AU8087994A - Inhibition of hiv mucosal infection - Google Patents
Inhibition of hiv mucosal infectionInfo
- Publication number
- AU8087994A AU8087994A AU80879/94A AU8087994A AU8087994A AU 8087994 A AU8087994 A AU 8087994A AU 80879/94 A AU80879/94 A AU 80879/94A AU 8087994 A AU8087994 A AU 8087994A AU 8087994 A AU8087994 A AU 8087994A
- Authority
- AU
- Australia
- Prior art keywords
- peptide
- mucosal
- hiv
- cells
- mucosa
- 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.)
- Abandoned
Links
- 206010065764 Mucosal infection Diseases 0.000 title description 4
- 230000005764 inhibitory process Effects 0.000 title description 4
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 90
- 210000004027 cell Anatomy 0.000 claims description 77
- 208000015181 infectious disease Diseases 0.000 claims description 28
- 210000002919 epithelial cell Anatomy 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 23
- 210000004877 mucosa Anatomy 0.000 claims description 23
- 102000014914 Carrier Proteins Human genes 0.000 claims description 21
- 108091008324 binding proteins Proteins 0.000 claims description 20
- 229960005486 vaccine Drugs 0.000 claims description 18
- 102000009016 Cholera Toxin Human genes 0.000 claims description 15
- 108010049048 Cholera Toxin Proteins 0.000 claims description 15
- 150000001413 amino acids Chemical class 0.000 claims description 15
- 150000002632 lipids Chemical class 0.000 claims description 15
- 239000003795 chemical substances by application Substances 0.000 claims description 11
- 101710146739 Enterotoxin Proteins 0.000 claims description 7
- 239000000147 enterotoxin Substances 0.000 claims description 7
- 231100000655 enterotoxin Toxicity 0.000 claims description 7
- 238000004519 manufacturing process Methods 0.000 claims description 7
- 241000588724 Escherichia coli Species 0.000 claims description 6
- 150000001875 compounds Chemical class 0.000 claims description 6
- 230000002401 inhibitory effect Effects 0.000 claims description 6
- 108020001507 fusion proteins Proteins 0.000 claims description 5
- 102000037865 fusion proteins Human genes 0.000 claims description 5
- 241000713772 Human immunodeficiency virus 1 Species 0.000 claims description 3
- 108020004511 Recombinant DNA Proteins 0.000 claims description 3
- 230000002708 enhancing effect Effects 0.000 claims description 3
- 108091033319 polynucleotide Proteins 0.000 claims description 3
- 102000040430 polynucleotide Human genes 0.000 claims description 3
- 239000002157 polynucleotide Substances 0.000 claims description 3
- 241000725303 Human immunodeficiency virus Species 0.000 description 65
- 102000004196 processed proteins & peptides Human genes 0.000 description 32
- 108020004414 DNA Proteins 0.000 description 22
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 14
- 241000282693 Cercopithecidae Species 0.000 description 13
- 230000000692 anti-sense effect Effects 0.000 description 13
- 239000002299 complementary DNA Substances 0.000 description 13
- 235000001014 amino acid Nutrition 0.000 description 12
- 241000700199 Cavia porcellus Species 0.000 description 11
- 239000000203 mixture Substances 0.000 description 11
- 238000006386 neutralization reaction Methods 0.000 description 11
- 210000002966 serum Anatomy 0.000 description 10
- 238000010790 dilution Methods 0.000 description 9
- 239000012895 dilution Substances 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 102100034349 Integrase Human genes 0.000 description 8
- 239000012634 fragment Substances 0.000 description 8
- 230000000521 hyperimmunizing effect Effects 0.000 description 8
- 108020004707 nucleic acids Proteins 0.000 description 8
- 102000039446 nucleic acids Human genes 0.000 description 8
- 150000007523 nucleic acids Chemical class 0.000 description 8
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 7
- 241000700605 Viruses Species 0.000 description 7
- 239000000427 antigen Substances 0.000 description 7
- 108091007433 antigens Proteins 0.000 description 7
- 102000036639 antigens Human genes 0.000 description 7
- 101710149279 Small delta antigen Proteins 0.000 description 6
- 102100022563 Tubulin polymerization-promoting protein Human genes 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 230000016379 mucosal immune response Effects 0.000 description 6
- 230000001566 pro-viral effect Effects 0.000 description 6
- 208000031886 HIV Infections Diseases 0.000 description 5
- 210000001744 T-lymphocyte Anatomy 0.000 description 5
- 230000003053 immunization Effects 0.000 description 5
- 238000002649 immunization Methods 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 210000001072 colon Anatomy 0.000 description 4
- 239000012894 fetal calf serum Substances 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 230000028993 immune response Effects 0.000 description 4
- 230000036039 immunity Effects 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 230000003472 neutralizing effect Effects 0.000 description 4
- 239000011541 reaction mixture Substances 0.000 description 4
- FWMNVWWHGCHHJJ-SKKKGAJSSA-N 4-amino-1-[(2r)-6-amino-2-[[(2r)-2-[[(2r)-2-[[(2r)-2-amino-3-phenylpropanoyl]amino]-3-phenylpropanoyl]amino]-4-methylpentanoyl]amino]hexanoyl]piperidine-4-carboxylic acid Chemical compound C([C@H](C(=O)N[C@H](CC(C)C)C(=O)N[C@H](CCCCN)C(=O)N1CCC(N)(CC1)C(O)=O)NC(=O)[C@H](N)CC=1C=CC=CC=1)C1=CC=CC=C1 FWMNVWWHGCHHJJ-SKKKGAJSSA-N 0.000 description 3
- 208000030507 AIDS Diseases 0.000 description 3
- 241000713311 Simian immunodeficiency virus Species 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000012228 culture supernatant Substances 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 210000000981 epithelium Anatomy 0.000 description 3
- 210000004392 genitalia Anatomy 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000002502 liposome Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 230000009885 systemic effect Effects 0.000 description 3
- 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 2
- 101100505161 Caenorhabditis elegans mel-32 gene Proteins 0.000 description 2
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 2
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 208000037357 HIV infectious disease Diseases 0.000 description 2
- YKUAGFAXQRYUQW-KKUMJFAQSA-N His-Tyr-Cys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N)O YKUAGFAXQRYUQW-KKUMJFAQSA-N 0.000 description 2
- 241000282412 Homo Species 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 108010058846 Ovalbumin Proteins 0.000 description 2
- 241000288906 Primates Species 0.000 description 2
- BUYHXYIUQUBEQP-AVGNSLFASA-N Ser-Phe-Glu Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CO)N BUYHXYIUQUBEQP-AVGNSLFASA-N 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000036436 anti-hiv Effects 0.000 description 2
- 230000002788 anti-peptide Effects 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 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 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000006260 foam Substances 0.000 description 2
- 230000002496 gastric effect Effects 0.000 description 2
- 210000001035 gastrointestinal tract Anatomy 0.000 description 2
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 229940099472 immunoglobulin a Drugs 0.000 description 2
- 238000011534 incubation Methods 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- 210000003563 lymphoid tissue Anatomy 0.000 description 2
- 108010017391 lysylvaline Proteins 0.000 description 2
- 238000012423 maintenance Methods 0.000 description 2
- 238000007857 nested PCR Methods 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 229940092253 ovalbumin Drugs 0.000 description 2
- 108091008399 peptide binding proteins Proteins 0.000 description 2
- 102000023856 peptide binding proteins Human genes 0.000 description 2
- 239000008194 pharmaceutical composition Substances 0.000 description 2
- 239000002953 phosphate buffered saline Substances 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 239000000523 sample Substances 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
- 239000000829 suppository Substances 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000011282 treatment Methods 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 230000003612 virological effect Effects 0.000 description 2
- 102100021690 60S ribosomal protein L18a Human genes 0.000 description 1
- VIGKUFXFTPWYER-BIIVOSGPSA-N Ala-Cys-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)N1CCC[C@@H]1C(=O)O)N VIGKUFXFTPWYER-BIIVOSGPSA-N 0.000 description 1
- XZWXFWBHYRFLEF-FSPLSTOPSA-N Ala-His Chemical compound C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 XZWXFWBHYRFLEF-FSPLSTOPSA-N 0.000 description 1
- JAQNUEWEJWBVAY-WBAXXEDZSA-N Ala-Phe-Phe Chemical compound C([C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 JAQNUEWEJWBVAY-WBAXXEDZSA-N 0.000 description 1
- IPZQNYYAYVRKKK-FXQIFTODSA-N Ala-Pro-Ala Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O IPZQNYYAYVRKKK-FXQIFTODSA-N 0.000 description 1
- XUUXCWCKKCZEAW-YFKPBYRVSA-N Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](N)CCCN=C(N)N XUUXCWCKKCZEAW-YFKPBYRVSA-N 0.000 description 1
- HQIZDMIGUJOSNI-IUCAKERBSA-N Arg-Gly-Arg Chemical compound N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O HQIZDMIGUJOSNI-IUCAKERBSA-N 0.000 description 1
- VUGWHBXPMAHEGZ-SRVKXCTJSA-N Arg-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCN=C(N)N VUGWHBXPMAHEGZ-SRVKXCTJSA-N 0.000 description 1
- 108010041397 CD4 Antigens Proteins 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- NQSUTVRXXBGVDQ-LKXGYXEUSA-N Cys-Asn-Thr Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NQSUTVRXXBGVDQ-LKXGYXEUSA-N 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 101710121417 Envelope glycoprotein Proteins 0.000 description 1
- BUZMZDDKFCSKOT-CIUDSAMLSA-N Glu-Glu-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O BUZMZDDKFCSKOT-CIUDSAMLSA-N 0.000 description 1
- JXYMPBCYRKWJEE-BQBZGAKWSA-N Gly-Arg-Ala Chemical compound [H]NCC(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O JXYMPBCYRKWJEE-BQBZGAKWSA-N 0.000 description 1
- FMVLWTYYODVFRG-BQBZGAKWSA-N Gly-Asn-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)CN FMVLWTYYODVFRG-BQBZGAKWSA-N 0.000 description 1
- IEFJWDNGDZAYNZ-BYPYZUCNSA-N Gly-Glu Chemical compound NCC(=O)N[C@H](C(O)=O)CCC(O)=O IEFJWDNGDZAYNZ-BYPYZUCNSA-N 0.000 description 1
- IKAIKUBBJHFNBZ-LURJTMIESA-N Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CN IKAIKUBBJHFNBZ-LURJTMIESA-N 0.000 description 1
- WMGHDYWNHNLGBV-ONGXEEELSA-N Gly-Phe-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 WMGHDYWNHNLGBV-ONGXEEELSA-N 0.000 description 1
- 229930186217 Glycolipid Natural products 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 101000752293 Homo sapiens 60S ribosomal protein L18a Proteins 0.000 description 1
- 101000756632 Homo sapiens Actin, cytoplasmic 1 Proteins 0.000 description 1
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 1
- 229930182816 L-glutamine Natural products 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- IAJFFZORSWOZPQ-SRVKXCTJSA-N Leu-Leu-Asn Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O IAJFFZORSWOZPQ-SRVKXCTJSA-N 0.000 description 1
- DCGXHWINSHEPIR-SRVKXCTJSA-N Leu-Lys-Cys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)O)N DCGXHWINSHEPIR-SRVKXCTJSA-N 0.000 description 1
- ILDSIMPXNFWKLH-KATARQTJSA-N Leu-Thr-Ser Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O ILDSIMPXNFWKLH-KATARQTJSA-N 0.000 description 1
- WLCYCADOWRMSAJ-CIUDSAMLSA-N Lys-Asn-Cys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(O)=O WLCYCADOWRMSAJ-CIUDSAMLSA-N 0.000 description 1
- ODUQLUADRKMHOZ-JYJNAYRXSA-N Lys-Glu-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCCCN)N)O ODUQLUADRKMHOZ-JYJNAYRXSA-N 0.000 description 1
- YQAIUOWPSUOINN-IUCAKERBSA-N Lys-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H](N)CCCCN YQAIUOWPSUOINN-IUCAKERBSA-N 0.000 description 1
- 241000282567 Macaca fascicularis Species 0.000 description 1
- 241000713869 Moloney murine leukemia virus Species 0.000 description 1
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 1
- 238000010222 PCR analysis Methods 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
- MWQXFDIQXIXPMS-UNQGMJICSA-N Phe-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC1=CC=CC=C1)N)O MWQXFDIQXIXPMS-UNQGMJICSA-N 0.000 description 1
- WOIFYRZPIORBRY-AVGNSLFASA-N Pro-Lys-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O WOIFYRZPIORBRY-AVGNSLFASA-N 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 239000012980 RPMI-1640 medium Substances 0.000 description 1
- FUMGHWDRRFCKEP-CIUDSAMLSA-N Ser-Leu-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O FUMGHWDRRFCKEP-CIUDSAMLSA-N 0.000 description 1
- GDUZTEQRAOXYJS-SRVKXCTJSA-N Ser-Phe-Asn Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CO)N GDUZTEQRAOXYJS-SRVKXCTJSA-N 0.000 description 1
- SNXUIBACCONSOH-BWBBJGPYSA-N Ser-Thr-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](CO)C(O)=O SNXUIBACCONSOH-BWBBJGPYSA-N 0.000 description 1
- 206010042971 T-cell lymphoma Diseases 0.000 description 1
- 208000027585 T-cell non-Hodgkin lymphoma Diseases 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- FDALPRWYVKJCLL-PMVVWTBXSA-N Thr-His-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CNC=N1)C(=O)NCC(O)=O FDALPRWYVKJCLL-PMVVWTBXSA-N 0.000 description 1
- UJMCYJKPDFQLHX-XGEHTFHBSA-N Val-Ser-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N)O UJMCYJKPDFQLHX-XGEHTFHBSA-N 0.000 description 1
- 241000607626 Vibrio cholerae Species 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 230000010530 Virus Neutralization Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 108010070944 alanylhistidine Proteins 0.000 description 1
- 108010011559 alanylphenylalanine Proteins 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 239000003242 anti bacterial agent Substances 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 230000005875 antibody response Effects 0.000 description 1
- 229940121375 antifungal agent Drugs 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000036755 cellular response Effects 0.000 description 1
- 210000003756 cervix mucus Anatomy 0.000 description 1
- 201000010897 colon adenocarcinoma Diseases 0.000 description 1
- 208000029742 colonic neoplasm Diseases 0.000 description 1
- 239000006071 cream Substances 0.000 description 1
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 241001493065 dsRNA viruses Species 0.000 description 1
- 210000003038 endothelium Anatomy 0.000 description 1
- 108700004025 env Genes Proteins 0.000 description 1
- 101150030339 env gene Proteins 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical compound [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 229960005542 ethidium bromide Drugs 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 210000003191 femoral vein Anatomy 0.000 description 1
- QPJBWNIQKHGLAU-IQZHVAEDSA-N ganglioside GM1 Chemical compound O[C@@H]1[C@@H](O)[C@H](OC[C@H](NC(=O)CCCCCCCCCCCCCCCCC)[C@H](O)\C=C\CCCCCCCCCCCCC)O[C@H](CO)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@]2(O[C@H]([C@H](NC(C)=O)[C@@H](O)C2)[C@H](O)[C@H](O)CO)C(O)=O)[C@@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O3)O)[C@@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](CO)O1 QPJBWNIQKHGLAU-IQZHVAEDSA-N 0.000 description 1
- 150000002339 glycosphingolipids Chemical class 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000004046 hyporesponsiveness Effects 0.000 description 1
- 229940042743 immune sera Drugs 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 210000002490 intestinal epithelial cell Anatomy 0.000 description 1
- 238000007918 intramuscular administration Methods 0.000 description 1
- 239000007927 intramuscular injection Substances 0.000 description 1
- 238000010255 intramuscular injection Methods 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 210000001821 langerhans cell Anatomy 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007108 local immune response Effects 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000004379 membrane Anatomy 0.000 description 1
- 102000006240 membrane receptors Human genes 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 210000004400 mucous membrane Anatomy 0.000 description 1
- 230000010807 negative regulation of binding Effects 0.000 description 1
- 239000002674 ointment Substances 0.000 description 1
- 239000002357 osmotic agent Substances 0.000 description 1
- 239000006072 paste Substances 0.000 description 1
- 244000052769 pathogen Species 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 238000010647 peptide synthesis reaction Methods 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000003755 preservative agent Substances 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 108020003175 receptors Proteins 0.000 description 1
- 102000005962 receptors Human genes 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 230000003248 secreting effect Effects 0.000 description 1
- 238000013207 serial dilution Methods 0.000 description 1
- 230000001568 sexual effect Effects 0.000 description 1
- 239000007790 solid phase Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000017960 syncytium formation Effects 0.000 description 1
- 239000003826 tablet Substances 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 238000002255 vaccination Methods 0.000 description 1
- 230000007502 viral entry Effects 0.000 description 1
- 230000009385 viral infection Effects 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K39/12—Viral antigens
-
- 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
- A61K39/12—Viral antigens
- A61K39/21—Retroviridae, e.g. equine infectious anemia virus
-
- 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
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
- A61K2039/541—Mucosal route
- A61K2039/542—Mucosal route oral/gastrointestinal
-
- 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
- A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
- A61K2039/6031—Proteins
- A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- 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
-
- 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
- C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
Description
INHIBITION OF HIV MUCOSAL INFECTION
FIELD OF THE INVENTION This invention relates to the inhibition of binding of HIV to the genital and rectal mucosal epithelium. BACKGROUND OF THE INVENTION
Dispersed aggregates of non-encapsulated lymphoid tissue are often localized to the submucosal areas of the gastrointestinal, respiratory and urogenital tracts. These tracts are a main means of entry into the body by foreign microorganisms. Secretory immunoglobulin A (IgA) is an antibody capable of crossing mucosal membranes and protecting them against invasion by pathogens. Mucosal lymphoid tissue thus plays an important role in the local immune response which occurs at mucosal surfaces. It is well established that mucosal epithelial cells, regardless of whether they express the cell surface CD4 receptor used by the HIV to enter T-cells, macrophages and Langerhans cells, can be latently infected by HIV (Fantini et al., (1992) J. Virol . , 66: 5805; Fantini et al . , (1991) Virology, 185: 904) . Although the receptor(s) for HIV entry into mucosal intestinal epithelial cells appear to be glycolipids (Yahi et al. , (1992) J. Virol . , 66: 4848) , there is no information regarding the HIV epitope(s) which mediates attachment to these cells. Such knowledge would be of paramount importance since this epitope(s) would be an ideal target against which the local mucosal immune system could act to prevent the mucosal entry of HIV.
The induction of a mucosal immune response to prevent entry of human immunodeficiency virus (HIV-1) through the rectal and genital (vaginal) mucosa has not been significantly explored as an approach in preventing AIDS. Conventionally administered vaccines derived from the viral glycoprotein gpl20 provide little immunity to HIV. Systemic immunization strategies have protected against intravenous challenge with simian immunodeficiency virus (SIV) , the monkey counterpart of HIV, in monkeys, but have failed to prevent infection by SIV introduced via the vaginal mucosa (Miller et al . , (1990) J.
Immunol . , 144 : 122) .
In general, mucosal delivery of antigens does not evoke a strong immune response. A notable exception, however, is cholera toxin (CT) , produced by the bacterium Vibrio chol era , which is among the strongest mucosal immunogens known. CT binds strongly to a glycosphingolipid called ganglioside GM1 on mucosal cell surfaces using its B subunit. Mucosal administration of minute amounts of antigens covalently linked to the B subunit (CTB) has been shown to elicit vigorous mucosal as well as extramucosal immune responses in experimental animals including nonhuman primates (Czerkinsky et al., (1989) Infect . Immun . , 57: 1072-1077; Liang et al . , (1988) J. Immunol., 141, 3781-3787; Lehner et al . , (1992) Sci ence, 258: 1365-1369; Holmgren et al . , (1993) Vaccine, 11: 1179-1184) . The possibility of disseminating a specific B- cell response from the gut to other mucosal tissues in orally immunized humans has also been documented (Czerkinsky et al . ,
(1991) Infect . Immun . , 59: 996-1001) . In addition, it has been demonstrated that mucosal immune responsiveness in HIV-1 infected individuals remains relatively stable compared to a dramatic hyporesponsiveness to parenterally administered vaccines (Eriksson et al. , (1993) AIDS, 7: 1087-1091) . This study not only underscores the relative independence of mucosal and systemic immunity, but also raises the possibility of inducing HIV-specific mucosal immunity in an already infected individual, thus interfering with subsequent mucosal transmission. Immunization strategies effective in inducing an immune response in the genital and rectal mucosa have been evaluated in nonhuman primates (Lehner et al . , (1992) Sci ence, 258 : 1365-1369) .
In view of the incidence of sexually transmitted HIV infection (over 75% of all cases) , the alarming increase in the number of new AIDS cases and the inability of systemic immunization strategies to induce a significant mucosal immune response, a vaccine able to produce an immune response at the mucosal .surfaces through which HIV gains access to the circulation would have significant value as part of an overall
approach to reducing HIV-1 infection.
SUMMARY OF THE INVENTION
One embodiment of the present invention is a method for inhibiting the infection of mucosal cells by HIV-1 by administering a vaccine to the mucosa, thereby delivering to the mucosa a peptide of HIV-1 gpl20 having from about 10 to about 50 amino acids, whereby antibodies against the peptide are generated in the mucosa, the peptide being selected such that the antibodies inhibit infection of HIV-1 in mucosal epithelial cells.
In another aspect of this preferred embodiment, the peptide includes an epitope effective to generate mucosal production of antibodies that inhibit infection of mucosal cells by HIV-1, the peptide consisting essentially of SEQ ID NOS: 9, 10, 11, 12, or 13. Advantageously, the vaccine further includes an agent for enhancing delivery of the peptide to the mucosa. Preferably, the agent is a mucosal binding protein; most preferably, it is either the binding subunit of cholera toxin or that of E. coli heat labile enterotoxin. The invention also provides that the peptide and the mucosal binding protein are bound together to form a chimeric protein which may advantageously be the expression product of recombinant DNA. In another embodiment of the invention, the agent is a lipid. Preferably, the lipid is in the form of a lipid vesicle. Another aspect of this preferred embodiment provides that the administering step comprises administering to the mucosa a polynucleotide operably encoding the peptide, whereby the peptide is produced by cells of the mucosa. A further embodiment of the invention provides a vaccine for inhibiting the infection of mucosal cells by HIV-1, comprising a 10 to 50 amino acid peptide of HIV-1 gpl20 having an epitope selected such that antibodies against this epitope inhibit the infection of mucosal epithelial cells by HIV-1, and a compound or structure associated with the peptide for facilitating delivery of the peptide to the mucosa. Preferably, this peptide consists essentially of SEQ ID NO 9,
10, 11, 12 or 13 and the compound or structure is a lipid vesicle. Most preferably, the compound or structure is a mucosal binding protein. In a particularly preferred embodiment, the binding protein is a cholera toxin protein which may advantageously be the binding subunit. In another aspect of this preferred embodiment, the binding protein is the binding subunit of E. coli heat labile enterotoxin.
DETAILED DESCRIPTION OF THE INVENTION
The present invention discloses the identification of synthetic peptides derived from the sequence of the envelope glycoprotein gpl20 of HIV-1. These peptides were used to generate neutralizing antibodies which inhibited infection of transformed human vaginal and colorectal cell lines in vi tro . These peptides will induce the production of a localized mucosal immune response, generating antibodies able to neutralize infection of human colorectal and vaginal epithelial cells by HIV-1. The peptides are set forth herein as SEQ ID NOS: 9-13. In one aspect of the invention, one or more of the peptides of SEQ ID NO: 9, 10, 11, 12, and 13 is used to generate antibodies. These antibodies can be generated in any conventional manner, including by intramuscular, intraperitoneal, subcutaneous, or mucosal administration to an animal. Generation of both monoclonal and polyclonal antibodies are contemplated. These antibodies are then used to prevent infection of cells of the mucosal epithelium by providing the antibodies in association with the mucosal cells and then challenging the cells with HIV-1. The antibodies inhibit or prevent binding of the virus to the cells, and thereby inhibit or prevent infection of the cells by the virus.
The antibodies can be exogenous or endogenous antibodies, and the cells can be in vi tro or in vivo . When the cells are in vi tro, the antibodies are typically generated in laboratory or domestic animals or are monoclonal antibodies. More importantly, it provides a valuable tool for analyzing the mechanism and structure involved in that binding.
When the cells are in vivo, the antibodies are preferably
endogenous mucosal antibodies that have been generated by administering one or more of the peptides of SEQ ID NOs 9-13 to the animal in which the cells are located. Mucosal vaccination, as described below, is particularly preferred. However, exogenous antibodies may also be administered to the animal to inhibit HIV-1 infection of mucosal cells. In all of the treatments described herein, the mucosal cells are preferably of human origin.
The peptides of the present invention can be utilized alone or in combination and can also be uncoupled or coupled to other epithelial cell binding proteins including CT, CTB and the binding subunit of E. coli heat labile enterotoxin. The peptides may be coupled by either chemical or recombinant means. DNA encoding the peptides can be joined to DNA encoding cholera toxin, or its B subunit, by well known methods, inserted into a eukaryotic expression vector and delivered to epithelial cells using lipid vesicles or lamellar structures. The production of these peptide-CT, CTB or enterotoxin conjugates in vivo will then elicit a localized mucosal immune response and will protect against subsequent infection by HIV. The inclusion of mucosal epithelial cell binding proteins, such as cholera toxin, will advantageously increase the efficiency of entry of peptides into these cells. Since the B subunit of the cholera toxin A-B dimer is responsible for binding to cell surface receptors, a peptide- CTB conjugate will also bind efficiently to epithelial cells. The literature also reports methods for forming compositions of immunogenic peptides and other gut binding proteins (Wenneras et al. , (1990) FEMS Microbiol . Lett . , 66: 107-112) . Techniques for forming peptide-CTB conjugates are well known (Liang et al . , (1988) J". Immunol . , 141: 3781-3787; Sanchez et al., (1990) Res . Mi crobiol . , 141: 971-979) . Liposome formation and delivery of peptides encapsulated in liposomes is also well known as described by Lowell (New Generation Vaccines , Woodrow, G.C. and Levine, M.M. , eds . , Marcel Dekker, Inc., New York, pp. 141-160) . These peptides are also useful in the production of monoclonal and polyclonal antibodies.
These antibodies have a distinct neutralizing effect on HIV-1. These peptides, either alone or after coupling to CT or other molecules, maybe administered orally, rectally, vaginally, or in a combination of these routes in an amount sufficient to generate a mucosal antibody response sufficient to inhibit HIV-1 entry into the mucosal epithelial cells. The amounts of peptides used will depend on their pharmaceutical formulation and the site and route of delivery; however, for an adult human, a suitable immunogenic amount of peptide is generally between about 50 μg and about 1 mg, administered one to four times over a period of two weeks to one year or longer.
The peptides, peptide-binding protein conjugates, and other compositions of the present invention can be administered orally to generate a localized gastrointestinal mucosal immune response or intravaginally or intrarectally to produce a localized mucosal immune response in these areas prone to viral entry by sexual contact. These peptides can be administered in unit dosage in an amount necessary to produce localized mucosal immunity against HIV-1 infection. Pharmaceutical compositions envisioned for oral administration include tablets, capsules, liquids, and the like and those contemplated for intravaginal or intrarectal administration include injectable carriers, suppositories, ointments, gels, creams, foams, sprays, dispersions, suspensions, pastes and the like in an amount from about 10 μg to about 10 mg or more. These preparations can be in any suitable form, and generally comprise the active ingredient in combination with any of the well known pharmaceutically acceptable carriers. The preparations may further advantageously include preservatives, antibacterials, antifungals, antioxidants, osmotic agents, and similar materials in composition and quantity as is conventional. For assistance in formulating the compositions of the present invention, one may refer to Remington's Pharmaceutical Sciences, 15th Ed., Mack Publishing Co., Easton, PA (1975) .
The contemplated modes of administration assume that the peptides or conjugates are able to be directly taken up by the
epithelial cells lining these areas. These peptides and conjugates may advantageously be enclosed in liposomes to facilitate delivery of these agents to cells. Direct injection of the peptides or peptide-binding protein conjugates, either alone or in combination with lipid vesicles or other lamellar structures, into the mucosal endothelium in a similar dose range is also envisioned as a method of eliciting an anti-HIV response in these tissues.
Example 1 Susceptibility of colorectal and vaginal epithelial cells to infection by HIV-1
HIV-1 infectious virus stocks of HTLV-IIIB-infected H9 T cell lymphoma cells (ATCC HTB-176) (Popovic et al . , (1984) Science, 224: 497-500) were used in the following experiments. The cells were maintained in RPMI-1640 medium containing 20% fetal calf serum (FCS) , 100 units/ml penicillin and 100 μg/ml streptomycin. Virus stocks were prepared using well known procedures and frozen at -90°C. One stock of HTLV-IIIB with endpoint titer of 1 x 104 tissue culture infectious doses (TCID50) was used for all experiments.
Endpoint titration of the HTLV-IIIB isolate of HIV-1 in two clones of transformed vaginal epithelial cells (Hs 760 T and Hs 769.Vg cells; ATCC CRL-7491 and 7499, respectively) and 12 subclones of colon adenocarcinoma HT-29 cells (ATCC HTB-38) were performed by inoculation of respective cell lines with serial 10-fold dilutions of virus with 100 μl/well (ranging from 1 TCID50/cell to 0.00001 TCIDS0/cell) in 24-well plates
(Costar) for 2 hours at 37°C. After adsorption, cells were washed five times with Modified Eagle's Medium (MEM) and supplemented with 1.5 ml growth medium (DMEM for vaginal cells and MEM for colon cells, both containing 10% fetal calf serum (FCS) , 1% L-glutamine, and antibiotics) . Seven days after infection, epithelial cells were washed five times with MEM and treated with 0.1% trypsin in phosphate buffered saline (PBS) for 5 min at 37°C. HTLV-IIIB-infected H9 cells (106 cells) in H9 maintenance medium were added to each well and cocultured with epithelial cells for 24 hours. The H9
cultures were microscopically followed for 7 days for the presence of HIV-induced syncytium formation and p24 antigen production using an ELISA able to detect as little as 100 pg p24/ml) . The vaginal cell lines Hs 760.T and Hs 769.Vg were permissive. Viral infection of Hs 760.T was detected by coculture at a high multiplicity (1 TCID50/cell) and 6 of the HT-29 colon cell clones were permissive at multiplicities ranging from 0.1-0.01 TCID50/cell. Of these clones, cloOne L20 was chosen for further study.
Table 1
Susceptibility of colorectal and vaginal epithelial cells to infection by HIV-1 (HTLV-IIIB) .
multiplicity of infection (TCID50/cell) cell Line subclone method* 1 10"1 10"2 xlO3
HT-29 EO coculture
" E5 coculture
E8 coculture
L2 coculture
L4 coculture
L16 - coculture
L18B coculture
L18A coculture
L12 coculture
" L10 coculture
L14 coculture
L20 coculture " LL2200 PPCCRR 2.5" 2 0.5 0.125
HS 769. Vg coculture
PCR 2.5 0.6 0.5 0.125
HS 760 .T coculture
PCR 10
* coculture with H9 cells and subsequent p24 antigen detection or detection of proviral DNA by PCR. ** copy number, xlO"2 per cell
HIV-1 RNA and DNA was detected both in epithelial cells and in the culture supernatants as described in the following examples .
Example 2 Detection of proviral DNA by PCR
Epithelial cells were harvested seven days post-infection and DNA was extracted (Sambrook et al . , (1989) Mol ecular Cloning, second edition, Cold Spring Harbor Laboratory Press, 2: 9.16-9.19) . Primers specific for the HIV-1 env gene (5'- GTAACGCACAGTTTTAATTGTGGAGGGGAA-3' ; SEQ ID NO: 1) and (5'- CCTCATATTTCCTCCTCCAGGTCT-3' ; SEQ ID NO: 2) were used for detection of proviral DNA. DNA (200 ng) was amplified on a DNA thermal cycler (Perkin-Elmer, Norwalk, CT) using o.-32P-dCTP to label the fragments. The reaction mixture consisted of 10 μl of lOx PCR buffer (Promega, Madison, WI) , 1.5 mM MgCl2, 20 pmol primers, 0.125 mM dNTPs, 5 μCi α.-32P-dCTP and 0.5 units Taq DNA polymerase (Promega) . The amplification was for 35 cycles and included denaturation at 94°C for 1 min, annealing at 55°C for 1 min and extension at 72°C for 1 min. One-tenth of the final reaction mixture was analyzed by electrophoresis on 5% polyacrylamide gels. The gels were dried and exposed to X-ray film (X-OMAT; Eastman Kodak, Rochester, NY) for 13-16 hours using an intensifying screen.
To measure HIV copy number (the number of HIV genomes) , two-fold serial dilutions of DNA isolated from ACH-2 cells, which contain one proviral copy per cell (Clouse et al . , (1989) J. Immunol . , 142: 431-438; Seshamma et al . , (1992) J. Virol . Methods , 40: 331-346; Graziosi et al . , (1993) Proc . Na tl . Acad . Sci . U. S. A . , 90: 6405-6409) . The total amount of DNA in each dilution was normalized to 200 ng using DNA extracted from H9 cells and PCR was performed as above. HIV copy number was estimated by comparison of the intensities of the amplified bands. PCR analysis using a pair of human β- actin primers was performed in parallel as 'an internal standard.
Example 3 Detection of HIV RNA expression by RT-PCR Epithelial cells were harvested 7 days post-infection and total RNA was extracted by the RNAzol method (Biotex Laboratories, Houston, TX) . For each sample, 500 ng of total RNA was incubated with 10 units RNase-free DNase I (Boehringer Mannheim, Mannheim, Germany) at 37°C for 1 hour. Samples were then heated to 80°C for 10 min to degrade the DNase. cDNA was synthesized in a reverse transcriptase (RT) reaction with 10 pmol downstream PCR primer (described below) , 0.625 mM dNTPs, 5 x reaction buffer (Promega) and 200 units Moloney murine leukemia virus RT (Promega) to a final volume of 20 μl . The mixture was incubated at 37°C for 1 hour. The cDNA was amplified for 30 cycles by PCR as described in Example 3. The primers used to detect HIV-1 regulatory RNA were as follows: 5' -GAAGAAGCGGAGACAGCGACG-3' (SEQ ID NO: 3) 5' -GGCCTGTCGGGTCCCCTCG-3' (SEQ ID NO: 4)
Primers specific for the HIV-1 major splice donor (MSD) site used to detect HIV-1 structural RNA were as follows: 5' -CTCTCGACGCAGGACTCGGC-3' (SEQ ID NO: 5) 5' -CTTTCCCCCTGGCCTTAACCG-3' (SEQ ID NO: 6)
32P-dCTP was incorporated into the amplified fragments and one-tenth of the final reaction mixture was analyzed by electrophoresis on 8% polyacrylamide gels. In each sample, RNA without reverse transcriptase was also amplified by PCR to demonstrate that the amplified fragments were from HIV cDNA, not from contamination of HIV DNA.
Example 4 Detection of HIV RNA in culture supernatants by RT-nested PCR RNA was extracted from 500 μl of culture supernatants by the RNAzol method. After DNase I treatment, cDNA was synthesized by a RT reaction with SEQ ID NO:8 as an RT primer. The synthesized cDNA was amplified by the nested PCR method. The primers for the first PCR were as follows: 5' -GAAGAAGAGATAGTAATTAGATCT-3' (SEQ ID NO: 7)
5' -GGTGGGTGCTACTCCTAATTGTTCAATTC-3' (SEQ ID NO: 8)
The primers used for the second (nested) PCR were SEQ ID
NO: 7 and SEQ ID NO: 2 . One-tenth of the first PCR product was added to the second PCR reaction. The PCR conditions were as described in Example 3, except that 40 cycles of amplification were performed. One-tenth of the final reaction mixture was analyzed by electrophoresis on 2% agarose gels and stained with ethidium bromide. RNA samples without RT were also amplified by the nested primers as a test for DNA contamination. DNA content and RNA expression in HIV-1 infected epithelial cells is shown in Table 2. Approximately 1% of HT29 L20 cells are infected with HIV-
1, if HIV-infected cells contain 1 copy of proviral DNA per cell. As can be seen in Table 2, expression of regulatory RNA in HIV-1 infected HT29 L20 cells is lower than that in HIV-1 infected H9 cells and ACH-2 cells. Expression of structural RNA is barely detectable.
Table 2
HIV-1 (HTLV-IIIB) RNA expression in colorectal and vaginal epithelial cells
cell line HIV-1 DNA HIV-1 RNA Virus in cell medium
(copy/cell) expression
Regulatory Structural HIV P24 RNA RNA RNA antigen
HT-29 0.025 clone L20
Hs 769.Vg 0.025
Hs 760.T 0.1
ACH-2 1
H-9 20
Example 6 Neutralization of HTLV-IIIB infectivitv in L20 and Hs769 cells by antipeptide antisera
Hyperimmune sera was isolated from monkeys immunized with the five peptides derived from the gpl20 sequence listed below (Table 3) .
Table 3 peptide no. sequence SEQ ID NO.
12 GEIKNCSFNISTSIRGKVQKEYAFF 9
15 LTSCNTSVITQACPKVSFEPIPIHYC 10 16 PKVSFEPIPIHYCAPAGFAILKCNN 1
19 THGIRPWSTQLLLNGSLAEEE 12
24 IRIQRGPGRAFVTIGKIGNMRQAH 13
Solid phase peptide synthesis was performed using an Applied Biosystems (Foster City, CA) 430A peptide synthesizer. An amino-terminal cysteine residue was added to each peptide to facilitate coupling to a carrier protein. Peptides were covalently coupled to ovalbumin, grade V (Sigma, St. Louis, MO) at an approximate 10:1 (peptide:ovalbumin) molar ratio using N-succinimidyl 3- (2-pyridyldithio) propionate (SPDP; Pharmacia, Uppsala, Sweden) . Three to five year old male and female monkeys (Macaca fascicularis) were immunized by three consecutive intramuscular injections of 100 μg ovalbumin- conjugated peptides emulsified in Freund's complete (first injection) or incomplete (second and third injections) given three weeks apart. Blood was collected from the femoral vein before immunization and one or two weeks after the final immunization. Pre- and post-immune sera were prepared and stored at -20°C. These peptides have been shown to elicit neutralizing antibodies to HIV in monkeys (Vahlne et al . , (1991) Proc . Na tl . Acad . Sci . U. S .A . , 88: 10744-10748) . These antibodies, a guinea pig hyperimmune serum with high neutralizing HTLV- IIIB capacity and a monoclonal antibody against gpl20 (the latter two kindly provided by L. Akerblom, Uppsala, Sweden) were assayed for their ability to neutralize HTLV-IIIB infectivity by primary inhibition of HIV-1 infectivity in HT- 29, clone L20, colon cells and in Hs 760.T vaginal cells and subsequently assayed by cocultivation with highly permissive H9 lymphoid cells.
Stock virus was diluted to 104 TCID50 for neutralization in colon cells and used undiluted (106 TCID50) for
neutralization in vaginal cells and mixed with serial four fold dilutions of heat-inactivated monkey sera starting at 1:5. The monkey sera were used at a final dilution of 1:10 or 1:20. The guinea pig hyperimmune serum served as a positive control. After incubation for 2 hours at 37°C, the serum virus mixture was incubated with the epithelial cells for 2 hours at 37°C. The cells were washed twice with medium and supplemented with 1.5 ml of respective maintenance medium/well. Seven days after infection the cells were washed five times and treated with 0.1% trypsin at 37°C for 5 minutes. H9 cells (106) were added to each well and cocultures were monitored for 7 days for syncytia formation and presence of p24 antigen. Results for HS 760.T cells, Hs769 cells and HT-29 L20 cells are indicated in Tables 4/5, 6, and 7, respectively, and are expressed as mean neutralization titers, defined as the reciprocal of the serum dilution that reduced the p24 antigen by at least 90%. The HIV-1 copy number is also shown for HIV-1 infected HS 760.T cells (Tables 4 and 5) .
Table 4
Neutralization of HIV-1 (HTLV-IIIB) infectivity in Hs 760.T cells by monkey hyperimmune sera against gpl20 peptides.
HIV-1 DNA neutralization assayed (copy/lO'cells) by cocultivation sera to peptides pre-immune post-immune pre-immune post-immune gpl20-12 125 <12 . 5 ND gpl20-15 ND <12 . 5 ND gpl20-16 100 25 ND gpl20-19 125 <12 . 5 ND gpl20-24 100 < 12 . 5 ND mixture gpl20- 125 < 12 . 5 (12+15+16+19+24)
Guinea pig antι-gp!20 125 <12 . 5
ND , not done
Table 5
Neutralization of HIV-1 (HTLV-IIIB) infectivity in Hs 760.T cells by guinea pig antι-gpl20 serum and monkey hyperimmune sera against gpl20 peptides.
HIV-1 DNA neutralization assayed (copy/SxlCcells) by cocultivation
Serum* Pre-immune Post-immune Pre- ■immune Po, St- -immune
Guinea pig anti-gpl20 375 <37.5 - + gpl20-12 375 <37.5 ND + gpl20-15 ND <37.5 ND + gpl20-16 300 75 ND + gpl20-19 375 <37.5 ND + gpl20-24 300 <37.5 ND + mixture gp 120- 375 <37.5 - + (12+15+16+19+24)
* Guinea pig antι-gpl20 serum and monkey hyperimmune sera against gpl20 peptides were tested at a final dilution of 1/40 and 1/10, respectively. Mixture of gpl20 peptides antisera was tested at a final dilution of 1/20. ND, not done.
Table 6
Neutralization of HIV-1 (HTLV-IIIB) infectivity in HS 769.Vg cells by guinea pig and monkey hyperimmune sera against gpl20.
Guinea pig anti crpl20 dilution HIV-1 copy number (copy/104 cells) pre-immune post-immune x40 500 <12.5 xl60 250 100 x640 250 100
Monkey anti-peptide 24 dilution HIV-1 copy number (copy/104 cells) pre-immune post-immune xlO 500 12.5 x40 250 25
Table 7
Neutralization of HIV-1 (HTLV-IIIB) infectivity in HT-29 L20 cells by guinea pig anti-gp 120 serum and monkey hyperimmune sera against gpl20 peptides.
Neutralization assayed by cocultivation by PCR
Serum Pre-immune Post-immune Post-immune Guinea pig anti-gpl20 + gpl20-l to gpl20-ll ND (aa 1-164) gpl20-12 (aa 152-176) gpl20-13 to gpl20-14 ND (aa 165-205) gpl20-15 (aa 193-218) gpl20-16 (aa 206-230) gpl20-17 to gpl20-18 ND (aa 219-257) gpl20-19 ND ND ND (aa 248-269) gpl20-20 to gpl20-23 ND (aa 258-320) gpl20-24 (aa 307-330) gpl20-25 to gpl20-40 ND (aa 321-511) mixture of gpl20- ND (12+15+16+19+24)
ND, not done.
The results indicated that the level of proviral DNA was markedly decreased by incubation of HT29 L20 cells with anti- gpl20 guinea pig serum. A decrease in viral load was also detected in cells incubated with the antisera to peptides corresponding to SEQ ID NOS: 9, 10, 11 and 13 (Table 3) . HIV- 1 copy number was also markedly decreased in HS769 vaginal epithelial cells by an antiserum to the peptide of SEQ ID NO: 13.
Example 7 Protection from HIV-1 mucosal infection in vivo with a vaccine against gpl20 epitopes
DNA corresponding to peptides having the sequence of SEQ ID NO: 9-13 is linked to DNA encoding the B subunit of cholera toxin by standard methods of molecular biology. The resulting chimeric construct is placed in a commercially available eukaryotic expression vector such as pGEX (Pharmacia, Piscataway, NJ) containing the appropriate translation initiation and termination signals. This construct is then incorporated into a lipid vesicle by methods well known in the art. The lipid vesicle is then formulated into a foam or suppository composition by well known pharmacolological preparation methods and administered vaginally and/or rectally to humans at high risk for HIV infection. The dose range administered is in the range of from about 10 μg to 10 mg. The administration is repeated at two week intervals for a total of three administrations. The presence of anti-HIV antibodies in the vaginal and rectal mucosa is assayed by isolating protein from vaginal secretions and feces (which contains cells shed from the vaginal and rectal epithelium, respectively) and performing a p24 ELISA to determine whether any antibodies are present. These antibodies can then be used in HIV-1 virus neutralization assays (Vahlne et al. , (1991) Proc . Na tl . Acad . Sci . U. S. A . , 88: 10744-10748) .
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(i) APPLICANT: SYNTELLO, Inc. (ii) TITLE OF INVENTION: Inhibition of HIV Mucosal Infection (iii) NUMBER OF SEQUENCES: 13
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: Knobbe, Martens, Olson & Bear
(B) STREET: 620 Newport Center Drive, Sixteenth Floor
(C) CITY: Newport Beach
(D) STATE: CA
(E) COUNTRY: U.S.A.
(F) ZIP: 92660
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: Patentin Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE:
(C) CLASSIFICATION:
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Israelsen, Ned A.
(B) REGISTRATION NUMBER: 29.655
(C) REFERENCE/DOCKET NUMBER: METRICS .036QPC
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: (619) 235-8550
(B) TELEFAX: (619) 235-0176
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 : GTAACGCACA GTTTTAATTG TGGAGGGGAA 30
(2) INFORMATION FOR SEQ ID NO: 2 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: : CCTCATATTT CCTCCTCCAG GTCT 24
(2) INFORMATION FOR SEQ ID NO: 3 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: GAAGAAGCGG AGACAGCGAC G 21
(2) INFORMATION FOR SEQ ID NO:4:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 19 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 : GGCCTGTCGG GTCCCCTCG 19 (2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 20 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: CTCTCGACGC AGGACTCGGC 20 (2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 21 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: CTTTCCCCCT GGCCTTAACC G 21 (2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GAAGAAGAGA TAGTAATTAG ATCT 24
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8 : GGTGGGTGCT ACTCCTAATT GTTCAATTC 29
(2) INFORMATION FOR SEQ ID NO:9 :
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Gly Glu He Lys Asn Cys Ser Phe Asn He Ser Thr Ser He Arg Gly 1 5 10 15
Lys Val Gin Lys Glu Tyr Ala Phe Phe 20 25
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 26 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
Leu Thr Ser Cys Asn Thr Ser Val He Thr Gin Ala Cys Pro Lys Val 1 5 10 15
Ser Phe Glu Pro He Pro He His Tyr Cys 20 25
(2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 25 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
Pro Lys Val Ser Phe Glu Pro He Pro He His Tyr Cys Ala Pro Ala 1 5 10 15
Gly Phe Ala He Leu Lys Cys Asn Asn 20 25
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 22 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
Thr His Gly He Arg Pro Val Val Ser Thr Gin Leu Leu Leu Asn Gly 1 5 10 15
Ser Leu Ala Glu Glu Glu 20
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 amino acids
(B) TYPE: amino acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: peptide
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: internal
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
He Arg He Gin Arg Gly Arg Gly Arg Ala Phe Val Thr He Gly Lys 1 5 10 15
He Gly Asn Met Arg Gin Ala His 20
Claims (29)
1. A method for inhibiting the infection of mucosal cells by HIV-1, comprising the step of administering a vaccine to the mucosa, thereby delivering to the mucosa a peptide of HIV-1 gpl20 having from about 10 to about 50 amino acids, whereby antibodies against said peptide are generated in said mucosa, said peptide being selected such that said antibodies inhibit infection of mucosal epithelial cells by HIV-l.
2. The method of Claim 1, wherein said peptide includes an epitope effective to generate mucosal production of antibodies that inhibit infection of said mucosal cells by HIV-l, said peptide consisting essentially of SEQ ID NOS : 9, 10, 11, 12, or 13.
3. The method of Claim 2, wherein said vaccine further comprises an agent for enhancing delivery of said peptide to the mucosa.
4. The method of Claim 3, wherein said agent comprises a mucosal binding protein.
5. The method of Claim 4, wherein said mucosal binding protein is the binding subunit of cholera toxin.
6. The method of Claim 4, wherein said mucosal binding protein is the binding subunit of E. coli heat labile enterotoxin.
7. The method of Claim 4, wherein said peptide and said mucosal binding protein are bound together to form a chimeric protein.
8. The method of Claim 7, wherein said chimeric protein is the expression product of recombinant DNA.
9. The method of Claim 3, wherein said agent comprises a lipid.
10. The method of Claim 9, wherein said lipid is in the form of a lipid vesicle.
11. The method of Claim 1, wherein said administering step comprises administering to the mucosa a polynucleotide operably encoding said peptide, whereby said peptide is produced by cells of the mucosa.
12. A vaccine for inhibiting the infection of mucosal cells by HIV-l, comprising: a 10 to 50 amino acid peptide of HIV-l gpl20 having an epitope selected such that antibodies against such epitope inhibit the infection of mucosal epithelial cells by HIV-l; and a compound or structure associated with said peptide for facilitating delivery of said peptide to the mucosa.
13. The vaccine of Claim 12, wherein said peptide consists essentially of SEQ ID NO: 9, 10, 11, 12 or 13.
14. The vaccine of Claim 12, wherein said compound or structure is a lipid vesicle.
15. The vaccine of Claim 12, wherein said compound or structure is a mucosal binding protein.
16. The vaccine of Claim 15, wherein said binding protein is a cholera toxin protein.
17. The vaccine of Claim 15, wherein said binding protein is the binding subunit of cholera toxin.
18. The vaccine of Claim 15, wherein said binding protein is the binding subunit of E. coli heat labile enterotoxin.
19. A peptide of HIV-l gpl20 having from about 10 to about 50 amino acids for use in inhibiting the infection of mucosal epithelial cells by HIV-l, wherein said peptide is administered to the mucosa, said peptide being selected such that antibodies against said peptide are generated in said mucosa, said antibodies inhibiting infection of said mucosal epithelial cells by said HIV-l.
20. The peptide of Claim 19, wherein said peptide includes an epitope effective to generate mucosal production of antibodies that inhibit infection of said mucosal cells by HIV-l, said peptide consisting essentially of SEQ ID NOS: 9, 10, 11, 12, or 13.
21. The peptide of Claim 20, wherein said peptide further comprises an agent for enhancing delivery of said peptide to the mucosa.
22. The peptide of Claim 21, wherein said agent comprises a mucosal binding protein.
23. The peptide of Claim 22, wherein said mucosal binding protein is the binding subunit of cholera toxin.
24. The peptide of Claim 22, wherein said mucosal binding protein is the binding subunit of E. coli heat labile enterotoxin.
25. The peptide of Claim 22, wherein said peptide and said mucosal binding protein are bound together to form a chimeric protein.
26. The peptide of Claim 25, wherein said chimeric protein is the expression product of recombinant DNA.
27. The peptide of Claim 21, wherein said agent comprises a lipid.
28. The peptide of Claim 27, wherein said lipid is in the form of a lipid vesicle.
29. The peptide of Claim 19, wherein said administering step comprises administering to the mucosa a polynucleotide operably encoding said peptide, whereby said peptide is produced by cells of the mucosa.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US14357793A | 1993-10-26 | 1993-10-26 | |
US143577 | 1993-10-26 | ||
PCT/US1994/012152 WO1995011701A1 (en) | 1993-10-26 | 1994-10-25 | Inhibition of hiv mucosal infection |
Publications (1)
Publication Number | Publication Date |
---|---|
AU8087994A true AU8087994A (en) | 1995-05-22 |
Family
ID=22504664
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
AU80879/94A Abandoned AU8087994A (en) | 1993-10-26 | 1994-10-25 | Inhibition of hiv mucosal infection |
Country Status (5)
Country | Link |
---|---|
EP (1) | EP0726776A1 (en) |
JP (1) | JPH09504296A (en) |
AU (1) | AU8087994A (en) |
CA (1) | CA2169453A1 (en) |
WO (1) | WO1995011701A1 (en) |
Families Citing this family (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6348449B1 (en) | 1993-09-21 | 2002-02-19 | The Trustees Of The University Of Pennsylvania | Methods of inducing mucosal immunity |
US7135191B2 (en) * | 1997-09-04 | 2006-11-14 | Zsolt Istvan Hertelendy | Urogenital or anorectal transmucosal vaccine delivery system |
AU2487300A (en) | 1998-12-31 | 2000-07-31 | Chiron Corporation | Polynucleotides encoding antigenic hiv type c polypeptides, polypeptides and uses thereof |
JP4701532B2 (en) * | 2001-04-26 | 2011-06-15 | 東ソー株式会社 | Amplification and detection of HIV-1 RNA |
CA2452015C (en) | 2001-07-05 | 2012-07-03 | Chiron Corporation | Polynucleotides encoding antigenic hiv type c polypeptides, polypeptides and uses thereof |
ITMI20021781A1 (en) * | 2002-08-06 | 2004-02-07 | San Raffaele Centro Fond | GP41 EPITOPE AND ITS USES IN THE TREATMENT OF HIV INFECTIONS. |
KR101012507B1 (en) * | 2008-01-08 | 2011-02-08 | (주)디딤바이오텍 | A composition for sanitary supplies comprising mucosal immunoadjuvant and a preparation method thereof |
CN104039350B (en) * | 2011-09-17 | 2019-07-05 | 源道隆(苏州)医学科技有限公司 | The induction of polypeptide epitope and its associated antibodies that any three or more amino acid residues are constituted |
US10660951B2 (en) | 2012-09-17 | 2020-05-26 | Zhiwei Allen Wu | Antibody recognizing arbitrarily designed epitope of three or more amino acid residues in a peptide and method of generating thereof |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4808700A (en) * | 1984-07-09 | 1989-02-28 | Praxis Biologics, Inc. | Immunogenic conjugates of non-toxic E. coli LT-B enterotoxin subunit and capsular polymers |
-
1994
- 1994-10-25 WO PCT/US1994/012152 patent/WO1995011701A1/en not_active Application Discontinuation
- 1994-10-25 CA CA002169453A patent/CA2169453A1/en not_active Abandoned
- 1994-10-25 AU AU80879/94A patent/AU8087994A/en not_active Abandoned
- 1994-10-25 EP EP94931996A patent/EP0726776A1/en not_active Ceased
- 1994-10-25 JP JP7512750A patent/JPH09504296A/en active Pending
Also Published As
Publication number | Publication date |
---|---|
JPH09504296A (en) | 1997-04-28 |
CA2169453A1 (en) | 1995-05-04 |
WO1995011701A1 (en) | 1995-05-04 |
EP0726776A1 (en) | 1996-08-21 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Matsushita et al. | Characterization of a human immunodeficiency virus neutralizing monoclonal antibody and mapping of the neutralizing epitope | |
US8110203B2 (en) | Adjuvant comprising non-toxic cross-linked muramyl dipeptide (MDP) microparticles derived from Propionibacterium acnes | |
Sasaki et al. | Monophosphoryl lipid A enhances both humoral and cell-mediated immune responses to DNA vaccination against human immunodeficiency virus type 1 | |
US5891994A (en) | Methods and compositions for impairing multiplication of HIV-1 | |
US7153509B2 (en) | Immunogenic peptides comprising a T-helper epitope and a B-cell neutralizing antibody epitope | |
McLain et al. | Stimulation of neutralizing antibodies to human immunodeficiency virus type 1 in three strains of mice immunized with a 22 amino acid peptide of gp41 expressed on the surface of a plant virus | |
Girard et al. | Vaccine-induced protection of chimpanzees against infection by a heterologous human immunodeficiency virus type 1 | |
US7172761B2 (en) | Polyvalent immunogen | |
JP4749481B2 (en) | Induction of immune responsiveness by attenuated non-functional vif protein | |
US7195768B2 (en) | Polyvalent immunogen | |
JP2009005706A (en) | Anti-feline immunodeficiency virus (fiv) vaccines | |
EP1466924B9 (en) | Synthetic peptide vaccines for HIV: the CBD epitope as an effective immunogen to elicit broadly neutralizing antibodies against HIV | |
JP3938935B2 (en) | Methods and compositions for inducing mucosal immune responses | |
AU8087994A (en) | Inhibition of hiv mucosal infection | |
VAN EENDENBURG et al. | Cell-mediated immune proliferative responses to HIV-1 of chimpanzees vaccinated with different vaccinia recombinant viruses | |
Cattozzo et al. | Expression and immunogenicity of V3 loop epitopes of HIV-1, isolates SC and WMJ2, inserted in Salmonella flagellin | |
AU2006200454B2 (en) | Compositions and methods for treating viral infections | |
OHKAWA et al. | Analysis of envelope glycoprotein-specific antibodies from SIV-infected and gp110-immunized monkeys in ACC and ADCC assays | |
EP1007687A1 (en) | Fiv vaccine | |
WO2007107597A2 (en) | Immunogenic construct and a method for the prophylactic or therapeutic treatment of aids | |
KR19980702834A (en) | Nontoxic immunogens derived from retroviral regulatory proteins, antibodies, methods for their preparation, and pharmaceutical compositions containing them | |
Moureau et al. | Specificity of anti-Nef antibodies produced in mice immunized with DNA encoding the HIV-1 nef gene product | |
US20030118601A1 (en) | FIV vaccine | |
WO2004014945A1 (en) | Gp41 epitope and uses thereof for the treatment of hiv infections | |
US20100316672A1 (en) | Vaccine |