glu Lys Gly Lys Ile Thr Lys Ser Ile Glu Leu Asp Ala Pro 260 265 270 Asn Ser His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Thr Gly Thr Val 275 280 285 Met Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val 290 295 300 Ser Phe Asn Gln Asn Leu Asp Tyr Gln Ile Gly Tyr Ile Cys Ser Gly 305 310 315 320 Val Phe Gly Asp Asn Pro Arg Pro Lys Asp Gly Lys Gly Ser Cys Asp 325 330 335 Pro Val Thr Val Asp Gly Ala Asp Gly Val Lys Gly Phe Ser Tyr Arg 340 345 350 Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Asn Ser Ser Arg 355 360 365 Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Asp Thr Asp 370 375 380 Ser Asn Phe Leu Val Lys Gln Asp Val Val Ala Met Thr Asp Trp Ser 385 390 395 400 Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp 405 410 415 Cys Met Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Arg 420 425 430 Glu Lys Thr Ile Trp Thr Ser Gly Ser Ser Ser Ile Ser Phe Cys Gly Val 435 440 445 Asn Ser Asp Thr Val Asn Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro 450 455 460 Phe Thr Ile Asp Lys 465 <210> 11 <211> 469 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 11 Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Val 1 5 10 15 Val Gly Ile Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile 20 25 30 Trp Ile Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile 35 40 45 Cys Asn Gln Ser Ile Ile Thr Tyr Lys Asn Ser Thr Trp Val Asn Gln 50 55 60 Thr Tyr Val Asn Ile Ser Asn Thr Asn Val Val Ala Gly Lys Asp Thr 65 70 75 80 Thr Ser Val Ile Leu Thr Gly Asn Ser Ser Leu Cys Pro Ile Arg Gly 85 90 95 Trp Ala Ile Tyr Ser Lys Asp Asn Gly Ile Arg Ile Gly Ser Lys Gly 100 105 110 Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu 115 120 125 Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His 130 135 140 Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Ala Leu Met Ser 145 150 155 160 Cys Pro Val Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser 165 170 175 Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr 180 185 190 Ile Gly Ile Ser Gly Pro Asp Asp Gly Ala Val Ala Val Leu Lys Tyr 195 200 205 Asn Gly Ile Ile Thr Glu Thr Ile Lys Ser Trp Arg Lys Asn Ile Leu 210 215 220 Arg Thr Gln Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr 225 230 235 240 Ile Met Thr Asp Gly Pro Ser Asp Gly Leu Ala Ser Tyr Lys Ile Phe 245 250 255 Lys Ile Glu Lys Gly Lys Val Thr Lys Ser Ile Glu Leu Asp Ala Pro 260 265 270 Asn Ser His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Thr Gly Lys Val 275 280 285 Met Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val 290 295 300 Ser Phe Asp Gln Asn Leu Asp Tyr Gln Ile Gly Tyr Ile Cys Ser Gly 305 310 315 320 Val Phe Gly Asp Asn Pro Arg Pro Lys Asp Gly Thr Gly Ser Cys Gly 325 330 335 Pro Val Tyr Val Asp Gly Ala Asn Gly Val Lys Gly Phe Ser Tyr Arg 340 345 350 Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Gln Ser Ser Arg 355 360 365 Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp 370 375 380 Ser Ser Phe Ser Val Lys Gln Asp Val Val Ala Met Thr Asp Trp Ser 385 390 395 400 Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp 405 410 415 Cys Met Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys 420 425 430 Glu Asp Thr Ile Trp Thr Ser Gly Ser Ser Ser Ile Ser Phe Cys Gly Val 435 440 445 Asn Ser Asp Thr Val Asp Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro 450 455 460 Phe Thr Ile Asp Lys 465 <210> 12 <211> 469 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 12 Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Val 1 5 10 15 Val Gly Ile Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile 20 25 30 Trp Ile Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile 35 40 45 Cys Asn Gln Ser Ile Ile Thr Tyr Lys Asn Ser Thr Trp Val Asn Gln 50 55 60 Thr Tyr Val Asn Ile Ser Asn Thr Asn Val Val Ala Gly Lys Asp Thr 65 70 75 80 Thr Ser Val Ile Leu Thr Gly Asn Ser Ser Leu Cys Pro Ile Arg Gly 85 90 95 Trp Ala Ile Tyr Ser Lys Asp Asn Gly Ile Arg Ile Gly Ser Lys Gly 100 105 110 Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu 115 120 125 Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His 130 135 140 Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Ala Leu Met Ser 145 150 155 160 Cys Pro Val Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser 165 170 175 Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr 180 185 190 Ile Gly Ile Ser Gly Pro Asp Asp Gly Ala Val Ala Val Leu Lys Tyr 195 200 205 Asn Gly Ile Ile Thr Glu Thr Ile Lys Ser Trp Arg Lys Lys Ile Leu 210 215 220 Arg Thr Gln Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr 225 230 235 240 Ile Met Thr Asp Gly Pro Ser Asp Gly Leu Ala Ser Tyr Lys Ile Phe 245 250 255 Lys Ile Glu Lys Gly Lys Val Thr Lys Ser Ile Glu Leu Asp Ala Pro 260 265 270 Asn Ser His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Thr Gly Lys Val 275 280 285 Met Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val 290 295 300 Ser Phe Asp Gln Asn Leu Asp Tyr Gln Ile Gly Tyr Ile Cys Ser Gly 305 310 315 320 Val Phe Gly Asp Asn Pro Arg Pro Lys Asp Gly Thr Gly Ser Cys Gly 325 330 335 Pro Val Tyr Val Asp Gly Ala Asn Gly Val Lys Gly Phe Ser Tyr Arg 340 345 350 Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Asp Ser Ser Arg 355 360 365 Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp 370 375 380 Ser Ser Phe Leu Val Lys Gln Asp Val Val Ala Met Thr Asp Trp Ser 385 390 395 400 Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp 405 410 415 Cys Met Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys 420 425 430 Glu Asp Thr Ile Trp Thr Ser Gly Ser Ser Ser Ile Ser Phe Cys Gly Val 435 440 445 Asn Ser Asp Thr Val Asp Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro 450 455 460 Phe Thr Ile Asp Lys 465 <210> 13 <211> 469 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 13 Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Cys Met Thr 1 5 10 15 Ile Gly Met Ala Asn Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile 20 25 30 Trp Ile Ser His Ser Ile Gln Leu Gly Asn Gln Asn Gln Ile Glu Thr 35 40 45 Cys Asn Gln Ser Val Ile Thr Tyr Glu Asn Asn Thr Trp Val Asn Gln 50 55 60 Thr Tyr Val Asn Ile Ser Asn Thr Asn Phe Ala Ala Gly Gln Ser Val 65 70 75 80 Val Ser Val Ile Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Ser Gly 85 90 95 Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly 100 105 110 Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu 115 120 125 Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His 130 135 140 Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser 145 150 155 160 Cys Pro Ile Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser 165 170 175 Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr 180 185 190 Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr 195 200 205 Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu 210 215 220 Arg Thr Gln Glu Ser Glu Cys Ala Cys Val Asn Gly Ser Cys Phe Thr 225 230 235 240 Val Met Thr Asp Gly Pro Ser Asp Gly Gln Ala Ser Tyr Lys Ile Phe 245 250 255 Lys Ile Glu Lys Gly Lys Ile Val Lys Ser Val Glu Met Asp Ala Pro 260 265 270 Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Ser Gly Lys Val 275 280 285 Val Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val 290 295 300 Ser Phe Asp Gln Asn Leu Asp Tyr Gln Ile Gly Tyr Ile Cys Ser Gly 305 310 315 320 Val Phe Gly Asp Asn Pro Arg Ser Asn Asp Gly Thr Gly Ser Cys Gly 325 330 335 Pro Val Ser Ser Asn Gly Ala Asn Gly Val Lys Gly Phe Ser Phe Arg 340 345 350 Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Ile Ser Ser Arg 355 360 365 Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp 370 375 380 Asn Ser Phe Ser Ile Lys Gln Asp Ile Val Ala Ile Asn Glu Trp Ser 385 390 395 400 Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Met Asn 405 410 415 Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Arg Pro Lys 420 425 430 Glu Asn Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val 435 440 445 Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp Gly Ala Asp Leu Pro 450 455 460 Phe Thr Ile Asp Lys 465 <210> 14 <211> 469 <212> PRT <213> Artificial Sequence <220> <223> Description of artificial sequence: synthetic polypeptide <400> 14 Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Val Cys Met Thr 1 5 10 15 Ile Gly Met Ala Asn Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile 20 25 30 Trp Ile Ser His Ser Ile Gln Leu Gly Asn Gln Asn Gln Ile Glu Thr 35 40 45 Cys Asn Gln Ser Val Ile Thr Tyr Glu Asn Asn Thr Trp Val Asn Gln 50 55 60 Thr Tyr Val Asn Ile Ser Asn Thr Asn Phe Ala Ala Gly Gln Ser Val 65 70 75 80 Val Ser Ile Ile Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Ser Gly 85 90 95 Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly 100 105 110 Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu 115 120 125 Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His 130 135 140 Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser 145 150 155 160 Cys Pro Ile Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser 165 170 175 Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr 180 185 190 Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr 195 200 205 Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu 210 215 220 Arg Thr Gln Glu Ser Glu Cys Ala Cys Ile Asn Gly Ser Cys Phe Thr 225 230 235 240 Val Met Thr Asp Gly Pro Ser Asp Gly Gln Ala Ser Tyr Lys Ile Phe 245 250 255 Lys Ile Glu Lys Gly Lys Ile Val Lys Ser Val Glu Met Asp Ala Pro 260 265 270 Asn Tyr His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Ser Gly Lys Val 275 280 285 Val Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val 290 295 300 Ser Phe Asp Gln Asn Leu Asp Tyr Gln Ile Gly Tyr Ile Cys Ser Gly 305 310 315 320 Val Phe Gly Asp Asn Pro Arg Ser Asn Asp Gly Thr Gly Ser Cys Gly 325 330 335 Pro Val Ser Ser Asn Gly Ala Asn Gly Val Lys Gly Phe Ser Phe Arg 340 345 350 Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Ile Ser Ser Arg 355 360 365 Arg Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp 370 375 380 Asn Ser Phe Ser Ile Lys Gln Asp Ile Val Ala Ile Thr Glu Trp Ser 385 390 395 400 Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Met Asn 405 410 415 Cys Ile Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Gln Pro Lys 420 425 430 Glu Asn Thr Ile Trp Thr Ser Gly Ser Ser Ile Ser Phe Cys Gly Val 435 440 445 Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp Gly Ala Asp Leu Pro 450 455 460 Phe Thr Ile Asp Lys 465 <210> 15 <211> 469 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 15 Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Ala 1 5 10 15 Ile Gly Ile Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile 20 25 30 Trp Val Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile 35 40 45 Cys Asn Gln Arg Ile Ile Thr Tyr Glu Asn Ser Thr Trp Val Asn Gln 50 55 60 Thr Tyr Val Asn Ile Asn Asn Thr Asn Val Val Ala Gly Lys Asp Thr 65 70 75 80 Thr Ser Val Ile Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Arg Gly 85 90 95 Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly 100 105 110 Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu 115 120 125 Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His 130 135 140 Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Ala Leu Met Ser 145 150 155 160 Cys Pro Val Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser 165 170 175 Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr 180 185 190 Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr 195 200 205 Asn Gly Ile Ile Thr Glu Thr Ile Lys Ser Trp Arg Lys Asn Ile Leu 210 215 220 Arg Thr Gln Glu Ser Glu Cys Val Cys Val Asn Gly Ser Cys Phe Thr 225 230 235 240 Ile Met Thr Asp Gly Pro Ser Asp Gly Leu Ala Ser Tyr Lys Ile Phe 245 250 255 Lys Ile Glu Lys Gly Lys Val Thr Lys Ser Ile Glu Leu Asp Ala Pro 260 265 270 Asn Ser His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Thr Gly Lys Val 275 280 285 Met Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val 290 295 300 Ser Phe Asn Gln Asn Leu Asp Tyr Gln Ile Gly Tyr Ile Cys Ser Gly 305 310 315 320 Val Phe Gly Asp Asn Pro Arg Pro Lys Asp Gly Thr Gly Ser Cys Gly 325 330 335 Pro Val Thr Val Asp Gly Ala Asn Gly Val Lys Gly Phe Ser Tyr Arg 340 345 350 Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Asn Ser Ser Arg 355 360 365 Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp 370 375 380 Ser Ser Phe Ser Val Lys Gln Asp Val Val Ala Ile Thr Asp Trp Ser 385 390 395 400 Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp 405 410 415 Cys Met Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Leu Pro Arg 420 425 430 Glu Asn Thr Ile Trp Thr Ser Gly Ser Ser Ser Ile Ser Phe Cys Gly Val 435 440 445 Asn Ser Asp Thr Val Asn Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro 450 455 460 Phe Thr Ile Asp Lys 465 <210> 16 <211> 469 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 16 Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Ala 1 5 10 15 Ile Gly Ile Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile 20 25 30 Trp Val Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile 35 40 45 Cys Asn Gln Arg Ile Ile Thr Tyr Glu Asn Ser Thr Trp Val Asn Gln 50 55 60 Thr Tyr Val Asn Ile Asn Asn Thr Asn Val Val Ala Gly Lys Asp Thr 65 70 75 80 Thr Ser Val Thr Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Arg Gly 85 90 95 Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly 100 105 110 Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu 115 120 125 Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His 130 135 140 Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Ala Leu Met Ser 145 150 155 160 Cys Pro Val Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser 165 170 175 Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr 180 185 190 Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr 195 200 205 Asn Gly Ile Ile Thr Glu Thr Ile Lys Ser Trp Arg Lys Arg Ile Leu 210 215 220 Arg Thr Gln Glu Ser Glu Cys Val Cys Val Asn Gly Ser Cys Phe Thr 225 230 235 240 Ile Met Thr Asp Gly Pro Ser Asp Gly Leu Ala Ser Tyr Lys Ile Phe 245 250 255 Lys Ile Glu Lys Gly Lys Val Thr Lys Ser Ile Glu Leu Asp Ala Pro 260 265 270 Asn Ser His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Thr Gly Lys Val 275 280 285 Met Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val 290 295 300 Ser Phe Asn Gln Asn Leu Asp Tyr Gln Ile Gly Tyr Ile Cys Ser Gly 305 310 315 320 Val Phe Gly Asp Asn Pro Arg Pro Lys Asp Gly Thr Gly Ser Cys Gly 325 330 335 Pro Val Thr Val Asp Gly Ala Asn Gly Val Lys Gly Phe Ser Tyr Arg 340 345 350 Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Asn Ser Ser Arg 355 360 365 Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp 370 375 380 Ser Ser Phe Leu Val Lys Gln Asp Val Val Ala Ile Thr Asp Trp Ser 385 390 395 400 Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp 405 410 415 Cys Met Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Leu Pro Arg 420 425 430 Glu Asp Thr Ile Trp Thr Ser Gly Ser Ser Ser Ile Ser Phe Cys Gly Val 435 440 445 Asn Ser Asp Thr Val Asn Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro 450 455 460 Phe Thr Ile Asp Lys 465 <210> 17 <211> 469 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 17 Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Ala 1 5 10 15 Ile Gly Ile Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile 20 25 30 Trp Val Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile 35 40 45 Cys Asn Gln Arg Ile Ile Thr Tyr Glu Asn Ser Thr Trp Val Asn Gln 50 55 60 Thr Tyr Val Asn Ile Asn Asn Thr Asn Val Val Ala Gly Lys Asp Thr 65 70 75 80 Thr Ser Val Ile Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Arg Gly 85 90 95 Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly 100 105 110 Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu 115 120 125 Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His 130 135 140 Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Thr Leu Met Ser 145 150 155 160 Cys Pro Val Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser 165 170 175 Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr 180 185 190 Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr 195 200 205 Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Asn Ile Leu 210 215 220 Arg Thr Gln Glu Ser Glu Cys Val Cys Val Asn Gly Ser Cys Phe Thr 225 230 235 240 Ile Met Thr Asp Gly Pro Ser Asp Gly Gln Ala Ser Tyr Lys Ile Phe 245 250 255 Lys Ile Glu Lys Gly Lys Val Thr Lys Ser Ile Glu Leu Asp Ala Pro 260 265 270 Asn Ser His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Thr Gly Lys Val 275 280 285 Val Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val 290 295 300 Ser Phe Asp Gln Asn Leu Asp Tyr Gln Ile Gly Tyr Ile Cys Ser Gly 305 310 315 320 Val Phe Gly Asp Asn Pro Arg Pro Asn Asp Gly Thr Gly Ser Cys Gly 325 330 335 Pro Val Thr Ser Asn Gly Ala Asn Gly Val Lys Gly Phe Ser Phe Arg 340 345 350 Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Asp Ser Ser Arg 355 360 365 Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp 370 375 380 Ser Ser Phe Ser Val Lys Gln Asp Ile Val Ala Ile Thr Asp Trp Ser 385 390 395 400 Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp 405 410 415 Cys Met Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Leu Pro Lys 420 425 430 Glu Asn Thr Ile Trp Thr Ser Gly Ser Ser Ser Ile Ser Phe Cys Gly Val 435 440 445 Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro 450 455 460 Phe Thr Ile Asp Lys 465 <210> 18 <211> 469 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 18 Met Asn Pro Asn Gln Lys Ile Ile Thr Ile Gly Ser Ile Cys Met Ala 1 5 10 15 Ile Gly Ile Ile Ser Leu Ile Leu Gln Ile Gly Asn Ile Ile Ser Ile 20 25 30 Trp Val Ser His Ser Ile Gln Thr Gly Ser Gln Asn His Thr Gly Ile 35 40 45 Cys Asn Gln Arg Ile Ile Thr Tyr Glu Asn Ser Thr Trp Val Asn Gln 50 55 60 Thr Tyr Val Asn Ile Asn Asn Thr Asn Val Val Ala Gly Lys Asp Thr 65 70 75 80 Thr Ser Val Thr Leu Ala Gly Asn Ser Ser Leu Cys Pro Ile Arg Gly 85 90 95 Trp Ala Ile Tyr Ser Lys Asp Asn Ser Ile Arg Ile Gly Ser Lys Gly 100 105 110 Asp Val Phe Val Ile Arg Glu Pro Phe Ile Ser Cys Ser His Leu Glu 115 120 125 Cys Arg Thr Phe Phe Leu Thr Gln Gly Ala Leu Leu Asn Asp Lys His 130 135 140 Ser Asn Gly Thr Val Lys Asp Arg Ser Pro Tyr Arg Ala Leu Met Ser 145 150 155 160 Cys Pro Val Gly Glu Ala Pro Ser Pro Tyr Asn Ser Arg Phe Glu Ser 165 170 175 Val Ala Trp Ser Ala Ser Ala Cys His Asp Gly Met Gly Trp Leu Thr 180 185 190 Ile Gly Ile Ser Gly Pro Asp Asn Gly Ala Val Ala Val Leu Lys Tyr 195 200 205 Asn Gly Ile Ile Thr Asp Thr Ile Lys Ser Trp Arg Asn Arg Ile Leu 210 215 220 Arg Thr Gln Glu Ser Glu Cys Val Cys Val Asn Gly Ser Cys Phe Thr 225 230 235 240 Ile Met Thr Asp Gly Pro Ser Asp Gly Gln Ala Ser Tyr Lys Ile Phe 245 250 255 Lys Ile Glu Lys Gly Lys Val Thr Lys Ser Ile Glu Leu Asp Ala Pro 260 265 270 Asn Ser His Tyr Glu Glu Cys Ser Cys Tyr Pro Asp Thr Gly Lys Val 275 280 285 Met Cys Val Cys Arg Asp Asn Trp His Gly Ser Asn Arg Pro Trp Val 290 295 300 Ser Phe Asp Gln Asn Leu Asp Tyr Gln Ile Gly Tyr Ile Cys Ser Gly 305 310 315 320 Val Phe Gly Asp Asn Pro Arg Pro Asn Asp Gly Thr Gly Ser Cys Gly 325 330 335 Pro Val Thr Ser Asn Gly Ala Asn Gly Val Lys Gly Phe Ser Phe Arg 340 345 350 Tyr Gly Asn Gly Val Trp Ile Gly Arg Thr Lys Ser Asp Ser Ser Arg 355 360 365 Lys Gly Phe Glu Met Ile Trp Asp Pro Asn Gly Trp Thr Glu Thr Asp 370 375 380 Ser Ser Phe Ser Val Lys Gln Asp Ile Val Ala Ile Thr Asp Trp Ser 385 390 395 400 Gly Tyr Ser Gly Ser Phe Val Gln His Pro Glu Leu Thr Gly Leu Asp 405 410 415 Cys Met Arg Pro Cys Phe Trp Val Glu Leu Ile Arg Gly Leu Pro Lys 420 425 430 Glu Asn Thr Ile Trp Thr Ser Gly Ser Ser Ser Ile Ser Phe Cys Gly Val 435 440 445 Asn Ser Asp Thr Val Gly Trp Ser Trp Pro Asp Gly Ala Glu Leu Pro 450 455 460 Phe Thr Ile Asp Lys 465 <210> 19 <211> 2 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 19 Ser Ala 1 <210> 20 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 20 Gly Ser Gly Ser Leu Val Pro Arg Gly Ser Pro Ser Arg Ser 1 5 10 <210> twenty one <211> 40 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(40) <223> This sequence may contain 1-8 'Gly Gly Gly Gly Ser' repeating units <400> twenty one Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 20 25 30 Gly Gly Ser Gly Gly Gly Gly Ser 35 40 <210> twenty two <211> 16 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> twenty two Gly Gly Ser Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> twenty three <211> 5 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> twenty three Gly Gly Gly Gly Ser 1 5 <210> twenty four <211> 20 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(20) <223> This sequence may contain 1-4 'Gly Gly Gly Gly Ser' repeating units <400> twenty four Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 1 5 10 15 Gly Gly Gly Ser 20 <210> 25 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 25 Gly Gly Gly Gly Gly Gly Gly Gly 1 5 <210> 26 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 26 Gly Gly Gly Gly Gly Gly 1 5 <210> 27 <211> 15 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <220> <221> MISC_FEATURE <222> (1)..(15) <223> This sequence may contain 1-3 'Glu Ala Ala Ala Lys' repeating units <400> 27 Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10 15 <210> 28 <211> 27 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <220> <221> MISC_FEATURE <222> (2) (26) <223> This region may encompass 2-5 'Glu Ala Ala Ala Lys' repeating units <400> 28 Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10 15 Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala 20 25 <210> 29 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 29 Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala 1 5 10 <210> 30 <211> 46 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 30 Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys 1 5 10 15 Glu Ala Ala Ala Lys Ala Leu Glu Ala Glu Ala Ala Ala Lys Glu Ala 20 25 30 Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Ala 35 40 45 <210> 31 <211> 5 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 31 Pro Ala Pro Ala Pro 1 5 <210> 32 <211> 18 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 32 Lys Glu Ser Gly Ser Val Ser Ser Glu Gln Leu Ala Gln Phe Arg Ser 1 5 10 15 Leu Asp <210> 33 <211> 14 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 33 Glu Gly Lys Ser Ser Gly Ser Gly Ser Glu Ser Lys Ser Thr 1 5 10 <210> 34 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequences: synthetic peptides <400> 34 Gly Ser Ala Gly Ser Ala Ala Gly Ser Gly Glu Phe 1 5 10 <210> 35 <211> 6 <212> PRT <213> Artificial sequence <220> <223> Description of artificial sequence: synthetic 6xHis tag <400> 35 His His His His His 1 5

Claims

1. A method for producing a recombinant influenza neuraminidase (NA) polypeptide comprising a consensus amino acid, wherein the method comprises: a. selecting more than one influenza NA polypeptide sequence and aligning the sequences; b. Calculate the pairwise similarity / dissimilarity matrix; c. identifying and creating clusters of similar sequences from the pairwise similarity / dissimilarity matrix; d. In each cluster, determining whether a common amino acid exists at each position in the sequence alignment using a pairwise alignment method, wherein if the frequency of the amino acid at a given position is 50% or greater, the amino acid is designated as a common amino acid, and if the frequency of the amino acid at a given position is less than 50%, the amino acid is designated as a variable amino acid; e. generating a first sequence including common amino acids and variable amino acids for each cluster; f. In the first sequence generated in step (e), the consensus amino acid for each variable amino acid position is determined by: i. generating a set of test sequences based on the first sequence, wherein the test amino acid is located at the variable amino acid position; ii. performing molecular modeling on each of the test sequences, wherein the molecular modeling comprises (i) comparison with the crystal structure of an influenza NA polypeptide or protein; (ii) using Rosetta or any other molecular modeling software; or both (i) and (ii); iii. determining a consensus amino acid for each variable amino acid position by selecting one or more amino acids that result in a polypeptide with a negative total energy value that is predicted to fold into a stable protein, while ignoring amino acids that result in a polypeptide with a positive energy value that is predicted to fold incorrectly; and g. Producing a recombinant influenza NA polypeptide comprising the consensus amino acid.

2. The method of claim 1, wherein aligning the sequences comprises using MAFFT, MUSCLE, CLUSTALOMEGA, FASTA, a combination thereof, or any other multiple sequence alignment software package.

3. The method according to claim 1 or 2, wherein calculating the pairwise similarity / dissimilarity matrix comprises using BLOSUM, PAM, IDENTITY substitution matrix or a combination thereof.

4. The method of claim 1 or 2, wherein identifying and creating clusters of similar sequences from the pairwise similarity / dissimilarity matrix comprises using K-means clustering, minimax clustering, principal component analysis (PCA), multidimensional scaling (MDS), or a combination thereof.

5. The method of claim 1 or 2, wherein the test amino acid comprises any natural or unnatural amino acid found in proteins.

6. The method of claim 1 or 2, further comprising after step (e) if a plurality of clusters are analyzed: i. aligning the sequences generated in step (e) for each cluster; ii. determining the presence or absence of a consensus amino acid at each position in the sequence alignment using a pairwise alignment method, wherein if the amino acid frequency at a given position is 50% or greater, the amino acid is designated as a consensus amino acid, and if the amino acid frequency at a given position is less than 50%, the amino acid is designated as a variable amino acid; as well as iii. generating a second sequence comprising a consensus amino acid and a variable amino acid; In step (f), in the generated second sequence, the consensus amino acid for each variable amino acid position is determined.

7. The method of claim 6, wherein in step (f)(i) a set of test sequences is generated based on the second sequence.

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