Abstract:
Pandemic A(H1N1) continues its global spread, and vaccine production is a serious problem. Protection by current vaccines is limited by the mutational differences that rapidly accumulate in the circulating strains, especially in the virus surface proteins. New vaccine strategies are focusing at conserved regions of the viral internal proteins to produce T cell epitope-based vaccines. T cell responses have been shown to reduce morbidity and promote recovery in mouse models of influenza challenge. We previously reported 54 highly conserved sequences of NP, M1 and the polymerases of all human H1N1, H3N2, H1N2, and H5N1, and avian subtypes over the past 30 years. Sixty-three T cell epitopes elicited responses in HLA transgenic mice (A2, A24, B7, DR2, DR3 and DR4). These epitopes were compared to the 2007-2009 human H1N1 sequences to identify conserved and variant residues. Seventeen T cell epitopes of PB1, PB2, and M1 were selected as vaccine targets by analysis of sequence conservation and variability, functional avidity, non-identity to human peptides, clustered localization, and promiscuity to multiple HLA alleles. The vaccines composed of these epitopes, being highly conserved and temporally stable, would be useful for any avian or human influenza A virus.

Description:
[0001]    This invention was made using funding from the U.S. government. Consequently, the U.S. government retains certain rights according to the terms of N01 AI-040085. 
     
    
     TECHNICAL FIELD OF THE INVENTION 
       [0002]    This invention is related to the area of influenza viruses. In particular, it relates to vaccines and constituents of vaccines. 
       BACKGROUND OF THE INVENTION 
       [0003]    Influenza A viruses are major pathogens of avian origin, affecting humans and other mammals, with global spread and rapid evolutionary mutational change. Of particular global concern are the several ways a human influenza pandemic could emerge. One is through the occurrence of a novel highly pathogenic zoonotic strain capable of infecting humans, such as the H5N1 avian pathogen that infected 436 humans with a 60% mortality rate (as of 1 Jul. 2009, WHO). Another possibility is through mutation from a mild to an increased pathogenic human transmissible strain, such as the current A(H1N1) pandemic. The most threatening is mutations giving rise to a new highly transmissible-and-pathogenic human strain where there is no human immunity, as occurred with the original 1918 Spanish influenza. In any event, history teaches us that a vaccine to prevent a new influenza A pandemic must be effective against all future forms of the virus. 
         [0004]    Influenza A viruses are single stranded, negative-sense RNA viruses belonging to the family Orthomyxoviridae. The genome is composed of 8 RNA strands of about 13,500 bases, encoding at least ten viral proteins. The viral envelope is a lipid bilayer, consisting of the interior matrix protein 1 (M1) and three exterior transmembrane proteins: hemagglutinin (HA), neuraminidase (NA), and matrix protein 2 (M2). The viral core contains viral ribonucleoprotein complex particles, consisting of viral RNA, nucleoprotein (NP), and three polymerase proteins (PB1, PB2, and PA). Mutation in the viral RNA genome occurs by two mechanisms, known as antigenic drift and antigenic shift. Antigenic drift is the frequent occurrence of point mutations resulting from defects in RNA replication mechanisms, while antigenic shift is less frequent, involving re-assortment of the RNA segments arising from exchanges between different strains in host cells infected by multiple viruses. 
         [0005]    Protection by current human influenza vaccines is achieved by use of inactivated or attenuated forms of the corresponding pathogen and appears to require the function of neutralizing antibodies against the external HA and NA glycoproteins. However, these glycoproteins mutate rapidly through antigenic drift and current vaccines become ineffective as mutational differences accumulate in the circulating strains. To overcome the antigenic variability of influenza external glycoproteins, new vaccine strategies are increasingly directed at conserved regions of the viral internal proteins for production of T cell epitope-based vaccines against all influenza A virus subtypes and to obviate the need for yearly vaccine update. Several animal model studies taking this approach have reported T cell responses that reduce morbidity and promote recovery in mouse models of influenza challenge [1-4]. Both CD8+ and CD4+ T cell responses are required; CD8+ T cells to kill infected cells [5,6] and CD4+ T cells for the development of an effective immune response and immune memory [7-9]. However, there is limited characterization of cellular viral antigens as vaccine targets. Very few human T cell epitopes of influenza proteins other than HA and NA are reported [10]. Moreover, even for the T cell epitope peptides that were identified, the actual epitope structures and the requirements of epitope amino- and carboxyl-termini for epitope processing and presentation in humans are for most, if not all, unknown. 
         [0006]    We previously reported a detailed study of the evolutionarily conserved sequences of all human and avian influenza A viruses that were recorded over the past 30 years (36,343 sequences) [11]. Fifty-four (54) sequences of 9 or more amino acids of the PB2, PB1, PA, NP, and M1 sequences, conserved in at least 80%, and in most cases 95-100% of all recorded human H1N1, H3N2, H1N2, and H5N1, and avian subtypes were identified. These sequences have remained evolutionarily stable for all recorded influenza A viruses during the past decades, and are thus prime candidates for the development of T cell epitope-based vaccines against multiple influenza strains. However, the function of these conserved sequences as HLA-restricted T cell epitopes and the incidence of variant sequences in association with the conserved sequences were not known. 
         [0007]    There is a continuing need in the art to identify and test influenza vaccines to reduce the incidence and/or severity of influenza A infections and/or pandemics. 
       SUMMARY OF THE INVENTION 
       [0008]    According to one aspect of the invention a polypeptide is provided. The polypeptide comprises: (a) a LAMP-1 lumenal sequence comprising SEQ ID NO: 19; (b) one or more segments of an influenza A protein, wherein said segments comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-15, wherein segments are linked together by 0-20 amino acid residues; and (c) a LAMP transmembrane and cytoplasmic tail comprising SEQ ID NO: 21, wherein the lumenal sequence is amino-terminal to the one or more segments of an influenza A protein which are amino-terminal to the LAMP transmembrane and cytoplasmic tail. The polypeptides may be combined to form compositions comprising a mixture of at least two polypeptides. 
         [0009]    Other polypeptides which are provided include polypeptides consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 8, 11, and 12, as well as polypeptides which comprise less than a full-length PB1 or PB2 protein of influenza A virus and comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 8, 11, and 12. The polypeptides may be combined to form compositions comprising a mixture of at least two polypeptides. 
         [0010]    Another aspect of the invention is a polynucleotide which encodes a polypeptide. The polypeptide comprises: (a) a LAMP-1 lumenal sequence comprising SEQ ID NO: 19; (b) one or more segments of an influenza A protein, wherein said segments comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-15, wherein segments are linked together by 0-20 amino acid residues; and (c) a LAMP transmembrane and cytoplasmic tail comprising SEQ ID NO: 21, wherein the lumenal sequence is amino-terminal to the one or more segments of an influenza A protein which are amino-terminal to the LAMP transmembrane and cytoplasmic tail. Such polynucleotides can be combined to form mixtures of at least two polynucleotides. 
         [0011]    Another aspect of the invention is a polynucleotide which encodes a polypeptide. The polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 8, 11, and 12, or the polypeptide comprises less than a full-length PB1 or PB2 protein of influenza A virus and comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 8, 11, and 12. Such polynucleotides can be combined to form mixtures of at least two polynucleotides. 
         [0012]    Yet another aspect of the invention is a nucleic acid vector that comprises the polynucleotide. The polynucleotide may encode a polypeptide which comprises: (a) a LAMP-1 lumenal sequence comprising SEQ ID NO: 19; (b) one or more segments of an influenza A protein, wherein said segments comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-15, wherein segments are linked together by 0-20 amino acid residues; and (c) a LAMP transmembrane and cytoplasmic tail comprising SEQ ID NO: 21, wherein the lumenal sequence is amino-terminal to the one or more segments of an influenza A protein which are amino-terminal to the LAMP transmembrane and cytoplasmic tail. Alternatively the polynucleotide may encode a polypeptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 8, 11, and 12, or it may encode a polypeptide which comprises less than a full-length PB1 or PB2 protein of influenza A virus and comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 8, 11, and 12. 
         [0013]    Still another aspect of the invention is a host cell. The host cell comprises the nucleic acid vector that comprises the polynucleotide that encodes a polypeptide. The polypeptide comprises: (a) a LAMP-1 lumenal sequence comprising SEQ ID NO: 19; (b) one or more segments of an influenza A protein, wherein said segments comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-15, wherein segments are linked together by 0-20 amino acid residues; and (c) a LAMP transmembrane and cytoplasmic tail comprising SEQ ID NO: 21, wherein the lumenal sequence is amino-terminal to the one or more segments of an influenza A protein which are amino-terminal to the LAMP transmembrane and cytoplasmic tail. Alternatively, the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 8, 11, and 12, or the polypeptide comprises less than a full-length PB1 or PB2 protein of influenza A virus and comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 8, 11, and 12. 
         [0014]    According to another aspect of the invention a method is provided for producing a polypeptide. A host cell is cultured under conditions in which the host cell expresses a polypeptide. The polypeptide comprises: (a) a LAMP-1 lumenal sequence comprising SEQ ID NO: 19; (b) one or more segments of an influenza A protein, wherein said segments comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-15, wherein segments are linked together by 0-20 amino acid residues; and (c) a LAMP transmembrane and cytoplasmic tail comprising SEQ ID NO: 21, wherein the lumenal sequence is amino-terminal to the one or more segments of an influenza A protein which are amino-terminal to the LAMP transmembrane and cytoplasmic tail. Alternatively, the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 8, 11, and 12, or the polypeptide comprises less than a full-length PB1 or PB2 protein of influenza A virus and comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 8, 11, and 12. 
         [0015]    Another aspect of the invention is a method of producing a cellular vaccine. An antigen presenting cell is transfected with a nucleic acid vector which comprises a polynucleotide which encodes a polypeptide. The antigen presenting cells thereafter express the polypeptide. The polypeptide comprises: (a) a LAMP-1 lumenal sequence comprising SEQ ID NO: 19; (b) one or more segments of an influenza A protein, wherein said segments comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-15, wherein segments are linked together by 0-20 amino acid residues; and (c) a LAMP transmembrane and cytoplasmic tail comprising SEQ ID NO: 21, wherein the lumenal sequence is amino-terminal to the one or more segments of an influenza A protein which are amino-terminal to the LAMP transmembrane and cytoplasmic tail. Alternatively, the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 8, 11, and 12, or the polypeptide comprises less than a full-length PB1 or PB2 protein of influenza A virus and comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 8, 11, and 12. 
         [0016]    An additional aspect of the invention is a method of making a vaccine. A polypeptide and an immune adjuvant are mixed together. The polypeptide comprises: (a) a LAMP-1 lumenal sequence comprising SEQ ID NO: 19; (b) one or more segments of an influenza A protein, wherein said segments comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-15, wherein segments are linked together by 0-20 amino acid residues; and (c) a LAMP transmembrane and cytoplasmic tail comprising SEQ ID NO: 21, wherein the lumenal sequence is amino-terminal to the one or more segments of an influenza A protein which are amino-terminal to the LAMP transmembrane and cytoplasmic tail. Alternatively, the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 8, 11, and 12, or the polypeptide comprises less than a full-length PB1 or PB2 protein of influenza A virus and comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 8, 11, and 12. 
         [0017]    A further aspect of the invention is a vaccine composition which comprises a polypeptide. The polypeptide comprises: (a) a LAMP-1 lumenal sequence comprising SEQ ID NO: 19; (b) one or more segments of an influenza A protein, wherein said segments comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-15, wherein segments are linked together by 0-20 amino acid residues; and (c) a LAMP transmembrane and cytoplasmic tail comprising SEQ ID NO: 21, wherein the lumenal sequence is amino-terminal to the one or more segments of an influenza A protein which are amino-terminal to the LAMP transmembrane and cytoplasmic tail. Alternatively, the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 8, 11, and 12, or the polypeptide comprises less than a full-length PB1 or PB2 protein of influenza A virus and comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 8, 11, and 12. 
         [0018]    A further aspect of the invention is a method of immunizing a human or other animal subject. A polypeptide or a nucleic acid vector or a host cell is administered to the human or other animal subject in an amount effective to elicit influenza A-specific T cell activation. The polypeptide comprises: comprises: (a) a LAMP-1 lumenal sequence comprising SEQ ID NO: 19; (b) one or more segments of an influenza A protein, wherein said segments comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-15, wherein segments are linked together by 0-20 amino acid residues; and (c) a LAMP transmembrane and cytoplasmic tail comprising SEQ ID NO: 21, wherein the lumenal sequence is amino-terminal to the one or more segments of an influenza A protein which are amino-terminal to the LAMP transmembrane and cytoplasmic tail. Alternatively, the polypeptide consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 8, 11, and 12, or the polypeptide comprises less than a full-length PB1 or PB2 protein of influenza A virus and comprise an amino acid sequence selected from the group consisting of SEQ ID NO: 3, 4, 5, 6, 8, 11, and 12. 
         [0019]    These and other embodiments which will be apparent to those of skill in the art upon reading the specification provide the art with 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0020]      FIG. 1  shows localization of HLA-restricted T-cell epitopes of conserved sequences of influenza polymerases, NP, and M1 proteins. Numbers represent amino acid positions. Highly conserved amino acids are shown as grey boxes. T cell epitopes were restricted by HLA-DR4 (black boxes), -DR3 (blue boxes), -DR2 (brown boxes), -A24 (green boxes), and -B7 (orange boxes). 
           [0021]      FIG. 2  shows predicted HLA-supertype-restricted T-cell epitopes of conserved sequences of influenza PB2, PB1, PA, NP, and M1 proteins. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
       [0022]    The inventors have identified and characterized peptide segments of influenza virus A/New York/348/2003 (H1N1) that contain conserved sequences and elicit HLA-restricted T cell responses. HLA transgenic mice (HLA-A2, -A24, -B7, -DR2, -DR3 and -DR4) were immunized with selected peptides. The peptides that elicited T cell activation by IFN-γ ELISpot assay and thus functioned as human T cell epitope peptides were selected and analyzed for properties relevant in vaccine development. The evolutionary variability and the relationship of the 2003 H1N1 T cell epitope peptide sequences to the corresponding 2007-2009 human H1N1 sequences were studied. The results identified (i) the H1N1 HLA-restricted T cell epitope peptides in the context of pathogenic influenza A conserved sequences and (ii) the variant amino acids (aa) and percentage representation of 2007-2009 H1N1 strains as compared to the 2003 A/New York/348 strain. 
         [0023]    At least 9, 11, 13, 15, 17, 19, 20, or 21 amino acids of at least two of peptide segments identified as highly conserved and highly non-variant can optionally be linked together using 0-20 amino acids residues, such as GPGPG (alternating glycine and proline residue) linkers. Where distances between conserved sequences are small (one or two residues) and not highly variant, one may optionally join the sequences together with a natural but non-conserved amino acid or two, making larger mostly conserved segments. The linked segments may be from the same peptide segment or from different peptide segments. They may be from the same viral protein or from different viral proteins. The segments are shown in SEQ ID NO: 1-15. The linked segments form a catenate. The catenate may be flanked by two portions of the human LAMP-1 protein, also known as CD107a. The N-terminal portion is the luminal portion of the LAMP-1 protein. The C-terminal portion is the transmembrane domain and the short cytoplasmic tail. Thus the segment or the catenate is inserted in the midst of the LAMP-1 protein forming a chimeric protein. The chimeric protein may comprise at least 9 amino acids of at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the peptide segments. If duplicates are used or more than one of the at least 9-amino acid stretches from a single peptide segment are used, then more than 15 of the at least 9-amino acid stretches may be in the catenate. LAMP-1 chimeric proteins are used for antigen processing and presentation to the immune systems. 
         [0024]    The polypeptides need not be in catenates and need not be in LAMP-1 chimeric proteins. The polyepeptides may be isolated and consist of a segment as shown in SEQ ID NO:1-15, such as any of SEQ ID NO:3, 4, 5, 6, 8, 11, and 12. Such polyeptides may be made synthetically or recombinantly. They may be isolated from natural sources and enzymatically digested and purified. Any manner of making them as is known in the art may be used. Typically the polypeptides are less than full-length influenza proteins. In the case of PB1 and PB2 polypeptides, the polypeptides are less than 150, less than 125, less than 100, less than 75, or less than 50 amino acid residues of PB 1 or PB2 in length. The polypeptides may also comprise other amino acid sequences linked to the influenza sequences. The linked sequences may be selected, e.g., to facilitate processing or production. The linked sequences may be used to improve physiological processing, like the LAMP-1 sequences. The sequences may be used to improve presentation to the immune system. 
         [0025]    An alternative to catenates is mixtures of polypeptides (or polynucleotides encoding them). The mixtures may comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 of the polypeptides of SEQ ID NO: 1-15. The mixtures may also comprise immune adjuvants, as are known in the art. 
         [0026]    Any linkers may be used between influenza polypeptides in catenates. They may have glycine and proline residues in a different pattern than alternating. They may have a different length of glycine and proline residues. Linkers with other natural or non-naturally occurring amino acid residues may be used. Particular properties may be imparted by the linkers. They may provide a particular structure or property, for example a particular kink or a particular cleavable site. Design is within the skill of the art. 
         [0027]    Polynucleotides which encode the polyeptides or chimeric proteins may be designed and made by techniques well known in the art. The natural sequences used by influenza virus A may be used. Alternatively non-natural sequences may be used, including in one embodiment, sequences that are codon-optimized for humans. Design of human codon optimized sequences is well within the skill of the ordinary artisan. Data regarding the most frequently used codons in the human genome are readily available. Optimization may be applied partially or completely. 
         [0028]    The polynucleotides which encode the polyeptides or chimeric proteins can be replicated and/or expressed in vectors, such as DNA virus vectors, RNA virus vectors, and plasmid vectors. Preferably these will contain promoters for expressing the polyeptides or chimeric proteins in human or other mammalian or other animal cells. An example of a suitable promoter is the cytomegalovirus (CMV) promoter. Promoters may be inducible or repressible. They may be constitutive. They may express at high or low levels, as desired in a particular application. The vectors may be propagated in host cells for expression and collection of chimeric protein. Suitable vectors will depend on the host cells selected. In one embodiment host cells are grown in culture and the polypeptide is harvested from the cells or from the culture medium. Suitable purification techniques can be applied to the polyeptides or chimeric proteins as are known in the art. In another embodiment one transfects antigen presenting cells for ultimate delivery to a vaccinee of a cellular vaccine which expresses and presents antigen to the vaccinee. Suitable antigen presenting cells include dendritic cells, B cells, macrophages, and epithelial cells. In another embodiment vectors are directly administered to a vaccinee for expression in the vaccinee. 
         [0029]    Immune adjuvants may be administered with the vaccines of the present invention, whether the vaccines are polypeptides, polynucleotides, nucleic acid vectors, or cellular vaccines. The adjuvants may be mixed with the specific vaccine substance prior to administration or may be delivered separately to the recipient, either before, during, or after the vaccine substance is delivered. Vaccines may be produced in any suitable manner, including in cells, in eggs, and synthetically. In addition to adjuvants, booster doses may be provided. Boosters may be the same or a complementary type of vaccine. Boosters may include a conventional live or attenuated influenza A viral vaccine. Typically a high titer of T cell activation and/or antibody is desired with a minimum of adverse side effects. 
         [0030]    Any of the conventional or esoteric modes of administration may be used, including oral, mucosal, or nasal. Additionally intramuscular, intravenous, intradermal, or subcutaneous delivery may be used. The administration efficiency may be enhanced by using electroporation. Optimization of the mode of administration for the particular vaccine composition may be desirable. 
         [0031]    Whole virus, including live, attenuated, or genetically inactivated, may be used as a booster or adjuvant. The virus may be administered at the same time as, before, after, or mixed with the polypeptide or polynucleotide vaccines. 
         [0032]    An enigma of the immunobiology of influenza A is that vaccines fail to provide long term protection against infection and natural infection does not prevent reinfection. The rapid mutation of the viral proteins, particularly the external HA and NA proteins that are targets for neutralizing antibodies, is credited with a significant role in this loss of immunity. Defective adaptive immunity is also observed with several RNA viruses (including HIV-1 and dengue viruses) with high rates of mutation that result in multiple genetic variants bearing mutated T cell epitope sequences. This has resulted in widespread attention to the use of T cell epitopes incorporating conserved sequences of non-structural viral internal proteins [25-28]. However, the occurrence of reinfection, despite the human T cell response to conserved sequences after natural infection, suggests the function of a viral mechanism that intervenes in the host immune response to influenza infection. One possibility is the dual immunosuppressor roles of the influenza A NS 1 protein that inhibit innate immunity by preventing type I IFN release, as well as adaptive immunity by attenuating human dendritic cell maturation and the capacity of dendritic cells to induce T cell responses [29]. There is also the concept of immunological “original sin” where mutations in or adjacent to T cell epitopes preserve binding to MHC molecules but present an altered surface to the T-cell antigen receptor, resulting in an impaired or modified T cell response, including T cell immunosuppression [30-36]. 
         [0033]    In the examples shown below, HLA transgenic mice, HLA-A2, -A24, -B7, -DR2, -DR3 and DR4, were immunized with 196 overlapping H1N1 peptides of the A/New York/348/2003 strain that contained the highly conserved sequences of the M1, NP, PB1, PB2, and PA proteins of all reported human and avian influenza A viruses of the past 30 years [11]. Fifty-four (54) of these peptides (22 PB1, 16 PB2, 9 NP, 4 PA, and 3 M1) elicited 63 HLA-restricted T cell responses by IFN-γ ELISpot assay, where 7 peptides were restricted by multiple alleles. Further, the conserved T cell epitope peptides contained reported human T cell epitopes shared among pathogenic H1N1, H3N2 and H5N1 viral strains and were restricted by a broad range of HLA class I and II alleles. Thus, it is reasonable to expect that the conserved peptides identified here can elicit human T cell epitope responses in the context of several HLA alleles and HLA-supertypes [37] and that the memory T cells can cross-react with epitopes from H1N1, H3N2, and H5N1 [26,38,39]. The class I alleles described herein HLA-A*0201, -A*2402 and -B*0702 belong to the distinct supertypes A2, A24 and B7, respectively [40,41]. HLA class II supertypes are not as well documented but the 3 alleles of the transgenic mice of this study are assigned to supertypes DR1, DR3 and DR4 [42] based on similar protein and three-dimensional structures. 
         [0034]    Analysis of the conservation and mutational variants of these H1N1 HLA-restricted epitope peptides revealed the marked effect that single aa mutations may have on the representation of T cell epitope peptides in evolving virus populations. Over the 3 years interval (2007 to 2009) between the database records analyzed by Heiny et al. (2006) to the current 2009 H1N1 sequence analysis, only 8 of the 54 highly conserved T cell epitope peptide sequences were without mutational change. These 8 peptides (M1175-191, 181-197, PB131-47, 120-136, 126-142, 489-505, 495-511, and 548-564) were representative of almost complete conservation, 95-100%, during the previous recorded history of human H1N1 virus sequences. All others of the identified HLA-restricted T cell epitope peptides contained at least 1 aa substitution, primarily but not exclusively, of the non-conserved aa of the H1N1 peptides. Our data suggest that the most favorable sequences for a T cell epitope-based vaccine are the 17 H1N1 T cell epitope peptides of the PB1, PB2, and M1 proteins (Table 6A). These were highly conserved over the 33 years (1977-2009) of the examined database records, representing 88 to 100% of all recorded avian and human influenza A viruses, including the H1N1 isolates. These 17 T cell epitopes are clustered and have distinct advantages in the design of an epitope-based genetic vaccine, including the retention of native sequences for the function of transporters associated with antigen processing (TAPs) [43] and for the flanking sequences that are reported to modulate epitope processing and function in the selection of immunodominant epitopes [44]. Each of these 17 sequences, except M1181-197 and PB1537-553, was also characterized by high apparent functional avidity at the lowest peptide concentration of 0.1 μg/ml in the IFN-γ ELISpot assay. Several studies showed that high avidity CD8+ T-cells were more effective in limiting viral replication in vitro [45-47]. Further, the 17 T-cell epitope peptides had no identity of 8 or more continuous aa to human peptides that might trigger onset of human autoimmune diseases. It is also noteworthy that several of the epitope peptides are located in described functional domains: PB1518-575 in the interacting domain of PB1 with PB2 (PB1506-659) [48]; and the overlapping PB2126-142 and PB2132-148 in the PB1- and NP-binding domain of PB21-269 [49]. T cell epitopes within functional domains would remain conserved over time as viral mutations useful towards immune escape may disrupt the function of the domains. Thus, a vaccine comprising these 17 highly conserved T cell epitope peptides, could greatly reduce, if not eliminate, the incidence of variant amino acids of the corresponding T cell epitopes of any future influenza A pathogen. 
         [0035]    The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention. 
       EXAMPLE 1 
     Materials and Methods 
     Ethics Statement 
       [0036]    Mice were maintained in a pathogen-free facility at the Johns Hopkins University according to IACUC guidelines. 
       Influenza Peptides 
       [0037]    Peptide arrays of PB2 (BEI Cat.: NR-2616), PB1 (NR-2617), PA (NR-2618), NP (NR-2611), and Ml (NR-2613) of influenza virus A/New York/348/2003 (H1N1) were obtained through the NIH Biodefense and Emerging Infections Research Resources Repository, NIAID, NIH (BEI). A total of 196 peptides (all 17 aa long) were selected to fully cover all highly conserved sequences under study. Where these sequences spanned two or more 17 aa peptides, the consecutive peptides overlapped by 11 aa. Two immunization peptide pools for immunization were formed: one composed of 84 PB2 and 13 M1 peptides (Table 1), and the second composed of 48 PB1, 23 PA, and 28 NP peptides (Table 2). Each of the 196 peptides was dissolved in 100% DMSO and constituted to 20% with sterile filtered water. The final concentration of each peptide was 2 μg/μl. The dissolved peptides were stored at −20° C. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
             
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 The first immunization peptide pool   
               
               
                 consisted of 13 M1 and 84 PB2 peptides 
               
               
                  of A/New York/348/2003 (H1N1) containing   
               
               
                 the highly conserved aa (boldface). 
               
             
          
           
               
                 Protein 
                   
               
               
                   
               
               
                   
                 Peptides 
               
             
          
           
               
                 M1 
                   1 
                   MSLLTEVETYVLSI VPS 
                  17 
               
               
                   
               
               
                   
                   7 
                   VETYVLSI VPSGPLKAE 
                  23 
               
               
                   
               
               
                   
                 115 
                 IALSYSA GALASCMGLI   
                 131 
               
               
                   
               
               
                   
                 121 
                 A GALASCMGLIYNRMG A 
                 137 
               
               
                   
               
               
                   
                 127 
                   CMGLIYNRMG AVTTESA 
                 143 
               
               
                   
               
               
                   
                 169 
                 TNPLIR HENRMVLASTT   
                 185 
               
               
                   
               
               
                   
                 175 
                 
                   HENRMVLASTTAKAMEQ 
                 
                 191 
               
               
                   
               
               
                   
                 181 
                 
                   LASTTAKAMEQMAGSSE 
                 
                 197 
               
               
                   
               
               
                   
                 187 
                 
                   KAMEQMAGSSEQAAEAM 
                 
                 203 
               
               
                   
               
               
                   
                 193 
                   AGSSEQAAEAME VAS QA   
                 209 
               
               
                   
               
               
                   
                 199 
                   AAEAME VAS QARQMVQA   
                 215 
               
               
                   
               
               
                   
                 205 
                 VAS QARQMVQAMRA IGT 
                 221 
               
               
                   
               
               
                   
                 210 
                   RQMVQAMRA IGTHPSSS 
                 226 
               
               
                   
               
               
                 PB2 
                   1 
                 MERIKELRN LMSQSRTR   
                  17 
               
               
                   
               
               
                   
                   7 
                 LRN LMSQSRTREILTKT   
                  23 
               
               
                   
               
               
                   
                  12 
                 
                   SQSRTREILTKTTVDHM 
                 
                  28 
               
               
                   
               
               
                   
                  18 
                 
                   EILTKTTVDHMAIIKKY 
                 
                  34 
               
               
                   
               
               
                   
                  24 
                 
                   TVDHMAIIKKYTSGRQE 
                 
                  40 
               
               
                   
               
               
                   
                  30 
                   IIKKYTSGRQEKNP S LR   
                  46 
               
               
                   
               
               
                   
                  36 
                   SGRQEKNP S LRMKWMMA   
                  52 
               
               
                   
               
               
                   
                  42 
                   NP S LRMKWMMAMKYPIT   
                  58 
               
               
                   
               
               
                   
                  48 
                   KWMMAMKYPITADKRI T 
                  64 
               
               
                   
               
               
                   
                  54 
                   KYPITADKRI TEMI PER   
                  70 
               
               
                   
               
               
                   
                  60 
                   DKRI TEMI PERNEQGQT   
                  76 
               
               
                   
               
               
                   
                  66 
                 MI PERNEQGQTLWSK VN 
                  82 
               
               
                   
               
               
                   
                  72 
                   EQGQTLWSK VNDAGSDR 
                  88 
               
               
                   
               
               
                   
                  78 
                   WSK VNDAGSDRVMI SPL   
                  94 
               
               
                   
               
               
                   
                  84 
                 AGSDRVMI SPLAVTWWN   
                 100 
               
               
                   
               
               
                   
                  90 
                 MI SPLAVTWWNRNGP VA 
                 106 
               
               
                   
               
               
                   
                  96 
                   VTWWNRNGP VANTIHYP 
                 112 
               
               
                   
               
               
                   
                 102 
                   NGP VANTIHYPKIYKTY 
                 118 
               
               
                   
               
               
                   
                 108 
                 TIHYPKIYKTYFE KVER   
                 124 
               
               
                   
               
               
                   
                 114 
                 IYKTYFE KVERLKHGTF   
                 130 
               
               
                   
               
               
                   
                 120 
                 E KVERLKHGTFGPVHFR   
                 136 
               
               
                   
               
               
                   
                 126 
                 
                   KHGTFGPVHFRNQVKIR 
                 
                 142 
               
               
                   
               
               
                   
                 132 
                   PVHFRNQVKIRRRVD IN 
                 148 
               
               
                   
               
               
                   
                 137 
                   NQVKIRRRVD INPGHAD 
                 153 
               
               
                   
               
               
                   
                 143 
                   RRVD INPGHADLSAKEA 
                 159 
               
               
                   
               
               
                   
                 215 
                 TRFLPVAGGTSSV YIEV   
                 231 
               
               
                   
               
               
                   
                 221 
                 AGGTSSV YIEVLHLTQG   
                 237 
               
               
                   
               
               
                   
                 227 
                 V YIEVLHLTQGTCWEQM   
                 243 
               
               
                   
               
               
                   
                 233 
                 
                   HLTQGTCWEQMYTPGGE 
                 
                 249 
               
               
                   
               
               
                   
                 239 
                   CWEQMYTPGGEV R NDDV   
                 255 
               
               
                   
               
               
                   
                 245 
                   TPGGEV R NDDVDQSLII   
                 261 
               
               
                   
               
               
                   
                 251 
                 R NDDVDQSLIIAARNIV   
                 267 
               
               
                   
               
               
                   
                 256 
                   DQSLIIAARNIVRRA AV 
                 272 
               
               
                   
               
               
                   
                 262 
                   AARNIVRRA AVSADPL A   
                 278 
               
               
                   
               
               
                   
                 268 
                   RRA AVSADPL ASLLEM   
                 283 
               
               
                   
               
               
                   
                 273 
                 SADPL ASLLEMCHSTQI   
                 289 
               
               
                   
               
             
          
           
               
                   
                 Sequences 
               
             
          
           
               
                   
                 279 
                   SLLEMCHSTQIGG TRMV 
                 295 
               
               
                   
               
               
                   
                 285 
                   HSTQIGG TRMVDILRQN 
                 301 
               
               
                   
               
               
                   
                 339 
                 KREEEV LTGNLQTLK LT 
                 355 
               
               
                   
               
               
                   
                 345 
                   LTGNLQTLK LT VHEGYE   
                 361 
               
               
                   
               
               
                   
                 351 
                   TLK LT VHEGYEEFTMVG   
                 367 
               
               
                   
               
               
                   
                 357 
                   HEGYEEFTMVG K RATAI   
                 373 
               
               
                   
               
               
                   
                 363 
                   FTMVG K RATAILRKATR   
                 379 
               
               
                   
               
               
                   
                 369 
                   RATAILRKATRR LIQLI 
                 385 
               
               
                   
               
               
                   
                 393 
                 SIVEAIV VAMVFSQED   
                 408 
               
               
                   
               
               
                   
                 398 
                 IV VAMVFSQEDCM V KAV   
                 414 
               
               
                   
               
               
                   
                 404 
                   FSQEDCM V KAVRGDLNF   
                 420 
               
               
                   
               
               
                   
                 410 
                   M V KAVRGDLNFVNRANQ   
                 426 
               
               
                   
               
               
                   
                 416 
                 
                   GDLNFVNRANQRLNPMH 
                 
                 432 
               
               
                   
               
               
                   
                 422 
                 
                   NRANQRLNPMHQLLRHF 
                 
                 438 
               
               
                   
               
               
                   
                 428 
                 
                   LNPMHQLLRHFQKDAKV 
                 
                 444 
               
               
                   
               
               
                   
                 434 
                   LLRHFQKDAKVLF LNWG 
                 450 
               
               
                   
               
               
                   
                 440 
                   KDAKVLF LNWGIEHIDN 
                 456 
               
               
                   
               
               
                   
                 458 
                 MGMIGILP DMTPSTEMS   
                 474 
               
               
                   
               
               
                   
                 464 
                 LP DMTPSTEMS MRGVRV 
                 480 
               
               
                   
               
               
                   
                 470 
                   STEMS MRGV RVSKMGVD   
                 486 
               
               
                   
               
               
                   
                 476 
                 RGV RVSKMGVDEYS NAE 
                 492 
               
               
                   
               
               
                   
                 482 
                   KMGVDEYS NAERVVVSI 
                 498 
               
               
                   
               
               
                   
                 500 
                 RFLRVRDQR GNVLLSPE   
                 516 
               
               
                   
               
               
                   
                 506 
                 DQR GNVLLSPEEVSETQ   
                 522 
               
               
                   
               
               
                   
                 512 
                   LLSPEEVSETQG TEKLT 
                 528 
               
               
                   
               
               
                   
                 518 
                   VSETQG TEK LTITYSSS   
                 534 
               
               
                   
               
               
                   
                 524 
                 TEK LTITYSSSMMWEIN   
                 540 
               
               
                   
               
               
                   
                 530 
                 
                   TYSSSMMWEINGPESVL 
                 
                 546 
               
               
                   
               
               
                   
                 536 
                   MWEINGPESVL I NTYQW   
                 552 
               
               
                   
               
               
                   
                 542 
                   PESVL I NTYQWIIRNWE   
                 558 
               
               
                   
               
               
                   
                 548 
                   NTYQWIIRNWE TVKIQW 
                 564 
               
               
                   
               
               
                   
                 554 
                   IRNWE TVKIQWSQNPTM 
                 570 
               
               
                   
               
               
                   
                 560 
                 VKIQWSQNPT MLYNKME   
                 576 
               
               
                   
               
               
                   
                 565 
                 SQNPT MLYNKMEFEPFQ   
                 581 
               
               
                   
               
               
                   
                 571 
                 
                   LYNKMEFEPFQSLVPKA 
                 
                 587 
               
               
                   
               
               
                   
                 577 
                   FEPFQSLVPKA IRGQYS 
                 593 
               
               
                   
               
               
                   
                 606 
                 VLGTFDTT QIIKLLPFA   
                 622 
               
               
                   
               
               
                   
                 612 
                 TT QIIKLLPFAAAPP K Q   
                 628 
               
               
                   
               
               
                   
                 618 
                   LLPFAAAPP K QSRMQFS   
                 634 
               
               
                   
               
               
                   
                 624 
                   APP K QSRMQFSSLTVNV   
                 640 
               
               
                   
               
               
                   
                 630 
                 
                   RMQFSSLTVNVRGSGMR 
                 
                 646 
               
               
                   
               
               
                   
                 636 
                   LTVNVRGSGMRIL VRGN 
                 652 
               
               
                   
               
               
                   
                 642 
                   GSGMRIL VRGNSPVFNY 
                 658 
               
               
                   
               
               
                   
                 678 
                 DPDEGTA GVESAVLRGF   
                 694 
               
               
                   
               
               
                   
                 684 
                 A GVESAVLRGFLI LGKE 
                 700 
               
               
                   
               
               
                   
                 690 
                   VLRGFLI LGKEDR RYGP   
                 706 
               
               
                   
               
               
                   
                 696 
                   I LGKEDR RYGPALSIN E 
                 712 
               
               
                   
               
               
                   
                 702 
                 R RYGPALSIN ELSNLAK 
                 718 
               
               
                   
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 The second immunization peptide pool 
               
               
                 consisted of 28 NP, 23 PA and 48 PB1 
               
               
                 peptides of A/New York/348/2003 (H1N1) 
               
               
                 containing the highly conserved aa 
               
               
                 (boldface). 
               
             
          
           
               
                   
                 Protein 
                 Sequences 
               
               
                   
                   
               
             
          
           
               
                   
                 NP 
                   1 
                   MASQGTKRSYEQMET DG 
                  17 
               
               
                   
                   
               
               
                   
                   
                   7 
                   KRSYEQMET DGERQNAT 
                  23 
               
               
                   
                   
               
               
                   
                   
                  25 
                 IRASVGRMIG GIGRFYI   
                  41 
               
               
                   
                   
               
               
                   
                   
                  31 
                 RMIG GIGRFYIQMCTEL   
                  47 
               
               
                   
                   
               
               
                   
                   
                  37 
                   GRFYIQMCTELKL NDYE 
                  53 
               
               
                   
                   
               
               
                   
                   
                  43 
                   MCTELKL NDYEGRLIQN 
                  59 
               
               
                   
                   
               
               
                   
                   
                  61 
                 LTIER MVLSAFDERRN K 
                  77 
               
               
                   
                   
               
               
                   
                   
                  67 
                   VLSAFDERRN K YLEEHP   
                  83 
               
               
                   
                   
               
               
                   
                   
                  73 
                   ERRN K YLEEHPSAGKDP   
                  89 
               
               
                   
                   
               
               
                   
                   
                  79 
                 
                   LEEHPSAGKDPKKTGGP 
                 
                  95 
               
               
                   
                   
               
               
                   
                   
                  85 
                   AGKDPKKTGGPIY KRVD 
                 101 
               
               
                   
                   
               
               
                   
                   
                  91 
                   KTGGPIY KRVDGKWVRE 
                 107 
               
               
                   
                   
               
               
                   
                   
                 103 
                 KWVRELV LYDKEEIRRI   
                 119 
               
               
                   
                   
               
               
                   
                   
                 109 
                 V LYDKEEIRRIWRQANN   
                 125 
               
               
                   
                   
               
               
                   
                   
                 115 
                   EIRRIWRQANNG DDATA 
                 131 
               
               
                   
                   
               
               
                   
                   
                 121 
                   RQANNG DDATAGLTHI M   
                 137 
               
               
                   
                   
               
               
                   
                   
                 127 
                 DDATAGLTHI MIWHSNL   
                 143 
               
               
                   
                   
               
               
                   
                   
                 133 
                 LTHI MIWHSNLND TTYQ 
                 149 
               
               
                   
                   
               
               
                   
                   
                 139 
                   WHSNLND TTYQRTRALV 
                 155 
               
               
                   
                   
               
               
                   
                   
                 234 
                 AQKAMM DQVRESRNPGN   
                 250 
               
               
                   
                   
               
               
                   
                   
                 240 
                 
                   DQVRESRNPGNAEIEDL 
                 
                 256 
               
               
                   
                   
               
               
                   
                   
                 246 
                   RNPGNAEIEDL TFLARS 
                 262 
               
               
                   
                   
               
               
                   
                   
                 402 
                 SAGQIST QPTFSVQRNL   
                 418 
               
               
                   
                   
               
               
                   
                   
                 408 
                 T QPTFSVQRNLPF DKTT 
                 424 
               
               
                   
                   
               
               
                   
                   
                 414 
                   VQRNLPF DKTTIMAAFT 
                 430 
               
               
                   
                   
               
               
                   
                   
                 450 
                 SARPEEVSFQ GRGVFEL   
                 466 
               
               
                   
                   
               
               
                   
                   
                 456 
                 VSFQ GRGVFELSDE RAT 
                 472 
               
               
                   
                   
               
               
                   
                   
                 462 
                   GVFELSDE RATNPIVPS 
                 478 
               
               
                   
                   
               
               
                   
                 PA 
                  24 
                 YGEDL KIETNKFAAICT   
                  40 
               
               
                   
                   
               
               
                   
                   
                  30 
                 
                   IETNKFAAICTHLEVCF 
                 
                  46 
               
               
                   
                   
               
               
                   
                   
                  36 
                 
                   AAICTHLEVCFMYSDFH 
                 
                  52 
               
               
                   
                   
               
               
                   
                   
                  42 
                   LEVCFMYSDFHFI NEQG 
                  58 
               
               
                   
                   
               
               
                   
                   
                  48 
                   YSDFHFI NEQGESIIVE 
                  64 
               
               
                   
                   
               
               
                   
                   
                 120 
                 IGVTRREVHI YYLEKAN   
                 136 
               
               
                   
                   
               
               
                   
                   
                 126 
                 EVHI YYLEKANKIKSE K 
                 142 
               
               
                   
                   
               
               
                   
                   
                 132 
                   LEKANKIKSE K THIHIF   
                 148 
               
               
                   
                   
               
               
                   
                   
                 138 
                   IKSE K THIHIFSFTGEE   
                 154 
               
               
                   
                   
               
               
                   
                   
                 144 
                   HIHIFSFTGEEMA TKAD 
                 160 
               
               
                   
                   
               
               
                   
                   
                 150 
                   FTGEEMA TKADYTLDEE 
                 166 
               
               
                   
                   
               
               
                   
                   
                 179 
                 RQEMAS RGLWDSFRQSE   
                 195 
               
               
                   
                   
               
               
                   
                   
                 185 
                 
                   RGLWDSFRQSERGEETI 
                 
                 201 
               
               
                   
                   
               
               
                   
                   
                 191 
                   FRQSERGEETIEE RFEI 
                 207 
               
               
                   
                   
               
               
                   
                   
                 197 
                   GEETIEE RFEITGTLRR 
                 213 
               
               
                   
                   
               
               
                   
                   
                 292 
                 IEDPN HEGEGIPLYDAI   
                 308 
               
               
                   
                   
               
               
                   
                   
                 298 
                   EGEGIPLYDAIKC MRTF 
                 314 
               
               
                   
                   
               
               
                   
                   
                 304 
                   LYDAIKC MRTFFGWKEP 
                 320 
               
               
                   
                   
               
               
                   
                   
                 404 
                 SSWIQN EFNKACELTDS   
                 420 
               
               
                   
                   
               
               
                   
                   
                 410 
                   EFNKACELTDS IWIELD 
                 426 
               
               
                   
                   
               
               
                   
                   
                 552 
                 SAIGQV SRPMFLYVRTN   
                 568 
               
               
                   
                   
               
               
                   
                   
                 558 
                   SRPMFLYVRTNGTSK IK 
                 574 
               
               
                   
                   
               
               
                   
                   
                 564 
                   YVRTNGTSK IKMKWGME 
                 580 
               
               
                   
                   
               
               
                   
                 PB1 
                   1 
                 
                   MDVNPTLLFLKVPAQNA 
                 
                  17 
               
               
                   
                   
               
               
                   
                   
                   7 
                   LLFLKVP A QNAISTTFP   
                  23 
               
               
                   
                   
               
               
                   
                   
                  13 
                   P A QNAISTTFPYTGDPP   
                  29 
               
               
                   
                   
               
               
                   
                   
                  19 
                 
                   STTFPYTGDPPYSHGTG 
                 
                  35 
               
               
                   
                   
               
               
                   
                   
                  25 
                 
                   TGDPPYSHGTGTGYTMD 
                 
                  41 
               
               
                   
                   
               
               
                   
                   
                  31 
                 
                   SHGTGTGYTMDTVNRTH 
                 
                  47 
               
               
                   
                   
               
               
                   
                   
                  37 
                   GYTMDTVNRTHQYSE RG 
                  53 
               
               
                   
                   
               
               
                   
                   
                  43 
                   VNRTHQYSE RGRWTKNT 
                  59 
               
               
                   
                   
               
               
                   
                   
                 108 
                 IETMEV VQQTRVDKLTQ   
                 124 
               
               
                   
                   
               
               
                   
                   
                 114 
                 
                   VQQTRVDKLTQGRQTYD 
                 
                 130 
               
               
                   
                   
               
               
                   
                   
                 120 
                 
                   DKLTQGRQTYDWTLNRN 
                 
                 136 
               
               
                   
                   
               
               
                   
                   
                 126 
                 
                   RQTYDWTLNRNQPAATA 
                 
                 142 
               
               
                   
                   
               
               
                   
                   
                 132 
                 
                   TLNRNQPAATALANTIE 
                 
                 148 
               
               
                   
                   
               
               
                   
                   
                 138 
                   PAATALANTIE VFRSNG 
                 154 
               
               
                   
                   
               
               
                   
                   
                 191 
                 VRDNV TKKMVTQRTIGK   
                 207 
               
               
                   
                   
               
               
                   
                   
                 197 
                   KKMVTQRTIGKKK HKLD 
                 213 
               
               
                   
                   
               
               
                   
                   
                 203 
                   RTIGKKK HKLDKRSYLI 
                 219 
               
               
                   
                   
               
               
                   
                   
                 328 
                 NQPEWFRNI LSIAPIMF   
                 344 
               
               
                   
                   
               
               
                   
                   
                 334 
                 RNI LSIAPIMFSNKMAR   
                 350 
               
               
                   
                   
               
               
                   
                   
                 340 
                 
                   APIMFSNKMARLGKGYM 
                 
                 356 
               
               
                   
                   
               
               
                   
                   
                 346 
                   NKMARLGKGYMFESK S M   
                 362 
               
               
                   
                   
               
               
                   
                   
                 352 
                   GKGYMFESK S MKLRTQI   
                 368 
               
               
                   
                   
               
               
                   
                   
                 358 
                   ESK S MKLRTQIPAEMLA   
                 374 
               
               
                   
                   
               
               
                   
                   
                 364 
                   LRTQIPAEMLA NIDLKY 
                 380 
               
               
                   
                   
               
               
                   
                   
                 465 
                 RFYRTCKLL GINMSKKK   
                 481 
               
               
                   
                   
               
               
                   
                   
                 471 
                 KLL GINMSKKKSYIN R T   
                 487 
               
               
                   
                   
               
               
                   
                   
                 477 
                   MSKKKSYIN R TGTFEFT   
                 493 
               
               
                   
                   
               
               
                   
                   
                 483 
                   YIN R TGTFEFTSFFYRY   
                 499 
               
               
                   
                   
               
               
                   
                   
                 489 
                 
                   TFEFTSFFYRYGFVANF 
                 
                 505 
               
               
                   
                   
               
               
                   
                   
                 495 
                 
                   FFYRYGFVANFSMELPS 
                 
                 511 
               
               
                   
                   
               
               
                   
                   
                 501 
                   FVANFSMELPSFGVSG V 
                 517 
               
               
                   
                   
               
               
                   
                   
                 507 
                   MELPSFGVSG V NESADM   
                 523 
               
               
                   
                   
               
               
                   
                   
                 513 
                   GVSG V NESADMSIGVTV   
                 529 
               
               
                   
                   
               
               
                   
                   
                 519 
                 
                   ESADMSIGVTVIKNNMI 
                 
                 535 
               
               
                   
                   
               
               
                   
                   
                 525 
                 
                   IGVTVIKNNMINNDLGP 
                 
                 541 
               
               
                   
                   
               
               
                   
                   
                 531 
                 
                   KNNMINNDLGPATAQMA 
                 
                 547 
               
               
                   
                   
               
               
                   
                   
                 537 
                 
                   NDLGPATAQMALQLFIK 
                 
                 553 
               
               
                   
                   
               
               
                   
                   
                 543 
                 
                   TAQMALQLFIKDYRYTY 
                 
                 559 
               
               
                   
                   
               
               
                   
                   
                 548 
                 
                   LQLFIKDYRYTYRCHRG 
                 
                 564 
               
               
                   
                   
               
               
                   
                   
                 554 
                 
                   DYRYTYRCHRGDTQIQT 
                 
                 570 
               
               
                   
                   
               
               
                   
                   
                 560 
                 
                   RCHRGDTQIQTRRSFEI 
                 
                 576 
               
               
                   
                   
               
               
                   
                   
                 566 
                   TQIQTRRSFE IKKLWDQ 
                 582 
               
               
                   
                   
               
               
                   
                   
                 650 
                 GPAKN MEYDAVATTHSW   
                 666 
               
               
                   
                   
               
               
                   
                   
                 656 
                   EYDAVATTHSW V PKRNR   
                 672 
               
               
                   
                   
               
               
                   
                   
                 662 
                   TTHSW V PKRNRSILNTS   
                 678 
               
               
                   
                   
               
               
                   
                   
                 668 
                 
                   PKRNRSILNTSQRGILE 
                 
                 684 
               
               
                   
                   
               
               
                   
                   
                 674 
                 
                   ILNTSQRGILEDEQMYQ 
                 
                 690 
               
               
                   
                   
               
               
                   
                   
                 680 
                   RGILEDEQMYQ RCCNLF 
                 696 
               
               
                   
                   
               
             
          
         
       
     
       HLA Transgenic Mice 
       [0038]    Six different strains of HLA transgenic mice were used to cover HLA alleles of class I and class II supertypes. The HLA class I supertypes studied were HLA-A2 (A*0201) mice expressing a chimeric heavy chain with murine α3 domain and human β2m. Both H-2Db and murine β2m genes were disrupted by homologous recombination [12], HLA-A24 (A*2402) mice express a chimeric heavy chain and human β2m; the H-2Kb, H-2Db, and murine β2m genes were disrupted by homologous recombination (Lemonnier et al., unpublished), HLA-B7 (B*0702) mice express a chimeric heavy chain with the HLA-B*0702 α1 and α2 domains and the H-2Kd murine α3 domain [13]. The H-2Kb and H-2Db genes in HLA-B7 mice were inactivated by homologous recombination. 
         [0039]    The HLA class II supertypes were DR2 (DRB1*1501), DR3 (DRB1*0301), and DR4 (DRB1*0401). The peptide-binding domain of HLA-DR2 transgenic mice is encoded by human sequences, while the membrane proximal portion containing the CD4-binding domain is encoded by mouse sequences (DRA1*0101: I-Eα and DRB1*1501: I-Eβ) [14]. HLA-DR3 transgenic mice express HLA-DRA*0101 and -DRB1*0301 [15]. HLA-DR4 transgenic mice express HLA-DRA*0101, -DRB1*0401, and human CD4 [16]. The derivation and validation of the above transgenic mice, which were bred onto C57BL/6 genetic background, had been described in the relevant publications. 
       Immunization 
       [0040]    Mice were immunized with the selected 196 peptides in 2 pools by use of a protocol which had been validated for T cell studies [17] and adapted for these transgenic mice studies. Peptides were pooled in matrixes as described [18] and injected in groups of 9 mice of each transgenic strain: two for matrix array screening, two for identifying individual peptides, four for characterizing apparent functional avidity of T cells to positive peptides at three titration points: 10, 1, and 0.1 μg/ml peptide concentrations, and one as a control (adjuvant alone). Mice were injected subcutaneously at the base of tail with 100 μl of the immunization peptide pool in TiterMax® Gold adjuvant (TiterMax, Norcross, Ga.) (1:1). The amount of each peptide injected was 1 μg/mouse. After two weeks, spleens were harvested for IFN-γ ELISpot assay. 
       IFN-γ ELISpot Assay 
       [0041]    Harvested spleens from immunized transgenic mice were selectively depleted of T cells by use of anti-CD8 or anti-CD4 antibody-coated immunomagnetic beads with LD columns (Miltenyi Biotec, Auburn, Calif.) according to the manufacturer&#39;s protocol. Flow cytometry analysis of the depleted cells indicated this method routinely achieved &gt;95% depletion of the targeted cells. The resulting MHC class I or II depleted splenocytes were tested individually by IFN-γ ELISpot assays against the 196 influenza peptides arranged in two 10×10 matrix arrays, resulting in 40 peptide pools, where each peptide was present in two different pools, as described [18]. Peptides identified as immunogenic in the matrix array screen were retested individually in a confirmatory assay and a peptide titration assay. Thus, each ELISpot positive response was confirmed three times: by matrix array screening, individually by confirmatory assay and by peptide titration. 
         [0042]    The ELISpot assays were performed using mouse IFN-γ ELISpot sets from BD Biosciences (San Jose, Calif.) according to the manufacturer&#39;s protocol. Briefly, the ELISpot plates were coated with anti-IFN-γ at 5 μg/ml and incubated at 4° C. overnight. The plates were blocked with RPMI 1640 containing 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 μg of streptomycin/ml, and 100 U of penicillin for 2 h at room temperature, and either CD8+- or CD4+-depleted splenocytes (0.5-1.0×10̂6 cells/well) were then added for assays of class II and I T cell epitopes, respectively. The cells were cultured at 37° C. in 5% CO2 in the presence of peptide pools (final concentration of each peptide was 10 μg/ml) or individual peptides at final concentrations of 10 μg/ml, 1 μg/ml, and 0.1 μg/ml. Wells with medium alone served as background; Concanavalin A (2.5 μg/ml; Sigma-Alrich, St. Louis, Mo.) was used as a polyclonal stimulator; and known HLA-restricted peptides from Dengue serotype 3 were included in each assay as positive controls. After 16 h of culture, the plates were washed and incubated with biotinylated anti-IFN-γ for 2 h at room temperature, followed by HRP-conjugated streptavidin for 1 h at room temperature. Reactions were developed with AEC substrate (Calbiochem-Novabiochem, San Diego, Calif.). Final enumeration of IFN-γ spot-forming cells (SFC) was performed using the Immunospot Series 3B Analyzer ELISPOT reader (Cellular Technologies, Shaker Heights, Ohio) with aid of the Immunospot software version 3.0 (Cellular Technologies), indicating the number of SFC/10̂6 cells. The results were considered positive if the number of SFC subtracted by those in the background (culture with medium alone) was above 10 and the number of SFC was higher than the background plus two standard deviations. The results shown are SFC minus background, which was consistently found to be less than 15 spots/10̂6 cells throughout the experiments. 
       Presence of Experimentally Identified T Cell Epitopes in the Influenza a Highly Conserved Sequences 
       [0043]    Published influenza T cell epitopes within the highly conserved sequences were identified by matching the curated T cell epitope sequences mapped in human from the Immune Epitope Database and Analysis Resource (IEDB, http://www.immuneepitope.org/) [19] with the highly conserved sequences. All these published epitope sequences were derived from various T cell assays that included T cell proliferation, IFN-γ ELISpot, HLA tetramer staining, and 51Cr release assays. Only epitope data from unique sequences and containing HLA restriction information were included. 
       Determination of Human Self-Peptide in Influenza Peptides 
       [0044]    The 196 influenza 17 aa peptides were compared using the blastp program against the non-redundant protein sequences database restricted to human (taxid:9606) at NCBI (http://ww.ncbi.nlm.nih.gov/BLAST/) to detect the presence of fragments identical to human peptides. As the default search parameters were not suitable to probe for short peptide sequences of length 30 or less, the following parameters were used: word size of 2, expectation value of 30,000, matrix was PAM30, low complexity filter was disabled, and composition-based statistics was set to ‘no adjustment’. We disregarded search results containing predicted sequences and human peptides with fewer than six contiguous identical residues as the probability of matching five or less residues is high and non-significant. 
       Conservation and Variability of Influenza A(H1N1) T Cell Epitope Peptides 
       [0045]    The dataset and methodology for identification of highly conserved influenza protein sequences among pathogenic influenza strains for the past 30 years had been described by Heiny et al. [11]. Briefly, 3763 NP, 3781 M1, 3111 PA, 3175 PB1, and 3144 PB2 sequences were extracted from the NCBI GenBank and GenPept databases (as of September 2006) and multiple sequence alignments of the individual proteins were performed. The Antigenic Variability Analyzer tool (AVANA) [20] was used to extract alignments of each 17 aa T cell epitope mapped in the transgenic mice and to identify the most frequent 17 aa sequence present in at least 80% of all recorded viruses. To compare 2007-2009 human H1N1 sequences with the T cell epitopes of A/New York/348/2003 (H1N1), aligned protein sequence records of human H1N1 M1, PB1, and PB2 retrieved from the NCBI Influenza Virus Sequence Database (http://www.ncbi.nlm.nih.gov/genomes/FLU/FLU.html, as of Jun. 17, 2009) were submitted into the AVANA tool to identify the most frequent sequence and its variants for each year. 
       EXAMPLE 2 
     Results 
     Immunogenicity of Human and Avian Influenza A Highly Conserved Peptide Sequences 
       [0046]    The previously described 54 highly conserved influenza A peptide sequences of 9 or more contiguous aa of the recorded human and avian influenza strains were represented by a total of 956 aa [11]. The majority of the conserved sequences, 650 aa, were in the PB1 and PB2 proteins; there were no conserved sequence in NA, M2, NS1, and NS2. A total of 196 peptides (BEI) of the A/New York/348/2003 (H1N1) M1, NP, PA, PB1, and PB2 proteins were selected based on the presence of the conserved sequences. The immunogenicity of these 196 conserved influenza peptides was studied by immunizing HLA-A2, -A24, -B7, -DR2, -DR3 and -DR4 transgenic mice. Organization of the 54 conserved sequences in the BEI 17 aa peptides depended on their length and position. Conserved sequences that spanned adjacent 17 aa peptides were repeated up to a maximum of 11 aa because of overlapping peptide synthesis (Table 1 and 2). Peptides with conserved sequences of less than 17 aa contained mixtures of conserved and non-conserved aa. Thirty-three (33) short conserved sequences (9 to 16 aa) were present in various lengths with adjacent non-conserved aa. Conserved sequences of greater length (22 sequences of 17 to 57 aa) were present as complete (65 of the 196 peptides) or partial sequences in the overlapping peptides. The longest conserved sequence was PB 1518-575 which was included as part of a cluster of completely conserved aa of 7 overlapping peptides. 
         [0047]    Immunization of the HLA transgenic mice with the 196 H1N1 peptides was carried out with 2 pools of about 100 peptides each, with groups of 9 mice of each transgenic strain. Interferon-γ (IFN-γ) ELISpot assays for HLA-restricted class I and class II responses were performed with splenocytes of the immunized mice that were depleted of CD4+ and CD8+ T cells, respectively, to identify the responding T cell subset. The initial assays contained matrix arrays of peptide pools followed by validation assays with individual peptides [18]. Of the 196 peptides, 54 contained T cell epitopes that elicited 63 ELISpot responses (8 A24, 2 B7, 16 DR2, 17 DR3, and 20 DR4) (Table 3). None of the 196 peptides tested induced T cell responses in mice expressing the HLA-A2 allele. Forty-seven (47) of the 54 epitope peptides were restricted by one HLA allele; eight class I and 39 class II. The remaining 7 peptides were presented by at least two HLA alleles of distinct supertypes i.e. they contained multiple or promiscuous T cell epitopes. PB1680-696 and PB2548-564 were presented by both HLA class I and II alleles. Sixteen (16) pairs of consecutive peptides were restricted by the same HLA allele, possibly because there were identical epitopes in the overlapping 11 aa sequence shared by the 2 adjacent peptides. Clusters of 2 or more T cell epitope peptides with at least 16 conserved aa were M1175-197, PB1120-142, 340-374, 489-576, and PB242-64, 126-146 (Table 3,  FIG. 1 ). 
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 HLA-A24, -B7, -DR2, -DR3 and -DR4 restriction of 54 peptides of influenza proteins M1, 
               
               
                 NP, PA, PB1 and PB2 that contain conserved sequences of 9 or more amino acids. 
               
             
          
           
               
                 Pro- 
                   
                   
                   
                   
                   
                   
               
               
                 tein 
                 ELISpot positive 17 aa peptide* 
                 A24# 
                 B7 
                 DR2 
                 DR3 
                 DR4 
               
               
                   
               
               
                 M1 
                 169 TNPLIR HENRMVLASTT  185 
                 — 
                 — 
                  56 ± 5(0.1) 
                 120 ± 4(0.1) 
                 — 
               
               
                   
                       175  HENRMVLASTTAKAMEQ  191 
                 — 
                 — 
                 — 
                 — 
                 165 ± 1(0.1) 
               
               
                   
                             181  LASTTAKAMEQMAGSSE  197 
                 — 
                 — 
                 — 
                 — 
                 115 ± 21(1) 
               
               
                   
               
               
                 NP 
                   7  KRSYEQMET DGERQNAT 23 
                 — 
                 — 
                 — 
                 — 
                  52 ± 29(0.1) 
               
               
                   
                  31 RMIG GIGRFYIQMCTEL  47 
                  45 ± 5 
                 — 
                 — 
                 — 
                 — 
               
               
                   
                   
                 (0.1) 
                   
                   
                   
                   
               
               
                   
                        37  GRFYIQMCTELKL NDYE 53 
                 — 
                 — 
                 — 
                  66 ± 7(1) 
                 — 
               
               
                   
                  73  ERRN K YLEEHPSAGKDP  89 
                 — 
                 — 
                 — 
                 — 
                 121 ± 1(0.1) 
               
               
                   
                 103 KWVRELV LYDKEEIRRI  119 
                 — 
                 — 
                 — 
                 614 ± 21(0.1) 
                 — 
               
               
                   
                       109 V LYDKEEIRRIWRQANN  125 
                 — 
                 — 
                 — 
                 501 ± 42(0.1) 
                 — 
               
               
                   
                 133 LTHI MIWHSNLND TTYQ 149 
                 — 
                 — 
                 238 ± 59(0.1) 
                 — 
                 — 
               
               
                   
                 402 SAGQIST QPTFSVQRNL  418 
                 — 
                 — 
                 207 ± 3(0.1) 
                 — 
                 — 
               
               
                   
                       408 T QPTFSVQRNLPF DKTT 424 
                 — 
                 — 
                 110 ± 14(1) 
                  41 ± 2(10) 
                 — 
               
               
                   
               
               
                 PA 
                  42  LEVCFMYSDFHFI NEQG 58 
                 — 
                 — 
                  64 ± 11(1) 
                 — 
                 — 
               
               
                   
                 126 EVHI YYLEKANKIKSE K 142 
                 — 
                 — 
                 — 
                 — 
                  37 ± 11(0.1) 
               
               
                   
                       132  LEKANKIKSE K THIHIF  148 
                 — 
                 — 
                 — 
                 — 
                  41 ± 10(0.1) 
               
               
                   
                 558  SRPMFLYVRTNGTSK IK 574 
                 — 
                 — 
                 — 
                 — 
                 114 ± 24(0.1) 
               
               
                   
               
               
                 PB1 
                  31  SHGTGTGYTMDTVNRTH  47 
                 — 
                 — 
                 — 
                 — 
                 106 ± 1(0.1) 
               
               
                   
                        37  GYTMDTVNRTHQYSE RG 53 
                 — 
                 — 
                 — 
                 — 
                 125 ± 11(0.1) 
               
               
                   
                 120  DKLTQGRQTYDWTLNRN  136 
                 — 
                 — 
                 — 
                 142 ± 6(0.1) 
                 — 
               
               
                   
                       126  RQTYDWTLNRNQPAATA  142 
                 — 
                 — 
                 — 
                  78 ± 0(0.1) 
                 — 
               
               
                   
                 328 NQPEWFRNI LSIAPIMF  344 
                 — 
                  60 ± 8 
                 — 
                 — 
                 — 
               
               
                   
                   
                   
                 (10) 
                   
                   
                   
               
               
                   
                 340  APIMFSNKMARLGKGYM  356 
                 — 
                 — 
                 — 
                 175 ± 0(0.1) 
                 — 
               
               
                   
                 352  GKGYMFESKSMKLRTQI  368 
                 — 
                 — 
                  52 ± 2(1) 
                 — 
                 — 
               
               
                   
                       358  ESK S MKLRTQIPAEMLA  374 
                 — 
                 — 
                  84 ± 20(0.1) 
                 — 
                 — 
               
               
                   
                 465 RFYRTCKLL GINMSKKK  481 
                 — 
                 — 
                 231 ± 73(1) 
                 — 
                 — 
               
               
                   
                       471 KLL GINMSKKKSYIN R T  487 
                 — 
                 — 
                 — 
                 116 ± 10(0.1) 
                 — 
               
               
                   
                 489  TFEFTSFFYRYGFVANF  505 
                 213 ± 9 
                 — 
                 — 
                 — 
                 — 
               
               
                   
                   
                 (0.1) 
                   
                   
                   
                   
               
               
                   
                       495  FFYRYGFVANFSMELPS  511 
                 210 ± 25 
                 — 
                 — 
                 — 
                 — 
               
               
                   
                   
                 (0.1) 
                   
                   
                   
                   
               
               
                   
                 507  MELPSFGVSG V NESADM  523 
                 — 
                 — 
                 — 
                 — 
                 274 ± 15(0.1) 
               
               
                   
                 519  ESADMSIGVTVIKNNMI  535 
                 — 
                 — 
                  75 ± 10(0.1) 
                 — 
                 — 
               
               
                   
                       525  IGVTVIKNNMINNDLGP  541 
                 — 
                 — 
                 159 ± 53(0.1) 
                 — 
                 — 
               
               
                   
                 537  NDLGPATAQMALQLFIK  553 
                  92 ± 2(1) 
                 — 
                 — 
                 — 
                 — 
               
               
                   
                 548  LQLFIKDYRYTYRCHRG  564 
                 — 
                 — 
                  61 ± 2(1) 
                 230 ± 23(0.1) 
                  97 ± 30(0.1) 
               
               
                   
                       554  DYRYTYRCHRGDTQIQT  570 
                 — 
                 — 
                 109 ± 13(1) 
                 166 ± 22(0.1) 
                  76 ± 2(0.1) 
               
               
                   
                             560  RCHRGDTQIQTRRSFE I 576 
                 — 
                 — 
                 194 ± 47(0.1) 
                 — 
                 — 
               
               
                   
                 650 GPAKN MEYDAVATTHSW  666 
                 — 
                 — 
                 — 
                 142 ± 45(0.1) 
                  41 ± 9(0.1) 
               
               
                   
                       656  EYDAVATTHSW V PKRNR  672 
                 — 
                 — 
                 — 
                 — 
                  59 ± 2(0.1) 
               
               
                   
                 680  RGILEDEQMYQ RCCNLF 696 
                  78 ± 4 
                 — 
                 — 
                 181 ± 10(0.1) 
                 — 
               
               
                   
                   
                 (0.1) 
                   
                   
                   
                   
               
               
                   
               
               
                 PB2 
                  42  NP S LRMKWMMAMKYPIT  58 
                 — 
                 — 
                 — 
                 — 
                 166 ± 3(0.1) 
               
               
                   
                        48  KWMMAMKYPITADKRI T 64 
                 — 
                 — 
                 — 
                 — 
                 161 ± 18(0.1) 
               
               
                   
                              54  KYPITADKRI TEMI PER  70 
                 — 
                 — 
                 — 
                 499 ± 4(0.1) 
                 — 
               
               
                   
                 126  KHGTFGPVHFRNQVKIR  142 
                 — 
                 — 
                 — 
                 — 
                 316 ± 20(0.1) 
               
               
                   
                       132  PVHFRNQVKIRRRVD IN 148 
                 — 
                 — 
                 — 
                 — 
                 311 ± 37(0.1) 
               
               
                   
                 256  DQSLIIAARNIVRRA AV 272 
                 — 
                 — 
                 — 
                 169 ± 12(0.1) 
                 — 
               
               
                   
                 369  RATAILRKATRR LIQLI 385 
                 — 
                 — 
                 — 
                 — 
                  54 ± 2(0.1) 
               
               
                   
                 434  LLRHFQKDAKVLF LNWG 450 
                 — 
                 — 
                 — 
                 444 ± 14(0.1) 
                 — 
               
               
                   
                 458 MGMIGILP DMTPSTEMS  474 
                 — 
                 — 
                 — 
                 — 
                 238 ± 5(0.1) 
               
               
                   
                       464 LP DMTPSTEMS MRGVRV 480 
                 — 
                 — 
                 — 
                 324 ± 28(0.1) 
                 — 
               
               
                   
                 500 RFLRVRDQR GNVLLSPE  516 
                 — 
                 184 ± 3 
                 — 
                 — 
                 — 
               
               
                   
                   
                   
                 (0.1) 
                   
                   
                   
               
               
                   
                 524 TEK LTITYSSSMMWEIN  540 
                 — 
                 — 
                 151 ± 67(0.1) 
                 — 
                 — 
               
               
                   
                 536  MWEINGPESVL I NTYQW  552 
                 289 ± 16 
                 — 
                 — 
                 — 
                 — 
               
               
                   
                   
                 (0.1) 
                   
                   
                   
                   
               
               
                   
                       542  PESVL I NTYQWIIRNWE  558 
                 226 ± 5 
                 — 
                 — 
                 — 
                 — 
               
               
                   
                   
                 (0.1) 
                   
                   
                   
                   
               
               
                   
                             548  NTYQWIIRNWE TVKIQW 564 
                 322 ± 44 
                 — 
                  96 ± 9(0.1) 
                 — 
                 — 
               
               
                   
                   
                 (0.1) 
                   
                   
                   
                   
               
               
                   
                 630  RMQFSSLTVNVRGSGMR  646 
                 — 
                 — 
                 104 ± 16(0.1) 
                 — 
                 — 
               
               
                   
               
               
                   
                 ELISpot responses 
                 8 
                 2 
                 16 
                 17 
                 20 
               
               
                   
               
               
                 *Conserved aa are in boldface. Consecutive peptides overlapping by 11 aa are aligned. 
               
               
                 #Numbers are representative average IFN-γ spots forming cells per million splenocytes of individual transgenic mice that were positive at 10 μg/ml of peptide concentration. Number (10, 1 or 0.1) in parenthesis represents the lowest concentration of peptide (μg/ml) giving positive ELISpot response in peptide titration. 
               
               
                 — represents no positive ELISpot response. 
               
             
          
         
       
     
         [0048]    The apparent functional avidity of T cells to each of the 54 peptides was titrated at three peptide concentrations of 10, 1 and 0.1 μg/ml in IFN-γ ELISpot assays. Of the 63 positive ELISpot responses, including the responses of peptides restricted by multiple HLA alleles, 52 activated IFN-γ secretion at each of the three concentrations used in the ELISpot assay, 9 elicited at concentrations of 10 and 1 μg/ml, and 2 peptides (NP408-424 and PB1328-344) elicited solely at the highest peptide concentration (Table 3). 
       EXAMPLE 3 
     Presence of Reported T Cell Epitopes in the Conserved Sequences of Influenza A 
       [0049]    The conserved peptides of this study were compared with reported T cell epitope sequences of humans infected with influenza A viruses extracted from the IEDB. Twenty-one (21) of about 800 reported human T cell epitopes of PB2, PB1, PA, NP, and M1 were found to contain sequences of 9 or more conserved amino acids of all recorded 1977-2006 influenza A viruses (Table 4). These were mainly from H1N1, H3N2, and H5N1 infections and included sequences restricted by a broad range of HLA class I and II alleles, including many not covered by the transgenic mice of this study. For example, the same T cell epitope “RMVLASTTAK” in M1178-187 was reported to be restricted by HLA-A3 and -A11 [21,22]. Clusters of overlapping epitopes were also observed within the conserved sequences, for example, M1123-137 had three overlapping epitopes (123 ALASCMGLIY 132 was restricted by A1; 125 ASCMGLIY 132 by B35; and 129 GLIYNRMGA 137 by A2) [21,23]. Thus, the highly conserved sequences contained common epitopes shared by pathogenic influenza strains and could be restricted by a broad range of HLA alleles. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Presence of reported human influenza A T cell epitopes in 21 highly conserved aa 
               
               
                 peptides of A/New York/348/2003 (H1N1). 
               
             
          
           
               
                   
                   
                 HLA allele 
                 Published 
                   
               
             
          
           
               
                 Highly conserved 17 aa eptide* 
                 this work #   
                 HLA alleles 
                 Influenza strain 
               
               
                   
               
             
          
           
               
                 M1 
                   1 
                 M           SI VPS 
                  17 
                 — 
                 A2 
                 A/Puerto Rico/8/34 (H1N1) 
               
               
                 M1 
                 121 
                 A           A   
                 137 
                 — 
                 A1, A2, B35, 
                 A/Vietnam/1203/2004 (H5N1), Influenza 
               
               
                   
                   
                   
                   
                   
                 DRB1*0404 
                 A (H3N2) 
               
               
                 M1 
                 169 
                 TNPL IR             
                 185 
                 DR2, DR3 
                 B39, DR2, DRB1*0103, 
                 A/Vietnam/1203/2004 (H5N1), Influenza 
               
               
                   
                   
                   
                   
                   
                 DRB1*1101,  
                 A 
               
               
                   
                   
                   
                   
                   
                 DRB1*0701, DRB5*0101 
                   
               
               
                 M1 
                 175 
                 
                           
                 
                 191 
                 DR4 
                 A3, A11, DRB1*0701 
                 A/Puerto Rico/8/34 (H1N1), 
               
               
                   
                   
                   
                   
                   
                   
                 A/Vietnam/1203/2004 (H5N1) 
               
               
                   
               
               
                 NP 
                  61 
                 LTIER          K 
                  77 
                 — 
                 A3 
                 Influenza A 
               
               
                 NP 
                  67 
                   VLSAFDERRN K YLEEHP   
                  83 
                 — 
                 DRB1*0101 
                 A/Vietnam/1203/2004 (H5N1) 
               
               
                 NP 
                  73 
                   ERRN K YLEEHPSAGKDP   
                  89 
                 DR4 
                 DR1, DRB1*0101 
                 A/NT/60/68 (H3N2), A/Vietnam/1203/2004 
               
               
                   
                   
                   
                   
                   
                   
                 (H5N1) 
               
               
                 NP 
                  91 
                            KRVDGKWVRE 
                 107 
                 DR3 
                 A68 
                 A/Texas/1/77 (H3N2) 
               
               
                 NP 
                 109 
                 V LYDKEEIRRIWRQANN   
                 125 
                 DR3 
                 DRB1*1101 
                 A/Vietnam/1203/2004 (H5N1) 
               
               
                 NP 
                 402 
                 SAGQIST QPTFSVQRNL   
                 418 
                 DR2 
                 DRB1*0101, 
                 A/Vietnam/1203/2004 (H5N1) 
               
               
                   
                   
                   
                   
                   
                 DRB1*0404 
                   
               
               
                   
               
               
                 PA 
                  42 
                            NEQG 
                  58 
                 DR2 
                 A2 
                 A/Puerto Rico/8/34 (H1N1) 
               
               
                   
               
               
                 PB1 
                   1 
                 
                           
                 
                  17 
                 — 
                 A2 
                 Influenza A 
               
               
                 PB1 
                  37 
                            RG 
                  53 
                 DR4 
                 A26 
                 Influenza A 
               
               
                 PB1 
                 346 
                 
                           
                 
                 362 
                 — 
                 B62, B27 
                 Influenza A 
               
               
                 PB1 
                 352 
                   GKGYM            S           RTQI   
                 368 
                 DR2 
                 B44 
                 Influenza A 
               
               
                 PB1 
                 489 
                 
                           
                   RYGFVANF 
                 
                 505 
                 A24 
                 A1, B44 
                 Influenza A 
               
               
                 PB1 
                 501 
                             VSG V 
                 517 
                 — 
                 A2 
                 Influenza A 
               
               
                 PB1 
                 537 
                 
                   NDL 
                           
                   QLFIK 
                 
                 553 
                 A24 
                 B7 
                 Influenza A 
               
               
                 PB1 
                 560 
                   RCHRGD            EI 
                 576 
                 DR2 
                 B62 
                 Influenza A 
               
               
                 PB1 
                 566 
                   TQIQT             KKL WDQ 
                 582 
                 — 
                 B27 
                 Influenza A (H3N2) 
               
               
                   
               
               
                 PB2 
                  48 
                   K             TADKR IT 
                  64 
                 DR4 
                 A2 
                 A/Puerto Rico/8/34 (H1N1) 
               
               
                   
               
               
                 *Conserved aa are in boldface. Published HLA epitopes were extracted from the IEDB. HLA class I epitopes are underlined and the first amino acid of each identified allele is italicized. HLA class II epitopes longer than 17aa are represented only by the corresponding residues in the 17aa peptides of A/New York/348/2003 (H1N1). 
               
               
                   # —represents no positive ELISpot response. 
               
             
          
         
       
     
       EXAMPLE 4 
     Analysis of the Presence of Human aa Sequences in Influenza Peptides 
       [0050]    Each of the 196 influenza 17 aa peptides used in this study was compared with the human proteome sequences to investigate the possibility of human antigens that could trigger an autoimmune response to immunization. Specifically, we screened for exactly identical sequences of at least 8 continuous aa, which is the minimum binding peptide length for MHC class I [24]. Many of the conserved sequences of the influenza peptides contained sequences of 6 aa found in human proteins such as voltage-gated sodium channel, dystrophin etc. The longest influenza A sequence with an identical human counterpart was 7 aa of PA131-137 but none contained sequences of 8 or more aa identical to the human proteome. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 Determination of human self-peptides in representative influenza 17aa peptides. 
               
             
          
           
               
                 Viral peptide* 
                 Human peptide 
                 Human protein name 
                 GenPept ID 
               
               
                   
               
             
          
           
               
                 M1 
                 169 
                 TNPLIR HENR             T   
                 185 
                   26 MVLAST    31 
                 Ring finger protein 220 
                 NP_060620 
               
               
                 M1 
                 175 
                 
                   HENRMVLAST 
                           
                   Q 
                 
                 191 
                  140 TAKAME   145 
                 Mediator of cell motility 1 
                 NP_057039 
               
               
                 M1 
                 181 
                 
                   LASTTAKAM 
                           
                   SSE 
                 
                 197 
                 1387 EQMAGS  1392 
                 MYST histone acetyltransferase 3 
                 NP_001092882 
               
               
                   
               
               
                 NP 
                   7 
                             T DGERQNAT 
                  23 
                  582 KRSYEQ   587 
                 Metastasis associated protein 
                 NP_004680 
               
               
                 NP 
                 103 
                 KWVRELV LYDK             
                 119 
                  121 EEIRRI   126 
                 Annexin IV 
                 NP_001144 
               
               
                 NP 
                 402 
                 SAGQIST Q             
                 418 
                   80 PTFSVQ    85 
                 Mucin 6, gastric 
                 NP_005952 
               
               
                 NP 
                 408 
                 T          DKTT 
                 424 
                 1805 QPTFSV  1810 
                 Chromodomain helicase DNA binding 
                 NP_079410 
               
               
                   
                   
                   
                   
                   
                 protein 9 
                   
               
               
                   
               
               
                 PA 
                 126 
                 EVHI Y            K 
                 142* 
                 1266 YLEKANK 1272 
                 Dystrophin Dp427c isoform 
                 NP_000100 
               
               
                   
                   
                   
                   
                 1274 YLEKANK 1280 
                 Dystrophin Dp427m isoform 
                 NP_003997 
               
               
                   
                   
                   
                   
                 1151 YLEKANK 1157 
                 Dystrophin Dp427l isoform 
                 NP_003998 
               
               
                   
                   
                   
                   
                 1270 YLEKANK 1276 
                 Dystrophin Dp427pl isoform 
                 NP_004000 
               
               
                   
               
               
                 PB1  
                  31 
                 
                   SHGTGT 
                           
                   NRTH 
                 
                  47 
                 3151 GYTMDT  3156 
                 Polydom 
                 NP_699197 
               
               
                 PB1 
                  31 
                 
                   SHGTG 
                           
                   TVNRTH 
                 
                  47 
                 2141 TGYTMD  2146 
                 Multiple EGF-like-domains 8 
                 NP_001401 
               
               
                 PB1 
                 471 
                 KLL GIN             YIN R T   
                 487 
                  609 MSKKKS   614 
                 Suppressor variegation 4-20 homolog 1 
                 NP_060105 
               
               
                   
                   
                   
                   
                   
                 isoform 1 
                   
               
               
                 PB1 
                 489 
                 
                   TFEFT 
                           
                   GFVANF 
                 
                 505 
                  561 SFFYRY   566 
                 Phosphatidylinositol glycan anchor 
                 NP_036459 
               
               
                   
                   
                   
                   
                   
                 biosynthesis 
                   
               
               
                 PB1 
                 537 
                 
                   NDLG 
                           
                   QLFIK 
                 
                 553 
                  919 PATAQM   924 
                 Rho GTPase-activating protein 
                 NP_055530 
               
               
                 PB1 
                 548 
                 
                   LQLFIK 
                           
                   RCHRG 
                 
                 564 
                  231 DYRYTY   236 
                 Syntaxin binding protein 5 isoform a 
                 NP_640337 
               
               
                   
               
               
                 PB2 
                 256 
                   DQSLIIA            AV 
                 272 
                  725 ARNIVR   730 
                 Akt substrate AS250 
                 NP_065076 
               
               
                 PB2 
                 256 
                   DQSLI            AV 
                 272 
                 1301 IAARNI  1306 
                 ATP-binding cassette, sub-family A, 
                 NP_525023 
               
               
                   
                   
                   
                   
                   
                 member 6 
                   
               
               
                 PB2 
                 458 
                 MGMIGILP           
                 474 
                 1964 DMTPST  1969 
                 Voltage-gated sodium channel Type II, 
                 NP_066287 
               
               
                   
                   
                   
                   
                   
                 isoform 1 
                   
               
               
                 PB2 
                 458 
                 MGMIGILP           
                 474 
                 1964 DMTPST  1969 
                 Voltage-gated sodium channel Type II, 
                 NP_001035233 
               
               
                   
                   
                   
                   
                   
                 isoform 2 
               
               
                   
               
               
                 *Conserved aa are in boldface. Italicized aa are found in human peptides. + PA131-137 shared 7aa identity with human Dystrophin Dp427 isoform proteins. 
               
             
          
         
       
     
       EXAMPLE 5 
     Variants of the Conserved T Cell Epitope Sequences 
       [0051]    The 54 HLA-restricted T cell epitope peptides of A/New York/348/2003 (H1N1) strain were analyzed by the Antigenic Variability Analyzer (AVANA) tool for identification of (a) the consensus sequence (most frequent sequence) in the context of influenza A conserved sequences over the past 30 years, and (b) variants and percentage representation of 2007-2009 human H1N1 strains as compared to the 2003 H1N1 strain. Based on their conservation and variability, the 54 T cell epitope peptides formed three groups: 
         [0052]    1) Seventeen (17) T cell epitope peptide sequences of the 2003 strain (11 PB1, 4 PB2, and 2 M1) had consensus sequences representing at least 88% and, for all but 2 consensus sequences represented at least 95% of all recorded human and avian influenza strains (Table 6A). In particular, PB1489-505 was 100% conserved in all H1N1 viruses. Several variant sequences within this group were recorded, but these were mostly single conservative amino acid substitutions representing a small fraction (less than 5%) of all the recorded 1977-2006 virus sequences. The major change in 2009 was the apparent complete replacement of 2 previous consensus sequences by variant sequences, each with 1 mutated aa (PB2132-148, 630-646). 
         [0000]    
       
         
               
             
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6(A) 
               
             
             
               
                   
               
               
                 Representation of 26 H1N1 T cell epitope peptide sequences among 
               
               
                 all influenza A 1977-2003 strains and H1N1 strains 2007-2009. 
               
               
                 A) 17 H1N1 sequences corresponding to the consensus sequences 
               
               
                 with at least 88% representation. B) 9 sequences with single 
               
               
                 amino acid substitutions from the consensus sequences 
               
               
                 (≧80% representation). 
               
             
          
           
               
                   
                   
                 1977-2006 
                 2007 
                 2008 
                 2009 
               
               
                   
                 A/New York/348/2003 H1N1 
                 Influenza 
                 human 
                 human 
                 human 
               
               
                 Protein 
                 ELISpot positive peptide§ 
                 A* 
                 H1N1″ 
                 H1N1{circumflex over ( )} 
                 H1N1+ 
               
               
                   
               
             
          
           
               
                 PB1 
                  31 
                 
                   SHGTGTGYTMDTVNRTH 
                 
                  47 
                  99 
                 100 
                 100 
                 100 
               
               
                   
               
               
                   
                 120 
                 
                   DKLTQGRQTYDWTLNRN 
                 
                 136 
                  97 
                 100 
                 100 
                 100 
               
               
                   
               
               
                   
                 126 
                 
                   RQTYDWTLNRNQPAATA 
                 
                 142 
                  99 
                 100 
                 100 
                 100 
               
               
                   
               
               
                   
                 340 
                 
                   APIMFSNKMARLGKGYM 
                 
                 356 
                  96 
                  98 
                 100 
                  92 
               
               
                   
               
               
                   
                   
                 -------------R--- 
                   
                   2 
                   2 
                 — 
                   8 
               
               
                   
               
               
                   
                 489 
                 
                   TFEFTSFFYRYGFVANF 
                 
                 505 
                 100 
                 100 
                 100 
                 100 
               
               
                   
               
               
                   
                 495 
                 
                   FFYRYGFVANFSMELPS 
                 
                 511 
                  99 
                 100 
                 100 
                 100 
               
               
                   
               
               
                   
                 519 
                 
                   ESADMSIGVTVIKNNMI 
                 
                 535 
                  97 
                 100 
                 100 
                  99 
               
               
                   
               
               
                   
                   
                 ----------------T 
                   
                 # 
                 — 
                 — 
                   1 
               
               
                   
               
               
                   
                 525 
                 
                   IGVTVIKNNMINNDLGP 
                 
                 541 
                  97 
                 100 
                 100 
                  99 
               
               
                   
               
               
                   
                 537 
                 
                   NDLGPATAQMALQLFIK 
                 
                 553 
                  98 
                 100 
                 100 
                  99 
               
               
                   
               
               
                   
                   
                 S---------------- 
                   
                   0.11 
                 — 
                 — 
                   1 
               
               
                   
               
               
                   
                 548 
                 
                   LQLFIKDYRYTYRCHRG 
                 
                 564 
                  98 
                 100 
                 100 
                 100 
               
               
                   
               
               
                   
                 554 
                 
                   DYRYTYRCHRGDTQIQT 
                 
                 570 
                  98 
                 100 
                 100 
                  99 
               
               
                   
               
               
                   
                   
                 ------------A---- 
                   
                   0.04 
                 — 
                 — 
                   1 
               
               
                   
               
               
                 PB2 
                 126 
                 
                   KHGTFGPVHFRNQVKIR 
                 
                 142 
                  96 
                  96 
                 — 
                  98 
               
               
                   
               
               
                   
                   
                 -Y--------------- 
                   
                 # 
                 — 
                 — 
                   1 
               
               
                   
               
               
                   
                   
                 ---S------------- 
                   
                 # 
                 — 
                 — 
                   1 
               
               
                   
               
               
                   
                   
                 -Q--------------- 
                   
                   0.14 
                   3 
                 100 
                 — 
               
               
                   
               
               
                   
                 132 
                 
                   PVHFRNQVKIRRRVDIN 
                 
                 148 
                  88 
                 100 
                 100 
                 — 
               
               
                   
               
               
                   
                   
                 ---------------T- 
                   
                   4 
                 — 
                 — 
                 100 
               
               
                   
               
               
                   
                 500 
                 RFLRVRDQR GNVLLSPE   
                 516 
                  92 
                 100 
                 100 
                 100 
               
               
                   
               
               
                   
                 630  
                 
                   RMQFSSLTVNVRGSGMR 
                 
                 646 
                  97 
                 100 
                 100 
                 — 
               
               
                   
               
               
                   
                   
                 ---------------L- 
                   
                   1 
                 — 
                 — 
                 100 
               
               
                   
               
               
                 M1 
                 175 
                 
                   HENRMVLASTTAKAMEQ 
                 
                 191 
                  98 
                 100 
                 100 
                 100 
               
               
                   
               
               
                   
                 181 
                 
                   LASTTAKAMEQMAGSSE 
                 
                 197 
                  95 
                 100 
                 100 
                 100 
               
               
                   
               
               
                 §Highly conserved aa of 1977-2006 influenza A subtypes are in boldface. 
               
               
                 *3175 PB1, 3144 PB2, and 3781 M1 human H1N1, H3N2, H1N2, H5N1, and avian H5N1 and other avian subtypes sequences circulating between 1977 and 2006 were extracted from NCBI GenBank and GenPept databases as of September 2006. Sequences representing less than 1% were not included unless they were also represented in the 2007-2009 strains. 
               
               
                 All human PB1, PB2, and M1 H1N1 sequences from 2007 to 2009 were extracted from the Influenza Virus Resource on Jun 17, 2009. 
               
               
                 +168 PB1, 171 PB2, and 280 M1 human H1N1 2009 sequences. 
               
               
                 {circumflex over ( )}31 PB1, 31 PB2, and 39 M1 human H1N1 2008 sequences. 
               
               
                 ″314 PB1, 314 PB2, and 393 M1 human H1N1 2007 sequences. 
               
               
                 #New sequence representation not found in the 1977-2006 influenza A subtypes sequences. 
               
             
          
         
       
     
         [0000]    
       
         
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 6 (B) 
               
               
                   
               
               
                   
                 A/New York/348/2003 
                 1977-2006 
                 2007 
                 2008 
                 2009 
               
               
                   
                 H1N1 ELISpot 
                 Influenza 
                 human 
                 human 
                 human 
               
               
                 Protein 
                 positive peptide§ 
                 A* 
                 H1N1″ 
                 H1N1{circumflex over ( )} 
                 H1N1+ 
               
               
                   
               
             
             
               
                   
               
             
          
           
               
                 PB1 
                   
                 ---------------K- 
                   
                 86 
                 — 
                 — 
                  99 
               
               
                   
               
               
                   
                  37 
                   GYTMDTVNRTHQYSE RG 
                 53 
                 13 
                  99 
                  84 
                 — 
               
               
                   
               
               
                   
                   
                 -----------R---K- 
                   
                 # 
                 — 
                 — 
                   1 
               
               
                   
               
               
                   
                   
                 -----------H----- 
                   
                 # 
                 — 
                  16 
                 — 
               
               
                   
               
               
                   
                   
                 ----------I------ 
                   
                 89 
                   1 
                 — 
                 — 
               
               
                   
               
               
                   
                 507 
                   MELPSFGVSG V NESADM   
                 523 
                 10 
                  99 
                 100 
                 100 
               
               
                   
               
               
                   
                   
                 ----------------L 
                   
                 86 
                 — 
                 — 
                  99 
               
               
                   
               
               
                   
                 560 
                   RCHRGDTQIQTRRSFE I 
                 576 
                 11 
                 100 
                 100 
                 — 
               
               
                   
               
               
                   
                   
                 ------A---------L 
                   
                  0.04 
                 — 
                 — 
                   1 
               
               
                   
               
               
                   
                   
                 ----S------------ 
                   
                 84 
                 — 
                 — 
                 100 
               
               
                   
               
               
                   
                 650 
                 GPAKN MEYDAVATTHSW   
                 666 
                 12 
                  99 
                  97 
                 — 
               
               
                   
               
               
                   
                   
                 -----I----------- 
                   
                  0.68 
                 — 
                   3 
                 — 
               
               
                   
               
               
                   
                   
                 ----T------------ 
                   
                  0.42 
                   1 
                 — 
                 — 
               
               
                   
               
               
                   
                   
                 -----------I----- 
                   
                 87 
                 — 
                 — 
                  96 
               
               
                   
               
               
                   
                 656 
                   EYDAVATTHSW V PKRNR   
                 672 
                 11 
                 100 
                 100 
                 — 
               
               
                   
               
               
                   
                   
                 -----------T----- 
                   
                  0.76 
                 — 
                 — 
                   4 
               
               
                   
               
               
                   
                   
                 -----------K----- 
                   
                 85 
                 — 
                 — 
                 100 
               
               
                   
               
               
                   
                 680 
                   RGILEDEQMYQ RCCNLF 
                 696 
                 10 
                  98 
                  87 
                 — 
               
               
                   
               
               
                   
                   
                 --V-------------- 
                   
                  0.23 
                   1 
                  10 
                 — 
               
               
                   
               
               
                   
                   
                 ----------L------ 
                   
                 # 
                 — 
                   3 
                 — 
               
               
                   
               
               
                 PB2 
                   
                 -------------Q--- 
                   
                 89 
                 — 
                 — 
                 100 
               
               
                   
               
               
                   
                 434 
                   LLRHFQKDAKVLF LNWG 
                 450 
                 7 
                  97 
                 100 
                 — 
               
               
                   
               
               
                   
                   
                 ---------R------- 
                   
                  0.03 
                   1 
                 — 
                 — 
               
               
                   
               
               
                   
                   
                 ----------I------ 
                   
                  0.03 
                   1 
                 — 
                 — 
               
               
                   
               
               
                   
                   
                 -----------V----- 
                   
                 90 
                 — 
                 — 
                  99 
               
               
                   
               
               
                   
                 536 
                   MWEINGPESVL I NTYQW   
                 552 
                  8 
                 100 
                 100 
                   1 
               
               
                   
               
               
                   
                   
                 -----V----------- 
                   
                 84 
                 — 
                 — 
                  99 
               
               
                   
               
               
                   
                 542 
                   PESVL I NTYQWIIRNWE   
                 558 
                  8 
                  99 
                 100 
                   1 
               
               
                   
               
             
          
         
       
     
         [0053]    2) A group of 9 PB1 and PB2 T cell epitope peptides of the New York/348/2003 H1N1 strain were variants of the 1977-2006 total recorded influenza A virus population at a single mutated aa position (Table 6B). These variant New York/348/2003 strain sequences represented less than 15% of the consensus sequences of the entire 1977-2006 avian and human virus population. One of these, PB1507-523, became the H1N1 consensus sequence of 2007-2009. For the others, a single aa modification to the BEI peptide would result in 96-100% representation in the 2009 human H1N1 population. 
         [0054]    3) The remaining 28 peptides were each represented in the dataset by 2 to 7 variant sequences with multiple mutations (Table 7). The New York/348/2003 2003 sequences were the consensus form in only 13 of the 28 peptides and at reduced representations of 6 to 72% of the recorded viruses. As the variant forms contained a mixture of the conserved sequences and variable amino acids, it is not possible to predict the immunogenicity of the variant sequences represented in nature and their use as vaccine sequences. These data demonstrated that when T cell epitopes contain mixtures of conserved and non-conserved aa, the occurrences of mutated sequences in a subsequent influenza A strain are greatly enhanced. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 Representation of 28 (9 NP, 4 PA, 9 PB2, 
               
               
                 5 PB1, and 1 M1) T cell epitope peptides  
               
               
                 of A/New York/348/2003 (H1N1) among human 
               
               
                 H1N1, H3N2, H1N2, H5N1, and other avian 
               
               
                 subtypes circulating between 1977 to 2006. 
               
             
          
           
               
                   
                 A/New York/348/2003 
                 1977-2006 
               
               
                   
                 H1N1 ELISpot 
                 influenza 
               
               
                 Protein 
                 positive peptide§ 
                 A* 
               
               
                   
               
             
          
           
               
                 NP 
                   
                 ---------G------- 
                   
                 39 
               
               
                   
               
               
                   
                   
                 -----------D----- 
                   
                 31 
               
               
                   
               
               
                   
                   7 
                   KRSYEQMET DGERQNAT 
                  23 
                 22 
               
               
                   
               
               
                   
                   
                 ---------G----D-- 
                   
                  3 
               
               
                   
               
               
                   
                   
                 ---------S------- 
                   
                  1 
               
               
                   
               
               
                   
                   
                 K-D-------------- 
                   
                 42 
               
               
                   
               
               
                   
                   
                 --V-------------- 
                   
                 28 
               
               
                   
               
               
                   
                   
                 --VS------------- 
                   
                 11 
               
               
                   
               
               
                   
                  31 
                 RMIG GIGRFYIQMCTEL   
                  47 
                  8 
               
               
                   
               
               
                   
                   
                 --V-------V------ 
                   
                  3 
               
               
                   
               
               
                   
                   
                 K---------------- 
                   
                  2 
               
               
                   
               
               
                   
                   
                 ---D------------- 
                   
                  2 
               
               
                   
               
               
                   
                   
                 ---S------------- 
                   
                  2 
               
               
                   
               
               
                   
                   
                 -------------S--- 
                   
                 75 
               
               
                   
               
               
                   
                   
                 -------------S-H- 
                   
                  9 
               
               
                   
               
               
                   
                  37 
                   GRFYIQMCTELKL NDYE 
                  53 
                  8 
               
               
                   
               
               
                   
                   
                 -------------S-Q- 
                   
                  1 
               
               
                   
               
               
                   
                   
                 ----V--------S--- 
                   
                  1 
               
               
                   
               
               
                   
                   
                 -----------Q-S--- 
                   
                  1 
               
               
                   
               
               
                   
                   
                 ----R------------ 
                   
                 49 
               
               
                   
               
               
                   
                  73 
                   ERRN K YLEEHPSAGKDP   
                  89 
                 45 
               
               
                   
               
               
                   
                   
                 ----R----N------- 
                   
                  2 
               
               
                   
               
               
                   
                   
                 ------I---------- 
                   
                 24 
               
               
                   
               
               
                   
                   
                 --M-------------- 
                   
                 22 
               
               
                   
               
               
                   
                   
                 R-M-------------- 
                   
                 21 
               
               
                   
               
               
                   
                   
                 --M---I---------- 
                   
                 16 
               
               
                   
               
               
                   
                 103 
                 KWVRELV LYDKEEIRRI   
                 119 
                  7 
               
               
                   
               
               
                   
                   
                 --M---I---------V 
                   
                  3 
               
               
                   
               
               
                   
                   
                 --I---I---------- 
                   
                  2 
               
               
                   
               
               
                   
                   
                 --M---I----D----- 
                   
                  1 
               
               
                   
               
               
                   
                 109 
                 V LYDKEEIRRIWRQANN   
                 125 
                 50 
               
               
                   
               
               
                   
                   
                 I---------------- 
                   
                 41 
               
               
                   
               
               
                   
                   
                 I---------V------ 
                   
                  3 
               
               
                   
               
               
                   
                   
                 I----D----------- 
                   
                  1 
               
               
                   
               
               
                   
                   
                 ---L---------A--- 
                   
                 38 
               
               
                   
               
               
                   
                   
                 ---M------------- 
                   
                 25 
               
               
                   
               
               
                   
                   
                 ---M---------A--- 
                   
                 17 
               
               
                   
               
               
                   
                   
                 -------------A--- 
                   
                 12 
               
               
                   
               
               
                   
                 133 
                 LTHI MIWHSNLND TTYQ 
                 149 
                  7 
               
               
                   
               
               
                   
                   
                 ------V---------- 
                   
                 69 
               
               
                   
               
               
                   
                   
                 ----T-V---------- 
                   
                 10 
               
               
                   
               
               
                   
                 402 
                 SAGQIST QPTFSVQRNL   
                 418 
                  6 
               
               
                   
               
               
                   
                   
                 ------I---------- 
                   
                  5 
               
               
                   
               
               
                   
                   
                 ------V-A-------- 
                   
                  5 
               
               
                   
               
               
                   
                   
                 ------V--------S- 
                   
                  3 
               
               
                   
               
               
                   
                   
                 V------------E-S- 
                   
                 41 
               
               
                   
               
               
                   
                   
                 V------------ERA- 
                   
                 35 
               
               
                   
               
               
                   
                 408 
                 T QPTFSVQRNLPF DKTT 
                 424 
                  6 
               
               
                   
               
               
                   
                   
                 I---------------- 
                   
                  3 
               
               
                   
               
               
                   
                   
                 V--------S---ERA- 
                   
                  3 
               
               
                   
               
               
                   
                   
                 V-A-----------P-- 
                   
                  2 
               
               
                   
               
               
                   
                   
                 V------------ERS- 
                   
                  1 
               
               
                   
               
               
                 PA 
                  42 
                   LEVCFMYSDFHF INEQG 
                  58 
                 58 
               
               
                   
               
               
                   
                   
                 -------------D-R- 
                   
                 27 
               
               
                   
               
               
                   
                   
                 -------------D-RS 
                   
                  9 
               
               
                   
               
               
                   
                   
                 -------------D--- 
                   
                  1 
               
               
                   
               
               
                   
                   
                 ---------------R- 
                   
                  1 
               
               
                   
               
               
                   
                   
                 --I----------D-R- 
                   
                  1 
               
               
                   
               
               
                   
                   
                 ---------------L- 
                   
                  1 
               
               
                   
               
               
                   
                   
                 ----------------N 
                   
                 47 
               
               
                   
               
               
                   
                 126 
                 EVHI YYLEKANKIKSE K 
                 142 
                 37 
               
               
                   
               
               
                   
                   
                 ---T------------- 
                   
                  9 
               
               
                   
               
               
                   
                   
                 ----------------R 
                   
                  1 
               
               
                   
               
               
                   
                   
                 -I--------------- 
                   
                  1 
               
               
                   
               
               
                   
                   
                 ----------------E 
                   
                  1 
               
               
                   
               
               
                   
                   
                 ---------S------- 
                   
                  1 
               
               
                   
               
               
                   
                   
                 ----------N------ 
                   
                 47 
               
               
                   
               
               
                   
                 132 
                 
                   LEKANKIKSEKTHIHIF 
                 
                 148 
                 47 
               
               
                   
               
               
                   
                   
                 ----------R------ 
                   
                  2 
               
               
                   
               
               
                   
                   
                 ----------E------ 
                   
                  1 
               
               
                   
               
               
                   
                   
                 ---S------------- 
                   
                  1 
               
               
                   
               
               
                   
                 558 
                   SRPMFLYVRTNGTSK IK 
                 574 
                 65 
               
               
                   
               
               
                   
                   
                 ---------------V- 
                   
                 32 
               
               
                   
               
               
                 PB2 
                  42 
                   NP S LRMKWMMAMKYPIT   
                  58 
                 60 
               
               
                   
               
               
                   
                   
                 --A-------------- 
                   
                 39 
               
               
                   
               
               
                   
                  48 
                   KWMMAMKYPITADKRI T 
                  64 
                 57 
               
               
                   
               
               
                   
                   
                 ----------------M 
                   
                 28 
               
               
                   
               
               
                   
                   
                 ----------------I 
                   
                  8 
               
               
                   
               
               
                   
                   
                 --------------K-- 
                   
                  2 
               
               
                   
               
               
                   
                   
                 -------------V--- 
                   
                 47 
               
               
                   
               
               
                   
                   
                 ----------M------ 
                   
                 25 
               
               
                   
               
               
                   
                  54 
                   KYPITADKRI TEMI PER   
                  70 
                  9 
               
               
                   
               
               
                   
                   
                 ----------I------ 
                   
                  7 
               
               
                   
               
               
                   
                   
                 --------K-------- 
                   
                  2 
               
               
                   
               
               
                   
                   
                 ----------MD----- 
                   
                  1 
               
               
                   
               
               
                   
                 256 
                   DQSLIIAARNIVRRA AV 
                 272 
                 61 
               
               
                   
               
               
                   
                   
                 ---------------T- 
                   
                 34 
               
               
                   
               
               
                   
                   
                 ----V------------ 
                   
                  2 
               
               
                   
               
               
                   
                   
                 ---------------I- 
                   
                  1 
               
               
                   
               
               
                   
                   
                 -------------V--- 
                   
                 47 
               
               
                   
               
               
                   
                 369 
                   RATAILRKATRR LIQLI 
                 385 
                 46 
               
               
                   
               
               
                   
                   
                 ------------MI--- 
                   
                  3 
               
               
                   
               
               
                   
                 458 
                 MGMIGILP DMTPSTEMS   
                 474 
                 43 
               
               
                   
               
               
                   
                   
                 ---V-V----------- 
                   
                 39 
               
               
                   
               
               
                   
                   
                 -----V----------- 
                   
                  5 
               
               
                   
               
               
                   
                   
                 ---V------------- 
                   
                  4 
               
               
                   
               
               
                   
                   
                 -------S--------- 
                   
                  1 
               
               
                   
               
               
                   
                   
                 --------------I-- 
                   
                 46 
               
               
                   
               
               
                   
                   
                 -----------L----- 
                   
                 25 
               
               
                   
               
               
                   
                   
                 -----------L--I-- 
                   
                 10 
               
               
                   
               
               
                   
                 464 
                 LP DMTPSTEMS MRGVRV 
                 480 
                 10 
               
               
                   
               
               
                   
                 524 
                 TEK LTITYSSSMMWEIN   
                 540 
                 46 
               
               
                   
               
               
                   
                   
                 --R-------------- 
                   
                 46 
               
               
                   
               
               
                   
                   
                 M---------------- 
                   
                  3 
               
               
                   
               
               
                   
                   
                 I-R-------------- 
                   
                  1 
               
               
                   
               
               
                   
                 548 
                   NTYQWIIRNWE TVKIQW 
                 564 
                 54 
               
               
                   
               
               
                   
                   
                 -----------A----- 
                   
                 35 
               
               
                   
               
               
                   
                   
                 -----V----------- 
                   
                  6 
               
               
                   
               
               
                   
                   
                 -----------I----- 
                   
                  1 
               
               
                   
               
               
                 PB1 
                 328 
                 NQPEWFRNI LSIAPIMF   
                 344 
                 55 
               
               
                   
               
               
                   
                   
                 --------V-------- 
                   
                 39 
               
               
                   
               
               
                   
                   
                 K-------V-------- 
                   
                  1 
               
               
                   
               
               
                   
                   
                 -----------M----- 
                   
                  1 
               
               
                   
               
               
                   
                 352 
                   GKGYMFESK S MKLRTQI   
                 368 
                 47 
               
               
                   
               
               
                   
                   
                 ---------R------- 
                   
                 47 
               
               
                   
               
               
                   
                   
                 -R--------------- 
                   
                  2 
               
               
                   
               
               
                   
                   
                 ----------------V 
                   
                  1 
               
               
                   
               
               
                   
                   
                 ---------N------- 
                   
                  1 
               
               
                   
               
               
                   
                   
                 --------R-------- 
                   
                  1 
               
               
                   
               
               
                   
                 358 
                   ESK S MKLRTQIPAEMLA   
                 374 
                 47 
               
               
                   
               
               
                   
                   
                 ---R------------- 
                   
                 46 
               
               
                   
               
               
                   
                   
                 ----------V------ 
                   
                  1 
               
               
                   
               
               
                   
                   
                 --R-------------- 
                   
                  1 
               
               
                   
               
               
                   
                   
                 --------V-------- 
                   
                 75 
               
               
                   
               
               
                   
                   
                 ----I---V-------- 
                   
                 13 
               
               
                   
               
               
                   
                 465 
                 RFYRTCKLL GINMSKKK   
                 481 
                 10 
               
               
                   
               
               
                   
                   
                 --V------------K- 
                   
                 46 
               
               
                   
               
               
                   
                   
                 --V-------------- 
                   
                 43 
               
               
                   
               
               
                   
                 471 
                 KLL GINMSKKKSYINRT   
                 487 
                 10 
               
               
                   
               
               
                 M1 
                 169 
                 TNPLIR HENRMVLASTT   
                 185 
                 72 
               
               
                   
               
               
                   
                   
                 -----K----------- 
                   
                 25 
               
               
                   
               
               
                   
                   
                 ------------I---- 
                   
                  1 
               
               
                   
               
               
                 §Highly conserved aa are in boldface. 
               
               
                 *3175 PB1, 3144 PB2, and 3781 M1 human H1N1, H3N2, H1N2, H5N1, and avian H5N1 and other avian subtypes sequences circulating between 1977 and 2006 were extracted from NCBI GenBank and GenPept databases as of September 2006. Sequences representing less than 1% of each dataset were excluded. 
               
             
          
         
       
     
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