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WO1997014436A1 - Vaccin de synthese pour la protection contre l'infection par le virus de l'immunodeficience humaine - Google Patents

Vaccin de synthese pour la protection contre l'infection par le virus de l'immunodeficience humaine Download PDF

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Publication number
WO1997014436A1
WO1997014436A1 PCT/US1996/016911 US9616911W WO9714436A1 WO 1997014436 A1 WO1997014436 A1 WO 1997014436A1 US 9616911 W US9616911 W US 9616911W WO 9714436 A1 WO9714436 A1 WO 9714436A1
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WO
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Prior art keywords
hiv
peptide
peptides
region
eiv
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PCT/US1996/016911
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English (en)
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WO1997014436A9 (fr
Inventor
Barton F. Haynes
Thomas J. Palker
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Duke University
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Priority claimed from US08/546,515 external-priority patent/US5993819A/en
Application filed by Duke University filed Critical Duke University
Priority to JP9516093A priority Critical patent/JPH11515006A/ja
Priority to AU74656/96A priority patent/AU7465696A/en
Priority to EP96936830A priority patent/EP0868196A4/fr
Publication of WO1997014436A1 publication Critical patent/WO1997014436A1/fr
Publication of WO1997014436A9 publication Critical patent/WO1997014436A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the prassnt invention relates, i general, to irsunoge ⁇ ic preparations and, in particular, to peptides comprising amir.c acid sequences corresponding tc a regicn cf the hua&n iasunedeficienc virus ( ⁇ TV) 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.
  • 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.
  • AIDS isaunede icienc syndrome
  • 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.
  • 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.
  • gpl20 external envelope glycoprotein
  • T4 CD4
  • 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 .
  • 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 .
  • 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 .
  • 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 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.
  • 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.
  • 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 ⁇ olecule, 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.
  • the present invention comprises an i. ⁇ -*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.
  • the present invention comprises a method of producing immunity to EIV comprising administering the above-described conjugate to a mammal.
  • the present invention comprises a method of detecting the presence of anti-gpl20 antibodies in biological test samples.
  • Figure 1 Recombinant proteins and relation to synthetic peptides.
  • FIG. 6 Binding of goat anti-S?-10 serum to ET V- ⁇ II a - but not to ET V- ⁇ II 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- ⁇ l-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.
  • Tl-SPlOHN( ⁇ ) , F-T1-SP10KN and F- Tl-S?10HN ( ⁇ ) peptides after 2 i suni rations induce higher levels of anti Tl-SPIOHH antibodies than did T1-SP10HN itself .
  • FIG. 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 ⁇ er 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 ( ⁇ ) 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 P ⁇ A.
  • 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 ( ⁇ ) 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.
  • FIG 17 shows the absorption of cross neutralizing antibodies induced by Tl-SPIOKN (A) peptide in rhesus monkey 18987 by peptides containing GFGPAJ sequence .
  • Tl-SPIOKN (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.
  • Neutralizing antibody titers determined in syncytium inhibition assay.
  • FIG. 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 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 ( ⁇ /C greater than 2.9).
  • 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 ii ⁇ tunodeficiency 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)) .
  • FIG. 27 Sequence of Tl-SPIO(A) Th-B-Tc peptides for human immunization.
  • FIG. 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 ⁇ g/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.
  • 8 molar urea treatment of the peptide denatured the peptide and preventive subsequent 48d binding to the peptide.
  • Figure 30 General scheme for an ELA-based vaccine for AIDS.
  • 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.l ⁇ g/lane) , cell lysate of HIVLAI/IIIB infected- or mock-infected CEM cells (0.5xlO ⁇ cells/lane) were fractionated on 4-20% SDS-PAGE gel, and transferred to nitrocellulose filters.
  • 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.
  • the present invention relates to peptides corresponding to iaaunege ⁇ ic epitopes of EIV and synthetic vaccines made therefrom.
  • 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.
  • 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.
  • 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-I ⁇ Ig 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.
  • 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- ⁇ IV 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-I ⁇ 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
  • 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 Z ⁇ immune response in mammals.
  • suitable carrier molecules include tetanus toxoid, keyhole limpet hemocyanin (XLH), and peptides correspcncing to T cell epitcpes (that is, Tl and T2) cf the ⁇ pl20 envelope glycoprotein that can substitute for nca- A ⁇ DS 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.
  • a 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.
  • the S?-10 sequence of ETLV- ⁇ II a can be conjugated to or synthesited with either the ST V- ⁇ I a g?120 envelope T cell epitope Tl (amino acids 428-443 cf gpl20), KQ ⁇ 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,
  • 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.
  • SP-10 Rf(A) and SP-10 C can also be covalently linked to carrier molecules, including ⁇ pi20 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
  • 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 ⁇ ).
  • 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.
  • 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
  • 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.
  • a carrier molecule such as tetanus toxoid
  • the present invention also relates to a polyvalent vaccine comprising S?-10-li' ⁇ e 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 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.
  • 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
  • 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. Taau ⁇ .1. 142:3612, 1989) and helper T cells (Ceasa et al Proc. Natl. lead. Sel.
  • 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 KTLVIII ⁇ ? (RJ) HIV isolates (Palker et al. £. iTtcaunol . 142:3612, 1939).
  • neutralizing antibodies raised against the Ti-SPlO-liXe peptides ' containing sequences fraz the Xi or J KIV isolate neutralize the hocclog ⁇ us isclate but do net neutralize any of the other tvo KIV isolates.
  • AHTI-Tl-SPIOHH 6/9 (67%) La Rosa et al f Sc ncg 249 : 932 , 1990) have ⁇ hcvn 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 .
  • 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.
  • 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 .
  • a synthetic peptide iaaunogen be desirable that induced cytotoxic T cell (CTL) responses to HIV in addition to inducing neutralizing antibody and T helper cell responses .
  • CTL induced cytotoxic T cell
  • 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 .
  • F-derivatized peptides are internalized in iaaune cells in such a vay as to induce the ⁇ aae 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 PCT ⁇ JS96/16911 derivati ⁇ d peptides interact vith ce lls of the iaaune systea_ ⁇ uch 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.
  • the present invention relates, in a preferred eabodiaent, to peptides of the general foraulae:
  • 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 ;
  • (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.
  • 7 sequences can be , for exaaple, C-terainal to Th-S?10 (X) sequences.
  • 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.
  • 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-( ⁇ ) . 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 fro ⁇ Tl-hoaologcus sequences froa any sequenced HIV isolate including those shovn in Table VI.
  • the F-liJce sequences can be selected froc
  • 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 ⁇ laaos, Hev Mexico, p. 11-90. WKJ1 sequence is froa Brasseur et al. AIDS Res. Hua. Retrovirol. 4:83-90, 1988.
  • 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. TA3L ⁇ VIII
  • 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.
  • 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
  • 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.
  • 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.
  • aaino acid seguence see, for exaaple, Table VII
  • 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.
  • b oquenc ⁇ num er* for gpl20 and g P 41 are from Ratner et al Nature 113- 2 -, nj , «may « * ** ' ' 19 ° 0)
  • 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 HL ⁇ antigen that the (A) peptide uses to be presented to cytotoxic T cells void develop cytotoxic T cells against HIV.
  • 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.
  • 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) .
  • T helper epitopes 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 HL ⁇ 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).
  • T helper cell epitope may be recognized by T cells in the context of multiple HL ⁇ 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 (" ⁇ T ⁇ JSU ⁇ OI .
  • the AIDS vaccine of the present invention has the general structure and composition of mixtures of peptides of the formulation:
  • 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 .
  • (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.
  • 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.
  • P, Th, SP10 and (X) can vary as lone as the above-indicated function of each is retained.
  • F-Th-SPlO(X) peptides 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 ⁇ pl20 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) .
  • 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.
  • 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 HL ⁇ 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) forsulatie ⁇ 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.
  • 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.
  • KIV gp41 F sequences for exaaple, aaino acids 519-530 froa the BK10/III3 HIV-l isclate or frca hccclogcus regions of
  • 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 .
  • 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 also contain regions that are specif ic fcr clones of B cells making antibodies .
  • sequence of peptides froa the region of the B cell immunoglobulin aolecule that binds antigen can be identified using, for example , recombinant DH ⁇ techniques . Further, sequences capable of inducing KHC Class I or Class II cytotoxic T cell responses can be identified.
  • 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 ⁇ .
  • 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.
  • Hepatitis C non- ⁇ , non-3 hepatitis
  • cytotoxic T cell epitope sequences of the Hepatitis C virus protein 36 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 .
  • 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.
  • 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
  • gp41 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)) .
  • ELDKWAS Master et al, J. Virol. 67:6542-6647 (1993); Conley et al, Proc. Natl. Acad. Sci. USA 91:3343-3352 (1994)
  • the peptides listed in Table XXVIII are against certain HIV strains (HIV MN or BAL) .
  • HIV MN or BAL HIV strains
  • 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.
  • TRPNNNTRKSIHIGPGRAFYTTG gp120 V3 loop region, aa301-324 SP10BAL
  • 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
  • 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
  • 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
  • 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.
  • 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.
  • Fcr preparation of affinity coluans synthetic peptides containing aaino acid sequences frca BTLV-i ⁇ 8 g?120 (SP-10, 10A, 11, 14, 15, 22/ see Figure 1) vere coupled to BSA and then covalently linked to CH ⁇ r-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).
  • 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).
  • par dii il i tff ⁇ i are the Inver.e t ⁇ ( aiir..* dllutl ⁇ ne that Inhibitad the nua-hcr of ayncytia ((0- ⁇ O) par well hy > ⁇ ll ⁇ .
  • SF-10-TT a_lo ⁇ e was as good an antige ⁇ -specif ic T cell activator as TT alone.
  • SP-10-TT and SP-10-3SA vhen added to TT alone did not inhibit TT induced proliferation by noraal T cells.
  • anti-SP-10 gcat seru ⁇ did not bind to peripheral blocd lymphocytes or monocytes in indirect immuno luorescence assay using flov cytofluorometry.
  • 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.
  • Synthetic peptide SP-10 has an amino acid sequence derived from and unique to the gpl20 envelope protein of HIV isolates ETLV- ⁇ II 3 and LAV, vhile other EIV isolates have varying degrees of differing amino acid sequences in their S?-10-like ⁇ pl20 envelope proteins.
  • Synthetic peptide SP-10 (that is, S?-10- ⁇ II a ) frcm the ETLV- ⁇ - 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
  • Coat antibodies raised to synthetic peptide SP-10 vere tested for the ability to neutralize four different E ⁇ V isolates (Pig. 5A: ET V-III a , Fig. 5H: ETLV- III jj p, fig. 5C: ET V- ⁇ I ⁇ , Fig. 5D: ST V-II ⁇ SC ).
  • Goat anti-S?-10 antiserum #, pre-immune goat serum (0) and A ⁇ DS patient serum (S) all at a 1/10 dilution vere first incubated vith dilutions (IO -1 , IO -2 , 10 "3 ) of each virus isolate.
  • 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.
  • Ta ⁇ ahashi 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) (Ta ⁇ ahashi et al J . Eys . Ked . 170 : 2023 , 1989) .
  • CTL cytotoxic T cell
  • one TI ⁇ SPIO variant peptide aade vas the Tl-S?10KH ( ⁇ ) 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) .
  • 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.
  • 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
  • ⁇ Ixperiaents vith coapleaent (C) represent the results froa pooled s lenocytes fron 3 aice iaaunized vith F-Tl-S?10KH( ⁇ ) . 51
  • Figure 7 shovs a conparison of the levels of anti-peptide antibody generated in the serua of
  • addition of either the (A) region or the ? region increased the level of anti-peptide antibodies to the T1-SP10KH peptide in ELISA assay.
  • 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 ⁇ r ⁇ s ⁇ 85:1932-1936, 1533; Palker et al., . Iaaunol..
  • T1SP10III3 peptides these mouse strains make no neutralizing antibodies to the HIVIII3 V3 loop neutralizing deterainants.
  • CS7BL6 and Balb/c mice make good anti- HIV neutralizing antibodies when iaaunized with TISPIO peptides containing sequences rca "the HIVMN V3 loop.
  • Tl-SrlO peptides were excellent iaaunogens in aniaals 834 and 1023, while T1-SP10 peptides with the KIV gp41 fusion (F) dc ⁇ ain 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.
  • aniaals 1045 and 1070 were challenged at ⁇ o ⁇ th 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( ⁇ ) 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.
  • Peptides T1-SP10III3 and T1-S?10III3(A) induced high levels cf proliferation of circulating P ⁇ MC 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 P ⁇ S alone vith no adjuvant.
  • aniaals 1045 and 1070 that vere iaaunized vith F-T1-S?10II: ⁇ (A) peptide, did net proliferate to T1-S?10III ⁇ (A) peptide.
  • 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.
  • 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) .
  • T1-SP10 peptides vere superb iaaunogens vith I? ⁇ 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) .
  • 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 .
  • selective proteclysis of the HIVIII3 V3 loop occurs by chiapanzee and nousa mcnenuclear- cells in be . cr mora likely , that genetic restriction of antibedy responses to neutralizing deterainants cf the V3 loop exist in chiapanzees and nice .
  • 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 ⁇ cn 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 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) .
  • 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 des ⁇ will be doubled and the regimen repeated after a three mc ⁇ th rest.
  • IFA Incomplete Freund's adjuvant
  • the immunogens will be administsrad by IM.
  • the immunccer.s will b ⁇ mixed in a total volume of 2c 60
  • Immunizations will be given at 0 month, 1 month and 3 months .
  • the patients will be monitored 4 weeks after each 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-IZJC ⁇ 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) .
  • 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
  • Th-B Th-B
  • 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 EIVIII ⁇ , 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
  • ELA A2 and A3 are 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.
  • 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.
  • mice immunized with mixtures of all 4 peptides 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) ⁇ .con. peptide (a Th-3-CTL peptide reflective of the consensus V3 locp sequence of Clave E in Thailand (Table XXV) .
  • the 17b and 48c human an i-gp!20 mabs were isolated from human F ⁇ MC 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)) .
  • 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) .
  • 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
  • Tl-SPIOCANO (A) synthetic peptide can mimic broadly neutralizing C4-V3 conformational determinants of native gpl20.
  • 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) ; Mc ⁇ re, J. personal communication (1994)) .
  • 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 ( ⁇ uman Retrovirus and AIDS, 1991, 1992, 1993 edited by G. Myers, J.A. Eerzofsky, B. Korber, B.F. Smith and G.N.
  • 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.
  • 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 h ⁇ terodimer expressed in the combinatorial library on the surface of phage.
  • an HLA-based HIV vaccine 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
  • 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)) .
  • 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.
  • a similar analysis for the other 5 epitopes would require an additional 54 peptides, for a total of 62.
  • 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)) .
  • Th immunodominant T helper epitopes
  • X MHC Class I CTL epitopes
  • HIV CTL epitope variant will be a potent agonist and trigger T cells to an optimum anti-HIV immune response.
  • vaccines designed in accordance with the present approach can be expected to be of significant clinical effectiveness.
  • HIV B cell immunogens that induce broadly neutralizing antibodies for HIV primary isolates can be added to a multivalent KIV T cell immunogen.
  • 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)) .
  • the most frequent restriction elements in the population under consideration for vaccination 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.
  • 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) .
  • 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) .
  • Table XXX ELISA analysis of serumend-point titers of guinea pig antisera against immunizing peptides

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Abstract

Préparations immunogènes de peptides comportant des séquences d'acides aminés correspondant aux déterminants antigéniques de la glycoprotéine d'enveloppe du VIH, couplées de manière covalente, et directement ou par l'intermédiaire d'une molécule intercalaire, à des molécules porteuses utilisables dans la vaccination des mammifères.
PCT/US1996/016911 1995-10-20 1996-10-18 Vaccin de synthese pour la protection contre l'infection par le virus de l'immunodeficience humaine WO1997014436A1 (fr)

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WO1999051750A1 (fr) * 1998-04-03 1999-10-14 Glaxo Group Limited Medicaments pour l'induction de cellules t cytotoxiques
EP1307130A4 (fr) * 2000-02-04 2005-01-12 Beth Israel Hospital Vaccin contre le virus de l'immunodeficience humaine
US7033593B2 (en) 2000-09-22 2006-04-25 Duke University Immunogen comprising an HIV envelope protein, a ligand and H2 peptide
US7070787B2 (en) 2000-09-22 2006-07-04 Duke University Method of inducing the production of antibodies to HIV
US7153509B2 (en) 2001-11-07 2006-12-26 Duke University Immunogenic peptides comprising a T-helper epitope and a B-cell neutralizing antibody epitope
US7172761B2 (en) 2001-11-07 2007-02-06 Duke University Polyvalent immunogen
US7195768B2 (en) 2001-11-07 2007-03-27 Duke University Polyvalent immunogen
EP1673449A4 (fr) * 2003-10-23 2008-08-06 Nmk Res Llc Composition immunogene et procede d'elaboration d'un vaccin a base de proteine de fusion
EP2091973A4 (fr) * 2006-11-09 2010-08-04 Sudhir Paul Anticorps reconnaissant des epitopes binaires et stimulants immunitaires des superantigenes des lymphocytes b
US7892562B2 (en) 2008-10-08 2011-02-22 Karp Nelson M Methods of inducing TH-1 immune responses to HIV-1 by administering UV/psoralen-treated desialated inactiviated HIV-1 virions deficient in CD55 and CD59
US11602559B2 (en) 2016-10-03 2023-03-14 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services HIV-1 Env fusion peptide immunogens and their use

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Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999051750A1 (fr) * 1998-04-03 1999-10-14 Glaxo Group Limited Medicaments pour l'induction de cellules t cytotoxiques
US7078039B2 (en) 2000-02-04 2006-07-18 Duke University Immunogenic composition
EP1307130A4 (fr) * 2000-02-04 2005-01-12 Beth Israel Hospital Vaccin contre le virus de l'immunodeficience humaine
US6982086B2 (en) 2000-02-04 2006-01-03 Duke University Human immunodeficiency virus immunogenic composition
US7052699B2 (en) 2000-02-04 2006-05-30 Duke University Immunogenic composition
US7033593B2 (en) 2000-09-22 2006-04-25 Duke University Immunogen comprising an HIV envelope protein, a ligand and H2 peptide
US7070787B2 (en) 2000-09-22 2006-07-04 Duke University Method of inducing the production of antibodies to HIV
US7101552B2 (en) 2000-09-22 2006-09-05 Duke University Immunogen comprising an HIV envelope protein, a ligand and H2 peptide
US7153509B2 (en) 2001-11-07 2006-12-26 Duke University Immunogenic peptides comprising a T-helper epitope and a B-cell neutralizing antibody epitope
US7172761B2 (en) 2001-11-07 2007-02-06 Duke University Polyvalent immunogen
US7195768B2 (en) 2001-11-07 2007-03-27 Duke University Polyvalent immunogen
EP1673449A4 (fr) * 2003-10-23 2008-08-06 Nmk Res Llc Composition immunogene et procede d'elaboration d'un vaccin a base de proteine de fusion
EP2091973A4 (fr) * 2006-11-09 2010-08-04 Sudhir Paul Anticorps reconnaissant des epitopes binaires et stimulants immunitaires des superantigenes des lymphocytes b
US7892562B2 (en) 2008-10-08 2011-02-22 Karp Nelson M Methods of inducing TH-1 immune responses to HIV-1 by administering UV/psoralen-treated desialated inactiviated HIV-1 virions deficient in CD55 and CD59
US11602559B2 (en) 2016-10-03 2023-03-14 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services HIV-1 Env fusion peptide immunogens and their use

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