Patent Application: US-201013516501-A

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:
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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . 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 . the apparent functional avidity of wnv t - cell epitope peptides in elispot assays of 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 . 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 ( fig1 ). 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 . 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 . 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 . 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 . 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 %. 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 . 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 . 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 ( fig2 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 ( fig4 ). 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 ( fig4 ). 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 . representation of wnv t - cell epitope peptides and their variants in major flaviviruses 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 . 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 . 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 . 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 ). 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 . 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 . 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 . 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 - 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