Abstract:
Flaviviruses represent an increasing global public health issue, with no prophylactic and therapeutic formulations currently available for many of them. The combination of factors such as evolutionary change, global warming and wide range of animal hosts suggest the possible occurrence of  Flavivirus  strains with greater distribution and human pathogenicity. There is, thus, a need for greater understanding of viral protein sequences that function in the human immune responses. The evolutionary diversity of the reported sequences of major flaviviruses, such as dengue virus, yellow fever virus, Japanese encephalitis virus, and West Nile virus were analyzed with a combination of experimental and bioinformatics methodologies. The analysis of all reported sequences revealed that these species-specific peptide tags are highly conserved and are potential T-cell epitopes due to correspondence to known or predicted epitopes. These peptide tags have direct relevance to the development of new-generation vaccines and diagnostic applications.

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 grant nos. R37A1041908 and U19 A1-056541. 
     
    
     TECHNICAL FIELD OF THE INVENTION 
       [0002]    This invention is related to the area of flaviviruses. In particular, it relates to  Flavivirus  species specific sequences for vaccines, constituents of vaccines, diagnostic, prophylactic, and therapeutic applications. 
       BACKGROUND OF THE INVENTION 
       [0003]    Flaviviruses, such as West Nile virus (WNV), dengue virus (DENY), Japanese encephalitis virus (JEV), and yellow fever virus (YFV), among others, are arthropod-borne RNA virus pathogens of the genus  Flavivirus  that have high sequence and structural homology (Kuno et al., 1998). The genome of these viruses is a positive-sense, single strand RNA, of approximately 11,000 to 12,000 nucleotides, encoding a polyprotein of approximately 3,430 amino acids that is cleaved to produce three structural proteins, capsid (C), pre-membrane (prM), membrane (M), and envelope (E), and seven non-structural (NS) proteins, NS1, 2a, 2b, 3, 4a, 4b and 5, with similar structural organization. They have become increasingly important human pathogens. For example, following the introduction of WNV in New York in 1999, the virus has become established throughout the United States as a new genotype (WN02) with multiple genetic and phenotypic changes and more efficient mosquito transmission (Davis et al., 2007; Moudy et al., 2007). Additionally, with global warming, the already widespread dengue viruses have the potential of even greater worldwide distribution. The combined problems of evolutionary change, increasing global distribution, wide range of animal as well as human hosts, and possible occurrence of strains with greater human pathogenicity, call for concerted studies with the goal of developing an effective means to combat future as well as current strains. 
         [0004]    As many RNA viruses are pathogens in humans, there is need for increased understanding of viral protein sequences that function in the human cellular immune responses to these viruses. Several reports have described roles of CD8 +  cytolytic T lymphocytes (CTL) and of CD4 +  helper T lymphocytes (HTL) in the immune response to a variety of viral infections in animal model systems (BenMohamed et al., 2000; Blaney et al., 1998; Brien et al., 2007; Castrucci et al., 1994; Del Val et al., 1991; La Posta et al., 1993; Lieberman et al., 2007; Oukka et al., 1996; Purtha et al., 2007; Stemmer et al., 1999; Tsuji et al., 1998). However, knowledge of the identities and properties of both CTL and HTL immunogenic peptide sequences of pathogens is limited because of the great diversity in the recognition of the antigen peptides by the host immune system and the thousands of human leukocyte antigen molecules (HLA; human MHC) (Robinson et al., 2006); approximately 3500 reported as of June 2009 (www.ebi.ac.uk/imgt/hla/). There is also the complexity of the genetic structure of single stranded RNA viruses that are among the most variable and adaptable of subcellular parasites resulting from high frequency of point mutations during RNA replication. The genetic change, short generation times, and large virus populations result in rapid evolution dependent upon virus fitness to the vector and host. In almost all cases, the specific genetic changes responsible for viral adaptation are not known because of the stochastic nature of mutagenesis and viral fitness and the complexities of biological responses of the host. Of the great variety of T cell antigenic determinants on WNV proteins, there are only a few for which the structure is known (McMurtrey et al., 2008; Parsons et al., 2008; Wang et al., 2006). However, the advances in pathogen genome sequence data, the development of HLA transgenic mice as a model system, and large-scale synthesis of pathogen peptides has now made possible the systematic analysis of viral proteomes for protein sequences that function as T cell epitopes. 
         [0005]    HLA Transgenic (Tg) mice are widely recognized as a leading model system for analysis of HLA-restricted T cell responses to human pathogens and disease states (Cheuk et al., 2002; Hu et al., 2005; Loirat et al., 2000; Pajot et al., 2004; Pajot et al., 2006; Pascolo, 2005; Richards et al., 2007; Sonderstrup et al., 1999; Taneja and David, 1999). 
         [0006]    There is a continuing need in the art to identify and test  Flavivirus  vaccines to reduce the incidence and/or severity of  Flavivirus  infections and/or epidemics. The selection of evolutionarily conserved protein sequences has widely been considered important to vaccine design in order to limit the selective loss of immunity resulting from mutation and protein modification. However, sequences conserved in the evolution of viruses can be present in many different forms in viruses of related species. It is clear that exposure to multiple flaviviruses by infection or immunization will risk immune responses to a large number of cross-reactive T-cell epitopes that may, as altered peptide ligands (APL), significantly affect the immune responses to the pathogens. Memory T cells selectively engaged by a variant epitope sequence may exhibit an impaired immune response, depending on the positions and types of amino acid substitutions surrounding or within T cell epitopes and the effect of these changes on the affinity of the interaction (Ferrante and Gorski, 2007). The possible effect in humans of APL inhibition or modification of human T cell immune responses has been widely recognized (Sloan-Lancaster and Allen, 1996), particularly in relation to secondary infection and the marked cross-reactivity of memory T cells induced by primary infection followed by re-infection by a second of the four dengue serotypes (Mongkolsapaya et al., 2003; Mongkolsapaya et al., 2006; Screaton and Mongkolsapaya, 2006). The consequences of this may be relevant to the occurrence of dengue hemorrhagic fever (DHF), the more serious manifestation of the dengue virus infection (Rothman, 2004). Thus, we propose that the selection of conserved sequences that are also virus specific should have precedence in vaccine design. 
       SUMMARY OF THE INVENTION 
       [0007]    According to one aspect of the invention a polypeptide is provided. The polypeptide comprises one or more discontinuous segments of one or more proteins of a  Flavivirus . The segments comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-206. 
         [0008]    Another aspect of the invention is a polynucleotide which encodes a polypeptide. The polypeptide comprises one or more discontinuous segments of one or more proteins of a  Flavivirus . The segments comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-206. 
         [0009]    Yet another aspect of the invention is a nucleic acid vector that comprises the polynucleotide. The polynucleotide encodes the polypeptide. The polypeptide comprises one or more discontinuous segments of one or more proteins of a  Flavivirus . The segments comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-206. 
         [0010]    Still another aspect of the invention is a host cell. The host cell comprises a nucleic acid vector that comprises the polynucleotide. The polynucleotide encodes the polypeptide. The polypeptide comprises one or more discontinuous segments of one or more proteins of a  Flavivirus . The segments comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-206. 
         [0011]    According to another aspect of the invention a method is provided for producing a polypeptide. A host cell is cultured. The host cell comprises a nucleic acid vector that comprises the polynucleotide. The polynucleotide encodes the polypeptide. The polypeptide comprises one or more discontinuous segments of one or more proteins of a  Flavivirus . The segments comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-206. The culturing is under conditions in which the host cell expresses the polypeptide. 
         [0012]    Another aspect of the invention is a method of producing a cellular vaccine. Antigen presenting cells are transfected with a nucleic acid vector that comprises the polynucleotide. The polynucleotide encodes the polypeptide. The polypeptide comprises one or more discontinuous segments of one or more proteins of a  Flavivirus . The segments comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-206. The antigen presenting cells thereby express the polypeptide. 
         [0013]    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 one or more discontinuous segments of one or more proteins of a  Flavivirus . The segments comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-206. 
         [0014]    A further aspect of the invention is a vaccine composition. The vaccine composition comprises a polypeptide or a polynucleotide encoding the polypeptide. The polypeptide comprises one or more discontinuous segments of one or more proteins of a  Flavivirus . The segments comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-206. 
         [0015]    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  Flavivirus -specific T cell activation. The polypeptide comprises one or more discontinuous segments of one or more proteins of a  Flavivirus . The segments each comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-206. The nucleic acid vector comprises a polynucleotide that encodes the polypeptide. The host cell comprises the nucleic acid vector. 
         [0016]    Another aspect of the invention is a method of identifying a  Flavivirus . A polynucleotide encoding a polypeptide comprising one or more discontinuous segments of one or more proteins of a  Flavivirus  or its complement is hybridized to the genome of a  Flavivirus . The segments comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-206. Hybridization of the genome to the polynucleotide indicates a species of the  Flavivirus.    
         [0017]    Yet another aspect of the invention is a method of identifying a  Flavivirus . Proteins from a virus-infected cell are contacted with an antibody which specifically binds to a polypeptide comprising one or more discontinuous segments of one or more proteins of a  Flavivirus . The segments comprise at least 9 contiguous amino acid residues selected from SEQ ID NO: 1-206. Specific binding to the proteins indicates a species of  Flavivirus.    
         [0018]    Still another aspect of the invention is a method of identifying a  Flavivirus . A polypeptide comprising one or more discontinuous segments of one or more proteins of a  Flavivirus  is contacted with a blood sample from a patient. Binding of the polypeptide to an antibody in the blood sample or T cells in the blood sample indicates a species of  Flavivirus.    
         [0019]    These and other embodiments which will be apparent to those of skill in the art upon reading the specification provide the art with new diagnostic and prophylactic reagents. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0020]      FIG. 1  shows a comparison of peptide and DNA immunizations. 
           [0021]      FIG. 2  shows the number of WNV T-cell epitope peptides conserved in other flaviviruses. 
           [0022]      FIG. 3  shows the number of flaviviruses shared by the individual WNV T-cell epitope peptides. 
           [0023]      FIG. 4  shows the distribution of WNV epitope peptides with complete full-length occurrence in other flaviviruses. 
       
    
    
       [0024]    Table 1 provides HLA-restricted T-cell epitope peptides of the WNV proteome. 
         [0025]    Table 2 provides West Nile Virus HLA-restricted T-cell epitope peptides, class I and II. SEQ ID NOs: 211-347, in the order as shown. 
         [0026]    Table 3A and 3B provide the apparent functional avidity of WNV T-cell epitope peptides in ELISpot assays of splenocytes from immunized HLA-transgenic mice. 
         [0027]    Table 4 provides highly conserved WNV T-cell epitope peptides, entropy 0.1 or lower. 
         [0028]    Table 5 provides WNV T-cell epitope peptides with high variants incidence. 
         [0029]    Table 6 provides an example of a non-zero entropy WNV epitope peptide site. It commonly includes multiple sequences variant to the epitope, with one or more different amino acid mutations, each of which represented in a small fraction, less than 10%, of the reported sequences. 
         [0030]    Table 7 provides WNV-specific epitope peptides. 
         [0031]    Table 8 provides WNV T-cell epitope peptides with full-length occurrence in other flaviviruses. 
         [0032]    Table 9 provides the distribution of cross-reactive WNV T-cell epitopes in major flaviviruses. 
         [0033]    Table 10 provides variants of highly shared WNV epitope peptides and their incidence in other selected flaviviruses. WNV variant sequences representing less than about 10% of the corresponding database sequences were omitted. 
         [0034]    Table 11 provides WNV HLA-restricted T-cell epitope peptides incidence and their variant incidence distribution. Entropy describing the diversity at the epitope peptide sites is also indicated. 
         [0035]    Table 12 provides list of highly conserved and specific sequences of  Flavivirus  species West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV) and Japanese encephalitis virus (JEV). SEQ ID NOs: 1-206, in the order as shown. 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0036]    The inventors have identified and characterized discontinuous peptide segments from the proteomes of a number of flaviviruses. These are sequences of nine or more contiguous amino acids (aa) are highly conserved in all reported populations of the respective virus species, and are specific to the species, with no matching identity of at least nine aa in any other  Flavivirus . These sequences are potential HLA-restricted epitope peptides, with many of them shown to be immunogenic in humanized HLA transgenic mice (see, e.g., Table 2 and 7) or predicted to contain T-cell epitope determinants. The identified sequences (in their nucleotide or protein form) have applications to diagnosis of virus infection and the development of new-generation vaccines. Such vaccines may be used either prophylactically or therapeutically, i.e., administered to a person who has not been infected yet or to a person who is already infected. 
         [0037]    Discontinuous segments of the  Flavivirus  may be strung together to form a concatamer, if desired. They may be separated by spacer residues, optionally. Discontinuous segments are those that are not adjacent in the naturally occurring virus isolates. Segments are typically at least 9 amino acid residues and up to about 15, 16, 17, 18, 19, 20, 25, or 30 residues of contiguous amino acid residues from the virus proteome. Single segments may also be used. Because the segments are less than the whole, naturally occurring proteins, and/or because the segments are adjacent to other segments to which they are not adjacent in the proteome, the polypeptides and nucleic acids described here are non-naturally occurring. 
         [0038]    Linkers or spacers with natural or non-naturally occurring amino acid residues may be used optionally. 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. 
         [0039]    Polynucleotides which encode the polypeptides may be designed and made by techniques well known in the art. The natural nucleotide sequences used by flaviviruses may be used. Alternatively non-natural nucleotide sequences may be used, including in one embodiment, human codon-optimized sequences. 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. 
         [0040]    The polynucleotides which encode the polypeptides 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 polypeptides 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 active in a tissue specific manner. 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. 
         [0041]    Suitable purification techniques can be applied to the chimeric protein as are known in the art. In another embodiment one transfects antigen presenting cells for ultimate delivery of the transfected cells to a vaccinee of a cellular vaccine which expresses and presents antigen to the vaccine. Suitable antigen presenting cells include dendritic cells, B cells, macrophages, and epithelial cells. 
         [0042]    Polynucleotides of the invention include diagnostic DNA or RNA oligonucleotides, i.e., short sequences of proven specificity to viral species; these are sufficient to uniquely identify the viral species. Polynucleotides include oliogonucleotides such as primers and probes, which may be labeled or not. These may contain all or portions of the coding sequences for an identified conserved and specific polypeptide. Polynucleotides of the invention and/or their complements, may optionally be attached to solid supports as probes to be used diagnostically, for example, through hybridization to viral genomic sequences. Similarly, epitopic polypeptides can be attached to solid supports to be used diagnostically. These can be used to screen for activated T cells or even antibodies. Suitable solid supports include without limitation microarrays, microspheres, and microtiter wells. Antibodies may be used that are directed against the peptides as disclosed. The antibodies may be used to specifically diagnose a species of  Flavivirus , for example. Polynucleotides may also be used as primers, for example, of length 18-30, 25-50, or 15-75 nucleotides, to amplify the genetic material of a specific  Flavivirus . Polynucleotide primers and probes may be labeled with a fluorescent or radioactive label, if desired. These polynucleotides can be used to amplify and/or hybridize to a test sample to determine the presence or species identity of a  Flavivirus . Such polynucleotides will typically be at least 18, 20, 22, 24, 26, 28, 30, 32, 34 bases in length. Any technique, including but not limited to amplification, hybridization, single nucleotide extension, and sequencing, can be used to identify the presence or species identity of a  Flavivirus.    
         [0043]    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. Some immune adjuvants which may be used include CpG oligodeoxynucleotides, GM-CSF, QS-21, MF-59, alum, lecithin, squalene, and Toll-like receptors (TLRs) adaptor molecules. These include the Toll-interleukin-1 receptor domain-containing adaptor-inducing beta interferon (TRIF) or myeloid differentiation factor 88 (MyD88). Vaccines may be produced in any suitable manner, including in cultured 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 flaviviral vaccine. Typically a high titer of antibody and/or T cell activation is desired with a minimum of adverse side effects. 
         [0044]    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. The vaccines can be administered to patients who are infected already or to patients who do not yet have an infection. The vaccines can thus serve as prophylactic or therapeutic agents. One must, however, bear in mind, that no specific level of efficacy is mandated by the words prophylactic or therapeutic. Thus the agents need not be 100% effective to be vaccines. Vaccines in general are used to reduce the incidence in a population, or to reduce the risk in an individual. They are also used to stimulate an immune response to lessen the symptoms and or severity of the disease. 
         [0045]    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 
       [0046]    We have applied this system to a large-scale analysis of HLA class I and II-restricted T cell epitopes of the WNV proteome by immunization of 6 mice transgenic for HLA proteins, 3 class 1 and 3 class II, with 452 WNV peptides covering the entire WNV proteome. WNV peptide-specific T-cell responses were assayed by IFN-γ ELISpot and the identified T cell epitope sequences were further analyzed for their apparent avidity in the ELISpot assay, conservation and diversity in the recorded WNV protein sequences, and homology to other  Flavivirus  pathogens. The identification and characterization of these HLA-restricted T cell epitope peptides of the WNV proteome will facilitate further analysis of the human immune response to WNV infection, including application of peptide-specific methodologies for diagnosis for virus infection and the development of new-generation vaccines. 
       HLA Transgenic Mice 
       [0047]    H-2 class II-deficient, HLA-DR2 (Vandenbark et al., 2003), HLA-DR3 (Madsen et al., 1999; Strauss et al., 1994) and -DR4/human CD4 (huCD4) (Cope et al., 1999; Fugger et al., 1994) Tg mice, and H-2 class I-deficient, HLA-A2.1 (HHD monochain) (Pascolo et al., 1997), HLA-A24/huCD8α (Lemonnier et al., unpublished) and HLA-B7 (Rohrlich et al., 2003) Tg mice were used. HLA-DR2 Tg mice express chimeric molecules, with α1 and β1 domains encoded by the HLA-DRA1*0101 and -DRB1*1501 sequences and the other domains encoded by I-Eα and I-Eβ sequences, from the I-E promoters (Vandenbark et al., 2003). HLA-DR3 Tg mice express the full-length HLA-DRA*0101 and -DRB1*0301 sequences (Madsen et al., 1999; Strauss et al., 1994). HLA-DR4/huCD4 Tg mice express the full-length HLA-DRA*0101 and -DRB1*0401 sequences from the I-Ea promoter, and the human CD4 sequence from the murine CD3δ promoter (Cope et al., 1999; Fugger et al., 1994). HLA-DR2 and -DR3 mice have a homologous deletion of the murine H-2 class II region, and HLA-DR4/huCD4 mice are deficient for I-Aβ and I-Eα. HLA-DR2 mice have a predominant C57BL/6 background, and HLA-DR3 and -DR4/huCD4 mice have mixed backgrounds (B6, B10.H2b, and DBA/1J, 129/Sv, C57BL/6, respectively). HLA-A2.1 (HHD) Tg mice express a chimeric monochain containing the HLA-A*0201α1 and α2 domains and the murine H-2D b  α3 domain linked to human β2-microglobulin (huβ2-m), from the HLA-A2.1 promoter, and are deficient for H-2D and murine β2-m (mβ2-m) (Pascolo et al., 1997). HLA-A24/huCD8α mice express the full-length HLA-A*2402, huβ2-m and huCD8α sequences, and are deficient for H-2K, H-2D, and mβ2-m (Lemonnier et al., unpublished). HLA-B7 mice express a chimeric heavy chain with the HLA-B*0702 α1 and α2 domains and the H-2K d  α3 domain, from the HLA-B7 promoter, and are deficient for H-2K and H-2D (Rohrlich et al., 2003). The three HLA class I Tg strains have been backcrossed for 6 to 12 generations on the C57BL/6 genetic background (Lemonnier, Pasteur Institute). Animals were bred and maintained at the Johns Hopkins School of Medicine Research Animal Resources facilities. Specific pathogen-free (SFP) Tg mice were derived through iodine immersion of neonates (&lt;1 day old) and transfer to outbrcd foster females (Thompson K and Watson J, The Johns Hopkins School of Medicine). All experiments were approved by the Johns Hopkins Animal Care and Use Committee and carried out according to IACUC guidelines. 
       Synthetic WNV Peptides 
       [0048]    A library of 452 overlapping peptides covering the entire WNV proteome (NY99-flamingo 382-99 strain), each 14 to 19 amino acids in length with an overlap of 10 residues (&gt;80% purity), was obtained as lyophilized powders from the Biodefense and Emerging Infections Research Resources Repository, NIAID, NTH (Manassas, Va.). Each was dissolved in 100% DMSO and constituted to 20% with sterile filtered water. The final concentration of each peptide was 2 μg/μl. Dissolved peptides were stored at −20° C. 
       Large Scale Peptide Pool Immunization 
       [0049]    The 6 HLA Tg mice were each immunized with the WNV peptides by use of a peptide pool protocol for large scale T cell epitope identification (Roederer and Koup, 2003). The peptides were divided into 4 immunization pools containing 1 μg of each peptide in groups of about 100 peptides each, as follows: pool 1, 88 peptides spanning the PrM/M and E proteins; pool 2, 107 peptides spanning the N, NS1, NS2a, and NS2b proteins; pool 3, 135 peptides spanning NS 3, NS4a; and NS4b proteins; pool 4, 122 peptides spanning the NS5 protein (data not shown). Each pool was mixed with 50 μl zymosan, 10 mg/ml (Sigma-Aldrich Co, St. Louis, Mo.) in PBS as adjuvant and administered subcutaneously at the base of the tail to groups of 9 to 12 mice of each Tg strain (de la Rosa et al., 2005; Goodridge et al., 2007). Initial matrix assays with peptide pools were performed on day 15-19, after one immunization. Two mice were sacrificed and their splenocytes were selectively depleted of CD8 or CD4 T cells for HLA class II and class I Tg strains, respectively (see below), and T cell responses to peptide pools (10 peptides, 1 μg peptide per pool) were assessed by IFN-γ ELISpot assays. On the following day 2 additional mice were sacrificed and the WNV peptide immunogens identified by the deconvolution analyses were individually tested by ELISpot assay. Experimental values reported herein were obtained with peptide concentration of 10 μg/ml, and a minimum of 3 assays in duplicate with different immunized mice. The remaining mice were immunized a second time on day 21 with the original peptide pool without zymosan. The mice were sacrificed on day 35 and splenocyte T cell responses to individual WNV peptides were further assessed by ELISpot assay with peptide concentrations of 10, 1 and 0.1 μg/ml. Positive control dengue virus peptides immunogenic in the relevant Tg mouse were included in each immunization protocol to evaluate the responses of the individual immunized mouse. 
       DNA Immunization 
       [0050]    The WNV strain NY99-flamingo382-99 NS3 sequence (GenBank Accession No. AF196835) with NheI and KpnI sites was optimized using the Leto 1.0 software, synthesized by Geneart Inc (Toronto, Canada), and inserted into the p43 vector (Kessler et al., 1996). A p43 vector encoding the dengue virus type 2 prM and E antigens (p-DENV-prM-E) (J. Salmon, unpublished) was used as a control. Four 6-8 weeks old female HLA-DR2 Tg mice were immunized subcutaneously at the base of the tail, twice at 3-week intervals, with 50 μg of the endotoxin-free DNA plasmid p-WNV-NS3, or p-DENV-prM-E. The mice were sacrificed on day 42 and the CD4 T cell responses to the WNV NS3 peptides were assessed by IFN-γ ELISpot assays. 
       IFN-γ ELISpot Assays 
       [0051]    Ex vivo IFN-γ ELISpot assays were performed using mouse IFN-γ ELISpot sets (BD Biosciences, San Jose, Calif.) following manufacturer&#39;s recommendations. Briefly, 96-well ELISpot plates were coated with anti-IFN-γ antibody (5 μg/ml) by incubation at 4° C. overnight, and then blocked with RPMI 1640 medium containing 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 100 μg/ml streptomycin, and 100 U penicillin, for 2 h at room temperature. Freshly isolated splenocytes from HLA class II and HLA class I Tg mice were depleted of CD8 or CD4 T cells, respectively, using magnetic beads according to the manufacturer&#39;s protocol (Miltenyi Biotech, Auburn, Calif.). Flow cytometry analysis of the depleted cells indicated this method routinely achieved &gt;95% depletion of the targeted cells. CD8 or CD4-depleted splenocytes (100 μl containing 0.5−1.0×10 6  cells/well) were plated together with WNV peptides. The final concentration of each peptide was 10 μg/ml in the peptide matrix pool and individual peptide validation assays, and 10, 1, and 0.1 μg/ml in the titration assays. Each peptide preparation was tested in duplicated wells. Cells plated without peptide (medium alone) served as negative controls, and concanavalin A (2.5 μg/ml; Sigma-Alrich, St. Louis, Mo.) and known HLA-restricted peptides from dengue virus serotype 3 were included as positive controls. The cells were incubated at 37° C., 5% CO 2  for 16 h. The plates were washed, incubated with biotinylated anti-IFN-γ antibody for 2 h at room temperature, followed by HRP-conjugated streptavidin for 1 h at room temperature. Detection was performed with AEC substrate (Calbiochem, San Diego, Calif.) following manufacturer&#39;s instructions. IFN-γ spot-forming cells (SFC) were counted using the Immunospot Series 3B Analyzer ELISPOT reader and Immunospot software version 3.0 (Cellular Technologies, Shaker Heights, Ohio). Experimental values were expressed as the mean numbers of SFC/10 6  CD8- or CD4-depleted splenocytes±SD, after subtraction of values from negative controls (background). Positive ELISpot responses were defined as values above 10, and above the background plus 2 SD. Each ELISpot positive response was confirmed by three assays: matrix screening, individually by the validation assay with the individual peptide, and by peptide titration. 
       WNV Sequence Data Collection and Processing for Bioinformatics Analysis 
       [0052]    Full-length and partial WNV sequences were retrieved from the NCBI Entrez protein database (Berman et al., 2000; Wheeler et al., 2005) through the NCBI Taxonomy Browser application (taxonomy ID 11082) (as of June 2007). The sequences of the individual WNV proteins were extracted from the collected dataset by performing BLAST (Altschul et al., 1990) search against the downloaded dataset by using the individual protein sequences in the annotated WNV record P06935 as queries. Multiple sequence alignments were performed for each protein with the MUSCLE v3.6 program (Edgar, 2004) and were manually corrected for misalignments when necessary. 
         [0000]    Entropy analysis of WNV T-cell epitope sequences 
         [0053]    The evolutionary conservation and variability of the identified T-cell epitope regions in the recorded WNV sequences was measured by use of Shannon entropy computations (Khan et al., 2008; Shannon, 1948) in the Antigenic Variability Analyzer (AVANA) software (Miotto et al., 2008). AVANA was also used to study the representation of the individual epitope sequence and its variants in the corresponding protein alignment. At any given position x in the alignment, variant peptides were defined as those that differed by at least one amino acid from the experimentally identified T cell epitope. 
         [0000]    T-Cell Epitope Sequence Homologies with Other Viruses 
         [0054]    Homologs of the WNV T-cell epitopes were searched by performing BLAST analyses of all protein sequences deposited at NCBI (as of January 2009). The parameters set was as follow: limit by Entrez query “Root[ORGN] NOT txid11082[Organism:exp] NOT txid 81077[ORGN]”, “automatically adjust parameters for short sequences” option disabled, “low-complexity” filter disabled, maximum number of aligned sequences to be displayed set to “20,000”, expect threshold set to “2,000”, word size set to “2”, matrix set to “PAM30”, gap costs set to “Existence: 9, Extension: 1”, compositional adjustments set to “no adjustment”. Artificial sequence hits were removed by the “NOT txid81077[ORGN]” keyword. 
       Example 2 
     Identification of HLA-Restricted T Cell Epitopes of the WNV Proteome 
       [0055]    Immunization of each of the 6 HLA transgenic mice with 4 peptide pools that comprised the 452 WNV peptides of the entire WNV proteome (data not shown), with zymosan as adjuvant, resulted in the identification of a total of 137 T cell epitope peptides, ˜30% of the 452 total, as assayed by IFN-γ ELISpot with splenocytes of the immunized mice (summarized in Table 1; complete data in Table 2). Many (43) of the 137 epitope peptides were immunogenic in multiple, 2 or more, transgenic strains, 25 of which elicited both class I and class II responses, resulting in a total of 200 individual HLA-restricted T cell responses of the 6 transgenic strains, 74 class I (40 A2, 24 A24, 10 B7) and 126 class II (50 DR2, 38 DR3, 38 DR4). These allele-specific T-cell responses to the 452 peptide immunogen thus ranged from ˜2% B7 to ˜11% DR2, with responses of 6% to 9% of the A2, A24, DR3, and DR4 mice. The M, NS3, and E proteins had the highest concentrations of immunogenic peptides; as a group they represented 160 of the 452 (35%) total peptides, but accounted for 117 (58%) of the 200 T-cell responses. The peptides of preM were non-immunogenic and the least immunogenic were peptides of NC, NS1, and NS5, which collectively elicited only 38 (19%) of the T-cell responses to 193 (43%) of the peptides. Many of the epitope peptides of M, E, and NS3 proteins were in a clustered localization (immunological hotspots). All of the M epitope peptides were in a single cluster of 17 HLA-restricted responses, E contained 3 clusters of the protein amino acids 39-85, 119-152, and 426-482, and almost all of the NS3 peptide epitopes were in clusters of amino acids 1-115, 138-282, 304-376, and 455-605. These 8 clustered regions collectively comprise 65, almost 50%, of the 137 epitope peptide sequences. 
         [0000]    
       
         
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 HLA-restricted T-cell epitope peptides of the WNV proteome. 
               
             
          
           
               
                   
                   
                 Protein 
                 ELISpot 
                   
               
               
                   
                 Protein 
                 peptides 
                 positive 
               
               
                 WNV 
                 size 
                 analyzed 
                 peptides 
                 HLA-restricted T cell activation 
               
             
          
           
               
                 protein 
                 (aa) a   
                 (#) 
                 # (%) 
                 A2 
                 A24 
                 B7 
                 DR2 
                 DR3 
                 DR4 
                 Total 
               
               
                   
               
             
          
           
               
                 C 
                 123 
                 15 
                 1 (~7%) 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 1 
               
               
                 prM 
                 167 
                 21 
                  6 (~29%) 
                 1 
                 2 
                 1 
                 6 
                 3 
                 4 
                 17 
               
               
                 E 
                 501 
                 67 
                 25 (~37%) 
                 8 
                 6 
                 2 
                 8 
                 12 
                 6 
                 42 
               
               
                 NS1 
                 352 
                 46 
                 10 (~22%) 
                 2 
                 1 
                 0 
                 1 
                 3 
                 5 
                 12 
               
               
                 NS2a 
                 231 
                 30 
                 10 (~33%) 
                 0 
                 3 
                 0 
                 8 
                 3 
                 3 
                 17 
               
               
                 NS2b 
                 131 
                 16 
                  6 (~38%) 
                 1 
                 3 
                 1 
                 2 
                 0 
                 2 
                 9 
               
               
                 NS3 
                 619 
                 84 
                 46 (~55%) 
                 26 
                 0 
                 1 
                 16 
                 11 
                 4 
                 58 
               
               
                 NS4a 
                 149 
                 18 
                  5 (~28%) 
                 0 
                 3 
                 0 
                 1 
                 1 
                 2 
                 7 
               
               
                 NS4b 
                 255 
                 33 
                  9 (~27%) 
                 0 
                 1 
                 0 
                 3 
                 3 
                 5 
                 12 
               
               
                 NS5 
                 905 
                 122 
                 19 (~16%) 
                 2 
                 5 
                 4 
                 5 
                 2 
                 7 
                 25 
               
               
                 Total 
                 3433 
                 452 
                 137 (~30%)  
                 40 
                 24 
                 10 
                 50 
                 38 
                 38 
                 200 
               
             
          
           
               
                 Frequency allele-specific epitopes per total 
                 9% 
                 5% 
                 2% 
                 11% 
                 8% 
                 8% 
                   
               
               
                 proteome peptides 
               
               
                   
               
               
                   a Size indicated in number of amino acids with respect to the flamingo strain (NCBI accession no. AAF20092.2). 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 West Nile virus HLA-restricted T-cell epitope peptides, class I and II. (SEQ ID NOs: 211-347) 
               
             
          
           
               
                 ELISpot positive peptides 
                 T cell activation of HLA transgenic mice 
               
             
          
           
               
                 Protein &amp; 
                   
                 (SFC/10 6 ) IFN-γ ELISpot 
               
             
          
           
               
                 position a   
                 Peptide sequence b,c   
                 A2 
                 A24 
                 B7 
                 DR2 
                 DR3 
                 DR4 
               
               
                   
               
               
                 C 9-25 
                 GKSRAVN 4 M 1 LKRGMPRVL 
                   
                   
                  43 ± 17 
                   
                   
                   
               
               
                   
               
               
                 prM 114-130 
                 STKATR 2 Y 1   L 4 V 5 KTESWILR   
                   
                   
                   
                 101 ± 33 
                   
                   
               
               
                   
               
               
                 prM 121-138 
                 
                   L 4 VKTES 4 WI 3 LRNPGYALVA 
                 
                   
                   
                   
                 130 ± 23 
                   
                  56 ± 6 
               
               
                   
               
               
                 prM 129-145 
                 
                   LRN 3 PG 2 YALVAAVIGWML 
                 
                   
                  71 ± 38 
                  31 ± 7 
                  62 ± 11 
                   
                   
               
               
                   
               
               
                 prM 136-153 
                 
                   LVAAVI 4 GW 6 M 5,6 LGSNTMQRV 
                 
                   
                   
                   
                 235 ± 9 
                  78 ± 35 
                 327 ± 162 
               
               
                   
               
               
                 prM 144-161 
                 
                   M 5,6 LGSN 1 T 1 MQRV 1 VFVVLLLL 
                 
                   
                  59 ± 18 
                   
                  84 ± 32 
                 134 ± 35 
                 315 ± 157 
               
               
                   
               
               
                 prM 152-167 
                   RV 1 VF 4,6 VV 1 LLLL VAPAYS 
                 318 ± 179 
                   
                   
                 269 ± 30 
                  90 ± 47 
                 117 ± 10 
               
               
                   
               
               
                 E 39-56 
                 P 5 TID 6 VKM 1 M 6 NMEAANL AEV   
                   
                 137 ± 124 
                   
                   
                   
                   
               
               
                   
               
               
                 E 54-71 
                   AEV R 1 SY 6 CY 6   LATVSDLSTK   
                   
                   
                   
                   
                   
                 205 ± 56 
               
               
                   
               
               
                 E 62-77 
                 
                   LATVSDLSTKAACPTM 
                 
                 465 ± 343 
                   
                   
                   
                   
                  47 ± 4 
               
               
                   
               
               
                 E 68-85 
                   LSTKAACPTM GEAHNDKR 
                 372 ± 242 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 E 99-113 
                 RGWGNGCGLFGKGSI 
                   
                   
                   
                   
                  79 ± 7 
                   
               
               
                   
               
               
                 E 119-136 
                 FACSTKAI GRTILKENIK   
                   
                 179 ± 136 
                   
                   
                   
                   
               
               
                   
               
               
                 E 127-144 
                 
                   GRTILKENI 4,6 KYEVAIFVH 
                 
                   
                   
                   
                 120 ± 1 
                 198 ± 21 
                   
               
               
                   
               
               
                 E 135-152 
                   I 4,6 KYEVAIF 6 VH GPTTVESH 
                   
                   
                   
                   
                  86 ± 13 
                   
               
               
                   
               
               
                 E 171-188 
                 PAAPSYTLKLGEYGEVTV 
                 279 ± 217 
                 533 ± 333 
                 996 ± 100 
                   
                   
                   
               
               
                   
               
               
                 E 195-212 
                 G 1 IDTNAYY 6 VMTVGTK TFL   
                   
                 137 ± 70 
                   
                   
                   
                  53 ± 16 
               
               
                   
               
               
                 E 210-225 
                   TF 1 L VHREW 5,6 FMDLNLPW 
                 323 ± 175 
                 454 ± 412 
                 519 ± 477 
                   
                   
                   
               
               
                   
               
               
                 E 238-255 
                 TLMEFEE 3 PHATKQSVIAL 
                   
                 122 ± 108 
                   
                   
                   
                   
               
               
                   
               
               
                 E 293-310 
                 EKLQ 1 LKGTT 2 YGVCSKAFK 
                   
                   
                   
                   
                   
                  47 ± 5 
               
               
                   
               
               
                 E 356-373 
                 VTV 4 NPF 6 VS 1 VATANA KVLI   
                   
                   
                   
                   
                   
                 217 ± 51 
               
               
                   
               
               
                 E 370-386 
                   KVLI ELEPPFGDSY IVV   
                   
                   
                   
                   
                   
                 148 ± 10 
               
               
                   
               
               
                 E 384-399 
                   IVV GRG EQQINHHWHK   
                   
                   
                   
                   
                  87 ± 16 
                   
               
               
                   
               
               
                 E 390-407 
                 
                   EQQINHHWHKSGSSIGKA 
                 
                   
                   
                   
                   
                  50 ± 6 
                   
               
               
                   
               
               
                 E 398-415 
                   HKSGSSIGKA FTTTLKGA 
                   
                   
                   
                 168 ± 18  
                  88 ± 1 
                   
               
               
                   
               
               
                 E 426-443 
                 WDFGS 1 VG 4 G VFTSVGKAVH   
                   
                   
                   
                   
                  83 ± 13 
                   
               
               
                   
               
               
                 E 434-451 
                 
                   VFTSVGKAVHQVFGGAFR 
                 
                   
                   
                   
                 116 ± 5 
                  79 ± 19 
                   
               
               
                   
               
               
                 E 442-459 
                 
                   VHQVF 5 G 4 GAF 6 RSLFGGMSW 
                 
                 442 ± 313 
                   
                   
                 117 ± 5 
                 101 ± 15 
                   
               
               
                   
               
               
                 E 450-467 
                 
                   FRS 1 L 4 FGG 1 MSWITQGLLGA 
                 
                 205 ± 145 
                   
                   
                 261 ± 13 
                 104 ± 21 
                   
               
               
                   
               
               
                 E 458-474 
                 
                   SWITQGL 1 LGALLLWMGI 
                 
                   
                   
                   
                  51 ± 4 
                   
                   
               
               
                   
               
               
                 E 465-482 
                   LGAL 1,4 L 6 LWMGI NARDRSIA 
                 310 ± 186 
                   
                   
                 490 ± 64 
                 330 ± 91 
                   
               
               
                   
               
               
                 E 486-501 
                 LA 1 VGGV 1,4 L 6 L 6 FLSVNVHA 
                 323 ± 173 
                   
                   
                 214 ± 27 
                 244 ± 16 
                   
               
               
                   
               
               
                 NS1 1-19 
                 DTGCAI 5 DIS RQELRCGSGV   
                   
                   
                   
                   
                 572 ± 20 
                   
               
               
                   
               
               
                 NS1 10-27 
                 
                   RQELRCGSGVFIHNDVEA 
                 
                   
                   
                   
                   
                   
                  63 ± 8 
               
               
                   
               
               
                 NS1 18-35 
                   GVFIHNDVEA WMDRYKYY 
                   
                   
                   
                   
                  52 ± 8 
                   
               
               
                   
               
               
                 NS1 161-178 
                 GLTSTRMFL 6 KVRESNTTE 
                   
                   
                   
                 134 ± 8 
                   
                   
               
               
                   
               
               
                 NS1 205-221 
                 RLNDTW 4,6 KLERAVLGEVK 
                   
                   
                   
                   
                   
                  61 ± 15 
               
               
                   
               
               
                 NS1 228-245 
                 THTLWGDG ILESDLIIPV   
                  84 ± 47 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NS1 236-251 
                   ILESDL 6 IIPV TLAGPR 
                  78 ± 42 
                  43 ± 18 
                   
                   
                   
                   
               
               
                   
               
               
                 NS1 270-287 
                 EGRVEIDFDYCPGTTVTL 
                   
                   
                   
                   
                   
                  54 ± 6 
               
               
                   
               
               
                 NS1 305-322 
                 GKL 5 ITDWCCRSCTLPPLR 
                   
                   
                   
                   
                 557 ± 25 
                 109 ± 22 
               
               
                   
               
               
                 NS1 327-343 
                 SGCWY 5 GMEIRPQRHDEK 
                   
                   
                   
                   
                   
                  78 ± 4 
               
               
                   
               
               
                 NS2a 6-23 
                 IDPFQ 1 LGLLV 1 VFLATQEV 
                   
                 437 ± 62 
                   
                 563 ± 41 
                  45 ± 7 
                 234 ± 8 
               
               
                   
               
               
                 NS2a 45-61 
                 VFGGI 5 TY TDVLRYVILV   
                   
                 243 ± 196 
                   
                 307 ± 119 
                   
                  74 ± 11 
               
               
                   
               
               
                 NS2a 52-66 
                   TDV 4 LRY 3,6 VILV GAA FA   
                   
                   
                   
                 516 ± 9 
                   
                   
               
               
                   
               
               
                 NS2a 65-81 
                   FA ESNSGGDVVHLAL MA   
                   
                   
                   
                   
                   
                  60 ± 13 
               
               
                   
               
               
                 NS2a 80-97 
                   MA TF 6 KIQ 3 PV 4 F 1 MVASFLKA 
                   
                   
                   
                 289 ± 83 
                   
                   
               
               
                   
               
               
                 NS2a 128-145 
                 EIPDVLNS 1   LAVAWMILRA   
                   
                   
                   
                 384 ± 125 
                   
                   
               
               
                   
               
               
                 NS2a 136-154 
                   LAVAW 4 MI 6 LRA I 1 TFTTTSNV 
                   
                   
                   
                  98 ± 22 
                   
                   
               
               
                   
               
               
                 NS2a 160-177 
                 ALLTPGLR C 1 L 5 NLDVYRIL   
                  63 ± 18 
                   
                   
                   
                  59 ± 3 
                   
               
               
                   
               
               
                 NS2a 168-183 
                   C 1 LN 1 LDV 4 YRIL LLMVGI 
                   
                   
                   
                 556 ± 33 
                 231 ± 16 
                   
               
               
                   
               
               
                 NS2a 211-229 
                 G 1 LFN 3 PMILAAGLIACDPNR 
                   
                   
                   
                  74 ± 23 
                   
                   
               
               
                   
               
               
                 NS2b 1-18 
                 GWPATEVM 1 TAVGLMFAIV 
                   
                   
                   
                   
                   
                 104 ± 18 
               
               
                   
               
               
                 NS2b 39-56 
                 MFAAFVISGKSTDMWIER 
                   
                   
                   
                  47 ± 18 
                   
                   
               
               
                   
               
               
                 NS2b 59-75 
                 DISWESDAEITGSSERV 
                   
                   
                   
                  59 ± 12 
                   
                   
               
               
                   
               
               
                 NS2b 84-101 
                 NF 1,6 QL 5 MNDPGAPWKIWMLR 
                   
                  35 ± 1 
                   
                   
                   
                   
               
               
                   
               
               
                 NS2b 107-124 
                 ISAYTPW 4,6 A ILPSVVGFWI   
                   
                  50 ± 11 
                  81 ± 8 
                   
                   
                 163 ± 4 
               
               
                   
               
               
                 NS2b 115-131 
                   ILPS 1 VVGFWI TLQYTKR 
                 108 ± 60 
                  56 ± 2 
                   
                   
                   
                   
               
               
                   
               
               
                 NS3 1-15 
                 GGV 5 LW DTPSPKEYKK   
                   
                   
                  45 ± 12 
                   
                   
                   
               
               
                   
               
               
                 NS3 6-23 
                   DTPSPKEYKK GDTTTGVY 
                   
                   
                   
                   
                   
                  43 ± 6 
               
               
                   
               
               
                 NS3 22-38 
                   V 4 Y RIMTRGL 1 LGSYQA GA   
                   
                   
                   
                 155 ± 22 
                   
                   
               
               
                   
               
               
                 NS3 37-54 
                   GA GV 1 MVEG VFHTLWHTTK   
                   
                   
                   
                 149 ± 13  
                 368 ± 66 
                   
               
               
                   
               
               
                 NS3 45-59 
                 
                   VFHTLW 6 HTTKGAALM 
                 
                   
                   
                   
                   
                  63 ± 10 
                   
               
               
                   
               
               
                 NS3 50-65 
                   WHTTKGAA 1 LM SGEGRL 
                   
                   
                   
                   
                 191 ± 37 
                   
               
               
                   
               
               
                 NS3 70-87 
                 GSVKEDRL 4   CYGGPWKLQH   
                 174 ± 13 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NS3 78-95 
                 
                   CYGGPWKLQHKWNGQDEV 
                 
                 150 ± 17 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NS3 85-100 
                 
                   LQHKWNG 1 QDEVQMIVV 
                 
                  72 ± 9 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NS3 91-107 
                 
                   G 1 QDEV 6 QMIVVEPGKNVK 
                 
                 156 ± 11 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NS3 98-115 
                   IVVEPGKNVK NVQTKPGV 
                 145 ± 25 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NS3 138-155 
                 PIVDKNGD V 4 IGLYGNGVI   
                 159 ± 10 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NS3 146-162 
                   V 4 I 4 GLY 6 GNGVI MPNGS YI   
                  58 ± 38 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NS3 161-177 
                   YI SAIVQGERMDEPI PA   
                  56 ± 28 
                   
                   
                  72 ± 15 
                   
                   
               
               
                   
               
               
                 NS3 176-192 
                   PA GFEPE M 1 LRKKQITVL   
                   
                   
                   
                  59 ± 8 
                   
                   
               
               
                   
               
               
                 NS3 183-200 
                   M 1 LRKKQITVL DLHPGA GK   
                  62 ± 24 
                   
                   
                  55 ± 2 
                   
                   
               
               
                   
               
               
                 NS3 199-216 
                   GK TRRIL 3   PQIIKEAINRR   
                   
                   
                   
                  52 ± 6 
                   
                   
               
               
                   
               
               
                 NS3 206-223 
                 
                   PQIIKEAIN 4 RRLRTAVLA 
                 
                 108 ± 22 
                   
                   
                 323 ± 21 
                   
                   
               
               
                   
               
               
                 NS3 214-230 
                 
                   NRRL 6 R 4 TA 3 VLAPTRVVAA 
                 
                   
                   
                   
                 146 ± 29 
                   
                   
               
               
                   
               
               
                 NS3 221-237 
                 
                   VLAPTRVVAAEMAEALR 
                 
                  26 ± 4 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NS3 228-243 
                   VAAEMA 4 EALR GLP IRY   
                   
                   
                   
                   
                   
                  46 ± 9 
               
               
                   
               
               
                 NS3 241-258 
                   IRY 6   QTSAV PREHNGNEIV   
                 127 ± 18 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NS3 249-266 
                   PREHNGNEIV DVMCHA TL   
                  79 ± 27 
                   
                   
                 139 ± 25 
                   
                  46 ± 16 
               
               
                   
               
               
                 NS3 265-282 
                   TL T 4 HRLMSPHRVPNYNLF 
                 150 ± 30 
                   
                   
                 169 ± 16 
                   
                   
               
               
                   
               
               
                 NS3 304-321 
                 KVELGEAAAIFMT ATPPG   
                  94 ± 22 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NS3 317-333 
                   AT 3 PPG TS DPFPESNSPI   
                 151 ± 18 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NS3 324-340 
                 
                   DPFPESNSPISDLQTEI 
                 
                 117 ± 20 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NS3 336-352 
                   LQTEI PD RAWNSGYEWI   
                 167 ± 35 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NS3 343-360 
                   RAWNSGYEWI TEYTGK TV   
                  55 ± 21 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NS3 359-376 
                   TV WF 5,6 VPSVKIAGNEIALCL 
                  75 ± 44 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NS3 413-430 
                 EM 5 G 4 ANF 6 KASR 3 VIDSRKSV 
                 130 ± 15 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NS3 455-469 
                 AAQRR GRIGRNPSQV   
                   
                   
                   
                 200 ± 27 
                   
                   
               
               
                   
               
               
                 NS3 460-475 
                   GRIGRNPSQV GDEY CY   
                   
                   
                   
                 123 ± 4 
                   
                   
               
               
                   
               
               
                 NS3 474-489 
                   CY GGHT NEDDSNFAHW   
                  92 ± 18 
                   
                   
                   
                  83 ± 23 
                   
               
               
                   
               
               
                 NS3 480-496 
                 
                   NEDDSNFAHWTEARIML 
                 
                   
                   
                   
                   
                 121 ± 1 
                   
               
               
                   
               
               
                 NS3 487-501 
                 
                   AHWTEARIMLDNINM 
                 
                 308 ± 188 
                   
                   
                   
                 431 ± 112 
                   
               
               
                   
               
               
                 NS3 492-509 
                 
                   ARIM 6 LDNINM 3 PNGLIAQF 
                 
                   
                   
                   
                   
                 216 ± 42 
                   
               
               
                   
               
               
                 NS3 500-517 
                   NMPNGLIAQF YQPER EKV   
                   
                   
                   
                  51 ± 10 
                 145 ± 49 
                   
               
               
                   
               
               
                 NS3 515-532 
                   EKV Y 1 TMDG EYRLRGEERK   
                 140 ± 24 
                   
                   
                  95 ± 27 
                   
                   
               
               
                   
               
               
                 NS3 523-539 
                 
                   EYRLRGEERKNFLELLR 
                 
                  57 ± 11 
                   
                   
                 160 ± 48 
                   
                   
               
               
                   
               
               
                 NS3 530-547 
                   ERKNFL 4 ELLR 1   TADLP VWL   
                 127 ± 10 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NS3 545-562 
                   VW 1 L AYKVAAAGVSYH DRR   
                   
                   
                   
                   
                 110 ± 9 
                   
               
               
                   
               
               
                 NS3 560-574 
                   DRR WC F 6 DGPRTNTIL   
                   
                   
                   
                   
                 225 ± 10 
                   
               
               
                   
               
               
                 NS3 565-582 
                   FDGPRTNT 1 IL EDNNEV EV   
                   
                   
                   
                   
                  59 ± 3 
                   
               
               
                   
               
               
                 NS3 581-597 
                   EV ITK 5 L GERKILRPRWI   
                   
                   
                   
                   
                   
                  52 ± 11 
               
               
                   
               
               
                 NS3 588-605 
                   ERKILR 3 PRWI DARVYSDH 
                   
                   
                   
                  42 ± 4 
                   
                   
               
               
                   
               
               
                 NS4a 1-17 
                 S 1 QIG 1 LIEVLGKMPE HFM   
                   
                   
                   
                   
                 132 ± 17 
                   
               
               
                   
               
               
                 NS4a 15-31 
                   HF 1 M GK 1 TWEA 1 LDTMYVVA 
                   
                   
                   
                 278 ± 26 
                   
                 187 ± 20 
               
               
                   
               
               
                 NS4a 54-71 
                 I 1 A 1 L 4 IAL 1 LS V 1 M 1,2 TMGVFFLL   
                   
                  70 ± 16 
                   
                   
                   
                   
               
               
                   
               
               
                 NS4a 62-79 
                   V 1 M 1,2 TMGVFFLL MQRKGIGK 
                   
                 117 ± 39 
                   
                   
                   
                   
               
               
                   
               
               
                 NS4a 86-103 
                 VLGVAT 1 FFCWMAEVPGTK 
                   
                  65 ± 30 
                   
                   
                   
                  63 ± 1 
               
               
                   
               
               
                 NS4b 30-47 
                 GEF 1,5,6 LLDL 4 R 3   PA 1 TAWSLYAV   
                   
                   
                   
                  89 ± 21 
                   
                   
               
               
                   
               
               
                 NS4b 38-55 
                   PA 1 TAWS 1 L 2,4 Y 6 AV TTAVLTPL 
                   
                   
                   
                  57 ± 11 
                  67 ± 13 
                   
               
               
                   
               
               
                 NS4b 63-80 
                 DY 1 I 6 N 4 TS 1 L 6 TSINVQASALF 
                   
                   
                   
                   
                 386 ± 60 
                  92 ± 13 
               
               
                   
               
               
                 NS4b 87-103 
                 PF 1 VDVGVSALLLAA GCW   
                   
                   
                   
                   
                   
                  59 ± 14 
               
               
                   
               
               
                 NS4b 101-118 
                   GCW G 1 QV 6 TLTVTVTAATLL 
                   
                   
                   
                   
                   
                 134 ± 8 
               
               
                   
               
               
                 NS4b 187-204 
                 VV 5 NPSVKTVREAGI LITA   
                   
                   
                   
                   
                   
                  83 ± 13 
               
               
                   
               
               
                 NS4b 201-218 
                   LITA AAV 4 TLWENGASSVW 
                   
                   
                   
                   
                  99 ± 26 
                  73 ± 8 
               
               
                   
               
               
                 NS4b 233-250 
                 GWLS 1 CL 6 SI TW 4,6 TLIKNMEK   
                   
                   
                   
                  62 ± 6 
                   
                   
               
               
                   
               
               
                 NS4b 241-255 
                   TW 4,6 TLIKNMEK PGLKR 
                   
                  53 ± 32 
                   
                   
                   
                   
               
               
                   
               
               
                 NS5 115-132 
                 L 1 V 4 QS 2 YGWNI 4 VTMKSGVDV 
                   
                   
                   
                   
                  79 ± 23 
                   
               
               
                   
               
               
                 NS5 145-162 
                 CDIGESSS SAEVEEHRTI   
                   
                   
                  50 ± 22 
                   
                   
                   
               
               
                   
               
               
                 NS5 153-168 
                   SAEV 4 EEHRTI RVLEMV 
                 332 ± 52 
                   
                 112 ± 72 
                 250 ± 24 
                   
                   
               
               
                   
               
               
                 NS5 181-198 
                 V 6 KV 1 LCPY 1 MPK 1 VIEKMELL 
                   
                   
                   
                 122 ± 40 
                   
                   
               
               
                   
               
               
                 NS5 212-229 
                 SRNSTHEMYWVSRASGNV 
                   
                   
                   
                 168 ± 13 
                   
                   
               
               
                   
               
               
                 NS5 448-463 
                 ECHTCIYNMMGKREKK 
                  26 ± 7 
                   
                   
                   
                   
                   
               
               
                   
               
               
                 NS5 470-486 
                 AKGSRA 1,4 I W 2 F 1,4 MWLGARFL   
                   
                 183 ± 19 
                   
                   
                   
                   
               
               
                   
               
               
                 NS5 477-494 
                   W 2 F 1,4 MWLGARFL EFEALGFL 
                   
                 198 ± 60 
                   
                   
                   
                   
               
               
                   
               
               
                 NS5 598-615 
                 REDQRGS GQVVTYALNTF   
                   
                   
                   
                 252 ± 41 
                   
                   
               
               
                   
               
               
                 NS5 605-622 
                 
                   GQVV 6 TY 1 A 1,6 L 4,6 NTFTNLAVQL 
                 
                   
                   
                   
                 556 ± 27 
                   
                 240 ± 76 
               
               
                   
               
               
                 NS5 613-631 
                   NTF 1 TNLAVQL VRMMEGEGV 
                   
                   
                   
                   
                   
                 119 ± 21 
               
               
                   
               
               
                 NS5 704-721 
                 GWYDWQQVPFCSNHFTEL 
                   
                   
                   
                   
                   
                  65 ± 27 
               
               
                   
               
               
                 NS5 755-772 
                 DTACLAK 1 S 2   Y 6 AQMWLLLYF   
                   
                 132 ± 62 
                   
                   
                   
                   
               
               
                   
               
               
                 NS5 763-780 
                 
                   Y 6 AQMWLLL 5 YFHRRDLRLM 
                 
                   
                   
                  36 ± 16 
                   
                   
                  91 ± 32 
               
               
                   
               
               
                 NS5 771-788 
                   YFH5RRDL4,6RL 1   M   ANAICSAV 
                   
                   
                   
                   
                   
                 135 ± 47 
               
               
                   
               
               
                 NS5 791-808 
                 NWVPTGRTTWSIHAGGEW 
                   
                   
                   
                   
                   
                  78 ± 17 
               
               
                   
               
               
                 NS5 828-843 
                 WMEDKTPVEKWSDV PY   
                   
                   
                  39 ± 14 
                   
                   
                   
               
               
                   
               
               
                 NS5 842-859 
                   PY SGKREDIWCGSLIGTR 
                   
                 180 ± 66 
                   
                   
                   
                   
               
               
                   
               
               
                 NS5 863-879 
                 TWAENI 4,5,6 QVAINQVRAII 
                   
                  79 ± 11 
                   
                   
                 121 ± 2 
                  33 ± 1 
               
               
                   
               
               
                   a Sequence positions in boldface are those with more than one FILA-restricted T-cell response. 
               
               
                   b Underlined amino acids represent overlapping sequences of adjacent epitope peptides. 
               
               
                   c The amino acid residues with the superscript numbers 1 to 6 refer to the first residue of the HLA class I nonamer binders (1: A*0201; 2: A*2402; and 3: B*0702) or the nonamer core of the HLA class II binders (4: DRB1*1501; 5: DRB1*0301; and 6: DRB1*0401) predicted by the NetMHC 3.2 [48] (www.cbs.dtu.dk/services//NetMHC-3.2/) and NetMHCIIPan 1.0 [49] (www.cbs.dtu.dk/services/NetMHClIpan/) Web-server immunionformatic algorithims. Those sequences without a superscript number 1 to 6 did not have a predicted binder. 
               
             
          
         
       
     
         [0056]    As further characterization of the epitope peptides, their avidity in the IFN-γ ELISpot assay, which can be attributed either to the binding reaction of either the HLA or T-cell receptor molecules, was measured by titration of the peptides over a 100-fold range of concentrations (10, 1, and 0.1 μg/ml). The majority of the peptide epitopes, 37 of 74 HLA-restricted class 1 responses and 83 of 126 class II, demonstrated high functional avidity (Table 3). Notably, while 10 μg/ml resulted in the greatest number (200) of responses, only 54 required the high peptide concentration (10 μg) to elicit T-cell activation following 2 immunizations with the peptide pools. A majority of the assays of the combined class I and class II T-cell responses (120 of 200) were positive at 0.1 μg/ml peptide, including all M and E class II T-cell responses and peptides of each protein except for class I NS3 and NS5, and class II NS3 and NS4b responses. Many of the peptides with high T-cell response scores (&gt;200 SFC per 1×106 splenocytes) demonstrated comparable T-cell responses in assays with 1.0 and 0.1 μg/ml peptide. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
               
             
               
               
               
               
               
             
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 3A AND 3B 
               
             
             
               
                   
               
               
                 The apparent functional avidity of WNV T-cell epitope peptides in ELISpot assays of 
               
               
                 splenocytes from immunized HLA-transgenic mice. 
               
             
          
           
               
                 WNV 
                 T-cell responses (#) a   
               
             
          
           
               
                 protein 
                 L 
                 I 
                 H 
                 Total 
                 L 
                 I 
                 H 
                 Total 
                 L 
                 I 
                 H 
                 Total 
                 L 
                 I 
                 H 
                 Total 
               
               
                   
               
             
          
           
               
                 3A. HLA class I-restricted T-cell responses 
               
             
          
           
               
                   
                 A2 
                 A24 
                 B7 
                 A2, A24, B7 
               
             
          
           
               
                 C 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
               
               
                 prM 
                 0 
                 0 
                 1 
                 1 
                 0 
                 0 
                 2 
                 2 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
                 3 
                 4 
               
               
                 E 
                 2 
                 2 
                 4 
                 8 
                 0 
                 0 
                 6 
                 6 
                 2 
                 0 
                 0 
                 2 
                 4 
                 2 
                 10 
                 16 
               
               
                 NS1 
                 1 
                 0 
                 1 
                 2 
                 0 
                 0 
                 1 
                 1 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 2 
                 3 
               
               
                 NS2a 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 3 
                 3 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 3 
                 3 
               
               
                 NS2b 
                 0 
                 0 
                 1 
                 1 
                 1 
                 0 
                 2 
                 3 
                 0 
                 1 
                 0 
                 1 
                 1 
                 1 
                 3 
                 5 
               
               
                 NS3 
                 7 
                 9 
                 10 
                 26 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 1 
                 8 
                 9 
                 10 
                 27 
               
               
                 NS4a 
                 0 
                 0 
                 0 
                 0 
                 1 
                 1 
                 1 
                 3 
                 0 
                 0 
                 0 
                 0 
                 1 
                 1 
                 1 
                 3 
               
               
                 NS4b 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 1 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 0 
                 1 
               
               
                 NS5 
                 1 
                 0 
                 1 
                 2 
                 1 
                 1 
                 3 
                 5 
                 2 
                 2 
                 0 
                 4 
                 4 
                 3 
                 4 
                 11 
               
               
                 Total 
                 11 
                 11 
                 18 
                 40 
                 4 
                 2 
                 18 
                 24 
                 6 
                 3 
                 1 
                 10 
                 21 
                 16 
                 37 
                 74 
               
             
          
           
               
                 3B. HLA class II-restricted T-cell responses 
               
             
          
           
               
                   
                 DR-2 
                 DR-3 
                 DR-4 
                 DR-2, -3, -4 
               
             
          
           
               
                 C 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
                 0 
               
               
                 prM 
                 0 
                 0 
                 6 
                 6 
                 0 
                 0 
                 3 
                 3 
                 0 
                 0 
                 4 
                 4 
                 0 
                 0 
                 13 
                 13 
               
               
                 E 
                 0 
                 0 
                 8 
                 8 
                 0 
                 0 
                 12 
                 12 
                 0 
                 0 
                 6 
                 6 
                 0 
                 0 
                 26 
                 26 
               
               
                 NS1 
                 0 
                 0 
                 1 
                 1 
                 0 
                 0 
                 3 
                 3 
                 2 
                 0 
                 3 
                 5 
                 2 
                 0 
                 7 
                 9 
               
               
                 NS2a 
                 0 
                 0 
                 8 
                 8 
                 2 
                 1 
                 0 
                 3 
                 1 
                 0 
                 2 
                 3 
                 3 
                 1 
                 10 
                 14 
               
               
                 NS2b 
                 0 
                 0 
                 2 
                 2 
                 0 
                 0 
                 0 
                 0 
                 1 
                 0 
                 1 
                 2 
                 1 
                 0 
                 3 
                 4 
               
               
                 NS3 
                 7 
                 3 
                 6 
                 16 
                 6 
                 2 
                 3 
                 11 
                 3 
                 0 
                 1 
                 4 
                 16 
                 5 
                 10 
                 31 
               
               
                 NS4a 
                 0 
                 0 
                 1 
                 1 
                 1 
                 0 
                 0 
                 1 
                 1 
                 0 
                 1 
                 2 
                 2 
                 0 
                 2 
                 4 
               
               
                 NS4b 
                 2 
                 0 
                 1 
                 3 
                 3 
                 0 
                 0 
                 3 
                 1 
                 1 
                 3 
                 5 
                 6 
                 1 
                 4 
                 11 
               
               
                 NS5 
                 1 
                 2 
                 2 
                 5 
                 0 
                 0 
                 2 
                 2 
                 2 
                 1 
                 4 
                 7 
                 3 
                 3 
                 8 
                 14 
               
               
                 Total 
                 10 
                 5 
                 35 
                 50 
                 12 
                 3 
                 23 
                 38 
                 11 
                 2 
                 25 
                 38 
                 33 
                 10 
                 83 
                 126 
               
               
                   
               
               
                   a Low (L) avidity T-cell determinants defined as those IFN-γ ELISpot positive only at 10 μg/ml; intermediate (I), 10 and 1 μg/ml; and high (H), 10, 1 and 0.1 μg/ml. 
               
             
          
         
       
     
       Example 3 
     Correspondence Between Peptide and DNA Plasmid Immunization 
       [0057]    T-cell epitopes elicited by peptide immunization with adjuvant may differ from those elicited by viral infection because of the many differences in antigen delivery and processing, and the mechanisms involved in activation of the cellular immune response system. As an evaluation of the extent of these possible differences, the T-cell responses of DR2 transgenic mice immunized with NS3 peptides were compared to the responses to a DNA plasmid immunogen encoding the NS3 protein. The DNA construct was designed to encode NS3 as a cytoplasmic protein lacking a signal sequence and a transmembrane domain and therefore possibly subject to a processing pathway comparable to that of the NS3 proteolytically released from the viral proteome polyprotein. All but two of the peptide-specific T-cell responses following peptide immunization were also detected after DNA immunization ( FIG. 1 ). The correspondence between the two immunizations was greatest with the stronger T-cell responses, especially those of &gt;100 SFC/10 6  CD8 depleted splenocytes. The major difference was a greater number of low T-cell responses (&lt;50 SFC/10 6  CD8 depleted splenocytes) with DNA immunization. The data suggest that while processing pathways can be important in epitope activation of T cells, there are common mechanisms for selection of epitope peptides delivered as extracellular peptides or encoded by DNA for cellular synthesis. 
       Example 4 
     Evolutionary Conservation and Diversity of the WNV T-Cell Epitope Peptides 
       [0058]    Analysis of the evolutionary conservation and the variability of the T-cell epitope peptides identified in this study was performed to determine the distribution of these sequences in the known sequence dataset of WNV. A total of 2,746 complete and partial WNV protein sequences were extracted from the NCBI Entrez protein database (as of June 2007): C, 264; prM, 417; E, 927; NS1, 164; NS2a, 143; NS2b, 146; NS3, 146; NS4a, 142; NS4b, 141; and NS5, 256. The majority of WNV T-cell epitope peptides were highly conserved. All but 12 had an entropy of less than 1.0 (average 0.48) (Table 11) and 31 peptides with entropies of 0.1 or less, mainly present in the E, NS3, and NS5 proteins were found as the unmodified sequence in 99% or more of all WNV (Table 4). There were only 7 epitope peptides of the E, NS2a, NS3, NS4A, and NS5 proteins with entropies greater than 0.7 that included variant sequences present in more than 10% (11 to 23%) of the recorded WNV protein sequences (Table 5). However, it is noteworthy that each peptide epitope, except for sequences with entropy of 0.0 (completely conserved epitopes), were represented by multiple variant sequences with one or more amino acid mutations in a small fraction, less than 10%, of the reported sequences, and in many cases, less than 1%. For example, envelope peptide from position 62 to 77 aa with an entropy of 0.5, was present in 94.3% of the database sequences, while the remaining 5.7% of the database sequences were represented by 16 different variants, 14 of which were each present in less than 1.0% of the recorded viral sequences (Table 6). The origin of these apparently rare sequences is uncertain. Possibly, they represent an under-sequenced clade that is common in nature but localized to a region where the virus was not widely studied. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 4 
               
             
             
               
                   
               
               
                 Highly conserved WNV T-cell epitope peptides, 
               
               
                 entropy 0.1 or lower. 
               
             
          
           
               
                 Protein &amp; 
                   
                 Incidence 
                   
               
               
                 position 
                 Peptide sequence 
                 (%) a   
                 Entropy 
               
               
                   
               
             
          
           
               
                 prM 114-130 
                 STKATRYLVKTESWILR 
                 &gt;99 
                 0.1 
               
               
                   
               
               
                 prM 121-138 
                 LVKTESWILRNPGYALVA 
                 &gt;99 
                 0.1 
               
               
                   
               
               
                 E 99-113 
                 RGWGNGCGLFGKGSI 
                 &gt;99 
                 0.1 
               
               
                   
               
               
                 E 135-152 
                 IKYEVAIFVHGPTTVESH 
                 99 
                 0.1 
               
               
                   
               
               
                 E 293-310 
                 EKLQLKGTTYGVCSKAFK 
                 99 
                 0.1 
               
               
                   
               
               
                 E 370-386 
                 KVLIELEPPFGDSYIVV 
                 &gt;99 
                 0.0 
               
               
                   
               
               
                 E 384-399 
                 IVVGRGEQQINHHWHK 
                 &gt;99 
                 0.1 
               
               
                   
               
               
                 E 390-407 
                 EQQINHHWHKSGSSIGKA 
                 99 
                 0.1 
               
               
                   
               
               
                 E 450-467 
                 FRSLFGGMSWITQGLLGA 
                 &gt;99 
                 0.1 
               
               
                   
               
               
                 E 458-474 
                 SWITQGLLGALLLWMGI 
                 &gt;99 
                 0.1 
               
               
                   
               
               
                 E 465-482 
                 LGALLLWMGINARDRSIA 
                 99 
                 0.1 
               
               
                   
               
               
                 NS1 305-322 
                 GKLITDWCCRSCTLPPLR 
                 99 
                 0.1 
               
               
                   
               
               
                 NS2b 1-18 
                 GWPATEVMTAVGLMFAIV 
                 &gt;99 
                 0.1 
               
               
                   
               
               
                 NS3 45-59 
                 VFHTLWHTTKGAALM 
                 99 
                 0.1 
               
               
                   
               
               
                 NS3 50-65 
                 WHTTKGAALMSGEGRL 
                 99 
                 0.1 
               
               
                   
               
               
                 NS3 138-155 
                 PIVDKNGDVIGLYGNGVI 
                 99 
                 0.1 
               
               
                   
               
               
                 NS3 183-200 
                 MLRKKQITVLDLHPGAGK 
                 99 
                 0.1 
               
               
                   
               
               
                 NS3 265-282 
                 TLTHRLMSPHRVPNYNLF 
                 100 
                 0.0 
               
               
                   
               
               
                 NS3 359-376 
                 TVWFVPSVKMGNEIALCL 
                 99 
                 0.1 
               
               
                   
               
               
                 NS3 487-501 
                 AHWTEARIMLDNINM 
                 &gt;99 
                 0.1 
               
               
                   
               
               
                 NS3 565-582 
                 FDGPRTNTILEDNNEVEV 
                 99 
                 0.1 
               
               
                   
               
               
                 NS5 115-132 
                 LVQSYGWNIVTMKSGVDV 
                 &gt;99 
                 0.1 
               
               
                   
               
               
                 NS5 470-486 
                 AKGSRAIWFMWLGARFL 
                 &gt;99 
                 0.1 
               
               
                   
               
               
                 NS5 477-494 
                 WFMWLGARFLEFEALGFL 
                 100 
                 0.0 
               
               
                   
               
               
                 NS5 598-615 
                 REDQRGSGQVVTYALNTF 
                 100 
                 0.0 
               
               
                   
               
               
                 NS5 605-622 
                 GQVVTYALNTFTNLAVQL 
                 99 
                 0.1 
               
               
                   
               
               
                 NS5 613-631 
                 NTFTNLAVQLVRMMEGEGV 
                 99 
                 0.1 
               
               
                   
               
               
                 NS5 704-721 
                 GWYDWQQVPFCSNHFTEL 
                 100 
                 0.0 
               
               
                   
               
               
                 NS5 755-772 
                 DTACLAKSYAQMWLLLYF 
                 &gt;99 
                 0.1 
               
               
                   
               
               
                 NS5 763-780 
                 YAQMWLLLYFHRRDLRLM 
                 &gt;99 
                 0.1 
               
               
                   
               
               
                 NS5 771-788 
                 YFHRRDLRLMANAICSAV 
                 100 
                 0.0 
               
               
                   
               
               
                 NS5 842-859 
                 PYSGKREDIWCGSLIGTR 
                 99 
                 0.1 
               
               
                   
               
               
                   a Percentage incidence (rounded off to the nearest whole number) of the epitope peptide sequence in all reported WNV sequences. Those with &gt;99% incidence do not include 100%. SEQ ID NOs for each peptide are identified in Table 2. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 5 
               
             
             
               
                   
               
               
                 WNV T-cell epitope peptides with 
               
               
                 high variants incidence. 
               
             
          
           
               
                 Protein &amp; 
                   
                 Incidence 
                 En- 
               
               
                 position a   
                 Peptide sequence b   
                 (%) c   
                 tropy 
               
               
                   
               
               
                 E 119-136 
                 
                   FACSTKAIGRTILKENIK 
                 
                 81 
                 0.9 
               
               
                   
                 .......T.......... 
                 16 
                   
               
               
                   
                 ...T...T.WI.Q..... 
                  2 
                   
               
               
                   
                 11 variants 
                 &lt;1 each 
                   
               
               
                   
               
               
                 NS2a 168-183 
                 
                   CLNLDVYRILLLMVGI 
                 
                 78 
                 1.0 
               
               
                   
                 ...............V 
                 18 
                   
               
               
                   
                 .........V...I.. 
                  1 
                   
               
               
                   
                 4 variants 
                 &lt;1 each 
                   
               
               
                   
               
               
                 NS2a 211-229 
                 
                   GLFNPMILAAGLIACDPNR 
                 
                 75 
                 1.3 
               
               
                   
                 .............T..... 
                 12 
                   
               
               
                   
                 .V..........M...... 
                  7 
                   
               
               
                   
                 .F..........V...... 
                  3 
                   
               
               
                   
                 .M.S.LV............ 
                  1 
                   
               
               
                   
                 3 variants 
                 &lt;1 each 
                   
               
               
                   
               
               
                 NS3 317-333 
                 
                   ATPPGTSDPFPESNSPI 
                 
                 87 
                 0.7 
               
               
                   
                 ..............A.. 
                 11 
                   
               
               
                   
                 4 variants 
                 &lt;1 each 
                   
               
               
                   
               
               
                 NS3 343-360 
                 
                   RAWNSGYEWITEYTGKTV 
                 
                 69 
                 1.3 
               
               
                   
                 .............I.... 
                 23 
                   
               
               
                   
                 ....T........V.... 
                  6 
                   
               
               
                   
                 .............V.... 
                  1 
                   
               
               
                   
                 1 variant 
                 &lt;1 
                   
               
               
                   
               
               
                 NS4a 54-71 
                 
                   IALIALLSVMTMGVFFLL 
                 
                 80 
                 1.1 
               
               
                   
                 ....T............. 
                 11 
                   
               
               
                   
                 ..........SL...... 
                  4 
                   
               
               
                   
                 .V........SL...... 
                  3 
                   
               
               
                   
                 4 variants 
                 &lt;1 each 
                   
               
               
                   
               
               
                 NS5 863-879 
                 
                   TWAENIQVAINQVRAII 
                 
                 71 
                 1.6 
               
               
                   
                 ..............S.. 
                 12 
                   
               
               
                   
                 ......H.......SV. 
                  9 
                   
               
               
                   
                 ....D.........S.. 
                  2 
                   
               
               
                   
                 ......H.......SL. 
                  2 
                   
               
               
                   
                 10 variants 
                 &lt;1 each 
               
               
                   
               
               
                   a Epitope peptide sites (7), each with entropy greater than 0.7, that included at least one variant that was present in more than 10% of the recorded WNV protein sequences. 
               
               
                   b Evolutionary variants of the WNV predominant epitope peptide sequences (in bold face) are indicated with only the variant amino acids. Variant(s) with less than 1% incidence are indicated by the sum of the number of such variants. 
               
               
                   c Percentage incidence (rounded off to the nearest whole number) of the epitope peptide sequence and its variants in all WNV sequences analyzed. Those with &lt;1% incidence do not include 0%. SEQ ID NOs for each peptide are identified in Table 2. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 6 
               
             
             
               
                   
               
               
                 An example of a non-zero entropy WNV epitope 
               
               
                 peptide site. It commonly includes multiple 
               
               
                 sequences variant to the epitope, with one or 
               
               
                 more different amino acid mutations, each of 
               
               
                 which represented in a small fraction, 
               
               
                 less than 10%, of the reported sequences. 
               
             
          
           
               
                 Protein &amp; 
                   
                 Incidence 
                   
               
               
                 position 
                 Peptide sequence a   
                 (%) b   
                 Entropy 
               
               
                   
               
               
                 E 62-77 
                 
                   LATVSDLSTKAACPTM 
                 
                 94 
                 0.5 
               
               
                   
                 ..S......R...... 
                  1 
                   
               
               
                   
                 ..............A. 
                  1 
                   
               
               
                   
                 .....E.......... 
                 &lt;1 
                   
               
               
                   
                 ..S..E...R...... 
                 &lt;1 
                   
               
               
                   
                 A.SATEI.SS...... 
                 &lt;1 
                   
               
               
                   
                 A............... 
                 &lt;1 
                   
               
               
                   
                 ..A............. 
                 &lt;1 
                   
               
               
                   
                 ..I............. 
                 &lt;1 
                   
               
               
                   
                 ..S............. 
                 &lt;1 
                   
               
               
                   
                 ......H......A.. 
                 &lt;1 
                   
               
               
                   
                 ......H...T..A.. 
                 &lt;1 
                   
               
               
                   
                 ........I....... 
                 &lt;1 
                   
               
               
                   
                 .............A.. 
                 &lt;1 
                   
               
               
                   
                 ...............V 
                 &lt;1 
                   
               
               
                   
                 .........N...... 
                 &lt;1 
                   
               
               
                   
                 S............... 
                 &lt;1 
               
               
                   
               
               
                   a Variant of the epitope sequence (in bold face) are shown with the variable amino acids. 
               
               
                   b Incidence of the epitope peptide sequence in all reported WNV sequence data. Those with &lt;1% incidence do not include 0%. 
               
             
          
         
       
     
       Example 5 
     WNV-Specific T Cell Epitope Peptide Sequences 
       [0059]    A notable finding was that, despite the high WNV protein conservation, only 51 of the 137 HLA-restricted WNV epitope peptides of this study were specific for WNV (Table 7), with the remaining 86 shared among a number of other flaviviruses. The concentration of WNV specific epitope sequences was greatest in the NS2a, NS2b, NS4a, and NS4b proteins, with 23 of the 30 total epitope peptides of these proteins were specific to WNV. In contrast, there were only 28 WNV specific peptides of the total of 107 epitope peptides present in the E, NS1 and NS3 proteins. The WNV specificity of the epitope peptides was not a function of the conservation, which ranged from 75 to 99% of the recorded sequences. Notably, none of the epitope peptides of NS5, which collectively were among the most highly conserved sequences, was WNV specific. 
         [0000]    
       
         
               
             
               
               
               
               
             
           
               
                 TABLE 7 
               
             
             
               
                   
               
               
                 WNV-specific epitope peptides. 
               
             
          
           
               
                   
                   
                 Inci- 
                   
               
               
                 Protein &amp; 
                   
                 dence 
                 En- 
               
               
                 position 
                 Peptide sequence 
                 (%) a   
                 tropy 
               
               
                   
               
               
                 prM 136-153 
                 LVAAVIGWMLGSNTMQRV 
                 94 
                 0.4 
               
               
                   
               
               
                 E 62-77 
                 LATVSDLSTKAACPTM 
                 94 
                 0.5 
               
               
                   
               
               
                 E 119-136 
                 FACSTKAIGRTILKENIK 
                 81 
                 0.9 
               
               
                   
               
               
                 E 195-212 
                 GIDTNAYYVMTVGTKTFL 
                 86 
                 0.9 
               
               
                   
               
               
                 E 210-225 
                 TFLVHREWFMDLNLPW 
                 95 
                 0.3 
               
               
                   
               
               
                 E 356-373 
                 VTVNPFVSVATANAICVLI 
                 96 
                 0.3 
               
               
                   
               
               
                 E 384-399 
                 IVVGRGEQQINHHWHK 
                 99 
                 0.1 
               
               
                   
               
               
                 E 486-501 
                 LAVGGVLLFLSVNVHA 
                 98 
                 0.2 
               
               
                   
               
               
                 NS1 1-19 
                 DTGCAIDISRQELRCGSGV 
                 90 
                 0.7 
               
               
                   
               
               
                 NS1 161-178 
                 GLTSTRMFLKVRESNTTE 
                 87 
                 0.9 
               
               
                   
               
               
                 NS1 228-245 
                 THTLWGDGILESDLIIPV 
                 87 
                 0.8 
               
               
                   
               
               
                 NS2a 45-61 
                 VFGGITYTDVLRYVILV 
                 96 
                 0.4 
               
               
                   
               
               
                 NS2a 52-66 
                 TDVLRYVILVGAAFA 
                 97 
                 0.3 
               
               
                   
               
               
                 NS2a 80-97 
                 MATFKIQPVFMVASFLKA 
                 89 
                 0.7 
               
               
                   
               
               
                 NS2a 128-145 
                 EIPDVLNSLAVAWMILRA 
                 85 
                 1.0 
               
               
                   
               
               
                 NS2a 136-154 
                 LAVAWMILRAITFTTTSNV 
                 89 
                 0.7 
               
               
                   
               
               
                 NS2a 160-177 
                 ALLTPGLRCLNLDVYRIL 
                 87 
                 0.8 
               
               
                   
               
               
                 NS2a 168-183 
                 CLNLDVYRILLLMVGI 
                 78 
                 1.0 
               
               
                   
               
               
                 NS2a 211-229 
                 GLFNPMILAAGLIACDPNR 
                 75 
                 1.3 
               
               
                   
               
               
                 NS2b 59-75 
                 DISWESDAEITGSSERV 
                 85 
                 0.9 
               
               
                   
               
               
                 NS2b 84-101 
                 NFQLMNDPGAPWKIWMLR 
                 94 
                 0.5 
               
               
                   
               
               
                 NS2b 107-124 
                 ISAYTPWAILPSVVGFWI 
                 88 
                 0.6 
               
               
                   
               
               
                 NS2b 115-131 
                 ILPSVVGFWITLQYTKR 
                 89 
                 0.6 
               
               
                   
               
               
                 NS3 22-38 
                 VYRIMTRGLLGSYQAGA 
                 98 
                 0.2 
               
               
                   
               
               
                 NS3 50-65 
                 WHTTKGAALMSGEGRL 
                 99 
                 0.1 
               
               
                   
               
               
                 NS3 70-87 
                 GSVKEDRLCYGGPWKLQH 
                 97 
                 0.3 
               
               
                   
               
               
                 NS3 78-95 
                 CYGGPWKLQHKWNGQDEV 
                 87 
                 0.8 
               
               
                   
               
               
                 NS3 85-100 
                 LQHKWNGQDEVQMIVV 
                 88 
                 0.7 
               
               
                   
               
               
                 NS3 98-115 
                 IVVEPGICNVKNVQTKPGV 
                 97 
                 0.3 
               
               
                   
               
               
                 NS3 161-177 
                 YISAIVQGERMDEPIPA 
                 86 
                 0.8 
               
               
                   
               
               
                 NS3 176-192 
                 PAGFEPEMLRKKQITVL 
                 86 
                 0.4 
               
               
                   
               
               
                 NS3 241-258 
                 IRYQTSAVPREHNGNEIV 
                 81 
                 1.1 
               
               
                   
               
               
                 NS3 324-340 
                 DPFPESNSPISDLQTEI 
                 86 
                 0.9 
               
               
                   
               
               
                 NS3 474-489 
                 CYGGHTNEDDSNFAHW 
                 95 
                 0.4 
               
               
                   
               
               
                 NS3 480-496 
                 NEDDSNFAHWTEARIML 
                 94 
                 0.4 
               
               
                   
               
               
                 NS3 487-501 
                 AHWTEARIMLDNINM 
                 99 
                 0.1 
               
               
                   
               
               
                 NS3 492-509 
                 ARIMLDNINMPNGLIAQF 
                 89 
                 0.6 
               
               
                   
               
               
                 NS3 500-517 
                 NMPNGLIAQFYQPEREKV 
                 90 
                 0.6 
               
               
                   
               
               
                 NS3 581-597 
                 EVITKLGERKILRPRWI 
                 91 
                 0.6 
               
               
                   
               
               
                 NS3 588-605 
                 ERKILRPRWIDARVYSDH 
                 90 
                 0.6 
               
               
                   
               
               
                 NS4a 1-17 
                 SQIGLIEVLGKMPEHFM 
                 87 
                 0.8 
               
               
                   
               
               
                 NS4a 15-31 
                 HFMGKTWEALDTMYVVA 
                 97 
                 0.2 
               
               
                   
               
               
                 NS4a 54-71 
                 IALIALLSVMTMGVFFLL 
                 80 
                 1.1 
               
               
                   
               
               
                 NS4a 62-79 
                 VMTMGVFFLLMQRKGIGK 
                 92 
                 0.5 
               
               
                   
               
               
                 NS4a 86-103 
                 VLGVATFFCWMAEVPGTK 
                 84 
                 1.0 
               
               
                   
               
               
                 NS4b 38-55 
                 PATAWSLYAVTTAVLTPL 
                 97 
                 0.2 
               
               
                   
               
               
                 NS4b 63-80 
                 DYINTSLTSINVQASALF 
                 98 
                 0.2 
               
               
                   
               
               
                 NS4b 87-103 
                 PFVDVGVSALLLAAGCW 
                 96 
                 0.3 
               
               
                   
               
               
                 NS4b 201-218 
                 LITAAAVTLWENGASSVW 
                 91 
                 0.5 
               
               
                   
               
               
                 NS4b 233-250 
                 GWLSCLSITWTLIKNMEK 
                 76 
                 1.5 
               
               
                   
               
               
                 NS4b 241-255 
                 TWTLIKNMEKPGLKR 
                 79 
                 1.3 
               
               
                   
               
               
                   a Percentage incidence (round off to the nearest whole number) in all reported WNV sequence data. Those with &gt;99% incidence do not include 100%. SEQ ID NOs for each peptide are identified in Table 2. 
               
             
          
         
       
     
       Example 6 
       Flavivirus -Shared WNV T-Cell Epitope Peptide Sequences 
       [0060]    Representation of WNV epitope peptide sequences of 9 or more contiguous amino acids in other proteins was searched by BLAST analyses of all protein sequences deposited at NCBI (as of January 2009). Almost all flaviviruses, and few other viruses such as sindbis, Simian immunodeficiency and Trichoplusia ni SNPV, shared 1 or more such sequences of the 86 non-WNV specific epitope peptides, from a single WNV sequence in 1  Flavivirus  to the presence of the NS5 598-615 sequence in 62 flaviviruses ( FIGS. 2 and 3 ). Sequences of other NS5 epitope peptides and E 99-113 were each present in over 30 flaviviruses and particularly in the closely related Murray Valley encephalitis, Japanese encephalitis, and Usutu viruses. While most of the 86 non-WNV specific epitope peptides were only partially identical by sequences of 9 or more amino acids, 19 of the WNV epitope peptides were represented by complete sequence match in 28 other flaviviruses (Table 8). These complete epitope peptides consisted mainly of the E, NS3, and NS5 sequences, and included several of the most highly conserved sequences with entropy from 0.1 to 0.0. Three WNV epitope peptides (3) (E 99-113, NS5 598-615, and NS5 771-788) with entropies of 0.1 to 0.0 and conserved in &gt;99% or all recorded WNV, were extensively represented in 12 to 14 other flaviviruses ( FIG. 4 ). Japanese encephalitis and Usutu viruses each contained 9; Koutango, 8; Murray Valley, 7; Ilheus and Cacipacore, 6; St Louis and Bagaza, 5; and several others, 3 or fewer. There was no direct correlation between the diversity of the peptide and the number of flaviviruses that shared the sequence, and epitope peptides with greater entropies were also shared with multiple viruses. Because the 19 WNV epitope peptides were completely conserved in other flaviviruses, it is very likely that these shared sequences are corresponding HLA-restricted T cell epitopes of these viruses ( FIG. 4 ). 
         [0000]    
       
         
               
             
               
               
               
               
               
             
           
               
                 TABLE 8 
               
             
             
               
                   
               
               
                 WNV T-cell epitope peptides with full-length 
               
               
                 occurrence in other flaviviruses. 
               
             
          
           
               
                   
                   
                 Shared 
                   
                   
               
               
                 Protein &amp; 
                   
                 flaviviruses 
                 Incidence 
                   
               
               
                 position 
                 Peptide sequence 
                 (#) a   
                 (%) b   
                 Entropy 
               
               
                   
               
               
                 prM 152-167 
                 RVVEVVLLLLVAPAYS 
                  1 
                  95 
                 0.4 
               
               
                   
               
               
                 E 99-113 
                 RGWGNGCGLFGKGSI 
                 14 
                 &gt;99 
                 0.1 
               
               
                   
               
               
                 E 370-386 
                 KVLIELEPPFGDSYIVV 
                  1 
                 &gt;99 
                 0.0 
               
               
                   
               
               
                 E 398-415 
                 HKSGSSIGKAFTTTLKGA 
                  1 
                  97 
                 0.3 
               
               
                   
               
               
                 E 450-467 
                 FRSLFGGMSWITQGLLGA 
                  1 
                 &gt;99 
                 0.1 
               
               
                   
               
               
                 NS1 305-322 
                 GKLITDWCCRSCTLPPLR 
                  2 
                  99 
                 0.1 
               
               
                   
               
               
                 NS3 221-237 
                 VLAPTRVVAAEMAEALR 
                  2 
                  90 
                 0.7 
               
               
                   
               
               
                 NS3 228-243 
                 VAAEMAEALRGLPIRY 
                  1 
                  90 
                 0.6 
               
               
                   
               
               
                 NS3 304-321 
                 KVELGEAAAIFMTATPPG 
                  6 
                  95 
                 0.3 
               
               
                   
               
               
                 NS3 530-547 
                 ERKNFLELLRTADLPVWL 
                  1 
                  92 
                 0.4 
               
               
                   
               
               
                 NS5 448-463 
                 ECHTCIYNMMGKREKK 
                  5 
                  92 
                 0.5 
               
               
                   
               
               
                 NS5 470-486 
                 AKGSRAIWFMWLGARFL 
                  5 
                 &gt;99 
                 0.0 
               
               
                   
               
               
                 NS5 477-494 
                 WFMWLGARFLEFEALGFL 
                  4 
                 100 
                 0.0 
               
               
                   
               
               
                 NS5 598-615 
                 REDQRGSGQVVTYALNTF 
                 12 
                 100 
                 0.0 
               
               
                   
               
               
                 NS5 605-622 
                 GQVVTYALNTFTNLAVQL 
                  4 
                  99 
                 0.1 
               
               
                   
               
               
                 NS5 613-631 
                 NTFTNLAVQLVRMMEGEGV 
                  1 
                  99 
                 0.1 
               
               
                   
               
               
                 NS5 763-780 
                 YAQMWLLLYFHRRDLRLM 
                  5 
                 &gt;99 
                 0.1 
               
               
                   
               
               
                 NS5 771-788 
                 YFHRRDLRLMANAICSAV 
                 11 
                 100 
                 0.0 
               
               
                   
               
               
                 NS5 842-859 
                 PYSGKREDIWCGSLIGTR 
                  1 
                  98 
                 0.1 
               
               
                   
               
               
                   a Number of shared flaviviruses other than WNV. 
               
               
                   b Percentage incidence (round off to the nearest whole number) of the epitope peptide sequence in all reported WNV sequence data. Those with &gt;99% incidence do not include 100%. SEQ ID NOs for each peptide are identified in Table 2. 
               
             
          
         
       
     
       Example 7 
     Representation of WNV T-Cell Epitope Peptides and their Variants in Major Flaviviruses 
       [0061]    Further analysis of the sequence representation of the WNV epitope peptides was performed with other flaviviruses that had adequate database information. These included the Japanese encephalitis group [JEV, LEV (St. Louis EV)]; the tick-borne encephalitis virus group [TBEV, PV (powassan virus)]; yellow fever virus (YFV); and dengue virus (DV). The representation of many of the epitope peptides ranged from low (˜1%) to high (100%) among known sequences of the highly studied flaviviruses (Table 9). In particular, the epitopes from the NS5 protein were observed to be highly represented among many of the major flaviviruses, with identical or mutated sequences highly specific to the individual flaviviruses (Table 10). For example, the epitope peptide NS5 212-229  is present as the dominant sequence of the recorded WNV sequences (130 of 143) and not in any of the other selected viruses; however, specific mutant variants of this sequence were predominant peptides in LEV (26 or 29) and YFV (19 or 22); and several forms were present in dengue viruses with significant representation. The NS5 448-463 peptide and other WNV NS5 epitope peptides were either unique to WNV or shared with members of the closely related JEV group (LEV and/or JEV), and mutated forms were predominant peptide sequences in members of other less related flaviviruses of the tick-borne encephalitis virus group (TBEV and Powassan virus), yellow fever virus, and dengue. It thus is apparent that multiple forms of many WNV epitope peptide variants, that have only minor representation in the WNV database, are not specific for WNV and are widely present as predominant peptide sequences in other flaviviruses. 
         [0000]    
       
         
               
             
               
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 9 
               
             
             
               
                   
               
               
                 The distribution of cross-reactive WNV T-cell epitope peptides in other major  flaviviruses . 
               
             
          
           
               
                   
                   
                 Percentage incidence (%)  b   
               
             
          
           
               
                 Protein &amp; position 
                 Peptide sequence  a   
                 LEV 
                 JEV 
                 TBEV 
                 PV 
                 YFV 
                 DENV 
               
               
                   
               
               
                 prM 114-130 
                 STKATRYLVKTESWILR 
                   
                 0|1 
                   
                   
                   
                   
               
               
                 prM 121-138 
                 LVKTESWILRNPGYALVA 
                  0|100 
                   
                   
                   
                   
                 0|9 
               
               
                 prM 129-145 
                 LRNPGYALVAAVIGWML 
                  0|100 
                   
                   
                   
                   
                   
               
               
                 prM 152-167 
                 RVVFVVLLLLVAPAYS 
                   
                  0|97 
                   
                   
                   
                   
               
               
                 E 99-113 
                 
                   
                     RGWGNGCGLFG 
                     K 
                     GSI 
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                    0|&gt;99 
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
               
               
                 E 127-144 
                 GRTILKENIKYEVAIFVH 
                  0|98 
                   
                   
                   
                   
                   
               
               
                 E 135-152 
                 IKYEVAIFVHGPTTVESH 
                  0|96 
                   
                   
                   
                   
                   
               
               
                 E 238-255 
                 TLMEFEEPHATKQSVIAL 
                  0|98 
                   
                   
                   
                   
                   
               
               
                 E 293-310 
                 EKLQLKGTTYGVCSKAFK 
                   
                 0|1 
                   
                   
                   
                   
               
               
                 E 370-386 
                 KVLIELEPPFGDSYIVV 
                  0|93 
                  0|99 
                   
                   
                   
                  0|41 
               
               
                 E 426-443 
                 WDFGSVGGVFTSVGKAVH 
                   
                   
                   
                  0|50 
                   
                  0|55 
               
               
                 E 434-451 
                 VFTSVGKAVHQVFGGAFR 
                  0|99 
                  0|98 
                   
                   
                   
                 0|8 
               
               
                 E 442-459 
                 VHQVFGGAFRSLFGGMSW 
                  0|98 
                  0|98 
                   
                   
                   
                   
               
               
                 E 450-467 
                 FRSLFGGMSWITQGLLGA 
                  0|100 
                  0|99 
                   
                   
                   
                   
               
               
                 E 458-474 
                 SWITQGLLGALLLWMGI 
                  0|100 
                 0|3 
                   
                   
                   
                   
               
               
                 E 465-482 
                 LGALLLWMGINARDRSIA 
                  0|100 
                 0|4 
                   
                   
                   
                   
               
               
                 NS1 205-221 
                 RLNDTWKLERAVLGEVK 
                   
                  0|94 
                   
                   
                   
                   
               
               
                 NS1 270-287 
                 EGRVEIDFDYCPGTTVTL 
                  0|97 
                 0|2 
                   
                   
                   
                   
               
               
                 NS1 305-322 
                 
                   
                     GKLITDWCCRSCTLPPLR 
                   
                 
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                   
                   
                   
                  0|97 
               
               
                 NS1 327-343 
                 SGCWYGMEIRPQRHDEK 
                  0|100 
                  0|98 
                   
                   
                   
                  0|100 
               
               
                 NS2a 6-23 
                 IDPFQLGLLVVFLATQEV 
                   
                  0|98 
                   
                   
                   
                   
               
               
                 NS2b 1-18 
                 GWPATEVMTAVGLMFAIV 
                   
                  0|94 
                   
                   
                   
                   
               
               
                 NS3 1-15 
                 GGVLWDTPSPKEYKK 
                   
                   
                   
                   
                   
                  0|36 
               
               
                 NS3 6-23 
                 DTPSPKEYKKGDTTTGVY 
                   
                  0|98 
                   
                   
                   
                   
               
               
                 NS3 37-54 
                 GAGVMVEGVFHTLWHTTK 
                   
                  0|97 
                   
                   
                   
                   
               
               
                 NS3 45-59 
                 VFHTLWHTTKGAALM 
                   
                  0|97 
                   
                   
                   
                   
               
               
                 NS3 138-155 
                 PIVDKNGDVIGLYGNGVI 
                   
                   
                   
                   
                   
                 0|3 
               
               
                 NS3 146-162  
                 VIGLYGNGVIMPNGSYI 
                   
                   
                   
                   
                   
                 0|3 
               
               
                 NS3 214-230 
                 NRRLRTAVLAPTRVVAA 
                  0|100 
                  0|100 
                   
                   
                   
                  0|66 
               
               
                 NS3 221-237 
                 
                   
                     VLAPTRVVAAEMAEALR 
                   
                 
                  0|100 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                   
                   
                   
                  0|66 
               
               
                 NS3 228-243 
                 VAAEMAEALRGLPIRY 
                   
                  0|95 
                   
                   
                   
                  0|41 
               
               
                 NS3 249-266  
                 PREHNGNEIVDVMCHATL 
                  0|100 
                  0|100 
                  0|100 
                   
                   
                   
               
               
                 NS3 265-282 
                 TLTHRLMSPHRVPNYNLF 
                   
                  0|100 
                   
                   
                   
                   
               
               
                 NS3 304-321  
                 
                   
                     KVELGEAAAIFMTATPPG 
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                   
                   
                   
                  0|100 
               
               
                 NS3 343-360 
                 RAWNSGYEWITEYTGKTV 
                   
                  0|75 
                   
                   
                   
                  0|24 
               
               
                 NS3 359-376  
                 TVWFVPSVKMGNEIALCL 
                  0|100 
                  0|95 
                   
                   
                   
                   
               
               
                 NS3 455-469 
                 AAQRRGRIGRNPSQV 
                  0|81 
                   
                   
                   
                  0|100 
                  0|73 
               
               
                 NS3 460-475  
                 GRIGRNPSQVGDEYCY 
                  0|81 
                 0|2 
                   
                   
                   
                   
               
               
                 NS3 515-532  
                 EKVYTMDGEYRLRGEERK 
                   
                  0|98 
                   
                   
                   
                  0|72 
               
               
                 NS3 523-539  
                 EYRLRGEERKNFLELLR 
                  0|100 
                 0|2 
                   
                   
                   
                   
               
               
                 NS3 530-547 
                 
                   
                     ERKNFLELLRTADLPVWL 
                   
                 
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                   
                   
                   
                   
               
               
                 NS3 545-562 
                 VWLAYKVAAAGVSYHDRR 
                   
                   
                   
                   
                   
                  0|20 
               
               
                 NS3 560-574  
                 DRRWCFDGPRTNTIL 
                   
                  0|100 
                   
                   
                   
                   
               
               
                 NS3 565-582 
                 FDGPRTNTILEDNNEVEV  
                  0|100 
                   
                   
                   
                   
                   
               
               
                 NS4b 30-47 
                 GEFLLDLRPATAWSLYAV 
                   
                  0|100 
                   
                   
                   
                   
               
               
                 NS4b 101-118 
                 GCWGQVTLTVTVTAATLL 
                   
                 0|7 
                   
                   
                   
                   
               
               
                 NS5 145-162  
                 CDIGESSSSAEVEEHRTI 
                   
                   
                   
                   
                  0|86 
                   
               
               
                 NS5 181-198  
                 VKVLCPYMPKVIEKMELL 
                  0|64 
                  0|100 
                   
                   
                   
                   
               
               
                 NS5 212-229 
                 SRNSTHEMYWVSRASGNV 
                  0|93 
                   
                  0|100 
                  0|100 
                  0|100 
                  0|99 
               
               
                 NS5 448-463  
                 
                   
                     ECHTCIYNMMGKREKK 
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                  0|100 
                  0|88 
                  0|100 
                    0|&gt;99 
               
               
                 NS5 470-486  
                 AKGSRAIWFMWLGARFL 
                   
                  0|100 
                   
                   
                   
                   
               
               
                 NS5 477-494  
                 WFMWLGARFLEFEALGFL 
                  0|100 
                  0|100 
                  0|100 
                   
                  0|100 
                    0|&gt;99 
               
               
                 NS5 598-615  
                 
                   
                     REDQRGSGQVVTYALNTF 
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                  0|100 
                  0|95 
                  0|100 
                 0|3 
               
               
                 NS5 605-622  
                 
                   
                     GQVVTYALNTFTNLAVQL 
                   
                 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                  0|100 
                  0|100 
                  0|90 
                  0|100 
                   
               
               
                 NS5 613-631 
                 NTFTNLAVQLVRMMEGEGV 
                  0|100 
                   
                   
                   
                   
                   
               
               
                 NS5 704-721  
                 GWYDWQQVPFCSNHFTEL 
                   
                  0|98 
                   
                   
                   
                  0|61 
               
               
                 NS5 755-772  
                 DTACLAKSYAQMWLLLYF 
                  0|97 
                  0|95 
                   
                   
                   
                   0|&lt;1 
               
               
                 NS5 763-780  
                 
                   
                     YAQMWLLLYFHRRDLRLM 
                   
                 
                   
                 
                   
                             
                     
                         
                         
                     
                   
                 
                   
                   
                   
                    0|&gt;99 
               
               
                 NS5 771-788 
                 
                   
                     YFHRRDLRLMANAICSAV 
                   
                 
                  0|97 
                 
                   
                             
                     
                         
                         
                     
                   
                 
                   
                   
                   
                    0|&gt;99 
               
               
                 NS5 791-808 
                 NWVPTGRTTWSIHAGGEW 
                  0|100 
                   
                  0|97 
                  0|100 
                   
                   
               
               
                 NS5 828-843 
                 WMEDKTPVEKWSDVPY 
                   
                   
                   
                   
                   
                  0|34 
               
               
                 NS5 842-859 
                 PYSGKREDIWCGSLIGTR 
                  0|100 
                  0|100 
                   
                   
                   
                   
               
               
                 NS5 863-879 
                 TWAENIQVAINQVRAII 
                   
                  0|25 
                   
                   
                   
                  0|34 
               
               
                   
               
               
                   a  The WNV epitope peptide sequences that had 9 or more consecutive amino acids shared with any of the six other  flaviviruses . WNV epitope peptide sequences that have a full-length match to the sequences of any one of the six other  flaviviruses  are shown in boldface and underlined. 
               
               
                   b  Percentage incidence is depicted as “X|Y” where “X” refers to the percentage of the total virus sequences analyzed with full-length match to the WNV epitope peptide, and “Y” refers to the percentage of total virus sequences studied with ≧9 consecutive amino acids match to the WNV epitope. The “X” value is shaded                          
 when there is a full-length match in the respective virus. The  Flavivirus  species abbreviations: LEV, St. Louis  encephalitis  virus; JEV, Japanese  encephalitis  virus; TBEV, Tick-borne  encephalitis  virus; PV,  Powassan  virus; YFV, Yellow fever virus and DENY, Dengue virus. SEQ ID NOs for each peptide are identified in Table 2. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
               
               
             
           
               
                 TABLE 10 
               
             
             
               
                   
               
               
                 Variants of highly shared WNV epitope peptides and their 
               
               
                 incidence in other selected flaviviruses. WNV variant 
               
               
                 sequences representing less than about 10% of the 
               
               
                 corresponding database sequences were omitted. 
               
             
          
           
               
                   
                 Representation  b  of the peptides in 
               
               
                 WNV epitope peptide 
                 the respective flaviviruses 
               
               
                 and its variant  a   
                 (# of sequences analyzed) 
               
               
                   
               
             
          
           
               
                   
                 WNV 
                 LEV 
                 TBEV 
                 PV 
                 YFV 
                 DENV 
                   
               
               
                 NS5 212-229 
                 (143) 
                 (29) 
                 (29) 
                 (21) 
                 (22) 
                 (1309) 
                 Total 
               
               
                   
               
               
                 
                   SRNSTHEMYWVSRASGNV 
                 
                 130 
                  0 
                  0 
                  0 
                  0 
                   0 
                 130 
               
               
                   
               
               
                 ............H....I 
                  11 
                  0 
                  0 
                  0 
                  0 
                   0 
                  11 
               
               
                   
               
               
                 ............CGT..I 
                   0 
                  0 
                  0 
                  0 
                  0 
                 471 
                 471 
               
               
                   
               
               
                 ..........I.NGT..I 
                   0 
                  0 
                  0 
                  0 
                  0 
                 290 
                 290 
               
               
                   
               
               
                 ............N.T..I 
                   0 
                  0 
                  0 
                  0 
                  0 
                 244 
                 244 
               
               
                   
               
               
                 ............N....I 
                   0 
                  0 
                  0 
                  0 
                  0 
                 243 
                 243 
               
               
                   
               
               
                 ............G.A..I 
                   0 
                 26 
                  0 
                  0 
                  0 
                   0 
                  26 
               
               
                   
               
               
                 .........Y..G.RS.. 
                   0 
                  0 
                  0 
                  0 
                 19 
                   0 
                  19 
               
               
                   
               
               
                   
                 WNV 
                 LEV 
                 JEV 
                 TBEV 
                 PV 
                 YFV 
                   
               
               
                 NS5 448-463 
                 (182) 
                 (45) 
                 (60) 
                 (33) 
                 (24) 
                 (22) 
                 Total 
               
               
                   
               
               
                 
                   ECHTCIYNMMGKREKK 
                 
                 167 
                 43 
                 56 
                  0 
                  0 
                   0 
                 266 
               
               
                   
               
               
                 Q.RH.V.......... 
                   0 
                  0 
                  0 
                  0 
                 21 
                   0 
                  21 
               
               
                   
               
               
                   
                 WNV 
                 LEV 
                 JEV 
                 TBEV 
                 YFV 
                 DENV 
                   
               
               
                 NS5 477-494 
                 (181) 
                 (48) 
                 (60) 
                 (33) 
                 (23) 
                 (1372) 
                 Total 
               
               
                   
               
               
                 
                   WFMWLGARFLEFRALGFL 
                 
                 181 
                  0 
                  0 
                  0 
                  0 
                   0 
                 181 
               
               
                   
               
               
                 .Y................ 
                   0 
                 46 
                  0 
                  0 
                  3 
                 541 
                 590 
               
               
                   
               
               
                 .Y...............M 
                   0 
                  0 
                  0 
                  0 
                  0 
                 474 
                 474 
               
               
                   
               
               
                 .Y......Y......... 
                   0 
                  0 
                  0 
                  0 
                 20 
                 344 
                 364 
               
               
                   
               
               
                 ........Y......... 
                   0 
                  0 
                 60 
                  0 
                  0 
                   0 
                  60 
               
               
                   
               
               
                 .Y....S........... 
                   0 
                  0 
                  0 
                 33 
                  0 
                   0 
                  33 
               
               
                   
               
               
                   
                 WNV 
                 LEV 
                 JEV 
                 TBEV 
                 PV 
                 DENV 
                   
               
               
                 NS5 598-615 
                 (182) 
                 (28) 
                 (59) 
                 (30) 
                 (21) 
                 (1379) 
                 Total 
               
               
                   
               
               
                 
                   REDQRGSGQVVTYALNTF 
                 
                 182 
                 28 
                 58 
                  0 
                  0 
                   0 
                 268 
               
               
                   
               
               
                 .K........G..G.... 
                   0 
                  0 
                  0 
                  0 
                  0 
                 477 
                 477 
               
               
                   
               
               
                   
                 WNV 
                 LEV 
                 JEV 
                 TBEV 
                 PV 
                 YFV 
                   
               
               
                 NS5 605-622 
                 (182) 
                 (28) 
                 (59) 
                 (30) 
                 (21) 
                 (22) 
                 Total 
               
               
                   
               
               
                 
                   GQVVTYALNTFTNLAVQL 
                 
                 180 
                 28 
                  0 
                  0 
                  0 
                   0 
                 208 
               
               
                   
               
               
                 ..............S... 
                   2 
                  0 
                  0 
                  0 
                  0 
                   0 
                  2 
               
               
                   
               
               
                 ..........L..IK... 
                   0 
                  0 
                  0 
                 30 
                  0 
                   0 
                  30 
               
               
                   
               
               
                 .............I.... 
                   0 
                  0 
                 58 
                  0 
                  0 
                   0 
                  58 
               
               
                   
               
               
                 ..........I...K... 
                   0 
                  0 
                  0 
                  0 
                  0 
                  21 
                  21 
               
               
                   
               
               
                 ..........I..MK... 
                   0 
                  0 
                  0 
                  0 
                 17 
                   0 
                  17 
               
               
                   
               
               
                   a  The epitope sequences are shown in bold face and the mutations in the variant peptides are shown by the respective variant amino acids. 
               
               
                   b  Data was collected from the NCBI Entrez Protein Database (as of January 2009) SEQ ID NOs for each peptide are identified in Table 2. 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 11 
               
             
             
               
                   
               
               
                 WNV HLA-restricted T-cell epitope peptides incidence and their variant 
               
               
                 incidence distribution. Entropy describing the diversity at the epitope 
               
               
                 peptide sites is also indicated. 
               
             
          
           
               
                   
                   
                 ELISpot positive peptides 
                 Variant peptides  c   
               
             
          
           
               
                 Protein &amp; 
                   
                   
                 Incidence 
                 &gt;10% 
                 1-10% 
                 &lt;1% 
               
               
                 position 
                 Entropy  a   
                 Peptide sequence 
                 (%)  b   
                 (#) 
                 (#) 
                 (#) 
               
               
                   
               
             
          
           
               
                 C 9-25 
                 0.7 
                 GKSRAVNMLKRGMPRVL 
                 88 
                 — 
                 1 
                 5 
               
               
                   
               
               
                 prM 114-130 
                 0.1 
                 STKATRYLVKTESWILR 
                 &gt;99 
                 — 
                 — 
                 3 
               
               
                   
               
               
                 prM 121-138 
                 0.1 
                 LVKTESWILRNPGYALVA 
                 &gt;99 
                 — 
                 — 
                 2 
               
               
                   
               
               
                 prM 129-145 
                 0.3 
                 LRNPGYALVAAVIGWML 
                 95 
                 — 
                 1 
                 2 
               
               
                   
               
               
                 prM 136-153 
                 0.4 
                 LVAAVIGWMLGSNTMQRV 
                 94 
                 — 
                 1 
                 4 
               
               
                   
               
               
                 prM 144-161 
                 0.6 
                 MLGSNTMQRVVFVVLLLL 
                 92 
                 — 
                 3 
                 4 
               
               
                   
               
               
                 prM 152-167 
                 0.4 
                 RVVFVVLLLLVAPAYS 
                 95 
                 — 
                 2 
                 3 
               
               
                   
               
               
                 E 39-56 
                 0.3 
                 PTIDVKMMNMEAANLAEV 
                 96 
                 — 
                 1 
                 9 
               
               
                   
               
               
                 E 54-71 
                 0.3 
                 AEVRSYCYLATVSDLSTK 
                 96 
                 — 
                 1 
                 11 
               
               
                   
               
               
                 E 62-77 
                 0.5 
                 LATVSDLSTKAACPTM 
                 94 
                 — 
                 2 
                 14 
               
               
                   
               
               
                 E 68-85 
                 0.4 
                 LSTKAACPTMGEAHNDKR 
                 95 
                 — 
                 2 
                 9 
               
               
                   
               
               
                 E 99-113 
                 0.1 
                 RGWGNGCGLFGKGSI 
                 &gt;99 
                 — 
                 — 
                 3 
               
               
                   
               
               
                 E 119-136 
                 0.9 
                 FACSTKAIGRTILKENIK 
                 81 
                 1(16%) 
                 1 
                 11 
               
               
                   
               
               
                 E 127-144 
                 0.3 
                 GRTILICENIKYEVAIFVH 
                 97 
                 — 
                 1 
                 7 
               
               
                   
               
               
                 E 135-152 
                 0.1 
                 IKYEVAIFVHGPTTVESH 
                 99 
                 — 
                 — 
                 8 
               
               
                   
               
               
                 E 171-188 
                 0.6 
                 PAAPSYTLKLGEYGEVTV 
                 91 
                 — 
                 2 
                 12 
               
               
                   
               
               
                 E 195-212 
                 0.9 
                 GIDTNAYYVMTVGTKTFL 
                 86 
                 — 
                 2 
                 11 
               
               
                   
               
               
                 E 210-225 
                 0.3 
                 TFLVHREWFMDLNLPW 
                 95 
                 — 
                 1 
                 7 
               
               
                   
               
               
                 E 238-255 
                 0.3 
                 TLMEFEEPHATKQSVIAL 
                 97 
                 — 
                 1 
                 8 
               
               
                   
               
               
                 E 293-310 
                 0.1 
                 EKLQLKGTTYGVCSKAFK 
                 99 
                 — 
                 — 
                 6 
               
               
                   
               
               
                 E 356-373 
                 0.3 
                 VTVNPFVSVATANAKVLI 
                 96 
                 — 
                 1 
                 8 
               
               
                   
               
               
                 E 370-386 
                 0 
                 KVLIELEPPFGDSYIVV 
                 &gt;99 
                 — 
                 — 
                 2 
               
               
                   
               
               
                 E 384-399 
                 0.1 
                 IVVGRGEQQINHHWHK 
                 &gt;99 
                 — 
                 — 
                 4 
               
               
                   
               
               
                 E 390-407 
                 0.1 
                 EQQINHHWHKSGSSIGKA 
                 99 
                 — 
                 — 
                 6 
               
               
                   
               
               
                 E 398-415 
                 0.3 
                 HKSGSSIGKAFTTTLKGA 
                 97 
                 — 
                 1 
                 5 
               
               
                   
               
               
                 E 426-443 
                 0.4 
                 WDFGSVGGVFTSVGKAVH 
                 95 
                 — 
                 2 
                 5 
               
               
                   
               
               
                 E 434-451 
                 0.3 
                 VFTSVGKAVHQVFGGAFR 
                 96 
                 — 
                 1 
                 5 
               
               
                   
               
               
                 E 442-459 
                 0.2 
                 VHQVFGGAFRSLFGGMSW 
                 97 
                 — 
                 1 
                 2 
               
               
                   
               
               
                 E 450-467 
                 0.1 
                 FRSLFGGMSWITQGLLGA 
                 &gt;99.3 
                 — 
                 — 
                 3 
               
               
                   
               
               
                 E 458-474 
                 0.1 
                 SWITQGLLGALLLWMGI 
                 &gt;99 
                 — 
                 — 
                 4 
               
               
                   
               
               
                 E 465-482 
                 0.1 
                 LGALLLWMGINARDRSIA 
                 99 
                 — 
                 — 
                 7 
               
               
                   
               
               
                 E 486-501 
                 0.2 
                 LAVGGVLLFLSVNVHA 
                 98 
                 — 
                 — 
                 5 
               
               
                   
               
               
                 NS1 1-19 
                 0.7 
                 DTGCAIDISRQELRCGSGV 
                 90 
                 — 
                 1 
                 5 
               
               
                   
               
               
                 NS1 10-27 
                 0.2 
                 RQELRCGSGVFIHNDVEA 
                 98 
                 — 
                 — 
                 3 
               
               
                   
               
               
                 NS1 18-35 
                 0.7 
                 GVFIHNDVEAWMDRYKYY 
                 89 
                 — 
                 2 
                 5 
               
               
                   
               
               
                 NS1 161-178 
                 0.9 
                 GLTSTRMFLKVRESNTTE 
                 87 
                 — 
                 3 
                 6 
               
               
                   
               
               
                 NS1 205-221 
                 0.8 
                 RLNDTWKLERAVLGEVK 
                 87 
                 — 
                 3 
                 2 
               
               
                   
               
               
                 NS1 228-245 
                 0.8 
                 THTLWGDGILESDLIIPV 
                 87 
                 — 
                 2 
                 5 
               
               
                   
               
               
                 NS1 236-251 
                 0.7 
                 ILESDLIIPVTLAGPR 
                 89 
                 — 
                 2 
                 5 
               
               
                   
               
               
                 NS1 270-287 
                 0.6 
                 EGRVEIDFDYCPGTTVTL 
                 91 
                 — 
                 2 
                 2 
               
               
                   
               
               
                 NS1 305-322 
                 0.1 
                 GKLITDWCCRSCTLPPLR 
                 99 
                 — 
                 — 
                 2 
               
               
                   
               
               
                 NS1 327-343 
                 0.8 
                 SGCWYGMEIRPQRHDEK 
                 87 
                 — 
                 3 
                 4 
               
               
                   
               
               
                 NS2a 6-23 
                 0.4 
                 IDPFQLGLLVVFLATQEV 
                 94 
                 — 
                 1 
                 3 
               
               
                   
               
               
                 NS2a 45-61 
                 0.4 
                 VFGGITYTDVLRYVILV 
                 96 
                 — 
                 — 
                 6 
               
               
                   
               
               
                 NS2a 52-66 
                 0.3 
                 TDVLRYVILVGAAFA 
                 97 
                 — 
                 — 
                 5 
               
               
                   
               
               
                 NS2a 65-81 
                 0.5 
                 FAESNSGGDVVHLALMA 
                 92 
                 — 
                 1 
                 1 
               
               
                   
               
               
                 NS2a 80-97 
                 0.7 
                 MATFKIQPVFMVASFLKA 
                 89 
                 — 
                 1 
                 5 
               
               
                   
               
               
                 NS2a 128-145 
                 1 
                 EIPDVLNSLAVAWMILRA 
                 85 
                 — 
                 4 
                 5 
               
               
                   
               
               
                 NS2a 136-154 
                 0.7 
                 LAVAWMILRAITFTTTSNV 
                 88 
                 — 
                 1 
                 5 
               
               
                   
               
               
                 NS2a 160-177 
                 0.8 
                 ALLTPGLRCLNLDVYRIL 
                 87 
                 — 
                 2 
                 4 
               
               
                   
               
               
                 NS2a 168-183 
                 1 
                 CLNLDVYRILLLMVGI 
                 78 
                 1(18%) 
                 1 
                 4 
               
               
                   
               
               
                 NS2a 211-229 
                 1.3 
                 GLFNPMILAAGLIACDPNR 
                 75 
                 1(12%) 
                 3 
                 3 
               
               
                   
               
               
                 NS2b 1-18 
                 0.1 
                 GWPATEVMTAVGLMFAIV 
                 &gt;99 
                 — 
                 — 
                 1 
               
               
                   
               
               
                 NS2b 39-56 
                 0.3 
                 MFAAFVISGKSTDMWIER 
                 94 
                 — 
                 1 
                 1 
               
               
                   
               
               
                 NS2b 59-75 
                 0.9 
                 DISWESDAEITGSSERV 
                 85 
                 — 
                 3 
                 3 
               
               
                   
               
               
                 NS2b 84-101 
                 0.5 
                 NFQLMNDPGAPWKIWMLR 
                 94 
                 — 
                 2 
                 4 
               
               
                   
               
               
                 NS2b 107-124 
                 0.6 
                 ISAYTPWAILPSVVGFWI 
                 88 
                 — 
                 2 
                 1 
               
               
                   
               
               
                 NS2b 115-131 
                 0.6 
                 ILPSVVGFWITLQYTKR 
                 89 
                 — 
                 1 
                 2 
               
               
                   
               
               
                 NS3 1-15 
                 0.5 
                 GGVLWDTPSPKEYKK 
                 94 
                 — 
                 2 
                 3 
               
               
                   
               
               
                 NS3 6-23 
                 0.5 
                 DTPSPKEYKKGDTTTGVY 
                 94 
                 — 
                 2 
                 3 
               
               
                   
               
               
                 NS3 22-38 
                 0.2 
                 VYRIMTRGLLGSYQAGA 
                 98 
                 — 
                 — 
                 3 
               
               
                   
               
               
                 NS3 37-54 
                 0.2 
                 GAGVMVEGVFHTLWHTTK 
                 97 
                 — 
                 1 
                 2 
               
               
                   
               
               
                 NS3 45-59 
                 0.1 
                 VFHTLWHTTKGAALM 
                 99 
                 — 
                 — 
                 2 
               
               
                   
               
               
                 NS3 50-65 
                 0.1 
                 WHTTKGAALMSGEGRL 
                 99 
                 — 
                 — 
                 2 
               
               
                   
               
               
                 NS3 70-87 
                 0.3 
                 GSVKEDRLCYGGPWKLQH 
                 97 
                 — 
                 1 
                 3 
               
               
                   
               
               
                 NS3 78-95 
                 0.8 
                 CYGGPWKLQHKWNGQDEV 
                 87 
                 — 
                 2 
                 4 
               
               
                   
               
               
                 NS3 85-100 
                 0.7 
                 LQHKWNGQDEVQMIVV 
                 88 
                 — 
                 1 
                 4 
               
               
                   
               
               
                 NS3 91-107 
                 0.7 
                 GQDEVQMIVVEPGKNVK 
                 88 
                 — 
                 1 
                 4 
               
               
                   
               
               
                 NS3 98-115 
                 0.3 
                 IVVEPGKNVKNVQTKPGV 
                 97 
                 — 
                 1 
                 3 
               
               
                   
               
               
                 NS3 138-155 
                 0.1 
                 PIVDKNGDVIGLYGNGVI 
                 99 
                 — 
                 — 
                 2 
               
               
                   
               
               
                 NS3 146-162 
                 0.2 
                 VIGLYGNGVIMPNGSYI 
                 97 
                 — 
                 — 
                 2 
               
               
                   
               
               
                 NS3 161-177 
                 0.8 
                 YISAIVQGERMDEPIPA 
                 86 
                 — 
                 2 
                 3 
               
               
                   
               
               
                 NS3 176-192 
                 0.4 
                 PAGFEPEMLRKKQITVL 
                 95 
                 — 
                 — 
                 7 
               
               
                   
               
               
                 NS3 183-200 
                 0.1 
                 MLRKKQITVLDLHPGAGK 
                 99 
                 — 
                 — 
                 2 
               
               
                   
               
               
                 NS3 199-216 
                 0.6 
                 GKTRRILPQIIKEAINRR 
                 90 
                 — 
                 2 
                 2 
               
               
                   
               
               
                 NS3 206-223 
                 0.6 
                 PQIIKEAINRRLRTAVLA 
                 91 
                 — 
                 1 
                 4 
               
               
                   
               
               
                 NS3 214-230 
                 0.6 
                 NRRLRTAVLAPTRVVAA 
                 90 
                 — 
                 1 
                 5 
               
               
                   
               
               
                 NS3 221-237 
                 0.7 
                 VLAPTRVVAAEMAEALR 
                 90 
                 — 
                 1 
                 5 
               
               
                   
               
               
                 NS3 228-243 
                 0.6 
                 VAAEMAEALRGLPIRY 
                 90 
                 — 
                 2 
                 2 
               
               
                   
               
               
                 NS3 241-258 
                 1.1 
                 IRYCITSAVPREHNGNEIV 
                 81 
                 — 
                 3 
                 5 
               
               
                   
               
               
                 NS3 249-266 
                 1.1 
                 PREHNGNEIVDVMCHATL 
                 81 
                 — 
                 3 
                 4 
               
               
                   
               
               
                 NS3 265-282 
                 0 
                 TLTHRLMSPHRVPNYNLF 
                 100 
                 — 
                 — 
                 — 
               
               
                   
               
               
                 NS3 304-321 
                 0.3 
                 KVELGEAAAIFMTATPPG 
                 95 
                 — 
                 1 
                 1 
               
               
                   
               
               
                 NS3 317-333 
                 0.7 
                 ATPPGTSDPFPESNSPI 
                 87 
                 1(11%) 
                 — 
                 4 
               
               
                   
               
               
                 NS3 324-340 
                 0.9 
                 DPFPESNSPISDLQTEI 
                 86 
                 — 
                 2 
                 6 
               
               
                   
               
               
                 NS3 336-352 
                 0.6 
                 LQTEIPDRAWNSGYEWI 
                 90 
                 — 
                 2 
                 3 
               
               
                   
               
               
                 NS3 343-360 
                 1.3 
                 RAWNSGYEWITEYTGKTV 
                 69 
                 1(23%) 
                 2 
                 1 
               
               
                   
               
               
                 NS3 359-376 
                 0.1 
                 TVWFVPSVKMGNEIALCL 
                 99 
                 — 
                 — 
                 2 
               
               
                   
               
               
                 NS3 413-430 
                 0.2 
                 EMGANFKASRVIDSRKSV 
                 97 
                 — 
                 — 
                 2 
               
               
                   
               
               
                 NS3 455-469 
                 0.4 
                 AAQRRGRIGRNPSQV 
                 95 
                 — 
                 2 
                 2 
               
               
                   
               
               
                 NS3 460-475 
                 0.4 
                 GRIGRNPSQVGDEYCY 
                 95 
                 — 
                 2 
                 2 
               
               
                   
               
               
                 NS3 474-489 
                 0.4 
                 CYGGHTNEDDSNFAHW 
                 95 
                 — 
                 2 
                 1 
               
               
                   
               
               
                 NS3 480-496 
                 0.4 
                 NEDDSNFAHWTEARIML 
                 94 
                 — 
                 2 
                 2 
               
               
                   
               
               
                 NS3 487-501 
                 0.1 
                 AHWTEARIMLDNINM 
                 &gt;99 
                 — 
                 — 
                 1 
               
               
                   
               
               
                 NS3 492-509 
                 0.6 
                 ARIMLDNINMPNGLIAQF 
                 89 
                 — 
                 2 
                 2 
               
               
                   
               
               
                 NS3 500-517 
                 0.6 
                 NMPNGLIAQFYQPEREKV 
                 90 
                 — 
                 2 
                 1 
               
               
                   
               
               
                 NS3 515-532 
                 0.2 
                 EKVYTMDGEYRLRGEERK 
                 97 
                 — 
                 — 
                 4 
               
               
                   
               
               
                 NS3 523-539 
                 0.5 
                 EYRLRGEERKNFLELLR 
                 92 
                 — 
                 1 
                 2 
               
               
                   
               
               
                 NS3 530-547 
                 0.4 
                 ERKNFLELLRTADLPVWL 
                 92 
                 — 
                 1 
                 1 
               
               
                   
               
               
                 NS3 545-562 
                 0.9 
                 VWLAYKVAAAGVSYHDRR 
                 87 
                 — 
                 2 
                 5 
               
               
                   
               
               
                 NS3 560-574 
                 0.5 
                 DRRWCFDGPRTNTIL 
                 91 
                 — 
                 1 
                 2 
               
               
                   
               
               
                 NS3 565-582 
                 0.1 
                 FDGPRTNTILEDNNEVEV 
                 99 
                 — 
                 — 
                 2 
               
               
                   
               
               
                 NS3 581-597 
                 0.6 
                 EVITKLGERKILRPRWI 
                 91 
                 — 
                 1 
                 4 
               
               
                   
               
               
                 NS3 588-605 
                 0.6 
                 ERKILRPRWIDARVYSDH 
                 90 
                 — 
                 1 
                 3 
               
               
                   
               
               
                 NS4a 1-17 
                 0.8 
                 SQIGLIEVLGKMPEHFM 
                 87 
                 — 
                 2 
                 3 
               
               
                   
               
               
                 NS4a 15-31 
                 0.2 
                 HFMGKTWEALDTMYVVA 
                 97 
                 — 
                 1 
                 1 
               
               
                   
               
               
                 NS4a 54-71 
                 1.1 
                 IALIALLSVMTMGVFFLL 
                 80 
                 1(11%) 
                 2 
                 4 
               
               
                   
               
               
                 NS4a 62-79 
                 0.5 
                 VMTMGVFFLLMQRKGIGK 
                 92 
                 — 
                 1 
                 2 
               
               
                   
               
               
                 NS4a 86-103 
                 1 
                 VLGVATFFCWMAEVPGTK 
                 84 
                 — 
                 4 
                 4 
               
               
                   
               
               
                 NS4b 30-47 
                 0.5 
                 GEFLLDLRPATAWSLYAV 
                 92 
                 — 
                 1 
                 3 
               
               
                   
               
               
                 NS4b 38-55 
                 0.2 
                 PATAWSLYAVTTAVLTPL 
                 97 
                 — 
                 — 
                 4 
               
               
                   
               
               
                 NS4b 63-80 
                 0.2 
                 DYINTSLTSINVQASALF 
                 98 
                 — 
                 1 
                 1 
               
               
                   
               
               
                 NS4b 87-103 
                 0.3 
                 PFVDVGVSALLLAAGCW 
                 96 
                 — 
                 1 
                 2 
               
               
                   
               
               
                 NS4b 101-118 
                 0.8 
                 GCWGQVTLTVTVTAATLL 
                 87 
                 — 
                 2 
                 4 
               
               
                   
               
               
                 NS4b 187-204 
                 0.6 
                 VVNPSVKTVREAGILITA 
                 90 
                 — 
                 1 
                 5 
               
               
                   
               
               
                 NS4b 201-218 
                 0.5 
                 LITAAAVTLWENGASSVW 
                 91 
                 — 
                 1 
                 3 
               
               
                   
               
               
                 NS4b 233-250 
                 1.5 
                 GWLSCLSITWTLIKNMEK 
                 76 
                 — 
                 5 
                 5 
               
               
                   
               
               
                 NS4b 241-255 
                 1.3 
                 TWTLIKNMEKPGLKR 
                 79 
                 — 
                 4 
                 5 
               
               
                   
               
               
                 NS5 115-132 
                 0.1 
                 LVQSYGWNIVTMKSGVDV 
                 &gt;99 
                 — 
                 — 
                 1 
               
               
                   
               
               
                 NS5 145-162 
                 0.7 
                 CDIGESSSSAEVEEHRTI 
                 88 
                 — 
                 2 
                 1 
               
               
                   
               
               
                 NS5 153-168 
                 0.7 
                 SAEVEEHRTIRVLEMV 
                 87 
                 — 
                 2 
                 1 
               
               
                   
               
               
                 NS5 181-198 
                 0.6 
                 VKVLCPYMPKVIEKMELL 
                 91 
                 — 
                 1 
                 4 
               
               
                   
               
               
                 NS5 212-229 
                 0.5 
                 SRNSTHEMYWVSRASGNV 
                 91 
                 — 
                 1 
                 2 
               
               
                   
               
               
                 NS5 448-463 
                 0.5 
                 ECHTCIYNMMGKREKK 
                 92 
                 — 
                 2 
                 1 
               
               
                   
               
               
                 NS5 470-486 
                 0.1 
                 AKGSRAIWFMWLGARFL 
                 &gt;99 
                 — 
                 — 
                 1 
               
               
                   
               
               
                 NS5 477-494 
                 0 
                 WFMWLGARFLEFEALGFL 
                 100 
                 — 
                 — 
                 — 
               
               
                   
               
               
                 NS5 598-615 
                 0 
                 REDQRGSGQVVTYALNTF 
                 100 
                 — 
                 — 
                 — 
               
               
                   
               
               
                 NS5 605-622 
                 0.1 
                 GQVVTYALNTFTNLAVQL 
                 99 
                 — 
                 1 
                 — 
               
               
                   
               
               
                 NS5 613-631 
                 0.1 
                 NTFTNLAVQLVRMMEGEGV 
                 99 
                 — 
                 1 
                 — 
               
               
                   
               
               
                 NS5 704-721 
                 0 
                 GWYDWQQVPFCSNHFTEL 
                 100 
                 — 
                 — 
                 — 
               
               
                   
               
               
                 NS5 755-772 
                 0.1 
                 DTACLAKSYAQMWLLLYF 
                 &gt;99 
                 — 
                 — 
                 1 
               
               
                   
               
               
                 NS5 763-780 
                 0.1 
                 YAQMWLLLYFHRRDLRLM 
                 &gt;99 
                 — 
                 — 
                 1 
               
               
                   
               
               
                 NS5 771-788 
                 0 
                 YFHRRDLRLMANAICSAV 
                 100 
                 — 
                 — 
                 — 
               
               
                   
               
               
                 NS5 791-808 
                 0.7 
                 NWVPTGRTTWSIHAGGEW 
                 88 
                 — 
                 2 
                 4 
               
               
                   
               
               
                 NS5 828-843 
                 1 
                 WMEDKTPVEKWSDVPY 
                 85 
                 — 
                 2 
                 9 
               
               
                   
               
               
                 NS5 842-859 
                 0.1 
                 PYSGKREDIWCGSLIGTR 
                 98 
                 — 
                 — 
                 3 
               
               
                   
               
               
                 NS5 863-879 
                 1.6 
                 TWAENIQVAINQVRAII 
                 71 
                 1(12%) 
                 3 
                 10 
               
               
                   
               
               
                   a  Entropy value indicating the diversity of the region in the protein alignment that contained the epitope peptide sequence. 
               
               
                   b  The percentage of sequences (round off to the nearest whole number) analyzed that contained the exact sequence of the epitope peptide. Those with &gt;99% do not include 100% 
               
               
                   c  The fraction of the variants of the epitope peptide sequence, greater than 10%, 1-10%, and less than 1%. The actual percentage representation of the major variant sequence is shown for the 7 epitope peptides with a variant that represents greater than 10% of the WNV sequences analyzed (see also Table 4). 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
             
           
               
                 TABLE 12 
               
             
             
               
                   
               
               
                 List of highly conserved and specific sequences of  Flavivirus  species 
               
               
                 West Nile virus (WNV), dengue virus (DENV), yellow fever virus (YFV) and 
               
               
                 Japanese encephalitis virus (JEV). (SEQ ID NOs: 1-206, in the order as shown) 
               
             
          
           
               
                 Virus 
                 Protein 
                 Conserved Virus Specific Sequence 
               
               
                   
               
               
                 Dengue Virus (DENV) 
                 E 
                 VLGSQEGAMH 
               
               
                   
                 NS1 
                 VHTWTEQYKFQ 
               
               
                   
                   
                 AVHADMGYWIES 
               
               
                   
                   
                 HTLWSNGVLES 
               
               
                   
                   
                 GPWHLGKLE 
               
               
                   
                 NS3 
                 GLYGNGVVT 
               
               
                   
                   
                 LTIMDLHPG 
               
               
                   
                   
                 LMRRGDLPVWL 
               
               
                   
                 NS4a 
                 QRTPQDNQL 
               
               
                   
                 NS4b 
                 PASAWTLYAVATT 
               
               
                   
                   
                 HYAIIGPGLQAKATREAQKR 
               
               
                   
                   
                 AAGIMKNPTVDGI 
               
               
                   
                   
                 FWNTTIAVS 
               
               
                   
                 NS5 
                 SGVEGEGLH 
               
               
                   
                   
                 IFKLTYQNKVV 
               
               
                   
                   
                 DQRGSGQVGTYGLNTFTNME 
               
               
                   
                   
                 PTSRTTWSIHA 
               
               
                   
               
               
                 Japanese Encephalitis 
                 C 
                 SVAMKHLTSFK 
               
               
                 Virus (JEV) 
                 NS1 
                 GGITYTDLARYVVL 
               
               
                   
                 NS2a 
                 LDTYRIILL 
               
               
                   
                   
                 MAKKKGAVL 
               
               
                   
                   
                 GLALTSTGWFSPTTI 
               
               
                   
                 NS2b 
                 AAITGSSRRLDVKLD 
               
               
                   
                 NS3 
                 GKLTPYWGSV 
               
               
                   
                   
                 YGGPWRFDRKWNGT 
               
               
                   
                   
                 DVQVIVVEPGK 
               
               
                   
                   
                 SYVSAIVQG 
               
               
                   
                   
                 LRGLPVRYQTSAVQREHQGN 
               
               
                   
                   
                 ASAAQRRGRV 
               
               
                   
                   
                 LDNIHMPNGLV 
               
               
                   
                   
                 QLYGPEREKA 
               
               
                   
                 NS4a 
                 LATFFLWAAEV 
               
               
                   
                   
                 GTKIAGTLL 
               
               
                   
                   
                 ALLLMVVLIPEP 
               
               
                   
                   
                 AANEYGMLE 
               
               
                   
                   
                 SQAGSLFVLPRGVP 
               
               
                   
                   
                 TDLDLTVGLV 
               
               
                   
                 NS5 
                 ERENHLRGECHT 
               
               
                   
                   
                 LARAIIELTY 
               
               
                   
                   
                 EIVMKDGRS 
               
               
                   
                   
                 RARISPGAG 
               
               
                   
               
               
                 Yellow Fever Virus (YFV) 
                 C 
                 MSGRKAQGKTLG 
               
               
                   
                 prM 
                 WCPDSMEYNCPNLS 
               
               
                   
                   
                 GRMGERQLQ 
               
               
                   
                   
                 ALLVLAVGPA 
               
               
                   
                 E 
                 KCVTVMAPDKPSLDISL 
               
               
                   
                   
                 DLTLPWQSGS 
               
               
                   
                   
                 HLVEFEPPH 
               
               
                   
                   
                 VLIEVNPPFGDS 
               
               
                   
                   
                 GDSRLTYQWHKEGSSI 
               
               
                   
                   
                 RNMTMSMSMI 
               
               
                   
                 NS1 
                 KRELKCGDG 
               
               
                   
                   
                 KYSYYPEDPVKLASI 
               
               
                   
                   
                 EEGKCGLNSVDSL 
               
               
                   
                   
                 HEMWRSRADEINAI 
               
               
                   
                   
                 YQRGTHPFSRIRDGLQYGWKTWGK 
               
               
                   
                   
                 FSPGRKNGSFIIDGKSRKECPFSNRVWNS 
               
               
                   
                   
                 ILGAAVNGKKSAHGSPTFWMGSHEVNGTWM 
               
               
                   
                   
                 LDYKECEWP 
               
               
                   
                   
                 THTIGTSVEE 
               
               
                   
                   
                 MFMPRSIGGPVSSHN 
               
               
                   
                   
                 IPGYKVQTNGPWMQVPLEV 
               
               
                   
                   
                 CTMPPVSFHG 
               
               
                   
                   
                 DGCWYPMEIRP 
               
               
                   
                 NS2a 
                 HAVPFGLVSMMIA 
               
               
                   
                   
                 LLGAMLVGQVT 
               
               
                   
                   
                 NNGGDAMYMALIA 
               
               
                   
                   
                 GFGLRTLWSPRERLV 
               
               
                   
                   
                 KDTSMQKTIP 
               
               
                   
                   
                 GLTQPFLGLCA 
               
               
                   
                 NS2b 
                 SIPVNEALAA 
               
               
                   
                   
                 GVLAGLAFQ 
               
               
                   
                   
                 MENFLGPIAVGG 
               
               
                   
                   
                 LMMLVSVAG 
               
               
                   
                   
                 WEEEAEISGSS 
               
               
                   
                   
                 EQGEFKLLSE 
               
               
                   
                   
                 VMTSLALVGAA 
               
               
                   
                 NS3 
                 SGDVLWDIPTPK 
               
               
                   
                   
                 GIFQSTFLGASQ 
               
               
                   
                   
                 TFLGASQRGVGVAQGGVFHTM 
               
               
                   
                   
                 PSWASVKEDLVAYGGSWKL 
               
               
                   
                   
                 WDGEEEVQLIAA 
               
               
                   
                   
                 VVNVQTKPS 
               
               
                   
                   
                 NGGEIGAVAL 
               
               
                   
                   
                 YGNGILVGDNSFVSAISQTE 
               
               
                   
                   
                 PGAGKTRRFLPQILAECARR 
               
               
                   
                   
                 AECARRRLRTL 
               
               
                   
                   
                 RRRLRTLVL 
               
               
                   
                   
                 GLDVKFHTQAFSAHGSG 
               
               
                   
                   
                 MCHATLTYRMLEPTR 
               
               
                   
                   
                 AHFLDPASIAAR 
               
               
                   
                   
                 ARGWAAHRARANESATILMTATPP 
               
               
                   
                   
                 ATPPGTSDEFPHSNGEIEDVQTDIPSEPW 
               
               
                   
                   
                 WILADKRPTAWFLP 
               
               
                   
                   
                 AWFLPSIRA 
               
               
                   
                   
                 LPSIRAANVMAASLRKAGK 
               
               
                   
                   
                 IAEMGANLCV 
               
               
                   
                   
                 KVAIKGPLRISA 
               
               
                   
                   
                 GRIGRNPNRDGDSYYYSEPTSE 
               
               
                   
                   
                 NAHHVCWLEASMLLDN 
               
               
                   
                   
                 MLLDNMEVRGGMVAPLYG 
               
               
                   
                   
                 KTPVSPGEMRLRDDQR 
               
               
                   
                   
                 GLKTNDRKWCF 
               
               
                   
                   
                 LRPRWCDERVSSDQSAL 
               
               
                   
                 NS4a 
                 RAYRNALSMMPEAMT 
               
               
                   
                 NS4b 
                 AAWTINVGI 
               
               
                   
                   
                 LSPMLHHWIK 
               
               
                   
                   
                 MLHHWIKVEYGNLSLSGI 
               
               
                   
                   
                 QSASVLSFMDKG 
               
               
                   
                   
                 PFMKMNISV 
               
               
                   
                   
                 LILPGIKAQQSKL 
               
               
                   
                   
                 DIEEAPEMP 
               
               
                   
                   
                 LYEKKLALYL 
               
               
                   
                   
                 KKLALYLLLALSL 
               
               
                   
                   
                 SVAMCRTPFSL 
               
               
                   
                   
                 PLIEGNTSLLWNGPMAVSMTGVMRGN 
               
               
                   
                 NS5 
                 GKTLGEVWKRELNLLD 
               
               
                   
                   
                 ARRHLAEGKV 
               
               
                   
                   
                 HLAEGKVDT 
               
               
                   
                   
                 AEGKVDTGVAVSRG 
               
               
                   
                   
                 VAVSRGTAK 
               
               
                   
                   
                 GTAKLRWFHERGYVKLEGRV 
               
               
                   
                   
                 WCYYAAAQKE 
               
               
                   
                   
                 YAAAQKEVSGVKG 
               
               
                   
                   
                 EKWLACGVDNFC 
               
               
                   
                   
                 EKLELLQRRFG 
               
               
                   
                   
                 LLQRRFGGT 
               
               
                   
                   
                 TFTVNQTSRLLMRRMRRPTGKVT 
               
               
                   
                   
                 LPIGTRSVETDKGPL 
               
               
                   
                   
                 DNDNPYRTWHYCGSY 
               
               
                   
                   
                 IEEVTRMAMTD 
               
               
                   
                   
                 RMAMTDTTP 
               
               
                   
                   
                 KEKVDTRAKDPPAGTRKIM 
               
               
                   
                   
                 VVNRWLFRHL 
               
               
                   
                   
                 QWKTANEAVQDPKFWE 
               
               
                   
                   
                 KLSEFGKAKG 
               
               
                   
                   
                 EDHWASRENSGGG 
               
               
                   
                   
                 GLQYLGYVI 
               
               
                   
                   
                 EADLDDEQEI 
               
               
                   
                   
                 MEMTYKNKVVK 
               
               
                   
                   
                 YKNKVVKVLR 
               
               
                   
                   
                 YALNTITNLKVQL 
               
               
                   
                   
                 ALSHLNAMSK 
               
               
                   
                   
                 MSKVRKDISEWQPSK 
               
               
                   
                   
                 GRGRVSPGNGW 
               
               
                   
                   
                 ACLSKAYANM 
               
               
                   
                   
                 SKAYANMWSLMYFHKRDMRLLS 
               
               
                   
                   
                 KRQDKLCGSL 
               
               
                   
               
               
                 West Nile Virus (WNV) 
                 prM 
                 LVAAVIGWMLGSNTMQRV 
               
               
                   
                 E 
                 LATVSDLSTKAACPTM 
               
               
                   
                   
                 FACSTKAIGRTILKENIK 
               
               
                   
                   
                 GIDTNAYYVMTVGTKTFL 
               
               
                   
                   
                 TFLVHREWFMDLNLPW 
               
               
                   
                   
                 VTVNPFVSVATANAKVLI 
               
               
                   
                   
                 IVVGRGEQQINHHWHK 
               
               
                   
                   
                 LAVGGVLLFLSVNVHA 
               
               
                   
                 NS1 
                 DTGCAIDISRQELRCGSGV 
               
               
                   
                   
                 RSVSRLEHQMW 
               
               
                   
                   
                 GLTSTRMFLKVRESNTTE 
               
               
                   
                   
                 THTLWGDGILESDLIIPV 
               
               
                   
                 NS2a 
                 VFGGITYTDVLRYVILV 
               
               
                   
                   
                 TDVLRYVILVGAAFA 
               
               
                   
                   
                 MATFKIQPVFMVASFLKA 
               
               
                   
                   
                 EIPDVLNSLAVAWMILRA 
               
               
                   
                   
                 LAVAWMILRAITFTTTSNV 
               
               
                   
                   
                 ALLTPGLRCLNLDVYRIL 
               
               
                   
                   
                 CLNLDVYRILLLMVGI 
               
               
                   
                   
                 GLFNPMILAAGLIACDPNR 
               
               
                   
                 NS2b 
                 GWPATEVMTA 
               
               
                   
                   
                 PMTIAGLMF 
               
               
                   
                   
                 DISWESDAEITGSSERV 
               
               
                   
                   
                 NFQLMNDPGAPWKIWMLR 
               
               
                   
                   
                 ISAYTPWAILPSVVGFWI 
               
               
                   
                   
                 ILPSVVGFWITLQYTKR 
               
               
                   
                 NS3 
                 VYRIMTRGLLGSYQAGA 
               
               
                   
                   
                 WHTTKGAALMSGEGRLDPYWGSV 
               
               
                   
                   
                 GSVKEDRLCYGGPWKLQH 
               
               
                   
                   
                 CYGGPWKLQHKWNGQDEV 
               
               
                   
                   
                 LQHKWNGQDEVQMIVV 
               
               
                   
                   
                 IVVEPGKNVKNVQTKPGV 
               
               
                   
                   
                 YISAIVQGERMDEPIPA 
               
               
                   
                   
                 PAGFEPEMLRKKQITVL 
               
               
                   
                   
                 IRYQTSAVPREHNGNEIV 
               
               
                   
                   
                 DPFPESNSPISDLQTEIPDRAWN 
               
               
                   
                   
                 RVIDSRKSVKP 
               
               
                   
                   
                 CYGGHTNEDDSNFAHW 
               
               
                   
                   
                 NEDDSNFAHWTEARIML 
               
               
                   
                   
                 AHWTEARIMLDNINM 
               
               
                   
                   
                 ARIMLDNINMPNGLIAQF 
               
               
                   
                   
                 NMPNGLIAQFYQPEREKV 
               
               
                   
                   
                 EVITKLGERKILRPRWI 
               
               
                   
                   
                 ERKILRPRWIDARVYSDH 
               
               
                   
                 NS4a 
                 SQIGLIEVLGKMPEHFM 
               
               
                   
                   
                 HFMGKTWEALDTMYVVA 
               
               
                   
                   
                 IALIALLSVMTMGVFFLL 
               
               
                   
                   
                 VMTMGVFFLLMQRKGIGK 
               
               
                   
                   
                 VLGVATFFCWMAEVPGTK 
               
               
                   
                 NS4b 
                 PATAWSLYAVTTAVLTPL 
               
               
                   
                   
                 DYINTSLTSINVQASALF 
               
               
                   
                   
                 PFVDVGVSALLLAAGCW 
               
               
                   
                   
                 LITAAAVTLWENGASSVW 
               
               
                   
                   
                 GWLSCLSITWTLIKNMEK 
               
               
                   
                   
                 TWTLIKNMEKPGLKR 
               
               
                   
                 NS5 
                 GGAKGRTLGE 
               
               
                   
                   
                 VEDWLHRGP 
               
               
                   
                   
                 EREAHLRGEC 
               
               
                   
               
             
          
         
       
     
       Example 8 
       [0062]    Large-scale analysis of the T-cell epitopes of WNV by immunization of HLA transgenic mice with 452 overlapping peptides spanning the entire WNV proteome has resulted in the identification of 137 peptides that elicited 200 HLA-restricted IFN-γ T-cell responses in 6 HLA transgenic mice strains: 74 for class I A2, A24, and B7, and 126 for class II DR2, DR3, and DR4. The multiple HLA responses to some of the peptides can be attributed to peptide promiscuity in T-cell activation, and to multiple T-cell epitope sequences in the same peptide. Many of these T-cell epitope peptides are likely dominant immunogens in nature, whether conveyed by natural pathogens or vaccines. Several mechanism(s) by which exogenous peptide immunogens are presented by antigen presenting cells in both HLA class I and II pathways have been described (Ackerman and Cresswell, 2004; Giodini and Cresswell, 2008; Lindner and Unanue, 1996; Nygard et al., 1994; Pathak and Blum, 2000; Pinet et al., 1995). Mechanistic studies have also shown that exogenous peptides compete for the presentation of endogenous antigens to MHC II-restricted T cells (Adorini et al., 1991) and that both endogenously processed peptide and the corresponding exogenous peptide act as ligands for a T-cell receptor (Gyotoku et al., 1998). There is also an abundance of evidence that support the HLA-transgenic mouse model for the efficient identification of peptides that contain sequences recognized both by the HLA molecules of the transgenic mice and by antigen receptors of the mouse T cells (Sonderstrup et al., 1999; Taneja and David, 1999). However, a limitation of this and most studies of T-cell epitopes is that because of the complexity of peptide HLA-processing and T-cell receptor recognition, the specific minimal epitope sequences are not known. As pointed out by Niels Jerne in 1960 (Jerne, 1960) processed peptides that would be recognized by T cells in association with MHC molecules are what he termed “cryptotopes,” hidden epitopes which become immunologically available only after cellular processing. “T cell epitopes” as is now commonly used, describes the peptide sequence of the original protein, not the form that it is recognized by the T cell. For this reason, we herein use the terms “T cell epitope peptide” or “T cell epitope determinant” to describe the 15-18 amino acid peptides that contain T-cell epitopes of unknown specific sequence. Moreover, this mouse data is not used to elucidate the functional properties of human T-cells because the mouse T cells are educated to the HLA transgene, and little is known of the nature of this response as compared to the response of naive human T cells. Thus, our basic interpretation of these studies is that they reveal WNV protein sequences that contain T-cell epitopes specific for the selected HLA molecules and T cell class I or class II activation, but not a more detailed understanding of the functional role of these sequences in pathogen infection of humans. 
         [0063]    A remarkable finding was the extensive identity of WNV epitope peptide sequences with other flaviviruses. WNV are among the more highly conserved RNA viruses with an average peptide sequence conservance of about 92% in all WNV in the public databases. In this study, there were only 7 epitope peptides that differed in more than 10% of the corresponding database sequences. However, and importantly, only 51 of the 137 epitope peptide sequences were specific for WNV and the remaining 86 contained sequences of 9 or more amino acids that collectively were identical to at least 67 other flaviviruses. Moreover, the entire sequences of 19 WNV epitope peptides, chiefly of the E, NS3 and NS5 proteins, were present in 26 viruses. Additionally, immune relevant homologous sequences of 9 or more amino acids of the 86 shared WNV epitope peptides were commonly found in other flaviviruses, with as many as 45 to 62 WNV sequences in Murray Valley encephalitis, Japanese encephalitis, and Usutu viruses. These mainly included E 99-113 and/or representatives of 8 NS5 sequences that each was found in 32 to 62 other flaviviruses. While the data presented are specific for WNV and other similar flaviviruses, we expect that the same would be true to some extent among other groups of phylogenetically related flaviviruses, certainly the 4 serotypes of dengue which contain many sequences with inter-DENV identity (Khan et al., 2008). There is high probability that many of the homologous sequences would act as epitopes or as altered peptide ligands in the event of multiple  Flavivirus  infections or immunization followed by infection with similar flaviviruses. 
         [0064]    These findings have strong bearing on the possible pathological consequences of exposure to altered peptide ligands. Many studies have shown that peptide analogs recognized by T-cell receptors are participants in many T cell biological phenomena with possible pathogenic consequences (reviewed in Sloan-Lancaster and Allen, 1996; Mongkolsapaya, J. 2003). The selection of evolutionarily conserved protein sequences has widely been considered important to vaccine design in order to limit the selective loss of immunity resulting from mutation and protein modification. However, as shown herein, in the evolution of viruses, conserved sequences can be present in many different forms in viruses of related species and our conclusion is that the selection of virus specific sequences should have precedance to conserved, non-virus specific sequences. These observations also question the use of “ChimeriVax” vaccines such as the use of a yellow fever virus vaccine platform to deliver the premembrane and envelope genes of WNV (Monath et al., 2006), which clearly would have the potential of exposing the vaccine recipient to a large number of altered peptide ligands in the event of infection by WNV or any other  Flavivirus.    
         [0065]    The methodology applied to this study of WNV sequences provides an experimental basis for identification of HLA-restricted T cell epitope peptides of any pathogen. The analysis of pathogen antigens may conveniently use the same overlapping peptides required for ELISpot analysis of peptide specific T cell activation, but experiments comparing peptide and DNA-encoded antigen shown in this study and other unpublished experiments uniformly suggest the preferred use of genetic immunogens as vaccines. Selection of T-cell epitope peptides for vaccine design would omit sequences that are highly conserved in other related viruses, and focus on pathogen-specific sequences present in 80% or more of all recorded evolutionary variants of the pathogen and have clustered or closely contiguous localization. Clustered epitopes has distinct advantages in the design of an epitope-based vaccine, including the retention of native sequences for the function of transporters associated with antigen processing (TAPs) (Niedermann, 2002) and for the flanking sequences that are reported to modulate epitope processing and function in the selection of immunodominant epitopes (Le Gall et al., 2007). We elsewhere describe the use of sequences of the lysososome-associated membrane protein (LAMP) in the vaccine construct to elicit enhanced antigen delivery to the MHC II compartment of antigen presenting cells (de Arruda et al., 2004; Marques et al., 2003; Ruff et al., 1997). 
       REFERENCES 
       [0066]    The disclosure of each reference cited is expressly incorporated herein.
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