AU1906592A - Peptides for use in induction of t cell activation against hiv-1 - Google Patents
Peptides for use in induction of t cell activation against hiv-1Info
- Publication number
- AU1906592A AU1906592A AU19065/92A AU1906592A AU1906592A AU 1906592 A AU1906592 A AU 1906592A AU 19065/92 A AU19065/92 A AU 19065/92A AU 1906592 A AU1906592 A AU 1906592A AU 1906592 A AU1906592 A AU 1906592A
- Authority
- AU
- Australia
- Prior art keywords
- peptide
- group
- ser
- ile
- phe
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims description 310
- 102000004196 processed proteins & peptides Human genes 0.000 title claims description 147
- 230000006044 T cell activation Effects 0.000 title description 14
- 230000006698 induction Effects 0.000 title description 7
- 210000001744 T-lymphocyte Anatomy 0.000 claims description 88
- 235000001014 amino acid Nutrition 0.000 claims description 73
- 150000001413 amino acids Chemical class 0.000 claims description 72
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 claims description 46
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 34
- 125000003275 alpha amino acid group Chemical group 0.000 claims description 24
- 230000008878 coupling Effects 0.000 claims description 22
- 238000010168 coupling process Methods 0.000 claims description 22
- 238000005859 coupling reaction Methods 0.000 claims description 22
- 239000000203 mixture Substances 0.000 claims description 20
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 16
- 125000003277 amino group Chemical group 0.000 claims 30
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims 30
- 241000282693 Cercopithecidae Species 0.000 description 60
- 229940024606 amino acid Drugs 0.000 description 58
- 210000004027 cell Anatomy 0.000 description 54
- 241000725303 Human immunodeficiency virus Species 0.000 description 42
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 41
- 108090000623 proteins and genes Proteins 0.000 description 31
- 102000004169 proteins and genes Human genes 0.000 description 30
- 241000700605 Viruses Species 0.000 description 29
- 235000018102 proteins Nutrition 0.000 description 29
- 102000036639 antigens Human genes 0.000 description 27
- 108091007433 antigens Proteins 0.000 description 27
- 239000000427 antigen Substances 0.000 description 26
- 238000000338 in vitro Methods 0.000 description 25
- 230000003472 neutralizing effect Effects 0.000 description 22
- 238000011534 incubation Methods 0.000 description 21
- 229940092253 ovalbumin Drugs 0.000 description 21
- 230000000875 corresponding effect Effects 0.000 description 20
- 229960005486 vaccine Drugs 0.000 description 20
- 108010058846 Ovalbumin Proteins 0.000 description 19
- 238000004519 manufacturing process Methods 0.000 description 18
- 108010002350 Interleukin-2 Proteins 0.000 description 16
- 102000000588 Interleukin-2 Human genes 0.000 description 16
- 230000003053 immunization Effects 0.000 description 16
- 238000012360 testing method Methods 0.000 description 15
- 230000000890 antigenic effect Effects 0.000 description 13
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 12
- 230000015572 biosynthetic process Effects 0.000 description 12
- 238000002649 immunization Methods 0.000 description 12
- 238000006386 neutralization reaction Methods 0.000 description 12
- 230000009696 proliferative response Effects 0.000 description 12
- 230000004044 response Effects 0.000 description 12
- 210000003719 b-lymphocyte Anatomy 0.000 description 11
- 238000000034 method Methods 0.000 description 10
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 9
- 230000003213 activating effect Effects 0.000 description 9
- 230000002163 immunogen Effects 0.000 description 9
- 230000001939 inductive effect Effects 0.000 description 9
- 208000015181 infectious disease Diseases 0.000 description 9
- 238000013102 re-test Methods 0.000 description 9
- 125000000539 amino acid group Chemical group 0.000 description 8
- 238000003556 assay Methods 0.000 description 8
- 239000006228 supernatant Substances 0.000 description 8
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 7
- 102100027723 Endogenous retrovirus group K member 6 Rec protein Human genes 0.000 description 7
- 208000031886 HIV Infections Diseases 0.000 description 7
- 239000002671 adjuvant Substances 0.000 description 7
- 230000000694 effects Effects 0.000 description 7
- 239000012894 fetal calf serum Substances 0.000 description 7
- 210000002443 helper t lymphocyte Anatomy 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- 241001430294 unidentified retrovirus Species 0.000 description 7
- 238000002255 vaccination Methods 0.000 description 7
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 6
- 230000000521 hyperimmunizing effect Effects 0.000 description 6
- 238000010166 immunofluorescence Methods 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- 239000007790 solid phase Substances 0.000 description 6
- JWDFQMWEFLOOED-UHFFFAOYSA-N (2,5-dioxopyrrolidin-1-yl) 3-(pyridin-2-yldisulfanyl)propanoate Chemical compound O=C1CCC(=O)N1OC(=O)CCSSC1=CC=CC=N1 JWDFQMWEFLOOED-UHFFFAOYSA-N 0.000 description 5
- 101710121417 Envelope glycoprotein Proteins 0.000 description 5
- 208000037357 HIV infectious disease Diseases 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 5
- 230000028993 immune response Effects 0.000 description 5
- 230000036039 immunity Effects 0.000 description 5
- JWYOAMOZLZXDER-UHFFFAOYSA-N 2-azaniumylcyclopentane-1-carboxylate Chemical group NC1CCCC1C(O)=O JWYOAMOZLZXDER-UHFFFAOYSA-N 0.000 description 4
- 102000014914 Carrier Proteins Human genes 0.000 description 4
- 108010078791 Carrier Proteins Proteins 0.000 description 4
- 108010010803 Gelatin Proteins 0.000 description 4
- 108090000288 Glycoproteins Proteins 0.000 description 4
- 102000003886 Glycoproteins Human genes 0.000 description 4
- 241000282567 Macaca fascicularis Species 0.000 description 4
- 241000124008 Mammalia Species 0.000 description 4
- IQFYYKKMVGJFEH-XLPZGREQSA-N Thymidine Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](CO)[C@@H](O)C1 IQFYYKKMVGJFEH-XLPZGREQSA-N 0.000 description 4
- 101800001690 Transmembrane protein gp41 Proteins 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 230000005859 cell recognition Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 230000018109 developmental process Effects 0.000 description 4
- 238000010790 dilution Methods 0.000 description 4
- 239000012895 dilution Substances 0.000 description 4
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 4
- 229920000159 gelatin Polymers 0.000 description 4
- 235000019322 gelatine Nutrition 0.000 description 4
- 235000011852 gelatine desserts Nutrition 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 239000000816 peptidomimetic Substances 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 230000035755 proliferation Effects 0.000 description 4
- 230000001681 protective effect Effects 0.000 description 4
- 230000009257 reactivity Effects 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000006467 substitution reaction Methods 0.000 description 4
- ITMCEJHCFYSIIV-UHFFFAOYSA-N triflic acid Chemical compound OS(=O)(=O)C(F)(F)F ITMCEJHCFYSIIV-UHFFFAOYSA-N 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 3
- 102000009016 Cholera Toxin Human genes 0.000 description 3
- 108010049048 Cholera Toxin Proteins 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 241000282412 Homo Species 0.000 description 3
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 3
- 241001465754 Metazoa Species 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 3
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 3
- 238000002835 absorbance Methods 0.000 description 3
- 230000005875 antibody response Effects 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 210000001217 buttock Anatomy 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000003795 chemical substances by application Substances 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 231100000433 cytotoxic Toxicity 0.000 description 3
- 230000001472 cytotoxic effect Effects 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 230000007812 deficiency Effects 0.000 description 3
- 108700004025 env Genes Proteins 0.000 description 3
- 210000003743 erythrocyte Anatomy 0.000 description 3
- 108700004026 gag Genes Proteins 0.000 description 3
- 239000008273 gelatin Substances 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 230000000652 homosexual effect Effects 0.000 description 3
- 230000016784 immunoglobulin production Effects 0.000 description 3
- 238000001727 in vivo Methods 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 238000010647 peptide synthesis reaction Methods 0.000 description 3
- 229920000642 polymer Polymers 0.000 description 3
- 230000003389 potentiating effect Effects 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000002062 proliferating effect Effects 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 208000011580 syndromic disease Diseases 0.000 description 3
- 210000002845 virion Anatomy 0.000 description 3
- 208000030507 AIDS Diseases 0.000 description 2
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 101710091045 Envelope protein Proteins 0.000 description 2
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 229940033330 HIV vaccine Drugs 0.000 description 2
- 229920000209 Hexadimethrine bromide Polymers 0.000 description 2
- 241000598436 Human T-cell lymphotropic virus Species 0.000 description 2
- 241000713340 Human immunodeficiency virus 2 Species 0.000 description 2
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- 229930182816 L-glutamine Natural products 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- KDXKERNSBIXSRK-UHFFFAOYSA-N Lysine Natural products NCCCCC(N)C(O)=O KDXKERNSBIXSRK-UHFFFAOYSA-N 0.000 description 2
- 239000004472 Lysine Substances 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- 101710188315 Protein X Proteins 0.000 description 2
- 239000006146 Roswell Park Memorial Institute medium Substances 0.000 description 2
- 229920005654 Sephadex Polymers 0.000 description 2
- 239000012507 Sephadex™ Substances 0.000 description 2
- 230000005867 T cell response Effects 0.000 description 2
- 108010003533 Viral Envelope Proteins Proteins 0.000 description 2
- 125000003368 amide group Chemical group 0.000 description 2
- RDOXTESZEPMUJZ-UHFFFAOYSA-N anisole Chemical compound COC1=CC=CC=C1 RDOXTESZEPMUJZ-UHFFFAOYSA-N 0.000 description 2
- 235000003704 aspartic acid Nutrition 0.000 description 2
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000003776 cleavage reaction Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 210000001151 cytotoxic T lymphocyte Anatomy 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 102000037865 fusion proteins Human genes 0.000 description 2
- 108020001507 fusion proteins Proteins 0.000 description 2
- 235000013922 glutamic acid Nutrition 0.000 description 2
- 239000004220 glutamic acid Substances 0.000 description 2
- -1 gpl20" Proteins 0.000 description 2
- 230000001900 immune effect Effects 0.000 description 2
- 238000003018 immunoassay Methods 0.000 description 2
- 230000005847 immunogenicity Effects 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 238000007918 intramuscular administration Methods 0.000 description 2
- 238000001990 intravenous administration Methods 0.000 description 2
- 235000018977 lysine Nutrition 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 210000003071 memory t lymphocyte Anatomy 0.000 description 2
- 230000007193 modulation by symbiont of host erythrocyte aggregation Effects 0.000 description 2
- 150000007523 nucleic acids Chemical group 0.000 description 2
- 239000013641 positive control Substances 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 125000006239 protecting group Chemical group 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 230000007017 scission Effects 0.000 description 2
- 238000013207 serial dilution Methods 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 125000005931 tert-butyloxycarbonyl group Chemical group [H]C([H])([H])C(OC(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- JOXIMZWYDAKGHI-UHFFFAOYSA-N toluene-4-sulfonic acid Chemical compound CC1=CC=C(S(O)(=O)=O)C=C1 JOXIMZWYDAKGHI-UHFFFAOYSA-N 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 235000002374 tyrosine Nutrition 0.000 description 2
- VRTXRNJMNFVTOM-ZDUSSCGKSA-N (2r)-3-[(4-methoxyphenyl)methylsulfanyl]-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound COC1=CC=C(CSC[C@H](NC(=O)OC(C)(C)C)C(O)=O)C=C1 VRTXRNJMNFVTOM-ZDUSSCGKSA-N 0.000 description 1
- ZQEBQGAAWMOMAI-ZETCQYMHSA-N (2s)-1-[(2-methylpropan-2-yl)oxycarbonyl]pyrrolidine-2-carboxylic acid Chemical compound CC(C)(C)OC(=O)N1CCC[C@H]1C(O)=O ZQEBQGAAWMOMAI-ZETCQYMHSA-N 0.000 description 1
- DMBKPDOAQVGTST-LBPRGKRZSA-N (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-3-phenylmethoxypropanoic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)COCC1=CC=CC=C1 DMBKPDOAQVGTST-LBPRGKRZSA-N 0.000 description 1
- ZYJPUMXJBDHSIF-NSHDSACASA-N (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-3-phenylpropanoic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 ZYJPUMXJBDHSIF-NSHDSACASA-N 0.000 description 1
- SOHLZANWVLCPHK-LBPRGKRZSA-N (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-4-oxo-4-phenylmethoxybutanoic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CC(=O)OCC1=CC=CC=C1 SOHLZANWVLCPHK-LBPRGKRZSA-N 0.000 description 1
- AJDUMMXHVCMISJ-ZDUSSCGKSA-N (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]-5-oxo-5-phenylmethoxypentanoic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CCC(=O)OCC1=CC=CC=C1 AJDUMMXHVCMISJ-ZDUSSCGKSA-N 0.000 description 1
- QVHJQCGUWFKTSE-YFKPBYRVSA-N (2s)-2-[(2-methylpropan-2-yl)oxycarbonylamino]propanoic acid Chemical compound OC(=O)[C@H](C)NC(=O)OC(C)(C)C QVHJQCGUWFKTSE-YFKPBYRVSA-N 0.000 description 1
- WBIIPXYJAMICNU-AWEZNQCLSA-N (2s)-5-[amino-[(4-methylphenyl)sulfonylamino]methylidene]azaniumyl-2-[(2-methylpropan-2-yl)oxycarbonylamino]pentanoate Chemical compound CC1=CC=C(S(=O)(=O)NC(N)=NCCC[C@H](NC(=O)OC(C)(C)C)C(O)=O)C=C1 WBIIPXYJAMICNU-AWEZNQCLSA-N 0.000 description 1
- VVNYDCGZZSTUBC-LURJTMIESA-N (2s)-5-amino-2-[(2-methylpropan-2-yl)oxycarbonylamino]-5-oxopentanoic acid Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CCC(N)=O VVNYDCGZZSTUBC-LURJTMIESA-N 0.000 description 1
- UHPQFNXOFFPHJW-UHFFFAOYSA-N (4-methylphenyl)-phenylmethanamine Chemical compound C1=CC(C)=CC=C1C(N)C1=CC=CC=C1 UHPQFNXOFFPHJW-UHFFFAOYSA-N 0.000 description 1
- DHBXNPKRAUYBTH-UHFFFAOYSA-N 1,1-ethanedithiol Chemical compound CC(S)S DHBXNPKRAUYBTH-UHFFFAOYSA-N 0.000 description 1
- VYMPLPIFKRHAAC-UHFFFAOYSA-N 1,2-ethanedithiol Chemical compound SCCS VYMPLPIFKRHAAC-UHFFFAOYSA-N 0.000 description 1
- 229940124718 AIDS vaccine Drugs 0.000 description 1
- 208000037859 AIDS-related disorder Diseases 0.000 description 1
- 206010067484 Adverse reaction Diseases 0.000 description 1
- 208000000044 Amnesia Diseases 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- DCXYFEDJOCDNAF-UHFFFAOYSA-N Asparagine Natural products OC(=O)C(N)CC(N)=O DCXYFEDJOCDNAF-UHFFFAOYSA-N 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 102100026189 Beta-galactosidase Human genes 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 101710205625 Capsid protein p24 Proteins 0.000 description 1
- 241000700199 Cavia porcellus Species 0.000 description 1
- 108010062580 Concanavalin A Proteins 0.000 description 1
- 101150105088 Dele1 gene Proteins 0.000 description 1
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 1
- 101100536354 Drosophila melanogaster tant gene Proteins 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 238000012286 ELISA Assay Methods 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 101000935043 Homo sapiens Integrin beta-1 Proteins 0.000 description 1
- 101001002657 Homo sapiens Interleukin-2 Proteins 0.000 description 1
- 102100025304 Integrin beta-1 Human genes 0.000 description 1
- QNAYBMKLOCPYGJ-REOHCLBHSA-N L-alanine Chemical compound C[C@H](N)C(O)=O QNAYBMKLOCPYGJ-REOHCLBHSA-N 0.000 description 1
- DCXYFEDJOCDNAF-REOHCLBHSA-N L-asparagine Chemical compound OC(=O)[C@@H](N)CC(N)=O DCXYFEDJOCDNAF-REOHCLBHSA-N 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- KDXKERNSBIXSRK-YFKPBYRVSA-N L-lysine Chemical compound NCCCC[C@H](N)C(O)=O KDXKERNSBIXSRK-YFKPBYRVSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- KZSNJWFQEVHDMF-BYPYZUCNSA-N L-valine Chemical compound CC(C)[C@H](N)C(O)=O KZSNJWFQEVHDMF-BYPYZUCNSA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 208000008771 Lymphadenopathy Diseases 0.000 description 1
- 241001430197 Mollicutes Species 0.000 description 1
- LSQXZIUREIDSHZ-ZJZGAYNASA-N Morphiceptin Chemical class C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N1[C@@H](CCC1)C(N)=O)C1=CC=C(O)C=C1 LSQXZIUREIDSHZ-ZJZGAYNASA-N 0.000 description 1
- FYYSQDHBALBGHX-YFKPBYRVSA-N N(alpha)-t-butoxycarbonyl-L-asparagine Chemical compound CC(C)(C)OC(=O)N[C@H](C(O)=O)CC(N)=O FYYSQDHBALBGHX-YFKPBYRVSA-N 0.000 description 1
- 229940123257 Opioid receptor antagonist Drugs 0.000 description 1
- 208000001388 Opportunistic Infections Diseases 0.000 description 1
- 241000282579 Pan Species 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 101710177166 Phosphoprotein Proteins 0.000 description 1
- ONIBWKKTOPOVIA-UHFFFAOYSA-N Proline Natural products OC(=O)C1CCCN1 ONIBWKKTOPOVIA-UHFFFAOYSA-N 0.000 description 1
- 239000012979 RPMI medium Substances 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 101710149279 Small delta antigen Proteins 0.000 description 1
- 101000874347 Streptococcus agalactiae IgA FC receptor Proteins 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 102100023935 Transmembrane glycoprotein NMB Human genes 0.000 description 1
- 241000251221 Triakidae Species 0.000 description 1
- 102100022563 Tubulin polymerization-promoting protein Human genes 0.000 description 1
- 206010046865 Vaccinia virus infection Diseases 0.000 description 1
- KZSNJWFQEVHDMF-UHFFFAOYSA-N Valine Natural products CC(C)C(N)C(O)=O KZSNJWFQEVHDMF-UHFFFAOYSA-N 0.000 description 1
- 241000607626 Vibrio cholerae Species 0.000 description 1
- 108010015780 Viral Core Proteins Proteins 0.000 description 1
- 108010067390 Viral Proteins Proteins 0.000 description 1
- 230000010530 Virus Neutralization Effects 0.000 description 1
- ZAFQHMFHEMVQEB-UHFFFAOYSA-N acetonitrile 2,2,2-trifluoroacetic acid Chemical compound CC#N.OC(=O)C(F)(F)F.OC(=O)C(F)(F)F ZAFQHMFHEMVQEB-UHFFFAOYSA-N 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 238000005903 acid hydrolysis reaction Methods 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 239000003463 adsorbent Substances 0.000 description 1
- 230000006838 adverse reaction Effects 0.000 description 1
- 235000004279 alanine Nutrition 0.000 description 1
- 230000009435 amidation Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000036436 anti-hiv Effects 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 229910052786 argon Inorganic materials 0.000 description 1
- 235000009582 asparagine Nutrition 0.000 description 1
- 229960001230 asparagine Drugs 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- VWYLMMNTUOUYCT-UHFFFAOYSA-N benzyl carbonobromidate Chemical compound BrC(=O)OCC1=CC=CC=C1 VWYLMMNTUOUYCT-UHFFFAOYSA-N 0.000 description 1
- HSDAJNMJOMSNEV-UHFFFAOYSA-N benzyl chloroformate Chemical compound ClC(=O)OCC1=CC=CC=C1 HSDAJNMJOMSNEV-UHFFFAOYSA-N 0.000 description 1
- 125000000051 benzyloxy group Chemical group [H]C1=C([H])C([H])=C(C([H])=C1[H])C([H])([H])O* 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- 230000001588 bifunctional effect Effects 0.000 description 1
- 238000004166 bioassay Methods 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 229920001222 biopolymer Polymers 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 238000001516 cell proliferation assay Methods 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000005094 computer simulation Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- 230000000120 cytopathologic effect Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 230000002939 deleterious effect Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000000432 density-gradient centrifugation Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000003503 early effect Effects 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 101150030339 env gene Proteins 0.000 description 1
- 230000007515 enzymatic degradation Effects 0.000 description 1
- 238000013213 extrapolation Methods 0.000 description 1
- 210000003191 femoral vein Anatomy 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 239000000417 fungicide Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 101150098622 gag gene Proteins 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- ZDXPYRJPNDTMRX-UHFFFAOYSA-N glutamine Natural products OC(=O)C(N)CCC(N)=O ZDXPYRJPNDTMRX-UHFFFAOYSA-N 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 210000004201 immune sera Anatomy 0.000 description 1
- 229940042743 immune sera Drugs 0.000 description 1
- 208000026278 immune system disease Diseases 0.000 description 1
- 230000006028 immune-suppresssive effect Effects 0.000 description 1
- 230000004957 immunoregulator effect Effects 0.000 description 1
- 230000003308 immunostimulating effect Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 239000012678 infectious agent Substances 0.000 description 1
- 230000001524 infective effect Effects 0.000 description 1
- 230000004073 interleukin-2 production Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 238000011005 laboratory method Methods 0.000 description 1
- 210000000265 leukocyte Anatomy 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 231100000863 loss of memory Toxicity 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 208000018555 lymphatic system disease Diseases 0.000 description 1
- 210000000207 lymphocyte subset Anatomy 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 210000004779 membrane envelope Anatomy 0.000 description 1
- UZKWTJUDCOPSNM-UHFFFAOYSA-N methoxybenzene Substances CCCCOC=C UZKWTJUDCOPSNM-UHFFFAOYSA-N 0.000 description 1
- 239000003226 mitogen Substances 0.000 description 1
- 238000001823 molecular biology technique Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 230000005298 paramagnetic effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- RGCLLPNLLBQHPF-HJWRWDBZSA-N phosphamidon Chemical compound CCN(CC)C(=O)C(\Cl)=C(/C)OP(=O)(OC)OC RGCLLPNLLBQHPF-HJWRWDBZSA-N 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 108700004029 pol Genes Proteins 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 125000001500 prolyl group Chemical class [H]N1C([H])(C(=O)[*])C([H])([H])C([H])([H])C1([H])[H] 0.000 description 1
- 229940070376 protein Drugs 0.000 description 1
- 230000009145 protein modification Effects 0.000 description 1
- 230000006337 proteolytic cleavage Effects 0.000 description 1
- 239000002516 radical scavenger Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000004062 sedimentation Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 238000004513 sizing Methods 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 229940031626 subunit vaccine Drugs 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000017960 syncytium formation Effects 0.000 description 1
- 125000006253 t-butylcarbonyl group Chemical group [H]C([H])([H])C(C(*)=O)(C([H])([H])[H])C([H])([H])[H] 0.000 description 1
- HNKJADCVZUBCPG-UHFFFAOYSA-N thioanisole Chemical compound CSC1=CC=CC=C1 HNKJADCVZUBCPG-UHFFFAOYSA-N 0.000 description 1
- 125000002088 tosyl group Chemical group [H]C1=C([H])C(=C([H])C([H])=C1C([H])([H])[H])S(*)(=O)=O 0.000 description 1
- 108091007466 transmembrane glycoproteins Proteins 0.000 description 1
- 208000007089 vaccinia Diseases 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 229940118696 vibrio cholerae Drugs 0.000 description 1
- 230000007501 viral attachment Effects 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 229960004854 viral vaccine Drugs 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/18—Antivirals for RNA viruses for HIV
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2740/00—Reverse transcribing RNA viruses
- C12N2740/00011—Details
- C12N2740/10011—Retroviridae
- C12N2740/16011—Human Immunodeficiency Virus, HIV
- C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
- C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
- Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
- Y10T292/00—Closure fasteners
- Y10T292/65—Braces
- Y10T292/67—Portable
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Molecular Biology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biophysics (AREA)
- General Chemical & Material Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Oncology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Genetics & Genomics (AREA)
- Communicable Diseases (AREA)
- Gastroenterology & Hepatology (AREA)
- Biochemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Tropical Medicine & Parasitology (AREA)
- AIDS & HIV (AREA)
- Peptides Or Proteins (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
Description
Description
Peptides for Use in Induction of T Cell Activation Against HIV-1
This application is a continuation-in-part of pending U.S. patent application Serial No. 07/571,080, filed August 22, 1990.
Background of the Invention
AIDS and AIDS-related disorders (ARC) are caused by a retrovirus, the human immunodeficiency virus (HIV). Barre-Sinoussi et al., "Isolation of a T-Lymphotropic Retrovirus from a Patient at Risk for Acguired Immune Deficiency Syndrome (AIDS) , Science, 220:868 (1983); and Gallo et al., "Frequent Detection and Isolation of Cytopathic Retroviruses (HTLV-III) From Patients with AIDS and at Risk for AIDS", Science, 224:500 (1984). Like most viruses, HIV often elicits the produc¬ tion of neutralizing antibodies. Unlike many other viruses and other infectious agents for which infection leads to protective immunity, however, HIV specific antibodies are insufficient to halt the progression of the disease. Therefore, in the case of HIV, a vaccine that elicits the immunity of natural infection could prove to be ineffective. In fact, vaccines prepared from the HIV protein gpl60 appear to provide little immunity to HIV infection although they elicit neu- tralizing antibodies. The failure to produce an effective anti-HIV vaccine has led to the prediction that an effective vaccine will not be available until the end of the 1990's.
The HIV genome has been well characterized. Its approximately 10Kb encodes sequences that contain
regulatory segments for HIV replication as well as the gag, pol and env genes coding for the core proteins, the reverse transcriptase-protease-endonuclease, and the internal and external envelope glycoproteins respectively.
The HIV env gene encodes the intracellular glyco- protein, gpl60, which is normally processed by proteo- lytic cleavage to form gpl20, the external viral gly- coprotein, and gp41, the viral transmembrane glyco- protein. The gpl20 protein remains associated with HIV virions by virtue of noncovalent interactions with gp41. These noncovalent interactions are weak, conse¬ quently most of the gpl20 is released from cells and virions in a soluble form. Previous studies have shown that the proteins encoded by the gag and especially the env regions of the HIV-1 genome are immunogenic since antibodies to the products of the gag and env genes are found in the sera of HIV infected, AIDS and ARC patients. It has previously been shown that some antibodies obtained from sera of AIDS and ARC patients, as well as asymptomatic individuals infected with the virus are specific to gpl20 and gpl60. Occasionally these anti¬ bodies are neutralizing. The envelope glycoproteins are the HIV-1 antigen most consistently recognized by antibodies in AIDS and ARC patient sera. Allan et al. , "Major Glycoprotein Antigens that Induce Antibodies in AIDS Patients are Encoded by HTLV-III", Science, 228:1091-1094 (1985); and Barin et al., "Virus Envelope Protein of HTLV-III Represents Major Target Antigen for Antibodies in AIDS Patients", Science, 228:1094-1096 (1985) . In addition, antibodies in patient sera also recognize epitopes of the. viral core proteins encoded by the gag gene. Im unologically important HIV-l antigens for use in diagnosis and as potential vaccine compositions have
been prepared by cloning portions of the HIV-1 genome in various expression systems such as bacteria, yeast or vaccinia. Cabradilla et al., "Serodiagnosis of Antibodies to the Human AIDS Retrovirus With a Bacterially Synthesized env Polypeptide", Bio¬ technology, 4:128-133 (1986); and Chang et al., "Detection of Antibodies to Human T-Cell Lymphotropic Virus-Ill (HTLV-III) With an Immunoassay Employing a Recombinant Escherichia coli - Derived Viral Antigenic Peptide", Biotechnology, 3:905-909 (1985). HIV-1 antigens produced by recombinant DNA methods, however, must still be exhaustively purified to avoid adverse reactions upon vaccination and false positive reactions in ELISA assays due to any antibody reactivity to antigens of the expression system which may contaminate the HIV-l antigen preparation. Also, denaturation of HIV-1 antigens during purification may destroy impor¬ tant antigen activity. Preparation of proteins from intact viruses can also result in contamination by the virus.
Several publications have presented data showing immunologic reactivity of selected synthetic peptides corresponding to portions of the antigenic proteins of HIV-l. In one study, a peptide having the amino acid sequence Tyr-Asp-Arg-Pro-Glu-Gly-Ile-Glu-Glu-Glu- Gly-Gly-Glu-Arg-Asp-Arg-Asp-Arg-Ser-Gly-Cys which corresponds to amino acid residues 735-752 of HIV-l was synthesized. Kennedy et al., "Antiserum to a Synthetic Peptide Recognizes the HTLV-III Envelope Glycoprotein", Science, 231:1556-1559 (1986). This peptide, derived from a portion of gp41, was used to immunize rabbits in an attempt to elicit a neutralizing antibody response to HIV-l. Furthermore, several sera from AIDS patients known to contain anti-gp41 antibodies were weakly reactive with this peptide, thus indicating that this peptide contains at least one epitope recognized, to
some extent, by antibodies to native gpl60/gp41. However, this peptide has not been shown to elicit neutralizing antibodies in mammals other than rabbits nor has it been suggested for use as a human vaccine. Longitudinal studies conducted on cohorts of
HIV-infected individuals have indicated that a stable clinical condition is associated with presence of high titers of neutralizing antibodies against the envelope glycoprotein gpl20 of HIV and especially against a specific segment of eight amino acids. Ranki et al., "Neutralizing Antibodies in HIV (HTLV-III) Infection: Correlation with Clinical Outcome and Antibody Response Against Different Viral Proteins", Clin. Exp. Immunol., 69:231 (1987); and Marx (1989). Achieving protective immunity against HIV is likely to lie on the induction of gpl20 specific neutralizing antibodies. Marx, "New Hope on the AIDS Vaccine Front", Science, 244:1254 (1989). Potent T cell help might also be critical to promote the gen- eration and the expansion of virus-specific cytotoxic T cells. Reinherz and Schlossman, "The Characteriza¬ tion and Function of Human Immunoregulatory T Lympho¬ cyte Subsets", Immunol. Today, 2:69 (1981); Burns et al, "Thy us Dependence of Viral Antigens", Nature, 256:654 (1975); and Askonas et al., "Cytotoxic T-memory Cells in Virus Infection and the Specificity of Helper T Cells", Immunology, 45:79 (1982). To be durable and broad, protective immunity should rely on induction of immunologic memory to structurally conserved antigenic moieties comprising epitopes displaying limited MHC restriction for T helper cell recognition. Askonas et al. (1982) .
Since production of antibodies, including neu¬ tralizing antibodies, by B cells is critically depen- dent on cognate T cell help, and antigenic determinants recognized by T cells are often distinct from the ones
recognized by B cells, identification of antigenic moieties recognized by T cells (so-called "T cell epitopes") , is important when considering vaccination strategies based on appropriate combinations of T and B cell epitopes.
It would therefore be useful in the treatment and prevention of AIDS and ARC to have an HIV vaccine cap¬ able of producing neutralizing antibodies and con- comitantly eliciting T cell help. Most antigenic determinants recognized by T cells are composed of continuous stretches of peptides. Streitcher et al., "Antigen Conformation Determines Processing Requirements for T-cell Activation", Proc. Natl. Acad. Sci. U.S.A., 79:4723 (1982) ; DeLisi and Berzofsky, "T Cell Antigen Sites Tend to be Amphipathic Structures", Proc. Natl. Acad. Sci. U.S.A., 82:7048 (1985); and Margalit et al., "Prediction of Immunodominant Helper T Cell Antigenic Sites From Primary Sequence", J. Immunol., 138:2213 (1987) . B and T cell recognition sites are often located in different regions of a complex antigen. Milich et al., "Nonoverlapping T and B Cell Deter¬ minants on an Hepatitis B Antigen pre-S(2) Region Synthetic Peptide", J. Exp. Med. , 164:532 (1986). Within the functional T cell repertoire, T helper cells, T cytotoxic cells, and T suppressor cells, appear to recognize structurally distinct determinants. Krzych et al., "Induction of Helper and Suppressor T Cells by Nonoverlapping Determinants on the Large Protein Antigen, β-galactosidase", FASEB J. , 2:141
(1988) . This functional separation may have important bearing on the development of vaccines, since particular determinants recognized by T suppressor cells may be ablated resulting in important benefits for immunogenicity.
AIDS and ARC are associated with progressive impairment of CD4+ T cells, and increased suscep¬ tibility to opportunistic infections. In this respect, HIV-infected persons show decreased T helper cell activity for polyclonal B cell differentiation and decreased T cell proliferative responses to antigens and mitogens associated with an early loss of CD29+ memory T cells. Terpstra et al. , "Longitudinal Study of Leukocyte Functions in Homosexual Men Seroconverted for HIV: Rapid and Persistent Loss of B Cell Function After HIV Infection", Eur. J. Immunol., 19:667 (1989); Fahey et al., "Quantitative Changes in T Helper or Suppressor/Cytotoxic Lymphocyte Subsets that Distin¬ guish Acquired Immune Deficiency Syndrome From Other Immune Subsets Disorders", JAMA, 76:95 (1984); Shearer et al., "Functional T Lymphocyte Immune Deficiency in a Population of Homosexual Men Who do not Exhibit Symptoms of Acquired Immune Deficiency Syndrome", J. Clin. Invest., 74:496-506 (1984); Giorgi et al., "Early Effects of HIV on CD4 Lymphocytes in vivo". J.
Immunol., 138:3725 (1987); and van Noesels et al., "Functional and Phenotypic Evidence for a Selective Loss of Memory T Cells in Asymptomatic Human Immuno¬ deficiency Virus-infected Men", 86:293 (1990). The use of synthetic peptides as artificial T cell recognition sites in the composition of candidate subunit vaccines, offers attractive prospects. In this regard, the possibility to educate T helper cells with synthetic peptides for the development of subsequent antibody responses against overlapping and non- overlapping B cell (antibody) recognition sites has been documented in several experimental systems. Streitcher et al. (1982) ;. DeLisi and Berzovsky (1985) ; and Milich et al. , "A Single 10-residue PreS(l) Peptide Can Prime T Cell Help for Antibody Production to
Multiple Epitopes Within the pre-S(l), pre-S(2), and S
regions of HBsAg", J. Immunol., 138:4457 (1987). It has now been found that peptides derived from two regions of the HIV genome elicit T cell activation. These peptides are also capable of inducing the production of neutralizing antibodies to HIV-l.
Summary of the Invention
In accordance with the present invention, novel peptides corresponding to epitopes of HIV-l gpl20 protein and analogues and homologs thereof are provided. These peptides can be utilized alone or in combination, uncoupled or coupled to other molecules or substrates. The peptides are useful in eliciting T cell activation, immunization against HIV infection, induction of a heightened immune response to HIV and in production of polyclonal and monoclonal antibodies. Forty synthetic peptides corresponding to the entire primary sequence of the envelope gpl20 of the human immunodeficiency virus type 1 (HIV-l) were examined for their ability to induce antibody formation and/or T cell activation antibody formation was determined by measuring the amount of peptide-specific antibody formed. T cell activation was measured by the ability of the peptides to induce in vitro proliferative responses and/or IL-2 production when added to cultures of unfractionated, T cell enriched, and/or CD4+ T cell enriched peripheral blood mononuclear cells (PBMC) from immune monkeys. Among four major areas of T cell recognition identified, two novel T cell activating regions were identified, both of which were also found to be capable of inducing, in vivo, the production of neutralizing antibodies to HIV-l. One of these two novel areas corresponds to a highly conserved region of HIV-l gp-120, the other area being located to a variable region of gp-120. Recognition of the latter variable region does not
appear to be restricted by MHC polymorphism, since all of six monkeys immunized with corresponding peptides were found to display in vitro proliferative responses to these peptides. The results of peptides thus have great utility for the development of synthetic subunit AIDS vaccines.
Brief Description of the Drawings
Fig. 1 is a graph depicting in vitro proliferative responses of monkey peripheral blood mononuclear cells (PBMC) to recall peptide after two and/or three peptide immunizations.
Fig. 2 is a graph depicting in vitro proliferative responses of monkey PBMC to half-overlapping peptides.
Detailed Description of the Invention A vaccine against AIDS, if an efficient one is to be found, is likely to contain components that are capable of inducing T helper cell activity to cognate B cells committed to the production of HIV neutralizing antibodies. The present invention provides peptides, some of which have previously been found to elicit production of HIV neutralizing antibodies by primate subjects and all of which have now been found to have the surprising property of eliciting T cell activation. The peptides correspond to regions of the gpl20 protein with amino acid coordinates as defined by Kennedy et al. , "Antiserum to a Synthetic Peptide Recognizes the HTLV- III Envelope Glycoprotein", Science, 231:1556-1559 (1986) . The peptides of the present invention are termed gpl20-ll (amino acid coordinates 141-164) , gpl20-12 (amino acid coordinates 151-176) , gpl20-13 (amino acid coordinates 164-192) , gpl20-16 (amino acid coordinates 205-230) and gpl20-19 (amino acid coor¬ dinates 247-269) , gpl20-29 (amino acid coordinates 366-
389) and gpl20-30 (amino acid coordinates 377-400) . The peptides of the present invention have been des¬ cribed for use as immunogens in vaccine compositions and to elicit polyclonal or monoclonal antibody pro- ductions in United States patent application Serial Number 07/589,422 filed Sept. 27, 1990 which is incorporated herein by reference.
Four topographically related groups of peptides derived from gpl20 have now been identified which display T cell activating properties. Two of the gpl20 regions found to elicit T cell activation are similar to previously identified T cell epitopes. Bolognesi, "HIV Antibodies and AIDS Design", AIDS 3:S111-S118 (1989) . The present study indicates that recognition of the area defined by amino acid coordinates 295-343 by immune T cells may be submitted to strong MHC- restriction as one of the 2 monkeys injected with peptide gpl20-25 failed to respond to that particular peptide in vitro. The T cell antigenic determinants in this area seem to be more or less exclusively located within each of the immunizing peptides as none of the overlapping peptides gave rise to jln vitro prolifera¬ tive responses. However, PBMC isolated from monkeys immunized with peptide gpl20-24 secreted IL-2 when cultured in the presence of peptide gpl20-25, indica¬ ting the existence of a minor epitope shared by these two peptides.
The region of gpl20 corresponding to amino acid coordinates 295 to 343 (peptides gpl20-23, gpl20-24 and gpl20-25) , is similar to a region (amino acid coordi¬ nates 301 to 338) which has previously been shown by other investigators to contain a major T cell recogni¬ tion site (amino acid coordinates 303-337) whose sequence encompasses that of the neutralizing loop. Bolognesi (1989); Javaherian et al., "Principal
Neutralizing Domain of the Human Immunodeficiency Virus
Type 1 Envelope Protein", Proc. Natl. Acad. Sci. USA, 86:6768 (1989); and Rusche et al., "Antibodies That Inhibit Fusion of Human Immunodeficiency Virus infected Cells Bind a 24-amino Acid Sequence of the Viral Envelope, gpl20", Proc. Natl. Acad. Sci. USA, 85:3198 (1988).
The area of gp-120 located between amino acid coordi¬ nates 409 and 466 (peptides gpl20-33, gpl20-34, gpl20-35 and gpl20-36), as described in the Examples presented below, was found to have potent T cell activating properties. The area between amino acid coordinates 409 och 466 has previously been shown to accommodate T cell activating domains. Bolog¬ nesi (1989). In this area, two T cell epitopes have been identified, one between amino acid coordinates 410 and 429, and one between amino acid coordinates 428 and 443. The latter area largely overlaps with the CD4-binding site (amino acid coordinates 420-463) of gpl20, the main site of virus attachment on permissive T cells. Several other epitopes with T cell activating pro¬ perties have now been identified in discrete areas of the gpl20 molecule. Thus, peptide gpl20-4 (amino acid coordina¬ tes 53-74), gpl20-5 (amino acid coordinates 64-89), gpl20-8 (amino acid coordinates 100-126), gp 120-7 (amino acid coordinates 218-247) and peptide gpl20-21 (amino acid coor¬ dinates 269-295), and at least on of their overlapping peptides, were shown to be capable of inducing T cell re¬ sponses in vitro. However, both of the monkeys immunized with peptide gpl20-21, when retested five months after the last booster dose, hade lost their in vitro T cell responsi¬ veness to recall peptide. Thus, not every peptide capable of eliciting
T cell activation is suitable for use in treatment and prevention of AIDS.
Surprisingly, peptide gpl20-19, has now been shown to have T cell immunogenic properties as defined by in vitro proliferative responses of simian PBMC to cognate peptide. Additionally, PBMC from a monkey immunized with OVA-conjugated peptide gpl20-16 has now been found to secrete IL-2 after in vitro exposure to peptide gpl20-16. Peptide gpl20-16 therefore represents an additional novel T cell epitope.
Two novel areas with in vitro T cell activating properties have now been identified. Peptides gpl20- 11, gpl20-12 and gpl20-13., corresponding to amino acid coordinates 141 to 192, induce the most potent .in vitro proliferative responses, with SI values exceeding some¬ times 20 in cultures of T cells from monkeys immunized with corresponding OVA-conjugated peptides. The fact that six out of six monkeys from an outbred population, responded to peptides gpl20-ll, gpl20-12 and gpl20-13 strongly indicates that recognition of this bona fide T cell epitope area by simian T cells is not under strict MHC control. Accordingly, at least three distinct epi¬ topes have now been recognized by immune monkey PBMC, one shared by peptides gpl20-ll and gpl20-12, one shared by peptides gpl20-12 and gpl20-13 and one addi¬ tional epitope within peptide gpl20-13. In addition, in vitro proliferative responses to peptides gpl20-12 and gpl20-13 have now been demonstrated in cultures of CD2+ T cells, and also CD4+ T cells, initiated as late as five months after the second immunization indicating the presence of memory T helper (CD4+) cell activating epitopes in that area.
Another novel area identified in this study, includes peptides gpl20-29 and gpl20-30 (amino acid coordinates 366 to 400) which induced T cell responses in 3 out of 4 monkeys. Recognition of this area
SUBSTITUTE SHEET
by immune T cells appears to be also under limited MHC restriction, or to involve epitope(s) associated with polymorphic MHC determinants. At least two epitopes would be expected within this area as the responses did not always overlap.
Importantly, among the novel T cell activating areas that have now been identified, three peptides were also found to be capable of inducing, in vivo, the production of neutralizing antibodies against HIV-l. Thus, sera obtained from all monkeys immunized with peptide gpl20-12, peptide gpl20-16 and peptide gpl20-19 inhibited in vitro HIV induced p-24 antigen release and syncytia formation by human permissive T cell lines exposed to HIV-l virions of the corresponding (BRU) isolate. Further, peptide gpl20-12 is derived from a partly conserved region of gpl20 and is associated with a site recognized by neutralizing antibodies. Peptide gpl20-16 represents a highly conserved area of gpl20 within all 14 different isolates investigated. The efficiency of the peptides, derived from a conserved region of HIV, at inducing the production of HIV- neutralizing antibodies as well as at triggering a T cell response is noteworthy. Peptides gpl20-l2 and gpl20-16 are thus the preferred embodiments of the present invention.
Less than 10% of HIV infected individuals produce antibodies capable of recognizing peptides gpl20-12, gpl20-15, gpl20-16 and gpl20-19. Since antibodies are generated in response to immunization with these pep- tides it is possible to induce an increase in the repertoire of neutralizing antibody producing B cells in HIV positive individuals.
Proteins contain a number of antigenic deter¬ minants or epitopes which are the regions of the proteins comprising the recognition and binding sites for specific antibodies. An epitope contains a
sequence of 6 to 8 amino acids. Epitopes can be either continuous wherein the sequence of 6-8 amino acids are linear or discontinuous in which case the amino acids are brought together by the three dimensional folding of the protein. Even though an epitope constitutes only a relatively few amino acids, its reactivity with an antibody may be influenced by the amino acids in the protein which surround the epitope.
Studies aimed at mapping antigenic sites or epitopes of proteins have been aided by the use of synthetic peptides corresponding to various regions of the proteins of interest. Lerner et al., in, The Biology of Immunological Disease: A Hospital Practice Book, (Dixon and Fisher, eds.) pp. 331-338 (1983); and Lerner, Adv. Immunol., 36:1 (1984). In addition to their usefulness in epitope mapping studies, synthetic peptides, if encompassing major antigenic determinants of a protein, have potential as vaccines and diagnostic reagents. Van Regenmortel, Ann. Inst. Pasteur Virol., 137E:497-528 (1986); and Van Regenmortel, Laboratory Techniques in Biochemistry and Molecular Biology, Buroden and Van Knippenburg eds. Vol. 19, Synthetic Peptides as Antigens, Elsevier ISBN 0-444-80974-0 (1988) . Synthetic peptides have several advantages with regard to specific antibody production and reactivity. The exact sequence of the synthesized peptide can be selected from the amino acid sequence of the protein as determined by amino acid sequencing of the protein or the predicted amino acid sequence determined from the
DNA sequence encoding the protein. The use of specific synthetic peptides eliminates the need for the full- length protein in vaccination and the production of or assay for antibodies. Furthermore, the solid phase peptide synthetic techniques of Merrifield and co- workers allow for essentially unlimited quantities of
the synthesized peptide of interest to be chemically produced. Erickson and Merrifield in The Proteins, 3rd Edit., Vol. 2 , Academic Press, New York, Chapter 3 (1976) . The availability of automated peptide syn- thesizers has further advanced such techniques.
Although a variety of criteria can be used to predict antigenic regions of proteins, peptides corres¬ ponding to such regions may not always be useful as vaccines. For example, antigenicity may be lost because the peptide is not in the proper spatial orientation to be recognized by antibodies which react with the protein. It has also been found that certain peptides derived from type C retroviruses and HIV act as immune-suppressive agents much as does HIV itself. Cianciolo et al., J. Immunol., 124:2900-2905 (1980); and Cianciolo et al., Proc. Natl. Acad. Sci. USA, 230:453-455 (1985) . Peptides such as these, which have a deleterious effect on the patient, would not be suitable for use as vaccines. Furthermore, as is particularly evident with HIV-l and HIV-2, there is significant genetic variability within each of these two virus groups leading to many serotypes, or isolates, of the viruses. This has put a significant constraint on choosing a region of a pro- tein from which to derive a peptide for use in formu¬ lating immunogens. However, certain immunodominant portions of HIV-l and HIV-2 proteins have been found to be relatively invariant. Synthetic peptides may also be key to viral vaccines in that they may induce an immune response against type common sequences not nor¬ mally immunogenic in the native molecule. These other¬ wise silent epitopes may be of broad protective speci¬ ficity. Stevard et al., Immunol. Today, 8:51-58 (1987) . Several experimental vaccines have been for- mulated with the aim of preventing infection in those people who are likely to be exposed to the virus.
Berman et al., "Protection of Chimpanzees from Infection by HIV-l After Vaccination With Recombinant Glycoprotein gpl20 but Not gpl60", Nature 345:622-625 (1990) . A number of neutralization epitopes on gpl20 have been found and defined by several investigators, for an overview see Bolognesi, AIDS (1989) 3(suppl 1): Slll-sllδ. In his overview Bolognesi refers to four different virus neutralization epitopes with the following amino acid coordinates: 254-274, 303-337, 458-484 and 491-523. The peptide with amino acid coordinates 254-274 was used to immunize rabbits and the resulting antiseru was found to neutralize HIV-l as described above. Ho et al., Science, 239:1021-1023 (1988) .
The peptides encompassed by the invention comprise amino acid sequences each containing at least one con¬ tinuous (linear) epitope that elicits production of activated T cells in the host in addition to eliciting the production of HIV specific antibodies.
The invention thus encompasses immunogenic pep¬ tides corresponding to regions of HIV gpl20 protein encoded by the envelope gene of HIV-l HTLV III-B described by Muesing et al., "Nucleic Acid Structure and Expression of the Human AIDS/Lymphadenopathy
Retrovirus", Nature, 313:450-458 (1985). The nucleo- tide sequence is given in Genbank Release 63 under the name HIVPV22. The invention further encompasses func¬ tionally equivalent variants of the peptides which do not significantly affect the immunogenic properties of the peptides. For instance, conservative substitution of amino acid residues, one or a few amino acid dele¬ tions or additions, and substitution of amino acid residues by amino acid analogues are within the scope of the invention.
Homologs are peptides which have conservatively substituted amino acid residues and peptides derived from corresponding regions of different HIV isolates. Amino acids which can be conservatively substituted for one another include but are not limited to: glycine/ alanine; valine/isoleucine/leucine; asparagine/ glutamine; aspartic acid/glutamic acid; serine/ threonine; lysine/arginine; and phenylalanine/tyrosine. Homologous peptides are considered to be within the scope of the invention if they are recognized by antibodies which recognize the peptides designated gpl20-12, gpl20-16 and gpl20-19 the sequences of which are shown below. Further, all homologous peptides corresponding to the peptides of the present invention but derived from different HIV isolates are also encompassed by the scope of this invention.
The invention also encompasses polymers of one or more of the peptides, and peptide analogues or homologs are within the scope of the invention. Also within the scope of this invention are peptides of fewer amino acid residues than the peptides but which encompass one or more immunogenic epitopes present in any one of the peptides and thus retain the immunogenic properties of the base peptide. The invention further encompasses functionally equivalent variants of the peptides which do not significantly affect the antigenic or T cell activating properties of the peptides. For instance, various analogues, or peptidomimetics, are known in the art and can be used to replace one or more of the amino acids in the peptides. Analogues are defined as peptides which are functionally equivalent to the peptides of the present invention but which contain certain non- naturally occurring or modified amino acid residues. Additionally, polymers of one or more of the peptides are within the scope of the invention.
The use of peptide analogues can result in pep¬ tides with increased activity, that are less sensitive to enzymatic degradation, and which are more selective. A suitable proline analogue is 2-aminocyclopentane carboxylic acid (j8Ac5c) which has been shown to increase activity of a native peptide more than 20 times. Mierke et al., "Morphiceptin Analogs Containing 2- aminocyclopentane Carboxylic Acid as a Peptidomimetic for Proline", Int. J. Peptide Protein Res., 35:35-45 (1990). See also Portoghese et al., "Design of
Peptidomimetic S Opioid Receptor Antagonists Using the Message-Address Concept", J. Med. Chem., 33:1714-1720 (1990); and Goodman et al., "Peptidomimetics: Synthesis, Spectroscopy, and Computer Simulations", Biopolymers, 26:S25-S32 (1987).
The peptides were synthesized by known solid phase peptide synthesis techniques. Merrifield and Barany, The Peptides: Analysis, Synthesis, Biology, Vol. 1, Gross and Meinenhofer, eds., Academic Press, New York, Chap. 1 (1980) . The synthesis also allows for one or more amino acids not corresponding to the original protein sequence to be added to the amino or carboxyl terminus of the peptide. Such extra amino acids are useful for coupling the peptides to another peptide, to a large carrier protein or to a solid support. Amino acids that are useful for these purposes include but are not limited to tyrosine, lysine, glutamic acid, aspartic acid, cysteine and derivatives thereof. Additional protein modification techniques may be used, e.g., NH2-acetylation or COOH-terminal amidation, to provide additional means for coupling the peptides to another protein or peptide molecule or a support. Procedures for coupling peptides to each other, carrier proteins and solid supports are well known in the art. Peptides containing the above-mentioned extra amino acid residues either carboxy or amino terminally,
uncoupled or coupled to a carrier or solid support are consequently within the scope of the invention. Refer¬ ence to the peptides of the present invention encom¬ passes all of the embodiments discussed herein. An alternative method of vaccine production is to use molecular biology techniques to produce a fusion protein containing one or more of the peptides of the present invention and a highly immunogenic protein. For instance, fusion proteins containing the antigen of interest and the B subunit of cholera toxin have been shown to induce an immune response to the antigen of interest. Sanchez et al., "Recombinant System For Overexpression of Cholera Toxin B Subunit in Vibrio cholerae as a Basis for Vaccine Development", Proc. Natl. Acad. Sci. USA, 86:481-485 (1989). It is thus implicit in the present invention that vaccine con¬ structs based on appropriate constructions of B and T cell epitopes fused to a carrier protein like cholera toxin would represent important benefits in vaccination.
The novel peptide amino acid sequences are set forth below and in Table 2. The amino acid residues are derived from the nucleotide sequence previously described by Kennedy et al. (1986) . The peptides may contain either an amido or carboxy group at their carboxy termini.
gpl20-ll
X-Ser-Ser-Ser-Gly-Arg-Met-Ile-Met-Glu-Lys-Gly-Glu-Ile-
Lys-Asn-Cys-Ser-Phe-Asn-Ile-Ser-Thr-Ser-Y-Z
gpl20-12
X-Gly-Glu-Ile-Lys-Asn-CysτSer-Phe-Asn-Ile-Ser-Thr- Ser-Ile-Arg-Gly-Lys-Val-Gln-Lys-Glu-Tyr-Ala-Phe-Phe-Y-Z
gpl20-13
X-Ile-Arg-Gly-Lys-Val-Gln-Lys-Glu-Tyr-Ala-Phe-Phe-Tyr- Lys-Leu-Asp-Ile-Ile-Pro-Ile-Asp-Asn-Asp-Thr-Thr-Ser- Tyr-Thr-Y-Z
gpl20-16
X-Pro-Lys-Val-Ser-Phe-Glu-Pro-Ile-Pro-Ile-His-Tyr-Cys- Ala-Pro-Ala-Gly-Phe-Ala-Ile-Leu-Lys-Cys-Asn-Asn-Y-Z
gpl20-19
X-Thr-His-Gly-Ile-Arg-Pro-Val-Val-Ser-Thr-Gln-Leu- Leu-Leu-Asn-Gly-Ser-Leu-Ala-Glu-Glu-Glu-Y-Z
gpl20-29
X-Gly-Asp-Pro-Glu-Ile-Val-Thr-His-Ser-Phe-Asn-Cys-Gly-
Gly-Glu-Phe-Phe-Tyr-Cys-Asn-Ser-Thr-Gln-Y-Z
gpl20-30 X-Cys-Gly-Gly-Glu-Phe-Phe-Tyr-Cys-Asn-Ser-Thr-Gln-Leu- Phe-Asn-Ser-Thr-Trp-Phe-Asn-Ser-Thr-Trp-Y-Z
wherein X is either a hydrogen atom of the amino terminal NH2 group of the peptide or an additional amino acid being selected to facilitate coupling of the peptide to a carrier; Y is absent or Cys; and Z is the carboxyl group of the carboxy terminal amino acid or an amido group. The amino acid abbreviations used are defined in Table 2.
In addition to eliciting T cell activation, several of the peptides are useful as vaccines to protect against future infection by HIV or to heighten the immune response to HIV in subjects already infected by HIV. Although any human subject could be vaccinated with the peptides, the most suitable subjects are people at risk for HIV infection. Such subjects include but are not limited to homosexuals,
prostitutes, intravenous drug users, hemophiliacs and those in the medical professions who have contact with patients or biological samples. The invention also provides monoclonal and polyclonal antibodies which specifically recognize the peptides. The invention further provides antibodies produced in response to vaccination with the peptides which neutralize HIV.
In the preferred embodiment of the present inven¬ tion, the peptides are formulated into compositions for use as immunogens. These immunogens can be used as vaccines in mammals including humans or to elicit T cell activation and/or production of polyclonal and monoclonal antibodies in animals. For formulation of such compositions, an amount sufficient to elicit T cell activation of at least one of the peptides (about 1-500 μg) is admixed with a physiologically acceptable carrier suitable for administration to mammals including humans.
The peptides may be covalently attached to each other, to other peptides, to a protein carrier or to other carriers, incorporated into liposomes or other such vesicles, and/or mixed with an adjuvant or adsorbent as is known in the vaccine art. For instance, the peptide or peptides can be mixed with immunostimulating complexes as described by Takahashi et al., "Induction of CD8+ Cytotoxic T Cells by Immunization With Purified HIV-l Envelope Protein and ISCOMS", Nature, 344:873-875 (1990). Alternatively, the peptides are uncoupled and merely admixed with a physiologically acceptable carrier such as normal saline or a buffering compound suitable for admin¬ istration to mammals including humans.
As with all immunogenic compositions for eliciting antibodies, the immunogenically effective amounts of the peptides of the invention must be determined empirically. Factors to be considered include the
immunogenicity of the native peptide, whether or not the peptide will be complexed with or covalently attached to an adjuvant or carrier protein or other carrier and route of administration for the compo- sition, i.e. intravenous, intramuscular, subcutaneous, etc., and the number of immunizing doses to be admin¬ istered. Such factors are known in the vaccine art and it is well within the skill of immunologists to make such determinations without undue experimentation. The invention is further illustrated by the following specific examples which are not intended in any way to limit the scope of the invention. In order to determine T cell activation, PBMC from monkeys immunized with OVA-conjugated HIV gpl20 peptides were tested for their ability to produce IL-2 and/or to proliferate when exposed jLn vitro to recall (immuniz¬ ing) , overlapping, and non overlapping peptide(s).
Example 1 Animals Used in Subsequent Examples Cynomolgus monkeys (Macaca fascicularis. were given 3 intramuscular doses of ovalbumin (OVA)- conjugated peptides (see below) , three weeks apart, each dose consisting of 100 μg of ovalbumin-coupled peptide emulsified in Freund's complete (first dose) or incomplete (booster doses) adjuvant.
Example 2 Peptide Synthesis 40 HIV-l gpl20 peptides (Table 1) , with an addi¬ tional carboxy-terminal cysteine residue, were syn- thesized on solid phase with an Applied Biosystems 430A peptide synthesizer (Applied Biosystems, Foster City, CA, USA) using the polymer p-methylbenzhydryl amine resin as solid phase (Peptides Int., Louisville, USA).
All amino acids for use in synthesis contained t-butylcarbonyl groups (t-Boc) protecting the α-NH2 group and were obtained from Novabiochem AG, Switzerland. Amino acids with reactive side chain groups contained additional protective groups to prevent unwanted and undesirable side chain reactions. The individual protected amino acids used in synthe¬ sizing all of the peptides are set forth in Table 1.
TABLE 1 AMINO ACIDS USED IN PEPTIDES SYNTHESIS
Boc-Ala-OH
Boc-Arg (Tos)-OH
Boc-Asn-OH
Boc-Asp (Obzl)-OH Boc-Cys (Pmeobzl)-Oh
Boc-Glu (Obzl)-OH
Boc-Gln-OH
(cryst.)
Boc-Phe-OH Boc-Pro-OH
Boc-Ser (Bzl)-OHΛDCHA
Tos: Tosyl or p-Toluene sulfonic acid Obzl = Benzyloxy Pmeobzl = p-Methylbenzyloxy 2-CL-Z = Carbobenzoxy chloride 2-Br-Z = Carbobenzoxy bromide
The peptides were synthesized using the t-Boc synthesis protocol as suggested by the manufacturer. All solvents were from Applied Biosystems and the side chain protected amino acids used were from Nova Biochem
(Switzerland) and Applied Biosystems. Following each amino acid coupling, a sample was taken and a quanti¬ tative ninhydrin assay was performed. Only if the coupling efficiency exceeded 99% for each amino acid coupled was the peptide accepted for further processing. Completed peptides were cleaved from the solid phase and amino acid side chains were deprotected by acidic hydrolysis using anisole and ethanedithiol (Merck, Germany) as scavengers. After completion of a particular synthesis, the protecting groups were removed from the synthesized peptide and the peptide was cleaved from the solid support resin by treatment with trifluoromethane sulfonic acid (TFMSA) according to the method des- cribed by Bergot et al., "Utility of Trifluoromethane Sulfonic Acid as a Cleavage Reagent in Solid Phase Peptide Synthesis", Applied Biosystems User Bulletin, Peptide Synthesizer, Issue No. 16, Sept. 2, 1986. The following is the detailed protocol used. 1. For 1 gram peptide-resin, 3 ml Thio-Anisol
1,2-Ethane-Dithiol (2:1) was added as scavenging agent and the mixture was incubated with continuous stirring for 10 min. at room temperature.
2. Trifluoracetic Acid (TFA) , 10 ml, was added and stirred continuously for 10 min. at room temperature.
3. TFMSA, 1 ml, was added dropwise with forceful stirring and reacted for 25 min. at room temperature.
4. Following cleavage, the peptides were pre- cipitated with and washed with anhydrous ether.
5. The precipitated and washed peptides were dissolved in a small volume of TFA.
6. The dissolved peptides were again precipitated and washed as above in step 4 and the precipitate was dried under a stream of N2.
Prior to use in specific assays, the peptides can be further purified, if desired, by reverse phase high performance liquid chromatography (HPLC) . A particu¬ larly suitable column for such purification is the reverse-phase Vydak® C-18 column using a water (TFA) - acetonitrile (TFA) gradient to elute the peptides. Forty peptides were synthesized having the amino acid sequences shown in Table 2.
The amino acid sequences of the peptides, 17-29 amino acids long, half overlapping each other and entirely encompassing gp-120, were obtained from the HIV-l BRU isolate. Muesing et al., "Nucleic Acid Structure and Expression of the Human AIDS/Lym- phadenopathy Retrovirus", Nature, 313:450 (1985).
** As previously described by Kennedy et al. (1986)
Example 3 Preparation of Peptides for Immunization Peptides according to the present invention were covalently coupled to ovalbumin grade V (Sigma, St. Louis, MO, USA) at an approximate 10:1 (peptide: ovalbumin) molar ratio using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) , (Pharmacia, Uppsala, Sweden) as bifunctional linker according to the manufacturer's instructions (Pharmacia) i.e. briefly as follows:
Ovalbumin was dissolved in coupling buffer (0.2 M NaH2P04, Ph 8.5) . The dissolved ovalbumin was then run through a Sephadex G-25M column (Pharmacia, Sweden) , using the same buffer. Protein concentration was measured at 280 nm and the recovery was determined. SPDP was dissolved in 99.5% ethanol to a final concentration of 40 mM. SPDP was then added dropwise to the ovalbumin solution under stirring. The SPDP-ovalbumin mixture was then left at room temperature for approximately 30 minutes. The ovalbumin- SPDP conjugate was separated from unconjugated SPDP by running the mixture through a Sephadex G-25M column, using water as eluent. The degree of substitution for the ovalbumin-SPDP conjugate was determined after diluting 50 μl conjugate in 2 ml of water, by measuring the diluted conjugate at 280 nm and the diluted conjugate plus 100 μl Dithiothreitol (DTT) (Sigma) at 343 nm, in order to determine the amount to be added to the peptide solution. Finally, the synthetic peptide to be coupled to the ovalbumin-SPDP conjugate was dissolved in 10% acetic acid to a final concentration of 1 mg/ml and a suitable amount of ovalbumin-SPDP conjugate (as determined by the substitution degree above) was added and allowed to stand overnight at room temperature.
Example 4 Immunization Protocols M. fascicularis were used to generate antibodies. Prior to the initial peptide injection, a blood sample was drawn from the monkeys. This initial blood sample is termed "pre-immune" (Tables 5-8) and is used as an internal control and analyzed simultaneously with respective immuneserum.
The monkeys were injected with 100 μg peptide- SPDP-ovalbumin suspended in 0.5 ml phosphate buffered saline (PBS) . The monkeys were immunized intramuscularly three times, three weeks apart. As adjuvant, 0.5 ml of Freund's complete adjuvant was used for all initial immunizations and Freund's incomplete adjuvant was used for booster shots. Two weeks after the final immunization the monkeys were bled by removing a 10 ml blood sample from the fossa and pre-immune and hyperimmune sera were subject to neutralization assays as described in Example 9.
Example 5
Isolation and Fractionation of Lymphocytes from Immunized M. fascicularis
Heparinized venous blood was collected from the femoral vein, at least two weeks after the second and/or the third injections. Peripheral blood mononuclear cells (PBMC) were obtained by gelatin sedimentation followed by density gradient centrifugation by the following method. A solution of 3% (weight/volume) gelatin (gelatin L936, PB Gelatins UK LTD, GB) in Hank's Balanced Salt Solution was mixed with the blood at a 1:3 ratio and erythrocytes were allowed to sediment for one hour at 37°C. The erythrocyte-free supernatant was layered onto a Ficoll-Hypaque cushion (Pharmacia, Sweden) and centrifuged for 15 minutes at 930 x g, at 20°C.
Interface PBMC were washed twice by centrifugation (500 xg, 20°C, 5 min) with isotonic phosphate-buffered saline (PBS, 0.01 M phosphate buffer in 0.15 M NaCl, pH 7.4) . In some Examples represented below, T cells were enriched by rosetting with AET-treated sheep red blood cells as described by Kaplan and Clare, "Improved Rosetting Assay for Detection of Human T Lymphocytes", J. Immunol. Met., 6:131 (1974) followed by density centrifugation on Ficoll-Hypaque. The rosetted cells (nominal T cells) were collected from the pellets and resuspended for 20 seconds with distilled water to lyse sheep red blood cells. Further enrichment into CD4+ T cells was obtained by paramagnetic depletion of CD8+ T cells using icrospheres coated with monoclonal anti-CD8 antibodies (Dynal AS, Norway) , according to the manufacturer's instructions.
Example 6 Lymphocyte Proliferation Assays Unfractionated PBMC were resuspended in complete medium (see below) and dispersed in round-bottomed 96 micro-well plates (Nunc, Denmark) at three different cell densities (2xl05, lxlO5 and 5xl04 cells per well) in Iscove's medium supplemented with 10% fetal calf serum (FCS, Biological Industries, Israel) , 3 μg/ml
L-Glutamine (Gibco, UK) and 0.1 mg/ml Gentamycinsulfate (Essex Lakemedel AB, Sweden) . Fractionated T cells (2xl05 or 1.2xl05 nominal T cells, or 4xl04 CD4+ T cells) were dispersed in separate sets of wells together with 4xl04 or 2xl04 autologous T cell-depleted irradiated (2500 rad) , PBMC as a source of accessory cells. Synthetic peptides were dissolved in dimethylsulfoxide (20 μg/ml) and further diluted in culture medium. Uncoupled peptides were added at different
concentrations (10, 1 and 0.1 μg/ml) to the culture wells. Concanavalin A (Sigma) (10 μg/ml) was added to separa- te cultures as a positive control. Cells, in a final volume of 0.2 ml, were incubated for five days at 37°C in a humid atmosphere with 7.5% C02. After four days, 25 μl of culture supernatant were collected from each well and frozen at -70°C until assayed for IL-2 activity according to the method described in example 5. 16 hours prior to the comple¬ tion of the culture period, 20 μl culture medium containing 1 μCi of [3H]thymidine (Amersham, UK) were added to each well. The harvesting and subsequent measurement of incorpo¬ rated radioactivity was performed on an automated filter cell harvester coupled to an argon activated β-scintillation counter (Inotech, Switzerland). Data were expressed as arithmetic mean stimulating indexes (SI), the latter SI being defined as the mean ratio of [3H]-thymidine incorpora¬ ted in peptide stimulated cultures (mean from three cultures divided by corresponding triplicate of control cultures (unstimulated). A mean SI of at least 2.4 is considered positive. SI values equal to at least 2.4 [i.e. twice the sum of the mean plus 3.3 times the SD of the replicate cultures exposed to irrelevant peptides (confidence inter- val, p < 0.001, Student's + test)] were considered as signi¬ ficantly increased.
As seen i Figure 1, a substantial number (18/40) of peptides that were injected into monkeys in an OVA-substitu¬ ted form, induced in vitro proliferation of PBMC from corre- sponding immune animals. In Figure 1, results are expressed as mean SI + SD of all triplicates tested % SD (if tested on two monkeys). Black bars indicate a positive result. The frequency of responding monkey(s) is indicated. Four major areas corresponding to the additive sequence of 2 to 3 overlapping peptides were found to accommodate this activi¬ ty. Peptides gpl20-ll, gpl20-12 and gpl20-13
(amino acid ccordinates 141-192) correspond to one such area. Five out of six monkeys immunized with one of these three peptides responded to recall peptide. Another major area comprises peptides gpl20-23, gpl20-24 and gpl20-25 (amino acid coordinates 295-343) which induced proliferative responses of PBMC from at least one out of 2 monkeys immuni- zed with the corresponding peptide. A third area, comprising peptides gpl20-29 and gpl20-30, accommodates a site(2) of proliferation inducing activity on PBMC from monkeys immuni¬ zed with the corresponding OVA-conjugated paptides. The fourth area consists of peptides gpl20-33, gpl20-34, gpl20- 35 and gpl20-36 (amino acid coordinates 409-466) where each peptide could induce profilerationof PBMC from at least one of the immunized monkeys.
Apart from these major areas, five peptides, i.e. peptides gpl20-4 (amino acid coordinates 53-74), gp 120-5 (amino acid coordinates 64-89), gpl20-17 (amino acid coordi¬ nates 218-247) and gpl20-21 (amino acid coordinates 269-295), were shown to induce in vitro profilerative responses when added to PBMC from monkeys immunized with the corresponding OVA-con ugated peptide. Peptides found to be capable of inducing a prolifera¬ tive response of PBMC from monkeys immunized with the corre¬ sponding OVA-coupled peptide were reassayed on PBMC from at least three other monkeys immunized with a non-cognate OVA- coupled peptide. Peptides gpl20-4, gpl20-13 and gpl20-34 induced proliferation of PBMC from 1 out of 3 monkeys and peptide gpl20-30 in 1 monkey out of 7 (SI ranging between 2.0 and 2.5) while the other peptides failed to induce any significant proliferative responses.
Peptides capable of inducing a proliferative response in one or two immunized monkeys were retested after the third immunization. On this occastion, the in vitro pro¬ liferative responses of PBMC from immune
SUBSTITUTESHEET
monkeys to each of 2 peptides containing a sequence half overlapping with the immunizing peptide were also evaluated. As seen in Figure 2, PBMC from both monkeys immunized with OVA-conjugated peptide gpl20-ll responded also in vitro to peptide gpl20-12, but none of the peptide gpl20-12-immu- nized monkeys responded to peptide gpl20-ll. Cells were obtained two weeks after the third immunization. The pepti¬ des tested were selected on the basis of in vitro responsi¬ veness to the immunizing peptide after two immunizations. In Figure 2, results are expressed as mean SI of all triplica¬ tes tested % SD (if tested on two monkeys). Black bars indicate a positive result.
Similarly, monkeys immunized with peptide gpl20-12 responded to peptide gpl20-13 but none of the peptide gpl20- 13 immunized monkeys responded to peptide gpl20-12. In the next area of in vitro profilerative activity, i.e. peptides gpl20-23, gpl20-24 and gpl20-25, none of the overlapping peptides induced in vitro profileration of PBMC from any of the monkeys immunized with OVA-conjugated peptides. The same holds true for PBMC from monkeys immunized with peptides gpl20-29 and gpl20-30 (OVA-conjugated) as no response to overlapping peptides is achieved. In the area consisting of peptides gpl20-33, gpl20-34, and gpl20-35, none of the overlapping peptides induced in vitro proliferation of PBMC from any of the monkeys immunized with OVA-conjugated pepti¬ des. PBMC from the other monkeys were also negative in this respect. Of the other epitopes identified, only PBMC from the peptide gpl20-4 immunized monkey responded in vitro to an overlapping peptide, i.e. to peptide gpl20-5.
The profilerative responses of different cell frac¬ tions from two monkeys (immunized with peptide gpl20-12 or peptide gpl20-13) were examined. As seen
in Table 3, enrichment of CD2+ T cells increased the proliferative response of PBMC obtained from both monkeys when cultured in the presence of immunized (but uncoupled) peptide. Also, existing responses to overlapping peptides remained relatively constant. After a further depletion of CD8+ T cells, the CD4+ T cell enriched fractions (containing 9 to 18% of the original CD2+ T cell fractions) still proliferated in response to incubation with immunized peptide. However, the CD4+ T cell enriched fraction from the peptide gpl20-12 immunized monkey did not proliferate in response to any of the overlapping peptides.
In Table 3, the various columns were obtained as follows. Immunized peptide: 100 μg of OVA-conjugated peptide was immunized at three occasions in Freund's
complete (1st dose) or incomplete (boosting doses) adjuvant;
In vitro peptide: unconjugated peptide;
Total PBMC: mean SI of four triplicates of different cell densities and peptide concentrations;
CD2+ enriched fraction: 2xl05 SRBC-rosetted PBMC incubated with 4xl04 irradiated, non-rosetted cells together with lOμg/ml of peptide(s) :
CD4+ enriched fraction: 1.25xl05 (peptide gpl20-12 immunized monkey) or 4xl04 (peptide gpl20-13 immunized monkey) SRBC-rosetted PBMC further enriched in CD4+ T cells by incubating with anti-CD8+ coated beads were incubated with 2xl04 irradiated, non-rosetted cells together with 10 μg/ml of peptide(s) .
Example 7
IL-2 assay The IL-2 content of individual cell microcultures was determined by the bioassay performed as described by Gillis et al., "T Cell Growth Factor: Parameters of Production and a Quantitative Microassay for Activity", J. Immunol., 120:2027 (1978). Briefly, supernatants were added at a final dilution of 1:4 to 104 CTLL-2 cells. Cells were incubated for 24 hours at 37°C in flat-bottomed 96 microwell plates (Nunc, Denmark) in Iscove's medium supplemented with 10% FCS, 3 μg/ml
L-Glutamine, 0.1 mg/ml Gentamycinsulfate and 5xl0"5 M S-Mercaptoethanol. Six hours prior to completion of the culture period, 1 μCi of [3H]-thymidine was added. The cells were harvested and [3H]-thymidine incorporation was determined as described in Example 6. IL-2 content in the supernatants was determined by extrapolation from a standard dose-response curve gen¬ erated by culturing CTLL-2 cells in the presence of
known amounts of recombinant human IL-2 (Genzyme, Boston, MA) .
As seen in Table 4, several cell culture super¬ natants contained detectable amounts of IL-2 in cultures of PBMC from monkeys immunized with
OVA-conjugated peptides gpl20-ll, gpl20-12, gpl20-13, gpl20-16, gpl20-21, gpl20-25, gpl20-30 and gpl20-34, secreted IL-2 could be detected after in vitro challenge with the corresponding, unconjugated, peptide. The ratio of secreted IL-2 found after 4 days of in vitro culturing ranged from 0.2 to l.0 U/ml. Cell culture supernatants of PBMC derived from monkeys immunized with peptides gpl20-ll, gpl20-12, gpl20-13, gpl20-30 and gpl20-34 also contained IL-2 after m vitro exposure to one or two of the overlapping peptides. Accordingly, PBMC from a monkey immunized with peptide gpl20-ll secreted detectable levels of IL-2 in the cell supernatant after 4 days of stimulation with peptide gpl20-12, and a monkey immunized with peptide gpl20-12 secreted detectable levels of IL-2 after stimulation with peptide gpl20-13. Cell culture supernatants containing IL-2 were identified from both PBMC cultures containing overlapping peptides (peptides gpl20-12 and gpl20-14) together with PBMC from a peptide gpl20-13 immunized monkey and the same holds true for peptides gpl20-33 and gpl20-35 when co-cultured with PBMC from a peptide gpl20-34 immunized monkey. Finally, PBMC obtained from a peptide gpl20-30 immunized monkey secreted detectable amounts of IL-2 not only when cultured in the presence of peptide gpl20-30, but also when peptide gpl20-29 had been added to the cultures.
Example 8 Cells and Virus Stocks All neutralization tests were performed using H-9 cells and HTLV-lllB virus (originating from R.C. Gallo and supplied by Dr. William Hall, North Shore Hospital, Manhasset, New York) . H-9 cells (designated H9 NY) were maintained in RPMI Medium (Gibco) supplemented with 20% fetal calf serum (FCS) , penicillin/streptomycin (PEN/STREP 50 μg/ml each and without any fungicides) . Cells were subcultured at a dilution of 1:3 every 4 days.
Cells were scraped from the plates and pelleted by centrifugation at 325 x g. Pelleted cells were resuspended in 1 ml of stock virus previously diluted 1/10 and allowed to adsorb for 60 min at 37°C with frequent stirring. After adsorption of the virus, the cells were recentrifuged and resuspended in 10 ml of RPMI with 20% FCS and polybrene (2 μg/ml) (giving a final concentration of 5x10s cells/ml) and incubated at 37°C in 5% C02.
Infected cells were shown to be detectable at 4-5 days post-infection (p.i.) by monitoring syncytia formation, positive cells in immunofluorescence and p-24 production (assayed by the Abbott p-24 antigen test) . The peak of HIV production was seen 10 - 15 days p.i. at which time virus was collected. After low speed centrifugation to remove debris, supernatants containing virus collected from infected cells were frozen in stocks at -90°C. One virus stock with endpoint titer of 40,000 50% tissue culture infective doses (TCID50) was used throughout the studies (referred to as NT3-NT19) .
Example 9 HIV-l Neutralization Assay Sera containing antibodies that neutralize HTLV 111-B infectivity were detected by their ability to prevent HIV-l syncytium formation, p-24 antigen production and decreased number of infected cells as determined by immunofluorescence markers, compared to control infections lacking peptide specific antisera. Stock virus, described in Example 8 was diluted to 100 TCID^ and mixed with serial fourfold dilutions (1/5, 1/20, and 1/80) of complement-inactivated immunesera obtained from the monkeys immunized as described in Example 4. As a positive control, a guinea pig hyperimmune serum (referred to as MSV) with known HIV neutralizing titer of 1/40 - 1/160 was included in all experiments (kindly provided by Prof. B. Morein, Dept. Veterinary Virology, BMC, Uppsala, Sweden) . After incubation for 60 min at 37°C or 16 hours at 4°C, the serum-virus mixture was added to lxlO6 H-9 cells and incubated for another 60 min at 37°C. Following incubation, the cells were washed once and placed in 24 well multidish plates with 2 ml of growth medium (RPMI, 10% FCS, 2 μg polybrene/ml) per well.
Cells were examined under the microscope (magni- fication x200) for the presence of syncytia on days 5- 12 p.i. Supernatants from infected cells were assayed for the presence of p-24 antigen according to the manufacturer's instructions (Abbott ag test HIVAG-1®, Enzyme Immunoassay for the Detection of Human Immunodeficiency Virus Type I (HIV-l) Antigen(s) in Human Serum or Plasma) in tenfold serial dilutions (1/10 - 1/1,000) at 10 days p.i. The results are presented as absorbance values at 454 nm with higher absorbance values indicating higher P-24 antigen concentration and hence HIV infection. Serial dilutions of the supernatants were made so as to detect
p-24 concentrations in the most accurate range (< 2.0 absorbance units) .
The number of infected cells were determined at the end of experiment (usually on day 15 p.i.) by acetone- fixation of cells on slides adopted for immunofluorescence (IF) . An indirect IF test was used according to standard procedures described in Jeansson et al., "Elimination of Mycoplasmas from Cell Cultures Utilizing Hyperimmune Sera", Ex. Cell Res., 161:181-188 (1985) , with 1/400 dilution hyperimmune sera from HIV-infected individuals and a fluorescein isothiocyanate (FITC) labeled antihuman IgG antibody (Bio-Merieux France) diluted 1/100. Tables 5-8 show the results obtained from screening of hyperimmune sera from monkeys immunized with peptides 1-40.
In Tables 5(A-D)-8 the p24 antigen content of the supernatants was analyzed by ELISA, indirect IF and syncytia formation as described above. The relative amount of antigen positive cells is depicted as AG POS cells wherein the percentages are represented by:
- = 0%, + = >0-2%, ++ = 3-10% and +++ = 11-20% where the percentage interval indicates the number of antigen positive cells.
Table 5A (HIVNT3P1.XLS) depicts the results obtained with sera derived from monkeys immunized with peptides gpl20-l - gpl20-10. The cells used were H9 NY and the virus used was HTLV-IIIB, Batch 18 described in Example 8. The incubation protocol was (virus plus serum) incubation at 37°C for one hour. Table 5B (HIVNT4P1.XLS) depicts the results obtained with sera derived from monkeys immunized with peptides gpl20-ll - gpl20-20. The cells used were H9 NY and the virus used was HTLV-IIIB, Batch 18 described in Example 8. The incubation protocol was (virus plus serum) incubation at 37°C for one hour.
Table 5C (HIVNT5P1.XLS) depicts the results obtained with sera derived from monkeys immunized with peptides gpl20-21 - gpl20-30. The cells used were H9 NY, and the virus used was HTLV-IIIB, Batch 18 described in Example 8. The incubation protocol used was virus plus serum incubated at 37°C for one hour.
Table 5D (HIVNT6P1.XLS) depicts the results obtained with sera derived from monkeys immunized with peptides gpl20-31 - gpl20-40. The cells used were H9 NY and the virus used was HTLV-IIIB, Batch 18 described in Example 8. The incubation protocol was (virus plus serum) incubation at 37°C for one hour.
Table 6 (HIVTAB4.XLS) shows the results of the first retest of putative neutralizing antibodies as determined by the first test (Tables 5A-D) . In each test the virus used was HTLV-IIIB, Batch 18 and the cells used were H9 NY. The first retest results in rows 1-19 are the results of neutralization test number 5. The incubation protocol was incubation at 37°C for one hour. The first retest results in rows 20-32 are the results of neutralization test number 7. The incubation protocol was incubation of at 37°C for one hour.
Table 7 (HIVTAB5.XLS) shows second, third and fourth retest results of the positive peptides. In each test the virus used was HTLV-IIIB Batch 18 and the cells used were H9 NY. The second retest results in rows 1-4 are the results of neutralization test number 7. The incubation protocol was incubation at 37°C for one hour. The second retest results in rows 5-13 are the results of neutralization test number 12. The third retest results are shown rows 14-16 are the results of neutralization test number 12. The incubation protocol was incubation at 37°C for one hour. The fourth retest results in rows 17-39 are the results of neutralization test number 16. The
incubation protocol was at 4°C for 16 hours. The second retest results in rows 40-53 are the result of neutralization test 19. The incubation protocol was cells plus virus at 4° for 16 hours. Table 8 (HIVKOMBP.XLS) shows the neutralization assay results with combined hyperimmune sera. Note that the incubation of virus and cells was at 4°C for 16 hours.
The results depicted in Tables 5(A-D)-8 indicate that peptides gpl20-12, gpl20-16, and gpl20-l9 elicit the production of HIV neutralizing antibodies in primate subjects. The use of the peptides in vaccination of human subjects is therefore applicable to prevent infection by HIV or to induce heightened immune response in subjects already infected by HIV.
Claims (1)
- Claimsl. A composition comprising (a) an amount of a peptide sufficient to elicit T cell activiation, the peptide having at least one epitope recognized by T cells said epitope being selected from the amino acid sequence: X-Ser-Ser-Ser-Gly-Arg-Met-Ile-Met-Glu-Lys- Gly-Glu-Ile-Lys-Asn-Cys-Ser-Phe-Asn-Ile-Ser- Thr-Ser-Y, analogues and homologs of said sequence and subfragments of said sequence which include an epitope recognized by T cells wherein X is either a hydrogen atom of the amino terminal NH2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group; and (b) a physiologically acceptable carrier therefor.2. A composition comprising (a) an amount of a peptide sufficient to elicit T cell activiation, the peptide having at least one epitope recognized by T cells said epitope being selected from the amino acid sequence: X-Gly-Glu-Ile-Lys-Asn-Cys-Ser-Phe-Asn-Ile- Ser-Thr-Ser-Ile-Arg-Gly-Lys-Val-Gln-Lys-Glu- Tyr-Ala-Phe-Phe-Y, analogues and homologs of said sequence and subfragments of said sequence which include an epitope recognized by T cells wherein X is either a hydrogen atom of the amino terminal NH2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group; and (b) a physiologically acceptable carrier therefor.composition comprising (a) an amount of a peptide sufficient to elicit T cell activiation, the peptide having at least one epitope recognized by T cells said epitope being selected from the amino acid sequence: X-Ile-Arg-Gly-Lys-Val-Gln-Lys-Glu-Tyr-Ala- Phe-Phe-Tyr-Lys-Leu-Asp-Ile-Ile-Pro-Ile-Asp- Asn-Asp-Thr-Thr-Ser-Tyr-Thr-Y, analogues and homologs of said sequence and subfragments of said sequence which include an epitope recognized by T cells wherein X is either a hydrogen atom of the amino terminal NH2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group; and (b) a physiologically acceptable carrier therefor. 4. A composition comprising (a) an amount of a peptide sufficient to elicit T cell activiation, the peptide having at least one epitope recognized by T cells said epitope being selected from the amino acid sequence: X-Pro-Lys-Val-Ser-Phe-Glu-Pro-Ile-Pro-Ile- His-Tyr-Cys-Ala-Pro-Ala-Gly-Phe-Ala-Ile-Leu- Lys-Cys-Asn-Asn-Y, analogues and homologs of said sequence and subfragments of said sequence which include an epitope recognised by T cells wherein X is either a hydrogen atom of the amino terminal NH2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group; and (b) a physiologically acceptable carrier therefor.]5. A composition comprising (a) an amount of a peptide sufficient to elicit T cell activiation, the peptide having at least one epitope recognized by T cells said epitope being selected from the amino acid sequence: X-Thr-His-Gly-Ile-Arg-Pro-Val-Val-Ser-Thr- Gln-Leu-Leu-Leu-Asn-Gly-Ser-Leu-Ala-Glu-Glu- Glu-Y, analogues and homologs of said sequence and subfragments of said sequence which include ari epitope recognized by T cells wherein X is either a hydrogen atom of the amino terminal NH2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group; and (b) a physiologically acceptable carrier therefor.6. A composition comprising (a) an amount of a peptide sufficient to elicit T cell activiation, the peptide having at least one epitope recognized by T cells said epitope being selected from the amino acid sequence: X-Gly-Asp-Pro-Glu-Ile-Val-Thr-His-Ser-Phe- Asn-Cys-Gly-Gly-Glu-Phe-Phe-Tyr-Cys-Asn-Ser- Thr-Gln-Y, analogues and homologs of said sequence and subfragments of said sequence which include an epitope recognized by T cells wherein X is either a hydrogen atom of the amino terminal NH2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group; and (b) a physiologically acceptable carrier therefor.7. A composition comprising (a) an amount of a peptide sufficient to elicit T cell activiation, the peptide having at least one epitope recognized by T cells said epitope being selected from the amino acid sequence: X-Cys-Gly-Gly-Glu-Phe-Phe-Tyr-Cys-Asn-Ser- Thr-Gln-Leu-Phe-Asn-Ser-Thr-Trp-Phe-Asn-Ser- Thr-Trp-Y, analogues and homologs of said sequence and subfragments of said sequence which include an epitope recognized by T cells wherein X is either a hydrogen atom of the amino terminal NH2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group; and (b) a physiologically acceptable carrier therefor.composition comprising (a) an amount of at least two peptides sufficient to elicit T cell activiation, wherein each peptide has at least one epitope recognized by T cells said epitope being selected from the amino acid sequences: X-Ser-Ser-Ser-Gly-Arg-Met-Ile-Met-Glu-Lys- Gly-Glu-Ile-Lys-Asn-Cys-Ser-Phe-Asn-Ile-Ser- Thr-Ser-Y;X-Gly-Glu-Ile-Lys-Asn-Cys-Ser-Phe-Asn-Ile- Ser-Thr-Ser-Ile-Arg-Gly-Lys-Val-Gln-Lys-Glu- Tyr-Ala-Phe-Phe-Y; X-Ile-Arg-Gly-Lys-Val-Gln-Lys-Glu-Tyr-Ala- Phe-Phe-Tyr-Lys-Leu-Asp-Ile-Ile-Pro-Ile-Asp- Asn-Asp-Thr-Thr-Ser-Tyr-Thr-Y;X-Pro-Lys-Val-Ser-Phe-Glu-Pro-Ile-Pro-Ile- His-Tyr-Cys-Ala-Pro-Ala-Gly-Phe-Ala-Ile-Leu- Lys-Cys-Asn-Asn-Y;X-Thr-His-Gly-Ile-Arg-Pro-Val-Val-Ser-Thr- Gln-Leu-Leu-Leu-Asn-Gly-Ser-Leu-Ala-Glu-Glu- Glu-Y;X-Gly-Asp-Pro-Glu-Ile-Val-Thr-His-Ser-Phe- Asn-Cys-Gly-Gly-Glu-Phe-Phe-Tyr-Cys-Asn-Ser- Thr-Gln-Y; andX-Cys-Gly-Gly-Glu-Phe-Phe-Tyr-Cys-Asn-Ser- Thr-Gln-Leu-Phe-Asn-Ser-Thr-Trp-Phe-Asn-Ser- Thr-Trp-Yanalogues and homologs of said sequences and subfragments of said sequence which include an epitope recognized by T cells wherein X is either a hydrogen atom of the amino terminal NH2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group; and (b) a physiologically acceptable carrier therefor. 9. A peptide having the amino acid sequence X-Ser-Ser-Ser-Gly-Arg-Met-Ile-Met-Glu-Lys- Gly-Glu-Ile-Lys-Asn-Cys-Ser-Phe-Asn-Ile-Ser- Thr-Ser-Y wherein X is either a hydrogen atom of the amino terminal NH2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group.10. A peptide having the amino acid sequence X-Gly-Glu-Ile-Lys-Asn-Cys-Ser-Phe-Asn-Ile- Ser-Thr-Ser-Ile-Arg-Gly-Lys-Val-Gln-Lys-Glu- Tyr-Ala-Phe-Phe-Y wherein X is either a hydrogen atom of the amino terminal NH2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group.11. A peptide having the amino acid sequence X-Ile-Arg-Gly-Lys-Val-Gln-Lys-Glu-Tyr-Ala- Phe-Phe-Tyr-Lys-Leu-Asp-Ile-Ile-Pro-Ile-Asp- Asn-Asp-Thr-Thr-Ser-Tyr-Thr-Y wherein X is either a hydrogen atom of the amino terminal NH2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group.12. A peptide having the amino acid sequence X-Pro-Lys-Val-Ser-Phe-Glu-Pro-Ile-Pro-Ile- His-Tyr-Cys-Ala-Pro-Ala-Gly-Phe-Ala-Ile-Leu- Lys-Cys-Asn-Asn-Y wherein X is either a hydrogen atom of the amino terminal NH2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group.13. A peptide having the amino acid sequence X-Thr-His-Gly-Ile-Arg-Pro-Val-Val-Ser-Thr- Gln-Leu-Leu-Leu-Asn-Gly-Ser-Leu-Ala-Glu-Glu- Glu-Y wherein X is either a hydrogen atom of the amino terminal NH2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group.1 . A peptide having the amino acid sequence X-Gly-Asp-Pro-Glu-Ile-Val-Thr-His-Ser-Phe- Asn-Cys-Gly-Gly-Glu-Phe-Phe-Tyr-Cys-Asn-Ser- Thr-Gln-Y wherein X is either a hydrogen atom of the amino terminal NH2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group.15. A peptide having the amino acid sequence X-Cys-Gly-Gly-Glu-Phe-Phe-Tyr-Cys-Asn-Ser- Thr-Gln-Leu-Phe-Asn-Ser-Thr-Trp-Phe-Asn-Ser- Thr-Trp-Y wherein X is either a hydrogen atom of the amino terminal NH2 group of said peptide or an additional amino acid selected to facilitate coupling of said peptide to a carrier and Y is selected from the group consisting of an amino group, a hydroxy group, a Cysteine residue, a Cysteine residue followed by an amino group and a Cysteine residue followed by a hydroxy group.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US70970991A | 1991-06-03 | 1991-06-03 | |
US709709 | 1991-06-03 | ||
PCT/SE1992/000373 WO1992021377A1 (en) | 1991-06-03 | 1992-06-03 | Peptides for use in induction of t cell activation against hiv-1 |
Related Child Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU71694/94A Division AU7169494A (en) | 1991-06-03 | 1994-09-07 | Peptides for use in induction of T cell activation against HIV-1 |
Publications (2)
Publication Number | Publication Date |
---|---|
AU1906592A true AU1906592A (en) | 1993-01-08 |
AU662534B2 AU662534B2 (en) | 1995-09-07 |
Family
ID=24851028
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU19065/92A Ceased AU662534B2 (en) | 1991-06-03 | 1992-06-03 | Peptides for use in induction of T cell activation against HIV-1 |
AU71694/94A Abandoned AU7169494A (en) | 1991-06-03 | 1994-09-07 | Peptides for use in induction of T cell activation against HIV-1 |
Family Applications After (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU71694/94A Abandoned AU7169494A (en) | 1991-06-03 | 1994-09-07 | Peptides for use in induction of T cell activation against HIV-1 |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0594638A1 (en) |
JP (1) | JPH06510025A (en) |
AU (2) | AU662534B2 (en) |
CA (1) | CA2109961A1 (en) |
WO (1) | WO1992021377A1 (en) |
Families Citing this family (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6210873B1 (en) | 1987-08-28 | 2001-04-03 | Board Of Regents, The University Of Texas System | Methods and compositions for the priming of specific cytotoxic T-lymphocyte response |
US5128319A (en) | 1987-08-28 | 1992-07-07 | Board Of Regents, The University Of Texas System | Prophylaxis and therapy of acquired immunodeficiency syndrome |
ES2194836T3 (en) * | 1990-09-27 | 2003-12-01 | Tripep Ab | PEPTIDES FOR USE IN VACCINATION AND INDUCTION OF NEUTRALIZING ANTIBODIES AGAINST THE VIRUS OF HUMAN IMMUNODEFICIENCY. |
WO1994023746A1 (en) * | 1993-04-16 | 1994-10-27 | Syntello Vaccine Development Ab | Peptides for use in vaccination and induction of neutralizing antibodies against human immunodeficiency virus |
US5603933A (en) * | 1993-08-31 | 1997-02-18 | Board Of Regents, The University Of Texas | CD4 peptides for binding to viral envelope proteins |
JPH09510437A (en) * | 1993-12-28 | 1997-10-21 | カイロン ミモトープス プロプライエトリー リミテッド | T cell epitope |
Family Cites Families (14)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE3587181T2 (en) * | 1984-10-18 | 1993-06-24 | Centre Nat Rech Scient | F ANTIGENS OF THE HUMAN IMMUNODEFICIENCY VIRUS AND THEIR USE. |
AU617088B2 (en) * | 1986-06-12 | 1991-11-21 | Biogen, Inc. | Peptides involved in the pathogenesis of hiv infection |
WO1988005051A1 (en) * | 1986-12-30 | 1988-07-14 | United States Of America, Represented By The Unite | Synthetic peptides which induce cellular immunity of the aids virus and aids viral proteins |
AU608294B2 (en) * | 1987-01-16 | 1991-03-28 | Institut Pasteur | Peptides having immunological properties 2-hiv-2 |
GB8714802D0 (en) * | 1987-06-24 | 1987-07-29 | Proteus Biotech Ltd | Synthetic polypeptides |
US5128319A (en) * | 1987-08-28 | 1992-07-07 | Board Of Regents, The University Of Texas System | Prophylaxis and therapy of acquired immunodeficiency syndrome |
WO1989005820A1 (en) * | 1987-12-21 | 1989-06-29 | Arch Development Corporation | Hiv-related antigens and antibodies |
EP0330359A3 (en) * | 1988-02-25 | 1991-06-05 | Bio-Rad Laboratories, Inc. | Composition useful in the diagnosis and treating of hiv-1 infection |
WO1989010416A1 (en) * | 1988-04-20 | 1989-11-02 | Trustees Of The University Of Pennsylvania | PROTECTIVE PEPTIDES DERIVED FROM HUMAN IMMUNODEFICIENCY VIRUS-1 gp160 |
EP0339504A3 (en) * | 1988-04-26 | 1990-09-12 | The Du Pont Merck Pharmaceutical Company | Human immunodeficiency virus (hiv) env-coded peptide capable of eliciting hiv-inhibiting antibodies in mammals |
US4943628A (en) * | 1988-06-13 | 1990-07-24 | Ortho Pharmaceutical Corporation | HIV peptide-inducted T cell stimulation |
AP237A (en) * | 1990-05-29 | 1993-04-29 | Cedars Sinai Medical Center | Immunoreagents reactive with a conserved epitope of human immunodeficiency virus type 1 (HIV-1) gp120 and methods of use. |
AU660422B2 (en) * | 1990-09-06 | 1995-06-29 | Immulogic Pharmaceutical Corporation | Pathogen-specific CTL therapy |
ES2194836T3 (en) * | 1990-09-27 | 2003-12-01 | Tripep Ab | PEPTIDES FOR USE IN VACCINATION AND INDUCTION OF NEUTRALIZING ANTIBODIES AGAINST THE VIRUS OF HUMAN IMMUNODEFICIENCY. |
-
1992
- 1992-06-03 AU AU19065/92A patent/AU662534B2/en not_active Ceased
- 1992-06-03 EP EP92911797A patent/EP0594638A1/en not_active Ceased
- 1992-06-03 JP JP4510823A patent/JPH06510025A/en active Pending
- 1992-06-03 WO PCT/SE1992/000373 patent/WO1992021377A1/en not_active Application Discontinuation
- 1992-06-03 CA CA 2109961 patent/CA2109961A1/en not_active Abandoned
-
1994
- 1994-09-07 AU AU71694/94A patent/AU7169494A/en not_active Abandoned
Also Published As
Publication number | Publication date |
---|---|
JPH06510025A (en) | 1994-11-10 |
CA2109961A1 (en) | 1992-12-10 |
WO1992021377A1 (en) | 1992-12-10 |
EP0594638A1 (en) | 1994-05-04 |
AU662534B2 (en) | 1995-09-07 |
AU7169494A (en) | 1994-11-24 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US5840313A (en) | Peptides for use in vaccination and induction of neutralizing antibodies against human immunodeficiency virus | |
US5589175A (en) | Peptides for induction of neutralizing antibodies against human immunodeficiency virus | |
CA1341285C (en) | Synthetic peptides for the detection of antibodies to hiv gp120 envelope protein for diagnosis of aids and pre-aids conditions and as vaccines | |
Lairmore et al. | Human T-lymphotropic virus type 1 peptides in chimeric and multivalent constructs with promiscuous T-cell epitopes enhance immunogenicity and overcome genetic restriction | |
WO1998015658A1 (en) | Compositions and methods for treating viral infections | |
JP3510249B2 (en) | Peptides for stimulating hepatitis C virus-specific cytotoxic T cells | |
US5346989A (en) | Peptides for use in induction of T cell activation against HIV-1 | |
US7374875B2 (en) | Peptides having affinity for the gp120 viral protein and use thereof | |
AU662534B2 (en) | Peptides for use in induction of T cell activation against HIV-1 | |
AU662954B2 (en) | Synthetic peptides for a rubella vaccine | |
EP0693938B1 (en) | Peptides for use in vaccination and induction of neutralizing antibodies against human immunodeficiency virus | |
US7319000B1 (en) | Compositions and methods for eliciting immune or anti-infective responses | |
AU666160B2 (en) | Compositions for eliciting cytotoxic T-lymphocyte responses against viruses | |
Syennerholm et al. | Vahlne et al. | |
Eriksson et al. | Systematic identification of T-cell activating epitopes on the human immunodeficiency virus type 1 envelope glycoprotein gp120 in primates immunized with synthetic peptides | |
IE871388L (en) | Small peptides which inhibit binding to t-4 receptors and¹act as immunogens |