Patent Publication Number: US-11642407-B2

Title: Identification of variable influenza residues and uses thereof

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application claims the benefit of U.S. Provisional Patent Application Ser. No. 62/983,519, filed Feb. 28, 2020, the entire contents of which is incorporated herein by reference. 
    
    
     GOVERNMENT SUPPORT 
     This invention was made with Government support under Grant No. FA8702-15-D-0001 awarded by the U.S. Air Force. The Government has certain rights in the invention. 
    
    
     SEQUENCE LISTING STATEMENT 
     A computer readable form of the Sequence Listing is filed with this application by electronic submission and is incorporated into this application by reference in its entirety. The Sequence Listing is contained in the file created on Aug. 18, 2022 having the file name “21-0895-US_SeqList2.txt” and is 170 in size. 
     BACKGROUND 
     Influenza viruses are members of the family Orthomyxoviridae and are divided into three genera: A, B, and C. Influenza A and B viruses cause respiratory infections in humans. Current vaccines are designed to induce immunity to hemagglutinin, one of two glycoproteins present on the surface of influenza viruses. Despite the availability of highly effective vaccines, influenza infection still results in up to 5,000,000 hospitalizations and 500,000 deaths annually worldwide. Currently available vaccines against influenza include up to four influenza hemagglutinin components intended to provide protection against H1N1, H3N2, and influenza B strains. Vaccine compositions are reassessed annually by the World Health Organization (WHO) to accommodate antigenic shift and drift in circulating virus strains. Such a strategy requires diligent surveillance of circulating influenza strains from year to year, and vaccine mismatches resulting from inaccurate predictions or unpredictable HA mutations arising during vaccine manufacture, which can result in increased morbidity and mortality even in vaccinated populations. 
     Given the shortcomings of the currently available vaccines, there remains a need for prophylactic and therapeutic compositions and methods that can be used to broadly target influenza in view of the high virus mutation rate amongst strains. 
     SUMMARY OF THE INVENTION 
     The present disclosure provides immunogenic compositions, methods for immunizing a subject against infection with an influenza virus, methods for inducing an immune response against influenza virus, and methods of reducing an influenza virus infection in a subject in need thereof by administering one or more immunogenic compositions of the disclosure. 
     In one aspect, the disclosure provides an immunogenic composition comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more hypervariable amino acid residues and one or more conserved amino acid residues, wherein the amino acid sequence of the polypeptide comprises a substitution of at least one hypervariable amino acid residue with a different, non-hypervariable amino acid residue. In one embodiment, the at least one non-hypervariable amino acid residue is a nonpolar, aliphatic R group amino acid selected from alanine, glycine, valine, leucine, isoleucine, and methionine. In one embodiment, the non-hypervariable amino acid residue is alanine or glycine. 
     In one aspect, the disclosure provides an immunogenic composition comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more hypervariable amino acid residues and one or more conserved amino acid residues, wherein the amino acid sequence of the polypeptide comprises a substitution of at least one hypervariable amino acid residue with an amino acid residue that is a hypervariable-substitute. In one embodiment, the hypervariable-substitute is a nonpolar, aliphatic R group amino acid selected from alanine, glycine, valine, leucine, isoleucine, and methionine. In one embodiment, the hypervariable-substitute is alanine or glycine. 
     In one aspect, the disclosure provides an immunogenic composition comprising two or more polypeptides each individually comprising an amino acid sequence of a viral protein comprising one or more hypervariable amino acid residues, wherein each polypeptide individually comprises an amino acid sequence having a substitution of at least one hypervariable amino acid residue with a different, non-hypervariable amino acid residue, and wherein the polypeptides are of the same or different influenza virus strains. In one embodiment, the at least one non-hypervariable amino acid residue is a nonpolar, aliphatic R group amino acid selected from alanine, glycine, valine, leucine, isoleucine, and methionine. In one embodiment, the non-hypervariable amino acid residue is alanine or glycine. 
     In one aspect, the disclosure provides an immunogenic composition comprising two or more polypeptides each individually comprising an amino acid sequence of a viral protein comprising one or more hypervariable amino acid residues, wherein each polypeptide individually comprises an amino acid sequence having a substitution of at least one hypervariable amino acid residue with an amino acid residue that is a hypervariable-substitute, and wherein the polypeptides are of the same or different influenza virus strains. In one embodiment, the hypervariable-substitute is a nonpolar, aliphatic R group amino acid selected from alanine, glycine, valine, leucine, isoleucine, and methionine. In one embodiment, the hypervariable-substitute is alanine or glycine. 
     In one aspect, the disclosure provides an immunogenic composition comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more hypervariable amino acid residues and one or more conserved amino acid residues, wherein the influenza virus is an influenza A virus strain or an influenza B virus strain, wherein the amino acid sequence of the polypeptide comprises a substitution of at least one hypervariable amino acid residue with a different, non-hypervariable amino acid residue. In one embodiment, the at least one non-hypervariable amino acid residue is a nonpolar, aliphatic R group amino acid selected from alanine, glycine, valine, leucine, isoleucine, and methionine. In one embodiment, the non-hypervariable amino acid residue is alanine or glycine. 
     In one aspect, the disclosure provides an immunogenic composition comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more hypervariable amino acid residues and one or more conserved amino acid residues, wherein the influenza virus is an influenza A virus strain or an influenza B virus strain, wherein the amino acid sequence of the polypeptide comprises a substitution of at least one hypervariable amino acid residue with an amino acid residue that is a hypervariable-substitute. In one embodiment, the hypervariable substitute is a nonpolar, aliphatic R group amino acid selected from alanine, glycine, valine, leucine, isoleucine, and methionine. In one embodiment, the hypervariable-substitute is alanine or glycine. 
     In one aspect, the disclosure provides an immunogenic composition comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more hypervariable amino acid residues and one or more conserved amino acid residues, wherein the virus influenza virus is H1N1, H3N2, B/Victoria/2/1987-like, B/Yamagata/16/1988-like, H5N1, or any combination thereof, wherein the amino acid sequence of the polypeptide comprises a substitution of at least one hypervariable amino acid residue with a different, non-hypervariable amino acid residue. In one embodiment, the at least one non-hypervariable amino acid residue is a nonpolar, aliphatic R group amino acid selected from alanine, glycine, valine, leucine, isoleucine, and methionine. In one embodiment, the non-hypervariable amino acid residue is alanine or glycine. 
     In one aspect, the disclosure provides an immunogenic composition comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more hypervariable amino acid residues and one or more conserved amino acid residues, wherein the virus influenza virus is H1N1, H3N2, B/Victoria/2/1987-like, B/Yamagata/16/1988-like, H5N1, or any combination thereof, wherein the amino acid sequence of the polypeptide comprises a substitution of at least one hypervariable amino acid residue with an amino acid residue that is a hypervariable-substitute. In one embodiment, the hypervariable substitute is a nonpolar, aliphatic R group amino acid selected from alanine, glycine, valine, leucine, isoleucine, and methionine. In one embodiment, the hypervariable-substitute is alanine or glycine. 
     In one aspect, the disclosure provides an immunogenic composition comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more hypervariable amino acid residues and one or more conserved amino acid residues, wherein the at least one viral protein is a hemagglutinin protein, a neuraminidase protein, a M2 matrix protein, or combinations thereof, wherein the amino acid sequence of the polypeptide comprises a substitution of at least one hypervariable amino acid residue with a different, non-hypervariable amino acid residue. In one embodiment, the at least one non-hypervariable amino acid residue is a nonpolar, aliphatic R group amino acid selected from alanine, glycine, valine, leucine, isoleucine, and methionine. In one embodiment, the non-hypervariable amino acid residue is alanine or glycine. 
     In one aspect, the disclosure provides an immunogenic composition comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more hypervariable amino acid residues and one or more conserved amino acid residues, wherein the at least one viral protein is a hemagglutinin protein, a neuraminidase protein, a M2 matrix protein, or combinations thereof, wherein the amino acid sequence of the polypeptide comprises a substitution of at least one hypervariable amino acid residue with an amino acid that is a hypervariable-substitute. In one embodiment, the hypervariable substitute is a nonpolar, aliphatic R group amino acid selected from alanine, glycine, valine, leucine, isoleucine, and methionine. In one embodiment, the hypervariable-substitute is alanine or glycine. 
     In one aspect, the disclosure provides an immunogenic composition comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more hypervariable amino acid residues and one or more conserved amino acid residues, wherein the amino acid sequence of the polypeptide comprises a substitution of at least one hypervariable amino acid residue with a different, non-hypervariable amino acid residue, and wherein the polypeptide comprises at least one B cell epitope. In one embodiment, the at least one non-hypervariable amino acid residue is a nonpolar, aliphatic R group amino acid selected from alanine, glycine, valine, leucine, isoleucine, and methionine. In one embodiment, the non-hypervariable amino acid residue is alanine or glycine. In one embodiment, the immunogenic composition elicits an immune response against the at least one B cell epitope. In one embodiment, the immune response comprises production of antibodies that bind the at least one B cell epitope. 
     In one aspect, the disclosure provides an immunogenic composition comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more hypervariable amino acid residues and one or more conserved amino acid residues, wherein the amino acid sequence of the polypeptide comprises a substitution of at least one hypervariable amino acid residue with an amino acid that is a hypervariable-substitute, and wherein the polypeptide comprises at least one B cell epitope. In one embodiment, the hypervariable substitute is a nonpolar, aliphatic R group amino acid selected from alanine, glycine, valine, leucine, isoleucine, and methionine. In one embodiment, the hypervariable-substitute is alanine or glycine. In one embodiment, the immunogenic composition elicits an immune response against the at least one B cell epitope. In one embodiment, the immune response comprises production of antibodies that bind the at least one B cell epitope. 
     In one aspect, the disclosure provides an immunogenic composition comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more hypervariable amino acid residues and one or more conserved amino acid residues, wherein the viral protein comprises an amino acid sequence selected from SEQ ID NOs: 1-6, and wherein the amino acid sequence of the polypeptide comprises a substitution of at least one hypervariable amino acid residue with a different, non-hypervariable amino acid residue. In one embodiment, the polypeptide comprises at least one B cell epitope. In one embodiment, the at least one non-hypervariable amino acid residue is a nonpolar, aliphatic R group amino acid selected from alanine, glycine, valine, leucine, isoleucine, and methionine. In one embodiment, the non-hypervariable amino acid residue is alanine or glycine. In one embodiment, the immunogenic composition elicits an immune response against the at least one B cell epitope. In one embodiment, the immune response comprises production of antibodies that bind the at least one B cell epitope. In one embodiment, the hypervariable amino acid which is substituted is selected from one or more underlined amino acid residues set forth in SEQ ID NOs: 1-6. 
     In one aspect, the disclosure provides an immunogenic composition comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more hypervariable amino acid residues and one or more conserved amino acid residues, wherein the viral protein comprises an amino acid sequence selected from SEQ ID NOs: 1-6, and wherein the amino acid sequence of the polypeptide comprises a substitution of at least one hypervariable amino acid residue with an amino acid that is a hypervariable-substitute. In one embodiment, the polypeptide comprises at least one B cell epitope. In one embodiment, the at least one non-hypervariable amino acid residue is a nonpolar, aliphatic R group amino acid selected from alanine, glycine, valine, leucine, isoleucine, and methionine. In one embodiment, the non-hypervariable amino acid residue is alanine or glycine. In one embodiment, the immunogenic composition elicits an immune response against the at least one B cell epitope. In one embodiment, the immune response comprises production of antibodies that bind the at least one B cell epitope. In one embodiment, the hypervariable amino acid which is substituted is selected from one or more underlined amino acid residues set forth in SEQ ID NOs: 1-6. 
     In any of the foregoing or related embodiments, the immunogenic composition further comprises an adjuvant. 
     In any of the foregoing and related aspects, the immunogenic composition comprises a nucleic acid encoding the at least one polypeptide. 
     In one aspect, the disclosure provides a method for immunizing a subject against infection with an influenza virus, comprising administering one or more immunogenic compositions of the disclosure. 
     In one aspect, the disclosure provides a method for inducing an immune response against influenza virus, comprising administering to a subject one or more immunogenic compositions of the disclosure. 
     In one aspect, the disclosure provides a method of reducing an influenza virus infection in a subject in need thereof, comprising administering to a subject one or more immunogenic compositions of the disclosure. 
     In any of the foregoing and related aspects, the administration of one or more immunogenic compositions to the subject results in the production of antibodies against the at least one B cell epitope in the polypeptide. 
     Other aspects of the disclosure relate to methods for generating an immunogenic composition comprising: 
     (i) obtaining two or more amino acid sequences of viral proteins from one or more strains of a particular type and/or subtype of influenza virus; 
     (ii) aligning the amino acid sequences to generate an alignment; 
     (iii) identifying one or more hypervariable amino acid residues between strains and one or more conserved amino acid residues; and 
     (iv) substituting at least one hypervariable amino acid residue identified in (iii) with a different, non-hypervariable amino acid residue. In some aspects, the alignment is generated with Dawn, or Clustal-Omega. In some aspects, the method further comprises performing site-specific mutagenesis at each hypervariable amino residue, or combinations thereof, and determining if the mutated viral protein elicits neutralizing antibodies against the multiple strains of influenza virus. 
     Other aspects of the disclosure relate to immunogenic compositions comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more amino acid residues which are conserved between one or more strains of a type and/or subtype of influenza virus, wherein the amino acid sequence of the polypeptide comprises an amino acid sequence comprising the one or more conserved amino acid residues. 
     In one aspect, the disclosure provides an immunogenic composition comprising two or more polypeptides, each individually comprising an amino acid sequence of a viral protein having one or more amino acid residues which are conserved between one or more strains of a type and/or subtype of influenza virus, wherein each polypeptide individually comprises an amino acid sequence comprising one or more conserved amino acid sequences, and wherein the polypeptides are of the same or different influenza virus strains. In some aspects, the two or more polypeptides are of the same viral protein. In some aspects the two or more polypeptides are of different viral proteins. 
     In one aspect the disclosure relates to immunogenic compositions comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more amino acid residues which are conserved between one or more strains of a type and/or subtype of influenza virus, wherein the amino acid sequence of the polypeptide comprises an amino acid sequence comprising the one or more conserved amino acid residues, and wherein the polypeptide comprises two or more T cell epitopes, wherein each T cell epitope is operably linked to one other, optionally via a linker. 
     In one aspect, the disclosure provides immunogenic compositions comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more amino acid residues which are conserved between one or more strains of a type and/or subtype of influenza virus, wherein the influenza virus is an influenza A virus strain or an influenza B virus strain, and wherein the amino acid sequence of the polypeptide comprises an amino acid sequence comprising the one or more conserved amino acid residues. 
     In one aspect, the disclosure provides immunogenic compositions comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more amino acid residues which are conserved between one or more strains of a type and/or subtype of influenza virus, wherein the influenza virus is H1N1, H3N2, B/Victoria/2/1987-like, B/Yamagata/16/1988-like, H5N1, or any combination thereof, and wherein the amino acid sequence of the polypeptide comprises an amino acid sequence comprising the one or more conserved amino acid residues. 
     In one aspect, the disclosure provides immunogenic compositions comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more amino acid residues which are conserved between one or more strains of a type and/or subtype of influenza virus, wherein the viral protein is a hemagglutinin protein, a neuraminidase protein, a M2 matrix protein, or combinations thereof, and wherein the amino acid sequence of the polypeptide comprises an amino acid sequence comprising the one or more conserved amino acid residues. 
     In one aspect, the disclosure provides immunogenic compositions comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more amino acid residues which are conserved between one or more strains of a type and/or subtype of influenza virus, wherein the at least one viral polypeptide comprises at least one conserved amino acid sequence selected from SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 151, 153 and 155, and any combination thereof, and wherein the amino acid sequence of the polypeptide comprises an amino acid sequence comprising the one or more conserved amino acid residues. 
     In any of the foregoing and related aspects, the immunogenic composition comprises a nucleic acid encoding the at least one polypeptide. 
     In any of the foregoing and related aspects, the immunogenic composition elicits an immune response against the virus. In some aspects, the immune response is a T cell response directed to the one of more T cell epitopes comprising the conserved amino acid residues of the viral protein. 
     In any of the foregoing and related aspects, the immunogenic composition further comprises an adjuvant. 
     In some aspects the disclosure provides methods of immunizing a subject against infection with an influenza virus, optionally a T cell or B cell response or both, comprising administering one or more immunogenic compositions of the disclosure. 
     In some aspects the disclosure provides methods for inducing an immune response against influenza virus, optionally a T cell or B cell response or both, comprising administering one or more immunogenic compositions of the disclosure. 
     In some aspects the disclosure provides methods of reducing an influenza virus infection in a subject in need thereof, optionally a T cell or B cell response or both, comprising administering one or more immunogenic compositions of the disclosure. 
     In some aspects, the composition elicits a T cell response against one or more T cell epitopes comprising the conserved amino acid residues of the viral protein. 
     In other aspects, the disclosure provides methods for generating an immunogenic composition comprising: 
     (i) obtaining two or more amino acid sequences of viral proteins from multiple strains of a particular type and/or subtype of influenza virus; 
     (ii) aligning the amino acid sequences to generate an alignment; 
     (iii) identifying a region of amino acid residues having conserved amino acid residues between strains; and 
     (iv) generating a polypeptide comprising the region of amino acids identified in (iii). In one embodiment, the alignment is generated with Dawn or Clustal-Omega. 
     In one embodiment, the methods of the disclosure further comprise determining if the immunogenic composition elicits a T cell response against the multiple strains of influenza virus. 
     In some aspects, the disclosure provides an immunogenic composition comprising: 
     (a) one or more immunogenic compositions comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more hypervariable amino acid residues and one or more conserved amino acid residues, optionally wherein the influenza virus is an influenza A virus strain or an influenza B virus strain, and wherein the amino acid sequence of the polypeptide comprises a substitution of at least one hypervariable amino acid residue with a different, non-hypervariable amino acid residue; and 
     (b) one or more immunogenic compositions comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more amino acid residues which are conserved between one or more strains of a type and/or subtype of influenza virus, optionally wherein the influenza virus is an influenza A virus strain or an influenza B virus strain, and wherein the amino acid sequence of the polypeptide comprises an amino acid sequence comprising the one or more conserved amino acid residues. In one embodiment, the at least one non-hypervariable amino acid residue in an immunogenic composition (a) is a nonpolar, aliphatic R group amino acid selected from alanine, glycine, valine, leucine, isoleucine, and methionine. In one embodiment, the non-hypervariable amino acid residue is alanine or glycine. 
     In one embodiment, the at least one polypeptide of (a) and the at least one polypeptide of (b) are from the same or different viral proteins from the same influenza virus type. In one embodiment, the at least one polypeptide of (a) and the at least on polypeptide of (b) are from the same or different proteins from different influenza virus types. In one embodiment, the at least one polypeptide of (b) comprises two or more polypeptides each individually comprising a T cell epitope. In one embodiment, the two or more polypeptides comprise same amino acid sequence. In one embodiment, the two or more polypeptides comprise different amino acid sequences. In one embodiment, the two or more polypeptides are derived from the same viral protein. In one embodiment the two or more polypeptides are derived from different viral proteins. In one embodiment, the influenza virus is H1N1, H3N2, B/Victoria/2/1987-like, B/Yamagata/16/1988-like, H5N1, or any combination thereof. In one embodiment, the two or more polypeptides are operably linked to each other, optionally comprising a linker and/or spacer between each polypeptide. In one embodiment, the one or more compositions of (a) and the one ore more compositions of (b) further comprise an adjuvant. 
     In some aspects, the disclosure provides an immunogenic composition comprising: 
     (a) one or more immunogenic compositions comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more hypervariable amino acid residues and one or more conserved amino acid residues, the viral protein is a hemagglutinin protein, a neuraminidase protein, a M2 matrix protein, or combinations thereof, and wherein the amino acid sequence of the polypeptide comprises a substitution of at least one hypervariable amino acid residue with a different, non-hypervariable amino acid residue; and 
     (b) one or more immunogenic compositions comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more amino acid residues which are conserved between one or more strains of a type and/or subtype of influenza virus, the viral protein is a hemagglutinin protein, a neuraminidase protein, a M2 matrix protein, or combinations thereof, and wherein the amino acid sequence of the polypeptide comprises an amino acid sequence comprising the one or more conserved amino acid residues. In one embodiment, the at least one non-hypervariable amino acid residue in an immunogenic composition (a) is a nonpolar, aliphatic R group amino acid selected from alanine, glycine, valine, leucine, isoleucine, and methionine. In one embodiment, the non-hypervariable amino acid residue is alanine or glycine. In one embodiment, the immunogenic composition elicits an immune response against at least one T cell epitope, at least one B cell epitope, or combinations thereof. In one embodiment, the immune response comprises production of antibodies that bind B cell epitopes, eliciting a T cell response against T cell epitopes, or both. 
     In some aspects, the disclosure provides an immunogenic composition comprising: 
     (a) one or more immunogenic compositions comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more hypervariable amino acid residues and one or more conserved amino acid residues, wherein the at least one polypeptide comprises an amino acid sequence selected from SEQ ID NOs: 1-6, or combinations thereof, and wherein the amino acid sequence of the polypeptide comprises a substitution of at least one hypervariable amino acid residue with a different, non-hypervariable amino acid residue; and 
     (b) one or more immunogenic compositions comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more amino acid residues which are conserved between one or more strains of a type and/or subtype of influenza virus, and wherein the amino acid sequence of the polypeptide comprises an amino acid sequence comprising the one or more conserved amino acid residues. In one embodiment, the at least one non-hypervariable amino acid residue in an immunogenic composition (a) is a nonpolar, aliphatic R group amino acid selected from alanine, glycine, valine, leucine, isoleucine, and methionine. In one embodiment, the non-hypervariable amino acid residue is alanine or glycine. In one embodiment, the immunogenic composition elicits an immune response against at least one T cell epitope, at least one B cell epitope, or combinations thereof. In one embodiment, the immune response comprises production of antibodies that bind B cell epitopes, eliciting a T cell response against T cell epitopes, or both. 
     In some aspects, the disclosure provides an immunogenic composition comprising: 
     (a) one or more immunogenic compositions comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more hypervariable amino acid residues and one or more conserved amino acid residues, wherein the amino acid sequence of the polypeptide comprises a substitution of at least one hypervariable amino acid residue with a different, non-hypervariable amino acid residue, and wherein the hypervariable amino acid which is substituted is selected from one or more underlined amino acid residues set forth in SEQ ID NOs: 1-6; and 
     (b) one or more immunogenic compositions comprising at least one polypeptide comprising an amino acid sequence of an influenza viral protein having one or more amino acid residues which are conserved between one or more strains of a type and/or subtype of influenza virus, and wherein the amino acid sequence of the polypeptide comprises an amino acid sequence comprising the one or more conserved amino acid residues. In one embodiment, the at least one non-hypervariable amino acid residue in an immunogenic composition (a) is a nonpolar, aliphatic R group amino acid selected from alanine, glycine, valine, leucine, isoleucine, and methionine. In one embodiment, the non-hypervariable amino acid residue is alanine or glycine. In one embodiment, the immunogenic composition elicits an immune response against at least one T cell epitope, at least one B cell epitope, or combinations thereof. In one embodiment, the immune response comprises production of antibodies that bind B cell epitopes, eliciting a T cell response against T cell epitopes, or both. 
     In one embodiment, the at least one polypeptide of (b) comprises at least one conserved amino acid sequence selected from SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 151, 153, and 155, or any combination thereof. 
     In any of the foregoing or related aspects, the immunogenic composition comprises a nucleic acid encoding the at least one polypeptide of (b). 
     Other aspects of the disclosure relate to methods for immunizing a subject against infection with an influenza virus, comprising administering one or more immunogenic composition of (a) and one or more immunogenic compositions of (b). 
     Other aspects of the disclosure relate to methods for inducing an immune response in a subject to protect against infection with an influenza virus, or reducing an influenza virus infection comprising administering one or more immunogenic composition of (a) and one or more immunogenic compositions of (b). 
     In one embodiment, the immunogenic composition of (a) is administered prior to, simultaneously with, or subsequently to administration of the immunogenic composition (b). 
     Other aspects of the disclosure relate to methods for inducing an immune response or reducing an influenza virus infection in a subject in need thereof who has received or is receiving one or more compositions of (a), the method comprising: administering to the subject an effective amount of one or more compositions of (b). 
     Other aspects of the disclosure relate to methods for inducing an immune response in a subject in need thereof who has received or is receiving one or more compositions of (b), the method comprising: administering to the subject an effective amount of one or more compositions of (a). 
     Other aspects of the disclosure relate to methods for reducing an influenza virus infection in a subject, comprising administering to the subject an immunogenic composition comprising: one or more compositions of any of (a); and one or more compositions of (b). In one embodiment, the immunogenic composition of (a) is administered prior to, simultaneously with, or subsequently to administration of the immunogenic composition (b). 
     In other aspects, the disclosure provides nucleic acid molecules comprising a nucleotide sequence having substantial complementarity to a nucleotide sequence encoding a polypeptide derived from a viral protein of influenza, wherein the at least one polypeptide comprises conserved amino acid sequence between multiple strains of a particular type and/or subtype of influenza virus. In some embodiments, the nucleic acid molecule is an RNA interference (RNAi) molecule. In some embodiments, the RNAi molecule is an siRNA or miRNA molecule. In some embodiments, the nucleic acid molecule is an antisense oligonucleotide. In some embodiments, the nucleic acid encodes for one or more polypeptides. In some embodiment, the one or more polypeptides comprise the same amino acid sequence. In some embodiments, the one or more polypeptides comprise different amino acid sequences. In some embodiments, wherein the one or more polypeptides are derived from the same viral protein. In some embodiments, wherein the one or more polypeptides are derived from different viral proteins. In some embodiments, the one or more polypeptides are operably linked to each other, optionally comprising a linker and/or spacer between each polypeptide. In some embodiments, the nucleic acid is formulated in a composition comprising an adjuvant. In some embodiments, the influenza virus is an influenza A virus strain or an influenza B virus strain. In some embodiments, the influenza virus is H1N1, H3N2, B/Victoria/2/1987-like, B/Yamagata/16/1988-like, H5N1, or any combination thereof. In some embodiments, the viral protein is a hemagglutinin protein, a neuraminidase protein, a M2 matrix protein, or combinations thereof. In some embodiments, the composition elicits an immune response against the virus. In some embodiments, the immune response is a T cell response directed to one of more T cell epitopes. In some embodiments, the nucleic acid encodes a conserved amino acid sequence selected from SEQ ID NOs: 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 151, 153 and 155, or any combination thereof. 
     In some embodiments, the disclosure provides method for immunizing a subject against infection with an influenza virus, methods for inducing an immune response against influenza virus, and methods of reducing an influenza virus infection in a subject in need thereof, comprising administering the nucleic acid molecule of the disclosure. In some embodiments, the nucleic acid molecule elicits a T cell response directed to one of more T cell epitopes. In some embodiments, the method further comprises determining if the nucleic acid molecule elicits a T cell response against the multiple strains of influenza virus. 
     In other aspects, the disclosure provides methods of treating an influenza infection, comprising administering the nucleic acid molecule of the disclosure, optionally in a delivery vehicle. 
    
    
     
       BRIEF DESCRIPTION OF FIGURES 
         FIG.  1    is a schematic showing yearly variations in hemagglutinin amino acid sequence from influenza A H1N1. 
         FIG.  2    provides the amino acid sequence (SEQ ID NO: 1) and nucleic acid sequence (SEQ ID NO: 169) of influenza A hemagglutinin. Hypervariable residues are indicated by a box whereas highly conserved regions are underlined. 
         FIG.  3    provides the amino acid sequence (SEQ ID NO: 157) and nucleic acid sequence (SEQ ID NO: 170) of influenza A hemagglutinin where hypervariable residues have been replaced with alanine (shown using a box). 
     
    
    
     DETAILED DESCRIPTION 
     The present disclosure provides compositions and methods for inducing an immune response across strains of influenza virus. Specifically, hypervariable residues and highly conserved regions of amino acid sequences have been identified in various influenza viral proteins that can be exploited to induce a universal immune response amongst strains. 
     Identification of Influenza Residues 
     Influenza is caused by a virus that attacks mainly the upper respiratory tract—the nose, throat and bronchi and rarely also the lungs. The infection usually lasts for about a week. It is characterized by sudden onset of high fever, myalgia, headache and severe malaise, non-productive cough, sore throat, and rhinitis. Most people recover within one to two weeks without requiring any medical treatment. However, in the very young, the elderly and people suffering from medical conditions such as lung diseases, diabetes, cancer, kidney or heart problems, influenza poses a serious risk. In these people, the infection may lead to severe complications of underlying diseases, pneumonia and death. Annual influenza epidemics are thought to result in between three and five million cases of severe illness and between 250,000 and 500,000 deaths every year around the world. 
     Influenza virus is a member of Orthomyxoviridae family. There are three subtypes of influenza viruses, designated influenza A, influenza B, and influenza C. The influenza virion contains a segmented negative-sense RNA genome, which encodes the following proteins: hemagglutinin (HA), neuraminidase (NA), matrix (MI), proton ion-channel protein (M2), nucleoprotein (NP), polymerase basic protein 1 (PBI), polymerase basic protein 2 (PB2), polymerase acidic protein (PA), and nonstructural protein 2 (NS2). The HA, NA, MI, and M2 are membrane associated, whereas NP, PBI, PB2, PA, and NS2 are nucleocapsid associated proteins. The MI protein is the most abundant protein in influenza particles. The HA and NA proteins are envelope glycoproteins, responsible for virus attachment and penetration of the viral particles into the cell. Specifically, HA binds the influenza virus to cells with sialic acid-containing on surface structures on their membranes. 
     Both HA and NA proteins are the sources of the major immunodominant epitopes for virus neutralization and protective immunity, making them important components for prophylactic influenza vaccines. The genetic makeup of influenza viruses allows frequent minor genetic changes, known as antigenic drift. Thus, the amino acid sequence of the major antigens of influenza, particularly HA, is highly variable across groups, subtypes and strains. For this reason, current seasonal influenza vaccines need to be revised every 1-3 years to account for mutations in HA and NA proteins (antigenic drift). A further limitation of the current vaccine approach is that the influenza strains used in the vaccine are selected by the WHO/CDC based on the agencies&#39; best guess as to the prevalent influenza strains for the upcoming flu season. Often times, the guess is not accurate, and the vaccine strains do not match the seasonal influenza strains, limiting the effectiveness of the seasonal vaccines. Seasonal vaccines are also not designed to provide protection against pandemic strains that can result from antigen shift. Further, as the name suggests, seasonal vaccines must be administered every year. 
     Pandemic outbreaks of influenza are caused by the emergence of a pathogenic and transmissible virus to which the human population is immunologically naive. Because the virus is new, the human population has little to no immunity against it. The virus spreads quickly from person-to-person worldwide. Three times in the last century, the influenza A viruses have undergone major genetic changes mainly in their H-component, resulting in global pandemics and large tolls in terms of both disease and deaths. The most infamous pandemic was “Spanish Flu” which affected large parts of the world population and is thought to have killed at least 40 million people in 1918-1919. More recently, two other influenza A pandemics occurred in 1957 (“Asian influenza”) and 1968 (“Hong Kong influenza”) and caused significant morbidity and mortality globally. In contrast to current influenza epidemics, these pandemics were associated with severe outcomes also among healthy younger persons, albeit not on such a dramatic scale as the “Spanish flu” where the death rate was highest among healthy young adults. More recently, limited outbreaks of a new influenza subtype A (H1N1) directly transmitted from swine to humans have occurred in Mexico in 2009 and are being detected in an increasing number of countries. Currently, the mortality rate associated with swine-origin H1N1 influenza viruses appears to be similar to that of seasonal influenza strains. However, increased surveillance and detection of swine-origin H1N1 influenza could push the mortality rates higher. Due to antigenic drift, and even more dramatic alterations known as antigenic shift, pandemic influenza antigens (e.g., the HA amino acid sequence of the pandemic strain) are highly unpredictable. Thus, vaccines have traditionally been unavailable until the later stages of a pandemic. 
     There is an unmet need for influenza vaccines that can better address the current problems of antigenic drift, antigenic shift, and virus mismatch by providing broader protection against multiple influenza strains, including both seasonal and pandemic strains. There is also an unmet need for influenza vaccines that provide longer lasting immunity, particularly vaccines that would not have to be administered every year. 
     In some embodiments, the present disclosure provides immunogenic compositions that direct the immune response to highly conserved areas, surface exposed areas of the viral proteins, e.g., the HA and/or NA proteins. In some embodiments, the immunogenic compositions additionally comprise the M2 ectodomain of the virus. In yet another embodiment, the immunogenic compositions additionally comprise additional influenza proteins including internal virus proteins, e.g., the M1, NEP, NS1, NS2, PA, PB1, and PB2 proteins. Specifically, by mutating (e.g., substituting) hypervariable amino acid residues and/or generating polypeptides comprising highly conserved amino acid sequences, the compositions and methods described herein can be used to induce an immune response against different strains of influenza, including future strains that may develop due to antigenic shift. 
     In one embodiment, the present disclosure provides immunogenic compositions comprising one or more polypeptides derived from influenza proteins, wherein at least one hypervariable amino acid residue is replaced by a conserved, non-hypervariable amino acid residue. In one embodiment, the non-hypervariable amino acid residue is selected from amino acid residues with non-polar or neutral side charge. In one embodiment, the non-hypervariable amino acid residue is selected from alanine, glycine, valine, leucine, isoleucine and methionine. 
     In one embodiment, the present disclosure provides immunogenic compositions comprising one or more polypeptides derived from influenza proteins, wherein at least one hypervariable amino acid residue is replaced by an amino acid residue that is a conserved, hypervariable-substitute. In one embodiment, the hypervariable-substitute is selected from amino acid residues with non-polar or neutral side charge. In one embodiment, the hypervariable-substitute is selected from alanine, glycine, valine, leucine, isoleucine and methionine. 
     In some embodiments, the immunogenic composition comprises an influenza protein or polypeptide having a highly conserved regions as described herein. In some embodiments, the immunogenic composition comprises an influenza protein or polypeptide having a highly conserved regions annotated in any one of SEQ ID NOs: 171-193. In some embodiments, the protein or polypeptide comprises a non-hypervariable amino acid residue at an amino acid residue that is a hypervariable amino acid residue as annotated in any one of SEQ ID NOs: 171-193. In some embodiments, the protein or polypeptide comprises a hypervariable-substitute at an amino acid residue that is a hypervariable amino acid residue as annotated in any one of SEQ ID NOs: 171-193. 
     Influenza A 
     In some embodiments, the methods and compositions described herein target influenza A. Influenza A virus is both best characterized and the most serious threat to public health, capable of inducing massive epidemics or pandemics. 
     In some embodiments, the methods and compositions described herein comprise a recombinant viral protein derived from influenza A. In some embodiments, the viral protein of an influenza A virus is selected from subtypes H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16. In some embodiments, the influenza virus is selected from the group consisting of H1N1, H3N2, H5N1, and H7N9. In some embodiments, the type A virus is a seasonal strain, such as, /Texas/36/1991, A/Singapore/1986, A/New Caledonia/20/1999, A/Solomon Islands/03/2006, A/Brisbane/59/2007, or A/Wisconsin/67/2005. In some embodiments, the type A virus is a pandemic strain such as A/California/07/2009, A/California/04/2009, A/Belgium/145/2009, A/South Carolina/01/1918, or A/New Jersey/1976. 
     Influenza B 
     In some embodiments, the methods and compositions described herein target influenza B. Influenza B viruses generally mutate slower than influenza A viruses. 
     In some embodiments, the methods and compositions described herein comprise a recombinant viral protein derived from influenza B. In some embodiments, the viral protein of an influenza B virus is selected from a Yamagata lineage strain or a Victoria lineage strain. In some embodiments, the viral protein of an influenza B virus is selected from B/Hong Kong/330/2001, B/Hong Kong/05/1972, B/Lee/40, B/Massachusetts/02/2012, B/Panama/45/1990, B/Singapore/222/79, B/Victoria/02/1987, B/Yamagata/16/1988, or B/Brisbane/60/2008. 
     Hemagglutinin (HA) 
     In some embodiments, an immunogenic composition described herein comprises a hemagglutinin (HA) recombinant protein, polypeptide or both. In some embodiments, the HA recombinant protein comprises a non-hypervariable amino acid substituted for a hypervariable amino acid residue. In some embodiments, the HA recombinant protein comprises a non-hypervariable amino acid replaced with an amino acid that is a hypervariable-substitute. In some embodiments, the HA polypeptide comprises a highly conserved region of amino acid sequences. 
     HA is a glycoprotein on the surface of influenza virus responsible for interaction of the virus with host cell receptors. HA proteins on the virus surface are trimers of hemagglutinin protein monomers that are enzymatically cleaved to yield amino-terminal HA1 and carboxy-terminal HA2 polypeptides. The globular head consists exclusively of the major portion of the HA1 polypeptide, whereas the stem that anchors the hemagglutinin protein into the viral lipid envelope is comprised of HA2 and part of HAL The globular head of a hemagglutinin protein includes two domains: the receptor binding domain (RBD), an  ˜ 148-amino acid residue domain that includes the sialic acid-binding site, and the vestigial esterase domain, a smaller  ˜ 75-amino acid residue region just below the RBD. The top part of the RBD adjacent to the 2,6-sialic acid recognition sites includes a large region (amino acids 131-143, 170-182, 205-215 and 257-262, 1918 numbering) (referred to herein as the RBD-A region) of over 6000 A2 per trimer that is 95% conserved between A/South Carolina/1/1918 (1918 SC) and A/California/04/2009 (2009 CA) pandemic strains. The globular head includes several antigenic sites that include immunodominant epitopes. Examples include the Sa, Sb, Ca1, Ca2 and Cb antigenic sites (see, for example, Caton A J et al, 1982, Cell 31, 417-427). The RBD-A region includes the Sa antigenic site and part of the Sb antigenic site. 
     H1N1 
     In some embodiments, the immunogenic composition comprises an HA recombinant protein or polypeptide derived from H1N1. In some embodiments, the recombinant H1N1 HA protein or polypeptide comprises a non-hypervariable amino acid residue at an amino acid position selected from Table 1, or any combination thereof. In some embodiments, the recombinant H1N1 HA protein or polypeptide comprises an amino acid that is a hypervariable-substitute at an amino acid position selected from Table 1, or any combination thereof. 
     
       
         
           
               
             
               
                 TABLE 1 
               
               
                   
               
               
                 List of Hypervariable Amino Acid Residues in H1N1 HA Protein 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                  13T 
               
               
                   
                  14T 
               
               
                   
                  52D 
               
               
                   
                  53K 
               
               
                   
                  78I 
               
               
                   
                  86S 
               
               
                   
                  88S 
               
               
                   
                 101N 
               
               
                   
                 114N 
               
               
                   
                 137T 
               
               
                   
                 145S 
               
               
                   
                 147K 
               
               
                   
                 155H 
               
               
                   
                 158A 
               
               
                   
                 159K 
               
               
                   
                 160S 
               
               
                   
                 163K 
               
               
                   
                 178L 
               
               
                   
                 179N 
               
               
                   
                 180Q 
               
               
                   
                 185D 
               
               
                   
                 187G 
               
               
                   
                 195G 
               
               
                   
                 200S 
               
               
                   
                 202T 
               
               
                   
                 203A 
               
               
                   
                 214A 
               
               
                   
                 220T 
               
               
                   
                 222R 
               
               
                   
                 225K 
               
               
                   
                 228K 
               
               
                   
                 233T 
               
               
                   
                 239D 
               
               
                   
                 241E 
               
               
                   
                 251V 
               
               
                   
                 256K 
               
               
                   
                 273T 
               
               
                   
                 274M 
               
               
                   
                 275E 
               
               
                   
                 277N 
               
               
                   
                 278A 
               
               
                   
                 287T 
               
               
                   
                 288P 
               
               
                   
                 293N 
               
               
                   
                 300E 
               
               
                   
                 315I 
               
               
                   
                 319K 
               
               
                   
                 331L 
               
               
                   
                 338V 
               
               
                   
                 362V 
               
               
                   
                 382L 
               
               
                   
                 391K 
               
               
                   
                 415N 
               
               
                   
                 419K 
               
               
                   
                 468N 
               
               
                   
                 490N 
               
               
                   
                 491T 
               
               
                   
                 516K 
               
               
                   
                 544V 
               
               
                   
                   
               
               
                   
                 *residue numbering based on straight numbering of SEQ ID NO: 1. SEQ ID NO: 1 indicates these residues in bold. 
               
            
           
         
       
     
     In some embodiments, the recombinant H1N1 HA polypeptide comprises a highly conserved region of amino acid sequences. In some embodiments, the highly conserved region of amino acid sequences is selected from Table 2, or any combination thereof. 
                     TABLE 2               Highly Conserved Regions in H1N1 HA Protein                                                 GYHANNST   NVTVTHS   SWSYIVE   QSRGLFGAIAGF       (SEQ ID NO 7)   (SEQ ID NO 9)   (SEQ ID NO 11)   (SEQ ID NO 13)               QGSGYAAD   ITNKVNS   WTYNAELL   GCFEFYH       (SEQ ID NO 15)   (SEQ ID NO 17)   (SEQ ID NO 19)   (SEQ ID NO 21)               LGNPEC   EGGWTG   LLENER           (SEQ ID NO 23)   (SEQ ID NO 25)   (SEQ ID NO 27)                    
H3N2
 
     In some embodiments, the immunogenic composition comprises an HA recombinant protein or polypeptide derived from H3N2. In some embodiments, the recombinant H3N2 HA protein or polypeptide comprises a non-hypervariable amino acid residue at an amino acid position selected from Table 3, or any combination thereof. In some embodiments, the recombinant H3N2 HA protein or polypeptide comprises an amino acid that is a hypervariable-substitute at an amino acid position selected from Table 3, or any combination thereof. 
     
       
         
           
               
             
               
                 TABLE 3 
               
               
                   
               
               
                 List of Hypervariable Amino Acid Residues in H3N2 HA Protein 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
               
               
               
               
               
               
               
               
               
               
            
               
                  7L 
                  26T 
                  73Q 
                 110Y 
                 151T 
                 172H 
                 189Q 
                 214S 
                 242I 
                 296E 
                 391D 
                 494I 
               
               
                  9Y 
                  41I 
                  78E 
                 117D 
                 153S 
                 173L 
                 202G 
                 215S 
                 243P 
                 315R 
                 394N 
                 495G 
               
               
                  14V 
                  47N 
                  91Q 
                 137N 
                 154A 
                 174N 
                 205K 
                 218I 
                 245R 
                 327Q 
                 400L 
                 500G 
               
               
                  16A 
                  49R 
                  94G 
                 138N 
                 156I 
                 175F 
                 206D 
                 219T 
                 277R 
                 328S 
                 402G 
                 505N 
               
               
                  18K 
                  61N 
                  98K 
                 140S 
                 158R 
                 176K 
                 208I 
                 228A 
                 278S 
                 342K 
                 422I 
                 506V 
               
               
                  19L 
                  64I 
                  99K 
                 144T 
                 160N 
                 179A 
                 209F 
                 238R 
                 291G 
                 362M 
                 466K 
                 509D 
               
               
                  22Y 
                  66E 
                 107S 
                 147T 
                 161S 
                 187N 
                 212A 
                 239I 
                 292K 
                 363V 
                 468K 
                 522E 
               
               
                  25S 
                  69D 
                 108K 
                 149N 
                 171T 
                 188E 
                 213Q 
                 241N 
                 294K 
                 377R 
                 469K 
                 545V 
               
               
                 546A 
                 560I 
                 561R 
                 562C 
                 563N 
                 559N 
               
               
                   
               
               
                 *residue numbering based on straight numbering of SEQ ID NO: 3. SEQ ID NO: 3 indicates these residues in hold 
               
            
           
         
       
     
     In some embodiments, the recombinant H3N2 HA polypeptide comprises a highly conserved region of amino acid sequences. In some embodiments, the highly conserved region of amino acid sequences is selected from Table 4, or any combination thereof. 
                     TABLE 4               Highly Conserved Regions       in H3N2 HA Protein                                                LCLGHHA   GNLIAPRGYF   LKLATGMRN   FGAIAGF       (SEQ ID NO 61)   (SEQ ID NO 63)   (SEQ ID NO 65)   IENGWEG                   (SEQ ID NO 67)               KFHQIEKEF   DLTDSEM   LRENAED           (SEQ ID NO 69)   (SEQ ID NO 71)   (SEQ ID NO 73)                    
Influenza B
 
     In some embodiments, the immunogenic composition comprises an HA recombinant protein or polypeptide derived from influenza B. In some embodiments, the recombinant influenza B HA protein or polypeptide comprises a non-hypervariable amino acid residue at an amino acid position selected from Table 5, or any combination thereof. In some embodiments, the recombinant influenza B HA protein or polypeptide comprises an amino acid that is a hypervariable-substitute at an amino acid position selected from Table 5, or any combination thereof. 
     
       
         
           
               
             
               
                 TABLE 5 
               
               
                   
               
               
                 List of Hypervariable Amino Acid Residues 
               
               
                 in Influenza B HA Protein 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                  55H 
               
               
                   
                  63E 
               
               
                   
                  71K 
               
               
                   
                  73L 
               
               
                   
                  86K 
               
               
                   
                  88T 
               
               
                   
                  90K 
               
               
                   
                  91I 
               
               
                   
                  95R 
               
               
                   
                  96V 
               
               
                   
                 123P 
               
               
                   
                 131H 
               
               
                   
                 132I 
               
               
                   
                 137H 
               
               
                   
                 141N 
               
               
                   
                 144N 
               
               
                   
                 151K 
               
               
                   
                 152I 
               
               
                   
                 161I 
               
               
                   
                 163N 
               
               
                   
                 164G 
               
               
                   
                 165N 
               
               
                   
                 177K 
               
               
                   
                 178N 
               
               
                   
                 180K 
               
               
                   
                 181N 
               
               
                   
                 183T 
               
               
                   
                 187P 
               
               
                   
                 188L 
               
               
                   
                 190I 
               
               
                   
                 195I 
               
               
                   
                 197T 
               
               
                   
                 213E 
               
               
                   
                 217A 
               
               
                   
                 218K 
               
               
                   
                 224K 
               
               
                   
                 245G 
               
               
                   
                 248N 
               
               
                   
                 267V 
               
               
                   
                 270S 
               
               
                   
                 277T 
               
               
                   
                 282I 
               
               
                   
                 314K 
               
               
                   
                 328E 
               
               
                   
                 494E 
               
               
                   
                 513R 
               
               
                   
                 520D 
               
               
                   
                 566I 
               
               
                   
                 570V 
               
               
                   
                   
               
               
                   
                 *residue numbering based on straight numbering of SEQ ID NO: 5. SEQ ID NO: 5 indicates these residues in bold. 
               
            
           
         
       
     
     In some embodiments, the recombinant influenza B HA polypeptide comprises a highly conserved region of amino acid sequences. In some embodiments, the highly conserved region of amino acid sequences is selected from Table 6, or any combination thereof. 
                     TABLE 6               Highly Conserved Regions in       Influenza B HA Protein                                                VKTATQG   NCTDLDVAL   TSGCFPIMH   NLLRGYE       EVNVTG   (SEQ ID NO 95)   DRTKIRQL   (SEQ ID NO 99)       (SEQ ID NO 194)       (SEQ ID NO 97)                   TMAWAVP   EDGGLPQS   LPLIGEAD   YGGLNKSKP       (SEQ ID NO 101)   GRIWDYM   CLHE   YYTG           (SEQ ID NO 103)   (SEQ ID NO 105)   (SEQ ID NO 107)               CPIWVKTPL   GFFGAIAGF   AGWHGYTSHGAHG   AVAADLKSTQEA       (SEQ ID NO 109)   LEGGWEGM   (SEQ ID NO 113   (SEQ ID NO 115)           (SEQ ID NO 111)                       KITKNLNSLSELE   KNLQRLS   EILELDEK   IGNGCFETKH       (SEQ ID NO 117)   (SEQ ID NO 119)   VDDLRADT   KCNQTCLD               ISSQIELA   (SEQ ID NO 123)               VLLSNEGI                   INSEDEHL                   LALERKLK                   KMLGPSA                   (SEQ ID NO 121)                   AGEFSLPTFD   HTILLYYSTA               SLNITAASL   ASSLAVTLM               (SEQ ID NO 125)   (SEQ ID NO 127)                    
Neuraminidase (NA)
 
     In some embodiments, an immunogenic composition described herein comprises a neuraminidase (NA) recombinant protein, polypeptide or both. In some embodiments, the NA recombinant protein comprises a non-hypervariable amino acid substituted for a hypervariable amino acid residue. In some embodiments, the NA recombinant protein comprises a non-hypervariable amino acid replaced with an amino acid that is a hypervariable-substitute. In some embodiments, the NA polypeptide comprises a highly conserved region of amino acid sequences. 
     NA is an enzyme found on the surface of influenza that enables the virus to be released from the host cells. Neuraminidases are enzymes that cleave sialic acid groups from glycoproteins and are required for virus replication. The NA protein also functions during entry of virus into the respiratory tract. The epithelial cells are bathed in mucus, a complex protective coating that contains may sialic acid-containing glycoproteins. When influenza virions enter the respirator tract, they are trapped in mucus where they bind sialic acids. This interaction would prevent the viruses from binding to a susceptible cell were it not for the action of the NA protein. When a virus particle encounters a cell, it binds the sialic acid-containing receptor and is rapidly taken into the cell before the NA protein can cleave the carbohydrate from the cell surface. 
     The NA is a tetramer of four identical polypeptides. Each polypeptide contains about 470 amino acids arranged in four domains, an N-terminal cytoplasmic sequence, followed by a membrane-anchoring hydrophobic transmembrane domain and a thin stalk of variable length, ending in a globular “head” domain that carries the enzyme active site. Crystal structures of NA encompass the catalytically active heads, either proteolytically cleaved from the virus or engineered as a soluble secreted protein. The intact NA has not been crystallized, but a cryoelectron microscopy study of the X-31 (A/Aichi/68, H3N2) reassortant virus has revealed considerable detail at near atomic resolution. The structure confirms that the N2 NA protrudes slightly further than the HA from the viral membrane, that there are 40-50 NA spikes per virion, and that these occur in clusters amid 300-400 HA spikes on an average sized virion of diameter 120 nm. 
     H1N1 
     In some embodiments, the immunogenic composition comprises an NA recombinant protein or polypeptide derived from H1N1. In some embodiments, the recombinant H1N1 NA protein or polypeptide comprises a non-hypervariable amino acid residue at an amino acid position selected from Table 7, or any combination thereof. In some embodiments, the recombinant H1N1 NA protein or polypeptide comprises an amino acid that is a hypervariable-substitute at an amino acid position selected from Table 7, or any combination thereof. 
     
       
         
           
               
             
               
                 TABLE 7 
               
               
                   
               
               
                 List of Hypervariable Amino Acid Residues in H1N1 NA Protein 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                  13I 
               
               
                   
                  14C 
               
               
                   
                  15M 
               
               
                   
                  16T 
               
               
                   
                  19M 
               
               
                   
                  20A 
               
               
                   
                  21N 
               
               
                   
                  23I 
               
               
                   
                  34I 
               
               
                   
                  40L 
               
               
                   
                  42N 
               
               
                   
                  44N 
               
               
                   
                  45Q 
               
               
                   
                  46I 
               
               
                   
                  47E 
               
               
                   
                  48T 
               
               
                   
                  52S 
               
               
                   
                  53V 
               
               
                   
                  59N 
               
               
                   
                  64Q 
               
               
                   
                  70S 
               
               
                   
                  74F 
               
               
                   
                  75A 
               
               
                   
                  77G 
               
               
                   
                  78Q 
               
               
                   
                  79S 
               
               
                   
                  80V 
               
               
                   
                  81V 
               
               
                   
                  82S 
               
               
                   
                  84K 
               
               
                   
                  86A 
               
               
                   
                  93P 
               
               
                   
                  94V 
               
               
                   
                 101S 
               
               
                   
                 106I 
               
               
                   
                 126P 
               
               
                   
                 130R 
               
               
                   
                 149I 
               
               
                   
                 157T 
               
               
                   
                 163I 
               
               
                   
                 166V 
               
               
                   
                 173R 
               
               
                   
                 188I 
               
               
                   
                 189N 
               
               
                   
                 200N 
               
               
                   
                 214D 
               
               
                   
                 220R 
               
               
                   
                 221N 
               
               
                   
                 222N 
               
               
                   
                 232A 
               
               
                   
                 234V 
               
               
                   
                 241I 
               
               
                   
                 248D 
               
               
                   
                 249G 
               
               
                   
                 250Q 
               
               
                   
                 257R 
               
               
                   
                 263I 
               
               
                   
                 264V 
               
               
                   
                 267V 
               
               
                   
                 269M 
               
               
                   
                 270N 
               
               
                   
                 274Y 
               
               
                   
                 275H 
               
               
                   
                 285S 
               
               
                   
                 286S 
               
               
                   
                 287E 
               
               
                   
                 288I 
               
               
                   
                 289T 
               
               
                   
                 311E 
               
               
                   
                 314I 
               
               
                   
                 321V 
               
               
                   
                 329N 
               
               
                   
                 331K 
               
               
                   
                 332T 
               
               
                   
                 336G 
               
               
                   
                 339S 
               
               
                   
                 340S 
               
               
                   
                 341N 
               
               
                   
                 344N 
               
               
                   
                 351F 
               
               
                   
                 354G 
               
               
                   
                 365I 
               
               
                   
                 366S 
               
               
                   
                 367S 
               
               
                   
                 369K 
               
               
                   
                 382G 
               
               
                   
                 385N 
               
               
                   
                 386N 
               
               
                   
                 389I 
               
               
                   
                 393I 
               
               
                   
                 395G 
               
               
                   
                 397N 
               
               
                   
                 398E 
               
               
                   
                 416D 
               
               
                   
                 427I 
               
               
                   
                 430R 
               
               
                   
                 432E 
               
               
                   
                 449N 
               
               
                   
                 450S 
               
               
                   
                 452T 
               
               
                   
                 453V 
               
               
                   
                 454G 
               
               
                   
                   
               
               
                   
                 *residue numbering based on straight numbering of SEQ ID NO: 2. SEQ ID NO: 2 indicates these residues in bold. 
               
            
           
         
       
     
     In some embodiments, the recombinant H1N1 NA polypeptide comprises a highly conserved region of amino acid sequences. In some embodiments, the highly conserved region of amino acid sequences is selected from Table 8, or any combination thereof. 
                     TABLE 8               Highly Conserved Regions in H1N1 NA Protein                                                 MNPNQKIITIGS   RIGSKGDVFV   REPFISCS   TFFLTQGAL       (SEQ ID NO 29)   (SEQ ID NO 31)   (SEQ ID NO 33)   LNDKHSNGT                   (SEQ ID NO 35)               KDRSPYR   FESVAWSASACHDG   WLTIGISGPD   GAVAVLKY       (SEQ ID NO 37)   (SEQ ID NO 39)   (SEQ ID NO 41)   (SEQ ID NO 155)               ILRTQESEC   YEECSCYPD   CVCRDNWHGS   NGVWIGRTKS       (SEQ ID NO 43)   (SEQ ID NO 45)   NRPWVSFNQNL   (SEQ ID NO 49)               (SEQ ID NO 47)                   GFEMIWDPNGWT   WSGYSGSFV   RPCFWVEL   WTSGSSISFCGV       (SEQ ID NO 51)   QHPELTGL   (SEQ ID NO 55)   (SEQ ID NO 57)           (SEQ ID NO 53)                       WSWPDGAELPF                   (SEQ ID NO 59)                    
H3N2
 
     In some embodiments, the immunogenic composition comprises an NA recombinant protein or polypeptide derived from H3N2. In some embodiments, the recombinant H3N2 NA protein or polypeptide comprises a non-hypervariable amino acid residue at an amino acid position selected from Table 9, or any combination thereof. In some embodiments, the recombinant H3N2 NA protein or polypeptide comprises an amino acid that is a hypervariable-substitute at an amino acid position selected from Table 9, or any combination thereof. 
     
       
         
           
               
             
               
                 TABLE 9 
               
               
                   
               
               
                 List of Hypervariable Amino Acid Residues in H3N2 NA Protein 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                  16T 
               
               
                   
                  18S 
               
               
                   
                  23F 
               
               
                   
                  26I 
               
               
                   
                  30I 
               
               
                   
                  40Y 
               
               
                   
                  41E 
               
               
                   
                  42F 
               
               
                   
                  43N 
               
               
                   
                  44S 
               
               
                   
                  45P 
               
               
                   
                  46P 
               
               
                   
                  51M 
               
               
                   
                  52L 
               
               
                   
                  56T 
               
               
                   
                  62I 
               
               
                   
                  73I 
               
               
                   
                  75K 
               
               
                   
                  81L 
               
               
                   
                  82A 
               
               
                   
                  93N 
               
               
                   
                 126P 
               
               
                   
                 127D 
               
               
                   
                 140L 
               
               
                   
                 143V 
               
               
                   
                 147D 
               
               
                   
                 149V 
               
               
                   
                 150R 
               
               
                   
                 155Y 
               
               
                   
                 161N 
               
               
                   
                 172K 
               
               
                   
                 176I 
               
               
                   
                 194V 
               
               
                   
                 197D 
               
               
                   
                 199K 
               
               
                   
                 208N 
               
               
                   
                 215I 
               
               
                   
                 216V 
               
               
                   
                 220K 
               
               
                   
                 221E 
               
               
                   
                 249K 
               
               
                   
                 263V 
               
               
                   
                 265T 
               
               
                   
                 267T 
               
               
                   
                 303V 
               
               
                   
                 307I 
               
               
                   
                 310Y 
               
               
                   
                 313V 
               
               
                   
                 329N 
               
               
                   
                 331S 
               
               
                   
                 332S 
               
               
                   
                 336H 
               
               
                   
                 338L 
               
               
                   
                 339D 
               
               
                   
                 344E 
               
               
                   
                 346G 
               
               
                   
                 356D 
               
               
                   
                 367S 
               
               
                   
                 369K 
               
               
                   
                 370L 
               
               
                   
                 372S 
               
               
                   
                 380I 
               
               
                   
                 381E 
               
               
                   
                 385N 
               
               
                   
                 386P 
               
               
                   
                 387N 
               
               
                   
                 392I 
               
               
                   
                 399D 
               
               
                   
                 400R 
               
               
                   
                 401G 
               
               
                   
                 402N 
               
               
                   
                 416S 
               
               
                   
                 432E 
               
               
                   
                 435E 
               
               
                   
                 437L 
               
               
                   
                 464I 
               
               
                   
                 468P 
               
               
                   
                   
               
               
                   
                 *residue numbering based on straight numbering of SEQ ID NO: 4. SEQ ID NO: 4 indicates these residues in bold. 
               
            
           
         
       
     
     In some embodiments, the recombinant H3N2 NA polypeptide comprises a highly conserved region of amino acid sequences. In some embodiments, the highly conserved region of amino acid sequences is selected from Table 10, or any combination thereof. 
                     TABLE 10               Highly Conserved Regions in H3N2 NA Protein                                                     QFALGQGTT   AWSSSSC   LRTQESEC           (SEQ ID NO 75)   (SEQ ID NO 77)   (SEQ ID NO 79)                   EECSCYP   CSGLVGDTPR   GVKGWAFD           (SEQ ID NO 81)   (SEQ ID NO 83)   (SEQ ID NO 85)                   NRCFYVELIRG   VFCGTSGTYG   GSWPDGA           (SEQ ID NO 87)   (SEQ ID NO 89)   (SEQ ID NO 91)                    
Influenza B
 
     In some embodiments, the immunogenic composition comprises an NA recombinant protein or polypeptide derived from influenza B. In some embodiments, the recombinant influenza B NA protein or polypeptide comprises a non-hypervariable amino acid residue at an amino acid position selected from Table 11, or any combination thereof. In some embodiments, the recombinant influenza B NA protein or polypeptide comprises an amino acid that is a hypervariable-substitute at an amino acid position selected from Table 11, or any combination thereof. 
     
       
         
           
               
             
               
                 TABLE 11 
               
               
                   
               
               
                 List of Hypervariable Amino Acid Residues 
               
               
                 in Influenza B NA Protein 
               
               
                   
               
             
            
               
                   
               
            
           
           
               
               
            
               
                   
                 42P 
               
               
                   
                 45I 
               
               
                   
                 49T 
               
               
                   
                 61Q 
               
               
                   
                 65R 
               
               
                   
                 67A 
               
               
                   
                 68T 
               
               
                   
                 73L 
               
               
                   
                 74L 
               
               
                   
                 107T 
               
               
                   
                 121V 
               
               
                   
                 126N 
               
               
                   
                 149G 
               
               
                   
                 172I 
               
               
                   
                 187K 
               
               
                   
                 199N 
               
               
                   
                 205V 
               
               
                   
                 220N 
               
               
                   
                 221N 
               
               
                   
                 236N 
               
               
                   
                 245S 
               
               
                   
                 249V 
               
               
                   
                 296R 
               
               
                   
                 321D 
               
               
                   
                 330N 
               
               
                   
                 341D 
               
               
                   
                 343D 
               
               
                   
                 344K 
               
               
                   
                 359K 
               
               
                   
                 372K 
               
               
                   
                 374E 
               
               
                   
                 385G 
               
               
                   
                 390A 
               
               
                   
                 393D 
               
               
                   
                 396A 
               
               
                   
                 397F 
               
               
                   
                 402V 
               
               
                   
                 403S 
               
               
                   
                 405K 
               
               
                   
                 437E 
               
               
                   
                 464D 
               
               
                   
                 466A 
               
               
                   
                   
               
               
                   
                 *residue numbering based on straight numbering of SEQ ID NO: 6. SEQ ID NO: 6 indicates these residues in bold. 
               
            
           
         
       
     
     In some embodiments, the recombinant influenza B NA polypeptide comprises a highly conserved region of amino acid sequences. In some embodiments, the highly conserved region of amino acid sequences is selected from Table 12, or any combination thereof. 
                     TABLE 12               Highly Conserved Regions in       Influenza B NA Protein                                                HFALTHYAAQPG   DRNKLRHL   AWSGSACHDG   KYGEAYT       (SEQ ID NO 131)   (SEQ ID NO 133)   (SEQ ID NO 135)   DTYHSY                   (SEQ ID NO 137)               LRTQESACNCI   CRFLKIREGR   HTEECTCGFA   YTAKRPFVKL       (SEQ ID NO 139)   (SEQ ID NO 141)   (SEQ ID NO 143)   (SEQ ID NO 145)               KGGFVHQR   GRWYSRT   EPGWYSFGFE   EMVHDGG       (SEQ ID NO 147)   (SEQ ID NO 149)   (SEQ ID NO 151)   (SEQ ID NO 153)               ALLISPHRFGE                   (SEQ ID NO 129)                    
M2 Ectodomain
 
     In some embodiments, an immunogenic composition described herein comprises an M2 ectodomain (M2e) recombinant protein, polypeptide or both. In some embodiments, the M2e recombinant protein comprises a non-hypervariable amino acid substituted for a hypervariable amino acid residue. In some embodiments, the M2e recombinant protein comprises a non-hypervariable amino acid replaced with an amino acid residue that is a hypervariable-substitute. In some embodiments, the M2e polypeptide comprises a highly conserved region of amino acid sequences. 
     The M2 protein is a surface protein on the influenza virion encoded by the M segment. The M segment encodes M1 from unspliced mRNA and M2 protein by mRNA splicing. M2 forms homotetramers and possesses ion channel activity that allows for acidification of the inside of the virion during endocytosis and facilitates the dissociation of the matrix protein M1 from viral ribonucleoprotein complexes. The M2e, which is the exposed portion of the M2 protein found on the virion membrane, is highly conserved among influenza strains. Accordingly, the M2e protein is a target for universal influenza vaccine approaches. 
     In some embodiments, M2e protein sequences are obtained and aligned using a method described herein (e.g., the Dawn method) to identify hypervariable amino acid residues subject to antigenic shift/drift, and highly conserved regions of amino acid sequences. 
     In some embodiments, the immunogenic composition comprises an M2e recombinant protein or polypeptide derived from H1N1. In some embodiments, the recombinant H1N1 M2e protein or polypeptide comprises a non-hypervariable amino acid residue at a hypervariable amino acid residue, or any combination thereof. In some embodiments, the recombinant H1N1 M2e polypeptide comprises a highly conserved region of amino acid sequences, or any combination thereof. 
     In some embodiments, the immunogenic composition comprises an M2e recombinant protein or polypeptide derived from H3N2. In some embodiments, the recombinant H3N2 M2e protein or polypeptide comprises a non-hypervariable amino acid residue at a hypervariable amino acid residue, or any combination thereof. In some embodiments, the recombinant H3N2 M2e polypeptide comprises a highly conserved region of amino acid sequences, or any combination thereof. 
     In some embodiments, the immunogenic composition comprises an M2e recombinant protein or polypeptide derived from influenza B. In some embodiments, the recombinant influenza B M2e protein or polypeptide comprises a non-hypervariable amino acid residue at a hypervariable amino acid residue, or any combination thereof. In some embodiments, the recombinant influenza B M2e polypeptide comprises a highly conserved region of amino acid sequences, or any combination thereof. 
     Additional Influenza Proteins 
     In some embodiments, an immunogenic composition described herein comprises at least one additional influenza protein, polypeptides or both. In some embodiments the at least one additional influenza protein is selected from NP, M1, PA, PB2, PB2, NS1, and NS2. In some embodiments, the additional influenza protein comprises a non-hypervariable amino acid substituted for a hypervariable amino acid residue. In some embodiments, the additional influenza protein comprises a non-hypervariable amino acid replaced with an amino acid residue that is a hypervariable-substitute. In some embodiments, the additional influenza protein comprises a highly conserved region of amino acid sequences. 
     The nucleoprotein molecules encapsidate the viral single-stranded RNAs. Nucleoprotein molecules also participate in the nuclear import and export of vRNPs and viral replication, and interact with host proteins. The influenza viral polymerase (P complex) is a heterotrimer of subunits PA, PB1 and PB2. The P complex carries out mRNA transcription and replication of the influenza virus. The PA subunit N domain has a cation-dependent endonuclease active-site core; the catalytic residues His41, Glu80, Asp108 and Glu119 are conserved among influenza A subtypes and strains. The nonstructural protein NS1 binds double-stranded RNA (dsRNA) in a non-sequence specific manner. The NS1 protein has a conserved residue, Arg39 that interact with dsRNA. Accordingly, the additional influenza proteins are also targets for universal influenza vaccine approaches. 
     In some embodiments, the additional influenza protein sequences are obtained and aligned using a method described herein (e.g., the Dawn method) to identify hypervariable amino acid residues subject to antigenic shift/drift, and highly conserved regions of amino acid sequences. 
     In some embodiments, the immunogenic composition comprises an additional influenza protein or polypeptide derived from H1N1. In some embodiments, the recombinant H1N1 protein or polypeptide comprises a non-hypervariable amino acid residue at a hypervariable amino acid residue, or any combination thereof. In some embodiments, the recombinant H1N1 polypeptide comprises a highly conserved region of amino acid sequences, or any combination thereof. 
     In some embodiments, the immunogenic composition comprises an additional influenza protein or polypeptide derived from H3N2. In some embodiments, the recombinant H3N2 protein or polypeptide comprises a non-hypervariable amino acid residue at a hypervariable amino acid residue, or any combination thereof. In some embodiments, the recombinant H3N2 polypeptide comprises a highly conserved region of amino acid sequences, or any combination thereof. 
     In some embodiments, the immunogenic composition comprises an additional influenza protein or polypeptide derived from influenza B. In some embodiments, the recombinant influenza B protein or polypeptide comprises a non-hypervariable amino acid residue at a hypervariable amino acid residue, or any combination thereof. In some embodiments, the recombinant influenza B polypeptide comprises a highly conserved region of amino acid sequences, or any combination thereof. 
     In some embodiments, the immunogenic composition comprises an additional influenza protein or polypeptide having a highly conserved regions as annotated in any one of SEQ ID NOs: 171-193. In some embodiments, the protein or polypeptide comprises a non-hypervariable amino acid residue at an amino acid residue that is a hypervariable amino acid residue as annotated in any one of SEQ ID NOs: 171-193. In some embodiments, the protein or polypeptide comprises a hypervariable-substitute at an amino acid residue that is a hypervariable amino acid residue as annotated in any one of SEQ ID NOs: 171-193. 
     Methods for Identifying Hypervariable and Conserved Influenza Residues 
     In some embodiments, the present disclosure provides methods for identifying hypervariable and conserved residues in an influenza viral protein between types and/or subtypes of strains. In some embodiments, the hypervariable amino acid residues identified by the methods described herein are substituted with a non-hypervariable amino acid (e.g., alanine). In some embodiments, the hypervariable amino acid residues identified by the methods described herein are substituted with an amino acid residue that is a hypervariable-substitute (e.g., alanine). In some embodiments, the highly conserved regions of amino acids are used to generate polypeptides for peptide vaccines and/or as targets of nucleic acids. 
     In some embodiments, the present disclosure provides methods for evaluating the role of hypervariable and conserved residues on the ability to induce an immune response (e.g., production of antibodies). 
     Sequence Alignment—Dawn Method 
     Protein sequence evolutionary conservation analysis improves understanding of protein structure, function, and disease. Multiple sequence alignments of different isolates, orthologs, paralogs, and functional domains provide essential insights into protein function and structure. Evolutionary conservation level is directly correlated with likelihood of missense mutations&#39; functional impact. 
     Missense mutations are typically either deleterious or neutral in regards to function impact. Deleterious mutations experience negative selection. Neutral mutations are not positively or negatively selected and can drift through populations. A few mutations experience positive selection and become fixed within populations. Aligning sequences from different species enable the estimation of residue functional importance based on sequence divergence of evolutionarily related proteins. Aligned residues that are identical are composed of a combination of functionally important residues and residues not observed to change due to stochastic chance. Aligned residue positions that are different can represent (1) functionally neutral residues, (2) positions that allow limited conservative residue changes of similar amino acid residues, and (3) positions with alignment errors (this varies by alignment tool used). 
     A protein enzyme typically consists of a globular domain with a conserved inner core with non-conserved residues observed on the solvent exposed surface. Protein folding includes structures like random coils, alpha helices, beta sheets, and loops/turns. Unlike a ball of yarn, protein peptide strands fold into the tertiary structure with the peptide strand traversing into the interior until typically turning in a solvent exposed loop. Residues in the inner core are typically conserved with amino acid substitutions likely impacting protein folding, structure, and/or function. These interior segments are typically what motif signature models such as, Profile analysis, Psi-Blast, and HAMMR, are derived from or trained on. See e.g., Gribskov, M. et al.,  Proc. Natl. Acad. Sci.,  84: 4355-4358 (1987); Altschul, S. F., et al.,  Nucl. Acids Res.  25:3389-3402 (1997); Eddy, S. R.,  Bioinformatics,  14:755-763 (1998). Ideally, these are the segments that should be aligned in a multiple sequence alignment without gapping allowed within each segment. Small insertions and gaps are observed in exterior turns/loops. Pascarella, S., and P. Argos, P., J Mol. Biol., 224: 461-471 (1992). 
     Multiple sequence alignment of protein sequences provides numerous insights into protein structures and functions. Available solutions for generating multiple sequence alignments is slow, and the resulting alignments are plagued by frequent over gapping. Scientists routinely realign sub-segments within alignments to enhance alignment quality. Algorithm developers have treated protein sequences as text strings for comparisons. Some advanced algorithms include knowledge extracted from motifs, profiles, and structures. 
     In some embodiments, protein sequences are aligned using the Dawn method. 
     The Dawn multiple sequence alignment and conservation analysis tool uses conserved residues as anchors such that evolutionarily related sequences can be added to the alignment incrementally. This approach reduces the complexity of creating multiple sequence alignment, or of comparing every sequence to every other sequence. This works for both evolutionarily close or distant sequences and combinations of both. 
     The ability to identify distant orthologs is directly correlated with the proportion of essential residues in a protein. Doolittle, R. F., OF URFS AND ORFS: A Primer on How to Analyze Derived Amino Acid Sequences: University Science Books, (1986), characterized sequence alignments below 25% identity as being the “Twilight Zone”—a limit on sequence alignment approaches. Below 20% identify is termed the “Midnight Zone”, an accepted theoretical limit to sequence analysis techniques. 
     Dawn aligns sequences based on the Divergence Model of protein evolution, and can align and characterize large numbers of related protein sequences rapidly. Using this tool, a performance improvement of at least two orders of magnitude improvement over current methods. Dawn is applied to three pressing challenges: identification of antiviral targets for therapeutics, multigene family alignment, and analysis of human missense mutations (variants). Dawn implements two concepts of (1) conserved core segments and (2) insertions in loops. Using the sequence analysis technique, Dawn is able to align some paralogs deep into the sequence alignment Twilight and Midnight Zones. 
     In some embodiments, the workflow for multiple sequence alignment strategy comprises:
     1. Identify highly conserved protein segments and use these as vertical segments throughout the multiple sequence alignment;   2. Place insertions and gaps in candidate loop segments to align conserved segments. To minimize alignment gaps, align insertions and gaps in a loop region in overlapping alignment positions unless local sequence identity indicate two likely independent mutation events have likely occurred;   3. Residues in two homologs share high sequence identity between conserved segments that are ordered within a domain. Unrelated sequences can share common simple sequence motifs, but these can be ignored;   4. Conserved segments can be approximated by common k-mers between sequences. Multiple homologs will share a common set of ordered k-mers. Multiple unrelated sequences will not share ordered k-mer sequences outside of expected random sharing.   

     In some embodiments, the following definitions are used to define conserved segments:
         Ai=Multiple sequence alignment position, i, for sequence of interest;   C(Ai)=−V with V&gt;0—nonconserved position with V different amino acids observed at this alignment position;   C(Ai)=0—nonconserved position with residue observed missing in sequences for this gene;   C(Ai)=1—conserved positions for all sequences for this gene for organisms of the same taxonomic class;   C(Ai)=1.T—conserved position for all sequences for this gene for taxonomic class of this sequence and T-1 additional taxonomic classes; and   C(Ai)=V with V&gt;1—conserved position with residue conserved in all sequences for V genes.       

     In some embodiments, the following definitions are used to define conserved variable or non-conserved segments:
         V(Ai)=number of nonconserved residues observed at alignment position, i, for the taxonomic class of interest. Allowable conservative substitutions defined by Bottema were used to define observed nonconservative substitutions. Bottema, C. D. K., et al.,  Am J Hum Genet , (49):820-838 (1991).       

     In some embodiments, an algorithm, MSAQ-compute.py (Multiple sequence alignment quality compute), developed in Python, is used to evaluate the quality of multiple sequence alignments. The algorithm accepts an MSA in Clustal format as an input, as well as optional parameters for the number of residues that should not be scored at the beginning and end of the alignment. This accommodates cases of partial sequence overlap and avoids imposing a penalty for otherwise good alignments with excess residues at the beginning or end. The algorithm generates an index of all scored positions within the MSA input file and tallies reported residues at each position to generate a consensus sequence for the alignment. 
     For each sequence in the MSA, the algorithm computes the number of residues that match the consensus sequence, the number of residues that are different from the corresponding position in the consensus sequence (mismatch), the total number of gap characters in the aligned sequence, and the total number of unique gaps in the aligned sequence. These values are reported in a “details” file generated by the algorithm. Additionally, these values are averaged across all sequences in the MSA to generate average match and average mismatch metrics. 
     The average length of all gaps in the MSA is also reported as well as the total number of gaps present in the alignment (summed across all sequences). Finally, based on the rationale that any gaps in a high quality alignment should overlap (i.e. input sequences should have alignment gaps at roughly the same positions), the number of non-overlapping gaps is computed. To generate this value, all gaps in the alignment are mapped to positions in the consensus sequence to generate ranges of gap positions. The number of such non-overlapping ranges is reported. 
     In some embodiments, viral protein sequences are selected from GenBank for influenza. For each selected virus protein, subsets are evaluated to measure execution runtimes using a single Linux core (no parallelization). 
     Alanine Scanning 
     The methods described supra are used to identify hypervariable amino acid residues. In some embodiments, the importance of a hypervariable amino acid residue for inducing an immune response is determined by alanine scanning. 
     As described herein, alanine scanning is a technique used to determine the contribution of a specific wild-type residue to the stability or function(s) (e.g., inducing an immune response) of given protein or polypeptide. The technique involves the substitution of an alanine residue for a wild-type residue in a polypeptide, followed by an assessment of the stability or function(s) (e.g., inducing an immune response) of the alanine-substituted derivative or mutant polypeptide and comparison to the wild-type polypeptide. In some embodiments, the residues identified as not critical are further evaluated to modulate the induction of an immune response. A non-limiting example of such analysis is deep mutational scanning. This method allows for the evaluation of large numbers of mutations. Other methods for analyzing the effect of amino acid residue mutations are known in the art. For example, arginine/glutamic acid scanning is employed to study the effects of bulky, charged amino acid residues on antigen binding. In an embodiment, an arginine amino acid in the hypervariable region is replaced by glutamic acid. 
     Inducing T Cell Responses with Highly Conserved Regions 
     T cell immune response plays an important role in eliciting and maintaining protective immunity against influenza virus. In a recent human study, repeated influenza virus boosted multifunctional memory CD4+ T cell populations. Specifically, IFN-γ and TNF-α secreting CD4 cell population have been shown to boost anti-virus antibody titers after repeated vaccination, and is correlated with maintenance of protective antibody titers. Trieu, M. C., et al., npj Vaccines, 3:37 (2018). doi:10.1038/s41541-018-0069-1. Similarly, administration of a combination vaccine comprising trivalent influenza vaccine and a VLP based vaccine showed enhanced CD8+ and CD4+ immune response, and CD4+ T-cell response is correlated with neutralization antibody titers. Skibinski, D. A. G., et al., Sci Rep. 8:18007 (2018). 
     In some embodiments, the present disclosure provides a polypeptide comprising highly conserved regions of amino acid sequences within a viral protein. In some embodiments, the conserved region is a continuous stretch of at least 7, 8, 9, 10, 11, or 12 invariant or minimally variable amino acid residues. In some embodiments, the polypeptide has 100% identity to a highly conserved region provided herein. In some embodiments, the polypeptide has 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91% or 90% identity to a highly conserved region provided herein. 
     In some embodiments, a polypeptide comprising a highly conserved region is operably linked to at least one additional polypeptide comprising a different highly conserved region. In some embodiments, a polypeptide comprising a highly conserved region is operably linked to at least one additional polypeptide comprising the same highly conserved region. In some embodiments, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 polypeptides comprising highly conserved regions are operably linked to each other, wherein each polypeptide is the same or different. In some embodiments, at least 10, at least 20, at least 30, at least 40 or at least 50 polypeptides comprising highly conserved regions are operably linked to each other, wherein each polypeptide is the same or different. 
     In some embodiments, a polypeptide or polypeptides operably linked to each other, induce a T cell response, such as virus-specific CD8+ or CD4+ T cell responses. In some embodiments, an virus-specific CD8+ T cell response comprises CD8+ T cell proliferation or CD8+ T cell cytokine production or both, are induced. In some embodiments, CD8 +  T cell cytokine production increases by at least 5% or at least 10% or at least 15% or at least 20% or at least 25% or at least 30% or at least 35% or at least 40% or at least 45% or at least 50%. In some embodiments, the percentage of CD8 +  T cells among the total T cell population increases by at least 5% or at least 10% or at least 15% or at least 20% or at least 25% or at least 30% or at least 35% or at least 40% or at least 45% or at least 50%. 
     In one embodiment, the disclosure provides a method for eliciting T cell response to conserved polypeptides of influenza viruses, the method comprising administering to a subject in need thereof an immunogenic composition comprising at least one influenza virus polypeptide comprising high conserved amino acid sequence, wherein the T cell response immune response to the highly conserved amino acid sequence is elicited in the subject. In one embodiment, eliciting T cell immune response in a subject comprises stimulating cytokine production (e.g., IFN-γ or TNF-α). 
     In another embodiment, eliciting an immune response in a subject comprises stimulating virus polypeptide-specific CD4+ or CD8+ T cell activity, e.g., priming, proliferation and/or survival (e.g., increasing the effector/memory T cell population). In one aspect, eliciting a T-cell immune response in a subject comprises stimulating virus-specific CD4+ T cell activity (e.g., increasing helper T cell activity). In other aspects, the CD4+ T cell immune response stimulates cell responses (e.g., increasing antibody production). In some embodiments, enhancing T cell immune response in a subject comprises stimulating cytokine production, stimulating antigen-specific CD8+ T cell responses, stimulating antigen-specific CD4+ helper cell responses, increasing the effector memory CD62Llo T cell population, stimulating B cell activity or stimulating virus-specific antibody production, or any combination of the foregoing responses. 
     In some embodiments, the enhanced immune response comprises an virus-specific CD8+ T cell response, wherein the CD8+ T cell response comprises an increase in the percentage of effector memory CD62Llo T cells among CD8+ T cells. 
     Inducing B Cell Responses by Targeting Hypervariable Amino Acid Residues 
     Most neutralizing antibodies bind to the loops that surround the virus receptor binding site and interfere with receptor binding and attachment. Since these loops are highly variable, most antibodies targeting these regions are strain-specific, and elicit limited, strain-specific immunity. Fully human monoclonal antibodies against influenza virus hemagglutinin with broad cross-neutralizing potency have been generated. Functional and structural analysis have revealed that these antibodies interfere with the membrane fusion process and are directed against highly conserved epitopes in the stem domain of the influenza HA protein (Throsby et al., Plos One 12(3): 1-15 (2008); Ekiert et al., Science 324:246-251 (2009), US2009/0311265, US2012/0039898, US2014/0120113). 
     In some embodiments, the present disclosure provides an influenza viral protein or fragment thereof, wherein hypervariable amino acid residues are replaced with a non-hypervariable amino acid. Non-hypervariable amino acid residues include, but are not limited to, alanine and glycine. In some embodiments, a non-hypervariable amino acid residue is referred to as a hypervariable-substitute. In some embodiments, the hypervariable amino acid residues are replaced with alanine, glycine, valine, leucine, isoleucine, and methionine. In some embodiments, the hypervariable amino acid residues are replaced with alanine and glycine. In some embodiments, the hypervariable amino acids are replaced with the exemplary and/or preferred amino acids to preserve the conformation of the viral protein and to minimize disruption to adjacent or overlapping conserved regions. In some embodiments, bulky and charged arginine amino acid residues are replaced with glutamic acid residues. In some embodiments, the polypeptide comprises a fragment of the amino acid sequence of the viral proteins. In some embodiments, the fragment comprises the entire amino acid sequence of the viral protein. In some embodiments, viral proteins and fragments thereof can be used in combination. 
     In some embodiments, the present disclosure provides immunogenic composition comprising at least viral protein or fragment thereof wherein 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55, 56-60 hypervariable amino acid residues are replaced with non-hypervariable amino acid residues. In some embodiments at one, two, three, four, five or more hypervariable amino acids are replaced with non-hypervariable amino acid residues. 
     In some embodiments, the present disclosure provides immunogenic composition comprising at least viral protein or fragment thereof wherein 1-5, 6-10, 11-15, 16-20, 21-25, 26-30, 31-35, 36-40, 41-45, 46-50, 51-55, 56-60 hypervariable amino acid residues are replaced with amino acid residues that are hypervariable-substitute. In some embodiments at one, two, three, four, five or more hypervariable amino acids are replaced with an amino acid residue that is a hypervariable-substitute. 
     In some embodiments, substituting the hypervariable amino acid residues with non-hypervariable amino acid residues directs the immune response away from the residues subject to antigenic drift/shift and induces an immune response to the highly conserved regions of amino acid sequences. By targeting the highly conserved regions, such polypeptides can be used for protection against current and yet to exist influenza strains. 
     In some embodiments, the polypeptides described herein induce a B cell response (e.g., antibody production). In some embodiments, the B cell response is an antigen-specific antibody response. In some embodiments, the B cell response elicit neutralizing antibodies directed to the highly conserved regions in the viral protein. In some embodiments, the neutralizing antibodies are neutralizing against multiple strains of influenza viruses. 
     In another aspect, the disclosure provides a method of directing the specificity of an B cell immune response in a subject by administering to a subject an immunogenic composition comprising the viral protein, wherein one or more hypervariable amino acid residues of the virus protein are replaced with non-hypervariable amino acid residues. 
     In another embodiment, administration of immunogenic composition having the amino acid residue substitution results in the immune response to be directed to an highly conserved B cell epitope, and thus eliciting one or more protective neutralizing antibodies. In some embodiments, the neutralizing antibodies provide protective immunity against multiple strains of influenza virus. 
     Targeting Highly Conserved Regions with Nucleic Acid Molecules 
     In some embodiments, the present disclosure provides nucleic acid molecules having substantial complementarity to a highly conserved region of amino acid residues. Such nucleic acid molecules are capable of disrupting the transcription and/or translation of a viral protein comprising the base sequence. 
     Exemplary nucleic acid molecules that can modulate protein function include antisense oligonucleotides and RNA interference molecules (e.g., small interfering RNA (siRNA), microRNA (miRNA) and shRNA). 
     Antisense oligonucleotides are capable of blocking or decreasing the expression of a desired target gene by targeting nucleic acids encoding the gene or subunit thereof. Methods are known to those of ordinary skill in the art for the preparation of antisense oligonucleotide molecules that will specifically bind one or more target gene(s) without cross-reacting with other polynucleotides. Exemplary sites of targeting include, but are not limited to, the initiation codon, the 5′ regulatory regions, including promoters or enhancers, the coding sequence, including any conserved consensus regions, and the 3′ untranslated region. In some embodiments, the antisense oligonucleotides are about 10 to about 100 nucleotides in length, about 15 to about 50 nucleotides in length, about 18 to about 25 nucleotides in length, or more. In certain embodiments, the oligonucleotides further comprise chemical modifications to increase nuclease resistance and the like, such as, for example, phosphorothioate linkages and 2′-O-sugarmodifications known to those of ordinary skill in the art. 
     RNA interference (RNAi) is a biological process in which RNA molecules inhibit gene expression or translation by neutralizing targeted mRNA molecules. Specifically, RNAi refers to a post-transcriptional silencing mechanism initiated by small double-stranded RNA molecules that suppress expression of genes with sequence homology. Key to the mechanism of RNAi are small interfering RNA (siRNA) strands, which have complementary nucleotide sequences to a targeted messenger RNA (mRNA) molecule. siRNAs are short, single-stranded nucleic acid molecules capable of inhibiting or down-regulating gene expression in a sequence-specific manner; see, for example, Zamore et al., Cell 101:25 33 (2000); Bass, Nature 411:428-429(2001); Elbashir et al., Nature 411:494-498 (2001); and Kreutzer et al., International PCT Publication No. WO 00/44895; Zernicka-Goetz et al., International PCT Publication No. WO 01/36646; Fire, International PCT Publication No. WO 99/32619; Plaetinck et al., International PCT Publication No. WO 00/01846; Mello and Fire, International PCT Publication No. WO 01/29058; Deschamps-Depaillette, International PCT Publication No. WO 99/07409; and Li et al., International PCT Publication No. WO 00/44914. Methods of preparing a siRNA molecule for use in gene silencing are described in U.S. Pat. No. 7,078,196, which is hereby incorporated by reference. Generally, one would prepare siRNA molecules that will specifically target one or more mRNAs without cross-reacting with other polynucleotides. siRNA molecules can be generated by methods known in the art, such as by typical solid phase oligonucleotide synthesis, and often will incorporate chemical modifications to increase half-life and/or efficacy of the siRNA agent, and/or to allow for a more robust delivery formulation. Alternatively, siRNA molecules are delivered using a vector encoding an expression cassette for intracellular transcription of siRNA. 
     Nucleic Acids Encoding Influenza Polypeptides 
     In some aspects, the polypeptides described herein are encoded by a nucleic acid molecule (e.g., DNA, RNA). 
     A “coding sequence” or a sequence which “encodes” a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or “control elements”). The boundaries of the coding sequence are determined by a start codon at the 5′ (amino) terminus and a translation stop codon at the 3′ (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, procaryotic or eucaryotic mRNA, genomic DNA sequences from viral or procaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3′ to the coding sequence. Transcription and translation of coding sequences are typically regulated by “control elements,” including, but not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3′ to the translation stop codon), sequences for optimization of initiation of translation (located 5′ to the coding sequence), and translation termination sequences. 
     A “promoter” is a nucleotide sequence which initiates transcription of a polypeptide-encoding polynucleotide. Promoters can include inducible promoters (where expression of a polynucleotide sequence operably linked to the promoter is induced by an analyte, cofactor, regulatory protein, etc.), repressible promoters (where expression of a polynucleotide sequence operably linked to the promoter is repressed by an analyte, cofactor, regulatory protein, etc.), and constitutive promoters. In addition, such promoters can also have tissue specificity, for example, the CD80 promoter is only inducible in certain immune cells, and the myoD promoter is only inducible in muscle cells. It is intended that the term “promoter” or “control element” includes full-length promoter regions and functional (e.g., controls transcription or translation) segments of these regions. A promoter is “derived from” a gene encoding a co-stimulatory molecule if it has the same or substantially the same basepair sequence as a region of the promoter region of the co-stimulatory molecule, complements thereof, or if it displays sequence identity as described below. 
     A “vector” is capable of transferring gene sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, “vector construct,” “expression vector,” and “gene transfer vector,” mean any nucleic acid construct capable of directing the expression of a gene of interest and which can transfer gene sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors. 
     Nucleotide sequences selected for use in the present disclosure can be derived from known sources, for example, by isolating the same from cells containing a desired gene or nucleotide sequence using standard techniques. Similarly, the nucleotide sequences can be generated synthetically using standard modes of polynucleotide synthesis that are well known in the art. See, e.g., Edge et al. (1981) Nature 292:756-762; Nambair et al. (1994) Science 223:1299-1301: Jay et al. (1984) J. Biol. Chem. 259:6311-6317. Generally, synthetic oligonucleotides can be prepared by either the phosphotriester method as described by Edge et al., supra, and Duckworth et al. (1981) Nucleic Acids Res. 9:1691-1706, or the phosphoramidite method as described by Beaucage et al. (1981) Tet. Letts. 22:1859, and Matteucci et al. (1981) J. Am. Chem. Soc.103:3185. 
     Another method for obtaining nucleic acid sequences for use herein is by recombinant means. Thus, a desired nucleotide sequence can be excised from a plasmid carrying the same using standard restriction enzymes and procedures. Site specific DNA cleavage is performed by treating with the suitable restriction enzyme (or enzymes) under conditions which are generally understood in the art, and the particulars of which are specified by manufacturers of commercially available restriction enzymes. If desired, size separation of the cleaved fragments may be performed by polyacrylamide gel or agarose gel electrophoresis using standard techniques. 
     Yet another convenient method for isolating specific nucleic acid molecules is by the polymerase chain reaction (PCR). Mullis et al. (1987) Methods Enzymol. 155:335-350. This technique uses DNA polymerase, usually a thermostable DNA polymerase, to replicate a desired region of DNA. The region of DNA to be replicated is identified by oligonucleotides of specified sequence complementary to opposite ends and opposite strands of the desired DNA to prime the replication reaction. The product of the first round of replication is itself a template for subsequent replication, thus repeated successive cycles of replication result in geometric amplification of the DNA fragment delimited by the primer pair used. This method also allows for the facile addition of nucleotide sequences onto the ends of the DNA product by incorporating these added sequences onto the oligonucleotide primers (see, e.g., PCR Protocols, A Guide to Methods and Applications, Innis et al (eds) Harcourt Brace Jovanovich Publishers, NY (1994)). PCR conditions used for each amplification reaction are empirically determined. A number of parameters influence the success of a reaction. Among them are annealing temperature and time, extension time, Mg2+ and ATP concentration, pH, and the relative concentration of primers, templates, and deoxyribonucleotides. 
     Once coding sequences for desired proteins have been prepared or isolated, such sequences can be cloned into any suitable vector or replicon. Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice. Ligations to other sequences are performed using standard procedures, known in the art. 
     In some aspects, a nucleic acid molecule described herein is provided in an expression vector. In some embodiments, the vector comprises the nucleic acid molecule that codes for the peptides operatively linked to appropriate expression control sequences. Methods of affecting this operative linking, either before or after the nucleic acid molecule is inserted into the vector, are well known. Expression control sequences include promoters, activators, enhancers, operators, ribosomal nuclease domains, start signals, stop signals, cap signals, polyadenylation signals, and other signals involved with the control of transcription or translation. 
     Viral vectors that are suitable for use include, for example, retroviral, adenoviral, and adeno-associated vectors, herpes virus, simian virus 40 (SV40), and bovine papilloma virus vectors (see, for example, Gluzman (Ed.), Eukaryotic Viral Vectors, CSH Laboratory Press, Cold Spring Harbor, N.Y.). 
     A number of viral based systems have been used for gene delivery. For example, retroviral systems are known and generally employ packaging lines which have an integrated defective provirus (the “helper”) that expresses all of the genes of the virus but cannot package its own genome due to a deletion of the packaging signal, known as the psi sequence. Thus, the cell line produces empty viral shells. Producer lines can be derived from the packaging lines which, in addition to the helper, contain a viral vector which includes sequences required in cis for replication and packaging of the virus, known as the long terminal repeats (LTRs). The gene of interest can be inserted in the vector and packaged in the viral shells synthesized by the retroviral helper. The recombinant virus can then be isolated and delivered to a subject. (See, e.g., U.S. Pat. No. 5,219,740.) Representative retroviral vectors include but are not limited to vectors such as the LHL, N2, LNSAL, LSHL and LHL2 vectors described in e.g., U.S. Pat. No. 5,219,740, incorporated herein by reference in its entirety, as well as derivatives of these vectors, such as the modified N2 vector described herein. Retroviral vectors can be constructed using techniques well known in the art. See, e.g., U.S. Pat. No. 5,219,740; Mann et al. (1983) Cell 33:153-159. 
     Adenovirus based systems have been developed for gene delivery and are suitable for delivering the nucleic acid molecules described herein. Human adenoviruses are double-stranded DNA viruses which enter cells by receptor-mediated endocytosis. These viruses are particularly well suited for gene transfer because they are easy to grow and manipulate and they exhibit a broad host range in vivo and in vitro. For example, adenoviruses can infect human cells of hematopoietic, lymphoid and myeloid origin. Furthermore, adenoviruses infect quiescent as well as replicating target cells. Unlike retroviruses which integrate into the host genome, adenoviruses persist extrachromosomally thus minimizing the risks associated with insertional mutagenesis. The virus is easily produced at high titers and is stable so that it can be purified and stored. Even in the replication-competent form, adenoviruses cause only low level morbidity and are not associated with human malignancies. Accordingly, adenovirus vectors have been developed which make use of these advantages. For a description of adenovirus vectors and their uses see, e.g., Haj-Ahmad and Graham (1986) J. Virol. 57:267-274; Bett et al. (1993) J. Virol. 67:5911-5921; Mittereder et al. (1994) Human Gene Therapy 5:717-729; Seth et al. (1994) J. Virol. 68:933-940; Barr et al. (1994) Gene Therapy 1:51-58; Berkner, K. L. (1988) BioTechniques 6:616-629; Rich et al. (1993) Human Gene Therapy 4:461-476. Adeno-associated viral vector (AAV) can also be used to administer the polynucleotides described herein. AAV vectors can be derived from any AAV serotype, including without limitation, AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAVX7, etc. AAV vectors can have one or more of the AAV wild-type genes deleted in whole or part, preferably the rep and/or cap genes, but retain one or more functional flanking inverted terminal repeat (ITR) sequences. Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV virion. Thus, an AAV vector includes at least those sequences required in cis for replication and packaging (e.g., functional ITRs) of the virus. The ITR sequence need not be the wild-type nucleotide sequence, and may be altered, e.g., by the insertion, deletion or substitution of nucleotides, so long as the sequence provides for functional rescue, replication and packaging. 
     AAV expression vectors are constructed using known techniques to at least provide as operatively linked components in the direction of transcription, control elements including a transcriptional initiation region, the DNA of interest and a transcriptional termination region. The control elements are selected to be functional in a mammalian cell. The resulting construct which contains the operatively linked components is bounded (5′ and 3′) with functional AAV ITR sequences. Suitable AAV constructs can be designed using techniques well known in the art. See, e.g., U.S. Pat. Nos. 5,173,414 and 5,139,941; International Publication Nos. WO 92/01070 (published 23 Jan. 1992) and WO 93/03769 (published 4 Mar. 1993); Lebkowski et al. (1988) Molec. Cell. Biol. 8:3988-3996; Vincent et al. (1990) Vaccines 90 (Cold Spring Harbor Laboratory Press); Carter, B. J. (1992) Current Opinion in Biotechnology 3:533-539; Muzyczka, N. (1992) Current Topics in Microbiol. and Immunol. 158:97-129; Kotin, R. M. (1994) Human Gene Therapy 5:793-801; Shelling and Smith (1994) Gene Therapy 1:165-169; and Zhou et al. (1994) J. Exp. Med. 179:1867-1875. 
     Models for Assessing Prophylactic and Therapeutic Efficacy 
     In Vitro Models 
     In some embodiments, in vitro evaluation are utilized to screen vaccine candidates. See e.g., Tapia-Calle, G., et al., Vaccines (Basel) 5(3) pii: E21 (2017). doi: 10.3390/vaccines5030021. Dendritic cells (DCs) play an important in the development of innate and adaptive immune responses. In a study, a DC line (MUT-3) and primary monocyte-derived DCs (Mo-DCs) were employed to screen whole inactivated and subunit influenza vaccines. The Mo-DCs were stimulated with both vaccines and showed upregulated protein expression of activation markers (MHC II, CD86 and CD40) and changes in cytokine secretion in response to whole inactivated vaccines. The Mo-DCs additionally showed increase in gene coding for surface markers of DC cells. The results show that Mo-DCs derived from either fresh or frozen/thawed PBMCs could be utilized to screen vaccine candidates. 
     In another embodiment, long-term cultures of unfractionated PBMCS were employed to assess recall T cell responses to vaccine candidates. See e.g., Tapia-Calle, G., et al., Vaccines (Basel) 7(4). pii: E181 (2019). doi: 10.3390/vaccines7040181. After stimulation with whole inactivated and subunit influenza vaccines. T cell-mediated immune responses, e.g., activation, proliferation, increase in cytotoxic potential and IFN-γ responses were evaluated. CD4+ and CD8+ phenotyping showed that effector and central memory T cells were activated. Additionally, vaccine induced follicular T helper cell responses (T FH ) were also elicited. 
     In some embodiments, long-term cultures human precision-cut lung slices (PCLS) from human donors are used as an ex vivo model to evaluate immune response to stimulation by influenza vaccine. See e.g., Temann, A., et al., Hum Vac Immunother 13(2):351-358 (2017). Upon stimulation with influenza vaccines, PCLS showed upregulation of cytokine secretions, e.g., IFN-γ, TNF-α and IL-2. 
     In Vivo Models 
     Various animal models for evaluating influenza vaccines are known in the art. Margine, I., Krammer, F., Pathogens 3(4):845-874 (2014). Immunogenicity and protective efficacy of candidate influenza vaccines have been tested in e.g., chicken, mouse, ferret, pigs, and non-human primates models. 
     Ferrets were the first species to be successfully infected with human influenza isolates, and is susceptible to a wide range of human isolates without prior adaptation. Ferrets display clinical symptoms similar to human disease when infected with human influenza, although the presence and severity of symptoms vary depending on the challenge viral strain and route of administration. 
     Wild mice are not natural hosts of the influenza viruses. However, mice are widely used in influenza research due to their small size, low cost, availability of immunological reagents, availability of laboratory mice strains that can be infected with certain influenza, and availability of transgenic mice strains with targeted gene disruptions to study host responses. Generally, influenza viruses require adaption in mice to be able to infect mice and replicate. The process of adaptation, i.e., repeated in vivo passage in mouse lungs will cause antigenic and phenotypic changes in the adapted virus. However, several pathogenic pandemic influenza strains, such as H1N1, H5N1, and H7, are able to cause disease in mice without prior adaption. 
     Pigs are an attractive model for influenza research as they are naturally infected by both human and avian influenza viruses. Innate and adaptive B- and T-cell immunity against influenza have been characterized in the pig model. Holzer, B., et al., Front. Immunol. 10:98 (2019). doi: 10.3389/fimmu.2019.00098. 
     Immunogenicity and challenge influenza studies have been conducted in pigs. For example cold adapted 2017-2018 Northern Hemisphere LAIV vaccine Fluenz Tetra (AstraZeneca) containing two type A viruses: H1N1 A/Slovenia/2903/2015, MEDI 279432 107.0±0.5 FFU [A/Michigan/45/2015 (H1N1) pdm09-like strain]; H3N2 A/New Caledonia/71/2014, MEDI 263122 107.0±0.5 FFU [A/Hong Kong/4801/2014 (H3N2)-like strain] and two type B (IBV) viruses; (B/Brisbane/60/2008, MEDI 228030) 107.0±0.5 FFU (B/Brisbane/60/2008-like strain) and B/Phuket/3073/2013, MEDI 254977) 107.0±0.5 FFU (B/Phuket/3073/2013-like strain) were administered intranasally to pigs. Holzer, B., et al.,  Front. Immunol  10:2625 (2019). doi: 10.3389/fimmu.2019.02625. Four weeks after immunization, the pigs were challenged intranasally with wild-type viruses contained in the LAIV vaccine. 
     Nasal swabs were collected to test virus load. Serum and bronchoalveolar lavage (BAL) fluid were collected and tested for antibody and neutralizing antibody titers in ELISA and microneutralization (MN) assays, respectively. Cellular response were tested in IFN-γ ELISPOT and intracellular cytokine staining of cells collected from peripheral blood, trachea bronchial lymph nodes (TBLNs) and BALs. 
     Nonhuman primates are naturally infected by human influenza virus, and are considered good models of human responses to influenza infection and vaccination. Although ethical and economical considerations limit the use of non-human primates in influenza vaccine research, their use is challenge experiments are useful in testing pandemic influenza virus strains. 
     In some embodiments, the immunogenic compositions herein are tested in immunogenicity and/or challenge studies in animal models. 
     Immunogenic Compositions 
     Also provided herein are immunogenic compositions (e.g., vaccines) comprising combinations or cocktails of the recombinant viral proteins and/or polypeptides described herein. In some embodiments, the immunogenic compositions comprise a nucleic acid molecule encoding the recombinant viral proteins and/or polypeptides described herein. In some embodiments, the compositions further comprise a pharmaceutically acceptable carrier. 
     In some embodiments, immunogenic compositions described herein further comprise one or more adjuvants. For example, alum, aluminum salts (Baylor et al., 2002, Vaccine, 20:S18; incorporated herein by reference) and monophosphoryl lipid A (MPL; Ribi et al., 1986, Immunology and Immunopharmacology of Bacterial Endotoxins, Plenum Publ. Corp., NY, p. 407; incorporated herein by reference) can be used as adjuvants in human vaccines. Alternatively or additionally, new compounds are currently being tested as adjuvants in human vaccines, such as: MF59 (See, e.g., Ott et al., “MF59—Design and Evaluation of a Safe and Potent Adjuvant for Human Vaccines” in Vaccine Design: The Subunit and Adjuvant Approach (Powell, M. F. and Newman, M. J. eds.) Plenum Press, New York, 1995, pp. 277-296; incorporated herein by reference); CpG oligodeoxynudeotide (ODN) adjuvants such as CPG 7909 (Cooper et al., 2004, Vaccine, 22:3136; incorporated herein by reference); Monophosphoryl lipid A (MPL) adjuvants and lipid A mimetis including AS04 (Didierlaurent, A. M. et al, J. Immunol., 2009, 183: 6186-6197; incorporated by reference herein), monophosphoryl lipid A (MPL, GSK) and glucopyranosyl lipid A GLA (Immune Design Corporation, IDC); AF03 (Klucker, M. F. et al, J. Pharm Sci., 2012, 101: 4490-4500; incorporated herein by reference); the TLR-3 ligand polyinosinic:polycytidylic acid [poly(I:C)]; TLR9 adjuvants such as IC31 (Riedl, K. et al., Vaccine, 2008, 26: 3461-3468; incorporated herein by reference); imidazoquinolines (double cyclic organic molecules that act as TLR-7/8 agonists) such as imiquimod (R837) or resiquimod (R848); saponins such as QS21 (Ghochikyan et al., 2006, Vaccine, 24:2275; incorporated herein by reference), ISCOMATRIX adjuvant (Duewell, P., et al., J. Immunol, 2011, 187: 55-63; incorporated herein by reference), and Matrix-M™ (Novavax). 
     Additionally, some adjuvants are known in the art to enhance the immunogenicity of influenza vaccines, such as poly[di(carboxylatophenoxy)phosphazene] (PCCP; Payne et al., 1998, Vaccine, 16:92; incorporated herein by reference), interferon-.gamma. (Cao et al., 1992, Vaccine, 10:238; incorporated herein by reference), block copolymer P1205 (CRL1005; Katz et al., 2000, Vaccine, 18:2177; incorporated herein by reference), interleukin-2 (IL-2; Mbwuike et al., 1990, Vaccine, 8:347; incorporated herein by reference), and polymethyl methacrylate (PMMA; Kreuter et al., 1981, J. Pharm. Sci., 70:367; incorporated herein by reference). 
     In some embodiments, the immunogenic compositions include one or more inactive agents such as a sterile, biocompatible carrier including, but not limited to, sterile water, saline, buffered saline, or dextrose solution. In some embodiments, the composition contains any of a variety of additives, such as stabilizers, buffers, excipients (e.g., sugars, amino acids, etc.), or preservatives. Pharmaceutically acceptable carriers used in particular embodiments include, but are not limited to, saline, buffered saline, dextrose, water, glycerol, ethanol, and combinations thereof. In some embodiments, the carrier and composition are sterile, and the formulation suits the mode of administration. In some embodiments, an immunogenic composition contains minor amounts of wetting or emulsifying agents, or pH buffering agents. In some embodiments, a pharmaceutical composition is a liquid solution, suspension, emulsion, tablet, pill, capsule, sustained release formulation, or powder. Oral formulations can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. Any of the common pharmaceutical carriers, such as sterile saline solution or sesame oil, can be used. The medium can also contain conventional pharmaceutical adjunct materials such as, for example, pharmaceutically acceptable salts to adjust the osmotic pressure, buffers, preservatives and the like. Other media that can be used with the compositions and methods provided herein are normal saline and sesame oil. 
     In some embodiments, an immunogenic composition is formulated for intradermal injection, intranasal administration or intramuscular injection. In some embodiments, injectables are prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection, or as emulsions. In some embodiments, injection solutions and suspensions are prepared from sterile powders, granules, and. General considerations in the formulation and manufacture of pharmaceutical agents for administration by these routes may be found, for example, in Remington&#39;s Pharmaceutical Sciences, 19.sup.th ed., Mack Publishing Co., Easton, Pa., 1995; incorporated herein by reference. At present the oral or nasal spray or aerosol route (e.g., by inhalation) are most commonly used to deliver therapeutic agents directly to the lungs and respiratory system. In some embodiments, compositions in accordance with the invention are administered using a device that delivers a metered dosage of composition (e.g., of an optimized HA polypeptide). Suitable devices for use in delivering intradermal pharmaceutical compositions described herein include short needle devices such as those described in U.S. Pat. Nos. 4,886,499, 5,190,521, 5,328,483, 5,527,288, 4,270,537, 5,015,235, 5,141,496, 5,417,662 (all of which are incorporated herein by reference). 
     Intradermal compositions may also be administered by devices which limit the effective penetration length of a needle into the skin, such as those described in WO1999/34850, incorporated herein by reference, and functional equivalents thereof. Also suitable are jet injection devices which deliver liquid vaccines to the dermis via a liquid jet injector or via a needle which pierces the stratum corneum and produces a jet which reaches the dermis. Jet injection devices are described for example in U.S. Pat. Nos. 5,480,381, 5,599,302, 5,334,144, 5,993,412, 5,649,912, 5,569,189, 5,704,911, 5,383,851, 5,893,397, 5,466,220, 5,339,163, 5,312,335, 5,503,627, 5,064,413, 5,520,639, 4,596,556, 4,790,824, 4,941,880, 4,940,460, WO1997/37705, and WO1997/13537 (all of which are incorporated herein by reference). Also suitable are ballistic powder/particle delivery devices which use compressed gas to accelerate vaccine in powder form through the outer layers of the skin to the dermis. Additionally, conventional syringes may be used in the classical mantoux method of intradermal administration. 
     Preparations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer&#39;s dextrose, dextrose and sodium chloride, lactated Ringer&#39;s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer&#39;s dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. 
     In some embodiments, the compositions are administered as a pharmaceutically acceptable acid- or base-addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines. 
     Methods of Use 
     In some embodiments, the polypeptides described herein are capable of eliciting an immune response against an influenza virus. In some embodiments, the polypeptides can be used as vaccines to protect individuals against influenza infection. In some embodiments, the nucleic acid molecules encoding polypeptides described herein are capable of eliciting an immune response against an influenza virus. In some embodiments, the nucleic acid molecules can be used as vaccines to protect individuals against influenza infection. 
     In some embodiments, the disclosure provides a method of vaccinating a subject against influenza, in particular, against various strains of influenza. Such methods employ the immunogenic compositions of the present disclosure. Accordingly, in some embodiments, the method comprises administering an immunogenic composition to a subject such that an immune response against influenza virus is produced in the subject. In some embodiments, the polypeptides described herein are capable of eliciting neutralizing antibodies to influenza. In some embodiments, the nucleic acid molecules encoding polypeptides described herein are capable of eliciting neutralizing antibodies to influenza. 
     Immunogenic compositions of the present disclosure can be used to vaccinate individuals using a prime/boost protocol. Such a protocol is described in U.S. Patent Publication No. 2011/0177122, which is incorporated herein by reference in its entirety. In such a protocol, a first immunogenic composition may be administered to the individual (prime) and then after a period of time, a second immunogenic composition may be administered to the individual (boost). Administration of the boosting composition is generally weeks or months after administration of the priming composition, preferably about 2-3 weeks or 4 weeks, or 8 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks. In one embodiment, the boosting composition is formulated for administration about 1 week, or 2 weeks, or 3 weeks, or 4 weeks, or 5 weeks, or 6 weeks, or 7 weeks, or 8 weeks, or 9 weeks, or 16 weeks, or 20 weeks, or 24 weeks, or 28 weeks, or 32 weeks after administration of the priming composition. 
     In some embodiments, the subject is at risk for infection with influenza. In some embodiments, the subject has been exposed to influenza. For example, the subject may be an elderly individual, a child, an infant or an immunocompromised individual. As used herein, the terms exposed, exposure, and the like, indicate the subject has come in contact with a person or animal that is known to be infected with influenza. Immunogenic compositions of the present disclosure may be administered using techniques well known to those in the art and described herein. 
     In some embodiments, the polypeptides and immunogenic compositions of the present disclosure is used to protect a subject against infection by antigenically divergent influenza. In some embodiments, the nucleic acid molecules and immunogenic compositions of the present disclosure is used to protect a subject against infection by antigenically divergent influenza. 
     Methods of preparing and administering immunogenic compositions to a subject in need thereof are well known in the art or readily determined by those skilled in the art. The dosage and frequency of administration may depend on whether the treatment is prophylactic or therapeutic. 
     The immunogenic composition and polypeptides of the disclosure are suitable for administration to mammals (e.g., primates, (e.g., humans, chimpanzees, monkeys, baboons), rats (e.g., cotton rats), mice, cows (e.g., calves), guinea pigs, ferrets and hamsters). In some embodiments, the disclosure provides a method of inducing an immune response in a mammal, comprising the step of administering a composition (e.g., an immunogenic composition) of the disclosure to the mammal. The compositions (e.g., an immunogenic composition) can be used to produce a vaccine formulation for immunizing a mammal. The mammal is typically a human, and the immunogenic composition typically comprises a polypeptide comprising an amino acid sequence of an influenza viral protein. In some embodiments, the mammal is a human, and the immunogenic composition comprises a nucleic acid molecule encoding a polypeptide comprising an amino acid sequence of an influenza viral protein. 
     The disclosure also provides a composition of for use as a medicament, e.g., for use in immunizing a patient against influenza infection. 
     The disclosure also provides the use of a polypeptide as described above in the manufacture of a medicament for raising an immune response in a patient. In some embodiments, the disclosure provides the use of a nucleic acid molecule described herein in the manufacture of a medicament for raising an immune response in a patient. 
     The immune response raised by these methods and uses will generally include an antibody response, preferably a protective antibody response. Methods for assessing antibody responses after influenza vaccination are well known in the art. 
     Compositions of the invention can be administered in a number of suitable ways, such as intramuscular injection (e.g., into the arm or leg), subcutaneous injection, intranasal administration, oral administration, intradermal administration, transcutaneous administration, transdermal administration, and the like. The appropriate route of administration will be dependent upon the age, health and other characteristics of the mammal A clinician will be able to determine an appropriate route of administration based on these and other factors. 
     Immunogenic compositions, and vaccine formulations, may be used to treat both children and adults, including pregnant women. Thus a subject may be less than 1 year old, 1-5 years old, 5-15 years old, 15-55 years old, or at least 55 years old. Preferred subjects for receiving the vaccines are the elderly (e.g., &gt;50 years old, &gt;60 years old, &gt;65 years, and preferably &gt;75 years), the young (e.g., &lt;6 years old, such as 4-6 years old, &lt;5 years old), and pregnant women. The vaccines are not limited to these groups, however, and may be used more generally in a population. 
     Treatment can be by a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and/or in a booster immunization schedule. In a multiple dose schedule the various doses may be given by the same or different routes, e.g., a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, etc. Administration of more than one dose (typically two doses) is particularly useful in immunologically naive patients. Multiple doses will typically be administered at least 1 week apart (e.g., about 2 weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about 10 weeks, about 12 weeks, about 16 weeks, and the like.) 
     Vaccine formulations produced using a composition of the disclosure may be administered to patients at substantially the same time as (e.g., during the same medical consultation or visit to a healthcare professional or vaccination center) other vaccines. 
     In some embodiments, the immunogenic compositions, polypeptides or nucleic acid molecules described herein are administered as a therapeutic to a subject infected with influenza. 
     Kits 
     The immunogenic composition or polypeptide of the disclosure can be provided in a kit. In some embodiments, a nucleic acid molecule of the disclosure is provided in a kit. In some embodiments, the kit includes (a) a container that contains a composition that includes one or more unit doses of the immunogenic composition or polypeptide, and optionally (b) instructions for use. In some embodiments, the kit includes (a) a container that contains a composition that includes one or more unit doses of the immunogenic composition or nucleic acid molecule, and optionally (b) instructions for use. The unit doses of the immunogenic composition or polypeptide are sufficient to cause an immunogenic response (e.g., antibody production) in a subject. In some embodiments, the unit doses of the immunogenic composition or nucleic acid molecule are sufficient to cause an immunogenic response (e.g., antibody production) in a subject. The kit can also include reagents and instructions useful in the testing (assaying) for an immunogenic response. Such methods of assaying for an immunogenic response include, but are not limited to, any of the testing methods described herein. In one embodiment, the kit includes one or more additional agents for treating influenza. For example, the kit includes a first container that contains a composition that includes the immunogenic composition, and a second container that includes the one or more additional agents. 
     In some embodiments, the instructions provide methods of administering the immunogenic composition, e.g., in a suitable dose, dosage form, or mode of administration (e.g., a dose, dosage form, or mode of administration described herein), to treat a subject who is infected with influenza, or who is at risk of being infected with influenza. 
     In addition to the immunogenic composition or polypeptide, the composition in the kit can include other ingredients, such as a solvent or buffer, a stabilizer, or a preservative. The agent can be provided in any form, e.g., liquid, dried or lyophilized form, preferably substantially pure and/or sterile. When the agents are provided in a liquid solution, the liquid solution preferably is an aqueous solution. When the agents are provided as a dried form, reconstitution generally is by the addition of a suitable solvent. The solvent, e.g., sterile water or buffer, can optionally be provided in the kit. 
     The kit can include one or more containers for the composition or compositions containing the agents. In some embodiments, the kit contains separate containers, dividers or compartments for the composition and informational material. For example, the composition can be contained in a bottle, vial, or syringe, and the informational material can be contained in a plastic sleeve or packet. In other embodiments, the separate elements of the kit are contained within a single, undivided container. For example, the composition is contained in a bottle, vial or syringe that has attached thereto the informational material in the form of a label. In some embodiments, the kit includes a plurality (e.g., a pack) of individual containers, each containing one or more unit dosage forms (e.g., a dosage form described herein) of the agents. The containers can include a combination unit dosage, e.g., a unit that includes both the polypeptide and the second agent, e.g., in a desired ratio. For example, the kit includes a plurality of syringes, ampules, foil packets, blister packs, or medical devices, e.g., each containing a single combination unit dose. The containers of the kits can be air tight, waterproof (e.g., impermeable to changes in moisture or evaporation), and/or light-tight. 
     The kit optionally includes a device suitable for administration of the composition, e.g., a syringe or other suitable delivery device. The device can be provided pre-loaded with one or both of the agents or can be empty, but suitable for loading. 
     Definitions 
     Terms used in the claims and specification are defined as set forth below unless otherwise specified. 
     It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. 
     As used herein, “about” will be understood by persons of ordinary skill and will vary to some extent depending on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill given the context in which it is used, “about” will mean up to plus or minus 10% of the particular value. 
     As used herein, the term “alanine scanning” refers to a technique used to determine the contribution of a specific wild-type residue to the stability or function(s) (e.g., binding affinity) of a given protein or polypeptide. The technique involves the substitution of an alanine residue for a wild-type residue in a polypeptide, followed by an assessment of the stability or function(s) (e.g., binding affinity) of the alanine-substituted derivative or mutant polypeptide and comparison to the wild-type polypeptide. Techniques to substitute alanine for a wild-type residue in a polypeptide are known in the art. 
     The term “ameliorating” refers to any therapeutically beneficial result in the treatment of a disease state, e.g., infection, lessening in the severity or progression, remission, or cure thereof. 
     As used herein, the term “amino acid” refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid. 
     Amino acids can be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, can be referred to by their commonly accepted single-letter codes. 
     As used herein, an “amino acid substitution” refers to the replacement of at least one existing amino acid residue in a predetermined amino acid sequence (an amino acid sequence of a starting polypeptide) with a second, different “replacement” amino acid residue. An “amino acid insertion” refers to the incorporation of at least one additional amino acid into a predetermined amino acid sequence. While the insertion will usually consist of the insertion of one or two amino acid residues, larger “peptide insertions,” can also be made, e.g., insertion of about three to about five or even up to about ten, fifteen, or twenty amino acid residues. The inserted residue(s) can be naturally occurring or non-naturally occurring as disclosed above. An “amino acid deletion” refers to the removal of at least one amino acid residue from a predetermined amino acid sequence. The following table provides exemplary and preferred substitutions for all 20 amino acids. 
     
       
         
           
               
               
               
               
             
               
                   
                   
               
               
                   
                 Original 
                 Exemplary 
                 Preferred 
               
               
                   
                 Residues 
                 Substitutions 
                 Substitutions 
               
               
                   
                   
               
             
            
               
                   
                 Ala 
                 Val, Leu, Ile 
                 Val 
               
               
                   
                 Arg 
                 Lys, Gln, Asn 
                 Lys 
               
               
                   
                 Asn 
                 Gln 
                 Gln 
               
               
                   
                 Asp 
                 Glu 
                 Glu 
               
               
                   
                 Cys 
                 Ser, Ala 
                 Ser 
               
               
                   
                 Gln 
                 Asn 
                 Asn 
               
               
                   
                 Glu 
                 Asp 
                 Asp 
               
               
                   
                 Gly 
                 Pro, Ala 
                 Ala 
               
               
                   
                 His 
                 Asn, Gln, Lys, Arg 
                 Arg 
               
               
                   
                 Ile 
                 Leu, Val, Met, Ala, Phe, 
                 Leu 
               
               
                   
                   
                 Norleucine 
               
               
                   
                 Leu 
                 Norleucine, Ile, Val, Met, 
                 Ile 
               
               
                   
                   
                 Ala, Phe 
               
               
                   
                 Lys 
                 Arg, 1,4 Diamino-butyric 
                 Arg 
               
               
                   
                   
                 acid, Gln, Asn 
               
               
                   
                 Met 
                 Leu, Phe, Ile 
                 Leu 
               
               
                   
                 Phe 
                 Leu, Val, Ile, Ala, Tyr 
                 Leu 
               
               
                   
                 Pro 
                 Ala 
                 Gly 
               
               
                   
                 Ser 
                 Thr, Ala, Cys 
                 Thr 
               
               
                   
                 Thr 
                 Ser 
                 Ser 
               
               
                   
                 Trp 
                 Tyr, Phe 
                 Tyr 
               
               
                   
                 Tyr 
                 Trp, Phe, Thr, Ser 
                 Phe 
               
               
                   
                 Val 
                 Ile, met, Leu, Phe, Ala, 
                 Leu 
               
               
                   
                   
                 norleucine 
               
               
                   
                   
               
            
           
         
       
     
     The term “antigen presenting cell” or “APC” is a cell that displays foreign antigen complexed with MHC on its surface. T cells recognize this complex using T cell receptor (TCR). Examples of APCs include, but are not limited to, dendritic cells (DCs), peripheral blood mononuclear cells (PBMC), monocytes (such as THP-1), B lymphoblastoid cells (such as C1R.A2, 1518 B-LCL) and monocyte-derived dendritic cells (DCs). Some APCs internalize antigens either by phagocytosis or by receptor-mediated endocytosis. 
     The term “antigen presentation” refers to the process by which APCs capture antigens and enables their recognition by T cells, e.g., as a component of an MHC-I and/or MHC-II conjugate. 
     As used herein, the term “base pair” refers to two nucleobases on opposite complementary nucleic acid strands that interact via the formation of specific hydrogen bonds. As used herein, the term “Watson-Crick base pairing”, used interchangeably with “complementary base pairing”, refers to a set of base pairing rules, wherein a purine always binds with a pyrimidine such that the nucleobase adenine (A) forms a complementary base pair with thymine (T) and guanine (G) forms a complementary base pair with cytosine (C) in DNA molecules. In RNA molecules, thymine is replaced by uracil (U), which, similar to thymine (T), forms a complementary base pair with adenine (A). The complementary base pairs are bound together by hydrogen bonds and the number of hydrogen bonds differs between base pairs. As in known in the art, guanine (G)-cytosine (C) base pairs are bound by three (3) hydrogen bonds and adenine (A)-thymine (T) or uracil (U) base pairs are bound by two (2) hydrogen bonds. Base pairing interactions that do not follow these rules can occur in natural, non-natural, and synthetic nucleic acids and are referred to herein as “non-Watson-Crick base pairing” or alternatively “non-complementary base pairing”. 
     A polypeptide or amino acid sequence “derived from” a designated polypeptide or protein refers to the origin of the polypeptide. Preferably, the polypeptide or amino acid sequence which is derived from a particular sequence has an amino acid sequence that is essentially identical to that sequence or a portion thereof, wherein the portion consists of at least 10-20 amino acids, preferably at least 20-30 amino acids, more preferably at least 30-50 amino acids, or which is otherwise identifiable to one of ordinary skill in the art as having its origin in the sequence. Polypeptides derived from another peptide can have one or more mutations relative to the starting polypeptide, e.g., one or more amino acid residues which have been substituted with another amino acid residue or which has one or more amino acid residue insertions or deletions. 
     A polypeptide can comprise an amino acid sequence which is not naturally occurring. Such variants necessarily have less than 100% sequence identity or similarity with the starting molecule. In certain embodiments, the variant has an amino acid sequence from about 75% to less than 100% amino acid sequence identity or similarity with the amino acid sequence of the starting polypeptide, more preferably from about 80% to less than 100%, more preferably from about 85% to less than 100%, more preferably from about 90% to less than 100% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%) and most preferably from about 95% to less than 100%, e.g., over the length of the variant molecule. 
     In some embodiments, there is one amino acid difference between a starting polypeptide sequence and the sequence derived there from. Identity or similarity with respect to this sequence is defined herein as the percentage of amino acid residues in the candidate sequence that are identical (i.e., same residue) with the starting amino acid residues, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. In certain embodiments, a polypeptide consists of, consists essentially of, or comprises an amino acid sequence selected from a sequence set forth in the sequence listing table. In certain embodiments, a polypeptide includes an amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to an amino acid sequence selected from a sequence set forth in the sequence listing table. In certain embodiments, a polypeptide includes a contiguous amino acid sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous amino acid sequence selected from a sequence set forth in the sequence listing table. In certain embodiments, a polypeptide includes an amino acid sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or any integer within these numbers) contiguous amino acids of an amino acid sequence selected from a sequence set forth in the sequence listing table. 
     In certain embodiments, the polypeptides of the disclosure are encoded by a nucleotide sequence. Nucleotide sequences of the disclosure can be useful for a number of applications, including: cloning, gene therapy, protein expression and purification, mutation introduction, DNA vaccination of a host in need thereof, antibody generation for, e.g., passive immunization, PCR, primer and probe generation, and the like. In certain embodiments, the nucleotide sequence of the invention comprises, consists of, or consists essentially of, a nucleotide sequence selected from a sequence set forth in the sequence listing table. In certain embodiments, a nucleotide sequence includes a nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence selected from a sequence set forth in the sequence listing table. In certain embodiments, a nucleotide sequence includes a contiguous nucleotide sequence at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to a contiguous nucleotide sequence selected from a sequence set forth in the sequence listing table. In certain embodiments, a nucleotide sequence includes a nucleotide sequence having at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, or 500 (or any integer within these numbers) contiguous nucleotides of a nucleotide sequence selected from a sequence set forth in the sequence listing table. 
     It will also be understood by one of ordinary skill in the art that the polypeptides suitable for use in the compositions and methods disclosed herein can be altered such that they vary in sequence from the naturally occurring or native sequences from which they were derived, while retaining the desirable activity of the native sequences. For example, nucleotide or amino acid substitutions leading to conservative substitutions or changes at “non-essential” amino acid residues can be made. Mutations can be introduced by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. 
     The polypeptides suitable for use in the compositions and methods disclosed herein can, in some embodiments, comprise conservative amino acid substitutions at one or more amino acid residues, e.g., at essential or non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a nonessential amino acid residue in a polypeptide is preferably replaced with another amino acid residue from the same side chain family. In some embodiments, a string of amino acids can be replaced with a structurally similar string that differs in order and/or composition of side chain family members. Alternatively, in some embodiments, mutations can be introduced randomly along all or part of a coding sequence, such as by saturation mutagenesis, and the resultant mutants can be incorporated into polypeptides of the disclosure and screened for their ability to induce an immune response. 
     As used herein, the term antigen “cross-presentation” refers to presentation of exogenous protein antigens to T cells via MEW class I and class II molecules on APCs. 
     As used herein, the term “cytotoxic T lymphocyte (CTL) response” refers to an immune response induced by cytotoxic T cells. CTL responses are mediated primarily by CD8 +  T cells. 
     As used herein, the term “effective dose” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term “therapeutically effective dose” is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease. Amounts effective for this use will depend upon the severity of the disorder being treated and the general state of the patient&#39;s own immune system. 
     As used herein, the term “epitope” or “antigenic determinant” refers to a determinant or site on an antigen (e.g., hemagglutinin) to which an antigen-binding protein (e.g., an immunoglobulin, antibody, or antigen-binding fragment) specifically binds. The epitopes of protein antigens can be demarcated into “linear epitopes” and “conformational epitopes”. As used herein, the term “linear epitope” refers to an epitope formed from a contiguous, linear sequence of linked amino acids. Linear epitopes of protein antigens are typically retained upon exposure to chemical denaturants (e.g., acids, bases, solvents, cross-linking reagents, chaotropic agents, disulfide bond reducing agents) or physical denaturants (e.g. thermal heat, radioactivity, or mechanical shear or stress). In some embodiments, an epitope is non-linear, also referred to as an interrupted epitope. As used herein, the term “conformational epitope” or “non-linear epitope” refers to an epitope formed from noncontiguous amino acids juxtaposed by tertiary folding of a polypeptide. Conformational epitopes are typically lost upon treatment with denaturants. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in a unique spatial conformation. In some embodiments, an epitope includes fewer than 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6 or 5 amino acids in a unique spatial conformation. An epitope that is recognized by a T cell receptor is generally referred to as a T-cell epitope. An epitope that is recognized by an antibody or a B cell receptor is generally referred to as a B-cell epitope. Generally, an antibody, or antigen-binding fragment thereof, specific for a particular target molecule will preferentially recognize and bind to a specific epitope on the target molecule within a complex mixture of proteins and/or macromolecules. As used herein, the T and/or B cell epitopes comprises conserved amino acid residues, hypervariable amino acid residues, or combinations thereof of a viral protein. In other embodiments, the T and/or B cell epitopes comprises conserved amino acid residues of the viral proteins. 
     As used herein, the term “epitope mapping” refers to a process or method of identifying the binding site, or epitope, of an antibody, or antigen-binding fragment thereof, on its target protein antigen. Epitope mapping methods and techniques are provided herein. 
     As used herein, the term “fragment” in the context of an amino acid sequence refers to an amino acid sequence comprising a portion of consecutive amino acid residues from a parent sequence. In a specific embodiment, the term refers to an amino acid sequence of 8 to 15, 10 to 20, 2 to 30, 5 to 30, 10 to 60, 25 to 100, 150 to 300 or more consecutive amino acid residues from a parent sequence. In another embodiment, the term refers to an amino acid sequence of at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 125, 150, 175, or 200 consecutive amino acid residues of a parent sequence. 
     As used herein, the term “hemagglutinin protein” (or “HA protein’) refers to a protein or polypeptide whose amino acid sequence includes at least one characteristic sequence of an influenza type A or B HA. A wide variety of HA sequences from influenza isolates are known in the art; indeed, the National Center for Biotechnology Information (NCBI) maintains a database (http://www.ncbi.nlm.nih.gov/genomes/FLU/) that, as of the filing of the present application includes approximately 40,000 HA sequences (for type A and B viruses). Those of ordinary skill in the art, referring to this database, can readily identify sequences that are characteristic of HA polypeptides generally, and/or of particular HA polypeptides (e.g., H1, H2, H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15, or H16 polypeptides; or of HAs that mediate infection of particular hosts, e.g., human, avian, seal etc.). For example, in some embodiments, an HA polypeptide includes one or more characteristic sequence elements found between about residues 97 and about 185, about 324 and about 340, about 96 and about 100, and/or about 130 and about 230 of an HA protein found in a natural isolate of an influenza virus. 
     As used herein, “conserved” or “highly conserved regions” are influenza virus sequences from different strains, or consensus sequences, which have been employed to provide an antigen with broad protective properties. Sequence alignments are relied on to yield a “consensus” sequence, where many genetic sequences are incorporated into a single sequence. A consensus sequence may thus minimize the genetic distance between vaccine strains and viruses and so may elicit more cross-reactive immune responses than an immunogen derived from any single influenza virus. 
     As used herein, the term “hybridization” refers to the process of a first single-stranded nucleic acid, or a portion, fragment, or region thereof, annealing to a second single-stranded nucleic acid, or a portion, fragment, or region thereof, either from the same or separate nucleic acid molecules, mediated by Watson-Crick base pairing to form a secondary and/or tertiary structure. Complementary strands of linked nucleobases able to undergo hybridization can be from either the same or separate nucleic acids. Due to the thermodynamically favorable hydrogen bonding interaction between complementary base pairs, hybridization is a fundamental property of complementary nucleic acid sequences. Such hybridization of nucleic acids, or a portion or fragment thereof, may occur with “near” or “substantial” complementarity, as well as with exact complementarity. 
     As used herein, the term “hypervariable” refers to amino acid residues and/or protein regions that are abundant and surface exposed, and is a primary target of the immune response against the standard influenza vaccine. The immune response to influenza is overwhelmingly driven against the hypervariable regions of the virus. Thus, in traditional influenza vaccination or natural infections, the protective immune response is overwhelmingly directed at a limited number of continuously evolving, strain-specific, primary antigenic determinants on the surface of the influenza proteins, and there is minimal cross reaction with or protection against other serotypes of influenza. 
     As used herein, the term “non-hypervariable” or “hypervariable-substitute” refers to an amino acid residue that is substituted for a hypervariable amino acid residue, wherein the substitution eliminates or substantially reduces a strain-specific immune response (e.g., antibody response) against the region containing the hypervariable amino acid residue. In some embodiments, the non-hypervariable residue is one that when substituted for the hypervariable amino acid residue, provides surface epitope with reduced antigenicity. In some embodiments, the non-hypervariable residue is selected from is a nonpolar, aliphatic R group amino acid, e.g., alanine, glycine, valine, leucine, isoleucine, and methionine. In some embodiments, the non-hypervariable residue is conserved at the same position in a plurality of influenza strains. 
     As used herein, the term “immune response” refers to a response of a cell of the immune system, such as a B cell, T cell, dendritic cell, macrophage or polymorphonucleocyte, to a stimulus such as an antigen or vaccine. An immune response can include any cell of the body involved in a host defense response, including for example, an epithelial cell that secretes an interferon or a cytokine. An immune response includes, but is not limited to, an innate and/or adaptive immune response. As used herein, a protective immune response refers to an immune response that protects a subject from infection (prevents infection or prevents the development of disease associated with infection). Methods of measuring immune responses are well known in the art and include, for example, measuring proliferation and/or activity of lymphocytes (such as B or T cells), secretion of cytokines or chemokines, inflammation, antibody production and the like. 
     The terms “inducing an immune response” and “enhancing an immune response” are used interchangeably and refer to the stimulation of an immune response (i.e., either passive or adaptive) to a particular antigen. The term “induce” as used with respect to inducing CDC or ADCC refer to the stimulation of particular direct cell killing mechanisms. 
     As used herein, the term “influenza strains” is based upon, e.g., the ability of influenza to agglutinate red blood cells (RBCS or erythrocytes). Influenza strains are typically categorized based upon their immunologic or antigenic profile. An HA1 titer is typically defined as the highest dilution of a serum that completely inhibits hemagglutination. See, e.g., Schild, et al., Bull. Wld Hlth Org., 1973, 48:269-278, etc. Those of skill in the art will be quite familiar with categorization and classification of influenza into strains and the methods to do so. Antibodies specific for particular influenza strains can bind to the virus and, thus, prevent such agglutination. Assays determining strain types based on such inhibition are typically known as hemagglutinin inhibition assays (HI assays or HA1 assays) and are standard and well known methods in the art to characterize influenza strains. Of course, those of skill in the art will be familiar with other assays, e.g., ELISA, indirect fluorescent antibody assays, immunohistochemistry, Western blot assays, etc. with which to characterize influenza strains and the use of and discussion herein of HI assays should not be necessarily construed as limiting. 
     As used herein “influenza types and subtypes” are influenza A and B virus typically associated with influenza outbreaks in human populations. The type A viruses are categorized into subtypes based upon differences within their hemagglutinin and neuraminidase surface glycoprotein antigens. Hemagglutinin in type A viruses has 14 known subtypes and neuraminidase has 9 known subtypes. In humans, currently only about 3 different hemagglutinin and 2 different neuraminidase subtypes are known, e.g., H1, H2, H3, N1, and N2. In particular, two major subtypes of influenza A have been active in humans, namely, H1N1 and H3N2. H1N2, however has recently been of concern. 
     As used herein, the term “influenza vaccine” refers to an immunogenic composition capable of stimulating an immune response, administered for the prevention, amelioration, or treatment of influenza virus infection. An influenza vaccine may include, for example, attenuated or killed influenza virus, virus-like particles (VLPs) and/or antigenic polypeptides (e.g., the engineered hemagglutinins described herein) or DNA derived from them, or any recombinant versions of such immunogenic materials. 
     As used herein, a subject “in need of prevention,” “in need of treatment,” or “in need thereof,” refers to one, who by the judgment of an appropriate medical practitioner (e.g., a doctor, a nurse, or a nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals), would reasonably benefit from a given treatment (such as treatment with an immunogenic composition). 
     The term “in vivo” refers to processes that occur in a living organism. 
     As used herein, “immunogenic composition” refers to a composition that comprises at least one antigen which elicits an immunological response in the host to which the immunogenic composition is administered. Such immunological responses can be a cellular and/or antibody-mediated immune response to the immunogenic composition. 
     As used herein, the terms “linked,” “operably linked,” “fused”, or “fusion”, are used interchangeably. These terms refer to the joining together of two more elements or components or domains, by whatever means including chemical conjugation or recombinant means. Methods of chemical conjugation (e.g., using heterobifunctional crosslinking agents) are known in the art. 
     As used herein, “MHC molecules” refers to two types of molecules, MHC class I and MHC class II. MHC class I molecules present antigen to specific CD8+ T cells and MHC class II molecules present antigen to specific CD4+ T cells. Antigens delivered exogenously to APCs are processed primarily for association with MHC class II. In contrast, antigens delivered endogenously to APCs are processed primarily for association with MHC class I. 
     As used herein, the terms “NA” and “neuraminidase” refer to any influenza neuraminidase, such as an influenza A neuraminidase, an influenza B neuraminidase, or an influenza C neuraminidase. A typical neuraminidase comprises domains known to those of skill in the art including a cytoplasmic domain, a transmembrane domain, a stalk domain, and a globular head domain. As used herein, the terms “neuraminidase” and “NA” encompass neuraminidase polypeptides that are modified by post-translational processing such as disulfide bond formation, glycosylation (e.g., N-linked glycosylation), 
     As used herein, the term “naturally-occurring” as applied to an object refers to the fact that an object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring. 
     As used herein, the term “nucleic acid” is used in its broadest sense and encompasses any compound and/or substance that includes a polymer of nucleotides. These polymers are often referred to as polynucleotides. Exemplary nucleic acids or polynucleotides of the disclosure include, but are not limited to, ribonucleic acids (RNAs), deoxyribonucleic acids (DNAs), DNA-RNA hybrids, RNAi-inducing agents, RNAi agents, siRNAs, shRNAs, miRNAs, antisense RNAs, ribozymes, catalytic DNA, RNAs that induce triple helix formation, threose nucleic acids (TNAs), glycol nucleic acids (GNAs), peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNA having a β-D-ribo configuration, α-LNA having an α-L-ribo configuration (a diastereomer of LNA), 2′-amino-LNA having a 2′-amino functionalization, and 2′-amino-α-LNA having a 2′-amino functionalization) or hybrids thereof. A polynucleotide can also contain one or more modified bases or DNA or RNA backbones modified for stability or for other reasons. “Modified” bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus, “polynucleotide” embraces chemically, enzymatically, or metabolically modified forms. 
     As used herein, the term “nucleoside” refers to a compound containing a sugar molecule (e.g., a ribose in RNA or a deoxyribose in DNA), or derivative or analog thereof, covalently linked to a nucleobase (e.g., a purine or pyrimidine), or a derivative or analog thereof (also referred to herein as “nucleobase”), but lacking an internucleoside linking group (e.g., a phosphate group). As used herein, the term “nucleotide” refers to a nucleoside covalently bonded to an internucleoside linking group (e.g., a phosphate group), or any derivative, analog, or modification thereof that confers improved chemical and/or functional properties (e.g., binding affinity, nuclease resistance, chemical stability) to a nucleic acid or a portion or segment thereof. 
     A nucleic acid is “operably linked” when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence. With respect to transcription regulatory sequences, operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame. For switch sequences, operably linked indicates that the sequences are capable of effecting switch recombination. 
     As used herein, “parenteral administration,” “administered parenterally,” and other grammatically equivalent phrases, refer to modes of administration other than enteral and topical administration, usually by injection, and include, without limitation, intravenous, intranasal, intraocular, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, intracerebral, intracranial, intracarotid and intrasternal injection and infusion. 
     As used herein, the term “patient” includes human and other mammalian subjects that receive either prophylactic or therapeutic treatment. 
     The term “percent identity,” in the context of two or more nucleic acid or polypeptide sequences, refer to two or more sequences or subsequences that have a specified percentage of nucleotides or amino acid residues that are the same, when compared and aligned for maximum correspondence, as measured using one of the sequence comparison algorithms described below (e.g., BLASTP and BLASTN or other algorithms available to persons of skill) or by visual inspection. Depending on the application, the “percent identity” can exist over a region of the sequence being compared, e.g., over a functional domain, or, alternatively, exist over the full length of the two sequences to be compared. For sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculates the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters. 
     Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith &amp; Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman &amp; Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson &amp; Lipman, Proc. Nat&#39;l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by visual inspection (see generally Ausubel et al., infra). In some embodiments, alignment of sequences is conducted by the Dawn method (Ricke, D. O. &amp; Shcherbina, A. 2015  IEEE High Performance Extreme Computing Conference  ( HPEC ), doi:10.1109.HPEC.2015.7322463 (2015)). 
     One example of an algorithm that is suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol. 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information website. 
     As generally used herein, “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues, organs, and/or bodily fluids of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit/risk ratio. 
     As used herein, a “pharmaceutically acceptable carrier” refers to, and includes, any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The compositions can include a pharmaceutically acceptable salt, e.g., an acid addition salt or a base addition salt (see, e.g., Berge et al. (1977)  J Pharm Sci  66:1-19). 
     As used herein, the terms “polypeptide,” “peptide”, and “protein” are used interchangeably to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. 
     As used herein, the term “preventing” when used in relation to a condition, refers to administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition. 
     As used herein, the term “purified” or “isolated” as applied to any of the proteins described herein refers to a polypeptide that has been separated or purified from components (e.g., proteins or other naturally-occurring biological or organic molecules) which naturally accompany it, e.g., other proteins, lipids, and nucleic acid in a prokaryote expressing the proteins. Typically, a polypeptide is purified when it constitutes at least 60 (e.g., at least 65, 70, 75, 80, 85, 90, 92, 95, 97, or 99) %, by weight, of the total protein in a sample. 
     As used herein, the term “recombinant influenza vaccine” refers to influenza-specific immunogenic composition comprising one or more of engineered influenza viral proteins described herein (e.g., hemaglutinin, neuraminidase), including, but not limited to whole influenza virus, subunit preparations thereof, virus-like particles, recombinant protein (i.e., preparations composed of recombinant HA purified to varying degree), and DNA- and viral vector-based vaccines. Recombinant influenza vaccines as described herein may optionally contain one or more adjuvants. 
     As used herein, the term “subject” includes any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat a subject with an immune disorder. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc. 
     As used herein, the term “substantially” refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. 
     For nucleic acids, the term “substantial homology” indicates that two nucleic acids, or designated sequences thereof, when optimally aligned and compared, are identical, with appropriate nucleotide insertions or deletions, in at least about 80% of the nucleotides, usually at least about 90% to 95%, and more preferably at least about 98% to 99.5% of the nucleotides. Alternatively, substantial homology exists when the segments will hybridize under selective hybridization conditions, to the complement of the strand. 
     The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=# of identical positions/total # of positions×100), taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm, as described in the non-limiting examples below. 
     The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ( J. Mol. Biol . (48):444-453 (1970)) algorithm which has been incorporated into the GAP program in the GCG software package (available at www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. 
     The nucleic acid and protein sequences of the present disclosure can further be used as a “query sequence” to perform a search against public databases to, for example, identify related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990)  J. Mol. Biol.  215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997)  Nucleic Acids Res.  25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See www.ncbi.nlm.nih.gov. 
     The term “T cell” refers to a type of white blood cell that can be distinguished from other white blood cells by the presence of a T cell receptor on the cell surface. There are several subsets of T cells, including, but not limited to, T helper cells (a.k.a. T H  cells or CD4 +  T cells) and subtypes, including T H 1, T H 2, T H 3, T H 17, T H 9, and T FH  cells, cytotoxic T cells (i.e., Tc cells, CD8 +  T cells, cytotoxic T lymphocytes, T-killer cells, killer T cells), memory T cells and subtypes, including central memory T cells (T CM  cells), effector memory T cells (T EM  and T EMRA  cells), and resident memory T cells (T RM  cells), regulatory T cells (a.k.a. T reg  cells or suppressor T cells) and subtypes, including CD4 +  FOXP3 +  T reg  cells, CD4 +  FOXP3 −  T reg  cells, Tr1 cells, Th3 cells, and T reg 17 cells, natural killer T cells (a.k.a. NKT cells), mucosal associated invariant T cells (MAITs), and gamma delta T cells (γδ T cells), including Vγ9/Vδ2 T cells. Any one or more of the aforementioned or unmentioned T cells may be the target cell type for a method of use of the invention. 
     As used herein, the terms “T cell activation” or “activation of T cells” refers to a cellular process in which mature T cells, which express antigen-specific T cell receptors on their surfaces, recognize their cognate antigens and respond by entering the cell cycle, secreting cytokines or lytic enzymes, and initiating or becoming competent to perform cell-based effector functions. T cell activation requires at least two signals to become fully activated. The first occurs after engagement of the T cell antigen-specific receptor (TCR) by the antigen-major histocompatibility complex (MEW), and the second by subsequent engagement of co-stimulatory molecules (e.g., CD28). These signals are transmitted to the nucleus and result in clonal expansion of T cells, upregulation of activation markers on the cell surface, differentiation into effector cells, induction of cytotoxicity or cytokine secretion, induction of apoptosis, or a combination thereof. 
     As used herein, the term “T cell-mediated response” refers to any response mediated by T cells, including, but not limited to, effector T cells (e.g., CD8 +  cells) and helper T cells (e.g., CD4 +  cells). T cell mediated responses include, for example, T cell cytotoxicity and proliferation. 
     As used herein, the terms “therapeutically effective amount” or “therapeutically effective dose,” or similar terms used herein are intended to mean an amount of an agent (e.g., a nucleic acid molecule) that will elicit the desired biological or medical response (e.g., an improvement in one or more symptoms of an infection). 
     The terms “treat,” “treating,” and “treatment,” as used herein, refer to therapeutic or preventative measures described herein. The methods of “treatment” employ administration to a subject, in need of such treatment, a human antibody of the present disclosure, for example, a subject in need of an enhanced immune response against a particular antigen or a subject who ultimately may acquire such a disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. 
     As used herein, the term “vaccination” refers to the administration of an immunogenic composition intended to generate an immune response, for example to a disease-causing agent such as influenza. Vaccination can be administered before, during, and/or after exposure to a disease-causing agent, and/or to the development of one or more symptoms, and in some embodiments, before, during, and/or shortly after exposure to the agent. Vaccines may elicit both prophylactic (preventative) and therapeutic responses. Methods of administration vary according to the vaccine, but may include inoculation, ingestion, inhalation or other forms of administration. Inoculations can be delivered by any of a number of routes, including parenteral, such as intravenous, subcutaneous or intramuscular. Vaccines may be administered with an adjuvant to boost the immune response. In some embodiments, vaccination includes multiple administrations, appropriately spaced in time, of an immunogenic composition. 
     Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the presently disclosed methods and compositions. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. 
     EXAMPLES 
     Example 1: Identification of Residues in H1N1 
     To identify highly conserved amino acid residues between strains of a particular type and/or subtype of influenza virus, amino acid sequences were obtained and aligned. Specifically, the Dawn method, described in Ricke, D. O &amp; Shcherbina, A.,  IEEE High Performance Extreme Computing Conference  ( HPEC ), doi:1031109/HPEC.2015.7322463 (2015), herein incorporated by reference, was used to align 52,443 influenza A H1N1 hemagglutinin amino acid sequences and 51,784 influenza A H1N1 neuraminidase amino acid sequences.  FIG.  1    shows an alignment of a section of amino acid residues in the H1N1 HA protein from strains in years 2009-2019. 
     SEQ ID NO: 1 provides the amino acid sequence for hemagglutinin from the A/Michigan/45/2015 H1N1 strain. SEQ ID NO: 2 provides the amino acid sequence for neuraminidase from the A/Michigan/45/2015 strain H1N1 strain. 
     Highly variable residues were identified for both proteins, along with residues having low variability. The following sequence for hemagglutinin indicates hypervariable residues in bold and conserved regions are underlined. 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 1) 
               
               
                   
                 MKAILVVLLYTF TT ANADTLCI GYHANNST DT 
               
               
                   
               
               
                   
                 VDTVLEK NVTVTHS VNLLE DK HNGKLCKLRGV 
               
               
                   
               
               
                   
                 APLHLGKCNIAGW I   LGNPEC ESLSTAS SWSYI   
               
               
                   
               
               
                   
                   VE TSNSDNGTCYPGDFI N YEELREQLSSVSSF 
               
               
                   
               
               
                   
                 ERFEIFPK T SSWPNHD S N K GVTAACP H AG AKS   
               
               
                   
               
               
                   
                 FY K NLIWLVKKGNSYPK LNQ SYIN D K G KEVLV 
               
               
                   
               
               
                   
                 LW G IHHP S T TA DQQSLYQNAD A YVFVG T S R YS 
               
               
                   
               
               
                   
                   K KF K PEIA T RPKVR D Q E GRMNYYWT L VEPGD K   
               
               
                   
               
               
                   
                 ITFEATGNLVVPRYAF TME R NA GSGIIISD TP   
               
               
                   
               
               
                   
                 VHDC N TTCQTP E GAINTSLPFQNIHP I TIG K C 
               
               
                   
               
               
                   
                 PKYVKSTKLR L ATGLRN V PSI QSRGLFGAIAG   
               
               
                   
               
               
                   
                   F I EGGWTG M V DGWYGYHHQNE QGSGYAAD   L KS 
               
               
                   
               
               
                   
                 TQNAID K   ITNKVNS VIEKMNTQFTAVGKEF N H 
               
               
                   
               
               
                   
                 LE K RIENLNKKVDDGFLDI WTYNAELL V LLEN   
               
               
                   
               
               
                   
                   ER TLDYHDSNVKNLYEKVR N QLKNNAKEIGN G   
               
               
                   
               
               
                   
                   CFEFYH KCD NT CMESVKNGTYDYPKYSEEAKL 
               
               
                   
               
               
                   
                 NRE K IDGVKLESTRIYQILAIYSTVASSLVL V   
               
               
                   
               
               
                   
                 VSLGAISFWMCSNGSLQCRICI 
               
            
           
         
       
     
       FIG.  2    provides the amino acid sequence and nucleic acid sequences for the H1N1 hemagglutinin protein with the nucleic acid sequences underlined for highly conserved regions and shown by boxes for the hypervariable amino acid residues. 
     The following sequence for neuraminidase indicates hypervariable residues in bold and conserved regions are underlined. 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 2) 
               
               
                   
                   MNPNQKIITIGS   ICMT IG MAN L I LQIGNIISI 
               
               
                   
               
               
                   
                 W V SHSIQ I G N Q SQIET CNQ SV ITYEN N TWVN Q   
               
               
                   
               
               
                   
                 TYVNI S NIN FA A GQSVVS V K L A GNSSLC PV SG 
               
               
                   
               
               
                   
                 WAIY S KDNS V   RIGSKGDVFVIREPFISC S P LE 
               
               
                   
               
               
                   
                 C R   TFFLTQGALLNDKHSNGT   I   KDRSPYR   T LMS 
               
               
                   
               
               
                   
                 CP I GE V PSPYNS R   FESVAWSASACHDG   IN   WLT   
               
               
                   
               
               
                   
                   IGISGPD   S   GAVAVLKY NGIIT D TIKSW RNN   IL   
               
               
                   
               
               
                   
                   RTQESEC   A C V NGSCFT I MIDGPS DGQ ASYKIF 
               
               
                   
               
               
                   
                   R IEKGK II KS V E MK APN YH Y EECSCYPD   SSEI   
               
               
                   
               
               
                   
                   T   CVCRDNWHGSNRPWVSFNQNL   E YQ M GYICSG 
               
               
                   
               
               
                   
                   V TGDNPRP N D KT GSC G PV SSN GA N GVKGFS F K 
               
               
                   
               
               
                   
                 Y G   NGVWIGRTKS   ISS R K   GFEMIWDPNGWT   G TD 
               
               
                   
               
               
                   
                   NK FS I KQD I VGI NE   WSGYSGSFVQHPELTGL   D   
               
               
                   
               
               
                   
                 CI RPCFWVEL   I RG R P E ENTI WTSGSSISFCGV   
               
               
                   
               
               
                   
                   NS D TVG W SWPDGAELPF TIDK 
               
            
           
         
       
     
     Example 2: Identification of Residues in H3N2 
     Using the same method described in Example 1, hypervariable amino acid residues and highly conserved regions were identified in the hemagglutinin and neuraminidase proteins of H3N2. Specifically, 42,653 hemagglutinin amino acid sequences and 29,491 neuraminidase amino acid sequences were aligned using the Dawn method. 
     SEQ ID NO: 3 provides the amino acid sequence for hemagglutinin from the A/Mississippi/27/2013 H3N2 strain. SEQ ID NO: 4 provides the amino acid sequence for neuraminidase from the Neuraminidase A/Miyagi/N1289/2005 H3N2 strain. 
     Highly variable residues were identified for both proteins, along with residues having low variability. The following sequence for hemagglutinin indicates hypervariable residues in bold and conserved regions are underlined. 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 3) 
               
               
                   
                 MKTIIA L S Y ILCL V F A Q KL PP Y GN ST AT LCLG   
               
               
                   
               
               
                   
                   HHA LPNGT I VKTIT N D R IEVTNATELVQ N SS I   
               
               
                   
               
               
                   
                 G E IC D SPH Q ILDG E NCTLIDALLGDP Q CD G FQ 
               
               
                   
               
               
                   
                 N KK WDLFVER SK A Y SNCYPY D VPDYASLRSLV 
               
               
                   
               
               
                   
                 ASSGTLEF NN E S FNW T GV T Q N G T S SA C I R R S N   
               
               
                   
               
               
                   
                   S SFFSRLNWL THLNFK YP A INVIMPN NEQ FDK 
               
               
                   
               
               
                   
                 LYIWGVHHP G TD KD Q IF LY AQSS GR IT VSTKR 
               
               
                   
               
               
                   
                 SQQ A VIPNIGSRP RI R NIP S R ISIYWTIVKPG 
               
               
                   
               
               
                   
                 DILLINST GNLIAPRGYF KI RS GKSSIMRSDA 
               
               
                   
               
               
                   
                 PI GK C K S E CITPNGSIPNDKPFQNVN R ITYGA 
               
               
                   
               
               
                   
                 CPRYVK QS T LKLATGMRN VPE K QTRGI FGAIA   
               
               
                   
               
               
                   
                   GFIENGWEG   MV DGWYGFRHQNSEG R GQAADLK 
               
               
                   
               
               
                   
                 STQAAI D QI N GKLNR L I G KTNEKFHQIEKEFS 
               
               
                   
               
               
                   
                 EVEGR I QDLEKYVEDTKIDLWSYNAELLVALE 
               
               
                   
               
               
                   
                 NQHTI DLTDSEM NKLFE K T KK Q LRENAED MGN 
               
               
                   
               
               
                   
                 GCFKIYHKCDNAC IG SIRN G TYDH NV YR D EAL 
               
               
                   
               
               
                   
                 NNRFQIKGV E LKSGYKDWILWISFAISCFLLC 
               
               
                   
               
               
                   
                   VA IKGFIMWACQKG NIRCN IRCNICI 
               
            
           
         
       
     
     The following sequence for neuraminidase indicates hypervariable residues in bold and conserved regions are underlined. 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 4) 
               
               
                   
                 MNPNQKIITIGSVSL T I S TICF F MQ I AIL I TT 
               
               
                   
               
               
                   
                 VTLHFKQ YEFNSPP NNQV ML CEP T IIERN I TE 
               
               
                   
               
               
                   
                 IVYLTNTT I E K EICPK LA EYRKWSKPQC N IT G   
               
               
                   
               
               
                   
                   FAPFS K DNS IRLSAGGDIWVTREPYVSCD PD K 
               
               
                   
               
               
                   
                 CY QFALGQGTT   L NN V HSN D I VR DRTP Y RTLLM 
               
               
                   
               
               
                   
                   N ELGVPFHLGT K QVC I   AWSSSSC HDGKAWLHV 
               
               
                   
               
               
                   
                 C V TG D DKNATASFIY N GRLVDS IV SWS KE I LR   
               
               
                   
               
               
                   
                   TQESEC VCINGTCTVVMTDG S A S G K ADTKILF 
               
               
                   
               
               
                   
                 IEEGKI V H T S T LSGSAQHV EECSCYP RYPGVR 
               
               
                   
               
               
                   
                 CV CRDNW K GSNRP I V DIN I KD Y SI V SSYV CSG   
               
               
                   
               
               
                   
                   LVGDTPR K N D SS SSS H C LD PNNE E G G H GVKGW   
               
               
                   
               
               
                   
                   AFD   D GNDVWMGRTI S E KL RS G YETFXV IE GWS 
               
               
                   
               
               
                   
                   NPN SKLQ I NRQVIV DRGN RSGYSGIFSVEGK S   
               
               
                   
               
               
                   
                 CI NRCFYVELIRG RK E ET E V L WTSNSIV VFCG   
               
               
                   
               
               
                   
                   TSGTYG T GSWPDGA D I NLM P I 
               
            
           
         
       
     
     Example 3: Identification of Residues in Influenza B 
     Using the same method described in Example 1, hypervariable amino acid residues and highly conserved regions were identified in the hemagglutinin and neuraminidase proteins of influenza B. Specifically, 20,906 hemagglutinin amino acid sequences and 14,546 neuraminidase amino acid sequences were aligned using the Dawn method. 
     SEQ ID NO: 5 provides the amino acid sequence for hemagglutinin from the B/Brisbane/60/2008 influenza B strain. SEQ ID NO: 6 provides the amino acid sequence for neuraminidase from the B/Wisconsin/05/2016 influenza B strain. 
     Highly variable residues were identified for both proteins, along with residues having low variability. The following sequence for hemagglutinin indicates hypervariable residues in bold and conserved regions are underlined. 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 5) 
               
               
                   
                 MKAIIVLLMVVTSNADRICTGITSSNSPHV VK   
               
               
                   
               
               
                   
                   TATQGEVNVTG VIPLTTTPTKS H FANLKGT E T 
               
               
                   
               
               
                   
                 RGKLCP K C L   NCTDLDVAL GRP K C T G KI PSA RV   
               
               
                   
               
               
                   
                 SILHEVRPV TSGCFPIMHDRTKIRQL   P   NLLRG   
               
               
                   
               
               
                   
                   YE   HI RLST H NVI N AE N APGGPY KI GTSGSCPN 
               
               
                   
               
               
                   
                   I T NGN GFFA TMAWAVP   KN D KN K T ATN PL T I EV 
               
               
                   
               
               
                   
                 PY I C T EGEDQITVWGFHSDD E TQM AK LYGDS K   
               
               
                   
               
               
                   
                 PQKFTSSANGVTTHYVSQIG G FP N QT EDGGLP   
               
               
                   
               
               
                   
                   QSGRIVVDYM   V QK S GKTGTI T YQRG I LLPQKV 
               
               
                   
               
               
                   
                 WCASGRSKVIKGS LPLIGEADCLHE   K   YGGLNK   
               
               
                   
               
               
                   
                   SKPYYTG   E HAKAIGNC PIWVKTPL KLANGTKY 
               
               
                   
               
               
                   
                 RPPAKLLKER GFFGAIAGFLEGGWEGM I AGWH   
               
               
                   
               
               
                   
                   GYTSHGAHG V AVAADLKSTQEA IN KITKNLNS   
               
               
                   
               
               
                   
                   LSELE V KNLQRLS GAMDELHN EILELDEKVDD   
               
               
                   
               
               
                   
                 
                   LRADTISSQIELAVLLSNEGIINSEDEHLLAL 
                 
               
               
                   
               
               
                   
                   ERKLKKMLGPSA V E   IGNGCFETKHKCNQTCLD   
               
               
                   
               
               
                   
                   R IAAGTF D   AGEFSLPTFDSLNITAASL NDDGL 
               
               
                   
               
               
                   
                 DN HTILLYYSTAASSLAVTLM   I AIF V VYMVSR 
               
               
                   
               
               
                   
                 DNVSCSICL 
               
            
           
         
       
     
     The following sequence for neuraminidase indicates hypervariable residues in bold and conserved regions are underlined. 
     
       
         
           
               
               
            
               
                   
                 (SEQ ID NO: 6) 
               
               
                   
                 MLPSTIQTLTLFLTSGGVLLSLYVSASLSYLL 
               
               
                   
               
               
                   
                 YSDILLKFS P TE I TAP T MPLDCANASNV Q AVN 
               
               
                   
               
               
                   
                   R S AT KGVT LL LLPEPEWTYPRLSCPGSTFQK A   
               
               
                   
               
               
                   
                   LLISPHRFGE   T KGNSAPLIIREPF V ACGP N EC 
               
               
                   
               
               
                   
                 K HFALTHYAAQPG GYYNGTR G   DRNKLRHL ISV 
               
               
                   
               
               
                   
                 KLGKIPTVENS I FHMA AWSGSACHDG   K EWTYI 
               
               
                   
               
               
                   
                 GVDGPD N NALLK V   KYGEAYTDTYHSY A NN I LR   
               
               
                   
               
               
                   
                   TQESACNCI GG N CYLMITDG S ASG V SE CRFLK   
               
               
                   
               
               
                   
                   IREGR IIKEIFPTGRVK HTEECTCGFA SNKTI 
               
               
                   
               
               
                   
                 ECACRDN R   YTAKRPFVKL NVETDTAEIRLMCT 
               
               
                   
               
               
                   
                   D TYLDTPRP N DGSITGPCES D G DK GSGGI KGG   
               
               
                   
               
               
                   
                   FVHQR M K SKIG RWYSRT MS K T E RMGMGLYVKY 
               
               
                   
               
               
                   
                   G GDPW A DSDAL AF SGVM VS M K   EPGWYSFGFE I 
               
               
                   
               
               
                   
                 KDKKCDVPCIGI EMVHDGG K E TWHSAATAIYC 
               
               
                   
               
               
                   
                 LMGSGQLLWDTVTGV D M A L 
               
            
           
         
       
     
     Example 4: Production of B Cell Immune Response 
     To determine whether the hypervariable residues identified in HA and NA proteins as described in Examples 1-3, alanine scanning of each residue and combinations of residues is performed.  FIG.  3    shows an exemplary sequence wherein each hypervariable residue identified in the H1N1 HA protein described in Example 1 is replaced with an alanine. 
     Each mutated HA and NA protein comprising an alanine is subjected to in vitro and in vivo testing to determine what mutations will elicit an immune response to highly conserved amino acid regions and provide protection against influenza infection. 
     In one study, mutated HA or NA proteins, or combinations thereof, are administered to a subject (e.g., a pig). Serum, BAL, and TBLN samples are collected and tested in ELISA or neutralization assays to determine antibodies titers to the highly conserved amino acid regions. Generation of such antibodies indicates the immune response has been directed to such regions and thus the mutated proteins are suitable as a universal influenza vaccine. In another study, after administration of the mutated HA or NA proteins, or combinations thereof, subjects are challenged with various influenza virus strains and infection levels are monitored. The ability of the mutated HA or NA proteins to prevent infection by different influenza strains indicates the mutated proteins are suitable as a universal influenza vaccine. 
     Example 5: Production of T Cell Immune Response 
     To determine whether the highly conserved regions of amino acids identified in Examples 1-3 are capable of eliciting a T cell immune response, immunogenic compositions comprising polypeptides having amino acid sequences of the conserved regions are generated and administered to subjects. In some studies, polypeptides comprising different regions are combined by operably linking the polypeptides together. 
     PBMCS are collected at various time points after immunization, and are cultured with 15-mer peptide pools encompassing the sequence of the polypeptide or operably linked polypeptides. T cell activation is measured by assessing proliferation, production of cytokines and/or the cytotoxic ability of the cells against different influenza virus strains. The ability of the polypeptide or operably linked polypeptides to induce cytokine induction or induce killing of different strains by T cells indicates the polypeptide(s) are suitable as a universal influenza vaccine. 
     In another study, polypeptide(s) or operably linked polypeptides are administered to a subject (e.g., a pig) which is then challenged with various influenza virus strains and infection levels are monitored. The ability of the polypeptide(s) to prevent infection by different influenza strains indicates they are suitable as a universal influenza vaccine. 
     Example 6: Therapeutic Efficacy of Nucleic Acids Targeting Highly Conserved Regions 
     To determine whether targeting the highly conserved regions identified in Examples 1-3 provides therapeutic efficacy, nucleic acid molecules (e.g., siRNA or miRNA) having substantial complementarity to nucleotide sequences encoding the highly conserved regions are generated. 
     In one study, a nucleic acid molecule targeting a highly conserved region is contacted with various influenza virus strains. Ability of the viruses to infect cells is assessed after contact. If the nucleic acid molecule disrupts the life cycle of the virus and prevents infection, the nucleic acid molecule may be suitable for treating influenza infection. 
     
       
         
           
               
            
               
                   
               
               
                 SEQUENCE LISTING TABLE 
               
            
           
           
               
               
               
            
               
                 SEQ 
                   
                   
               
               
                 ID 
                   
                   
               
               
                 NO 
                 Description 
                 Sequence 
               
               
                   
               
            
           
           
               
               
               
            
               
                 1 
                 H1N1 
                 MKAILVVLLYTF TT ANADTLCI GYHANNST DT 
               
               
                   
                 Hemagglutinin 
                 VDTVLEK NVTVTHS VNLLE DK HNGKLCKLRGV 
               
               
                   
                 A/Michigan/ 
                 APLHLGKCNIAGW I   LGNPEC ESLSTAS SWSYI   
               
               
                   
                 45/2015 strain 
                   VE TSNSDNGTCYPGDFI N YEELREQLSSVSSF 
               
               
                   
                 (amino acid) 
                 ERFEIFPK T SSWPNHD S N K GVTAACP H AG AKS   
               
               
                   
                 GenBank: 
                 FY K NLIWLVKKGNSYPK LNQ SYIN D K G KEVLV 
               
               
                   
                 MK622940.1 
                 LW G IHHP S T TA DQQSLYQNAD A YVFVG T S R YS 
               
               
                   
                   
                   K KF K PEIA T RPKVR D Q E GRMNYYWT L VEPGD K   
               
               
                   
                   
                 ITFEATGNLVVPRYAF TME R NA GSGIIISD TP   
               
               
                   
                   
                 VHDC N TTCQTP E GAINTSLPFQNIHP I TIG K C 
               
               
                   
                   
                 PKYVKSTKLR L ATGLRN V PSI QSRGLFGAIAG   
               
               
                   
                   
                   F I EGGWTG M V DGWYGYHHQNE QGSGYAAD   L KS 
               
               
                   
                   
                 TQNAID K   ITNKVNS VIEKMNTQFTAVGKEF N H 
               
               
                   
                   
                 LE K RIENLNKKVDDGFLDI WTYNAELL V LLEN   
               
               
                   
                   
                   ER TLDYHDSNVKNLYEKVR N QLKNNAKEIGN G   
               
               
                   
                   
                   CFEFYH KCD NT CMESVKNGTYDYPKYSEEAKL 
               
               
                   
                   
                 NRE K IDGVKLESTRIYQILAIYSTVASSLVL V   
               
               
                   
                   
                 VSLGAISFWMCSNGSLQCRICI 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 2 
                 H1N1 
                   MNPNQKIITIGS   ICMT IG MAN L I LQIGNIISI 
               
               
                   
                 Neuraminidase 
                 W V SHSIQ I G N Q SQIET CNQ SV ITYEN N TWVN Q   
               
               
                   
                 A/Michigan/ 
                 TYVNI S NIN FA A GQSVVS V K L A GNSSLC PV SG 
               
               
                   
                 45/2015 strain 
                 WAIY S KDNS V   RIGSKGDVFVIREPFISC S P LE 
               
               
                   
                 (amino acid) 
                 C R   TFFLTQGALLNDKHSNGT   I   KDRSPYR   T LMS 
               
               
                   
                 GenBank: 
                 CP I GE V PSPYNS R   FESVAWSASACHDG   IN   WLT   
               
               
                   
                 MK622934.1 
                   IGISGPD   S   GAVAVLKY NGIIT D TIKSW RNN   IL   
               
               
                   
                   
                   RTQESEC   A C V NGSCFT I MIDGPS DGQ ASYKIF 
               
               
                   
                   
                   R IEKGK II KS V E MK APN YH Y EECSCYPD   SSEI   
               
               
                   
                   
                   T   CVCRDNWHGSNRPWVSFNQNL   E YQ M GYICSG 
               
               
                   
                   
                   V TGDNPRP N D KT GSC G PV SSN GA N GVKGFS F K 
               
               
                   
                   
                 Y G   NGVWIGRTKS   ISS R K   GFEMIWDPNGWT   G TD 
               
               
                   
                   
                   NK FS I KQD I VGI NE   WSGYSGSFVQHPELTGL   D   
               
               
                   
                   
                 CI RPCFWVEL   I RG R P E ENTI WTSGSSISFCGV   
               
               
                   
                   
                   NS D TVG W SWPDGAELPF TIDK 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 3 
                 H3N2 
                 MKTIIA L S Y ILCL V F A Q KL PP Y GN ST AT LCLG   
               
               
                   
                 Hemagglutinin 
                   HHA LPNGT I VKTIT N D R IEVTNATELVQ N SS I   
               
               
                   
                 A/Mississippi/ 
                 G E IC D SPH Q ILDG E NCTLIDALLGDP Q CD G FQ 
               
               
                   
                 27/2013 strain 
                 N KK WDLFVER SK A Y SNCYPY D VPDYASLRSLV 
               
               
                   
                 (amino acid) 
                 ASSGTLEF NN E S FNW T GV T Q N G T S SA C I R R S N   
               
               
                   
                 GenBank: 
                   S SFFSRLNWL THLNFK YP A INVIMPN NEQ FDK 
               
               
                   
                 AIK26600.1 
                 LYIWGVHHP G TD KD Q IF LY AQSS GR IT VSTKR 
               
               
                   
                   
                 SQQ A VIPNIGSRP RI R NIP S R ISIYWTIVKPG 
               
               
                   
                   
                 DILLINST GNLIAPRGYF KI RS GKSSIMRSDA 
               
               
                   
                   
                 PI GK C K S E CITPNGSIPNDKPFQNVN R ITYGA 
               
               
                   
                   
                 CPRYVK QS T LKLATGMRN VPE K QTRGI FGAIA   
               
               
                   
                   
                   GFIENGWEG   MV DGWYGFRHQNSEG R GQAADLK 
               
               
                   
                   
                 STQAAI D QI N GKLNR L I G KTNEKFHQIEKEFS 
               
               
                   
                   
                 EVEGR I QDLEKYVEDTKIDLWSYNAELLVALE 
               
               
                   
                   
                 NQHTI DLTDSEM NKLFE K T KK Q LRENAED MGN 
               
               
                   
                   
                 GCFKIYHKCDNAC IG SIRN G TYDH NV YR D EAL 
               
               
                   
                   
                 NNRFQIKGV E LKSGYKDWILWISFAISCFLLC 
               
               
                   
                   
                   VA IKGFIMWACQKG NIRCN IRCNICI 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 4 
                 H3N2 
                 MNPNQKIITIGSVSL T I S TICF F MQ I AIL I TT 
               
               
                   
                 Neuraminidase 
                 VTLHFKQ YEFNSPP NNQV ML CEP T IIERN I TE 
               
               
                   
                 A/Miyagi/N12 
                 IVYLTNTT I E K EICPK LA EYRKWSKPQC N IT G   
               
               
                   
                 89/2005 strain 
                   FAPFS K DNS IRLSAGGDIWVTREPYVSCD PD K 
               
               
                   
                 (amino acid) 
                 CY QFALGQGTT   L NN V HSN D I VR DRTP Y RTLLM 
               
               
                   
                 GenBank: 
                   N ELGVPFHLGT K QVC I   AWSSSSC HDGKAWLHV 
               
               
                   
                 AB271522.1 
                 C V TG D DKNATASFIY N GRLVDS IV SWS KE I LR   
               
               
                   
                   
                   TQESEC VCINGTCTVVMTDG S A S G K ADTKILF 
               
               
                   
                   
                 IEEGKI V H T S T LSGSAQHV EECSCYP RYPGVR 
               
               
                   
                   
                 CV CRDNW K GSNRP I V DIN I KD Y SI V SSYV CSG   
               
               
                   
                   
                   LVGDTPR K N D SS SSS H C LD PNNE E G G H GVKGW   
               
               
                   
                   
                   AFD   D GNDVWMGRTI S E KL RS G YETFXV IE GWS 
               
               
                   
                   
                   NPN SKLQ I NRQVIV DRGN RSGYSGIFSVEGK S   
               
               
                   
                   
                 CI NRCFYVELIRG RK E ET E V L WTSNSIV VFCG   
               
               
                   
                   
                   TSGTYG T GSWPDGA D I NLM P I 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 5 
                 Influenza B 
                 MKAIIVLLMVVTSNADRICTGITSSNSPHV VK   
               
               
                   
                 Hemagglutinin 
                   TATQGEVNVTG VIPLTTTPTKS H FANLKGT E T 
               
               
                   
                 B/Brisbane/60/ 
                 RGKLCP K C L   NCTDLDVAL GRP K C T G KI PSA RV   
               
               
                   
                 2008 strain 
                 SILHEVRPV TSGCFPIMHDRTKIRQL   P   NLLRG   
               
               
                   
                 (amino acid) 
                   YE   HI RLST H NVI N AE N APGGPY KI GTSGSCPN 
               
               
                   
                 GenBank: 
                   I T NGN GFFA TMAWAVP   KN D KN K T ATN PL T I EV 
               
               
                   
                 KX058884.1 
                 PY I C T EGEDQITVWGFHSDD E TQM AK LYGDS K   
               
               
                   
                   
                 PQKFTSSANGVTTHYVSQIG G FP N QT EDGGLP   
               
               
                   
                   
                   QSGRIVVDYM   V QK S GKTGTI T YQRG I LLPQKV 
               
               
                   
                   
                 WCASGRSKVIKGS LPLIGEADCLHE   K   YGGLNK   
               
               
                   
                   
                   SKPYYTG   E HAKAIGNC PIWVKTPL KLANGTKY 
               
               
                   
                   
                 RPPAKLLKER GFFGAIAGFLEGGWEGM I AGWH   
               
               
                   
                   
                   GYTSHGAHG V AVAADLKSTQEA IN KITKNLNS   
               
               
                   
                   
                   LSELE V KNLQRLS GAMDELHN EILELDEKVDD   
               
               
                   
                   
                 
                   LRADTISSQIELAVLLSNEGIINSEDEHLLAL 
                 
               
               
                   
                   
                   ERKLKKMLGPSA V E   IGNGCFETKHKCNQTCLD   
               
               
                   
                   
                   R IAAGTF D   AGEFSLPTFDSLNITAASL NDDGL 
               
               
                   
                   
                 DN HTILLYYSTAASSLAVTLM   I AIF V VYMVSR 
               
               
                   
                   
                 DNVSCSICL 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 6 
                 Influenza B 
                 MLPSTIQTLTLFLTSGGVLLSLYVSASLSYLL 
               
               
                   
                 Neuraminidase 
                 YSDILLKFS P TE I TAP T MPLDCANASNV Q AVN 
               
               
                   
                 B/Wisconsin/ 
                   R S AT KGVT LL LLPEPEWTYPRLSCPGSTFQK A   
               
               
                   
                 05/2016 strain 
                   LLISPHRFGE   T KGNSAPLIIREPF V ACGP N EC 
               
               
                   
                 (amino acid) 
                 K HFALTHYAAQPG GYYNGTR G   DRNKLRHL ISV 
               
               
                   
                 GenBank: 
                 KLGKIPTVENS I FHMA AWSGSACHDG   K EWTYI 
               
               
                   
                 KX007164.1 
                 GVDGPD N NALLK V   KYGEAYTDTYHSY A NN I LR   
               
               
                   
                   
                   TQESACNCI GG N CYLMITDG S ASG V SE CRFLK   
               
               
                   
                   
                   IREGR IIKEIFPTGRVK HTEECTCGFA SNKTI 
               
               
                   
                   
                 ECACRDN R   YTAKRPFVKL NVETDTAEIRLMCT 
               
               
                   
                   
                   D TYLDTPRP N DGSITGPCES D G DK GSGGI KGG   
               
               
                   
                   
                   FVHQR M K SKIG RWYSRT MS K T E RMGMGLYVKY 
               
               
                   
                   
                   G GDPW A DSDAL AF SGVM VS M K   EPGWYSFGFE I 
               
               
                   
                   
                 KDKKCDVPCIGI EMVHDGG K E TWHSAATAIYC 
               
               
                   
                   
                 LMGSGQLLWDTVTGV D M A L 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 7 
                 H1N1 
                 GYHANNST 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 8 
                 H1N1 
                 ggttatcatgcgaacaattcaaca 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 9 
                 H1N1 
                 NVTVTHS 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 10 
                 H1N1 
                 aatgtaacagtaacacactct 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 11 
                 H1N1 
                 SWSYIVE 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 12 
                 H1N1 
                 tcatggtcctacattgtggaa 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 13 
                 H1N1 
                 QSRGLFGAIAGF 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 14 
                 H1N1 
                 caatctagaggcctattcggggccattgccggcttc 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 15 
                 H1N1 
                 QGSGYAAD 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 16 
                 H1N1 
                 caggggtcaggatatgcagccgac 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 17 
                 H1N1 
                 ITNKVNS 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 18 
                 H1N1 
                 attactaacaaagtaaattct 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 19 
                 H1N1 
                 WTYNAELL 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 20 
                 H1N1 
                 tggacttacaatgccgaactgttg 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 21 
                 H1N1 
                 GCFEFYH 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 22 
                 H1N1 
                 gcctgctttgaattttaccac 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 23 
                 H1N1 
                 LGNPEC 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 24 
                 H1N1 
                 ctgggaaatccagagtgt 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 25 
                 H1N1 
                 EGGWTG 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 26 
                 H INI 
                 gaaggggggtggacaggg 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 27 
                 H1N1 
                 LLENER 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 28 
                 H1N1 
                 ctattggaaaatgaaaga 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 29 
                 H1N1 
                 MNPNQKI1TIGS 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 30 
                 H1N1 
                 atgaatccaaaccaaaagataataaccattggttcg 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 31 
                 H1N1 
                 RIGSKGDVFV 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 32 
                 H1N1 
                 agaatcggttccaagggggatgtgtttgtc 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 33 
                 H1N1 
                 REPFISCS 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 34 
                 H1N1 
                 agggaaccattcatatca 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 35 
                 H1N1 
                 TFFLTQGALLNDKHSNGT 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 36 
                 H1N1 
                 accttcttcttgactcaaggggccttgctaaa 
               
               
                   
                 Neuraminidase 
                 tgacaaacattccaatggaacc 
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 37 
                 H1N1 
                 KDRSPYR 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 38 
                 H1N1 
                 aaagacaggagcccataccga 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 39 
                 H1N1 
                 FESVAWSASACHDG 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 40 
                 H1N1 
                 tttgagtcagtcgcttggtcagcaagtgcttgtcatgatggc 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 41 
                 H1N1 
                 WLTIGISGPD 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 42 
                 H1N1 
                 tggctaacaattggaatttctggcccagac 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 43 
                 H1N1 
                 ILRTQESEC 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 44 
                 H1N1 
                 atattgagaacacaagagtctgaatgt 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 45 
                 H1N1 
                 YEECSCYPD 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 46 
                 H1N1 
                 tatgaggaatgctcctgttaccctgat 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 47 
                 H1N1 
                 CVCRDNWHGSNRPWVSFNQNL 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 48 
                 H1N1 
                 tgtgtgtgcagggataactggcatggctcga 
               
               
                   
                 Neuraminidase 
                 atcgaccgtgggtgtctttcaaccagaatctg 
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 49 
                 H1N1 
                 NGVWIGRTKS 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 50 
                 H1N1 
                 aatggtgtttggatagggagaactaaaagc 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 51 
                 H1N1 
                 GFEMIWDPNGWT 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 52 
                 H1N1 
                 ggttttgagatgatttgggatccgaatggatggact 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 53 
                 H1N1 
                 WSGYSGSFVQHPELTGL 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 54 
                 H1N1 
                 tggtcagggtatagcgggagttttgttcagcatcc 
               
               
                   
                 Neuraminidase 
                 agaactaacagggctg 
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 55 
                 H1N1 
                 RPCFWVEL 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 56 
                 H1N1 
                 agaccttgcttctgggttgaacta 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 57 
                 H1N1 
                 WTSGSS1SFCGV 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 58 
                 H1N1 
                 tggactagcgggagcagcatatccttttgtggtgta 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 59 
                 H1N1 
                 WSWPDGAELPF 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 60 
                 H1N1 
                 tggtcttggccagacggtgctgagttgccattt 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 61 
                 H3N2 
                 LCLGHHA 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 62 
                 H3N2 
                 ctgtgccttgggcaccatgcatta 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 63 
                 H3N2 
                 GNLIAPRGYF 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 64 
                 H3N2 
                 gggaatctaattgctcctaggggttacttc 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 65 
                 H3N2 
                 LKLATGMRN 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 66 
                 H3N2 
                 ctgaaattggcaacaggaatgcgaaat 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 67 
                 H3N2 
                 FGAIAGFIENGWEG 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 68 
                 H3N2 
                 tttggcgcaatagcaggtttcatagaaaatggttgggagggg 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 69 
                 H3N2 
                 KFHQIEKEF 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 70 
                 H3N2 
                 aaattccatcagattgaaaaagaattc 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 71 
                 H3N2 
                 DLTDSEM 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 72 
                 H3N2 
                 gatctaactgactcagaaatg 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 73 
                 H3N2 
                 LRENAED 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 74 
                 H3N2 
                 ctgagggaaaatgctgaggat 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 75 
                 H3N2 
                 QFALGQGTT 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 76 
                 H3N2 
                 caatttgcccttggacagggaacaaca 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 77 
                 H3N2 
                 AWSSSSC 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 78 
                 H3N2 
                 gcatggtccagctcaagttgt 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 79 
                 H3N2 
                 LRTQESEC 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 80 
                 H3N2 
                 ctcaggacccaggagtcagaatgc 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 81 
                 H3N2 
                 EECSCYP 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 82 
                 H3N2 
                 gaggagtgctcctgctatcct 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 83 
                 H3N2 
                 CSGLVGDTPR 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 84 
                 H3N2 
                 tgctcaggacttgttggagacacacccaga 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 85 
                 H3N2 
                 GVKGWAFD 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 86 
                 H3N2 
                 ggagtgaaaggctgggcctttgat 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 87 
                 H3N2 
                 NRCFYVELIRG 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 88 
                 H3N2 
                 aatcggtgcttttatgtggagttgataagggga 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 89 
                 H3N2 
                 VFCGTSGTYG 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 90 
                 H3N2 
                 gtgttttgtggcacctcaggtacatatgga 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 91 
                 H3N2 
                 GSWPDGA 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 92 
                 H3N2 
                 ggctcatggcctgatggggcg 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 93 
                 Influenza B 
                 VKTATQEVNVTG 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 94 
                 Influenza B 
                 gtcaaaactgctactcaaggggaggtcaatgtgactggt 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 95 
                 Influenza B 
                 NCTDLDVAL 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 96 
                 Influenza B 
                 aactgcacagatctggacgtagccttg 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 97 
                 Influenza B 
                 TSGCFPIMHDRTKIRQL 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 98 
                 Influenza B 
                 acatctgggtgctttcctataatgcacgac 
               
               
                   
                 Hemagglutinin 
                 agaacaaaaattagacagctg 
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 99 
                 Influenza B 
                 NLLRGYE 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 100 
                 Influenza B 
                 aaccttctccgaggatacgaa 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 101 
                 Influenza B 
                 TMAWAVP 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 102 
                 Influenza B 
                 acaatggcttgggccgtccca 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 103 
                 Influenza B 
                 EDGGLPQSGRIVVDYM 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 104 
                 Influenza B 
                 gaagacggaggactaccacaaagtggta 
               
               
                   
                 Hemagglutinin 
                 gaattgttgttgattacatg 
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 105 
                 Influenza B 
                 LPLIGEADCLHE 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 106 
                 Influenza B 
                 ttgcctttaattggagaagcagattgcctccacgaa 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 107 
                 Influenza B 
                 YGGLNKSKPYYTG 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 108 
                 Influenza B 
                 tacggtggattaaacaaaagcaagccttactacacaggg 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 109 
                 Influenza B 
                 CPIWVKTPL 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 110 
                 Influenza B 
                 tgcccaatatgggtgaaaacacccttg 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 111 
                 Influenza B 
                 GFFGAIAGFLEGGWEGM 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 112 
                 Influenza B 
                 ggtttcttcggagctattgctggtttcttag 
               
               
                   
                 Hemagglutinin 
                 aaggaggatgggaaggaatg 
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 113 
                 Influenza B 
                 AGWHGYTSHGAHG 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 114 
                 Influenza B 
                 gcaggttggcacggatacacatcccatggggcacatgga 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 115 
                 Influenza B 
                 AVAADLKSTQEA 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 116 
                 Influenza B 
                 gcggtggcagcagaccttaagagcactcaagaggcc 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 117 
                 Influenza B 
                 KITKNLNSLSELE 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 118 
                 Influenza B 
                 aagataacaaaaaatctcaactctttgagtgagctggaa 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 119 
                 Influenza B 
                 KNLQRLS 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 120 
                 Influenza B 
                 aagaatcttcaaagactaagc 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 121 
                 Influenza B 
                 EILELDEKVDDLRADTISSQIELAVLLSNEGIINSED 
               
               
                   
                 Hemagglutinin 
                 EHLLALERKLKKMLGPSA 
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 122 
                 Influenza B 
                 gaaatactagaactagatgagaaagtggatga 
               
               
                   
                 Hemagglutinin 
                 tctcagagctgatacaataagctcacaaatag 
               
               
                   
                 conserved 
                 aactcgcagtcctgctttccaatgaaggaata 
               
               
                   
                 region 
                 ataaacagtgaagatgaacatctcttggcgct 
               
               
                   
                 (nucleic 
                 tgaaagaaagctgaagaaaatgctgggcccct 
               
               
                   
                 acid) 
                 ctgct 
               
               
                   
               
               
                 123 
                 Influenza B 
                 IGNGCFETKHKCNQTCLD 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 124 
                 Influenza B 
                 atagggaatggatgctttgaaaccaaac 
               
               
                   
                 Hemagglutinin 
                 acaagtgcaaccagacctgtctcgac 
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 125 
                 Influenza B 
                 AGEFSLPTFDSLNITAASL 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 126 
                 Influenza B 
                 gcaggagaattttctctccccacctttg 
               
               
                   
                 Hemagglutinin 
                 attcactgaatattactgctgcatcttta 
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 127 
                 Influenza B 
                 HTILLYYSTAASSLAVTLM 
               
               
                   
                 Hemagglutinin 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 128 
                 Influenza B 
                 catactatactgctttactactcaactgc 
               
               
                   
                 Hemagglutinin 
                 tgcctccagtttggctgtaacactgatg 
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 129 
                 Influenza B 
                 ALLISPHRFGE 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 130 
                 Influenza B 
                 gcactcctaattagccctcatagattcggagaa 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 131 
                 Influenza B 
                 HFALTHYAAQPG 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 132 
                 Influenza B 
                 cactttgctttaacccattatgcagcccaaccaggg 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 133 
                 Influenza B 
                 DRNKLRHL 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 134 
                 Influenza B 
                 gacagaaacaagctgaggcatcta 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 135 
                 Influenza B 
                 AWSGSACHDG 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 136 
                 Influenza B 
                 gcatggagcgggtccgcgtgccatgatggt 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 137 
                 Influenza B 
                 KYGEAYTDTYHSY 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 138 
                 Influenza B 
                 aaatatggagaagcatatactgacacataccattcctat 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 139 
                 Influenza B 
                 LRTQESACNCI 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 140 
                 Influenza B 
                 ctaagaacacaagaaagtgcctgcaattgcatc 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 141 
                 Influenza B 
                 CRFLKIREGR 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 142 
                 Influenza B 
                 tgcagatttcttaagattcgagagggccga 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 143 
                 Influenza B 
                 HTEECTCGFA 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 144 
                 Influenza B 
                 cacactgaggaatgcacatgcggatttgcc 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 145 
                 Influenza B 
                 YTAKRPFVKL 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 146 
                 Influenza B 
                 tacacagcaaaaagaccttttgtcaaatta 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 147 
                 Influenza B 
                 KGGFVHQR 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 148 
                 Influenza B 
                 aagggaggatttgttcatcaaaga 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 149 
                 Influenza B 
                 GRWYSRT 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 150 
                 Influenza B 
                 ggaaggtggtactctcgaacg 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 151 
                 Influenza B 
                 EPGWYSFGFE 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 152 
                 Influenza B 
                 gaacctggttggtattcctttggcttcgaa 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 153 
                 Influenza B 
                 EMVHDGG 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 154 
                 Influenza B 
                 gagatggtacatgatggtgga 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 155 
                 H1N1 
                 GAVAVLKY 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (amino 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 156 
                 H1N1 
                 ggggcagtggctgtgttaaagtac 
               
               
                   
                 Neuraminidase 
                   
               
               
                   
                 conserved 
                   
               
               
                   
                 region 
                   
               
               
                   
                 (nucleic 
                   
               
               
                   
                 acid) 
                   
               
               
                   
               
               
                 157 
                 Variant of 
                 MKAILVVLLYTFAAANADTLCIGYH 
               
               
                   
                 H1N1 
                 ANNSTDTVDTVLEKNVTVTHSVNLL 
               
               
                   
                 Hemagglutinin 
                 EAAHNGKLCKLRGVAPLHLGKCNIA 
               
               
                   
                 A/Michigan/45/ 
                 GWALGNPECEALATASSWSYIVETS 
               
               
                   
                 2015 strain 
                 ASDNGTCYPGDFIAYEELREQLSSV 
               
               
                   
                 (amino acid) 
                 SSFERFEIFPKASSWPNHDANAGVT 
               
               
                   
                 GenBank: 
                 AACPAAGAAAFYANLIWLVKKGNSY 
               
               
                   
                 MK622940.1 
                 PKAAASYINAKAKEVLVLWAIHHPA 
               
               
                   
                 Hypervariable 
                 TAADQQSLYQNADAYVFVGASAYSA 
               
               
                   
                 residues 
                 KFAPEIAARPKVRAQAGRMNYYWTL 
               
               
                   
                 substituted 
                 AEPGDAITFEATGNLVVPRYAFAAA 
               
               
                   
                 with Ala 
                 RAAGSGIIISDAAVHDCATTCQTPA 
               
               
                   
                   
                 GAINTSLPFQNIHPATIGACPKYVK 
               
               
                   
                   
                 STKLRAATGLRNAPSIQSRGLFGAI 
               
               
                   
                   
                 AGFIEGGWTGMADGWYGYHHQNEQG 
               
               
                   
                   
                 SGYAADAKSTQNAIDAITNKVNSVI 
               
               
                   
                   
                 EKMNTQFTAVGKEFAHLEARIENLN 
               
               
                   
                   
                 KKVDDGFLDIWTYNAELLVLLENER 
               
               
                   
                   
                 TLDYHDSNVKNLYEKVRAQLKNNAK 
               
               
                   
                   
                 EIGNGCFEFYHKCDAACMESVKNGT 
               
               
                   
                   
                 YDYPKYSEEAKLNREAIDGVKLEST 
               
               
                   
                   
                 RIYQILAIYSTVASSLVLAVSLGAI 
               
               
                   
                   
                 SFWMCSNGSLQCRICI* 
               
               
                   
               
               
                 158 
                 Influenza A 
                 SNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYIN 
               
               
                   
                 H1N1 
                 DKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGSSRYS 
               
               
                   
                 Hemagglutinin 
                 KKFKP 
               
               
                   
                 2009 residues 
                   
               
               
                   
                 145-229 
                   
               
               
                   
               
               
                 159 
                 Influenza A 
                 SNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYIN 
               
               
                   
                 H1N1 
                 DKGKEVLVLWGIHHPPTSADQQSLYQNADAYVFVGTSRYS 
               
               
                   
                 Hemagglutinin 
                 KKFKP 
               
               
                   
                 2010 residues 
                   
               
               
                   
                 145-229 
                   
               
               
                   
               
               
                 160 
                 Influenza A 
                 TTRGTTVACSHSGANSFYRNLLWIVKKGNSYPKLSKSYTNN 
               
               
                   
                 H1N1 
                 KGKEVLVIWGVHHPPTDSDQQTLYQNNHTYVSVGSSKYYK 
               
               
                   
                 Hemagglutinin 
                 RLTP 
               
               
                   
                 2011 residues 
                   
               
               
                   
                 145-229 
                   
               
               
                   
               
               
                 161 
                 Influenza A 
                 SNKGVTAACPHAGAKGFYKNLIWLVKKGNSYPKLSKSYIN 
               
               
                   
                 H1N1 
                 DKGKEVLVLWGIHHPSTTADQQSLYQNADTYWVGTSRYS 
               
               
                   
                 Hemagglutinin 
                 KKFKP 
               
               
                   
                 2012 residues 
                   
               
               
                   
                 145-229 
                   
               
               
                   
               
               
                 162 
                 Influenza A 
                 SNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYIN 
               
               
                   
                 H1N1 
                 DKGKEVLVLWGIHHPSTTADQQSLYQNANAYVFVGTSKYS 
               
               
                   
                 Hemagglutinin 
                 KKFKP 
               
               
                   
                 2013 residues 
                   
               
               
                   
                 145-229 
                   
               
               
                   
               
               
                 163 
                 Influenza A 
                 SNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYIN 
               
               
                   
                 H1N1 
                 DKGKEVLVLWGIHHPSTSADQQSLYQNADAYVFVGTSRYS 
               
               
                   
                 Hemagglutinin 
                 KKFKP 
               
               
                   
                 2014 residues 
                   
               
               
                   
                 145-229 
                   
               
               
                   
               
               
                 164 
                 Influenza A 
                 SNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLSKSYIN 
               
               
                   
                 H1N1 
                 DKGKEVLVLWGIHHPSTSADQQSLYQNADAYWVGTSRYS 
               
               
                   
                 Hemagglutinin 
                 KKFKP 
               
               
                   
                 2015 residues 
                   
               
               
                   
                 145-229 
                   
               
               
                   
               
               
                 165 
                 Influenza A 
                 SNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLNQSYIN 
               
               
                   
                 H1N1 
                 DKGKEVLVLWGIHHPSTTADQQSLYQNADAYVFVGTSRYS 
               
               
                   
                 Hemagglutinin 
                 KKFKP 
               
               
                   
                 2016 residues 
                   
               
               
                   
                 145-229 
                   
               
               
                   
               
               
                 166 
                 Influenza A 
                 SNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLNQTYIN 
               
               
                   
                 H1N1 
                 DKGKEVLVLWGIHHPPTTADQQSLYQNADAYVFVGTSRYS 
               
               
                   
                 Hemagglutinin 
                 KKFKP 
               
               
                   
                 2017 residues 
                   
               
               
                   
                 145-229 
                   
               
               
                   
               
               
                 167 
                 Influenza A 
                 SDKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKLNQTYIN 
               
               
                   
                 HIN1 
                 DKGKEVLVLWGIHHPPTIADQQSLYQNADAYVFVGTSRYS 
               
               
                   
                 Hemagglutinin 
                 KKFKP 
               
               
                   
                 2018 residues 
                   
               
               
                   
                 145-229 
                   
               
               
                   
               
               
                 168 
                 Influenza A 
                 SNKGVTAACPHAGAKSFYKNLIWLVKKGNSYPKINQTYIND 
               
               
                   
                 H1N1 
                 KGKEVLVLWGIHHPPTTADOQSLYQNADAYVFVGTSRYSK 
               
               
                   
                 Hemagglutinin 
                 KFKP 
               
               
                   
                 2019 residues 
                   
               
               
                   
                 145-229 
                   
               
               
                   
               
               
                 169 
                 H1N1 
                 atgaaggcaatactagtagttctgctatat 
               
               
                   
                 Hemagglutinin 
                 acatttacaaccgcaaatgcagacacatta 
               
               
                   
                 A/Michigan/ 
                 tgtataggttatcatgcgaacaattcaaca 
               
               
                   
                 45/2015 strain 
                 gacactgtagacacagtactagaaaagaat 
               
               
                   
                 (nucleotide) 
                 gtaacagtaacacactctgttaaccttctg 
               
               
                   
                 GenBank: 
                 gaagacaagcataacggaaaactatgcaaa 
               
               
                   
                 MK622940.1 
                 ctaagaggggtagccccattgcatttgggt 
               
               
                   
                   
                 aaatgtaacattgctggctggatcctggga 
               
               
                   
                   
                 aatccagagtgtgaatcrctctccacagca 
               
               
                   
                   
                 agttcatggtcctacattgtggaaacatct 
               
               
                   
                   
                 aattcagacaatggaacgtgttacccagga 
               
               
                   
                   
                 gatttcatcaattatgaggagctaagagag 
               
               
                   
                   
                 caattgagctcagtgtcatcatttgaaagg 
               
               
                   
                   
                 tttgagatattccccaagacaagttcatgg 
               
               
                   
                   
                 cccaatcatgactcgaacaaaggtgtaacg 
               
               
                   
                   
                 gcagcatgtcctcacgctggagcaaaaagc 
               
               
                   
                   
                 ttctacaaaaacttgatatggctagttaaa 
               
               
                   
                   
                 aaaggaaattcatacccaaagcttaaccaa 
               
               
                   
                   
                 tcctacattaatgataaagggaaagaagtc 
               
               
                   
                   
                 ctcgtgctgtggggcattcaccatccatct 
               
               
                   
                   
                 actactgctgaccaacaaagtctctatcag 
               
               
                   
                   
                 aatgcagatgcatatgtttttgtggggaca 
               
               
                   
                   
                 tcaagatacagcaagaagttcaagccggaa 
               
               
                   
                   
                 atagcaacaagacccaaagtgagggatcaa 
               
               
                   
                   
                 gaagggagaatgaactattactggacacta 
               
               
                   
                   
                 gtagagccgggagacaaaataacattcgaa 
               
               
                   
                   
                 gcaactggaaatctagtggtaccgagatat 
               
               
                   
                   
                 gcattcacaatggaaagaaatgctggatct 
               
               
                   
                   
                 ggtattatcatttcagatacaccagtccac 
               
               
                   
                   
                 gattgcaatacaacttgtcagacacccgag 
               
               
                   
                   
                 ggtgctataaacaccagcctcccatttcag 
               
               
                   
                   
                 aatatacatccgatcacaattggaaaatgt 
               
               
                   
                   
                 ccaaagtatgtaaaaagcacaaaattgaga 
               
               
                   
                   
                 ctggccacaggattgaggaatgttccgtct 
               
               
                   
                   
                 attcaatctagaggcctattcggggccatt 
               
               
                   
                   
                 gccggcttcattgaaggggggtggacaggg 
               
               
                   
                   
                 atggtagatggatggtacggttatcaccat 
               
               
                   
                   
                 caaaatgagcaggggtcaggatatgcagcc 
               
               
                   
                   
                 gacctgaagagcacacaaaatgccattgac 
               
               
                   
                   
                 aagattactaacaaagtaaattctgttatt 
               
               
                   
                   
                 gaaaagatgaatacacagttcacagcagtg 
               
               
                   
                   
                 ggtaaagagttcaaccacctggaaaaaaga 
               
               
                   
                   
                 atagagaatctaaataaaaaagttgatgat 
               
               
                   
                   
                 ggtttcctggacatttggacttacaatgcc 
               
               
                   
                   
                 gaactgttggttctattggaaaatgaaaga 
               
               
                   
                   
                 actttggactatcacgattcaaatgtgaag 
               
               
                   
                   
                 aacttgtatgaaaaagtaagaaaccagtta 
               
               
                   
                   
                 aaaaacaatgccaaggaaattggaaacggc 
               
               
                   
                   
                 tgctttgaattttaccacaaatgcgataac 
               
               
                   
                   
                 acgtgcatggaaagtgtcaaaaatgggact 
               
               
                   
                   
                 tatgactacccaaaatactcagaggaagca 
               
               
                   
                   
                 aaattaaacagagaaaaaatagatggggta 
               
               
                   
                   
                 aagctggaatcaacaaggatttaccagatt 
               
               
                   
                   
                 ttggcgatctattcaactgtcgccagttca 
               
               
                   
                   
                 ttggtactggtagtctccctgggggcaatc 
               
               
                   
                   
                 agcttctggatgtgctctaatgggtctcta 
               
               
                   
                   
                 cagtgtagaatatgtatttaa 
               
               
                   
               
               
                 170 
                 Variant of 
                 atgaaggcaatactagtagttctgctatat 
               
               
                   
                 H1N1 
                 acatttgcagccgcaaatgcagacacatta 
               
               
                   
                 Hemagglutinin 
                 tgtataggttatcatgcgaacaattcaaca 
               
               
                   
                 A/Michigan/ 
                 gacactgtagacacagtactagaaaagaat 
               
               
                   
                 45/2015 
                 gtaacagtaacacactctgttaaccttctg 
               
               
                   
                 strain 
                 gaagccgcgcataacggaaaactatgcaaa 
               
               
                   
                 (nucleotide) 
                 ctaagaggggtagccccattgcatttgggt 
               
               
                   
                 GenBank: 
                 aaatgtaacattgctggctgggccctggga 
               
               
                   
                 MK622940.1 
                 aatccagagtgtgaagcrctcgccacagca 
               
               
                   
                 Hypervariable 
                 agttcatggtcctacattgtggaaacatct 
               
               
                   
                 residues 
                 gcttcagacaatggaacgtgttacccagga 
               
               
                   
                 substituted 
                 gatttcatcgcttatgaggagctaagagag 
               
               
                   
                 with Ala 
                 caattgagctcagtgtcatcatttgaaagg 
               
               
                   
                   
                 tttgagatattccccaaggcaagttcatgg 
               
               
                   
                   
                 cccaatcatgacgcgaacgcaggtgtaacg 
               
               
                   
                   
                 gcagcatgtcctgccgctggagcagcagcc 
               
               
                   
                   
                 ttctacgcaaacttgatatggctagttaaa 
               
               
                   
                   
                 aaaggaaattcatacccaaaggctgccgca 
               
               
                   
                   
                 tcctacattaatgctaaagcgaaagaagtc 
               
               
                   
                   
                 ctcgtgctgtgggccattcaccatccagct 
               
               
                   
                   
                 actgctgctgaccaacaaagtctctatcag 
               
               
                   
                   
                 aatgcagatgcatatgtttttgtggggaca 
               
               
                   
                   
                 tcagcatacagcgcgaagttcgcgccggaa 
               
               
                   
                   
                 atagcagcaagacccaaagtgagggctcaa 
               
               
                   
                   
                 gcagggagaatgaactattactggacacta 
               
               
                   
                   
                 gcagagccgggagacgcaataacattcgaa 
               
               
                   
                   
                 gcaactggaaatctagtggtaccgagatat 
               
               
                   
                   
                 gcattcgcagcggcaagagctgctggatct 
               
               
                   
                   
                 ggtattatcatttcagatgcagcagtccac 
               
               
                   
                   
                 gattgcgctacaacttgtcagacacccgcg 
               
               
                   
                   
                 ggtgctataaacaccagcctcccatttcag 
               
               
                   
                   
                 aatatacatccggccacaattggagcatgt 
               
               
                   
                   
                 ccaaagtatgtaaaaagcacaaaattgaga 
               
               
                   
                   
                 gcggccacaggattgaggaatgctccgtct 
               
               
                   
                   
                 attcaatctagaggcctattcggggccatt 
               
               
                   
                   
                 gccggcttcattgaaggggggtggacaggg 
               
               
                   
                   
                 atggcagatggatggtacggttatcaccat 
               
               
                   
                   
                 caaaatgagcaggggtcaggatatgcagcc 
               
               
                   
                   
                 gacgcgaagagcacacaaaatgccattgac 
               
               
                   
                   
                 gcgattactaacaaagtaaattctgttatt 
               
               
                   
                   
                 gaaaagatgaatacacagttcacagcagtg 
               
               
                   
                   
                 ggtaaagagttcgcccacctggaagcaaga 
               
               
                   
                   
                 atagagaatctaaataaaaaagttgatgat 
               
               
                   
                   
                 ggtttcctggacatttggacttacaatgcc 
               
               
                   
                   
                 gaactgttggttctattggaaaatgaaaga 
               
               
                   
                   
                 actttggactatcacgattcaaatgtgaag 
               
               
                   
                   
                 aacttgtatgaaaaagtaagagcccagtta 
               
               
                   
                   
                 aaaaacaatgccaaggaaattggaaacggc 
               
               
                   
                   
                 tgctttgaattttaccacaaatgcgatgcc 
               
               
                   
                   
                 gcgtgcatggaaagtgtcaaaaatgggact 
               
               
                   
                   
                 tatgactacccaaaatactcagaggaagca 
               
               
                   
                   
                 aaattaaacagagaagcaatagatggggta 
               
               
                   
                   
                 aagctggaatcaacaaggatttaccagatt 
               
               
                   
                   
                 ttggcgatctattcaactgtcgccagttca 
               
               
                   
                   
                 ttggtactggcagtctccctgggggcaatc 
               
               
                   
                   
                 agcttctggatgtgctctaatgggtctcta 
               
               
                   
                   
                 cagtgtagaatatgtatttaa 
               
               
                   
               
               
                 171 
                 H3N2 M1 
                 
                   MSLLTEVETYVLSI 
                   V 
                   PSGPLKAEIAQRLE 
                   D 
                   VFAGKNTDLEAL 
                 
               
               
                   
                 Protein 
                 
                   MEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRFVQNAL 
                 
               
               
                   
                   
                   NGNGDPNNMD   K AVKLY R   KLKREITFHGAKE   IA LSYS A   GAL   
               
               
                   
                   
                   ASCMGLIYNRMG   A VTTE V   AFGLVCATCEQIADSQHRSHRQ   
               
               
                   
                   
                   M   VA TTNPLI K HENRMVL ASTTAKAMEQMAGSSEQAAEAM   
               
               
                   
                   
                   E   I ASQ A RQMV Q AMR AI   GTHPSSS   T GL R   DDLLENLQ   T   YQKR   
               
               
                   
                   
                 
                   MGVQMQRFK 
                 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 172 
                 H3N2 NEP 
                 MDSNTV SSFQDIL   L   RMSKMQL GSSSE D LNGM I T Q FESLK I   Y   
               
               
                   
                 Protein 
                   RDSLGE   A V MRMGDLH   L LQNRN G KWREQLG QKFEEIRWLIE   
               
               
                   
                   
                   E VRHRL RT TEN SFEQITFMQALQLL   F   EVEQEIR TFSFQLI 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 173 
                 H3N2 NP 
                   MASQGTKRSYEQMET   D G D RQNATEIRASVG K MI D GIG R   FYI   
               
               
                   
                 Protein 
                   QMCTELKLSD   H   EGRLIQNS   L TIE K   MVLSAFDERRN   K YLEEH 
               
               
                   
                   
                 PS AGKDPKKTGGPIY   R RVDG K WMREL V   LYDKEEIRR IWRQ 
               
               
                   
                   
                 ANNGEDAT S GLTH I   MIWHSNLND   A   TYQRTRALVRTG M DPR   
               
               
                   
                   
                   MCSLMQGSTLPRRSGAAGAAVKG   I GT MV MELIRMV KRGIN   
               
               
                   
                   
                   DRNFWRGENGR   K TRS AYERMCNILKGKFQTAAQRAM   V   DQ   
               
               
                   
                   
                   VRESRNPGNAEIEDLIF LA RSALILRGSVAHKSCLPAC AYG P   
               
               
                   
                   
                 AV S SG Y DFE K   EGYSLVGIDPF KLLQNSQ IY SLI RPNENPAHK   
               
               
                   
                   
                   SQLVWMACHSAAFEDLR   LL SFIRG T KV S PRGKLSTRGV QIA   
               
               
                   
                   
                   SNEN   MDN M G S S TLELRS G   YWAIRTRSGGNTNQQ   R ASAGQ 
               
               
                   
                   
                   T SVQPTFSVQRNLPFE K ST IM AAF T GN T   EGRTSDMR   A E I IR 
               
               
                   
                   
                 MME G AKPE EV SF R G RGVFELSDEKA   TN PIVPSFDMS NEGSY   
               
               
                   
                   
                 
                   FFGDNAEEYD 
                   N 
                 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 174 
                 H3N2 NS1 
                 MDSNTV SSFQVDCFLWH IRK QVV D QK LSD A   PFLDRLRRDQ   
               
               
                   
                 Protein 
                   R SL R GRGNTL G LDI KA AT HV GKQI V E K IL KE ES D E A LKMT 
               
               
                   
                   
                   MV S T P A SRY IT DMT I EE L SR N WFMLMP K QK VE GPLC I R M D 
               
               
                   
                   
                 QAI ME K N I M LKANFNVIF G RLET IV L LRAFTEEGAIVGEISPL   
               
               
                   
                   
                   PS   F PGHT I EDVKNAIGV LIGGLEWN   D NTVR V S KN LQRFAWR 
               
               
                   
                   
                   SS NE N G G P P L T P K   
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 175 
                 H3N2 NS2 
                 MDSNTV SSFQDIL   L   RMSKMQL GSSSE D LNGMITQFESLK I   YR   
               
               
                   
                 Protein 
                   DSLGE AVMRMGDLHLLQNRNGKWREQLG QKFEEIRWLIEE   
               
               
                   
                   
                 VRHRL KT   TENSFEQITFMQALQLL   F EVEQE I   RTFSFQLI   
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 176 
                 H3N2 PA 
                   MEDFVRQCFNPMIVELAEKA MKEYGED L   KIETNKFAAICTH   
               
               
                   
                 Protein 
                   LEVCFMYSDFHFI NE Q GESI V VE LD   DPNALLKHRFEIIEGRD   
               
               
                   
                   
                   R T MAWTVVNSICNTT G AG   KPKFLPDLYDYKENRF   I   EIGVTR   
               
               
                   
                   
                   R EVHI YYLEKANKIKSE   N   THIHIFSF TGEEMATK ADYTLDEE   
               
               
                   
                   
                   SRARIKTRLFTIRQEMA NRG LWDSFRQSERGEETIEE   K FEI T G 
               
               
                   
                   
                 TMRRLAD QSLPPNFS   C   LENFRAYV DGFEPNGCIE GKLSQMS   
               
               
                   
                   
                   KEV   N A Q IEPFL K TTPRP IK LPSGPPC Y   QRSKFLLMDALKLSIE   
               
               
                   
                   
                   DPSHEGEGIPLYDAIKC   I K TFFGWKEP   Y I V KPHEKGINSNYLL 
               
               
                   
                   
                 SWKQVLSELQD I ENEEKIP R TKNMK K TSQLKWA LGENMAP   
               
               
                   
                   
                   EKVDF   E NC R D I SDLKQYDS E EPE L RSLSSW I QS EFNKACELT   
               
               
                   
                   
                   DS V WIELDEIGED VAPIE H IASM RRNYFTAEVSHCRATEYIM   
               
               
                   
                   
                   KGVYINTALLNASCAAMDDFQLIPMISKCRTKEGRR K TNLY   
               
               
                   
                   
                   GFI I KGRSHLRNDTDVVNFVSMEFSLTDPRLEPHKWEKYCV   
               
               
                   
                   
                   LEIGDMLLR   S AIGQ I   SRPMFLYVRTNGTSK   V   KMKWGMEMR   
               
               
                   
                   
                   RCLLQSLQQIESMIEAESS   V KEKDMTKEFFENKSE A   WPIGES   
               
               
                   
                   
                   P K GVEEGSIGKVCR T LLAKSVFNSLYASPQLEGFSAESRKLL   
               
               
                   
                   
                   L   I VQALRD K   LEPGTFDL G GLYEAIEECLINDPWVLLNASWF   
               
               
                   
                   
                   NSFL THALK 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 177 
                 H3N2 PA-X 
                   MEDFVRQCFNPMIVELAEK A MKEYGED   L   KIETNKFAAICTH   
               
               
                   
                 Protein 
                   LEVCFMYSDFHFI   N E Q GESI V VE LD   DPNALLKHRFEIIEGRD   
               
               
                   
                   
                   R T MAWTVVNSICNTTG   AG   KPKFLPDLYDYKENRF   I   EIGVTR   
               
               
                   
                   
                   R EVHI YYLEKANKIKSE   N   THIHIFSFTGEEMAT K ADYTLDEE   
               
               
                   
                   
                   SRARIKTRLFTIRQEMA NRG LWDSFVSP   K EAK K QLKK N LK S   
               
               
                   
                   
                 QE L C A GLPTKVSHRTSPAL RI   LEPMWMDSNRT AALRASFLK 
               
               
                   
                   
                 CPKK 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 178 
                 H3N2 PB1 
                   MDVNPTLLFLK V PAQNAISTTFPYTGDPPYSHGTGTGYTMD   
               
               
                   
                 Protein 
                   TVNRTHQYSE   R GK WTTNTETGAPQLNPIDGPLPEDNEPSGY   
               
               
                   
                   
                   AQTDCVLEAMAFLEESHPGIFENSC LETME A   VQQTRVD K LT   
               
               
                   
                   
                   QGRQTYDWTLNRNQPAATALANTIEVFRSN GLTA NESGRLI   
               
               
                   
                   
                   DFLKDV MESMDKEE M EI TTHFQRKRR VRDNMTKKMV TQR   
               
               
                   
                   
                   TIGKKKQ R V NKR G YL IRALTLNTMTKDAERGKLKRRAIATP   
               
               
                   
                   
                 
                   GMQIRGFVYFVETLARSICEKLEQSGLPVGGNEKKAKLANV 
                 
               
               
                   
                   
                   VRKMMTNSQDTE L SFTITGDNTKWNENQNPRMFLAMITYIT   
               
               
                   
                   
                   K NQPEWFRNILS I APIMFSNKMARLGKGYMFESK R MK L RT 
               
               
                   
                   
                 QIPAEMLASIDLKYFNESTR KKIEKIRPLL I DGTASLSPGMM   
               
               
                   
                   
                   MGMFNMLSTVLGVSILNLGQK KYT K   TTYWWDGLQSSDDF   
               
               
                   
                   
                   ALIVNAPNHEGIQAGV DRFYR TCKLVGINMSKKKSYIN   K   TG   
               
               
                   
                   
                 
                   TFEFTSFFYRYGFVANFSMELPSFGVSGINESADMSIGVTVIK 
                 
               
               
                   
                   
                 
                   NNMINNDLGPATAQMALQLFIKDYRYTYRCHRGDTQIQTR 
                 
               
               
                   
                   
                   RSFE   I KKLW D QT Q S RT   GLLVSDGGPNLYNIRNLHIPEVCLK   
               
               
                   
                   
                   WELMD   E NY R   GRLCNPLNPFV SHKEI E SVN N   AVVMPAHGPA   
               
               
                   
                   
                   K S MEYDAVATTHSWIPKRNRSILNTSQRGILEDEQMYQKCC   
               
               
                   
                   
                   NLFEKFFPSSSYRRP   I GISSMV EAMVSRA RIDARI DFESGRIK   
               
               
                   
                   
                 KEEFSEIMK ICSTIEELRRQK   
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 179 
                 H3N2 PB2 
                 
                   MERIKELR 
                   N 
                   LMSQSRTREILTKTTVDHMAIIKKYTSGRQEKN 
                 
               
               
                   
                 Protein 
                   P   S   LRMKWMMAM KYPITAD K R ITE M V   PERNEQGQ TLWSK 
               
               
                   
                   
                   MS   DAGSDRVMVSPLAVTWWNRNGP   V T S TV HYPKVYKTYF   
               
               
                   
                   
                   D   KVERLKHGTFGPVHFRNQVKIRRRVD   I   NPGHADLSA K EA   
               
               
                   
                   
                   QDVIMEVVFPNEVGARILTSESQL T ITKEKKEEL   R DCKI S   PL   
               
               
                   
                   
                   MVAYMLERELVRKTRFLPV AGGTSS I   YIEVLHLTQGTCWEQ   
               
               
                   
                   
                   MYTPGG   G VRNDD V DQSLI IAARNIVRRA A VSADPLASLLEM   
               
               
                   
                   
                   CHSTQIGG   TR MVDIL R   QNPTEEQAVDICKAA M GLRISSSFSF   
               
               
                   
                   
                   GGFTFKRT SGSSVK K EEE VLTGNLQTL   R IR VHEGYEEFT MV 
               
               
                   
                   
                 G K   RATAILRKATRRL   V QLIVSG R D E   QSIAEAIIVAMVFSQED   
               
               
                   
                   
                   C M IKAVRGDLNFVNRANQRLNPMHQLLRHFQKDAKVLFQ   
               
               
                   
                   
                   NWG   V E H IDSVMGM V G V   LPDMTP STEMS M RGIRVSKM GVD   
               
               
                   
                   
                   EYSSTERVVVSIDRFLRVRDQRGN V LLSPEEVSETQG TE R   LT   
               
               
                   
                   
                   ITYSSSMMWEINGPESVLVNTYQW IIRNWE AV KIQWSQ N P A   
               
               
                   
                   
                 MLYNKM EFEPFQSLVPKA   T R SQ YSGFVRT LFQQMRDVLGT   
               
               
                   
                   
                 
                   FDT 
                   A 
                   QIIKLLPFAAAPP 
                   K 
                   QSRMQFSSLTVNVRGSG 
                   M 
                   RILVRG 
                 
               
               
                   
                   
                   NSPVFNYNK   T TKRLT I LGKDAG T L I EDPDE S T S   GVESAVLRG   
               
               
                   
                   
                   FL I I GKED R   RYGPALSINELSNL A KGEKANVLIGQGD V VLV   
               
               
                   
                   
                 
                   MKRKRDSSILTDSQTATKRIRMAIN 
                 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable 
               
               
                   
               
               
                 180 
                 Influenza B 
                 M L EPFQIL S   ICSFILSALHF M AWTIGHLNQIK R GV NMKIRIK G   
               
               
                   
                 bm2 Protein 
                 PNKETI N   REVSILRH S YQKEIQAKE   A MKEVLS D NMEVLSDHI 
               
               
                   
                   
                   V   IEGLSAEEIIKMGET VLEVEELH 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 181 
                 Influenza B 
                 
                   MSLFGDTIAYLLSLTEDGEGKAELAEKLHCWFGGKEFDLDS 
                 
               
               
                   
                 bm1 Protein 
                   ALEWIKNKRCLTDIQKALIGASICFLKPKDQERKR R FITEPLS   
               
               
                   
                   
                 
                   GMGTTATKKKGLILAERKMR 
                   K 
                   CVSFHEAFEIAEGHESSALL 
                 
               
               
                   
                   
                 
                   YCLMVMYLNPGNYSMQVKLGTLCALCEKQASHSHRAHSR 
                 
               
               
                   
                   
                 
                   AARSSVPGVRREMQMVSAMNTAKTMNGMGKGEDVQKLA 
                 
               
               
                   
                   
                 
                   EELQSNIGVLRSLGASQKNGEGIAKDVMEVLKQSSMGNSAL 
                 
               
               
                   
                   
                 
                   VKKYL 
                 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 182 
                 Influenza B 
                   MAD NM TTTQIEWRMK K MAIGSS   I HSSSV L MKDIQS QFEQL   
               
               
                   
                 nep Protein 
                   KLRWESYPNLVKSTDYHQKRETI RLVTE ELYLLSKRIDDNIL   
               
               
                   
                   
                 FHKT V   IANSSIIADM V VSLSLLETLYEMKDVVEVYSRQCL   
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 183 
                 Influenza B 
                   MAD   NMTTTQIEVGPGAT NA TINFEAGILECYERLSWQ R ALD   
               
               
                   
                 ns1 Protein 
                   YPG QDRLN RLKRKLESRI KTHNKS EPESKRMSLEERK AIGV 
               
               
                   
                   
                 KMMKVLLFMNPSAGIEGFEPYCM K SSSNSNC P KY N W TD YP 
               
               
                   
                   
                 STP G RCLDDIEEEP D DVDGPT EIVLRDM NN K   DARQKIKEEV   
               
               
                   
                   
                   NTQKEGKFRLT I K RD M RNVL SLRVLVNGTF LKHPNGYKSLS 
               
               
                   
                   
                 TLHRLNAYDQSGRL VAKLVATDDLTVEDE EDGHRILNSLFE 
               
               
                   
                   
                 RLNEGHSKPIRAAETA V   GVLSQFGQEHRLSPEE GDN 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 184 
                 Influenza B 
                 M AD NMT TTQIEVVRMKKM AIGSS T HSSSVL MKDIQSQFEQL   
               
               
                   
                 ns2 Protein 
                   KLRWESYPNLVKSTDYHQ KRETIRLVT EELYLLSKRIDD NIL 
               
               
                   
                   
                 FHKTVIANSSIIADM V   VSLSLLETLYEM KDVV EVYSRQCL   
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 185 
                 Influenza B 
                   MDTFITRNFQTTIIQKA K NTMAEFSEDPELQPAMLFNICVHL   
               
               
                   
                 pa 
                   EVCYVISDMNFLDEEG KAYT ALEGQGKEQNLRPQYEVIEG   
               
               
                   
                 Protein 
                 
                   MPRTIAVVMVQRSLAQEHGIETPKYLADLFDYKTKRFIEVGI 
                 
               
               
                   
                   
                 
                   TKGLADDYFWKKKEKLGNSMELMIFSYNQDYSLSNESSLD 
                 
               
               
                   
                   
                   EEGKGRVLSRLTELQAELSLKNLWQ VLIGEEDVEKGIDF K L 
               
               
                   
                   
                 GQTISR LRDISVPAGFSNFEGMRSYIDNI DP KGAIERNLARMS   
               
               
                   
                   
                   P LVSVTPKKL K   WEDLRPIGPHIY   N HELPEVPYN AFLLMSDEL   
               
               
                   
                   
                   GLAN M TEGKSKKPKTLAKECLEKYSTLRDQTDPILI   M   KSEK   
               
               
                   
                   
                 
                   ANENFLWKLWRDCVNTISNEE 
                   M 
                   SNELQKTNYAKWATGDG 
                 
               
               
                   
                   
                 
                   LTYQKIMKEVAIDDETMCQEEPKIPNKCRVAAWVQTEMNL 
                 
               
               
                   
                   
                   LSTLT S KRALDLPEIGPD   V   APVEHVGSERRKYFVNEINYCKA   
               
               
                   
                   
                 
                   STVMMKYVLFHTSLLNESNASMGKYKVIPITNR 
                   V 
                   VNEKGES 
                 
               
               
                   
                   
                   FDMLYGLAVKGQSHLRGDTDVVTVVTFEFS STDPRVD S   GK   
               
               
                   
                   
                 
                   WPKYTVFRIGSLFV 
                   S 
                   GREKSVYLYCRVNGTNKIQMKWGME 
                 
               
               
                   
                   
                   ARRCLLQSMQQMEAIVEQESS I QGYDMTKACFKGDR VNSP 
               
               
                   
                   
                 KTFSI GTQEGKLVKGSFGKALRVIFTKCLMHYVFGNAQLEG   
               
               
                   
                   
                   FSAESRRLLLLIQALKDRK G PWVFDLEG M YSGIEECISNNPW   
               
               
                   
                   
                   VIQS A YWFNEWL GFEKEGSKVLESVDEIMDE 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 186 
                 Influenza B 
                 
                   MNINPYFLFIDVP 
                   I 
                   QAAISTTFPYTGVPPYSHGTGTG 
                   Y 
                   TIDTVI 
                 
               
               
                   
                 pb1 Protein 
                   RTHEYSNKGKQY   V SD I TGCTM V DPT NGPLPEDNEPSAYAQL   
               
               
                   
                   
                 DCVLEALDRMDEEHPGLFQAASQNAMEALMVTTVDKLTQ 
               
               
                   
                   
                 
                   GRQTFDWTVCRNQPAATALNTTITSFRLNDLNGADKGGL V   
                 
               
               
                   
                   
                 
                   PFCQDIIDSLD 
                   K 
                   PEMTFFSVKNIKKKLPAKNRKGFLIKRIPMK 
                 
               
               
                   
                   
                   V KD R IS R   VEYIKRALSLNTMTKDAERGKLKRRAIATAGIQIR   
               
               
                   
                   
                 
                   GFVLVVENLAKNICENLEQSGLPVGGNEKKAKLSNAVAKM 
                 
               
               
                   
                   
                 
                   LSNCPPGGISMTVTGDNTKWNECLNPRIFLAMTERITRDSP 
                   I 
                 
               
               
                   
                   
                   WFRDFCS I APVLFSNKIARLGKGFM   I   TSKTKRLKAQIPCPDLF   
               
               
                   
                   
                   SI P LERYNEETRAKL   KK   LKPFFNEEGTASLSPGMMMGMFN   
               
               
                   
                   
                 
                   MLSTVLGVAALGIKNIGNKEYLWDGLQSSDDFALFVNAKD 
                 
               
               
                   
                   
                   EE T CMEGINDFYRTCKLLG I NMSKKKSYCNETGMFEFTSMF   
               
               
                   
                   
                 
                   YRDGFVSNFAMEIPSFGVAGVNESADMAIGMTIIKNNMINN 
                 
               
               
                   
                   
                 
                   GMGPATAQTAIQLFIADYRYTYKCHRGDSKVEGKRMKIIKE 
                 
               
               
                   
                   
                   LWENTKGRDGLLV A DGGPNIYNLRNLHIPEIVLKY NL MDPE   
               
               
                   
                   
                   YKGRLLHPQNPFVGHLSIEGIKEADITPAHGPV K KMDYDAV   
               
               
                   
                   
                 
                   SGTHSWRTKRNRSILNTDQRNMIEEEQCYAKCCNLFEACFN 
                 
               
               
                   
                   
                   SASYRKPVGQHSMLEAMAHRL R MDARLDYESGRMSKDDF   
               
               
                   
                   
                   EKAM A HLGEIGY T 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 187 
                 Influenza B 
                 
                   MTLAKIELLKQLLRDNEAKTVLKQTTVDQYNIIRKFNTSRIE 
                 
               
               
                   
                 pb2 Protein 
                 
                   KNPSLRMKWAMCSNFPLALTKGDMANRIPLEYKGIQLKTN 
                 
               
               
                   
                   
                   AEDIGTKGQMCSIAAVTWWNTYGPIG DTEGFE K   VYESFFLR   
               
               
                   
                   
                   KMRLD N ATWGRITFGPVERVRKRVLLNPLTKEMPPDEASN   
               
               
                   
                   
                 
                   VIMEILFPKEAGIPRESTWIHRELIKEKREKLKGTMITPIVLAY 
                 
               
               
                   
                   
                 
                   MLERELVARRRFLPVAGATSAEFIEMLHCLQGENWRQIYHP 
                 
               
               
                   
                   
                 
                   GGNKLTESRSQSMIVACRKIIRRSIVASNPLELAVEIANKTVI 
                 
               
               
                   
                   
                 
                   DTEPLKSCL 
                   T 
                   AIDGGDVACDIIRAALGLKIRQRQRFGRLELK 
                 
               
               
                   
                   
                 
                   RISGRGFKNDEEILIGNGTI 
                   Q 
                   KIGIWDGEEEFHVRCGECRGIL 
                 
               
               
                   
                   
                 
                   KKSKM 
                   RM 
                   EKLLINSAKKEDM 
                   K 
                   DLIILCMVFSQDTRMFQGV 
                 
               
               
                   
                   
                 
                   RGEINFLNRAGQLLSPMYQLQRYFL 
                   S 
                   RSNDLFDQWGYEESP 
                 
               
               
                   
                   
                   KASELHGINE   L   MNASDYTLKG   V VVT K   NVIDDFSSTETEKVS   
               
               
                   
                   
                   ITKNLSL I KRTGEVIMGANDVSELESQAQLMITYDTPKMWE   
               
               
                   
                   
                 
                   MGTTKELVQNTYQWV 
                   L 
                   KNLVTEKAQFLLGKEDMFQWDAF 
                 
               
               
                   
                   
                 
                   EAFESIIPQKMAGQYSGFARAVLKQMRDQEVMKTDQFIKLL 
                 
               
               
                   
                   
                   PFCFSPPKLRSNGEPYQFL RLV LKGGGENFIEVRKGSPLFSY   
               
               
                   
                   
                 
                   NPQTEVLTICGRMMSLKGKIEDEERNRSMGNAVLAGFLVSG 
                 
               
               
                   
                   
                   KYDPDLGDFKTIEELE K LKPGEKANILLYQGKPVKVVKRKR   
               
               
                   
                   
                 
                   YSALSNDISQGIKRQRMTVESMGWALS 
                 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 188 
                 H1N1 M 
                   MSLLTEVETYVLSI   I   PSGPLKAEIAQRLE   S   VFAGKNTDLE A L   
               
               
                   
                 Protein 
                 
                   MEWLKTRPILSPLTKGILGFVFTLTVPSERGLQRRRF 
                   I 
                   QNAL 
                 
               
               
                   
                   
                   NGNGDPNNMD RAVKLY K   KLKREITFHGAKE   VS LSYS T   GAL   
               
               
                   
                   
                   ASCMGLIYNRMG   T VTTE A AFGL V   CATCEQIADSQH   R SHRQ 
               
               
                   
                   
                 M A   TTTNPLIRHENRMVLASTTAKAMEQ   V   AGSSEQAAEAME   
               
               
                   
                   
                   VA   NQT RQMV H AMR T   IGTHPSSS   A GL RD DLLE NLQAYQKR   
               
               
                   
                   
                 
                   MGVQMQRFK 
                 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 189 
                 H1N1 ns1 
                 M D S N T M SSFQVDCFLWH I RK RF AD NG LGDAPFL DRLRRDQ   
               
               
                   
                 Protein 
                   K SL K GRG N TL G L D I E TAT LV GKQIVE W IL K EESSE T L R M TI   
               
               
                   
                   
                 AS VPT SRY LS DMT L EEMSRDWFMLMP R QK II G P LC V R L DQ 
               
               
                   
                   
                 A IME KNI V L K ANFSVIF N RLE T L I LLRAFTEEGAIVGEISP 
               
               
                   
                   
                 LPS L PGHT Y EDVKNA V GVLIGGLEWN G NTVR V SE NI QRFA 
               
               
                   
                   
                 WR NCD ENG R P SLPPE QK 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 190 
                 H1N1 pa 
                 MEG FVRQCFNPM I V ELAEKAMKEYGED P KI ETNKFAAICTH   
               
               
                   
                 Protein 
                 LEV CFMYSDFHF I D E R GESIIVES G DPNA LLKHRFEIIEGRDR   I   
               
               
                   
                   
                 MAWTV VNSICNTT G V EKPKF LPDLYDYKE NRFI EIGVTRRE   
               
               
                   
                   
                 VHI YYLEKANKIK SEK THIHIFSFTGEEMAT KADY TLDEESR   
               
               
                   
                   
                   ARIKTRLFTIRQEMA SRSL WDSFRQSE RGEETIEE K FEI T GTM 
               
               
                   
                   
                 R K LAD QSLPPNF SS L ENFRAYVDGFEPNG C   IEGKLSQMS KE 
               
               
                   
                   
                 VNA K IEPFL RT TPRP LR LP D GP L C H QRSKFLLMD A LKLSIED 
               
               
                   
                   
                 P S   HEGEGIPLYDA IKCM K   TFFGWKEP   NIV KPHEKG I NPNYL 
               
               
                   
                   
                   MT WKQVL A ELQDI E NEEKIP R TKNMK R TSQLKWA LGENM   
               
               
                   
                   
                   APEK VDF D DCKD VG DLKQYDSDEPE P RS LA SW V Q N   EFNKA   
               
               
                   
                   
                   CELTDS SW IELDEIGED VAPIEHIASMRRNYFTA EVSHCRAT   
               
               
                   
                   
                   EY IM KGVYINTALLNASCAAMD D FQLIPMISKCRTKEGRR K 
               
               
                   
                   
                   TNLYGFI I KGRSHLRNDTDVVNF VS MEFSLTDPRLE PHKWE 
               
               
                   
                   
                 KYCVLEIGDM L LR T AIGQVSRPMFLYV RTNGTSKIKMKWG   
               
               
                   
                   
                   ME M RRCLLQSLQQIESMIEAESS VKEKDMTKEFFENKSETW 
               
               
                   
                   
                 PIGESPRGVEEG SIGKVCR T LLAKSVFN SLYASP QLEGFSAES   
               
               
                   
                   
                   RKLLL IV QALRDNLEPGTFDL GGLYEA IEECLINDPWVLLNA   
               
               
                   
                   
                   SWFNS FLTHALK 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 191 
                 H1N1 pb1 
                   MDVNPTLLFLK IPAQNAIST TFPYTGDPPYSHGTGTGYT M DT   
               
               
                   
                 Protein 
                   VNRTHQYSE KGKWTTN TETGAPQLNPIDGPLP EDNEPS GYA   
               
               
                   
                   
                   QTDCVLEAMAFLE E SHPGIFENSC LETMEVV QQTRVDKLTQ   
               
               
                   
                   
                   GRQT Y DWTLNRNQPAATALANTIEVF RSNGLTAN ESGRLID   
               
               
                   
                   
                   FLKDV MESMNKEEIEIT THFQRKRR VRDNMTKKMVTQRTI 
               
               
                   
                   
                 GKKKQRLNKRGYLI RALTLNTMTKDAHRGKLKRRAIATPG   
               
               
                   
                   
                   MQIRGFV YFVETLARSI CEKLEQSGLPVGGNEKKAKLANVV   
               
               
                   
                   
                   RKMM TNSQDTEI SFTITGDNTKWNENQNPR MFLAMITYITR 
               
               
                   
                   
                 NQPEWFRNILSM APIMFSNK MARLGR GYMFESK RMKIRTQI 
               
               
                   
                   
                 PAEMLASIDLKYFNESTKKKIEKIRPLLIDG TASLSPGMMMG   
               
               
                   
                   
                   MFNMLSTVLGVSILNLGQK KYTKTI YWWDGLQSSDDFALI   
               
               
                   
                   
                   VNA PNHE GIQAGV DRFYRTCKLV GINMSKKKSYIN K TGTFE   
               
               
                   
                   
                   FTSFFYRYGFVANFSMELPSFGVSG V NESADMSIG V TVIKNN   
               
               
                   
                   
                   MINNDLGPATAQMALQLF I KDYRYTYRCH RG DTQIQT RRSF 
               
               
                   
                   
                 ELKKLWDQTQSKVGLLV SDGGPNLYNIRNLHIPEVCLKWEL   
               
               
                   
                   
                   MD DDYR GRLCNPLNPFV SHKEIDSVNNAVVMPAHGPAKSM 
               
               
                   
                   
                 
                   EYDAVATTHSWIPKRNRSILN 
                 
               
               
                   
                   
                   TSQ RGILEDEQMYQKCCN LFEKFFPSSSYRRP V GISSMVEAM   
               
               
                   
                   
                 V SRARIDAR VDFESGRIKKEEFSEIMKICSTIEELRRQK 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 192 
                 H1N1 pb2 
                 AMGL RISSSFSFGGFTFKRTSGSS VKREEEV LTGNLQ TLKLT 
               
               
                   
                 Protein 
                 V HEGYEEFT MVGKRATAILRKATRRLIQLIVSGRDEQSIVEA 
               
               
                   
                   
                 IV VAMVFSQED CMV KAVRGDLNFVNRANQRLNPMHQLLR   
               
               
                   
                   
                   HFQKDA KVLFLNWGVEPIDNVMGMIGILPDMTPSTEMSMR 
               
               
                   
                   
                 GVRVSKM GVDEYS NAERVV VSIDRFLR VRDQRGNV LLSPE   
               
               
                   
                   
                   EVSETQG TEK LTITYSSS MMWEI NGPESVL I NTYQWIIRNWE   
               
               
                   
                   
                 TVKIQWSQNPTMLYNKM EFEPFQSL VPKAIRGQ YSGFVRTL   
               
               
                   
                   
                   FQQMRDVLGTFDT T QIIKLLPFAAAPP K QSRMQFSS LT VNVR   
               
               
                   
                   
                   GSG MKILVRGNSPVFNYNKTTKRLTVLGKDAGTLTEDPDE 
               
               
                   
                   
                 GTA GVESAVLRGFLI LGKEDRR YGPALSINEL SNLAK GEKA   
               
               
                   
                   
                   NVLIGQGD V VLVMKRKR DS SILTDSQTATKR IRMAIN 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable) 
               
               
                   
               
               
                 193 
                 H1N1 NS2 
                 MD SN T M SSFQDIL M RMSKMQLGSSS ED LNGM V T R FESLK I   
               
               
                   
                 Protein 
                 YRDSLGE T VMR M GDLH Y LQ S RN E KWREQL G QKFEEIRWLI 
               
               
                   
                   
                 EE M RH R LK A TENSFEQITFMQALQLL L EVEQE I RAFSFQLI 
               
               
                   
                   
                 (underline = highly conserved; 
               
               
                   
                   
                 bold = hypervariable)