Patent Application: US-201013501339-A

Abstract:
pandemic a continues its global spread , and vaccine production is a serious problem . protection by current vaccines is limited by the mutational differences that rapidly accumulate in the circulating strains , especially in the virus surface proteins . new vaccine strategies are focusing at conserved regions of the viral internal proteins to produce t cell epitope - based vaccines . t cell responses have been shown to reduce morbidity and promote recovery in mouse models of influenza challenge . we previously reported 54 highly conserved sequences of np , m1 and the polymerases of all human h1n1 , h3n2 , h1n2 , and h5n1 , and avian subtypes over the past 30 years . sixty - three t cell epitopes elicited responses in hla transgenic mice . these epitopes were compared to the 2007 - 2009 human h1n1 sequences to identify conserved and variant residues . seventeen t cell epitopes of pb1 , pb2 , and m1 were selected as vaccine targets by analysis of sequence conservation and variability , functional avidity , non - identity to human peptides , clustered localization , and promiscuity to multiple hla alleles . the vaccines composed of these epitopes , being highly conserved and temporally stable , would be useful for any avian or human influenza a virus .

Description:
the inventors have identified and characterized peptide segments of influenza virus a / new york / 348 / 2003 ( h1n1 ) that contain conserved sequences and elicit hla - restricted t cell responses . hla transgenic mice ( hla - a2 , - a24 , - b7 , - dr2 , - dr3 and - dr4 ) were immunized with selected peptides . the peptides that elicited t cell activation by ifn - γ elispot assay and thus functioned as human t cell epitope peptides were selected and analyzed for properties relevant in vaccine development . the evolutionary variability and the relationship of the 2003 h1n1 t cell epitope peptide sequences to the corresponding 2007 - 2009 human h1n1 sequences were studied . the results identified ( i ) the h1n1 hla - restricted t cell epitope peptides in the context of pathogenic influenza a conserved sequences and ( ii ) the variant amino acids ( aa ) and percentage representation of 2007 - 2009 h1n1 strains as compared to the 2003 a / new york / 348 strain . at least 9 , 11 , 13 , 15 , 17 , 19 , 20 , or 21 amino acids of at least two of peptide segments identified as highly conserved and highly non - variant can optionally be linked together using 0 - 20 amino acids residues , such as gpgpg ( alternating glycine and proline residue ) linkers . where distances between conserved sequences are small ( one or two residues ) and not highly variant , one may optionally join the sequences together with a natural but non - conserved amino acid or two , making larger mostly conserved segments . the linked segments may be from the same peptide segment or from different peptide segments . they may be from the same viral protein or from different viral proteins . the segments are shown in seq id no : 1 - 15 . the linked segments form a catenate . the catenate may be flanked by two portions of the human lamp - 1 protein , also known as cd107a . the n - terminal portion is the luminal portion of the lamp - 1 protein . the c - terminal portion is the transmembrane domain and the short cytoplasmic tail . thus the segment or the catenate is inserted in the midst of the lamp - 1 protein forming a chimeric protein . the chimeric protein may comprise at least 9 amino acids of at least 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , or 15 of the peptide segments . if duplicates are used or more than one of the at least 9 - amino acid stretches from a single peptide segment are used , then more than 15 of the at least 9 - amino acid stretches may be in the catenate . lamp - 1 chimeric proteins are used for antigen processing and presentation to the immune systems . the polypeptides need not be in catenates and need not be in lamp - 1 chimeric proteins . the polyepeptides may be isolated and consist of a segment as shown in seq id no : 1 - 15 , such as any of seq id no : 3 , 4 , 5 , 6 , 8 , 11 , and 12 . such polyeptides may be made synthetically or recombinantly . they may be isolated from natural sources and enzymatically digested and purified . any manner of making them as is known in the art may be used . typically the polypeptides are less than full - length influenza proteins . in the case of pb1 and pb2 polypeptides , the polypeptides are less than 150 , less than 125 , less than 100 , less than 75 , or less than 50 amino acid residues of pb 1 or pb2 in length . the polypeptides may also comprise other amino acid sequences linked to the influenza sequences . the linked sequences may be selected , e . g ., to facilitate processing or production . the linked sequences may be used to improve physiological processing , like the lamp - 1 sequences . the sequences may be used to improve presentation to the immune system . an alternative to catenates is mixtures of polypeptides ( or polynucleotides encoding them ). the mixtures may comprise at least 2 , 3 , 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , or 15 of the polypeptides of seq id no : 1 - 15 . the mixtures may also comprise immune adjuvants , as are known in the art . any linkers may be used between influenza polypeptides in catenates . they may have glycine and proline residues in a different pattern than alternating . they may have a different length of glycine and proline residues . linkers with other natural or non - naturally occurring amino acid residues may be used . particular properties may be imparted by the linkers . they may provide a particular structure or property , for example a particular kink or a particular cleavable site . design is within the skill of the art . polynucleotides which encode the polyeptides or chimeric proteins may be designed and made by techniques well known in the art . the natural sequences used by influenza virus a may be used . alternatively non - natural sequences may be used , including in one embodiment , sequences that are codon - optimized for humans . design of human codon optimized sequences is well within the skill of the ordinary artisan . data regarding the most frequently used codons in the human genome are readily available . optimization may be applied partially or completely . the polynucleotides which encode the polyeptides or chimeric proteins can be replicated and / or expressed in vectors , such as dna virus vectors , rna virus vectors , and plasmid vectors . preferably these will contain promoters for expressing the polyeptides or chimeric proteins in human or other mammalian or other animal cells . an example of a suitable promoter is the cytomegalovirus ( cmv ) promoter . promoters may be inducible or repressible . they may be constitutive . they may express at high or low levels , as desired in a particular application . the vectors may be propagated in host cells for expression and collection of chimeric protein . suitable vectors will depend on the host cells selected . in one embodiment host cells are grown in culture and the polypeptide is harvested from the cells or from the culture medium . suitable purification techniques can be applied to the polyeptides or chimeric proteins as are known in the art . in another embodiment one transfects antigen presenting cells for ultimate delivery to a vaccinee of a cellular vaccine which expresses and presents antigen to the vaccinee . suitable antigen presenting cells include dendritic cells , b cells , macrophages , and epithelial cells . in another embodiment vectors are directly administered to a vaccinee for expression in the vaccinee . immune adjuvants may be administered with the vaccines of the present invention , whether the vaccines are polypeptides , polynucleotides , nucleic acid vectors , or cellular vaccines . the adjuvants may be mixed with the specific vaccine substance prior to administration or may be delivered separately to the recipient , either before , during , or after the vaccine substance is delivered . vaccines may be produced in any suitable manner , including in cells , in eggs , and synthetically . in addition to adjuvants , booster doses may be provided . boosters may be the same or a complementary type of vaccine . boosters may include a conventional live or attenuated influenza a viral vaccine . typically a high titer of t cell activation and / or antibody is desired with a minimum of adverse side effects . 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 . whole virus , including live , attenuated , or genetically inactivated , may be used as a booster or adjuvant . the virus may be administered at the same time as , before , after , or mixed with the polypeptide or polynucleotide vaccines . an enigma of the immunobiology of influenza a is that vaccines fail to provide long term protection against infection and natural infection does not prevent reinfection . the rapid mutation of the viral proteins , particularly the external ha and na proteins that are targets for neutralizing antibodies , is credited with a significant role in this loss of immunity . defective adaptive immunity is also observed with several rna viruses ( including hiv - 1 and dengue viruses ) with high rates of mutation that result in multiple genetic variants bearing mutated t cell epitope sequences . this has resulted in widespread attention to the use of t cell epitopes incorporating conserved sequences of non - structural viral internal proteins [ 25 - 28 ]. however , the occurrence of reinfection , despite the human t cell response to conserved sequences after natural infection , suggests the function of a viral mechanism that intervenes in the host immune response to influenza infection . one possibility is the dual immunosuppressor roles of the influenza a ns 1 protein that inhibit innate immunity by preventing type i ifn release , as well as adaptive immunity by attenuating human dendritic cell maturation and the capacity of dendritic cells to induce t cell responses [ 29 ]. there is also the concept of immunological “ original sin ” where mutations in or adjacent to t cell epitopes preserve binding to mhc molecules but present an altered surface to the t - cell antigen receptor , resulting in an impaired or modified t cell response , including t cell immunosuppression [ 30 - 36 ]. in the examples shown below , hla transgenic mice , hla - a2 , - a24 , - b7 , - dr2 , - dr3 and dr4 , were immunized with 196 overlapping h1n1 peptides of the a / new york / 348 / 2003 strain that contained the highly conserved sequences of the m1 , np , pb1 , pb2 , and pa proteins of all reported human and avian influenza a viruses of the past 30 years [ 11 ]. fifty - four ( 54 ) of these peptides ( 22 pb1 , 16 pb2 , 9 np , 4 pa , and 3 m1 ) elicited 63 hla - restricted t cell responses by ifn - γ elispot assay , where 7 peptides were restricted by multiple alleles . further , the conserved t cell epitope peptides contained reported human t cell epitopes shared among pathogenic h1n1 , h3n2 and h5n1 viral strains and were restricted by a broad range of hla class i and ii alleles . thus , it is reasonable to expect that the conserved peptides identified here can elicit human t cell epitope responses in the context of several hla alleles and hla - supertypes [ 37 ] and that the memory t cells can cross - react with epitopes from h1n1 , h3n2 , and h5n1 [ 26 , 38 , 39 ]. the class i alleles described herein hla - a * 0201 , - a * 2402 and - b * 0702 belong to the distinct supertypes a2 , a24 and b7 , respectively [ 40 , 41 ]. hla class ii supertypes are not as well documented but the 3 alleles of the transgenic mice of this study are assigned to supertypes dr1 , dr3 and dr4 [ 42 ] based on similar protein and three - dimensional structures . analysis of the conservation and mutational variants of these h1n1 hla - restricted epitope peptides revealed the marked effect that single aa mutations may have on the representation of t cell epitope peptides in evolving virus populations . over the 3 years interval ( 2007 to 2009 ) between the database records analyzed by heiny et al . ( 2006 ) to the current 2009 h1n1 sequence analysis , only 8 of the 54 highly conserved t cell epitope peptide sequences were without mutational change . these 8 peptides ( m1175 - 191 , 181 - 197 , pb131 - 47 , 120 - 136 , 126 - 142 , 489 - 505 , 495 - 511 , and 548 - 564 ) were representative of almost complete conservation , 95 - 100 %, during the previous recorded history of human h1n1 virus sequences . all others of the identified hla - restricted t cell epitope peptides contained at least 1 aa substitution , primarily but not exclusively , of the non - conserved aa of the h1n1 peptides . our data suggest that the most favorable sequences for a t cell epitope - based vaccine are the 17 h1n1 t cell epitope peptides of the pb1 , pb2 , and m1 proteins ( table 6a ). these were highly conserved over the 33 years ( 1977 - 2009 ) of the examined database records , representing 88 to 100 % of all recorded avian and human influenza a viruses , including the h1n1 isolates . these 17 t cell epitopes are clustered and have distinct advantages in the design of an epitope - based genetic vaccine , including the retention of native sequences for the function of transporters associated with antigen processing ( taps ) [ 43 ] and for the flanking sequences that are reported to modulate epitope processing and function in the selection of immunodominant epitopes [ 44 ]. each of these 17 sequences , except m1181 - 197 and pb1537 - 553 , was also characterized by high apparent functional avidity at the lowest peptide concentration of 0 . 1 μg / ml in the ifn - γ elispot assay . several studies showed that high avidity cd8 + t - cells were more effective in limiting viral replication in vitro [ 45 - 47 ]. further , the 17 t - cell epitope peptides had no identity of 8 or more continuous aa to human peptides that might trigger onset of human autoimmune diseases . it is also noteworthy that several of the epitope peptides are located in described functional domains : pb1518 - 575 in the interacting domain of pb1 with pb2 ( pb1506 - 659 ) [ 48 ]; and the overlapping pb2126 - 142 and pb2132 - 148 in the pb1 - and np - binding domain of pb21 - 269 [ 49 ]. t cell epitopes within functional domains would remain conserved over time as viral mutations useful towards immune escape may disrupt the function of the domains . thus , a vaccine comprising these 17 highly conserved t cell epitope peptides , could greatly reduce , if not eliminate , the incidence of variant amino acids of the corresponding t cell epitopes of any future influenza a pathogen . 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 . mice were maintained in a pathogen - free facility at the johns hopkins university according to iacuc guidelines . peptide arrays of pb2 ( bei cat . : nr - 2616 ), pb1 ( nr - 2617 ), pa ( nr - 2618 ), np ( nr - 2611 ), and ml ( nr - 2613 ) of influenza virus a / new york / 348 / 2003 ( h1n1 ) were obtained through the nih biodefense and emerging infections research resources repository , niaid , nih ( bei ). a total of 196 peptides ( all 17 aa long ) were selected to fully cover all highly conserved sequences under study . where these sequences spanned two or more 17 aa peptides , the consecutive peptides overlapped by 11 aa . two immunization peptide pools for immunization were formed : one composed of 84 pb2 and 13 m1 peptides ( table 1 ), and the second composed of 48 pb1 , 23 pa , and 28 np peptides ( table 2 ). each of the 196 peptides was dissolved in 100 % dmso and constituted to 20 % with sterile filtered water . the final concentration of each peptide was 2 μg / μl . the dissolved peptides were stored at − 20 ° c . six different strains of hla transgenic mice were used to cover hla alleles of class i and class ii supertypes . the hla class i supertypes studied were hla - a2 ( a * 0201 ) mice expressing a chimeric heavy chain with murine α3 domain and human β2m . both h - 2db and murine β2m genes were disrupted by homologous recombination [ 12 ], hla - a24 ( a * 2402 ) mice express a chimeric heavy chain and human β2m ; the h - 2kb , h - 2db , and murine β2m genes were disrupted by homologous recombination ( lemonnier et al ., unpublished ), hla - b7 ( b * 0702 ) mice express a chimeric heavy chain with the hla - b * 0702 α1 and α2 domains and the h - 2kd murine α3 domain [ 13 ]. the h - 2kb and h - 2db genes in hla - b7 mice were inactivated by homologous recombination . the hla class ii supertypes were dr2 ( drb1 * 1501 ), dr3 ( drb1 * 0301 ), and dr4 ( drb1 * 0401 ). the peptide - binding domain of hla - dr2 transgenic mice is encoded by human sequences , while the membrane proximal portion containing the cd4 - binding domain is encoded by mouse sequences ( dra1 * 0101 : i - eα and drb1 * 1501 : i - eβ ) [ 14 ]. hla - dr3 transgenic mice express hla - dra * 0101 and - drb1 * 0301 [ 15 ]. hla - dr4 transgenic mice express hla - dra * 0101 , - drb1 * 0401 , and human cd4 [ 16 ]. the derivation and validation of the above transgenic mice , which were bred onto c57bl / 6 genetic background , had been described in the relevant publications . mice were immunized with the selected 196 peptides in 2 pools by use of a protocol which had been validated for t cell studies [ 17 ] and adapted for these transgenic mice studies . peptides were pooled in matrixes as described [ 18 ] and injected in groups of 9 mice of each transgenic strain : two for matrix array screening , two for identifying individual peptides , four for characterizing apparent functional avidity of t cells to positive peptides at three titration points : 10 , 1 , and 0 . 1 μg / ml peptide concentrations , and one as a control ( adjuvant alone ). mice were injected subcutaneously at the base of tail with 100 μl of the immunization peptide pool in titermax ® gold adjuvant ( titermax , norcross , ga .) ( 1 : 1 ). the amount of each peptide injected was 1 μg / mouse . after two weeks , spleens were harvested for ifn - γ elispot assay . harvested spleens from immunized transgenic mice were selectively depleted of t cells by use of anti - cd8 or anti - cd4 antibody - coated immunomagnetic beads with ld columns ( miltenyi biotec , auburn , calif .) according to the manufacturer &# 39 ; s protocol . flow cytometry analysis of the depleted cells indicated this method routinely achieved & gt ; 95 % depletion of the targeted cells . the resulting mhc class i or ii depleted splenocytes were tested individually by ifn - γ elispot assays against the 196 influenza peptides arranged in two 10 × 10 matrix arrays , resulting in 40 peptide pools , where each peptide was present in two different pools , as described [ 18 ]. peptides identified as immunogenic in the matrix array screen were retested individually in a confirmatory assay and a peptide titration assay . thus , each elispot positive response was confirmed three times : by matrix array screening , individually by confirmatory assay and by peptide titration . the elispot assays were performed using mouse ifn - γ elispot sets from bd biosciences ( san jose , calif .) according to the manufacturer &# 39 ; s protocol . briefly , the elispot plates were coated with anti - ifn - γ at 5 μg / ml and incubated at 4 ° c . overnight . the plates were blocked with rpmi 1640 containing 10 % heat - inactivated fetal calf serum , 2 mm l - glutamine , 100 μg of streptomycin / ml , and 100 u of penicillin for 2 h at room temperature , and either cd8 +- or cd4 +- depleted splenocytes ( 0 . 5 - 1 . 0 × 10 ̂ 6 cells / well ) were then added for assays of class ii and i t cell epitopes , respectively . the cells were cultured at 37 ° c . in 5 % co2 in the presence of peptide pools ( final concentration of each peptide was 10 μg / ml ) or individual peptides at final concentrations of 10 μg / ml , 1 μg / ml , and 0 . 1 μg / ml . wells with medium alone served as background ; concanavalin a ( 2 . 5 μg / ml ; sigma - alrich , st . louis , mo .) was used as a polyclonal stimulator ; and known hla - restricted peptides from dengue serotype 3 were included in each assay as positive controls . after 16 h of culture , the plates were washed and incubated with biotinylated anti - ifn - γ for 2 h at room temperature , followed by hrp - conjugated streptavidin for 1 h at room temperature . reactions were developed with aec substrate ( calbiochem - novabiochem , san diego , calif .). final enumeration of ifn - γ spot - forming cells ( sfc ) was performed using the immunospot series 3b analyzer elispot reader ( cellular technologies , shaker heights , ohio ) with aid of the immunospot software version 3 . 0 ( cellular technologies ), indicating the number of sfc / 10 ̂ 6 cells . the results were considered positive if the number of sfc subtracted by those in the background ( culture with medium alone ) was above 10 and the number of sfc was higher than the background plus two standard deviations . the results shown are sfc minus background , which was consistently found to be less than 15 spots / 10 ̂ 6 cells throughout the experiments . presence of experimentally identified t cell epitopes in the influenza a highly conserved sequences published influenza t cell epitopes within the highly conserved sequences were identified by matching the curated t cell epitope sequences mapped in human from the immune epitope database and analysis resource ( iedb , http :// www . immuneepitope . org /) [ 19 ] with the highly conserved sequences . all these published epitope sequences were derived from various t cell assays that included t cell proliferation , ifn - γ elispot , hla tetramer staining , and 51cr release assays . only epitope data from unique sequences and containing hla restriction information were included . the 196 influenza 17 aa peptides were compared using the blastp program against the non - redundant protein sequences database restricted to human ( taxid : 9606 ) at ncbi ( http :// ww . ncbi . nlm . nih . gov / blast /) to detect the presence of fragments identical to human peptides . as the default search parameters were not suitable to probe for short peptide sequences of length 30 or less , the following parameters were used : word size of 2 , expectation value of 30 , 000 , matrix was pam30 , low complexity filter was disabled , and composition - based statistics was set to ‘ no adjustment ’. we disregarded search results containing predicted sequences and human peptides with fewer than six contiguous identical residues as the probability of matching five or less residues is high and non - significant . the dataset and methodology for identification of highly conserved influenza protein sequences among pathogenic influenza strains for the past 30 years had been described by heiny et al . [ 11 ]. briefly , 3763 np , 3781 m1 , 3111 pa , 3175 pb1 , and 3144 pb2 sequences were extracted from the ncbi genbank and genpept databases ( as of september 2006 ) and multiple sequence alignments of the individual proteins were performed . the antigenic variability analyzer tool ( avana ) [ 20 ] was used to extract alignments of each 17 aa t cell epitope mapped in the transgenic mice and to identify the most frequent 17 aa sequence present in at least 80 % of all recorded viruses . to compare 2007 - 2009 human h1n1 sequences with the t cell epitopes of a / new york / 348 / 2003 ( h1n1 ), aligned protein sequence records of human h1n1 m1 , pb1 , and pb2 retrieved from the ncbi influenza virus sequence database ( http :// www . ncbi . nlm . nih . gov / genomes / flu / flu . html , as of jun . 17 , 2009 ) were submitted into the avana tool to identify the most frequent sequence and its variants for each year . immunogenicity of human and avian influenza a highly conserved peptide sequences the previously described 54 highly conserved influenza a peptide sequences of 9 or more contiguous aa of the recorded human and avian influenza strains were represented by a total of 956 aa [ 11 ]. the majority of the conserved sequences , 650 aa , were in the pb1 and pb2 proteins ; there were no conserved sequence in na , m2 , ns1 , and ns2 . a total of 196 peptides ( bei ) of the a / new york / 348 / 2003 ( h1n1 ) m1 , np , pa , pb1 , and pb2 proteins were selected based on the presence of the conserved sequences . the immunogenicity of these 196 conserved influenza peptides was studied by immunizing hla - a2 , - a24 , - b7 , - dr2 , - dr3 and - dr4 transgenic mice . organization of the 54 conserved sequences in the bei 17 aa peptides depended on their length and position . conserved sequences that spanned adjacent 17 aa peptides were repeated up to a maximum of 11 aa because of overlapping peptide synthesis ( table 1 and 2 ). peptides with conserved sequences of less than 17 aa contained mixtures of conserved and non - conserved aa . thirty - three ( 33 ) short conserved sequences ( 9 to 16 aa ) were present in various lengths with adjacent non - conserved aa . conserved sequences of greater length ( 22 sequences of 17 to 57 aa ) were present as complete ( 65 of the 196 peptides ) or partial sequences in the overlapping peptides . the longest conserved sequence was pb 1518 - 575 which was included as part of a cluster of completely conserved aa of 7 overlapping peptides . immunization of the hla transgenic mice with the 196 h1n1 peptides was carried out with 2 pools of about 100 peptides each , with groups of 9 mice of each transgenic strain . interferon - γ ( ifn - γ ) elispot assays for hla - restricted class i and class ii responses were performed with splenocytes of the immunized mice that were depleted of cd4 + and cd8 + t cells , respectively , to identify the responding t cell subset . the initial assays contained matrix arrays of peptide pools followed by validation assays with individual peptides [ 18 ]. of the 196 peptides , 54 contained t cell epitopes that elicited 63 elispot responses ( 8 a24 , 2 b7 , 16 dr2 , 17 dr3 , and 20 dr4 ) ( table 3 ). none of the 196 peptides tested induced t cell responses in mice expressing the hla - a2 allele . forty - seven ( 47 ) of the 54 epitope peptides were restricted by one hla allele ; eight class i and 39 class ii . the remaining 7 peptides were presented by at least two hla alleles of distinct supertypes i . e . they contained multiple or promiscuous t cell epitopes . pb1680 - 696 and pb2548 - 564 were presented by both hla class i and ii alleles . sixteen ( 16 ) pairs of consecutive peptides were restricted by the same hla allele , possibly because there were identical epitopes in the overlapping 11 aa sequence shared by the 2 adjacent peptides . clusters of 2 or more t cell epitope peptides with at least 16 conserved aa were m1175 - 197 , pb1120 - 142 , 340 - 374 , 489 - 576 , and pb242 - 64 , 126 - 146 ( table 3 , fig1 ). * conserved aa are in boldface . consecutive peptides overlapping by 11 aa are aligned . # numbers are representative average ifn - γ spots forming cells per million splenocytes of individual transgenic mice that were positive at 10 μg / ml of peptide concentration . number ( 10 , 1 or 0 . 1 ) in parenthesis represents the lowest concentration of peptide ( μg / ml ) giving positive elispot response in peptide titration . the apparent functional avidity of t cells to each of the 54 peptides was titrated at three peptide concentrations of 10 , 1 and 0 . 1 μg / ml in ifn - γ elispot assays . of the 63 positive elispot responses , including the responses of peptides restricted by multiple hla alleles , 52 activated ifn - γ secretion at each of the three concentrations used in the elispot assay , 9 elicited at concentrations of 10 and 1 μg / ml , and 2 peptides ( np408 - 424 and pb1328 - 344 ) elicited solely at the highest peptide concentration ( table 3 ). presence of reported t cell epitopes in the conserved sequences of influenza a the conserved peptides of this study were compared with reported t cell epitope sequences of humans infected with influenza a viruses extracted from the iedb . twenty - one ( 21 ) of about 800 reported human t cell epitopes of pb2 , pb1 , pa , np , and m1 were found to contain sequences of 9 or more conserved amino acids of all recorded 1977 - 2006 influenza a viruses ( table 4 ). these were mainly from h1n1 , h3n2 , and h5n1 infections and included sequences restricted by a broad range of hla class i and ii alleles , including many not covered by the transgenic mice of this study . for example , the same t cell epitope “ rmvlasttak ” in m1178 - 187 was reported to be restricted by hla - a3 and - a11 [ 21 , 22 ]. clusters of overlapping epitopes were also observed within the conserved sequences , for example , m1123 - 137 had three overlapping epitopes ( 123 alascmgliy 132 was restricted by a1 ; 125 ascmgliy 132 by b35 ; and 129 gliynrmga 137 by a2 ) [ 21 , 23 ]. thus , the highly conserved sequences contained common epitopes shared by pathogenic influenza strains and could be restricted by a broad range of hla alleles . * conserved aa are in boldface . published hla epitopes were extracted from the iedb . hla class i epitopes are underlined and the first amino acid of each identified allele is italicized . hla class ii epitopes longer than 17aa are represented only by the corresponding residues in the 17aa peptides of a / new york / 348 / 2003 ( h1n1 ). each of the 196 influenza 17 aa peptides used in this study was compared with the human proteome sequences to investigate the possibility of human antigens that could trigger an autoimmune response to immunization . specifically , we screened for exactly identical sequences of at least 8 continuous aa , which is the minimum binding peptide length for mhc class i [ 24 ]. many of the conserved sequences of the influenza peptides contained sequences of 6 aa found in human proteins such as voltage - gated sodium channel , dystrophin etc . the longest influenza a sequence with an identical human counterpart was 7 aa of pa131 - 137 but none contained sequences of 8 or more aa identical to the human proteome . * conserved aa are in boldface . italicized aa are found in human peptides . + pa131 - 137 shared 7aa identity with human dystrophin dp427 isoform proteins . the 54 hla - restricted t cell epitope peptides of a / new york / 348 / 2003 ( h1n1 ) strain were analyzed by the antigenic variability analyzer ( avana ) tool for identification of ( a ) the consensus sequence ( most frequent sequence ) in the context of influenza a conserved sequences over the past 30 years , and ( b ) variants and percentage representation of 2007 - 2009 human h1n1 strains as compared to the 2003 h1n1 strain . based on their conservation and variability , the 54 t cell epitope peptides formed three groups : 1 ) seventeen ( 17 ) t cell epitope peptide sequences of the 2003 strain ( 11 pb1 , 4 pb2 , and 2 m1 ) had consensus sequences representing at least 88 % and , for all but 2 consensus sequences represented at least 95 % of all recorded human and avian influenza strains ( table 6a ). in particular , pb1489 - 505 was 100 % conserved in all h1n1 viruses . several variant sequences within this group were recorded , but these were mostly single conservative amino acid substitutions representing a small fraction ( less than 5 %) of all the recorded 1977 - 2006 virus sequences . the major change in 2009 was the apparent complete replacement of 2 previous consensus sequences by variant sequences , each with 1 mutated aa ( pb2132 - 148 , 630 - 646 ). § highly conserved aa of 1977 - 2006 influenza a subtypes are in boldface . * 3175 pb1 , 3144 pb2 , and 3781 m1 human h1n1 , h3n2 , h1n2 , h5n1 , and avian h5n1 and other avian subtypes sequences circulating between 1977 and 2006 were extracted from ncbi genbank and genpept databases as of september 2006 . sequences representing less than 1 % were not included unless they were also represented in the 2007 - 2009 strains . all human pb1 , pb2 , and m1 h1n1 sequences from 2007 to 2009 were extracted from the influenza virus resource on jun 17 , 2009 . + 168 pb1 , 171 pb2 , and 280 m1 human h1n1 2009 sequences . { circumflex over ( )} 31 pb1 , 31 pb2 , and 39 m1 human h1n1 2008 sequences . ″ 314 pb1 , 314 pb2 , and 393 m1 human h1n1 2007 sequences . # new sequence representation not found in the 1977 - 2006 influenza a subtypes sequences . 2 ) a group of 9 pb1 and pb2 t cell epitope peptides of the new york / 348 / 2003 h1n1 strain were variants of the 1977 - 2006 total recorded influenza a virus population at a single mutated aa position ( table 6b ). these variant new york / 348 / 2003 strain sequences represented less than 15 % of the consensus sequences of the entire 1977 - 2006 avian and human virus population . one of these , pb1507 - 523 , became the h1n1 consensus sequence of 2007 - 2009 . for the others , a single aa modification to the bei peptide would result in 96 - 100 % representation in the 2009 human h1n1 population . 3 ) the remaining 28 peptides were each represented in the dataset by 2 to 7 variant sequences with multiple mutations ( table 7 ). the new york / 348 / 2003 2003 sequences were the consensus form in only 13 of the 28 peptides and at reduced representations of 6 to 72 % of the recorded viruses . as the variant forms contained a mixture of the conserved sequences and variable amino acids , it is not possible to predict the immunogenicity of the variant sequences represented in nature and their use as vaccine sequences . these data demonstrated that when t cell epitopes contain mixtures of conserved and non - conserved aa , the occurrences of mutated sequences in a subsequent influenza a strain are greatly enhanced . * 3175 pb1 , 3144 pb2 , and 3781 m1 human h1n1 , h3n2 , h1n2 , h5n1 , and avian h5n1 and other avian subtypes sequences circulating between 1977 and 2006 were extracted from ncbi genbank and genpept databases as of september 2006 . sequences representing less than 1 % of each dataset were excluded . 1 . epstein s l , kong w p , misplon j a , lo c y , tumpey t m , et al . 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