Patent Application: US-41114295-A

Abstract:
the invention relates to the use of peptides representing a portion of the htlv - i envelope protein in diagnostic assays for exposure to htlv - i . the peptides are also useful as components of compositions for eliciting a t - cell response to htlv - i in an immunized subject .

Description:
it is the purpose of this invention to provide immunogenic peptides for use as vaccines to elicit t - cell immunity against htlv - i . it is a further purpose of the invention to provide peptides for use in priming antibody response against pathogenic proteins of the htlv - i virus as treatment for already - infected individuals . peptides of the invention are also useful as diagnostic agents to evaluate patient t - cell responses . the particular peptides taught herein may be conjugated to each other or to other moieties to specifically generate desired responses in the patient . examples of such moieties are b cell epitopes to produce neutralizing antibodies and cytotoxic t - cell epitopes to induce cytotoxic t - cells . the information relating to sequences disclosed indicates that individual mouse phenotypes respond with varying specificity to different immunogens in the same manner that individual human response varies . however , it is known that peptide epitopes which elicit t - cell responses in mice also elicit immune responses in humans . ( see references 9 - 16 in the list of references .) as in mice , different phenotypes may vary somewhat in response to a given epitope . therefore , preferred vaccines contain more than one immunogenic dominant epitope . two independent series of htlv - i envelope peptides were synthesised in two laboratories , based on two different strategies . one set was chosen based on prediction of amphipathic helical conformation to look for t - cell epitopes , and the other set was made originally to search for b - cell epitopes , and so had been chosen on the basis of hydrophilicity of amino acid sequence . the antigenicity of these peptides was tested on native htlv - i envelope protein - immunized murine lymph node cells and strong antigenicity was predominantly detected in peptides predicted to be amphipathic , α - helical sites . furthermore , three antigenic peptides could be used to immunize mice to elicit t - cells proliferating in response to the native envelope protein , and to prime helper t cells for an enhanced antibody response to the native envelope protein . these antigenic and / or immunogenic peptides identified in the murine system should be helpful for analysing the difference in immune responses to htlv - i among patients with tsp / ham and atll and their healthy seropositive relatives and also maybe useful for developing a peptide vaccine protecting against htlv - i transmission and htlv - i - associated disease development . the computer algorithm , amphi , was used for identifying predicted t - cell sites , based on segments with potential amphipathic helical structure , in the htlv - i - envelope protein whose primary amino acid sequence was identified by seiki et al . 28 !. the algorithm identified 15 predicted sites ; nine of them were amphipathic α - helical sites and 5 of them were 3 10 - helical sites as shown in fig1 a . in one predicted site , 387 - 411 , α - helicity changes to 3 10 - helical character in the c - terminal half . one series of peptides , v1e1 - 5 and v1e7 - 9 , were synthesized , based on the analysis , and v1e10 was synthesized as a control peptide with non - predicted sequence ( fig1 b ). although another series of peptides , sp1 - 6 and sp8 - 11 , were synthesized independent of these predictions , 7 of 10 peptides ( sp1 , sp2 , sp4a , sp5 , sp8 , sp9 , and sp10 ) corresponded to either α or 3 10 predicted sites coincidentally . furthermore , complete sharing or partial overlap of amino acid sequence was observed between some of the peptides in the two series : sp4a ( 190 - 209 ) and v1e9 ( 268 - 286 ) included v1e8 ( 191 - 209 ) and sp5 ( 269 - 280 ), respectively and v1e7 and sp2 , v1e4 sp8 , and v1e5 and sp 10 overlapped partially ( fig1 b , 1c , and table 1 ). identification of sites after immunization with native htlv - i envelope protein . table 2 shows the proliferative response of htlv - i envelope protein - immune lymph node cells to the synthetic peptides . congenic mice representing four independent mhc types were studied . moreover , these strains differ only in their mhc region . peptides v1e8 and sp4a induced prominant proliferative responses of b10 . br and b10 . a ( 5r ) lymph node cells and v1e1 elicited remarkable response only in b10 . br nice . these two sites , v1e8 / sp4a and v1e1 , were potent antigenic sites in htlv - i envelope protein and elicited proliferative response in these strains of mice even at 0 . 01 μm of antigen concentration ( fig2 .). v1e2 and v1e9 were also antigenic in envelope - immune b10 . br mice and v1e10 in b10 . a ( 5r ) mice ( table 2 ), but these were much less potent on a molar basis in dose - response curves ( fig2 ). no site was identified for b10 . d2 and b10 . htt ( and b10 . s ( 9r )) mice that was as highly antigenic as v1e1 and v1e8 / sp4a for b10 . br and b10 . a ( 5r ) mice . however , sp1 , sp5 , sp10 , v1e5 , and v1e9 were immunodominant for b10 . htt and sp1 , sp2 , sp4a , sp5 , v1e4 , v1e7 , v1e9 , and v1e10 were for b10 . d2 as shown in table 2 . the responses of b10 . s ( 9r ) mice were the same as those of b10 . htt ( data not shown ). the fact that sp4a , but not v1e8 which is contained within sp4a , stimulated b10 . d2 envelope - immune t cells suggests that the determinant seen by h - 2 d mice is slightly different from that seen by the other strains that respond to both peptides , and requires the additional nh 2 - terminal leucine residue present in sp4a but not in v1e8 . interestingly , all of these antigenic sites , except for v1e10 , were amphipathic α - helical portions of the htlv - i envelope and no amphipathic 3 10 - helical site was involved , as summarised in table 3 . responses to native envelope after immunization with peptides . for developing a peptide - vaccine , the candidate peptide should have the capability to elicit in vivo a t cell response to the whole native molecule from which the peptide derives . the peptides , v1e1 , v1e8 , and v1e10 , were found to be immunodominant sites by the results of the above experiments in which the native envelope protein was used as immunogen and synthetic peptides were used as antigens in secondary in vitro culture ( a native immunogen / peptide test antigen or &# 34 ; np &# 34 ; experiment ). therefore b10 . br , b10 . d2 , and b10 . a ( 5r ) mice were immunized with these three peptides individually and tested the response of peptide - immune lymph node cells to the native envelope protein ( a peptide immunogen / native test antigen or &# 34 ; pn &# 34 ; experiment ). as shown in fig3 the peptide v1e8 , antigenic in b10 . br and b10 . a ( 5r ) in the np direction , could immunize mice to elicit the response of lymph node cells to the native envelope protein as well as the immunogen ( v1e8 ) itself in the same strains of mice . likewise , the peptides v1e1 and v1e10 , antigenic in b10 . br and b10 . d2 , respectively , in the np experiments , were also immunogenic in the corresponding strains of mice for eliciting t cells responding to the native protein in pn experiments ( fig3 ). the concentrations of the secondary antigens producing the maximum responses in these pn experiments were similar for the peptide immunogens and the native protein antigen , the htlv - i envelope protein . ability of peptides to prime helper t cells for antibody responses to the whole envelope protein . in order to assess whether the peptides that could prime mice for a t - cell proliferative response to the native protein could also prime helper t cells for an enhanced antibody response to the whole envelope protein , four groups of five mice each of two strains were immunized first i . p . with 10 nmoles of one of three peptides ( without any carrier ) or pbs as a control in cfa . fifteen days later , the mice were boosted i . p . with a suboptimal dose of 5 . 6 μg of affinity - purified envelope protein in ifa , and bled on days 14 and 21 after the boost . antibodies were measured to the affinity - purified envelope protein by radiobinding assay as described in materials and methods . mice preimmunized with only pbs uniformly failed to produce any measurable binding activity ( fig4 ), consistent with the suboptimal immunization with whole protein . in b10 . br mice , preimmunization with peptide v1e8 and to a lesser extent with peptide v1e1 , both of which are immunogenic in this strain , produced significant titers of antibodies binding the envelope protein . in contrast , preimmunization with peptide v1e10 failed to enhance the antibody response in b 10 . br mice which also do not manifest a proliferative response to this peptide . in b10 . a ( 5r ) mice , which give a proliferative response to peptides v1e8 and v1e10 but not v1e1 , an enhanced antibody response to the envelope protein was observed in those mice immunized with v1e8 and v1e10 , and a weaker response on day 14 but not day 21 in those immunized with v1e1 . thus , the enhancement of antibody responses followed the same genetic ir gene control as the t - cell proliferative response . therefore , these data were interpreted as reflecting the priming of helper t cells so that a secondary response occurs when the mice are first immunized with whole protein . we noted the presence of some anti - htlv - i envelope antibodies in the mice immunized with peptides two weeks earlier , but before they were boosted with whole protein ( data not shown ). no antibodies were found in pbs - immune mice . this result suggests that the peptides may also contain b - cell epitopes . in particular , peptide sp4a , which coincides with v1e8 , has been shown to be a b - cell epitope in humans ( palker et . al . j . immunol . 142 : 971 - 978 ). however , even if some priming of b cells occurs , such priming of b cells by uncoupled small peptides without a carrier implies that these peptides contain effective helper t - cell determinants . thus , in any case , the results of immunization with uncoupled peptide and the ir gene control of the antibody response parallel to that of the t - cell proliferative response indicate that the three peptides tested can induce t - cell help as well as t - cell proliferation . the overlap of amino acid sequences useful as immunogens and as antibody primors has been observed in several cases , possibly due to the relatively high hydrophilic character of some amphipathic helical segments . however , as disclosed herein , hydrophilicity of amino acid sequence of peptides is not significantly correlated with the immunodominance of peptides for proliferative t - cell responses ( j . l . spouge , h . r . guy , and j . a . berzofsky , unpublished observations ). in fact , one of the two most potent immunodominant sites , v1e1 in gp21 , was not identified by the algorithm that selected peptide sequences because of their high hydrophilicity . hence , the preneficial peptides are not obvious in view of prior trackings in the art . table 3a shows the relation between immunodominance and the potential amphipathicity of the htlv - i envelope peptides . interestingly , ten of the eleven immunodominant sites ( all except for v1e10 ) were amphipathic α - helical sites and no amphipathic 3 10 - helical sites were identified as immunodominant . only one of four non - helical sites were immunodominant . the conformation of 3 10 - helices is energetically less stable than that of α - helices and usually short pieces of 3 10 - helix tend to be at the n - and c - termini of α - helices . although the original study ( margalit et . al . j . immunol . 138 : 2213 - 2229 ) of published immunodominant sires did not detect a difference in antigenicity between amphipathic α - helices and 3 10 - helices , p . hale et al . ( manuscript submitted ) recently found that amphipathic 3 10 - helical sites in the hiv envelope protein were rarely immunogenic as compared to the high tendency toward immunodominance of the amphipathic α - helical sites . it is noteworthy that the difference in antigenicity between amphipathic α - helices and 3 10 - helices is revealed in the envelope protein of two human retroviruses . the efficacy of the prediction of amphipathic α - helical segments as immunodominant sites in htlv - i envelope protein is demonstrated in table 3b . of 11sites found to be immunogenic , 10 sites were segments predicted to be amphipathic α - helices . furthermore , no site predicted to be an amphipathic α - helical site is among the 6 sites found to be non - immunogenic . the proliferative responses of native envelope protein - immunized lymph node cells to the synthetic peptides were clearly dependent on the mhc haplotype . four congenic strains of mice were used , differing only in their mhc type . they represent eight different i - a and i - e class ii mhc molecules . sites 190 - 209 ( v1e8 / sp4a ) and 342 - 363 ( v1e1 ) were found to be especially potent sites in the htlv - i envelope protein : the responses of b10 . br and b10 . a ( 5r ) mice to v1e8 / sp4a and of b10 . br mice to v1e1 occurred even at the low concentration of 0 . 01 μm in np experiments . although the maximum response of envelope protein - immune lymph node cells was not different among the four congenic strains of mice , b10 . br , b10 . d2 , b10 . a ( 5r ), and b10 . htt , no site was identified to be so potent at low concentrations for b10 . d2 and b10 . htt mice as v1e1and v1e8 / sp4a were for b10 . br and b 10 . a ( 5r ) mice . it was also found that three of the epitope peptides of the htlv - i envelope ( v1e8 / sp4a , v1e1 , and v1e10 ) primed mice for memory t cell help to enhance antibody responses to b - cell epitopes of the envelope protein . asymptomatic seropositive carriers of htlv - i and patients with atll and tsp / ham have comparable anti - htlv - i antibody titers in their sera . in contrast , a difference in hla haplotype and in lymphocyte proliferative response to htlv - i virions among the patients with these two distinct diseases was reported from southwestern japan , an endemic area for htlv - i ( usuku et . al . ann . neurol 23 : s143 - s150 ). the patients with ham were high responders to htlv - i virions in peripheral blood lymphocyte ( pbl ) cultures in vitro , whereas atll patients were low responders in their analysis . moreover , this difference in lymphocyte response was correlated to the difference in hla haplotype : healthy htlv - i carriers with ham - associated hla haplotypes tended to show higher htlv - i - specific lymphocyte responses than healthy carriers with hla haplotypes correlated with development of atll . when the response of human pbl to the synthetic htlv - i peptides identified in the current study is analysed , it should be possible to detect more distinct mhc - associated differences in the t - cell responses among the carriers and patients with atll and ham because individual peptide epitopes should show clearer genetic restriction than the whole envelope protein , as shown here in mice . also , responses to particular peptides may be associated with the diseases tsp / ham , especially if these diseases have autoimmune mechanisms . recently , palker et al . ( j . immunol . 142 : 971 - 978 ) reported that the central region of htlv - i gp46 envelope glycoprotein defined by synthetic peptide sp4a ( residues 190 - 209 ) bound antibodies from 78 % of htlv - i seropositive humans . thus , the sp4a / v1e8 region contains an immunodominant t - cell site in mice of three of four mhc types tested ( current study ) and also a major b - cell epitope in man . while sites of b and t cell recognition frequently differ in location within a protein and in structural and sequence features ( berzofsky et . al . immunol . rev . 98 : 9 - 52 ), major t - and b - cell sites can coincide within the same molecule ( takahashi et . al . proc . natl . acad . sci . usa 85 : 3105 - 2109 ., thomas et . al . eur . j . immunol . 17 : 133 - 136 ., palker et . al . j . immunol . 142 : in press ). as the incidence of disease among htlv - i infection is low compared with aids virus infection , it is difficult to test a vaccine for htlv - i even if it is developed . however , this observation also suggests that in htlv - i infection , the immune system is working more effectively for protection against the development of the diseases than in aids virus infection . table 1______________________________________sequence of synthetic htlv1 envelope peptides ( seq id nos 1 - 19 ) number ofpeptides amino acids amino acid sequence . sup . a______________________________________sp1 33 - 47 vssyhskpcnpaqpvsp2 86 - 107 ( c ) phwtkkpnrngggyysasysdpsp3 176 - 189 ( c ) lntepsqlpptapp ( y ) sp4a 190 - 209 ( c ) llphsnldhilepsipwksk ( y ) sp5 269 - 280 ( y ) lpfnwthcfdpq ( c ) sp6 296 - 312 ( c ) ppfslspvptlgsrsrrsp8 400 - 415 crfpnitnshvpilqesp9 411 - 422 ( c ) pileqrpplenrsp10 462 - 480 cirqlrhlpsrvryphyssp11 475 - 488 ( c ) ryphyslikpesslv1e1 342 - 363 sgksllhevdkdivsqltqaikv1e2 353 - 370 disqltqaivknhknllkv1e3 362 - 378 vknhknllkiaqyaaqnv1e4 388 - 402 eqgglckalqeqcrfv1e5 458 - 475 lagpcilrqlrhlpsrvrv1e7 97 - 111 ggyysasysdpcslkv1e8 191 - 209 lphsnldhilepsipwkskv1e9 268 - 286 tlpfnwthcfdpqiqaivsv1e10 141 - 156 ftqevsrlninlhfsk______________________________________ . sup . a amino acids in parentheses have been added to peptides to facilitate coupling to carrier protein ( c ) and iodination of peptide ( y ) for another study 24 !. these residues in parentheses are not present in the predicted amino acid sequence of htlvi envelope glycoprotein reported by seiki et al . 28 !. table 2__________________________________________________________________________the proliferative response of htlv1 envelope protein - immunized lymph nodecellsto the synthetic peptides and the immunogenb10 . br b10 . d2 b10 . a ( 5r ) b10 . httantigensδcpm ( sem ) s . i . δacpm ( sem ) s . i . δcpm ( sem ) s . i . δcpm ( sem ) s . i . __________________________________________________________________________sp1 - 2 , 792 ( 1 . 05 ) 0 . 57 16 , 121 ( 1 . 19 ) 2 . 99 - 1 , 886 ( 1 . 34 ) 0 . 52 11 , 919 ( 1 . 22 ) 5 . 15sp2 686 ( 1 . 13 ) 1 . 11 23 , 724 ( 1 . 21 ) 3 . 93 - 106 ( 1 . 37 ) 0 . 97 * 5 , 008 ( 1 . 18 ) 2 . 74sp3 359 ( 1 . 14 ) 1 . 06 * 11 , 148 ( 1 . 32 ) 2 . 38 - 1 , 405 ( 1 . 08 ) 0 . 64 2 , 733 ( 1 . 13 ) 1 . 95sp4a 26 , 184 ( 1 . 16 ) 5 . 04 17 , 561 ( 1 . 18 ) 3 . 17 34 , 607 ( 1 . 08 ) 7 . 50 867 ( 1 . 29 ) 1 . 30sp5 - 1 , 159 ( 1 . 03 ) 0 . 82 28 , 985 ( 1 . 18 ) 4 . 58 552 ( 1 . 12 ) 1 . 14 6 . 433 ( 1 . 24 ) 3 . 24sp6 544 ( 1 . 36 ) 1 . 08 3 , 313 ( 1 . 22 ) 1 . 41 2 , 037 ( 1 . 04 ) 1 . 51 2 , 069 ( 1 . 21 ) 1 . 72sp8 - 948 ( 1 . 25 ) 0 . 85 4 , 890 ( 1 . 49 ) 1 . 60 2 , 601 ( 1 . 07 ) 1 . 66 2 , 145 ( 1 . 21 ) 1 . 70sp9 3 , 240 ( 1 . 17 ) 1 . 50 4 , 074 ( 1 . 30 ) 1 . 46 1 , 990 ( 1 . 10 ) 0 . 91 2 , 279 ( 1 . 18 ) 1 . 74sp10 2 , 218 ( 1 . 10 ) 1 . 34 * 14 , 813 ( 1 . 44 ) 2 . 83 - 79 ( 1 . 14 ) 0 . 98 12 , 173 ( 1 . 05 ) 4 . 96sp11 3 , 946 ( 1 . 20 ) 1 . 61 * 11 , 811 ( 1 . 30 ) 2 . 46 - 367 ( 1 . 20 ) 0 , 91 3 , 343 ( 1 . 18 ) 2 . 09v1e1 30 , 621 ( 1 . 11 ) 5 . 73 957 ( 1 . 41 ) 1 . 12 - 194 ( 1 . 17 ) 0 . 95 862 ( 1 . 13 ) 1 . 28v1e2 21 , 191 ( 1 . 27 ) 4 . 27 1 , 252 ( 1 . 17 ) 1 . 15 104 ( 1 . 20 ) 1 . 03 2 , 155 ( 1 , 29 ) 1 . 70v1e3 7 , 444 ( 1 . 09 ) 2 . 15 1 , 910 ( 1 . 21 ) 1 . 22 - 2 , 440 ( 1 . 11 ) 0 . 38 792 ( 1 . 27 ) 1 . 26v1e4 1 , 289 ( 1 . 07 ) 1 . 20 25 , 568 ( 1 . 06 ) 3 . 97 - 1 , 862 ( 1 . 13 ) 0 . 53 1 , 111 ( 1 . 19 ) 1 . 37v1e5 2 , 498 ( 1 . 16 ) 1 . 39 7 , 186 ( 1 . 46 ) 1 . 84 - 1 , 990 ( 1 . 30 ) 0 . 50 4 , 669 ( 1 . 48 ) 2 . 55v1e7 - 1 , 072 ( 1 . 16 ) 0 . 83 13 , 155 ( 1 . 08 ) 2 . 53 - 221 ( 1 . 39 ) 0 . 94 2 , 885 ( 1 . 06 ) 1 . 95v1e8 26 , 747 ( 1 . 11 ) 5 . 13 - 2 , 009 ( 1 . 12 ) 0 . 77 25 , 993 ( 1 . 07 ) 7 . 56 1 , 928 ( 1 . 22 ) 1 . 64v1e9 19 , 605 ( 1 . 28 ) 3 . 76 26 , 721 ( 1 . 10 ) 4 . 11 8 . 005 ( 1 . 39 ) 2 . 31 8 , 196 ( 1 . 25 ) 3 . 71v1e107 , 438 ( 1 . 07 ) 1 . 43 27 , 183 ( 1 . 26 ) 4 . 16 20 . 477 ( 1 , 20 ) 5 . 92 1 , 149 ( 1 . 20 ) 1 . 38env . prot . 106 , 677 ( 1 . 16 ) 17 . 5 108 , 617 ( 1 . 10 ) 13 . 6 91 , 367 ( 1 . 04 ) 24 . 1 48 , 222 ( 1 . 10 ) 17 . 0__________________________________________________________________________ a . the native envelope protein - immune lymph node cells were cultured with three different concentrations of peptides : 0 . 3 μm , 1 . 0 μm , and 3 . 0 μm . the 3 h !- tdr incorporation into the responding cells at highest concentration of the antigens ( 3 μm ) is shown as δ geometric mean ( cpm ), sem , and stimulation indices ( s . i .) ( ratio of experimental mean to control mean without antigen ) b . the values underlined were highly statistically significant ( p & lt ; 0 . 01 ) also these peptides stimulated significantly at lower antigen concentrations . these peptides were identified to be immunodominant for the corresponding mouse strains . the values with asterisks were statistically significant ( 0 . 01 & lt ; p & lt ; 0 . 05 ) only at the highest antigen concentration , 3 . 0 μm . table 3______________________________________correlation of experimental immunodominance withα - helical amphipathicity predictiona . type predicted α - helix 3 . sub . 10 helix non - helical______________________________________tested sites 10 3 4immunodominant sites 10 0 1 % 100 0 25______________________________________b . found found immunogenic non - immunogenic______________________________________total sites 11 6predicted to be 10 0amphipathic alpha helix______________________________________ the peptides of the invention may be administered as immunogens in the usual carriers . the compositions for use as immunogens may contain adjuvants such as alum . compositions containing vie1 , vie8 / sp4a and vie10 are preferred . however , it is advisable that more than one of the epitopes taught herein be given . compositions containing at least three epitopes are preferred . compositions may be administered in the usual manner . however , preferred routes of administration for vaccine use are subcutaneous , intradermal , or intramuscular routes . the compositions of the invention can also be used as primers to elicit t - 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780 . 17 . delisi , c ., and j . a . berzofsky . 1985 . t cell antigenic sites tend to be amphipathic structures . proc . natl . acad . sci . u . s . a . 82 : 7048 - 7052 . 18 . margalit , h ., j . l . spouge , j . l . cornette , k . cease , c . delisi , and j . a . berzofsky . 1987 . prediction of immunodominant helper t - cell antigenic sites from the primary sequence . j . immunol . 138 : 2213 - 2229 . 19 . palker , t . j ., m . e . clark , m . g . sarngadharan , and t . j . matthews . 1987 . purification of envelope glycoproteins of human t cell lymphotropic virus type i ( htlv - i ) by affinity chromatography . j . virol . methods 18 : 243 . 20 . spouge , j . l ., h . r . guy , j . l . cornette , h . margalit , k . cease , j . a . berzofsky , and c . delisi . 1987 . strong conformational propensities enhance t - cell antigenicity . j . immunol . 138 : 204 - 212 . 21 . berzofsky , j . a ., k . b . cease , j . l . cornette , j . l . spouge , h . margalit , i . j . berkower , m . f . good , l . h . miller , and c . delisi . 1987 . protein antigenic structures recognized by t cells : potential applications to vaccine design . immunol . rev . 98 : 9 - 52 . 22 . cornette , j . l ., h . margalit , c . delisi , and j . a . berzofsky . 1989 . concepts and methods in the identification of t cell epitopes and their use in the construction of synthetic vaccines . methods in enzymol . 23 . kyte , j ., and r . f . doolittle . 1982 . a simple method for displaying the hydrophilic character of a protein . j . mol . biol . 157 : 105 . 24 . palker , t . j ., m . e . tanner , r . m . scearce , r . d . streilein , m . e . clark , and b . f . haynes . 1989 . mapping of immunogenic regions of human t cell leukemia virus type i ( htlv - i ) gp46 and gp 21 envelope glycoproteins with env - encoded synthetic peptides and a monoclonal antibody to gp46 . j . immunol . 142 : 971 - 978 . 25 . merrifield , r . b . 1965 . automated synthesis of peptides . science 150 : 178 - 185 . 26 . stewart , j . m ., and j . d . young . 1984 . solid phase peptide synthesis . pierce chemical company , rockford , ill ., 27 . corradin , g ., h . m . etlinger , and j . m . chiller . 1977 . lymphocyte specificity in protein antigens . i . characterization of the antigen - induced in vitro t cell - dependent proliferative response with lymph node cells from primed mice . j . immunol . 119 : 1048 . 28 . seiki , m ., s . hattori , and m . yoshida . 1982 . human adult t - cell leukemia virus : molecular cloning of the provirus dna and the unique terminal structure . proc . natl . acad . sci . usa 79 : 6899 - 6902 . 29 . takahashi , h ., j . cohen , a . hosmalin , k . b . cease , r . houghten , j . cornette , c . delisi , b . moss , r . n . germain , and j . a . berzofsky . 1988 . an immunodominant epitope of the hiv gp160 envelope glycoprotein recognized by class i mhc molecule - restricted murine cytotoxic t lymphocytes . proc . natl . acad . sci . usa 85 : 3105 - 3109 . 30 . thomas , d . b ., j . j . skehel , k . h . g . mills , and c . m . graham . 1987 . a single amino acid substitution in influenza haemagglutinin abrogates recognition by a monoclonal antibody and a spectrum of subtype - specific l3t4 + t cell clones . eur . j . immunol . 17 : 133 - 136 . 31 . palker , t . j ., t . j . matthews , a . langlois , m . e . tanner , m . e . martin , r . m . scearce , j . e . kim , j . a . berzofskjy , d . p . bolognesi , and b . f . haynes . 1989 . polyvalent human immunodeficiency virus synthetic immunogen comprised of envelope gp120t helper cell sites and b cell neutralization epitopes . j . immunol . 142 : in press . 32 . malik , k . t . a ., j . evan , and a . karpas . 1988 . molecular cloning and complete nucleotide sequence of an adult t cell leukaemia virus / human t cell leukaemia virus type i ( atlv / htlv - i ) isolate of caribbean origin : relationship to other members of the atlv / htlv - i subgroup . j . gen . virol . 69 : 1695 . __________________________________________________________________________sequence listing ( 1 ) general information :( iii ) number of sequences : 19 ( 2 ) information for seq id no : 1 :( i ) sequence characteristics :( a ) length : 15 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vii ) immediate source :( b ) clone : htlv - i ( xi ) sequence description : seq id no : 1 : valsersertyrhisserlysprocysasnproalaglnproval151015 ( 2 ) information for seq id no : 2 :( i ) sequence characteristics :( a ) length : 23 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vii ) immediate source :( b ) clone : htlv - i ( xi ) sequence description : seq id no : 2 : cysprohistrpthrlyslysproasnargasnglyglyglytyrtyr151015seralasertyrserasppro20 ( 2 ) information for seq id no : 3 :( i ) sequence characteristics :( a ) length : 16 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vii ) immediate source :( b ) clone : htlv - i ( xi ) sequence description : seq id no : 3 : cysleuasnthrgluproserglnleuproprothralaproprotyr151015 ( 2 ) information for seq id no : 4 :( i ) sequence characteristics :( a ) length : 22 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vii ) immediate source :( b ) clone : htlv - i ( xi ) sequence description : seq id no : 4 : cysleuleuprohisserasnleuasphisileleugluproserile151015protrplysserlystyr20 ( 2 ) information for seq id no : 5 :( i ) sequence characteristics :( a ) length : 14 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vii ) immediate source :( b ) clone : htlv - i ( xi ) sequence description : seq id no : 5 : tyrleupropheasntrpthrhiscyspheaspproglncys1510 ( 2 ) information for seq id no : 6 :( i ) sequence characteristics :( a ) length : 18 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vii ) immediate source :( b ) clone : htlv - i ( xi ) sequence description : seq id no : 6 : cyspropropheserleuserprovalprothrleuglyserargser151015argarg ( 2 ) information for seq id no : 7 :( i ) sequence characteristics :( a ) length : 16 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vii ) immediate source :( b ) clone : htlv - i ( xi ) sequence description : seq id no : 7 : cysargpheproasnilethrasnserhisvalproileleuglnglu151015 ( 2 ) information for seq id no : 8 :( i ) sequence characteristics :( a ) length : 13 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vii ) immediate source :( b ) clone : htlv - i ( xi ) sequence description : seq id no : 8 : cysproileleuglngluargproproleugluasnarg1510 ( 2 ) information for seq id no : 9 :( i ) sequence characteristics :( a ) length : 19 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vii ) immediate source :( b ) clone : htlv - i ( xi ) sequence description : seq id no : 9 : cysileleuargglnleuarghisleuproserargvalargtyrpro151015histyrser ( 2 ) information for seq id no : 10 :( i ) sequence characteristics :( a ) length : 15 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vii ) immediate source :( b ) clone : htlv - i ( xi ) sequence description : seq id no : 10 : cysargtyrprohistyrserleuilelysprogluserserleu151015 ( 2 ) information for seq id no : 11 :( i ) sequence characteristics :( a ) length : 22 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vii ) immediate source :( b ) clone : htlv - i ( xi ) sequence description : seq id no : 11 : serglylysserleuleuhisgluvalasplysaspileserglnleu151015thrglnalailevallys20 ( 2 ) information for seq id no : 12 :( i ) sequence characteristics :( a ) length : 18 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vii ) immediate source :( b ) clone : htlv - i ( xi ) sequence description : seq id no : 12 : aspileserglnleuthrglnalailevallysasnhislysasnleu151015leulys ( 2 ) information for seq id no : 13 :( i ) sequence characteristics :( a ) length : 17 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vii ) immediate source :( b ) clone : htlv - i ( xi ) sequence description : seq id no : 13 : vallysasnhislysasnleuleulysilealaglntyralaalagln151015asn ( 2 ) information for seq id no : 14 :( i ) sequence characteristics :( a ) length : 15 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vii ) immediate source :( b ) clone : htlv - i ( xi ) sequence description : seq id no : 14 : gluglnglyglyleucyslysalaleuglngluglncysargphe151015 ( 2 ) information for seq id no : 15 :( i ) sequence characteristics :( a ) length : 18 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vii ) immediate source :( b ) clone : htlv - i ( xi ) sequence description : seq id no : 15 : leualaglyprocysileleuargglnleuarghisleuproserarg151015valarg ( 2 ) information for seq id no : 16 :( i ) sequence characteristics :( a ) length : 15 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vii ) immediate source :( b ) clone : htlv - i ( xi ) sequence description : seq id no : 16 : glyglytyrtyrseralasertyrseraspprocysserleulys151015 ( 2 ) information for seq id no : 17 :( i ) sequence characteristics :( a ) length : 19 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vii ) immediate source :( b ) clone : htlv - i ( xi ) sequence description : seq id no : 17 : leuprohisserasnleuasphisileleugluproserileprotrp151015lysserlys ( 2 ) information for seq id no : 18 :( i ) sequence characteristics :( a ) length : 19 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vii ) immediate source :( b ) clone : htlv - i ( xi ) sequence description : seq id no : 18 : thrleupropheasntrpthrhiscyspheaspproglnileglnala151015ilevalser ( 2 ) information for seq id no : 19 :( i ) sequence characteristics :( a ) length : 16 amino acids ( b ) type : amino acid ( d ) topology : linear ( ii ) molecule type : peptide ( iii ) hypothetical : no ( iv ) anti - sense : no ( v ) fragment type : internal ( vii ) immediate source :( b ) clone : htlv - i ( xi ) sequence description : seq id no : 19 : phethrglngluvalserargleuasnileasnleuhispheserlys151015__________________________________________________________________________