SYNTHETIC VACCINE FOR PROTECTION AGAINST HUMAN IMMUNODEFICIENCY VIRUS INFECTION

Abstract: The present invention relates to immunogenic preparations of peptides comprising amino acid sequences corresponding to antigenic determinants of the envelope glycoprotein of HIV, covalently coupled, directly or through a spacer molecule, to carrier molecules suitable for vaccination of mammals.

Description: SYNTHETIC VACCINE FOR PROTECTION AGAINST HUMAN IMMUNODEFICIENCY VIRUS INFECTION . , . . „ This is a continuation-in-part of Application No. 08/546,515, filed October 20, 1995, which is a continuation-in-part of Application No. 08/235,305, filed April 29, 1994, which is a continuation-in-part of Application Serial No. 07/858,361, filed March 27, 1992, which is a continuation-in-part of Application Serial No. 07/832,849, filed February 10, 1992, which is a continuation-in-part of Application Serial No. 07/591,109, filed October 1, 1990, which is a continuation-in-part of Application Serial No. 07/093,854, filed September 8, 1987, the entire contents of which are hereby incorporated by reference. BASC50C1TO C? TT-T TyVTNTION The prassnt invention relates, i general, to irsunogeαic preparations and, in particular, to peptides comprising amir.c acid sequences corresponding tc a regicn cf the hua&n iasunedeficienc virus (ΞTV) enveloce protein, a ainst which neutralizing antibodies are produced. The invention further relates to a vaccine comprising the peptide coupled, either directly cr through a spacer mclecule, to a carrier molecule, suitable fcr vaccination cf humans. BACRSaOCTP TN?ep A IQW The husan retrovirus ETV has been demonstrated to be the causative agent ef acquired isaunede icienc syndrome (AIDS) , a disease for which there is currently no cure. Tbe epidemiologic pattern saeng AIDS-related cases indicates that it is a transmissible disease. The virus is frequently found in saliva, semen, vhcle blocd and plasma from individuals -in high risk categories, including male homosexuals, intravenous drug users, patients receiving blood products, and individuals fron Eaiti and Central Africa. The rapid rise in sercpcsitivity among individuals in high risk categories, the virulence cf the disease, and its grovir. world-vide distribution, underscore ar. cvervhelmir.g and immediate need fcr a vaccine camable cf inducir.tr coztleta trctactive immur.irv • - non-infected individuals . The need fcr diagnostic reagents to be used in testing for the presence of antibodies against HIV in biological samples is also clear. Previous work has demonstrated that HIV infects T lymphocytes cf the immune system by attaching its external envelope glycoprotein (gpl20) to the CD4 (T4) molecule on the surface cf T lymphocytes, thus using the C04 (T4) molecule as a receptor to enter and infect T cells . After infecting the cell, the virus subverts the ability of the T cell to fend off the virus. Retroviral envelope glycoproteins have been shown to be important in evoking a virus- neutralizing antibody response, as determined by the ability of sera containing anti-envelcte antibodies to inhibit HIV infection in vitro . Specifically , the KIV external envelope glycoprotein gpl20 has been shown to be capable of inducing neutralizing antibodies in goats and in man (Robey et al . , Proc . Nat ' l . Acad . Sci . tUSAft 83 : 7023 , 1986) . Little is known cf the precise location of epitopes on gpl20 that are either immunogenic in KlV-infected patients or that give rise to neutralizing antibodies . However, the recombinant protein PB1 (Putney et al . , Science . 234 : 1392 , 1986) , which encodes aDoraxi ately one-third of the entire ctl20 molecule , has been shewn to include the part of the envelope protein that induces the formation of neutralizing antibodies . The data accunlated to date suggest that neither P31 nor intact gpl20 are appropriate for use in a vaccine against EIV infection. Studies involving the use of goats and chimpanzees demonstrate that neither molecule has the ability to induce the production of high titers cf neutralizing antibodies . In addition, it has been shown that the intact cpl20 molecule binds to the T4 molecule of normal T cells and is capable of disrupting normal immune function. Specifically, whole gp!20 envelope molecules interfere with normal CD4 (74) function and suppress T cell activation in vitro (Mann et al., J. Taununol. 138:2640, 1987). Thus, the administration cf vaccines comprising large pieces of the external envelope glycoprotein may actually be detrimental to the normal immune system. It has become clear that HIV sequence diversity in the principle neutralizing domain of gpl20 (the V3 gpl20 envelope loop region) and rapid V3 loco seguence mutation rate is a major obstacle to overcome for vaccine development (Myers et al., Human Retroviruses and AIDS 1991? La Rosa et al., Science. 249:932-935, 1990; and Holley et al., PNAS (USX) . 88:6300-6304, 1991). Nonetheless, studies continue to shew the critical role that the gpl20 V3 region plays in generating anti-HIV neutralizing antibodies (Jiang et al., J. Era. Med. 174:1557- 1593, 1990). Moreover, it has recently been shown that approximately 50% of current HIV isolates share a consensus cf V3 sequences that is similar to the HIV MN isolate, and that approximately 80% of HIV isolates in the US share one of the 4 most common KIV sequences (Myers et al., Human Retroviruses and AIDS 1991; La Rosa et al., Science. 249:932-935, 1990; and Eollev et al., PMAS fUS T . 88:6300-6804, 1991) . Further, two of these sequences, GPGRAF and. IEIGPGRA, have induced widely cross-reactive HIV neutralizing antibodies in animals (Jahaverian et al., Science. 250:1590-1593, 1990 and Haynes et al., AIDS Res. Kumans. Retroviral. 6:38-39, 1990). Thus, critical to the development of a vaccine against HIV, is the generation of an antibody response against gpl20 that will interfere with cpl20 interaction with the CD4 (T4) molecule, but will not interfere with normal CD4 (T4) interaction with class II major histocapatibility molecules, a major normal function of the CD4 (T4) molecule in the mediation of a vriad cf staces cf normal T ceil response . In addition, an effective vaccine against HIV will induce protective immune responses in primates and in nan, that is, will prevent subsequent HIV infection from occurring . An immunogen that induced salutory (protective) anti-HIV immune responses for about 80% of HIV strains would be of great clinical use in at least three settings . First, the successful immunization of HIV negative IV drug users, prison inmates and homosexual populations thought to be at high risJc for contracting HIV in ection would significantly blunt the progression of the AIDS epidemic . Second, if immunization of HIV- infected mothers during the first trimester of pregnancy could boost salutory anti-HIV virus responses and decrease transmission cf KIV by 80% , then maternal- fetal HIV transmission would decrease fcr 30% to 6% of children born to KIV- infected mothers . Third, an i muncgen against HIV that induced salutory and net pathogenic anti-HIV responses , would be useful for immunization of ElV-infected assymptomatic individuals to boost anti-HIV immune responses , and promote the maintenance cf the assymptoisatic HIV- infected state. OBJ- CTS OF TSS INVENTION It is an obj ect of the present invention to provide a peptide that, when linked to a carrier molecule and/or polymerized to form molecular aggregates, is capable of inducing the production in mammals of high titers of neutralizing antibodies against HIV, which peptide does not distrub normal immune function. It is another obj ect of the invention to provide a synthetic vaccine conprising a peptide having an amino acid sequence corresponding to an antigenic determinant of the EIV envelope protein that is capable of inducing protective immunity in ma mala against EIV. It is a further object of the invention to provide a vaccine capable of inducing protective immunity in mammals against various forms of HIV. It is an addi tional object of the invention to provide a method of detecting the presence of anti-gplZO antibodies in biological test samples . SUKXAΛY OF THS INVENTION The invention relates to immunogenic preparations and vaccines Bade therefrom. Peptides having amino acid sequences corresponding to antigenic determinants of the envelope protein cf EIV are covalently coupled, either directly cr through spacer molecules, to suitable carrier molecules. Synthetic vaccines comprising one or core such peptides are disclosed. In one embodiment, the present invention comprises an essentially pure form of a peptide having an an:no acid sequence corresponding to an antigenic determinant of the envelope glycoprotein of HIV, which peptide is capable, when covalently linked to a carrier εolecule, of inducing in a mammal high titers c! protective antibodies against EIV. The pep ide can have, for example, the sequence CT?.?NNNT?.XSIRIQRG?G, corresponding to amino acids 303-321 of the envelope glycoprotein of the ETLV-III a isolate (Ratner et al., Nature 313:277, 1985), cr any portion thereof. In another embodiment, the present invention comprises an i.τ-*auncgenic conjugate capable cf inducing in a macwal high titers of protective antibodies against EIV, said conjugate comprising: (i) a carrier molecule covalently attached to (ii) a. peptide comprising an amino acid sequence corresponding to an antigenic determinant of the envelope glycoprotein of EIV. In yet another embodiment, the present invention comprises a method of producing immunity to EIV comprising administering the above-described conjugate to a mammal. In another embodiment, the present invention comprises a method of detecting the presence of anti-gpl20 antibodies in biological test samples. BRIEF DESCRIPTION Of TEE DRAWIHGS Figure 1. Recombinant proteins and relation to synthetic peptides. Figure 2. Reactivity of AIDS patient antibodies to synthetic peptides. Figure 3. Reactivity to gpl20 of antibodies frcm EIV* patient purified ever synthetic peptide affinity columns. Figure 4. Neutralization of E7 V-ΣII B by - goat anti-S?-10 antisera. Figure 5. Isolate specific neutralization of EIV. Figure 6. Binding of goat anti-S?-10 serum to ET V-ΣII a - but not to ET V-ΣII Rr -infected E9 T cells. Figure 7. Comparison of the ability of various T1-SP10 peptides frca the envelope of EIV HN to induce anti-ϊl-S?10HN peptide antibodies in Balb/c aice. Each point represents the mean level of anti-Tl- S?10 ser s antibody in 4-5 sica as determined by ELISA assay in 96 veil plates using the peptide T1-SP10 HN as antigen on the plate. Data are expressed as ratio (Z/C) of postbleed iaaunization (Z) optical density (OD) to prebleed (control) OD. Data shcv that Tl-SPlOHN(λ) , F-T1-SP10KN and F- Tl-S?10HN (λ) peptides after 2 i suni rations induce higher levels of anti Tl-SPIOHH antibodies than did T1-SP10HN itself . Figure 8. Coaparison of the ability of various T1-SP10 peptides fron the envelope of EIV HN to induce antibodies i Balb/c nice that neutralize EIV HN in syncytiua inhibitiua assays in vitro . Each bar indicates the results of βer a frca bleed 3 froa one aouse iaaunized with the indicated fors of T1-SP10. Eeight of bar indicates the percent of syncytiua formation inhibited by a 1 : 10 dilution of serun coapared to prebleed serua at the sase dilution. Figure 9 shews antibody titers in ELISA assay against immunizing peptide over time in chimpanzees immunized with HIV env synthetic peptides . Figure 10 shows peripheral blocd mononuclear cell proliferative responses to the Tl-SrlOIIIB (λ) peptide in 7 day tritiated thymidine incorporation assays . Figure 11 shows PBMC proliferative responses of chimpanzees immunized with T1-SP10 peptides and F- T1-SP10 peptides to PΞA. Figure 12 shows goats immunized with the same batch of peptides used to immunize chimpanzees 834 , 1028 , 1045 and 1070. The peptides were immunogenic in goats and induced high titers of anti-KIVIII3 neutral izing antibodies . Figure 13 shews anti-KIVKN neutralizing antibodies in Rhesus monkeys immunized with Tl-SPIOHN peptides . Data represent S0% neutralization titers in syncytium inhibition assay . Figure 14 shews antibody to immunizing peptide in Rhesus monkeys immunized with T1-SP10MN (λ) peptide . Figure 15 shews neutralizing antibody levels in syncytium inhibition assay in serum cf Rhesus monkeys immunized with F-Tl-SPIOHN (A) peptide . Figure 16 shews serum antibody titers to immunizing peptide in Rhesus monkeys immunized with F- Tl-SPIOMN (A) peptide. Figure 17 shows the absorption of cross neutralizing antibodies induced by Tl-SPIOKN (A) peptide in rhesus monkey 18987 by peptides containing GFGPAJ sequence . As shown, a peptide containing Tl did not absorb out neutralizing antibodies nor did a peptide with a sequence not in Tl-SPIOKN (A) . Only peptides with GPGP.AF absorbed the neutralizing activity proving that this animal selectively recognized the GPGRAF region of the V3 EIV gp!20 loop as i aunc enic and made cross-reactive antibodies to this region. 8a Figure 18. Neutralizing antibody titers against EIV III3/LAI (solid lines) and HIV HN (dotted lines) in serum of the four chimpanzees immunized with T1-SP10III3 or F-T1-ST10III3(A) peptides then immunized with Tl-SPIOMN(A) peptide. Neutralizing antibody titers determined in syncytium inhibition assay. Figure 19. For details, see legend to Figure 9. Solid lines indicate antibody titer against T1-SP10III3 peptide; dotted line indicates antibody response against Tl-SPlOMN(A) peptide. Figure 20. Absorption cf chimpanzee 1070 serum neutralizing antibodies against the EIV MN isolate by SPIOMN(A) peptides a d partial absorption by DP2 peptide. Figure 21. Induction of high levels of neutralizing antibodies against EIV MN with Tl- SPIOMN(A) peptide in Rhesus monkeys. Figure 22. Induction of anti-Tl-SPlOMN(A) peptide antibodies with T1-SP10MN(A) peptide in Rhesus monkeys. Figure 23. Induction of high levels of anti-E V MN neutralizing antibodies with Tl- SPIOMN(A) peptide. Figure 24. Induction of antibodies against F-Tl-SPIOMN(A) peptide using F-Tl-SPIOMN(A) peptide as immunogen in Rhesus monkeys. Assay used in Figs. 22, 24 was end-point ELISA against immunizing peptide (Ξ/C greater than 2.9). Figure 25. Absorption of serum neutralizing antibodies against the EIV II Ξ isolate by S?i0M (A) and DF2 peptides. 8b Figure 26. Panel A is a general prototype design of the C4-V3 peptide called Tl-SPIO(A) from the EIV isolate MN with 2 T helper determinants in the hybrid peptide, one MEC Class I CTL epitope restricted by B7, and a second CTL epitope restricted by ELA-A2. Panel B shows the Th-CTL peptide designed from simian iiππtunodeficiency virus envelope and simian immunodeficiency virus gag protein. This peptide was used to show the ability of the peptide to generate Class I restricted anti-SIV CTL in primates as described in Yasutomi et al (J. Immunol. 151:5096 (1993)) . Figure 27. Sequence of Tl-SPIO(A) Th-B-Tc peptides for human immunization. Figure 28. Mab 48d binds to the C4-V3 peptide Tl-SPIOCANO(A) whereas monoclonal antibody 17b does not. Increasing amounts of monoclonal antibodies were added to ELISA plates on which the Tl-SPIOCANO(A) C4-V3 peptide was coated (2 μg/well) as described in detail in Eay.es et al (J. Immunol. 151:1645 (1993), J. Exp. Med. 177:717 (1593)). Figure 28 shows that mab 43d bound to the Tl-SPIOCANO(A) peptide, and the 17b antibody did not. This plate was stripped with 8 molar urea (a treatment previously shown not to affect antibody binding to linear V3 determinants of peptides on the plate) and demonstrated that 8 molar urea treatment of the peptide denatured the peptide and preventive subsequent 48d binding to the peptide. These data strongly suggested that 48d antibody bound to a conformational determinant on the C4-V3 peptide Tl-SPIOCANO(A) . Figure 25. The whole Tl-SPIOCANO(A) peptide is required for maximal peptide binding to mab 8d. 8c Either the Tl peptide (C4 region alone) the V3 peptide [SPIOCANO(A) ] , C4-V3 peptide [Tl-SPIOCANO(A) 3 or a mixture of equal amounts of C4 (Tl) + V3 [SPIOCANO(A) ] peptide were incubated on an ELISA plate with the total concentration of 2 μg/well. It can be seen in Figure 29 that control monoclonal antibodies DU.ΞP20 did not bind to any of these peptides, whereas the 48d mabs bound to SPIOCANO(A) peptide and significantly better to the C4-V3 version of the Tl-SPIOCANO(A) peptide. Mixing the Tl + the SPIOCANO(A) peptide together did not increase 48d binding. Figure 30. General scheme for an ELA-based vaccine for AIDS. 8d Figure 31. Schematic representation of the possible interaction of functional native HIV-1 envelope regions. Figure shows that HIV-1 envelope protein gp4l on the viral surface has been proposed to interact with the V3 loop and C5 regions of HIV gpl20 envelope protein. Amino acids are shown in single letter code, and numbers represent the positions of amino acids in the HIVBAL envelope protein gpl60. Figure 32. Western blot analysis of guinea pig antisera against HIV gpl20 and proteins. Recombinant gpl20, gp41-MBP fusion proteins (0.lμg/lane) , cell lysate of HIVLAI/IIIB infected- or mock-infected CEM cells (0.5xlO δ cells/lane) were fractionated on 4-20% SDS-PAGE gel, and transferred to nitrocellulose filters. The filters were blocked overnight with 10% dry milk at 4°C, and incubated with guinea pig pre- or post-immune sera at 1:400 dilution for lh at room temperature, followed by incubation with goat anti-guinea pig IgG labeled with horseradish peroxidase for lh at room temperature. Results were visualized by using chemiluminescence technique. Panel A. Western blot with pig serum from guinea pig immunized with HIV-1 peptide GTH1SP10MN(A) . The pre-immune serum from the same guinea pig was used as control. Panel B. Western blot with sera from guinea pig immunized with HIV-l peptide HIV-1 gp41 peptide SP400-BAL. The pre-immune serum from the same guinea 8e pigs were used as control. Panel C. Western blot with sera from guinea pig immunized with HIV-l peptide HIV-l gpl20 peptide SP410-BAL. The pre-immune serum from the same guinea pigs were used as control. Figure 33. Indirect immunofluorescence and flow cytometric analysis of guinea pig antisera against HIV peptide on the HIVLAI/IIIB-infected CEM cells. Sera from guinea pig before and after immunization with HIV envelope peptides were incubated with HIVLAI/IIIB- infected or mock-infected CEM T cells (10 s cells) for 45 min at 4°C, followed by incubation with goat anti- guinea pig IgG labeled with FITC for additional 45 min at 4°C. Then, cells were washed, fixed with 1% paraformaldehyde, and analyzed by a flow cytometry profiler. Results were expressed as mean fluorescence channel number to reflect the fluorescence intensity. Data represent average value of two experiments. DETAIL-SO DESCRIPTION OF THΣ INVENTION The present invention relates to peptides corresponding to iaaunegeαic epitopes of EIV and synthetic vaccines made therefrom. These novel immunogenic agents are prepared by chemically synthesizing peptides sharing antigenic determinants with the envelope protein of HIV. The peptides are linked tc carrier molecules (and/or are polymerized) rendering them suitable as vaccines. These vaccines are useful fcr immunization against AIDS when administered to mammals , for example / by the parenteral route. It was determined that peptides that should be_ studied fcr immunogenic potential included those corresponding to hydrophilic, charged regicns of the HIV envelope glycoprotein. It was further deter¬ mined that, of such peptides, those with predicted beta turns would likely be cf particular importance. It was recognized that the formation of intrapeptide disulfide bonds would be useful in establishing native con igurational determinants. Also, it was recognized that formation of interchain disulfide bonds would be useful in polymerizing peptide molecules so as to form larger, core immunogenic peptide aggregates. Computer analysis cf the predicted amino acid sequence of the envelope protein cf the ETV- III 3 and ARV-2 isolates of EIV established the secondary structure and location of hydrophilic regions. Secondary structure was determined from the computer analysis using the method of Chou and Fas an (Biochemistry 13:211 and 13:222, 1974; Advances in Enzy clcov 47:42, 1978). ?o:ential areas of beta turns were localized using the method of Rose (Nature 272:535, 1978). Eydrcchilic regions cf the envelope protein were identified by the technique of Rose and Rcy (Proc. Hat'l. Acad. Sci. USA 77:4643, 1980). The peptides cf the instant invention correspond to, or are homologous with, B-cell epitopes present within the central region of the EIV isolate ETLV-IΣIg envelope protein, cr envelope protein cf related EIV isolates. The peptides of the present invention are about 35 amino acids (units) or less in length, are hydrophilic, and when conjugated to appropriate carrier molecules, evoke the production in mammals cf high titers (that is, advantageously, a reduction in infectivity of 100 infectious units cf approximately 80* in vitro at 1:600 dilution cf serum) of type (or isolate) specific neutralizing antibodies against EIV. Unlike the intact gpiΣO molecule, the peptides themselves are net capable cf inhibiting interaction between the C04 (T4) molecule cn the surface cf T lymphocytes and macrc hage K A class II molecules, and thus do not interfere with normal immune function. That is, peptides of the instant invention capable cf inducing anti-ΞIV neutralizing antibodies, o not inhibit antigen-specific normal T cell proliferative responses in vitro. Peptides cf the instant invention can have, for example, the sequence CTR SNNTRXSIRICRGrG (designated SP-10), corresponding to amino acids 303-321 cf the KTLV-IΣ∑ a envelope glycoprotein gpl20 (Ratner et al.. Nature 313:277, 1985), or some portion cf that sequence. Peptides cf the invention can also have sequences corresponding to the analogous SP-10 regions of EIV isolates c her than ET 7-III-, or portions thereof, these sequences 11 being des ignated "SP-lO-l ike " ( see , fcr example, sequences in Table I ) . TΛSLZ Σ SP-10 and SPlO-LiSce Sequences s?-io ιι∑ a CTRPNNNTRXSΣRIC J RGPG S?-10 HH CTHPHYHKRXRΣHIGPGRA? SP-10 RT CTRPNNNTRXS ΣTKCPCΛVIY SP-10 SZ CTRPHNNTrRSΣKIGPCSΛrY SP-10 WXJ-1 CT3?HNNVRRRHΣ KΣG?GSAΓY SP-10 WXJ-2 CTRPYHNVRRS SIGPGRATR SP-10 KXJ-3 CTa?NDIARΛRΣHIG?CRA?Y SP-10 ARV-2 CTλPNNNTRXSΣYIGPGSAJH SP-IO LAV- 1 CTRPNNNTRKS ΣRIQRGPG sp-io EIV-2 ( LAv-2 ) CXXPGNXTVXQ I H.LHSGHVΓKSEY The expression "S?-10-lixe" includes within its meaning the S?-10 sequence itself. Carrier molecules to which peptides of the invention are covalently linked (conjugated) are advantageously, non-toxic pharmaceutically acceptable and of a size sufficient to produce ZΛ immune response in mammals. Examples cf suitable carrier molecules include tetanus toxoid, keyhole limpet hemocyanin (XLH), and peptides correspcncing to T cell epitcpes (that is, Tl and T2) cf the ςpl20 envelope glycoprotein that can substitute for nca- AΣDS virus-derived carrier molecules (Cease, Proc. Nat'l. Acad. Sci. (OSA) 84:4249, 1987; Kennedy ec al., J. 5iol. Chem. 262:5769, 1987 J . Peptides can also be administered with a pharmaceutically acceptable adjuvant, fcr example, alua, cr conjugated to other carrier molecules sore immunogenic than tetanus toxoid. Linkage of a carrier molecule to a peptide of the invention can be direct or through a spacer molecule. Spacer molecules are, advantageously, non-toxic and reactive. Two glycine residues added to the amino terminal end of the peptide can provide a suitable spacer molecule for linking S?-10-like sequences, or portions thereof, to a carrier molecule; alternatively, S?-10-like se uences, or portions thereof, can for example be synthesized directly adjacent to, for example, another immunogenic HIV envelope sequence, for example, Ti or T2. Cysteines can be added either at the K or C terminus cf the S?-10-iike peptide for conjugation to the carrier molecule or tc both ends to facilitate interchain polymerization via di-sulfide bond foraation to form larger molecular aggregates. Conjugation of the carrier molecule to the peptide is accomplished using a coupling agent. Advantageously, the hetercfunctionai coupling agent K-maleimidobensoyl-N-hydroxysuccinimide ester (HBS) or the water soluble compound m-aaleimido- benzoylsulfosuccinimide ester (sulfo-.HSS) is used, as described by Green et al (Cell, 28:477; 1982) and by Falker et al. (Proc. Hat'l Acad. Sci. (U.S.A. ) 84:2479, 1987). Vaccines of the instant invention comprise one or more S?-10-li e peptides, or portion thereof, each S?-10-like peptide being derived from a. different EIV strain, which peptides are conjugated to carrier molecules. A polyvalent vaccine comprising a mixture cf synthetic pepcides, acvancacec siy about 2 to about 10, cc responding ia sequence to, for example, the isolates indicated in Tables I, can be used to provide immunity in man against various forms of HIV. Advantageously, the S?-10 sequence of ETLV- ΣII a (see Table Σ) can be conjugated to or synthesited with either the ST V-ΣΣI a g?120 envelope T cell epitope Tl (amino acids 428-443 cf gpl20), KQΣΣ∞WQEVGXAHYA, or to the T2 epitope (amino acids 112-124 of ETLV-III a gpl20), EZDIISLWNCSLX (Cease et al., Proc. at'l. Acad. Sci (USA) 84:4249, 1987) to form a single polypeptide (in the case cf Tl-SP-10 from the ETLV-III fl isolate of EIV, KQΣINXWQEVCX MYACTRrNNNTRXSIRIQRGPG). Similarly, Tl or T2 sequences from other EIV isolates can be linked to synthetic peptides derived fro- the SP-10 region of the corresponding isolates (set Table I), advantageously, at the N terminus of the Sr-10-li e peptide, to make a Ti(cr T2-)-S?-10-like peptide capable of inducing neutralizing antibody titers against a specific strain of EIV. Linkage at the C terminus of the Sr-10-like peptide is also possible. Smaller portions of S?-10-like peptides, fcr example, SP-10 Rf(A) and SP-10 C (Table II) can also be covalently linked to carrier molecules, including ςpi20 T ceil epitopes, and used in a vaccine. The present invention also relates to an effective protective vaccine against strains c! EIV comprising, in addition to S?-10-iike sequences and appropriate carrier molecule(s) additional sequences from the gp!20 envelope molecule. Since there is a major hypervariable region that is carbcxy terainal to peptides designated as S?-10-like in Table I (envelope amino acids 322-233, Ratner et al, Nature 313:277, 1935), and since the hypervariable region may play a role in enhancing the ability cf SF-10- 14 like peptides to raise type-specific neutralizing antibodies, amino acid sequences corresponding to a hypervariable region (approximately amino acids 322- 333) of EIV isolates can be included as vaccine components / in part or in whole, as described for other S?-10-like peptides {see, for example, sequences in Table ΣΣ). Eypervariable sequences are linked advantageously C-terminal to the S?-10-like peptide. Linkage H-terminal to the S?-10-like peptide is also possible. Table ΣI SP-10 and S?-10-llke sequences conta ini ng an addi t ional car boxy erminal hype rva r iab l e doma i n and sho r tened SP-10-l lke s ecuences . s?-ιo ma CTRPNNNTRX≤ IRIQRGPGRAFV'ΓIGXIGH SP-10 H.M CTRPHYNXRXRIKIGPGRAfYTTXNI IGT S?-10 R7 CTR?HNNTRXSITXG?GRVI YATG^I IGO S?-iO SC CTRPS rTRS IKIGPGRAFYATGDI IGD SP-10 WCJ-l CTRPNNNVRRRH iKIGPGRAr YTGilRGN s?-io κχj-2 CTRPYNNVRRSLSIGPGRAFRTRIIIGI SP-10 κχ -3 CTRPHDIARRRIKΓGPGRAΓYTGXIIGX SP-10 LAV-I CTRPNNNTRXSIRIQRGPGRAfVTIGXIGN SP-10 EIV-2 CXRPGNXTVXQIKLHSGKVFKSKYQPINXRPRQ (LAV-2) SP-10 C CTRXSIRIQRCPGR(Y) SP-10 R?(λ) CRXSITXGPGRVIY The present invention also relates to an effective protective vaccine against strains of EIV comprising, in addition to a S?-10-like sequence and a carrier molecule, a peptide correspo ding to the EIV gp41 transmembrane region that is involved in viral-induced cell fusion, FLGFLG, (Gallagher, Cell 15 50:327, 1987). The FLGFLG sequence is added, advantageously, at the C terminus of the S?-10-like peptide. Addition at the N terminus of the SP-10- like peptide is also possible. The present invention also relates to an effective vaccine against EIV formed frc cysteine- Tl-(or T2-)S?-10-llke, cysteine-Tl-(or T2-)S?-10- like-hypervariable region, or cysteine-Tl-(or T2-) S?-10-like-?LG?LG polypeptides; and/or S?-10-like- cysteine or S?-10-like-hypervariable regicn-cysteine polypeptides. The polypeptides can be treated with oxidizing agents to induce disulfide bonds between polypeptide chain cysteines, to effect polymerized and therefore, highly immunogenic antigens. The molecular aggregates thus formed advantage usly ccoprise S?-10-like peptides derived frcm (corresponding tc) at least 2 EIV isolates. A polyvalent EIV vaccine of the instant invention comprises, advantageously, two cr more ccnjugates comprising an S?-10-like sequence, or portion thereof (see, fcr example, sequences in Table 1) derived from 2 cr more EIV isolates, and a carrier molecule such as tetanus toxoid, c: two or more Tl- cr T2-S?-10-like peptide conjugates, wherein both the Tl (or T2) and the S?-I0-like sequences correspond to sequences present in a specific EIV isolate. The advantage c∑ using, as a carrier molecule, a synthetic peptide reflecting a portion of the gpl20 molecule recognized by helper T cells, is that no other carrier molecule, such as tetanus toxoid, would be required, and the B and T cell response to EIV would be specific. Combining in a pcylvaleπt vaccine several peptides reflecting sequences from the SP-10 region of different isolates, and possibly the T cell recognition region of the ςpl20 envelope, overcomes the problem of isolate-specific neutralization. The present invention also relates to a polyvalent vaccine comprising S?-10-li'κe peptides linked to hypervariable sequences described above (see, fcr example, Table II). A mixture of such polypeptides, coupled to appropriate carrier molecules and/cr polymerized via disulfide bond formation (Harington, C.S., et al., Sicche . J., 30:1598, 1530; E ing ten, C. ., et al., Biochem. J. , 38; 417, 1544; Keygand et al., I. Haturforsch. , 176:807, 1962), can be used as a vaccine to evoke a protective antibody response to multiple isolates of EIV. S?-10-!ike peptides can be used in a solid phase radioimmunoassay (5alke: et al. J. Immunol 136:2393, 1986; ibid., Proc. Hat'l. Acad. Sci. (USA) 84:2475, 1587) to (i) detect the presence and titers of neutralizing antibodies of EIV; and (ii) to determine with which strain of EIV the patient is infected. Thus, in addition to S?-10-like peptides being used as a vaccine or a component cf a vaccine, the peptide can be used, as described above, for diagnostic purposes. Peptides cf the instant invention can also be used in standard enzyme linked immunosorbent assays to detect the presence of EIV antibodies . 17 Summarizing and supplementing specific aspects of the foregoing, the present invention relates, at least in part, to a synthetic peptide comprising at least two regions of HIV proteins, the Tl gpl20 env region, reported to be recognized by both B cells (Palker et al J. Taauτκ.1. 142:3612, 1989) and helper T cells (Ceasa et al Proc. Natl. lead. Sel. fUS Ϊ 84:4249, 1987), and the S?10-lJJce gpl20 env region, a region that is also recognized by helper T cells and as veil as by B cells -and induces antibodies that are capable of neutralizing the human immunodeficiency virus EIV} (see reference of Palker et al cited immediately above; Palker et al Proc. Katl. Acad. Sci. /USA 85:1932, 1988; and also Rusche et al Proc. Natl. Acad. Sci. fUS ) 85:3198-3202, 1988 and Gcudsait et al Proc. Katl. Acad. Sci. (USA> 85:4478, 1983) (see Tables III and IV). TABLE III VARIANTS OF THI T1-SP10 PEPTIDE DΞ3IVZD FROH HIV KH ENVΣLOPZ SΣQUZNCZS F Tl S?10 A Tl - SP10 (A) KOIINHV. ' QEVG3 A-HYAC7R?NYNXRXXIHIG?GRAFYT7> F-T1 -SP10 AVGIGALf GFLXOIIJWrrOEVGXA.MYACτS? ΥNXRXSIHIG?GRA F-71 -SP10 (A) AVGIGALFLGF LXOII>^OEVGX>i-rr CTapyYNXaX3IHIG?Ca rY7TX Sequences frcm Heyers et al . Human Retroviruses and AIDS, 1988, Los Alamos National Laboratory, Los Alamos , New Hexico, p . 11 68 - 92. WO 97/14436 PCT/US96/16911 TA3LZ IV VARIANTS CF THZ T1-SP10 PΞPTIDE DΣP.IVΣD FROM HIV III3 EHVΣLOPΣ SΣ UΣ.CΣ5 P τi s p io T1-SP10 KQII } MrrjEVGXAJ«ACTRPKWi RXSIRIQRG?G Tl'SPlO (A ) K0H2«WQEVGXAKYACTRPX>tN7RXSIRIQRC?GRAFVTI F-T1-SP10 AVGIGALFLG?LXOII»WQEVGXAHyACTR?NX rRKSIRIORG?G F-Tl-SPlO ( A ) AVGIGALF G? XQII»WQEVGX.YACTRP >t , yrRXSIRI0RG?GRA?V7I Sequences frca Ratner et al. Katur*. 313:277, 1985. Heutralizing antibodies produced by TI- S?10-li3e peptides are type-specific, in that antibodies raised against the KIV ETLVIIIB (III3) isolate do not neutralize the KIV KTLVIIIXN (XH) or KTLVIIIΛ? (RJ) HIV isolates (Palker et al. £. iTtcaunol . 142:3612, 1939). Similarly, neutralizing antibodies raised against the Ti-SPlO-liXe peptides ' containing sequences fraz the Xi or J KIV isolate neutralize the hocclogαus isclate but do net neutralize any of the other tvo KIV isolates. Ecvever, when goat anti-71-S?I0-liXe antisera vere tested against 9 HIV field isolates in Kcrth Carolina, anti-TI-S?10III3 se ^ vas observed ta neutralize l of 9 HIV isolates, anti- T1-SP10R? neutralized 3 cf 9 HIV isolates, and anti-TI-S?10HH seru= neutralized 6 cf 9 HIV isolates (Haynes et al AIDS Res. Retrol. 6:38, 1990) (see Table V) . TA3LZ V A3ILITY OP λ I-TI-S?10 SΞRUM TO NΞ RALIZZ NCRTH CAROLINA FIILD ISOLATES 0? HIV λnττ-τι-s?ιoιιi3 1/9 (m) A-HTI-T1-SP10R? 3/9 (33%) AHTI-Tl-SPIOHH 6/9 (67%) La Rosa et al f Sc ncg 249 : 932 , 1990) have εhcvn that the HIV KH aotif described by Haynes et al in AIDS Kea . Petrol, , (above) is one of the prodoninant motifs of KIV isolates cultured froa AIDS patients around the United States . Pal er et al fJ. T-^unal . 142 : 3612 , 1989) vere the first to report that the strategy of nixing peptides fros various isolates could be a successful approach to the problea of raising antibodies against numerous strains of HIV with divergent aaino acid sequences in the 303-337 region of the HIV envelope. Moreover, PaUcer et .al reported that the Tl-SPlO-like peptide was advantageous over synthetic peptides coupled to carrier solecules such as XLH cr tetanus toxoid. Whereas carrier coupled peptides only induced large accents cf antibody against the carrier in pclyvale.-.t aixtures o f peptides , when the Tl sequence cf HIVIIIB env (aaino acids 429-443 ) vas covalently linked N-terainal to the S?10 sequence ( aaino acids 303 -321) , this carrier-free iaauncgen induced high titers of neutraliz ing antibodies to all three KIV isolates vhose sequences vere present in the T1-SP10 peptides . Horeaver, Kart et al IJ . Taaunol . 1990) have recently shewn that the T1-SP10 peptide is non-toxic to iaaune cells in rhesus aenxeys and is capable of inducing high- titered neutraliz ing antibodies and T helper cells in vivo in these prinates. Thus , the T1-SP10- lixe synthetic peptide construct is a sisple , non- toxic and highly efficacious aolecule for inducing high titered anti-HIV neutraliz ing antibody responses and T-helper cell responses in goats and priaates . One of the aajor prcbleas in developing a vaccine for AIDS has been the question of vhether antibody responses alone can protect a individual against both cell-free HIV and HΣV-in acted cells , cr vhether cell aediated iaaune responses (antigen-speci ic cytatcxic T calls) are needed as veil . Certainly, aany other viral infections require both antibody and cellular anti-viral iaaune responses for the generation cf protective iaaunity (Long et al T=.-unol . Today 10 : 45, 1989) . In addition, local iaaunity at aucosai surfaces consisting of IgG and Igλ antibody responses and mucosal surface-associated cytotoxic T cell activity aay be required to protect against transaission of HIV via sexual contact or via exposure of aucosai surfaces vit infected blood. Thus, a synthetic peptide iaaunogen vould be desirable that induced cytotoxic T cell (CTL) responses to HIV in addition to inducing neutralizing antibody and T helper cell responses . In addition to the eabodiaents disclosed and suaaarized above, the present invention relates to such an iaa ncce.n . The F region ( for exaaple , aaino acids 519-530 of the BK10/III3 HIV isolate and hcaologous regions of ether HIV-l , K V-2 and siaian iaaunode iciency virus (SIV) isolates) has sequence hcaolegy to the 71 (fusion) peptides of paraayxoviruses (Galla er Cel l 50 : 327 , 1937) . The F region has been postulated to fora a hydrophobic helical structure capable of inserting into lipid biiayers of cell aeabranes and inducing cell fusion . Bosch et al tScl ence 244 : 654 , 1989) have deaonstrated that the F region in SIV (that is, the region hoaologous to the 519-530 env gp41 region of the BH10/III3 HIV isolate) dees indeed aediate cell fusion of SIV-infected cells. It has been deterained that F-derivatized peptides are internalized in iaaune cells in such a vay as to induce the εaae type of cytotoxic T cell response that is necessary f or control of aany viral infections , naaely, the generation of HLA-restricted CD3+ cytotoxic T cel ls . P- 14436 PCTΛJS96/16911 derivati∑βd peptides interact vith ce lls of the iaaune systea_ βuch that vhen inj ected into a aaaaal , they induce anti-HIV aeaory T helper call activity, anti-HIV neutralizing antibodies, and aeaory anti-HIV CD8+, HLA-Class I restricted cytotoxic T cell responses. Accordingly, the present invention relates, in a preferred eabodiaent, to peptides of the general foraulae: ?-Th-s?ιo (X) Th-S?10 (X) and F (X) vherein: P sequences are fron the putative fusogenic doeain of HIV env gp41 ( for exaaple, aaino acids 519-530 in HIV isclate BK10/ III3 or hcaologcus regions in ether HIV-l , KIV-2 or SIV isolates , or sequences functionally equivalent thereto) ; Th sequences are either the Tl or T2 T helper epitopes or alternatively are any of the T helper cell epitopes listed in Table X (belov) or aaino acid sequences frcs other regions of HIV proteins not listed but that function as T helper epitopes ; S?10-lixe sequences are frca Tables I or II (see also Table VIII belov) or free any SPlO- like sequence frca HIV field isolates (see, for exaaple , LaP.osa et al Science 249 : 932 , 1990) ; and (X) sequences are HIV protein sequences recognized by XKC Class I or Class II restricted cytotoxic T cells . Examples of (X) region sequences are given in Tables VIII and IX belcv. Alternatively, 7 sequences can be , for exaaple, C-terainal to Th-S?10 (X) sequences. Moreover , Th , S?10 and (X) sequences can be arranged in any order in the peptide construct. The synthetic peptide iaaunogen of this eabodiaent of the invention is capable of inducing anti-HIV neutralizing antibodies, anti-HIV helper T calls, and anti-HIV cytotoxic (killer) T calls. One skilled in the art vill appreciate that this iaaunogen (vhich is a fusion protein} can either be synthesized cheaically or by recoabinant aeans known in the art. The iaaunogen can have, for exaaple, the structure: ?-Tl-S?10-(λ) . While exaaples of such iaaunogens are given in Tables III and IV, one skilled in the art vill appreciate that any S?10- ,like sequence froa field or laboratory HIV isolates (for exaaple, LaRosa et al Scierce 249:932, 1990) can be substituted for the SP10 sequences shovn in Tables III and IV (see also Tables I and II) . The Tl-like sequences can be selected froπ Tl-hoaologcus sequences froa any sequenced HIV isolate including those shovn in Table VI. 6 23 TA3LΣ VI HIV Envelope gp!20 Tl Sequences From Multiple HIV Isolaces Sequences for BHIO are a=ino acids 428-443 froa Ratner , L. et al , Nature 313 : 277-284 , 1985. Sequences for the resainder of the HIV-l and HIV- 2 isolates fron Hyers, et al, Kuπan Retroviruses and AID$ . 1988 , Lcs λlaaos National Laborator/, Los λlaaos , Nev Mexico, p. 11-89. : >= no aaino acid. The F-liJce sequences can be selected froc F-hcπolo<oous sequences fro2 any sequenced HIV isolate , including these shovn in Table VII . 436 24 TλflLΣ VII HIV Envelope gp4l Fusion Procein {FJ Sequences Frca Multiple HIV Isolates Isolate Sequence HIV-l BHIO A V G : Σ G A L F L G F L HN SC sri 0:4 WSJ2 RF ELI KAL 25 Z321 JY1 WU1 HIV -2 RCO R G V F V L G F L G F L NIKZ Sequences for BHIO are aaino acids 519-530 froa Ratner, L. et al Nature 313:277-284, 1985. Sequences for the reaainder of the HIV-l and H2V- 2 isolates froa Hyers, et al, puman Retroviruses and AIDS. 1988, Los Alaaos National Laboratory, Los λlaaos, Hev Mexico, p. 11-90. WKJ1 sequence is froa Brasseur et al. AIDS Res. Hua. Retrovirol. 4:83-90, 1988. : ■ no aaino acid. The (A) region-like sequences can be selected froa (A) -hoaolocous sequences froa any HIV isolate, including those shown in Tables II and V II. TA3LΣ VIII S? 10 AND SPiO- IXΣ and (A) RSGIOH gpl20 SBGβa S FROM MULTI PLZ HIV ΣS0LAT2S isolate S?10 Region ( } Region ■ IIV-I " ' 3H10 {1213) C T R P N N NT R X S I R I Q R G P G Rλ FVTI G KI C «ϊ C T R P N Y N K R X R I H I G P G λ F Y TT KN I I G T IF C T R P N N N T R X S I T K G P G R V I Y A TG Q I I G D SC C T R P N N N T T R S I H I G P G R A P Y A TG D I I G D P N N N V R R R H I E I G P G R A F Y T G B Σ R G K P Y N H V R R S L S I G P G R A P R T R B I I G I P N D I A R R R I H I G P G R A F Y T G K I I G N P N N N T R X S I Y I G P G R A F H T T G R I I G D P N N NT R X S I R I Q R G P G R A F VT I G X I C P G N X T V X Q I M L K S G K V P H S K G P Σ N X R P R Q Sequences froa BHIO (III3) are froa Ratner et al Kature 313:270-284, 1985. The invention further relates to a peptide ccnprising the F region sequence (that is, for exaaple, aaino acids 519-530 of the BHIO/IIIB isolate or other hoaologous region in other HIV- 1, HIV-2 or SIV isolates) froa HIV gp41 placed (covalently linked) H terainal to SP10 or SPlO- like regions froa any KTV sequence (see, for exaaple, Table II) froa field isolates such that the resulting construct can induce neutralizing antibodies and cytotoxic T cells against HIV. One skilled in the art vill appreciate froa a reading of the present disclosure that KHC Class I restricted cytotoxic T cells can be induced by adainistering in vivo, as an exaaple, 26 the 519-530 aaino acid region of HIV g?41, consisting of the 12 aaino acids: AVGIGALFLGFL (F) or F-region sequences of other HIV-l, HIV-2 or SIV isolates (see, for exasple, Table VII) that are hcaologous to the 519-530 aaino acid sequence of isolate BH10/III3 (Table VII), covalently linked to any other peptide ranging in length froa, for exaaple, 3 to 50 aaino acids, such that the F-linked peptide vill associate with antigen- presenting cells in such a vay as to effect the processing and presentation of the synthetic peptide that is covalently linked to F so that the peptide is presented to T cells in the context of XKC Class I aolecules and generates the developaent of CD8+ cytotoxic T cells i« vivq. In the context of an efficacious AIDS vaccine, several F-derivatized hybrid HIV peptides can be constructed ccnprising the ? aaino acid seguence (see, for exaaple, Table VII) coupled K or C terminal to an azino acid seguence of KIV proteins that are capable of inducing cytotoxic T cells in vivo. Exaaples cf described HIV peptides that are capable of being recognized by KLA Class I cytotoxic T cells are shovn in Table IX. TABLE IX CYTOTOXΪC T CEM. BPITOPES OP IIIV PROTEINS λλ Popti o Namo IIIV Protein So icnco (Roπtricting HLA) Ref, 315- 329 in gpl20 nionopoRAP TiOK 350-360 P21 gpl20 KQIDSKUUϊQKGHNK 410-429 gpl20 GSDTITLPCIU KQPΪNMWQE 644-650 P 1 OP«l NYTSLΪIISi.IEKSQM 664-678 P42 9P41 EOEL KLDK ASI.WN 707-001 P47 ΪIP«1 RIVEMX^RΠ WEA K 172- 196 40 pol (rt) I lπ pVKl.KPGMDGPICVKQ PLTEB 325- 349 49 pol(rt) AIFQSSMTKILBPPRKQNPDIVIYQ 342- 366 50 pol (rt) NPDIVIYQYMDD YVOSDLEIGQIIR 359-303 51 pol(rt) DI.BIGOIIHTKIEELROIILLR GLTT 461-405 57 pol (rt) PI.TBKλEL.ELΛB tUILKEPVIIGVY 495-519 59 poλ(rt) BIQKQGQGOWTYOIYQEPPKMLICPα 265-279 gag p24 KRWIII/ΪLNKΪVnMY(C) b oquenc β num er* for gpl20 and g P 41 are from Ratner et al Nature 113- 2 -, nj , « „ « * ** ' ' 19 ° 0) 28 This latter strategy is important in that cytotoxic T cell epitopes are recognized by specific polymorphic HLA Class I or Class II molecules. Ii only 1 such epitope [represented by one linear sequence of a peptide , such as the (A) peptide] is present in the vaccine, then only these individuals vith the specific HLλ antigen that the (A) peptide uses to be presented to cytotoxic T cells vould develop cytotoxic T cells against HIV. However, if numerous F-derivatized peptides, each containing a peptide that is capable of being recognized in the context of a distinct HLA Class I cr Class II molecule by cytotcxic T cells, are contained in an immunogen, then individuals vith a vide spectrum cf HLA-types vill m e cytotoxic T cells against KIV. Thus , an i .-ur.cgen capable cf inducing anti-HIV cytotoxic T cells in the majority of people in a population, advantageously contains a mixture of peptides , each recognized by a distinct HLA Class I type ( for instance) such that together, the mixture includes peptides that are immunogenic a recognized by Class I types of molecules that, taken together, are expressed by the majority cf individuals in a given population. Table IX shovs examples of described cytotoxic T cell epitopes and their HLA restricting elements,- if 3 nαvn, that are the types of peptides that can be derivati∑ed by F-sequences and used as a mixture vith F-Tl-S?10 (λ) peptides . Alternatively, sequences in Table IX can be covalently linked C- terminal to S?10 sequences in F-Tl-SrlO peptides instead of (A) sequences and a mixture of F-Tl- S?10 (X) peptides used as an AIDS vaccine (in the formulation F-T1-S?10 (X) , X is either an (A) sequence (see Tables II and VIII) or ether cytotc xic T cell-inducing sequence such as are listed in Table IX) . 29 The same considerations of KHC restriction that apply to T cytotoxic epitopes also apply to T helper epitopes. That is, recognition of antigens by T helper cells is HLλ restricted and for the majority of members of a population cohort to respond to an immunogen and generate a T helper call response to the immxinogen, sufficient T helper cell epitopes vill need to be present in order to have available sufficient varieties of T helper epitopes within vhich each patients' T cells may be able to see processed antigen in the context of their ovn HIA Class II solecules. Table X shovs T helper cell epitopes of HIV proteins that can be substituted for the Tl or T2 sequences in the F-Th-S?10(X) construct to provide alternative T helper cell epitopes in the construct. Sequences frcm the first 20 peptides above are from Schrier et al (J . Tmnunol . 142:1166-1176, 1939) and seguences Tl / T2, Th4 and p 18 are from Clerici et al feature 339:383-385, 1989). Studies indicate that the same T helper cell epitope may be recognized by T cells in the context of multiple HLλ Class II specificities and, therefore, only a fev T helper epitopes are needed to formulate an effective synthetic peptide based AIDS vaccine. Clerici et al (na re 339:333-385, 1939) have provided data that T cells of 85* of the population studied could recognize either T2 or Tl T helper cell epitopes (see Table X) . Schrier et al ("■ TΓJSUΠOI . 142:1166-1176, 1985) hive identif ied a nuab-er o f he lper T call epitopes in HIV proteins and demonstrated that T cells of 93* of the population studied responded to at least 1 of 4 T helper cell epitcpes (see Table X) . Thus , in a preferred embodiment, the AIDS vaccine of the present invention has the general structure and composition of mixtures of peptides of the formulation: 7-Th-S?10 (X) τh-s?ιo (X) Th-S?10 and ? (X) where , as indicated above, P sequences are from the putative fuscgenic domain cf HIV env gp41 (for example, a ino acids 515-530 in HIV isolate BH10/ III3 cr homologous regions in other HIV-l, KIV- 2 cr SIV iso lates , cr sequences functionally equivalent thereto) ( see, for example, Table VII) ; Th sequences are either the Tl cr T2 T helper epitcpes or alternatively are any of the T helper cell epitopes listed in Table X or amino acid sequences frcm other regions of KIV proteins not listed but that function as T helper epitopes , SPlO-like sequences are from Tables I , II or VIII or froa any SPlO-like sequence from HIV field isolates ( see , for example, LaRosa , G . et al . Science 249 : 932-935 , 1990) ; and (X) sequences are HIV protein sequences recognized by KKC Class I or Class II restricted cytotoxic T cells . Examples of (X) region sequences are given in Tables VIII and IX. The exact sequences to be included in the F-Th-SPlO (X) , Th-SPIO (X) , Th-SPIO and 7(X) peptides and the number of different peptides comprising the AIDS vaccine of the invention is determined by the number of cytotoxic T cell (X) and Th epitcpes needed to induce cytctcxic T cells 32 and T helper call responses in the majority of subjects in a given population cohort. One skilled in the art vill appreciate that the order of P, Th, SP10 and (X) can vary as lone as the above-indicated function of each is retained. For the induction of protective anti-HIV neutralizing antibodies, the specific SPlO-like sequences necessary to be present in F-Th-SPlO(X) peptides vill depend upon the number of variations of HIV isolates in a given population at a given time. One skilled in the art vill appreciate that this information vill need to be actively and continuously monitored in the population and the formulation of the AIDS vaccine changed frcm time to time depending cn changes in the above variables. The induction cf protective anti-HIV neutralizing ancibecies in populations that include a number of different KIV isolates can. be effected using the vaccine strategy described above and/or by employing at least one peptide construct that mimics a conserved conformational determinant cf σpl20 and thus is capable of inducing broadly cross-reactive anti-HIV antibodies. One such construct takes the form cf a mi eotope of a conformational determinant of the native HIV cpi20 C4-V3 region and is exemplified by Tl- SPIOCANO(A) (see Table XXIII) . While the primary V3 sequence of the CANO envelope is widely disparate from other HIV envelop V3 sequences (see again Table XXIII) , the Tl-SPIOCANO(A) peptide induces cross-reactive anti- V3 antibodies against a variety of HIV V3 motifs (see Example 11) . This induction of cross-reactivity is ue to secondary and higher order structures of the V3 leep of the EIV CANO isclate that result in the Tl- SPIOCANO(A) C4-V3 hybrid mirroring a broadly neutralizing determinant of HIV cp!20. This is demonstrated by the fact that the human anti-cpl20 mcncclonal antibody 43d (which blocks mouse mcncclonal antibodies that prevent C 4 binding to gpi20 but does not itself block gpi20-C 4 binding (Thali et al, J. Virol. 67:3978-3935 (1993)) binds Tl-SPIOCANO (A.) . 32a λs an alternative eabodiaent (strategy) , an effective vaccine can be formulated by determining the HIA Class I and Class II types for a particular individual by, for exaaple, either polymerase chain reaction analysis or by conventional HLλ tissue typing analysis. Based on that information, the specific iaaunc a.-.s that need to be included in the P-Th-S?10 (X) , Th- S?10 (X) , Th-S?10 and F (X) forsulatieπ can be determined. Thus , in this latter eabc-dime t, the peptides given to the subject are these necessary for eliciting the desired anti-HIV B a T cell responses. Frca a reading of the foregoing, one skilled in the art vill appreciate that this is a general strategy fcr developaent of a vaccine for any infectious disease . Moreover, the ability to conjugate the F-regicn fron the HIV gU envelope protein to any sequence capable of being recognized by cytotoxic T cells (thereby creating a linear peptide suitable for injection and capable of being recognized by cytotoxic T cells in the context of KKC Class I molecules) provides a s imple and effective method of inducing HHC Class I restricted cytotoxic T cells to any peptide bearing cytotoxic T call epitcpes. This is the case regardless cf vhether the sequence of the cytotoxic T cell epitope is derived froa proteins in an invading crganisa or vhether the cytotoxic T call epitope sequences are derived frca host proteins . λs an exaaple of the use of F-derivatized peptides that include sequences froa host proteins, it is contaaplated that F-derivatized peptides can be used comprising KIV gp41 F sequences (for exaaple, aaino acids 519-530 froa the BK10/III3 HIV-l isclate or frca hccclogcus regions of other HIV-l , HIV-2 or SIV isolates, or sequences functionally equivalent thereto) conjugated either H- or C-terainal to peptides capable of being recognized by cytotoxic T calls in the context of HHC Class I or Class II, the sequences for such peptides being derived frcm the variable region of T cell receptor for antigen (TC3) molecules expressed on the surface of autoreactive T cells that aediate host tissue destruction in various autoimmune diseases, infectious diseases and in the setting cf organ transplantation. Sun et al fNature 332 : 843 , 1933; Eur . J . Twπunol . 18 : 1993 , 1933) have reported the isolation of cytotoxic T cell clones that are specific fcr idiotypic determinants on encephalitcgenic T cells and vhich adoptively transfer resistance to experimental autaiaaune encephaloayelitis . The concept cf immunization of subjects vith autoimmune disease vith immunogens that vould induce an iaaune response against the autoiaaune clone of T cells has recently been recognized as an important experimental approach (Sevieved on Cohen et al Ianuricl . Toda 332 , 1988 ; Eovell et al Science 246 : 663 , 1939 ; Wraith et al 3 Cel T, 57 : 709 , 1939) . Thus , the pres ent invention provides a si=ple and ef fective aethod for inducing EC-rastricted Clasa I or Class II cytotoxic T calls to peptides of host antigens and thus represents a major advance in the developaent of vaccines for autoimmune disease . Using standard recombinant DHλ techniques and existing probes and sequences for T molecule antigen binding regions , sequences can be obtained froa unique regions cf the TCR aolecules (Barns et al J. 'gyn . ed . 69 ! 27 r 1989) . 7-derivati∑ed peptides can be used to induce a cytotoxic T cell iaaune response targeted to the specific clones of T cells bearing TCR≤ responsible for antigen- specif ic T call-mediated hcst tissue damage in the above disease categories . Cnce induced , such an F-peptide-ind ced anti-TCR-targeted cytotoxic T cell response can eliminate the autoreactive clone or T cells , thereby providing a novel, highly specific strategy for the control of T cell- mediated tissue destruction . λ second example cf the use cf F- derivatized hcst peptides is to similarly control antibcdy-mediated tissue damage that occurs in the context of autoimmune diseases , infectious diseases , and in the setting of organ transp lantation. B cell surface receptors for antigen (surface immunoglobulin) also contain regions that are specif ic fcr clones of B cells making antibodies . By identifying clones of B cells producing antibodies responsible for tissue- specific damage in the setting of the above disease categories, the sequence of peptides froa the region of the B cell immunoglobulin aolecule that binds antigen can be identified using, for example , recombinant DHλ techniques . Further, sequences capable of inducing KHC Class I or Class II cytotoxic T cell responses can be identified. By derivatizi.-.g such an immunoglobul n antigen- 35 binding region peptide vith F sequences and inj ecting the F-darivati∑ed peptide into the subject Baking the autoantibody, a cytotoxic T cell response against an autoantibcdy-producing B cell can be induced , thereby eliminating a tissue damaging autoantibody response that occurs in the context of the above disease categories . λ third example of the use of F- derivatized non-HIV proteins is the use of the principles described above for specific elimination of autoreactive T and B call types for the treataent of clonal B and T cell malignancies that express on their surface clonal immunc lobulin or TCR molecules . Anti-tumor therapeutic strategies have been described that employ antibodies against variable regions of either B cell surface immunoglobulin molecules (Kamblin et al Brit . J . Cancer 42 : 495 , 1530 ; Miller ' et al H . Enc . J . Ke . 306 : 517 , 1982) or antibodies against variable TCR regions in the case cf treatment cf T ceil tumors (Sanagava, 0. J . ftca . Ked . 170 : 1513-1519 , 1539} . Thus , F- derivatized synthetic peptides containing the seguences of variable regions of the TCR cr immunoglobulin molecules expressed cn the surface cf T or B cell malignant cells respectively, can be inj ected into the tumor-bearing host to induce anti-TCR or anti-immuncglcbulin-specif ic cytotoxic T cell responses that icill the tumor cells . A fourth example of the use of F- derivatized non-HIV proteins is the creation of an immunogen that xills pathogen-infected cells and thus facilitates the elimination of pathogen- infected cells from the host. For example, Hepatitis C (non-λ, non-3 hepatitis) is a disease that is caused by the transfer of viral particles in cells or in serum from cne individual to another . By F-derivatizing cytotoxic T cell epitope sequences of the Hepatitis C virus protein 36 and inj ecting such sequences into individuals , aemory anti-Kepatitis C specific cytotoxic T call responses can be induced that protect the individual fron infection vith live Hepatitis C virus, thus providing a novel Hepatitis C vaccine . Such a strategy can also be used to create a vaccine for other infectious pathogens . 36a Much of the foregoing disclosure focusses on the design of T helper cell epitope (Th) -B cell epitope (B) peptides derived from non-continuous regions of HIV gpl20 (Palker et al, J. Immunol. 142:3612-3619 (1989) ; Haynes et al, J. Immunol. 151:1646-1653 (1993)) . The Tl epitope from the gpl20 C4 region has served as a potent Th epitope in Th-B synthetic peptide design (Palker et al, J. Immunol. 142:3612-3619 (1989); Cease et al, Proc. Natl. Acad. Sci. USA 84:4249-4253 (1987)) (see Table XXVIII) . A region from aa 262-281 from HIV p24 core protein, YKRWIILGLNKIVRMYS (designated GTH1 in Table XXVIII) , has been reported to be a potent cytotoxic T cell determinant (Johnson et al, J. Immunol. 147:1512-1521 (1991) ; Nixon et al, Nature 336:484-486 (1988) ; Meyerhans et al, Eur. J. Immunol. 21:2637-2640 (1991) and a Th determinant (Mills et al, Vaccines 90:213 (1990)) . The presently described embodiment relates to a polyvalent mixture of synthetic peptides that are highly immunogenic and that induce antibodies against native HIV env gpl20 or gp41. The present embodiment results, at least in part, from the realization that immunogenic peptides reflective of points of contact between HIV gpl20 and gp41 (termed "gpl20/gp41 touchpoints") together provide antibodies against multiple sites on native gpl20 and gp41, and thereby facilitate the dissociation of gp41 and gpl20. Dissociation of gp41 and gpl20 promotes the neutralization of HIV primary isolates by these antibodies. A number of sites have been identified on gpl20 or gp4l that: are involved in, or regulate, the interactions of gpi20 with gp41 (see Figure 31, Table XXVIII) . These sites include: the gpl20 V3 loop region (Willey, R.L. and Martin, M.A. , J. Virol. 67:3639- 3643 (1993)), the gpl20 C2 region cencered around asparagine ac aa 267 (Willey, R.L. and Martin, M.A. , J. Virol. 67:3639-3643 36b (1993)), the C-terminus of gpl20 (Neurath et al, Virology 188:1- 13 (1992) ; Schulz et al, Aids Res. Human Retrovirol. 8:1571-1580 (1992); Lopalco et al, Aids Res. Human Retrovirol. 9:33-39 ( 1993) ; Lopalco et al, Eur. J. Immunol. 23:2016-2021 (1993)) , and a region of gp41 centered around the aa sequence AVERY (Reitz et al, Cell 54:57-63 (1988) ; Neurath et al, Virology 188:1-13 (1992); Schulz et al, Aids Rεs. Human Retrovirol. 8:1571-1580 (1992) ; Lopalco et al, Aids Res. Human Retrovirol. 9:33-39 (1993)) . The "AVERY" region of gp41 has been demonstrated to interact with the C5 C-terminal region of gpl20 (Neurath et al, Virology 188:1-13 (1992) ; Schulz et al, Aids Res. Human Retrovirol. 8:1571-1580 (1992) ; Lopalco ec al, Aids Res. Human Retrovirol. 9:33-39 (1993)) and A. Beretta has reported that antibodies against the C5 gp!20 region can "loosen" the gp41/gpl20 interaction and thereby inactivate HIV (Fust et al, Immunol. Today 16:167-169 (1995)) . Whether the C-terminus of gpl20 by itself induces anti-HIV neutralizing antibodies is controversial. Some investigators have found anti-gpl20 C-terminus antibodies neutralize HIV (Broliden et al, Proc. Natl. Acad. Sci. USA 89:461-465 (1992) ; Kennedy et al, J. Biol. Chem. 262:5769-5774 (1987) ; Chanh et al, EMBO J. 5:3065-3071 (1986) while others have not (Palker et al, Proc. Natl. Acad. Sci. USA 84:2479-2483 (1987) ; Laal et al, J. Virol. 68:4001-4008 (1994) ; Karwowska et al, AIDS Res. Human Retrovirol. 8:1099-1106 (1992) ; Vahlne et al, Proc. Natl. Acad. Sci. USA 88:10744-10748 (1991)) . An important new region for neutralizing both laboratory- adapted and primary HIV isolates is located in gp41 near the membrane spanning region, containing the sequence, ELDKWAS (Muster et al, J. Virol. 67:6542-6647 (1993); Conley et al, Proc. Natl. Acad. Sci. USA 91:3343-3352 (1994)) . Although human monoclonal antibodies against this region of gp41 reportedly 36c neutralize primary HIV isolates, this region has been poorly immunogenic in humans and animals (Conley et al, Proc. Natl. Acad. Sci. USA 91:3348-3352 (1994)) . Muster et al (J. Virol. 69:6678-6686 (1995)) have succeeded in raising anti-ELDKWAS antibodies but only after expressing the ELDKWAS sequence in a chimeric influenza virus and using the resulting virus for immunization. The peptides listed in Table XXVIII are against certain HIV strains (HIV MN or BAL) . For effective neutralization of HIV primary isolates, it is advantageous, for each peptide specificity, to also construct peptides reflective of known mutations in the indicated epitope. Such a listing can be found in the current Los Alamos Database (G. Myers and B. Korber, Eds. 1993, Los Alamos National Laboratory, Los Alamos, NM) , portions of that information also being accessible through Genbank) . Table XXIX described a peptide combination that induces anti-HIV antibody responses that synergize in neutralizing HIV laboratory and clinical isolates. TABLE XXVIII Peptide Sequences Used to Produce Immunogenic Peptides That Induce Antibodies Against Touchpoints Between HIV gp120 and gp41 Sequence HIV Protein Peptide Name YKRWIILGLNKIVRMYS p24, aa262-281 GTH1 KQIINMWQEVGRAMYA gp120, C4 region, aa421-436 T1 TRPNYNKRKRIHIGPGRAFYTTK gp120. V3 loop region, aa301-324 SP10MN(A) TRPNNNTRKSIHIGPGRAFYTTG gp120. V3 loop region, aa301-324 SP10BAL QQEKNEQELLELDKWAS gp 1. ELDKWAS region, aa652-668 SP61 RVLAVERYLRDQQLLGIWGCSGKLICTTAVPWNASWSNKSLNKI gp 1. AVERY region, aa579-622 SP400-BAL PGGGDMRDNWRSELYKYKWKIEPLGVAPTKAKRRWQR gp120. aa472-513 SP 10-BAL PWSTQLLLNGSLAEEEWIRS gp120. C2 region. aa257-279 SP420-BAL Hybrid Peptides Used To Induce Antibodies Against Native HIV Envelope Proteins ω a. GTH1-SP10MN(A) T1-SP10BAL GTH1-SP61 T1-SP420-BAL HIV sequences taken from the Los Alamos Data Base. 1993, B Korber, G Myers, Eds , Los Alamos National Laboratory, Los Alamos, NM 36e TABLE XXIX Peptide Combination that Induces Anti-HIV Antibody Responses that Synergize in Neutralizing HIV Laboratory and Clinical Isolates. GTH1-SP10(A) OR T1-SP10(A) (TO INDUCE ANTl-gp120 V3 REGION ANTIBODIES) GTH1-SP61 (TO INDUCE ANTI-ELDKWAS gp41 REGION ANTIBODIES) SP400 (TO INDUCE ANTI-AVERY REGION gp41 ANTIBODIES) SP-410 (TO INDUCE ANTI-C5 REGION gp120 ANTIBODIES) T1-SP420 or GTH1-SP420 (To Induce Anti-C2 gp120 Region Antibodies) 36f The follcvinc ncπlitai inς ex m les illustrate the invention iα tac re detail. EXAMPLE 1 Synthesis of Paotides and Preparati o n cf Ccnjucates Synthetic essantially pure peptices containing hydrophilic acinc acid sequences £rc= the E7LV-III- envelope clycccrcteir. cpi20 (Ratner, et 37 al. Mature , 313:277, 1985} were synthesited cn an Applied Biosystems 430A peptide synthesiser using chemical and program cycles supplied by the manufacturer. Sequences of synthetic peptides are given in Table XI • TASLE χι SYNTHETIC PEPTIDES WITH EYDROPSILIC AH ISO ACID SEQUENCES OF ETLV-IIIg ENVELOPE PF.CTΞIN Sequene β b ACV?TC?N?C;EV{Y) cτaPNNNτaxs ∑a:ςriG?G ( Y ) (YJGXZGNHSQASCHISRAX ( Y J SRA XnNNTLXQ I OSXLREQFGNN ( C ) (Y)NSt3LFNSrn?HSr«STXGSNNT£GSOTI{C) {Ϊ)LT?.CCGNSNNESΪIF(C) APTXAX « -?. WQP.EX-. ( C ) a According to Ratner et. al., Nature, 313:277, 1985. Amine acids in parentheses were added for icdination of peptide (Y) and coupling to carrier protein (C). The relationship cf the peptides synthesized to known reccmbinant proteins PE3, PSI and PΞNV9 is shown in Figure 1 (Putney et al., Science, 234:1392, 1986; Petteway et al., Viruses and Human Cancer: UCLA Svπocsia cn Molecular and Cellular Biology 1987). Peptides were cen gatad to carrier molecules such as bevine serum albumin (BSA) or tetanus toxoid (TT) with H3S, as described by Green et al. (Cell, 23:477, 1932; Palker et al, Proc. 38 Katl. Acad. Sci. (USA) 84:2479, 1387). Fcr the ccupling procedure/ 24 eg of tetanus toxoid (for exaaple] in O.S al of phcsshate buffered saline, pH 7.2, vas incubated vith 1 ag of HBS dissolved in lOOvl of dime hylforaaaide for 1 hr. at 23*C. Tetanus toxoid treated with HΞS (TT-H3S) vas then subjected to sieving chromato raphy on a PO-10 (Pharaacia) coluran to reaove unreacted HSS frca TT- H3S, and fracticns containing TT-HBS vere recovered in the void voluae of the coluan as deterained by spectrophotcmetric assay at an optical density cf 280 na. TT-.HSS was ther. incubated vith recking at 23*C for 3 hr. vith 6-3 ag cf synthetic peptide (aclar ratio 30:1, peptide carrier protein) in PBS containing reduced cysteine at either the carboxyl cr aaino terminus. TT-peptide conjugates vere dialyzed overnight at 4*C against PSS or again desalted cn a FO-10 coluan and were used as an ir.aunogen. Conjugaticn ' cf peptides to BSA .cr tetanus tcsoid vas monitored by subjecting conjugates to scdium dodecylsulfate-polyacrylaaide gel electrophoresis (SDS-PAGE) under non-reducing conditions and by measuring the increase in apparent aclecular veights over that cf ESA and TT treated vith HΞS. Coupling efficiencies also monitored by trace iodinaticn cf peptides varied from 10-30* depending cn the peptide. EXAMPLE 2 Reactivi ty of AIDS Pat ient Antibod ies To Synthe tic Peot ides Synthe tic peptides derived f rca hydrcphilic reg ions o f gpl20 coupled to BSA were us ed as a t igens in a radic iaauncassay ( RIA ) v i th EIV+ patient sera (N=12) and noraal serum control (N « 4), to evaluate the AIDS patient antibody response to epitopes on gpl20 (Figure 2) (Paller et al., J^ Iaaunol. 136:2393, 1986; ibid, Prec. Hat'l. Acad. Sci (USA), 84:2479/ 1987). The majority of EIV+ patient sera reacted vith two synthetic peptides, SP-10 (9/12/ 75%) and SP-22 (8/12, 671). Results are expressed as a ratio {t/C) of duplicate cpm values obtained vith experimental (E) AIDS sera and control (C) serua samples. E/C > 3.0 * positive. EXA.HP Σ 3 Reactivit y of cgl20 to Antibodies frca HIV* Patient Purified Over Synthetic Peptide Affinity Columns Fcr preparation of affinity coluans ; synthetic peptides containing aaino acid sequences frca BTLV-iπ 8 g?120 (SP-10, 10A, 11, 14, 15, 22/ see Figure 1) vere coupled to BSA and then covalently linked to CHΞr-activated Sepharose. Serus aliquots (2al) frca an EIV seropositive patient vere then passed over each coluan and the antibodies that bound to the affinity columns vere then tested for reactivity to purified 12S I-labelled ET V-III- gpl20 in RI? assay (Figure 3A) and for reactivity to the surface cf E-5 cells infected vith ET V-III- in indirect iaaunoflucrescer.es assays (Figure 3B). A) In RI? assay (Pa er et al., ?rcc. Nat'l. Acad. Sci (OSA) 84:2479, 1987; ibid, J^ Iaaunol . 136:2393, 1986), bound antibodies frca the SP-10 (lane 1), SP-lOA (lane 2), SP-li (lane 3), and SP-22 (lane 6) affinity coluans reacted vith g?120- III a in R ? assay, vith antibodies from the SP-10 40 coluan shoving the greatest reactivity to g?12Q- B) When tested in FACS analysis (Shapiro, Practiced Flov Cvtometrv, Alan R. iss Pub., NY, NY, 1985), antibodies reactive vith synthetic peptide SP-10 bound to the surface of ElV-infected cells vhile binding of affinity purified antibodies to S?- 14 or to SP-lOA, 11, 15 or 22 (not shovn) vas not detected. These data suggest that the antigenic s # ite(s) defined by SP-10 are accessible tc antibody binding vhen gpi20 is present cn the surface of EIV+ cells. EXAMPLE 4 Neutralisation of EIV bv Goat Ar.ti-S?-10 Antisera Goats vere immunized subcutaneously vith 28mg of tetanus toxoid SP-10 conjugates (S?-10-TT) in Freunds complete adjuvant (days 0) followed by bivee'xly inoculations in inconolete Freunds adjuvant (days 14 and 21). Serum samples vere collected after the second immunization and tested for the ability to inhibit (i.e. neutralise) EIV infection of E-9 T cells i_π vitro as measured by the presence of reverse transcriptase (RT) activity in ceil culture supernatants (Figure 4). Decreased cpta values obtained in RT assays reflect decreased levels of EIV after cocultivation of virus and cells for 10 days. When pre-incubated vith 100 infectious units of ETLV-III-, gcat anti-S?-10 antisarun neutralized the ability cf ΞIV isolate HTLV-III B to infect E-9 T cells (• - ♦, S0\ neutralization titer = 1/145). In contrast, se ve collected frcm the same goat prior to immunization did not appreciably 41 neutralize ETLV-IIIg (O- , SOt neutralization titer * 1/15). The original animal injected vith SP-10-TT (vhose serum neutralized ET V-III a in reverse transcriptase assay) vas subsequently injected vith additional doses of SP-10-TT (0.5 mgΛg body veight). The 50* neutralization titer rose to 1:1600 after tvo injections. Neutralization data frcm these and other experiments vith SP-10-lixe peptides are shovn in Table XII as the sar si dilutions that result in 80* rather than 50t neutralizations cf EIV. In addition, a second goat vas injected twice with O.S mς/xg doses cf SP-10-TT. Serum from the second goat neutralized ETLV-III B at a titer of 1:100. Importantly, both sera against SP-10-TT raised in goats also inhibited ETLV-III 2 infectivity of T cells in the syncytiua-inhibition assay (Table XII) . The syncytium-inhibition assay ( ifson et al., Nature 323:725, 1986) measures the ability of antibodies tc inhibit the fusion cf ElV-infected T cells, these expressing EIV gpl20 envelope protein . on the cell surface, vith CD4 (T4)+ uninfected T cells. The CD4 (T4) molecule serves as the receptor for the AIDS virus (Haddcn et al., Cell 47:333, 1986). The result of fusion of these two cell types is the formation of giant cells that are infected vith EIV. In many instances, the result cf EIV infection of cells and giant ceil formation is death of the infected cell (Zagary et al.. Science 231:850, 1986). X XII εrrccr or AWTI-SP-IO ANTI sen A, OH ιιιrecτιvιτv or mv IIO TEI IIT V-III B> IITLV-IIl^p, AMD HTLV-!1I MM Da * Poet Syncytium tlon Haul rai IKNI Ian ttt Cu.t f llliiϋiiliua laiauitiaallotta •it aat I on IIIV laolatea III, II! nr TΪT TΠ nr in MM >o si'-io πiii-rr 0 39 72 ♦(AO) 90 ♦<>B0) 111 * (>ao) at sp-io mn-rr 0 0 I 19 1 ii ♦ do) 3 ♦ Oo) (9 si'-io ιιιn(c) 0 0 3 A3 ♦(10) It. SP-IO ur(A)-TT • A sp-ai t I IB-IT 0 0 (com rul) I 23 1 A8 HO TT-IIIIS 0 0 (cont ol ) 2 II S y nthetic peπildca uara coupl β .l tβ tetanus to-old (TT) vith .--aL IdoUen.oyl-ll-hydro.yaueelrt lde .ater (MBS). jp -ιo ιιr (A) -cι sι Tr rcpcnviYi si— io mn(r)ιrrnκsiMQRcrcitY i /.l.m. In par dii il i tff βi are the Inver.e tι( aiir..* dllutl β ne that Inhibitad the nua-hcr of ayncytia ((0-βO) par well hy >βllϊ. Il i alion u. i dclcrnliiϋd liy cvnluai inn nf revert* tranacripta β e activity in aupemainn a of 11-9 culls cultured Inr IM t y β in ll i u presence ur 100 ii.lnclinui units of IIIV iaol.tca. V.luaa are Inver.e of nnli-acr iM a .lll.it Ion. that Inhibited r..u..r #ϋ tranβcrlpmβe activity liy >HOX. 43 Therefore / the above-described ability of goat anti-S?-10 sera to inhibit ETLV-ΣII B infectivity in the syncytiua inhibition assay and in the reverse transcriptase assay / indicated that anti-S?-10 antibodies are capable of blocking the binding of HIV gpl20 protein to T cell CD4 (T4) molecules. In addition / ςcat antiserum raised to a peptide [SP-10 RT(A)] containing S?-10-like sequence from HIV isolate ET V-ΣII BP inhibited syncytium formation by ET V-III-,, but net by ETLV-III-/ indicating that type-specific antigens contained in SP-10 R-f(A) are suitable as vaccine components to raise antibodies that inhibit the interaction of ETLV-III^ j . gpl20 and T cell CD4 (T4J molecules. EXAHSLE 5 Induction of Antibodies Capable o Inhibiting EIV col20-CD4 (T4) Interactions A series of studies were undertaken to deteraine 1) vhether the SP-10 peptide conjugated either to bovine albumin or to tetanus toxoid, exerted any inhibitory effect on antigen-specific, CD4 (T4) dependent / T cell responses in vitro; and 2) whether the anti-S?-10 antiserum (described in Example 4) bound to human white blood ceil populations not infected with HIV. When the SP-10 peptide was added directly in vitro human uninfected peripheral blood lymphocyte cultures stimulated with tetanus toxoid, no inhibition of normal T cell response to tetanus toxoid was observed (Table XIII) . 44 Table XIII SP-10-TT and SP-10-3SA DO HOT IHHIIIT AHTICEH SPECIFIC PiOLIFEaATIVE RESPOHSΣS OF HCRXAL HUKΛH ?E2X?HE2AL BLCCO LΩtPSOCYTES Additive CO Horsal fcJuman Peripheral Bleed Exp. No. I E:?. Ho. 2 Lymphocytes • in Culture CPM X IO 6 Lyraohocγt ts 3,100 61,000 100,400 35,800 144,200 rr 1:32 T7=ϊetanus toxoid (Wyeth Laboratories, Philadelphia, Pa.), 8SA=bovine serve albusia. CPM » cauacs per ainute of tritiated thvaidiae incorporation as described (Deaninj ec al J. Iaaunol. 138:630, 1987). 45 As seen in Table XIII, SF-10-TT a_loπe was as good an antigeπ-specif ic T cell activator as TT alone. Moreover SP-10-TT and SP-10-3SA vhen added to TT alone did not inhibit TT induced proliferation by noraal T cells. In addition, anti-SP-10 gcat seruα did not bind to peripheral blocd lymphocytes or monocytes in indirect immuno luorescence assay using flov cytofluorometry. These data indicate that the SP-10 peptide dees not perturb normal human T cell function that is dependent cn a functional CD A (T4) molecule but dees induce antibodies that will inhibit EIV epl20- CD4 (T4) interactions and neutralize EIV in reverse transcriptase inhibition assays. Thus, vaccines comprising the sr.ail synthetic S?-10-iike peptides (less than cr equal to about 35 amino acids in length) have distinct advantages ever EIV vaccines comprising recombinant cpl20, cr large subunits thereof, as the latter may interfere vith normal immune function. EXAMPLE 6 Isolate specific neutralization of EIV Synthetic peptide SP-10 has an amino acid sequence derived from and unique to the gpl20 envelope protein of HIV isolates ETLV-ΣII 3 and LAV, vhile other EIV isolates have varying degrees of differing amino acid sequences in their S?-10-like ςpl20 envelope proteins. Synthetic peptide SP-10 (that is, S?-10-∑II a ) frcm the ETLV-ΣΣΣ- isolate of EIV vas coupled to tetanus toxoid and used to raise anybodies in goats (0.5 mg cf conjugate per kg gcat body weight) as described by Palker et al. (Proc. 46 Nat'l. Acad. Sci. (USA) 84:2479, 1987). Coat antibodies raised to synthetic peptide SP-10 vere tested for the ability to neutralize four different EΣV isolates (Pig. 5A: ET V-III a , Fig. 5H: ETLV- III jj p, fig. 5C: ET V-ΣΣI^, Fig. 5D: ST V-II∑ SC ). Goat anti-S?-10 antiserum (#, pre-immune goat serum (0) and AΣDS patient serum (S) all at a 1/10 dilution vere first incubated vith dilutions (IO -1 , IO -2 , 10 "3 ) of each virus isolate. Next, these virus isolates were tested for the ability to infect E-9 T cells by cccultivaticn of virus and cells for 10 days in vitro. Levels of EIV present in cell culture supernatants after 10 days in culture vere estimated by measuring RT activity in supernatants, and results are expressed as cpm values obtained in RT assay. Increased cpc values in RT assay reflect increased levels ef EIV in culture. As shewn in Fig. SA, gcat anti-S?-10 antiserum inhibited {i.e. neutralized) ETiV-XIIg infection of E-9 cells at a virus dilution of IO "2 . Pre-immune gcat serum did not inhibit Ξ7LV- Illg infection at the same dilution of virus, ∑n contrast, gcat anti-S?-10 antiserum did net neutralize other isolates of EIV (Figs. 5 E-D). AIDS patient antibodies neutralized all fcur isolates of EIV (Figs. 5 A-0) .. The data indicate that gcat antiserum to synthetic peptide S?-10 neutralizes the ETLV-II∑ a isolate that contains in its gp!20 envelope protein the amino acid sequence present in SP-10. These data, alcng with data in Table XII,indicate that a vaccine comprising SP-10- like amino acid sequences frcm a variety cf EIV isolates will be effective against a wide spectrum cf EIV isolates. 47 EXM E 7 . Blndlno of Gcat Aπti-S?-10 Serum To ET V-IIIq- But Not To ETLV-Σ∑:..,- Infected E9 T Cells The reactivity cf goat anti-S?-10 serum and autologous prebleed control serum were ccapared on either uninfected E9 T cells, E9 T cells infected vith EIV isolate ETLV-III S , or E9 T ceils infected with EIV isolate ET V-IΣI 5Ϊ , using flow cytofluorcmetry and a Coulter Z?ICS V cytofluorcgraph (Ξaynes, Immunol. Rev. 57: 127, 1981; Eaynes et al.. New Snc. J. Ked. 304:319, 19 1). Goat anti-S?-10 serum (1:200) reacted vith 40. of ETLV-ΣI∑ a -infected Ξ9 T ceils coopered to ET V-IΣΣg-infected E9 cells incubated with control - (prebleed) goat serum (1:200) (Figure 6A) . Neither gcat anti-S?-10 nor control (prebleed) serum (1:50) reacted with noninfected E3 T cells (Figure 63). Neither control (prebleed) nor anti-S?-10 serum (1:50) bound to E9 T cells infected with the ETLV- HlfP isolate of EΣV (figure 6C) . 48 EXAMPLE 8 Develoeaent of a Synthetic Taaunocen Compris ing Mul t ip le Becions of the Human Tmauπodef lciencv Virus Envelope that Induces T He lper Cell . CDβ+ Cvtotoxic T Cel l and B Cell Neutral izing Responses Tn Vivo In order to develop a synthetic peptide immunogen that induces cytotoxic T cell responses to HIV in addition to inducing neutralizing antibody and T helper cell responses , a series of peptides vere prepared reflective of regions of the KIV EH isolate that have included therein a efined cytotoxic T cell epitope (see Table III) . These studies vere performed vith the HN EIV isolate since it appears that this is the Best cocao n prctotypic virus in the US at present (La Rosa et al Sc i ence 249 : 932 , 1990) . Taλahashi et al . I Science 246 : US , 1939) have defined a cytotoxic T cell (CTL) epitope that includes aaino acids 322-326 (FYTTX) frca the KH KIV isolate and includes aaino acids 323-329 of the KIVIII3 isolates (see Table IV) (Taλahashi et al J . Eys . Ked . 170 : 2023 , 1989) . Thus , one TI¬ SPIO variant peptide aade vas the Tl-S?10KH (λ) peptide vith the (A) signifying that aaino acids 322-326 vere added to the existing KH S?10 region ' cf aaino acids 303 -321 (see Table III) . Secondly, to aaJ e a synthetic peptide that might insert into the cell aeabrane of antigen-presenting cells and therefore potentially be processed and expressed via KHC Class I aolecules and therefore be recognized by CD3+ CTL, the first 12 aaino acids of the gp41 HIV envelope protein (aaino acids 519- 530 AVGIGALFLGFL in HIV isolate BH10/III3) vere covalently linked N-terainal to T1-SP10 peptides. These aaino acids (519-530) of HIV gp41 are highly hydrophobic . They have been postulated to be the priaary aaino acids that are capable cf inserting 436 49 into lipid seabranes and to play a role in the ability cf HIV to induce cell fusion (Brasseur βt al ΛTQS Sea , ffua . Retrovirol . 4 ; 83 . 1988) . Peptides vith this 12 aaino acid gp4 l sequence have the prefix ?- before the naae of the peptide (? for fusogenie region) (see Tables III and IV) . Bosch et al (Science 244 : 694 , 1989) have deaonstrated that the hoaologous region in SIV (GV7VLGFLGFLATAG) to the P region in HIV (AVGIGALFLGFL) is indeed the SIV fusion envelope peptide. Thus, it vas postulated that F- derivati∑ed peptides eight also insert into antigen presenting cell aeabranes , the F- deriYatized peptides vould be internalized and CD8÷ KKC Class I restricted CTL vould be generated 12 vivo follcvinc iaaunization vith F-derivatized peptides . (Deres et al fVature 342 : 561 , 1989) have shovn the conjugation of a fatty acid tripalai cyl-S-glycerylcysteinyl-seryl-serine aoiety to synthetic peptides can proaote synthetic peptide processing and presentation in the context cf KHC Class I aclecules and lead to generation of CD8r CTL n vivo . ) A series of studies vere perforaed in Balb/c aice vith the KH series of T1-SP10 peptides (Table III) to coapare their ability to induce anti-peptide antibodies (see Figure 7) , to coapare their ability to induce anti-HIV neutralizing antibodies (Figure 8) , and to determine if any of these peptides could induce KHC Class I restricted CD8 CTL vhen injected in nice in o (Tables XIV and XV) . 50 TA3L2 XIV ABILITY OF F-TI-S?IOO*(A) PSFΠDB TO ISDOC3 MS CLASS I-XESTJUCTED OΒ+ CYTOTOXIC T C2 3 Ef VTV3 DJ Ba2b/c 2CC2 % Specific S*Cr Release Target Cell (Claai I Type) et 50:1 E7 Ratio EΛI>. 1 fco.2 fccp. 51-78Y {S2-) Tl-S?10KΪ(λ) * L51-7SY (H2*) EL4 (H2*> Tl -STICKS (A. ( t ΣLA (E2 b l Tl-S210«ι{Λ) + L51 « 7aτ + C Tl 'SPICK* Cλ) ♦ LS1-7SY * Tfcy2.2 + C » Tl«S?l»»CA) + L51-7£ + Ly- ? ;.2{ 3S} ♦ C Tl«S?10Wf(A> ♦ LS1-7£Y + 172.4 ( CD4 ) * C « Soluble F-Tl-SrlOKH(λ) peptide (lOμg) in P3S vas injected subcutaneously in Balb/c mice in coaplete Freund's adjuvant (1st injection) and then incoaplete Freund's adjuvant (2nd through 5th injections) . Elec or cells vere splenocytes of iaaunized animals grcvn for 7 d ys in vitro vith 25μg/al F- TI-S?10HH(A) peotide in coaplete T cell aedia (Takahashi et al. J. T . Xed, 170:2023, 1989). On day 3 cf culture 10* v/v Con A supernatant vas added. • Con A supernatant vas derived frca Balb/c spleen cell stiaulated A days vith Concanavalin A lOμg/al in coaolete T cell aediua (Takahashi et al. J. E*a. We . 170:2023, 1939), and the supernatants reacved and used in activation of spleen cytotoxic T cells. ♦Ixperiaents vith coapleaent (C) represent the results froa pooled s lenocytes fron 3 aice iaaunized vith F-Tl-S?10KH(λ) . 51 TA3L2 XV A2mτγ o? π-snoni(A) PSPTI S rs UPOSQSS TO rsccs vac CLASS I HZSTI TZD C&+ CYTOTOXIC T CELLS TW VT 3 TH Balb/C KTC3 Tarςβt Cell (Class 3 Type) % Specific ^x Xeleaa« at 50:1 ET Satio Bxa. 1 tea. 2 S1-78Y C£C*1 IS 0 Tl-SPlOHiiA. -cβeted L51-78Y (H?*) 39 35 EL4 or RL-12 (K2 b )« 10 2 Tl-S?lO0i'(A) -caated EL4 oc RL-12 (H2-) * U 4 Tl-S?10«H{λ; + 51-78Y * C NS 27 T1-S?1CKH(A. ♦ 51-78Y + Lyt2.2{C€) ♦ C >B 2 Tl-S?lC*yfA ) + L51-78T + \12.ΛtCZA) * C W 31 Liposoaes vere prepared using Octylglucoside 7 " t (0.7g/10 al PBS) L-alpha dioleoyl lecithin, 20 ac/al, Tl-S?10JOϊ(λ) peptide and cholesterol 3.1 ag/ai using standard techniques (Hiaas et al. Biccheaistry 20:833, 1931; Lipcsoae Technology Vol. Ill 2d. G. Gregcriidis Chapter 14 po. 205- 224, 19S4. Liposoaes containing T1-S?10KH(A) peptide vere injected into Ealb/c sice in a total dose of lOμg of T1-SP10MN (A) peptide in liposoaes in 2 sites - subcutaneously in coaplete Freund's adjuvant (1st dose) followed by inccrplete Freund's adjuvant (2nd through 5th doses). Effector cells vere splenocytes frca iaaunized aniaals prepared and shovn and described in Table XIV. *∑xp. 1 used RL-12 targets cells and Exp. 2 used ZL4 target cells. Ccacariscn of the abili y of various Tl-S?10 peotides o induce anti-ceOtide antibodies in Balb/c mice: Figure 7 shovs a conparison of the levels of anti-peptide antibody generated in the serua of Balb/c aice after 1, 2 and 3 iaaunizaticns vith 10 μ/al of the various peptides. Figure 7 shovs that 52 after the second iaaunization, addition of either the (A) region or the ? region increased the level of anti-peptide antibodies to the T1-SP10KH peptide in ELISA assay. Coaoarisoπ of the abil itv of various T1-SP10 peotides to Induce an i-HTV neutral izing antibodies In Balb/c alee. Figure 8 shovs the percent inhibition of HIV syncytiua foraation in vitro vhen antisera froa bleed 3 vere added to a HIV syncytiua inhibition assay. Whereas only 1 animal in the Tl-S?lθ-inj ected group and none cf the aniaals in the F-T1-SF10 and the Tl-S?10 (λ) groups had serua antibodies that inhibited syncytiua foraation by greater than 50* , 3 cf 5 aniaals in the group of a iaals inj ected vith F-T1-S?10KH (A) had antibody levels that neutralized syncytiua foraation by greater than 50* (Figure 8) . Abilitv of various T1-SP10 oeotides to induce CP8+ CTL ln vivo : When injected into Balb/c aice , neither T1-SP10XX peptide nor F-T1-SP10HN peptide induced aeasurable CTL in Balb/c nice. Kcvever, soluble . F-Tl-S?10KH(λ) peptide (Table XIV) and Tl- S?10H (A) peptide in liposoaes (Table XV) vhen injected n vivo vere capable of inducing anti- HIV CTL in Balb/c aice that killed T1-S?10KX(A) coated D' target cells in vivo . Table XIV shovs that the cytotoxic T cells induced in Balb/c aice by the soluble F-T1-5?10KH(A) peptide in vo vere Thyl+, Ly2 (CD8) +. CD8+ anti-Tl-SPlO (A) cytotoxic T cells killed only B2* targets and did not kill H2* targets . Table XV shovs that the anti-Tl- S?10 (λ) cytotoxic T ceils induced by Tl-S?10 (λ) peptide in liposoaes vere CD8+ and KHC Class I restricted . 436 53 Thus , by addition of the F sequence and the (A) sequence (Table III) to the Tl-SPIOMN sequence , it was possible to construct a 52 aaino acid peptide " [7-Tl-S?10MN (λ) ] capable of inducing not only neutralizing antibodies and helper T cell responses , but also capable of inducing CD8+ anti- HIV MHC Class I restricted cytotoxic T cells as well . In addition, MHC Class I restricted anti-HXV cytotoxic T cells have been induced _ n vjvc by a 40 aaino acid peptide, T1-S?10M (A) incorporated into liposomes . EXAMr E 9 As has been shewn, the cons c ion of a synthetic peptide Ti-≤?10(A) containing aa303-327 of EIV gpI20 V3 loop [S?10(AJ] and aa 428-43 cf HIV gp!20 (Tl) serves as a potent T cell iaaunogen for induction of activation cf anti-HIV meaory T helper cells and B cell iaauncgen fcr anti-HIV neutralizing antibodies in vivo (Palker et al., PH ≤ rαs ■ 85:1932-1936, 1533; Palker et al., . Iaaunol.. 142:3612-3619, 1539; Hart et al., J. Iaaunol.. 145:2677-2635, 1590 and Hart et al., P AS rusAi . 83:9443-9452, 1551). The TlS?10(λ) peptide induces anti-HIV neutralizing antibodies in mice, goats and - rhesus monkeys (Palker et al., PNAS ΠIS I . 85:1932- 1936, 1933; Palker et al., J. Iaaunol.. 142:3612- 3619, 1985; Hart et al., J. Iaaunol.. 145:2677-2635, 1950 and Hart et al., λS rusAi . 88:5443-9452, 1991) , induces anti-HIV MHC Class I-restricted CTL in aice (Hart et al., PKAS ΓUSAT . 88:9443-5452, 1991) , and induces anti-HIV T helper cell responses in mice, goats, rhesus nenkeys, and chiapanzees (Palker et al., FKAS rυsλ . 85:1932-1936, 1938; Palker et al., J. Iaaunol .. 142:3612-3615, 1989; Hart et al. , J. Iaaunol.. 145:2677-2635, 1550 and Hart et al., PHAS fϋS K 83:9448-9452, 1591) . In a recently ccapietad study in aice, it was found that while CS7SL/5 and Balb/c aica πia a high titers cf 54 anti-peptide antibodies to T1SP10III3 peptides, these mouse strains make no neutralizing antibodies to the HIVIII3 V3 loop neutralizing deterainants. In contrast, CS7BL6 and Balb/c mice make good anti- HIV neutralizing antibodies when iaaunized with TISPIO peptides containing sequences rca "the HIVMN V3 loop. Immunization trials in chimpanzees vere conducted to test the synthetic peptides. The results discussed below show that the chiapanzees at Holloπan AFT, New Mexico, made good antibody and good helper T cell responses to T1S?10III3(A) peptides, but like Ealb/c mice, did net make antibodies against the neutralizing antibody deterainants cn the HIVIIIB V3 lecp. In Figure S the chiapanzees were iaaunized with EIV env synthetic peptides and their antibedy titers tested using an ELISA assay. Aniaals 834 and 1023 vere iaaunized vith peptide Tl- SP10III3 which vas alsc used in the ELISA assay. Peptide F-Tl-S?lQΣII3(λ) was used in the iaaunization and ELISA assays for aniaals 1045 and 1070. All iaaunizations vere in IFA ÷ ?ΞS (1:1) except for aniaal 1023 that developed IM abscesses after the third iaaunization and had one iaaunization held. Subsequent iaaunizaticns were given in PBS only. As can be seen, Tl-SrlO peptides were excellent iaaunogens in aniaals 834 and 1023, while T1-SP10 peptides with the KIV gp41 fusion (F) dcπain synthesized N-tarainai to the T1-SP10 peptide did not induce antibedy titers as high or as cf long duration as did peptides without the F dcaain. It should be noted that aniaals 1045 and 1070 were challenged at πoπth 16 with the iaaunogen T1-S?10III3(A) that induced good antibedy titers in aniaals 834 and 1023. Aniaals 1045 and 1023 did not respond to Tl-S?10III3(λ) in IFA, thus daacnstrating that they were tolerant tc the T1-S?10(A) peptide froa their prior iaaunizations with F-T1-S?10III3(A) peptide. It is also iapcrcant to note that while boost of aniaal 884 at week 14 gave a rise in titer to T1-S?10III3(A) peptide, boost of aniaal 1028 at the saae tiae did not. Aniaal 884 was boosted vith IFA, while boost of 1023 was vith no adjuvant, but rather only PSS. The peripheral blood mcncnuclear cell (PSHC) proliferative response to the ianunizing peptides was also studied (see Figure 10) . Peptides T1-SP10III3 and T1-S?10III3(A) induced high levels cf proliferation of circulating PΞMC in aniaals 884 and 1028. These levels fell to non-detectable levels after a 6 aonth rest (aonth 14) but rose again in aniaals 834 and 1028. Proliferative responses in aniaal 1023 rose with each boost after the 6 aonth rest even thcuch the iaaunizaticns were in PΞS alone vith no adjuvant. As vith E cell responses, aniaals 1045 and 1070, that vere iaaunized vith F-T1-S?10II:Ξ(A) peptide, did net proliferate to T1-S?10IIIΞ(A) peptide. When these latter two aniaals were iaaunized vith the T1-S?I0III3(A) peptide that vas a coed iaaunogen in 834 and 102S, neither of the aniaals 1045, 1070 developed a proliferative response to T1-S?10III3(A) which proves that the addition of the F-dcaain N-terainal to the T1-SP10 peptide created a toleragen that tolerized aniaals 1045 and 1070 to the Ti and SP10 regions cf gp!20. As shewn in Table XVI, vhile aniaals 884 and 1023 both responded in proliferative assays to native g?120, aniaals 1045 and 1070 vere tolerant to native gp!20 as well as tc iaaunizing peptides. The PSMC proliferative responses of chiapanzees iaaunized vith the synthetic peptides to PHA was also studied (see Figure 11) . The data shew that while aniaals 1045 and 1070 were tolerant to Tl and SP10 regions of EIV gp!2C, PΞHC FI-Lλ responses in these aniaals throughout the iaaunization period vere ncraal . The saae T1-SP10III3 peptide batches used in the chiapanzee study, vere also used as iaauncgens in goats, and good anti-HIV neutralization titers in goats vere obtained (See Figure 12) . Thus , T1-SP10 peptides vere superb iaaunogens vith I?λ in chiapanrees , vith reaarkaale anti-peptide serua antibody titers of >1 : 102 , 400 (See Figure 9) , and induction of T cell response to T1-SP10 and to native KIV g l20 (See Figure 10 and Table XVI belov) . TA3LΞ XVI .ijtre! TΛγmX.iXΛΛ t.-> srρsr it Lea ot Peri heral 3 loo; Λonooucl «»r Ca'mm' l rolievϋβ. T» f.'.rn Stijuiatioo WltΛ UV lϊv jpW •Sata rapraiant the paat ϊpl»° rβaponjβi ob»arv«d during I * iaαun^zatian TA2LΞ XVII Anti-IXVIχa(iAΣ, HawaiHia, Antibody ?it.r> la O ^n:*.. ba «!*,< „ lta JynW «. c ,„. -„ Cantaiaia β T and l CU Dat ni.-.ajιu of KVI∑Z3<UL. ) 57U0 tav,*^. >«P*-*a O 97/14436 TA3LZ XVIII Svncytiua Inhibition Serum Neutralizing Ar.tibc y Titers cf Rhesus * Jicnkay 13S78 Iaaunized with SOOuc ' Ti-SrlOM (λ) Peptide ∑gancniza ion No . ( H Η 1 HA Titer f∑rvi 1 0 ND 2 20 40 40 40 10 40 20 10 0* Nd « Ne tr lising Antibedy Pd s Fencing ND = Not co e * ■ = 68% inhibition of syncytia at 1 : 10 dilution. T h e high neutralizing antibedy responses of coats to the saae T1-SP10 peptide batches used in chir.par.zees danenst rated that chimpanzees selectively did not recognize the neutralizing V3 sequences as iaauncge.nic, while other non- neutral izaticr. T1-SP10III3 peptide sequences were imr.ur.cce.nic in chiapanzees . Thus , it is possible that selective proteclysis of the HIVIII3 V3 loop occurs by chiapanzee and nousa mcnenuclear- cells in vive . cr mora likely , that genetic restriction of antibedy responses to neutralizing deterainants cf the V3 loop exist in chiapanzees and nice . In rhesus aenkeys , the inj ection of 500 ug of purified T1S?10MN (A) peptides vas shcvr. to yield very high levels cf anti-KIVKJ neutralizing antibodies in 4/4 aniaals (see Figure 13-15) . In addition, in 1 cut cf 4 monkeys , iaaunization yielded cross-reactive anti-HIV neutralizing antibodies that neutralized the EIVIII3 and HIVMN viruses (see Table XVIII belcv) . Thus , if 25* chiapanzees a.-.d huaar.s respond to the Tl-SPIOMN (λ) peptide and aa a cross-neutralizing anti-HIV env anti b odies , t h en an additional 5* cf subjects 436 challenged vith an cthervise ncπ-MN-like KIV isolate couid he protected froa HIV challenge. See figures 16 and 17 for additional monkey data. Because T1-S?10III3(A) peptides did not induce anti-HIV II3 neutralizing antibodies in aniaals 834 and 1028, and because F-T1-S?:0III(3)A peptides induced tolerance in aniaals 1045 and 1070, all cf the chiapanzees vere iaaunized at either πcn h 16 (aniaals 884, 1028) or month 17 (aniaals 1045, 1070) vith Tl-SPIOMN(A) peptide. The rationale here vas to determine A) if the II- SPIQMN(A) peptide could break tolerance ir. aniaals 1045 and 1070, and E) if any cf the aniaals could genetically see the V3 neutralizing deterainants cf EIV MN V3 loop, since it appeared that none cf the aniaals could see the V2 deterainants cf EIV III3 as presented by T1-SPI0III3 peptides. Figure 13 shovs that after iaaunization of all 4 chiapanzees vith 0.lag/kg of T1-SP10M (A) peptide, three cf the 4 aniaals (334, 102S and 1045 shoved the appearance cf weak serua anti-HIV MN neutralizing antibodies (dotted lines) , while aniaal 1070 developed high levels of anti-HIV MN neutralizing antibodies that titered to >30% neutralization at 1:20, and also cross-neutralized EIV III3 (Table XIX, solid lines, Figure 13) . This break in tolerance can also be seen in the rise in titer to Tl-SPIOMN(A) peptide in the serua cf aniaals 834, 1045 and 1070 (Figure 19). Aniaal 1023 had an early abscess associated vith the iaaunizaticns and did not receive IFA after month 4 of the study, and never had an antibody rise to peptide cf_ EIV after the initial iaaunizaticn of peptide vith IFA. 5B = TA3LΞ XIX a-zzilizx-ti n β j JJV IΛZ/.ZZ3 And 317 «f ia S ccyxiss usϋitisn Assay la C X Z-ΛΛZ—3 j—my- d wiϋ 71-S710 »^— « Prtsanca ai Mumϋaas sn la Syscytisa Inhiaisisn Assay f*«cisr3cal Tlsar ia HT rasi-ii ion Jtsmavt III - - - 102S - - 1045 - - */- (23) 1030 • »/. , 9 ~ , . (221 ♦ f 100 ) « (?6 ) «/- HO *- 1350) - • < 4βt iafiiβisiss oi syne/ϊia »/- - > «J* and < !ϊ\ iasϋSitiSB βi syβeysia • • * SOt iafcώitisr. o. sγseyua, titir X; 10 — ■ a 101 Λaϋiϊiϊ.- oi syneysia, Siϊtr 1:20 The observation that neutralizing antibodies in chiapanzee 1070 neutralized both EIV HN and III3 isolates could be cue to the presence of either type-speci ic neutralizinc antibodies induced by both EIV M and EIV III3 peptides (Susche et al Proc. Natl. Acad. Sci. USA S5:31S£ (1983)), cr be due to the induction of cross-neutralizing anti- GrGRA antibodies by the Tl-SPIOMN(λ) peptides. Antibedy titers against a truncated SP10III3 peptide, SP1CD, IP.IQRGPGP, vas used in ELISA assay vith serua froa chiapanzee 1070. Ξnd-point ELISA titers against this peptide vere 1:800 or less froa 10-23-50 through 12-3-51 (aonths 3 through 17 of study) . Folloving the first immunization of aniaal 1070 vith T1-SP10M (A) cn 12-3-52, the titers of antibody to SP10D peptide rose froa 1:800 to 1:3200 on 1-7-92 and 1:12,800 c 2-4-52. During the saae tiae pericd, antibedy titers cf 1070 to T1-SP10M (A) peptide rose froa 1:12,800 to 1:102,400, vhile titers to the T1-ΞP10III3 peptide rose froa 1:3200 to 1:25,600. Absorption studies to absorb cut the neutralizing antibodies in aniaal 10 7 0 serua 4436 58b demonstrated that ail of the anti-Ξ V MN neutralizinc activity couid be absorbed cut with the S?10KN(A) peptide, and pur-, of the EIV KN neutralizinc activity couid be absorbed cut with a peptide with the sequence IG?GaλIG?GRAΣG?G2λC (DP2) (Janaverian et al Science, 250:1590 (1950)) that only contains sequences from the tip cf the V3 loop that are common to both EIV KN and EIV IIIB (Figure 20) . Thus, a portion of the chimpanzee antibedy response induced by Tl-S?l0KN(λ) peptides cross-neutralize EIV KN and EIV III3 and are directed against the conserved sequences at the tip cf the EIV g 120 V3 loop. Ir.pcrt-.ntly, i- rhesus monkeys it was demonstrated that injection of 500ug of purified T1S?10KN(A) peptides yielded very high levels of anti-HIVM neutralizing ar.tibcdies in 4/4 animals (Figures 20-24) and in 1 cf 4 rhesus monkeys, yielded cross-reactive anti-HIV neutralizing antibodies that neutralized the EIVIII3 and EIVMN viruses (Tables XVIII and XX) . Figure 25 shows that the D?2 (IGSGaλlGFGSAIGrrSAC) peptide absorbs the anti-HIVIIIΞ neutralizing activity in serum from rhesus monkey 185S7. Table XXI shows the sequences of peptides used in the chimpanzee and rhesus menkey studies described. TA3 Ξ XX Cross Neutralisation of EIV LAI/III3 In RT Inhibition λssav Ev immune Rhesus Kcrcev Sera From Animals " immunized with Tl-S?10 Peptides D e Animal No. 13987 17336 : /HIS ∞ LAI /III3 1GT_ r T i ar ia RT inhibition^ IΛ • E-ΞΪΞ xcn Saquaπct of Full Ltnςth T 1-S?10(A) Peptldat For Human Immunization KCIlNMWCεVGXAMYACTnPNYNXRKniHIGPGSAFYTTX 71-S?10 N(A) KQllNMWCEYGXAMYACTnFNN TRKSrrXGPGnVlYATG Tl-S?10RF(A) KQllNMWCEYGXAMYACTnPGNNTRXSIFlGPGnAFlATS T1-S?10EY91(A>- QilNMWCEVGXAMYACTRPHNNTn SiHMGPGKAFYTTG T 1-S?10CanOA(A) Human patients, both K A 2A- a d HLA 2A-, will be studied for cp tc two years. During treatment, the generation cf neutralizing antibodies against EIVMM and other EIV isolates as veil as the generation cf T helper and/or Class I-restricted anti-HIV CTL will be measured. The immunccer.s to be used will be TISPIO(A) peptides which are expected to give rise to antibodies against 80% cf the EIV isolates in the Los Alamcs Data Set (Myers et al., Human Retroviruses ar.d AIDS 1?5H . Some patients will receive the immunogens in Table XX and sere will receive the immunogens in Table XIX. Each patient will receive as immunogen dose about O.OSmg/kg/peptide cr l g cf each peptide. If no responses to the original dose schedule result, the desε will be doubled and the regimen repeated after a three mcπth rest. Incomplete Freund's adjuvant (IFA) will be nixed with the immunogen in a 1:1 v/v mixture (Hart al Immunol :2677-2S35, 1990) Total volume fcr each immunization should be 2cc. The immunogens will be administsrad by IM. The immunccer.s will bε mixed in a total volume of 2c 60 and given IM, lcc in each of two sites ( right or left upper arm, right or left thigh) . Immunizations will be given at 0 month, 1 month and 3 months . The patients will be monitored 4 weeks after each immunization. After the third immunization, the titer of responses to EIV will be tested and a decision made regarding immunization with a larger dose of peptide to begin after a three month rest. Routine blood and urine tests will be conducted on the patients . The following blood samples will be required. Serum ( 10 ml) (approximately 20co blood) will be used to study TISPIO and S?10 peptide binding in RIA and EIV g?120 binding in RIP/Western blot assays . Serum will also be used to determine neutral ization titers of ET V-III3, KTLV-IZJCϊ and field EIV isolates in reverse transcriptase and/or syncytium inhibition assays . Routine serum chemistries for toxicity ( liver function tests , renal functions and chem 13 panel ) and a complete blood count ( lOcc heparinized bleed) will be performed. Peripheral blcod cells ( 60 ml blood) will be used to study 7 cell proliferative responses to PEA, TT candidate TISPIO and SP10 peptides , gp!20 and OXT3 (about 30 ml heparinized blood} . T cell , B cell , NX cell , CD4 and CD8 call numbers will also be measured (about 5 ml heparinized blood) . Finally, CTL assays will be performed on autologous cr ELA- identical E3V-transformed B cell lines cr autologous E3V-trans ormed B cell lines using vaccinia gplSO infected targets and peptide coated targets . 60a EXAMPLE 11 A strategy has been developed for the design of experimental synthetic peptide immunogens for induction of T helper (Th) cells, neutralizing antibodies, and MHC Class I-restricted cytotoxic T lymphocytes (CTL) against EIV native proteins or EIV protein-expressing target cells (Palker et al, Proc. Natl. Acad. Sci. USA 85:1932 (1988), Palker et al, J. Immunol. 142:3612 (1989), Hart et al, J. Immunol. 145:2677 (1990), Hart et al, Proc. Natl. Acad. Sci. USA 88:9448 (1991), Eayaβs et al, AIDS Res. & Human Retroviruses 6:35 (1990), Eaynes et al, J. Immunol. 151:1646 (1993), Eaynes et al, J. Exp. Med. 177:717 (1993), Eaynes et al, Trans. Amer. Assoc. Physician 106:31 (1993), Yasutomi et al, J. Immunol. 151:5096 (1993)) . (A general scheme for an HLA-based vaccine for AIDS is set forth in Figure 30. Th 1> - -:1 - B 1- > >n includes the construct Th-SPIO and, by example, C4-V3. ^l.-.n" Tc l ...n is equivalent to Th-CTL ( CTL = X) ) . A general immunogen design for induction of neutralizing antibodies requires the synthesis cf one or more Th epitopes of EIV proteins N-terminal to the gp!20 envelope V3 loop neutralizing domain (Th-B, Figure 26) . For MHC Class I-restricted anti-HIV or anti-SIV CTL induction, both Th-3-CTL and Th-CTL peptide designs have been successful (Figure 25) (Hart et al, Proc. Natl. Acad. Sci. USA 88:9443 (1991), Yasutomi et al, J. Immunol. 151:5096 (1993)). Prototype synthetic peptide immunogens comprised of Th-3-CTL epitopes of EIVIIIΞ, MN or RF env g?120 have: a) induced Th responses to native gp!20 in mice, goats, rhesus monkeys and chimpanzees (Palker et al, Proc. Natl. Acad. Sci. USA 85:1932 (1983), Palker et al, J. Immunol. 142:3612 (1989), Hart et al, J. Immunol. 145:2677 (1990), Eaynes et a , J. Ex?. Med. 177:717 60b (19S3)) and b) induced in goats, rhesus monkeys and chimpanzees B cell neutralizinc antibody responses that have neutralized laboratory EIV isolates in a type- specific manner (Palker et al, Proc. Natl. Acad. Sci. USA 65:1932 (1988), Palker et al, J. Immunol. 142:3612 (1985), Hart et al, J. Immunol. 145:2677 (1990) , Haynes et al, J. Immunol. 151:1646 (1993), Eaynes et al, J. Exp. Med. 177:717 (1993), Eaynes et al, Trans. Amer. Asscc. Physician 106:31 (1993)) and c) induced in mice and rhesus monkeys anti-HIV or SIV MHC Class I- restricted CTL that kill target cells expressions EIV cr SIV proteins (Hart et al, Proc. Natl. Acad. Sci. USA 88:5443 (1951), Yasutomi et al, J. Immunol. 1=1:5096 (1953)) . In rhesus monkeys, it has been demonstrated ' that the Tl-SPIOMN(A) peptide induced in select animals antibodies that reacted primarily with the IGPGΛA? sequence at the tip of the V3 loop, and cross- neutralized EIVIIIΞ, EIVMN, EIVΛF as well as EIV primary isolates crown in CΞM cells (Haynes et al, J. Immunol. 151:1646 (1993)) . Dssicr. of A Prstotvos Polwalsr. EIV Immuncσer. Eecause cf the extreme variability that exists in EIV isolates both in geographic locations and among patients, a multivalent EIV immunogen design tailored to EIV isolates in specific geographic locations, will likely be required for successful preventive and therapeutic EIV immunogens (Falker et al, J. Immunol. 142:3612 (1585) , Eaynes et al, Trans. Amer. Asscc. Physician 106:31 (1993)) . To this end, a prototype polyvalent EIV immunogen has been designed containing Th-Ξ-CTL epitcpes reflective cf 4 common EIV isolate motifs in Clave E, EIVMN, HIVRF, EIVΞVS1, and EIVCANO (Figures 2S and 27) . In each of these prototype peptides resides at least two Th deterτ.inants, two Class I-restricted CTL deterr.ir.ants, 60c one restricted by ELA A2 and A3 (Clerici et al, Nature 339:383 (1939)) and another restricted by E7 (Safrit et al, Characterization of E A-37-Restricted cytotoxic T lymphocyte clones specific for the third variable region EIV cpl20, isolated from two patients during acute seronversion. Presented at the 6th NCVDG meeting Oct. 30 - Nov. 4, 1993)), and three or more epitopes recognized by anti-EIV neutralizing antibodies (Palker et al, Proc. Natl. Acad. Sci. USA 85:1932 (1933), Rusche et al, Froc. Natl. Acad. Sci. USA 85:3193 (1988), Jahavarian et al, Science 250:1590 (1590)) (Figures 2£ and 27) . Preclinial studies of this prototype peptide mixture in mice have demonstrated that two cf the components CTI-S IORF(A) and Tl- • SP10ΞV9KA) ] induced type-specific anti-V3 peptide [anti-S?10 (A) ] responses (Table XXIV) , while two of the components [T1-SP10MN(A) and Tl-SPIOCANO(A) ] induced broadly cross-reactive anti-V3 peptide antibody resoonse (Table XXIV) . 60c TA3LZ XXIV Ability of HIV Envelope gp120 Synthetic Peptides To Induce Antl-Peptide Antibodies After Three immunizations Synthetic Peptides Synthetic Peptides Used In ELISA Assays Used To Immunize Mice Balb c mice were immunized three with 50 ug of monovalent peptides subcutaneously in IFA (Seppic ISA 51). Animals were bled 2 weeks after the immunization, and antibody titers determined uslncj er.d-point ELISA assays (E/C 3.0). Data represent the geometric mean titers of ser m antibodies of three mice for each point. Ξliza method are described in Eaynes et al, J. Immun 1. 151.-1S4S (1593), Eaynes ec al, J. Exp. Med. 177:717 ( 1953 ) , Eayr.es et al, Trans. Aπier. Asscc. Physician 106:31 (1953) . 60e Abilitv of EIV Env Pentides to Induce Cross-Reactive Peotide Responses to African (Clave A) and Thailand (Clave E) EIV Isolate V3 Looo Peotidss In addition, sera from goats and mice immunized with mixtures of all 4 peptides (Table XXV) , contained antibodies that also cross-reacted with the T1-SP10(A)A. con. peptide (a Th-3-CTL peptide reflective of the consensus V3 loop sequence of Clave A in Africa) , and to a lesser extent, with the TI¬ SPIO (A) Ξ.con. peptide (a Th-3-CTL peptide reflective of the consensus V3 locp sequence of Clave E in Thailand (Table XXV) . When sera frcm mice immunized with cr.ly one of each of the 4 components of the polyvalent mixture were tested for their ability to bind tc the African T1-SP10 (A)A.con. peptide, it was found that the Tl-SPIOCANO (A) peptide was responsible for generating all of the cross-reactive antibodies to the African Clave A consensus sequence. Thus, although the primary V3 sequences of the CANO envelope is widely disparate from other EIV env V3 sequences (Figure 27) , secondary and possibly higher order structures of the V3 loop of the ΞIVCANC isolate appear to have the ability to induce cross-reactive anti-V3 antibodies against many different EIV V3 motifs. 60f IA3LΞ XXV Ability of Th-3 HIV Env Polyvalent Peptide Mixture To Prijne and Boost Mouse and Gcat Serum Antibodies That Cross-React With The Th-3 Peptide With V3 Clave A CVΞ2GPGQAFΪAT) Consensus Sequences Th-B Peptide V3 Sequence Data represent the gecπetric mean titers of either 3 mice or 2 gceits injected with the polyvalent Th-3 KlVer.v peptide mixture. Method used are described in Eaynes et al, J. Immunol. 151:1646 (19S3) , Eaynes et al, J. Exp. Med. 177:717 (1953f, Eaynes et al, Tra s. Amer. Assoc. Physician ,3 K V «oΞra F0R AFRICAN (A'C0N) AND SOUT »^ ST AS . AN Tl SP10 A KQIIN W Q RVGQAMYAT PNNNTRKSVHIGPG Q AFYATGDI T1-SP10(A)A.coπ K^N W Q GAG Q AMYATRPSNNTHTSITIGPGQVFYRTGDI Tl-SP10(A)E.con Human Retroviruses and AIDS 19 • edited by G. Myers, J. A. Bβrzcfsky, B. Korber, R. F. Smith, and G. N. Paviakis published by the Theoretical Biclogy and Biophysics Group T-10 Mail Stop K710 Las Alamos National Laboratory Los Alamos, NM 87545 60g Neutralizinc Antibody Responses Generated Bv The Polyvalent EIV Env Irπmur.oσer. Regarding neutralizing antibody responses, sera from animals immunized with the polyvalent immunogen (Figure 27) bind to EIV gpi20III3 and cpi20SF2 in either raάicitπmunoprecipitation assay cr in ELISA assay. Sera from these animals have neutralized HIVMN and R? isolates in syncytium inhibition assays . PCTSπ.s raς;or; ςf a N^; i;j c CD4-V3 Con.forma-jo al Determinant in EIV cnl20 The 17b and 48c human an i-gp!20 mabs were isolated from human FΞMC B cells frcm patients infected with EIV (Thali et al, J. Virol. €7:3978-3988 (1993); Moore et al, AIDS Res. Kumar.. Retroviral. 9:1185 (1993)) . The 17b and 48c mabs cross-block mouse mabs that block CD4 binding to g?12C, broadly neutralize disparate EIV isolates, but do not in and of themselves block g?120-O4 binding (Mccre et al, personal communication, 1994; Thali et al, J. Virol. 67:3978 (1993)) . Rather, bindinc cf the 48d mab is unregulated to native g?i20 following ligation of gpl20 by CD4. It has been found that one peptide, Tl-SP OCANO(A) , binds to the 48c mab (Figure 28), and the optimal binding of mab 43d to EIV env hybrid peptide Tl-SPIOCANO(A) depended on the preser.se of the CD4 peptide, Tl N- ter inal to the SPIOCANO(A) peptide (Figure 29) . Thus, the Tl-SPIOCANO(A) hybrid C4-V3 petide mirrors a conformational determinant cf EIV gpl2Q recognized by a potent broadly neutralizing human mab. It is interest that Wyatt et al, J. Virol. 6c:6997 (1992) and Mcore et al, J. Virol. 67:4785 (1993) have suggested that the V3 loop [S?10(A)] and the C4, Tl regicn are in close physical proximity tc each ether ir. native gpi20. Thus, the present data directly demonstrate that the 97/14436 60h Tl-SPIOCANO (A) synthetic peptide can mimic broadly neutralizing C4-V3 conformational determinants of native gpl20. General stratecv for identification of multiple CD4-V3 fTl-SPlOCAn peptides that mirror other EIV isolate C4-V3 conformational determinants Whereas the EIV V3 loop by itself induces primarily type-specific anti-EIV neutralizing antibodies, the C4-V3 determinant as defined by the Tl- SPIOCANO(A) peptide will induce more broadly, cross- reactive neutralizing antibodies. This is known from the fact that the 43d human moncclonal antibody derived frcm a EIV seropositive patient binds to a complex conformational determinant on the surface of gpl20, binds to a wide spectrum cf EIV isolates and neutralizes disparate EIV isolates such as EIVIII3 and EIVMN (Thali et al, J. Virol. 67:3978 (1993) ; Mcαre, J. personal communication (1994)) . Thus, a general strategy for identifying multiple C4-V3 peptides would be to construct a large number cf C4-V3 peptides derived from C4 sequences (for example, from amino acids 419 to 428 from the EIVMN isolate and from homologous regions in other EIV isolates) linked N-terminal to SP10 or SP10 (A) regions (such as amino acids 301-327 of EIVMN and from homologous regions in other EIV isolates) from sequences listed in the Los Alamos database (Ξuman Retrovirus and AIDS, 1991, 1992, 1993 edited by G. Myers, J.A. Eerzofsky, B. Korber, B.F. Smith and G.N. Paviakis, published by the Theoretical Eiolocy and Biophysic Group T-10, Mail Stop K710, Los Alamos National Laboratory, Los Alamos, NM 87545) (see Table XXVI for other examples) . One would then screen approximately 40 tc 100 of these different C4-V3 peptides against a combinatorial library cf heavy and licht chain immuncciobuiin variable genes expressed 7/14436- 60i on the surface of phage (Borbas et al, Proc. Natl. Acad. Sci. USA 88:7978 (1991)). The work of Ecrbas et al provides a method for screening a large number (IO 7 to 10 8 ) of human monoclonal antibodies derived frcm a patient with EIV infection making a screen possible of a wide spectrum of antibody responses to search for antibodies species against complex conformational determinants on epi20. Using this technology C4-V3 peptides can be identified that are in such a conformation to fit into the Fab notch of the variable region of the heavy and light chain hεterodimer expressed in the combinatorial library on the surface of phage. These Fab monoclonals can be isolated and cloned (Ξcrbas et al, Proc. Natl. Aca . Sci. USA, 88:7978 (1991)). Most importantly, C4-V3 peptide designs can be identified that reflect native σpl20 C4- V3 conformational determinants of a wide variety of EIV strains. When this type of protein based selection is performed with combinatorial libraries derived frcm a large number of EIV-infected individuals frcm many different geographic sites of EIV infection around the world, a wide selection of C4-V3 peptides that mimic broadly reactive neutralizing determinants from the C4- V3 region of native gpl20 can be identified and, for example, combined together with the Tl-SPIOCANO(A) prototype C4-V3 peptide into a multivalent C4-V3 peptide immunogen for induction of highly cross- reactive, broadly-neutralizing antibodies against C4-V3 conformational determinants of multiole EIV strains. ΛH' ' • XXVI C4-V3 Imnr .. ηen Constructs Tl SP10 C4-V3LAI KQFΪNMWQEVGKΛMYATRPNNNTRKSIRIQRGPGRΛFVTIG C4-V3IIXD2R KQ I I NMWQK VGKAMY AT RP MNNTR K S I R I Q RG P G R AFVT IG C4-V3 L43 KQF INMWQEVGKAMYATRPNNNTR K 3 I R I Q RG P G R AFVTIG C4-V3 FA KQFINMWQEVGKΛMYA PNNNTRK3I IQRGPGRAFVTIG C4-V3 N KQI I NMWQEVG KAMY AT R PN YNKR K R I II I G P G RA FYTTK C4-V3DRVA KQI INMWQEVGKAMYATRPNNNTR KRI MGPGRVYYTTG C4-V3SC K I INMWQEVGKAMYATR NNNTTRBIHIGPORAFYATG C4-V3JH3 KQIINM QEVGKAMYATRPSKTTRR IHIGPGRAFYTTK C4-V3 ALA1 KQIVNMWQEVGKΛMYAT NIYRKGRIIII GPGRAFIITTR C4-V3BΛL1 KQ I I NMWQEVG AMY AT P NN TRK 3 IHIGPG A YTTG C4-V3JnCSF KQIIHM QEVGKAMYATRPSNNTRKaiHI GPG FYTTG C4-V3JRFL KQI I NMWQ EVGKΛMY ATR P NNNT R K 31111 G P OR A F YTTG S C4-V3OYI KQIVNMWQEVGKAMYATRPNNNTRNRISIGPG AFHTTK C4-V3SF2 KQI I NMWQ E VG K AMY AT R P HNNT R K 3 I Y I G P G R A FHTTG C4-V3NY5CQ KQ I I NRWQE VGKAMY AT RP MNNT RK G I A I G P G R T h Y ARE C4-V3 SF162 KQIINRWQEVGKAMYATRPNNNTRK3I IGPGRAFYA G C4-V3JFL KQI I NRWQEVG K AMY AT RP NNNTRK 3 I T L 0 P G R A F YTTG C4-V3CDC4 KQIINRWQVVGKAMYA R NNHTRKRVTLGPGRVWYTTG C4-V3 SF33 KQTTNMWQEVGI(ΛMYΛTnPNNNnnnπiTSOPακV[,YTTfi C4-V3 HAN KQI I NM WQ E VG KΛM YΛT R P NNNTR K G I II I GPGRAVYTTG C4-V3ADA KQI I NMWQ E VGKΛMYΛT R P NNNT R K fl I II I G P O RA F YTTG C4-V3WMJ2 KQI I NMWQG VGKΛMYΛTR P YNN VR R 3 L 3 IOPGRAFRTRE C4-V3RF KQIVNMWQRVGKΛMYATRPNNNTRKSITKOPGRVIYΛTG Sequences taken Irom sequences taken liomlhe Loβ Alamos Dotβhβao, Hii on nelrovlriwee and AIDS Iftβl milled by O. Mywa. J. A. llono ky. θ. Kotber. ft F. Smiili. and Ct. N. Puvlakia published by Ihe llieorαllcal Qlology and Biopliyalc* Group T- to Mail Slop K/10, Loβ Alamo* National Laboratory. Los Alamos. NM 87646 60 k EXAMPLE 12 In the design of an HLA-based HIV vaccine, the following variables are taken into account: a) the HLA molecules that are expressed in the population or cohort to be immunized, b) the CTL or T helper epitopes present in the immunogen and their respective HLA-restricting elements, and c) the HIV variants present in the geographic location of the cohort to be vaccinated. An HLA-based vaccine for induction of anti-HIV T cell immunity is a multivalent mixture cf immunogens reflective of the most common HIV variants in a geographic location, and containing immunogenic CTL and T helper epitopes that bind to the HLA molecules expressed on antigen-presenting cells of subjects of the cohort to be vaccinated. The mixture of immunogens can range from a mixture of non-HIV vectors expressing HIV proteins, to mixtures of HIV recombinant proteins and/or synthetic peptides (Palker et al, J. Immunol. 142:3612 (1989); Hart et al, Proc. Natl. Acad. Sci. USA 88:9443 (1991); Berzofsky, FASEB J. 5:2412 (1991) ; Haynes et al, Trans. Assoc. Amer. Phys. 106:33 (1993) ; Haynes et al, AIDS Res. Hum. Retroviral 11:211 (1995) ; Cease et al, Ann. Rev. Immunol. 12:923 (1994); Walfield et al, Vaccines 92, Cold Spring Harbor Laboratory Press pp. 211-215 (1992)) . Data sets that can be used to develop HLA-based AIDS vaccines include: 1) a compilation of CD8- CTL and CD4+ T helper epitopes in HIV proteins that can be derived from the available literature (see particularly Nixon et al, Immunology 76:515 (1992)), 2) a listing of the HLA restricting antigens that present KIV CTL and T helper epitcpes which also car. be derived from the available literature, 3) a compilation of the HIV 60 1 variants present in specific geographic locations that can be derived from the available literature (see particularly Human Retroviruses and AIDS 1993, Myers et al (eds) published by Theoretical Biology and Biophysics Group, Los Alamos National Laboratory, Los Alamos, NM 87545), and 4) a listing of HLA types for ethnic groups in geographic locations (HLA 1991 Tsuji et al (eds) , Proceedings of the 11th International Histocompatibility Workshop and Conference, Oxford Unv. Press, Oxford, England 1992) . Table XXVI shows an HLA-based KIV vaccine design for CTL induction for African-Americans, and shows analyses of the most common HLA types present in the cohort to be immunized, the HLA Class I CTL epitopes restricted by the common HLA types in the cohort, and the HIV variants in the geographic location under consideration. The most useful HIV preventive immunogens will be those designed for all members of a cohort to be immunized in a geographic area regardless of ethnic background, and such immunogens can be designed by expanding the number of ELA types used in the analysis, and by choosing HIV CTL immunogenic epitopes presented by several disparate HLA molecules (see available literature including Haynes et al, AIDS Res. Human Retroviral. 11:211 (1995)) . Since a number of HIV CTL epitopes are presented by more than one allotype of HLA Class I molecules, by using nine CTL epitopes (as well as variant peptides reflective of HIV mutations at these sites) 95% of African-Americans, 97.5% of Caucasian Americans, 97% of Native Americans and 99% of Thais can be expected to respond to such an HIV immunogen (see Table XXVII below) (Haynes et al, AIDS Res. Human Retroviral 11:211 (1995)) . 60m TABLE XXVI Steps in the Development of an HLA-Based Subunit HIV Vaccine For Induction of Anti-HIV CTL in African-Americans in the United States 1. Analysis of common MHC types in cohort to be immunized. Example: For African-Americans in the United States, 80% will express HLA A2, A3, A28, A30, B7, or B8. 2. Analysis of the HLA Class I HIV CTL epitopes restricted by these common HLA types in African- Americans in the cohort to be immunized. Example: For HLA-A2, aa77-85, from pl7 gag (Johnson et al, J. Immunol. 147:1512 (1991)) ; for HLA-A3, aa73-82 from nef (Culmann et al, J. Immunol. 146:1560 (1991)); for HLA-A28, aa583-592, from gp41 (Lieberman et al, J. Immunol. 148:2738 (1992)); for HLA-A30, aa844-863, from gp41 (Lieberman et al, J. Immunol. 148:2738 (1992)) for HLA-B7, aa302-312, from gpl20 McMichael et al, AIDS 85.-S155 (1994)) ,- for HLA-B8, aa586-593 from gp41 (Sutton et al, Eur. J. Immunol. 23:447 (1993)) . 3. Analysis of the HIV clade variants in the geographic locations of the cohort to be immunized. Example: The most common clade in the United States and Western E roDe is Clade E. Fcr Clade B, 60n epitope aa77-85 from p!7, of 26 isolates analyzed in the Los Alamos database, these are 8 variants, necessitating including 8 peptides for this sequence. A similar analysis for the other 5 epitopes would require an additional 54 peptides, for a total of 62. 4. T cell help can be obtained for anti-HIV CTL induction by peptides by synthesizing immunodominant T helper determinants N terminal to the CTL epitopes or includsion of T helper determinants in larger subunits of HIV immunogens* (Palker et al, J. Immunol. 142:3612 (1989) ; Milich, Nature 329:547 (1987)) . Example: Th -X , Th : -X 2 , Th 3 -X ; ., Th.,-X N , etc: where Th = immunodominant T helper epitopes and X = MHC Class I CTL epitopes. ♦ Specific CTL epitopes are included because of the restricted nature of CTL-epitope presentation by HLA molecules (Palker et al, j " . Immunol. 142:3612 (1989) ; Berzofsky, FASEB J. 5:2412 (1991) ; Berzofsky, Sem. Immunol. 3:203 (1991)) . Only the most common immunodominant T helper epitopes need to be included for T cell help for CTL generation without regard to immunized cohort restriction element expression, since many HIV T helper epitopes are presented by multiple HLA Class II types " (rev. in (Berzofsky, FASEB J. 5:2412 (1991); Berzofsky, Sem. Immunol. 3:203 (1991)) , and some retrovirus CTL epitopes also been suggested to have T helper cell stimulating activity (rev. in Berzofsky, FASΞ3 J. 5:2412 (1991); Berzofsky, Sem. Immunol. 3:203 (1991)) . It will be appreciated that the actual degree of HIV variability may not be reflected by the degree of variability seen in existing databases . It will also 60 o be appreciated that it is not certain that every variant of every HLA type will equally present each peptide to T cells. Further, it is not certain that every HIV CTL epitope variant will be a potent agonist and trigger T cells to an optimum anti-HIV immune response. However, vaccines designed in accordance with the present approach can be expected to be of significant clinical effectiveness. Further, for design of an HLA-based HIV preventive immunogen that induces both anti-HIV T cell responses and anti-HIV neutralizing antibodies, HIV B cell immunogens that induce broadly neutralizing antibodies for HIV primary isolates can be added to a multivalent KIV T cell immunogen. By way of example, set forth below is an HLA-based HIV vaccine formulated based on the International Histocompatibility Workshop data presented in Histocompatibility Testing 1994, Albert et al eds Springer-Veriag, Berlin (1994) and HLA 1991, 2 volumes, Tsuji et al (eds) Oxford University Press, Oxford England (1992)) supplemented with other published data (Williams et al, Human Immunol. 33:39 (1992); Chandanayingyong et al, HLA antigens in Thais, In Proceedings of the Second Asia and Oceania Histocompatibility Workshop Conference, Simons and Tait (eds) I munopublishing Toorak pp. 276-87 (1983)) . Using these data and the Hardy-Weinberg theorem (Hardy, Science 28:49 (1908)), an estimate was made of the proportion cf each of four populations that would be expected to present peptides to the immune system if a limited number of HIV epitopes were included in a vaccine designed specifically for that population. Similar calculations were made for a vaccine designed to be immu cgenic for ail four populations. These results are presented in Table XXVII. TABLE XXVII TΛBI /II (cont ' d) (1'ropoι lioiifcif Thais expecled lo present Ihese 2 epilopes is 93.6%) e) African Americans Λ2, A3, All, B35 nef 73-82 QVPLRPMTYK USA Caucasians Λ28, 111 β p41 583-592 VERYLKDQQL North American Λ30, 1 ) 8 gp41 844-863 RRIRQGLERALL Indians Thais 1)17, 1)37 nef 117-128 TQGYFPQWQNYT C 4 B pl20 376-383 (S)FNCCGEFF 1)7 gp!20 302-312 RPNNNTRKSr o nef 126-138' NYTPGPGVRYPLT 1)12 p24 169-184 IPMFSΛISI.CATPQDL Λ31 β p41 770-780 RLRDLLL1VTR Λ24 gp4l 584-591 YLKDQQLL or nef 120-144 YFPDWQNYTPGPGIRYPLTFGWCYK (Piopυrtions of African Americans, USA Caucasians, North American Indians, and 'Iliais expected to present these 9 epitopes are 95.4%, 97.5%, 99.4%, and 97.2%, respectively) 7/14436 60 r The most frequent restriction elements in the population under consideration for vaccination (or common to the 4 populations) are first identified, peptides that are presented by more than one HLA allele are next identified, and commonality between the two lists is then determined. Probability calculations utilize the frequencies of the commonality alleles supplemented by those of additional high frequency alleles in the population. Alleles are added until the proportion of the individuals in the population carrying one or more of the alleles in the list is at an acceptable level, eg greater than 90%. The sum of the HLA gene frequencies that recognize the fewest number of different HIV peptides to be included in the HIV immunogen is thus maximized. The next step is to choose the peptides associated with the restricting allele. In some instances only one peptide is associated with an allele while in others, multiple peptides are presented by the same allele. For the four population cohorts considered in this analysis, as few as 2 and as many as 5 epitopes are required to achieve a theoretical protection level of at least 90% (Table XXVII) . The different numbers of , required epitopes reflect, in part, the relative amounts of HLA Class I polymorphism observed in the different ethnic groups and presentation of a peptide by multiple HLA ciass I molecules. A comparison between the individual and combined population (Table XXVII) demonstrates that relatively little is gained by including epitopes that are associtated with low frequence alleles . The proportion of individuals protected approaches 100% asymptotically so that even adding on epitopes associated with high frequency alleles adds little to the proportion as this level is approached. This is illustrated by the North 60 s American Indians where including 6 more epitopes associated with 5 very low frequency alleles and one intermediate frequency allele in the combined theoretical vaccine added only 3.0% protection. 60 t EXAMPLE 13 Induction of Antibodies That React With Native HIV Envelope Proteins Table XXVIII shows peptide sequences that were designed to induce antibodies against the C-terminus of gpl20 (SP410-BAL) , the AVERY region of gp41 (SP400-BAL) , the ELDKWAS region of gp41 (GTH1- SP61) , and the C2 region of gpl20 (T1-SP420-BAL) . In addition, Tl-SPIO(A) or GTHI-SPIO(A) peptides were included in the mixture of HIV env peptide designs (the Tl-SPIO(A) and GTHI-SPIO(A) peptides induce potent neutralizing antibodies against HIV laboratory adapted strains) . The following peptides were injected into guinea pigs with Freund's adjuvant to induce anti-peptide antibodies: SP400-3AL, SP410-BAL, and the Th-B design peptides, GTH1-SP61, GTHI-SPIOMN(A) , and T1-SP10 (A) -BAL. The SP400-BAL, SP410-BAL and GTHI-SPIOMN(A) peptides induced antibodies that bound to recombinant gpl20IIIB or to recombinant gp41 in Western blot assays (Figure 32A, B and C) , and bound to the surface of HIVIIIB/LAI-infected CEM T cells (Figure 33) . Antibodies against peptide GTH1-SP61 also bound to the surface of HIVIIIB/LAI infected CEM T cells (Figure 33) . All anti-peptide antisera bound to the immunizing peptide in ELISA assays (Table XXX) . Thus, the peptide designs listed in Table XXVIII induce antibodies against native HIV env proteins and, upon incorporation into a polyvalent immunogen, potentiate each other and particularly anti-V3 antibodies in neutralizing HIV laboratory adapted and primary isolates. Table XXX. ELISA analysis of serumend-point titers of guinea pig antisera against immunizing peptides Peptide ELISA Immunogen Peptide sequence Guinea pig # Titer E/C GHT1 -SP 10MN(A) YKRWIILGLNKIVRMYSTRPNYNKRKRIHIGPGRAFYTT 2 GTH-SP60 YKRW1ILGLNKIVRMYSELDKWAS 3 4 GTH-SP61 YKRWIILGLNKIVRMYSQQEKN EQELLELDKWAS 5 6 SP400-BAL RVLAVERYLRDQQLLGIWGCSGKLICTTAVPWNASWSNKSLNKI 13 14 SP410-BAL PGGGDMRDNWRSELYKYKWKIEPLGVAPTKAKRRWQREKR 15 16 61 All documents cited above are hereby incorporated in their entirety by reference. The foregoing invention has been described in some detail by way of examples for purposes of clarity and understanding. It will be obvious to those skilled in the art from a reading of the disclosure that the synthetic peptides of the instant invention may differ slightly in amino acid sequence from the sequences of SP-10 regions of specific EIV isolates, without departing from the scope of the invention.

Claims: 62 WEA? IS CLAIMED IS; 1. An essentially pure fora of a hydrophilic peptide consisting essentially of an aaino acid sequence of up to about 35 units in length and corresponding to at least one antigenic deterainant of the envelope glycoprotein of HIV recognized by 3 lymphocytes, said peptide being capable, when covalently linked to a carrier m lec-jle, of inducing in a mammal the production of high titers of protective, type-speci c, antibodies against EIV. 2. The peptide according to claim 1, wherein said aaino acid sequence corresponds to the SP-10 region cf the enveloce glycoprotein of HIV, or seme portion thereof. 3. The peptide according to claim 2, wherein said amino acid sequence consists essentially cf CTRPNNNTRKS IRIQRGPC, or seme portion thereof. 4. An immunogenic conjugate capable of inducing in a mammal the production of high titers of protective, type-speci ic, antibodies against EIV, said conjugate comprising: (i) a carrier molecule covalently attached to (ii) a hydrophilic peptide consisting essentially cf an amino acid sequence of up to about 35 units in length and corresponding to at least one antigenic determinant of the envelope glycoprotein of EIV recognized by B lymphocytes. 63 5. The conjugate according to claim 4, wherein said carrier molecule comprises an amino acid sequence corresponding to a region of the envelope glycoprotein of EIV, which region is distant from the SP-10 region and is recognized by T cells. 6. The conjugate according to claim 5, wherein said region distant from said SP-10 region is T cell epitope Tl or T cell epitope T2, or some portion thereof. 7. The conjugate according to claim 4, wherein said carrier molecule is tetanus toxoid. 8. The conjugate according to claim 4, wherein said carrier molecule is covalently attached to said peptide through at least one spacer molecule. S. The conjugate according tc claim 8, wherein said spacer molecule consists of the dipeptide glycine-glycine. 10. A conjugate according to ciaim 4 wherein said amine acid sequence corresponds to the SP-10 region of the envelope glycoprotein of EΣV, or seme portion thereof. 11. A conjugate according to claim.10 wherein said amino acid sequence consists essentially cf CTRPNNNTRXSI IQP.GPG, or seme portion thereof. 64 12. The conjugate according to ciaim 4, further comprising the amino acid sequence FLGFLG covalently linked to the S?-10-like peptide, or portion of the S?-10-like peptide. 13. The conjugate according to claim 4 further comprising an amino acid sequence corresponding to a hypervariable region of the envelope protein of EIV isolates located C terminal .to the SP-10 region. 14. A conjugate according to ciaim 13 wherein said sequence corresponding to said hypervariable region is RAFVTIGXIGN and is directly linked to the C terminus of SP-10. 15. A methcd of producing immunity to EIV in a mammal co-mprisinς administering at least one conjugate according to claim 4 to said mammal. 16. A methcd of producing immunity to EIV in a mammal comprising administering to said mammal at least one covalently linked aggregate cf at least two conjugates, each of said conjugates having (i) a carrier molecule covalently attached to (ii) at least one S?-10-like peptide, or portion thereof. 17. The methcd according to claim 16 wherein said aggregate comprises at least tvo SP-10- like peptides, or portions thereof, each cf which corresccπds to a different isolate of EIV. 18. The methcd according to ciaim 16 wherein said conjugates in said aggregate are covalently linked to each other via at least one disulfide bond. 19. An immunogenic covalently bended aggregate capable of producing a protective immune response to HIV in a mammal comprising at least two conjugates each composed of (i) a carrier molecule covalently attached to (il) a hydrophilic peptide consisting essentially cf an amino acid sequence of up to about 35 units in length and corresponding to at least one antigenic determinant of the envelope glycoprotein cf EIV recognized by E lymphocytes, said conjugates being linked to each ether through at least cne disulfide bend. 20. A methcd cf determining the presence and titers in mammalian serum of neutralizing antibodies against a specific strain cf EIV comprising the steps of: (i) contacting a SP-10- like peptide, c: portion thereof, with antibodies from mammalian serum; and (ii) measuring the formation cf S?-10-like peptide-antibo-dy complexes by radioimmunca≤say, or enzyme linked immunoscrbent assay. 65 21. A peptide of the general formula: ?-τh-s?ιo(X) Th-S?10(X) T -s?ιo or F(X) wherein: ? represents an aaino acid sequence derivable froa the putative fusogenic doaain of HIV env gp41, or sequence functionally equivalent thereto; Th represents an aaino acid sequence comprising a T helper epitope; S?10 represents a peptide as defined in claia 1; and (X) represents an aaino acid sequence corresponding to a KIV protein sequence recognized by KHC Class I or Class II restricted cytotoxic T cells. 22. A method of inducing, in a primate, antibodies that neutralize human immuncdeficiency virus (EIV) comprising administering to said primate a mi eotope of a conformational determinant of the native HIV cpl20 C4-V3 region. 23. The methcd according to claim 22 wherein said mimetope is Tl-SPIOCANO(A) . 24. A methcd of screening said peptides according to claim 21 fcr the ability to produce neutralizing antibodies against more than 1 EIV isolate comprising: i) contacting one of said peptides with a monoclonal antibody that recognizes a conformational determinant of the native EIV gpi20 C4-V3 region under conditions such, that binding can occur, and ii) determining whether said peptide binds to said monoclonal antibccv. 66a 25. A method of inducing, in a primate, the production of antibodies that neutralize human immunodeficiency virus (HIV) comprising administering to said primate a composition comprising HIV envelope peptides that disrupt gpl20/gp41 interactions. 26. A composition comprising human immunodeficiency virus envelope peptides that disrupt gpl20/gp41 interactions, together with a pharmaceutically acceptable carrier.

Inventors: HAYNES BARTON F, PALKER THOMAS J, HAYNES, BARTON, F., PALKER, THOMAS, J.
Applicants: UNIV DUKE, DUKE UNIVERSITY
Application year: 1996
Main classification: A61K39
IPC codes: A61K31, A61K38, A61K39, A61P31, C07K7, C07K14, G01N33