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EP0868196A1 - Synthetic vaccine for protection against human immunodeficiency virus infection - Google Patents

Synthetic vaccine for protection against human immunodeficiency virus infection

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Publication number
EP0868196A1
EP0868196A1 EP96936830A EP96936830A EP0868196A1 EP 0868196 A1 EP0868196 A1 EP 0868196A1 EP 96936830 A EP96936830 A EP 96936830A EP 96936830 A EP96936830 A EP 96936830A EP 0868196 A1 EP0868196 A1 EP 0868196A1
Authority
EP
European Patent Office
Prior art keywords
hiv
peptide
peptides
region
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP96936830A
Other languages
German (de)
French (fr)
Other versions
EP0868196A4 (en
Inventor
Barton F. Haynes
Thomas J. Palker
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Duke University
Original Assignee
Duke University
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Filing date
Publication date
Priority claimed from US08/546,515 external-priority patent/US5993819A/en
Application filed by Duke University filed Critical Duke University
Publication of EP0868196A1 publication Critical patent/EP0868196A1/en
Publication of EP0868196A4 publication Critical patent/EP0868196A4/en
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • 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

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.
BACKGROUND OF THE INVENTION TECHNICAL FIELD
The present invention relates, in general, to immunogenic preparations and, in particular, to peptides comprising amino acid sequences
corresponding to a region of the human
immunodeficiency virus (HIV) envelope protein,
against which neutralizing antibodies are produced.
The invention further relates to a vaccine
comprising the peptide coupled, either directly or through a spacer molecule, to a carrier molecule, suitable for vaccination of humans.
BACKGROUND INFORMATION
The human retrovirus HIV has been
demonstrated to be the causative agent of acquired immunodeficiency syndrome (AIDS), a disease for
which there is currently no cure. The epidemiologic pattern among AIDS-related cases indicates that it is a transmissible disease. The virus is frequently found in saliva, semen, whole blood and plasma from individuals in high risk categories, including male homosexuals, intravenous drug users, patients
receiving blood products, and individuals from Haiti and Central Africa. The rapid rise in
seropositivity among individuals in high risk
categories, the virulence of the disease, and its growing world-wide distribution, underscore an
overwhelming and immediate need for a vaccine
capable of inducing complete protective immunity :- non-infected individuals. The need for 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 of the immune system by attaching its external envelope glycoprotein (gp120) to the CD4 (T4) molecule on the surface of T
lymphocytes, thus using the CD4 (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-envelope antibodies to inhibit HIV infection in vitro. Specifically, the HIV external envelope glycoprotein gp120 has been shown to be capable of inducing neutralizing antibodies in goats and in man (Robey et al., Prog. Nat'l. Acad. Sci. (USA) 83: 7023, 1986). Little is known of the precise location of epitopes on gp120 that are either immunogenic in HIV-infected patients or that give rise to neutralizing antibodies.
However, the recombinant protein PBl (Putney et al., Science, 234:1392, 1986), which encodes
approximately one-third of the entire gp120
molecule, has been shown to include the part of the envelope protein that induces the formation of neutralizing antibodies.
The data accumlated to date suggest that neither PBl nor intact gp120 are appropriate for use in a vaccine against HIV infection. Studies involving the use of goats and chimpanzees
demonstrate that neither molecule has the ability to induce the production of high titers of neutralizing antibodies. In addition, it has been shown that the intact gp120 molecule binds to the T4 molecule of normal T cells and is capable of disrupting normal immune function. Specifically, whole gp120 envelope molecules interfere with normal CD4 (T4) function and suppress T cell activation in vitro (Mann et al., J. Immunol. 138:2640, 1987). Thus, the administration of 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 gp120 (the V3 gp120 envelope loop region) and rapid V3 loop sequence 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 (USA), 88:6300-6304, 1991). Nonetheless, studies continue to show the critical role that the gp120 V3 region plays in generating anti-HIV neutralizing antibodies (Jiang et al., J. Exp. Med. 174:1557-1593, 1990). Moreover, it has recently been shown that approximately 50% of current HIV isolates share a consensus of 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 HIV sequences (Myers et al., Human Retroviruses and AIDS 1991; La Rosa et al., Science. 249:932-935, 1990; and Holley et al., PNAS (USA), 88:6800-6804, 1991). Further, two of these sequences, GPGRAF and. IHIGPGRA, have induced widely cross-reactive HIV neutralizing antibodies in animals (Jahaverian et al., Science, 250:1590-1593, 1990 and Haynes et al., AIDS Res. Humans. Retroviral, 6:38-39, 1990).
Thus, critical to the development of a vaccine against HIV, is the generation of an antibody response against gp120 that will interfere with gp120 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 myriad of stages of normal T cell response. In addition, an effective vaccine against HIV will induce protective immune responses in primates and in man, 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 risk for contracting HIV infection 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 of HIV by 80%, then maternalfetal HIV transmission would decrease form 30% to 6% of children born to HIV-infected mothers. Third, an immunogen against HIV that induced salutory and net pathogenic anti-HIV responses, would be useful for immunization of HIV-infected assymptomatic
individuals to boost anti-HIV immune responses, and promote the maintenance of the assymptoisatic HIV-infected state.
OBJECTS OF THE INVENTION
It is an object 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 object of the invention to provide a synthetic vaccine comprising a peptide having an amino acid sequence corresponding to an antigenic determinant of the HIV envelope protein that is capable of inducing protective immunity in mammals against HIV.
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 additional object of the invention to provide a method of detecting the presence of anti-gp120 antibodies in biological test samples.
SUMMARY OF THE INVENTION
The invention relates to immunogenic preparations and vaccines made therefrom. Peptides having amino acid sequences corresponding to
antigenic determinants of the envelope protein of HIV are covalently coupled, either directly or through spacer molecules, to suitable carrier molecules. Synthetic vaccines comprising one or more such peptides are disclosed.
In one embodiment, the present invention comprises an essentially pure form of a peptide having an amino acid sequence corresponding to an antigenic determinant of the envelope glycoprotein of HIV, which peptide is capable, when covalently linked to a carrier molecule, of inducing in a mammal high titers of protective antibodies against HIV. The peptide can have, for example, the sequence CTRPNNNTRKSIRIQRGPG, corresponding to amino acids 303-321 of the envelope glycoprotein of the HTLV-IIIB isolate (Ratner et al., Nature 313:277, 1985), or any portion thereof.
In another embodiment, the present invention comprises an immunogenic conjugate capable of inducing in a mammal high titers of protective antibodies against HIV, 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 HIV.
In yet another embodiment, the present invention comprises a method of producing immunity to HIV comprising administering the above-described conjugate to a mammal.
In another embodiment, the present invention comprises a method of detecting the presence of anti-gp120 antibodies in biological test samples.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Recombinant proteins and relation to synthetic peptides.
Figure 2. Reactivity of AIDS patient antibodies to synthetic peptides.
Figure 3. Reactivity to gp120 of antibodies from HIV+ patient purified over synthetic peptide affinity columns.
Figure 4. Neutralization of ETLV-IIIB by goat anti-SP-10 antisera.
Figure 5. Isolate specific neutralization of HIV.
Figure 6. Binding of goat anti-SP-10 serum to ETLV-IIIB- but not to ETLV-IIIRF-infected E9 T cells. Figure 7. Comparison of the ability of various T1-SP10 peptides from the envelope of HIV MN to induce anti-T1-SP10MN peptide antibodies in Balb/c mice. Each point represents the mean level of anti-T1-SP10 serum antibody in 4-5 mica 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 (E/C) of postbleed immunization (E) optical density (OD) to prebleed (control) OD. Data show that T1-SP10MN(A), F-T1-SP10MN and F-T1-SP10MN(A) peptides after 2 immunizations induce higher levels of anti T1-SP10MN antibodies than did T1-SP10MN itself.
Figure 8. Comparison of the ability of various T1-SP10 peptides from the envelope of HIV MN to induce antibodies in Balb/c nice that neutralize HIV HN in syncytium inhibitium assays in vitro. Each bar indicates the results of serum from bleed 3 from one mouse immunized with the indicated form of T1-SP10. Height of bar indicates the percent of syncytium formation inhibited by a 1:10 dilution of serum compared to prebleed serum at the same dilution.
Figure 9 shows antibody titers in ELISA assay against immunizing peptide over time in chimpanzees immunized with HIV env synthetic peptides.
Figure 10 shows peripheral blood mononuclear cell proliferative responses to the T1-SP10IIIB(A) 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 PHA.
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-HIVIIIB
neutralizing antibodies.
Figure 13 shows anti-HIVMN neutralizing antibodies in Rhesus monkeys immunized with T1-SP10MN peptides. Data represent 90% neutralization titers in syncytium inhibition assay.
Figure 14 shows antibody to immunizing peptide in Rhesus monkeys immunized with T1-SP10MN (A) peptide.
Figure 15 shows neutralizing antibody levels in synyytium inhibition assay in serum of Rhesus monkeys immunized with F-T1-SP10MN(A) peptide.
Figure 16 shows serum antibody titers to immunizing peptide in Rhesus monkeys immunized with F-T1-SP10MN(A) peptide.
Figure 17 shows the absorption of cross neutralizing antibodies induced by T1-SP10MN(A) peptide in rhesus monkey 18987 by peptides containing GPGRAF sequence. As shown, a peptide containing T1 did not absorb out neutralizing antibodies nor did a peptide with a sequence not in T1-SP10MN(A). Only peptides with GPGRAF absorbed the neutralizing activity proving that this animal selectively recognized the GPGRAF region of the V3 HIV gp120 loop as immunogenic and made cross-reactive antibodies to this region. Figure 18. Neutralizing antibody titers against HIV IIIB/LAI (solid lines) and HIV MN
(dotted lines) in serum of the four chimpanzees immunized with T1-SP10IIIB or F-T1-ST10IIIB (A) peptides then immunized with T1-SP10MN(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-SP10IIIB peptide; dotted line indicates antibody response against T1-SP10MN(A) peptide.
Figure 20. Absorption of chimpanzee 1070 serum neutralizing antibodies against the HIV MN isolate by SP10MN(A) peptides and partial absorption by DP2 peptide.
Figure 21. Induction of high levels of neutralizing antibodies against HIV MN with T1-SP10MN(A) peptide in Rhesus monkeys.
Figure 22. Induction of anti-T1-SP10MN(A) peptide antibodies with T1-SP10MN(A) peptide in Rhesus monkeys.
Figure 23. Induction of high levels of anti-HIV MN neutralizing antibodies with T1-SP10MN (A) peptide. Figure 24. Induction of antibodies against F-T1-SP10MN(A) peptide using F-T1-SP10MN(A) peptide as immunogen in Rhesus monkeys. Assay used in Figs. 22, 24 was end-point ELISA against
immunizing peptide (E/C greater than 2.9). Figure 25. Absorption of serum neutralizing antibodies against the HIV IIIB isolate by SP10MN(A) and DP2 peptides. Figure 26. Panel A is a general prototype design of the C4-V3 peptide called T1-SP10(A) from the HIV isolate MN with 2 T helper determinants in the hybrid peptide, one MHC Class I CTL epitope restricted by B7, and a second CTL epitope restricted by HLA-A2. Panel B shows the Th-CTL peptide designed from simian immunodeficiency 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 T1-SP10(A) Th-B-Tc peptides for human immunization.
Figure 28. Mab 48d binds to the C4-V3 peptide T1-SP10CANO (A) whereas monoclonal antibody 17b does not. Increasing amounts of monoclonal antibodies were added to ELISA plates on which the T1-SP10CANO (A) C4-V3 peptide was coated (2 μg/well) as described in detail in Haynes et al (J. Immunol. 151:1645 (1993), J. Exp. Med. 177:717 (1593)). Figure 28 shows that mab 43d bound to the T1-SP10CANO(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 T1-SP10CANO (A).
Figure 25. The whole T1-SP10CANO (A) peptide is required for maximal peptide binding to mab 48d. Either the T1 peptide (C4 region alone) the V3 peptide [SP10CANO (A)], C4-V3 peptide [T1-SP10CANO (A) 3 or a mixture of equal amounts of C4 (T1) + V3 [SP10CANO (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.HP20 did not bind to any of these peptides, whereas the 48d mabs bound to SP10CANO (A) peptide and significantly better to the C4-V3 version of the T1-SP10CANO (A) peptide. Mixing the T1 + the SP10CANO (A) peptide together did not increase 48d binding.
Figure 30. General scheme for an HLA-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 gp120 envelope protein.
Amino acids are shown in single letter code, and numbers represent the positions of amino acids in the HIVBAL envelope protein gp160.
Figure 32. Western blot analysis of guinea pig antisera against HIV gp120 and proteins. Recombinant gp120, gp41-MBP fusion proteins (0.1μg/lane), cell lysate of HIVLAI/IIIB infected- or mock-infected CEM cells
(0.5×106 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 1h at room temperature, followed by incubation with goat anti-guinea pig IgG labeled with horseradish peroxidase for 1h 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-1 peptide HIV-1 gp41 peptide SP400-BAL. The pre-immune serum from the same guinea pigs were used as control. Panel C. Western blot with sera from guinea pig immunized with HIV-1 peptide HIV-1 gp120 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 (106 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.
DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to peptides corresponding to immunegenic epitopes of HIV 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 to carrier molecules (and/or are polymerized) rendering them suitable as vaccines. These vaccines are useful for immunization against AIDS when administered to mammals , for example, by the parenteral route.
It was determined that peptides that should be studied for immunogenic potential included those corresponding to hydrophilic, charged regions of the HIV envelope glycoprotein. It was further determined that, of such peptides, those with predicted beta turns would likely be of particular
importance. It was recognized that the formation of intrapeptide disulfide bonds would be useful in establishing native configurational 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 of the predicted amino acid sequence of the envelope protein of the ETLV-IIIB and ARV-2 isolates of HIV established the secondary structure and location of hydrophilic regions. Secondary structure was determined from the computer analysis using the method of Chou and Fasman (Biochemistry 13:211 and 13:222, 1974;
Advances in Enzymology 47:42, 1978). Potential areas of beta turns were localized using the method of Rose (Nature 272:535, 1978). Hydrophilic regions of the envelope protein were identified by the technique of Rose and Roy (Proc. Nat'l. Acad. Sci. USA 77:4643, 1980).
The peptides of the instant invention correspond to, or are homologous with, B-cell epitopes present within the central region of the HIV isolate ETLV-IIIB envelope protein, or envelope protein of related HIV 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 of high titers (that is, advantageously, a reduction in infectivity of 100 infectious units of approximately 80% in vitro at 1:600 dilution of serum) of type (or isolate) specific neutralizing antibodies against HIV.
Unlike the intact gp120 molecule, the peptides themselves are net capable of inhibiting interaction between the CD4 (T4) molecule on the surface of T lymphocytes and macrophage HLA class II molecules, and thus do not interfere with normal immune function. That is, peptides of the instant
invention capable of inducing anti-HIV neutralizing antibodies, do not inhibit antigen-specific normal T cell proliferative responses in vitro.
Peptides of the instant invention can have, for example, the sequence CTRPSNNTRXSIRICRGPG
(designated SP-10), corresponding to amino acids 303-321 of the HTLV-IIIB envelope glycoprotein gp120 (Rather et al.. Nature 313:277, 1985), or some portion of that sequence. Peptides of the invention can also have sequences corresponding to the analogous SP-10 regions of HIV isolates other than ETLV-IIIB, or portions thereof, these sequences being designated "SP-10- like " ( see , for example, sequences in Table I ) .
The expression "SP-10-like" includes within its meaning the SP-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 an immune response in mammals. Examples of suitable carrier molecules include tetanus toxoid, keyhole limpet hemocyanin (KLH), and peptides corresponding to T cell epitopes (that is, T1 and T2) of the gp120 envelope glycoprotein that can substitute for non- AIDS virus-derived carrier molecules (Cease, Proc. Nat'l. Acad. Sci. (USA) 84:4249, 1987; Kennedy et al., J. Biol. Chem. 262:5769, 1987).. Peptides can also be administered with a pharmaceutically acceptable adjuvant, for example, alum, or conjugated to other carrier molecules sore
immunogenic than tetanus toxcid.
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 SP-10-like sequences, or portions thereof, to a carrier molecule; alternatively, SP-10-like sequences, or portions thereof, can for example be synthesized directly adjacent to, for example, another
immunogenic HIV envelope sequence, for example, T1 or T2. Cysteines can be added either at the N or C terminus of the SP-10-like peptide for conjugation to the carrier molecule or to both ends to
facilitate interchain polymerization via di-sulfide bond formation to form larger molecular aggregates.
Conjugation of the carrier molecule to the peptide is accomplished using a coupling agent.
Advantageously, the heterofunctional coupling agent M-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) or the water soluble compound m-maleimidobenzoylsulfosuccinimide ester (sulfo-MBS) is used, as described by Green et al (Cell, 28:477; 1982) and by Palker et al. (Proc. Nat'l Acad. Sci. (U.S.A.) 84:2479, 1987).
Vaccines of the instant invention comprise one or more SP-10-like peptides, or portion thereof, each SP-10-like peptide being derived from a.
different HIV strain, which peptides are conjugated to carrier molecules. A polyvalent vaccine
comprising a mixture of synthetic peptides,
advantageously about 2 to about 10, corresponding in 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 SP-10 sequence of ETLV-IIIB (see Table I) can be conjugated to or
synthesized with either the ETLV-IIIB gp120 envelope T cell epitope T1 (amino acids 428-443 of gp120), KQIINMWQEVGKAMYA, or to the T2 epitope (amino acids 112-124 of ETLV-IIIB gp120), HEDIISLWNQSLK (Cease et al., Proc. Nat'l. Acad. Sci (USA) 84:4249, 1987) to form a single polypeptide (in the case of T1-SP-10 from the ETLV-IIIB isolate of HIV,
KQIINMWQEVGKAMYACTRPNNNTRKSIRIQRGPG). Similarly, T1 or T2 sequences from other HIV isolates can be linked to synthetic peptides derived from the SP-10 region of the corresponding isolates (set Table I), advantageously, at the N terminus of the SP-10-like peptide, to make a T1(or T2-)-SP-10-like peptide capable of inducing neutralizing antibody titers against a specific strain of HIV. Linkage at the C terminus of the SP-10-like peptide is also possible.
Smaller portions of SP-10-like peptides, for example, SP-10 RF(A) and SP-10 C (Table II) can also be covalently linked to carrier molecules, including gp120 T ceil epitopes, and used in a vaccine.
The present invention also relates to an effective protective vaccine against strains of HIV comprising, in addition to SP-10-like sequences and appropriate carrier molecule(s) additional sequences from the gp120 envelope molecule. Since there is a major hypervariable region that is carboxy terminal to peptides designated as SP-10-like in Table I (envelope amino acids 322-233, Rather et al, Nature 313:277, 1935), and since the hypervariable region may play a role in enhancing the ability of SP-10- like peptides to raise type-specific neutralizing antibodies, amino acid sequences corresponding to a hypervariable region (approximately amino acids 322-333) of HIV isolates can be included as vaccine components, in part or in whole, as described for other SP-10-like peptides (see, for example,
sequences in Table II). Eypervariable sequences are linked advantageously C-terminal to the SP-10-like peptide. Linkage H-terminal to the SP-10-like peptide is also possible.
The present invention also relates to an effective protective vaccine against strains of HIV comprising, in addition to a SP-10-like sequence and a carrier molecule, a peptide corresponding to the HIV gp41 transmembrane region that is involved in viral-induced cell fusion, FLGFLG, (Gallagher, Cell 50:327, 1987). The FLGFLG sequence is added, advantageously, at the C terminus of the SP-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 HIV formed from cysteine-T1-(or T2-)SP-10-llke, cysteine-T1-(or T2-)SP-10-like-hypervariable region, or cysteine-T1-(or T2-) SP-10-like-FLGFLG polypeptides; and/or SP-10-like-cysteine or SP-10-like-hypervariable region-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 advantageously comprise SP-10-like peptides derived from
(corresponding to) at least 2 HIV isolates.
A polyvalent HIV vaccine of the instant invention comprises, advantageously, two or more conjugates comprising an SP-10-like sequence, or portion thereof (see, for example, sequences in Table 1) derived from 2 or more HIV isolates, and a carrier molecule such as tetanus toxoid, or two or more T1- or T2-SP-10-like peptide conjugates, wherein both the T1 (or T2) and the SP-10-like sequences correspond to sequences present in a specific HIV isolate.
The advantage of using, as a carrier molecule, a synthetic peptide reflecting a portion of the gp120 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 HIV would be specific. Combining in a poylvalent vaccine several peptides reflecting sequences from the SP-10 region of different isolates, and possibly the T cell recognition region
of the gp120 envelope, overcomes the problem of isolate-specific neutralization.
The present invention also relates to a polyvalent vaccine comprising SP-10-like peptides linked to hypervariable sequences described above (see, for example, Table II). A mixture of such polypeptides, coupled to appropriate carrier molecules and/or polymerized via disulfide bond formation (Harington, C.R., et al., Biochem. J., 30:1598, 1530; Harington, C.R., et al., Biochem. J., 38; 417, 1544; Weygand et al., Z. Naturforsch., 176:807, 1962), can be used as a vaccine to evoke a protective antibody response to multiple isolates of HIV.
SP-10-like peptides can be used in a solid phase radioimmunoassay (Palker et al. J. Immunol
136:2393, 1986; ibid., Proc. Nat'l. Acad. Sci. (USA) 84:2475, 1587) to (i) detect the presence and titers of neutralizing antibodies of HIV; and (ii) to determine with which strain of HIV the patient is infected. Thus, in addition to SP-10-like peptides being used as a vaccine or a component of a vaccine, the peptide can be used, as described above, for diagnostic purposes. Peptides of the instant invention can also be used in standard enzyme linked immunosorbent assays to detect the presence of HIV antibodies. 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 T1 gp120 env region, reported to be recognized by both B cells (Palker et al J. Immunol.
142:3612, 1989) and helper T cells (Ceasa et al Proc. Natl. Acad. Sci. (USA) 84:4249, 1987), and the SP10-like gp120 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 (HIV) (see reference of Palker et al cited immediately above; Palker et al Proc. Natl. Acad. Sci. (USA) 85:1932, 1988; and also Rusche et al Proc. Natl. Acad. Sci. (USA) 85:3198-3202, 1988 and Goudsmit et al Proc. Natl. Acad. Sci. (USA) 85:4478, 1983) (see Tables III and IV).
Sequences from Ratner et al. Nature, 313:277, 1985.
Heutralizing antibodies produced by TI- SP10-like peptides are type-specific, in that antibodies raised against the HIV HTLVIIIB (IIIB) isolate do not neutralize the HIV HTLVIIIMN (MN) or HTLVIIIRF (RF) HIV isolates (Palker et al. J. Immunol, 142:3612, 1939). Similarly, neutralizing antibodies raised against the T1-SP10-like peptides containing sequences from the Xi or RJ HIV isolate neutralize the hocclogαus isolate but do net neutralize any of the other two HIV isolates. However, when goat anti-T1-SP10-like antisera were tested against 9 HIV field isolates in North Carolina, anti-T1-SP10IIIB serum vas observed to neutralize l of 9 HIV isolates, anti- T1-SP10RF serum neutralized 3 of 9 HIV isolates, and anti-TI-SP10MN serum neutralized 6 of 9 HIV isolates (Haynes et al AIDS Res. Retrol. 6:38, 1990) (see Table V).
La Rosa et al (Science 249:932, 1990) have shown that the HIV HN motif described by
Haynes et al in AIDS Res, Retrol, (above) is one of the prodominant motifs of HIV isolates cultured from AIDS patients around the United States.
Palker et al (J. Immunol, 142:3612, 1989) were the first to report that the strategy of mixing peptides from various isolates could be a successful approach to the problem of raising antibodies against numerous strains of HIV with divergent amino acid sequences in the 303-337 region of the HIV envelope. Moreover, Palker etal reported that the T1-SP10-like peptide was advantageous over synthetic peptides coupled to carrier molecules such as KLH or tetanus toxoid. Whereas carrier coupled peptides only induced large accents of antibody against the carrier in polyvalent mixtures of peptides, when the T1 sequence of HIVIIIB env (amino acids 429-443) was covalently linked N-terminal to the SP10 sequence (amino acids 303-321), this carrier-free immunogen induced high titers of neutralizing antibodies to all three HIV isolates whose sequences were present in the T1-SP10 peptides. Moreaver, Hart et al (J. Immunol. 1990) have recently shown that the T1-SP10 peptide is non-toxic to immune cells in rhesus monkeys and is capable of inducing high-titered neutralizing antibodies and T helper cells in vivo in these primates. Thus, the T1-SP10- like synthetic peptide construct is a simple, non-toxic and highly efficacious molecule for inducing high titered anti-HIV neutralizing antibody responses and T-helper cell responses in goats and primates.
One of the major problems in developing a vaccine for AIDS has been the question of whether antibody responses alone can protect an individual against both cell-free HIV and HIV-infected cells, or whether cell mediated immune responses (antigen-specific cytotoxic T calls) are needed as well. Certainly, many other viral infections require both antibody and cellular anti-viral immune responses for the generation of protective immunity (Long et al Immunol. Today 10:45, 1989). In addition, local immunity at mucosal surfaces consisting of IgG and IgÅ antibody responses and mucosal surface-associated cytotoxic T cell activity may be required to protect against transmission of HIV via sexual contact or via exposure of mucosal surfaces with infected blood. Thus, a synthetic peptide immunogen would 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 embodiments disclosed and summarized above, the present invention relates to such an immunogen.
The F region (for example, amino acids 519-530 of the BH10/IIIB HIV isolate and
homologous regions of ether HIV-1, HIV-2 and simian immunodeficiency virus (SIV) isolates) has sequence homolegy to the F1 (fusion) peptides of paramyxoviruses (Gallaher Cell 50:327, 1987). The F region has been postulated to form a hydrophobic helical structure capable of inserting into lipid bilayers of cell membranes and inducing cell fusion. Bosch et al (Science 244:694, 1989) have demonstrated that the F region in SIV (that is, the region homologous to the 519-530 env gp41 region of the BH10/IIIB HIV isolate) does indeed mediate cell fusion of SIV-infected cells.
It has been determined that F-derivatized peptides are internalized in immune cells in such a way as to induce the same type of cytotoxic T cell response that is necessary for control of many viral infections, namely, the generation of HLA-restricted CD8+ cytotoxic T cells. F- derivatized peptides interact with cells of the immune system such that when injected into a mammal, they induce anti-HIV memory T helper call activity, anti-HIV neutralizing antibodies, and memory anti-HIV CD8+, HLA-Class I restricted cytotoxic T cell responses.
Accordingly, the present invention relates, in a preferred embodiment, to peptides of the general formulae:
F-Th-SP10 (X)
Th-SP10 (X)
Th-SP10
and
F(X)
wherein:
P sequences are from the putative fusogenic domain of HIV env gp41 (for example, amino acids 519-530 in HIV isolate BH10/III3 or homologous regions in ether HIV-1, KIV-2 or SIV isolates, or sequences functionally equivalent thereto);
Th sequences are either the T1 or T2 T helper epitopes or alternatively are any of the T helper cell epitopes listed in Table X (belov) or amino acid sequences from other regions of HIV proteins not listed but that function as T helper epitopes;
SP10-like sequences are from Tables I or II (see also Table VIII below) or free any SP10- like sequence from HIV field isolates (see, for example, LaRosa et al Science 249:932, 1990); and
(X) sequences are HIV protein sequences recognized by HHC Class I or Class II restricted cytotoxic T cells. Examples of (X) region sequences are given in Tables VIII and IX below.
Alternatively, F sequences can be, for example, C-terminal to Th-SP10(X) sequences.
Moreover, Th, SP10 and (X) sequences can be arranged in any order in the peptide construct. The synthetic peptide immunogen of this embodiment 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 will appreciate that this immunogen (which is a fusion protein) can either be synthesized cheaically or by recombinant means known in the art.
The immunogen can have, for example, the structure: F-T1-SP10-(A). While examples of such immunogens are given in Tables III and IV, one skilled in the art will appreciate that any SP10-like sequence from field or laboratory HIV isolates (for example, LaRosa et al Science
249:932, 1990) can be substituted for the SP10 sequences shown in Tables III and IV (see also Tables I and II).
The T1-like sequences can be selected from T1-homologous sequences from any sequenced HIV isolate including those shown in Table VI.
.
The F-like sequences can be selected from
F-homologous sequences from any sequenced HIV isolate, including these shown in Table VII.
The (A) region-like sequences can be selected from (A)-homologous sequences from any HIV isolate, including those shown in Tables II and VIII.
Sequences from BH10 (IIIB) are from Ratner et al Nature 313:270-284, 1985.
The invention further relates to a peptide comprising the F region sequence (that is, for example, amino acids 519-530 of the BH10/IIIB isolate or other homologous region in other HIV- 1, HIV-2 or SIV isolates) from HIV gp41 placed (covalently linked) H terminal to SP10 or SP10- like regions from any HIV sequence (see, for example, Table II) from field isolates such that the resulting construct can induce neutralizing antibodies and cytotoxic T cells against HIV.
One skilled in the art will appreciate from a reading of the present disclosure that MHC Class I restricted cytotoxic T cells can be induced by administering in vivo, as an example, the 519-530 amino acid region of HIV gp41, consisting of the 12 amino acids: AVGIGALFLGFL (F) or F-region sequences of other HIV-1, HIV-2 or SIV isolates (see, for example, Table VII) that are homologous to the 519-530 amino acid sequence of isolate BH10/III3 (Table VII), covalently linked to any other peptide ranging in length from, for example, 3 to 50 amino acids, such that the F-linked peptide will associate with antigen- presenting cells in such a way 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 molecules and generates the
developaent of CD8+ cytotoxic T cells in vivo. In the context of an efficacious AIDS vaccine, several F-derivatized hybrid HIV peptides can be constructed comprising the F amino acid sequence (see, for example, Table VII) coupled K or C terminal to an azino acid sequence of HIV proteins that are capable of inducing cytotoxic T cells in vivo. Examples of described HIV peptides that are capable of being recognized by KLA Class I cytotoxic T cells are shown in Table IX.
This latter strategy is important in that cytotoxic T cell epitopes are recognized by specific polymorphic HLA Class I or Class II molecules. If 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 with the specific HLA antigen that the (A) peptide uses to be presented to cytotoxic T cells would 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 or Class II molecule by cytotoxic T cells, are contained in an immunogen, then individuals with a wide spectrum of HLA-types will make cytotoxic T cells against HIV.
Thus, an immunogen capable of 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 and recognized by Class I types of molecules that, taken together, are expressed by the majority of individuals in a given population. Table IX shows examples of described cytotoxic T cell epitopes and their HLA restricting elements, if known, that are the types of peptides that can be derivatized by F-sequences and used as a mixture with F-T1-SP10(λ) peptides. Alternatively, sequences in Table IX can be covalently linked C-terminal to SP10 sequences in F-T1-SP10 peptides instead of (A) sequences and a mixture of F-T1-SP10(X) peptides used as an AIDS vaccine (in the formulation F-T1-SP10(X), X is either an (A) sequence (see Tables II and VIII) or ether cytotoxic T cell-inducing sequence such as are listed in Table IX). The same considerations of MHC
restriction that apply to T cytotoxic epitopes also apply to T helper epitopes. That is, recognition of antigens by T helper cells is HLA 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
immunogen, sufficient T helper cell epitopes will need to be present in order to have available sufficient varieties of T helper epitopes within which each patients' T cells may be able to see processed antigen in the context of their own HLA Class II molecules. Table X shows T helper cell epitopes of HIV proteins that can be substituted for the T1 or T2 sequences in the F-Th-SP10(X) construct to provide alternative T helper cell epitopes in the construct.
Studies indicate that the same T helper cell epitope may be recognized by T cells in the context of multiple HLA Class II specificities and, therefore, only a few T helper epitopes are needed to formulate an effective synthetic peptide based AIDS vaccine. Clerici et al (nature
339:333-385, 1939) have provided data that T cells of 85% of the population studied could recognize either T2 or T1 T helper cell epitopes (see Table X). Schrier et al (J. Immunol. 142:1166-1176, 1985) have identified a number of helper 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 epitopes (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:
F-Th-SP10 (X)
Th-SP10(X)
Th-SP10
and
F(X)
where, as indicated above, P sequences are from the putative fuscgenic domain of HIV env gp41 (for example, amino acids 515-530 in HIV isolate
BH10/IIIB or homologous regions in other HIV-1, HIV-2 or SIV isolates, or sequences functionally equivalent thereto) (see, for example, Table
VII); Th sequences are either the T1 or T2 T helper epitopes or alternatively are any of the T helper cell epitopes listed in Table X or amino acid sequences frcm other regions of HIV proteins not listed but that function as T helper epitopes, SP10-like sequences are from Tables I, II or VIII or from any SP10-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-SP10(X), Th-SP10(X), Th-SP10 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 epitopes needed to induce cytotoxic T cells and T helper call responses in the majority of
subjects in a given population cohort. One
skilled in the art will appreciate that the order of P, Th, SP10 and (X) can vary as long as the
above-indicated function of each is retained. For the induction of protective anti-HIV neutralizing antibodies, the specific SP10-like sequences
necessary to be present in F-Th-SP10(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 will appreciate that this information will need to be actively and
continuously monitored in the population and the formulation of the AIDS vaccine changed from time to time depending on changes in the above
variables.
The induction of protective anti-HIV neutralizing antibodies in populations that include a number of different HIV 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 of gp120 and thus is capable of inducing broadly cross-reactive anti-HIV antibodies. One such construct takes the form of a mimeotope of a conformational determinant of the native HIV gp120 C4-V3 region and is exemplified by T1-SP10CANO(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 T1-SP10CANO(A) peptide induces cross-reactive anti-V3 antibodies against a variety of HIV V3 motifs (see Example 11). This induction of cross-reactivity is due to secondary and higher order structures of the V3 loop of the HIV CANO isolate that result in the T1-SP10CANO(A) C4-V3 hybrid mirroring a broadly
neutralizing determinant of HIV gp120. This is demonstrated by the fact that the human anti-gp120 monoclonal antibody 43d (which blocks mouse monoclonal antibodies that prevent CD4 binding to gp120 but does not itself block gp120-CD4 binding (Thali et al, J. Virol. 67:3978-3935 (1993)) binds T1-SP10CANO(A). As an alternative embodiment (strategy), an effective vaccine can be formulated by
determining the HLA Class I and Class II types for a particular individual by, for example, either polymerase chain reaction analysis or by
conventional HLA tissue typing analysis. Based on that information, the specific immunogens that need to be included in the P-Th-SP10(X), Th-SP10(X), Th-SP10 and F(X) formulation can be determined. Thus, in this latter embodiment, the peptides given to the subject are these necessary for eliciting the desired anti-HIV B and T cell responses.
From a reading of the foregoing, one skilled in the art will appreciate that this is a general strategy for development of a vaccine for any infectious disease. Moreover, the ability to conjugate the F-region from the HIV g41 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 MHC Class I molecules) provides
a simple and effective method of inducing MHC Class I restricted cytotoxic T cells to any peptide bearing cytotoxic T call epitopes. This is the case regardless of whether the sequence of the cytotoxic T cell epitope is derived from proteins in an invading organism or whether the cytotoxic T call epitope sequences are derived from host proteins.
As an example of the use of F-derivatized peptides that include sequences from host
proteins, it is contemplated that F-derivatized peptides can be used comprising HIV gp41 F
sequences (for example, amino acids 519-530 from the BK10/IIIB HIV-1 isolate or from homologous regions of other HIV-1, HIV-2 or SIV isolates, or sequences functionally equivalent thereto)
conjugated either H- or C-terminal to peptides capable of being recognized by cytotoxic T calls in the context of MHC Class I or Class II, the sequences for such peptides being derived from the variable region of T cell receptor for antigen (TCR) molecules expressed on the surface of autoreactive T cells that mediate host tissue destruction in various autoimmune diseases, infectious diseases and in the setting of organ transplantation.
Sun et al (Nature 332:843, 1933; Eur.
J. Immunol. 18:1993, 1933) have reported the isolation of cytotoxic T cell clones that are specific for idiotypic determinants on
encephalitogenic T cells and which adoptively transfer resistance to experimental autoimmune encephaloayelitis. The concept of immunization of subjects with autoimmune disease with immunogens that would induce an immune response against the autoimmune clone of T cells has recently been recognized as an important experimental approach (Seviewed on Cohen et al Immunol. Today 332, 1988; Eowell et al Science 246:668, 1939; Wraith et al Cell 57:709, 1939). Thus, the present invention provides a simple and effective method for
inducing MHC-restricted Class I or Class II cytotoxic T calls to peptides of host antigens and thus represents a major advance in the development of vaccines for autoimmune disease.
Using standard recombinant DHA techniques and existing probes and sequences for TCR molecule antigen binding regions, sequences can be obtained from unique regions of the TCR molecules (Barns et al J. Exp. Med. 169: 27, 1989). 7-derivatized peptides can be used to induce a cytotoxic T cell immune response targeted to the specific clones of T cells bearing TCRs responsible for antigen-specific T call-mediated host tissue damage in the above disease categories. Once induced, such an F-peptide-induced 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 of the use of F-derivatized host peptides is to similarly control antibody-mediated tissue damage that occurs in the context of autoimmune diseases, infectious diseases, and in the setting of organ
transplantation. B cell surface receptors for antigen (surface immunoglobulin) also contain regions that are specific for 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 from the region of the B cell immunoglobulin molecule that binds antigen can be identified using, for example, recombinant DHA techniques. Further, sequences capable of inducing KHC Class I or Class II cytotoxic T cell responses can be identified. By derivatizing such an immunoglobulin antigen- binding region peptide with F sequences and injecting the F-derivatized peptide into the subject Baking the autoantibody, a cytotoxic T cell response against an autoantibody-producing B cell can be induced, thereby eliminating a tissue damaging autoantibody response that occurs in the context of the above disease categories.
A 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
immuncglobulin 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. Eng. J. Med. 306:517, 1982) or antibodies against variable TCR regions in the case of treatment of T ceil tumors (Sanagava, O. J. Exp. Med. 170:1513-1519, 1539). Thus, F-derivatized synthetic peptides containing the seguences of variable regions of the TCR or immunoglobulin molecules expressed cn the surface of T or B cell malignant cells respectively, can be injected into the tumor-bearing host to induce anti-TCR or anti-immunoglobulin-specific 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-A, non-B 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 and injecting such sequences into individuals, memory anti-Hepatitis C specific cytotoxic T call responses can be induced that protect the
individual from infection with 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.
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 gp120 (Palker et al, J. Immunol. 142:3612-3619 (1989); Haynes et al, J. Immunol. 151:1646-1653 (1993)). The T1 epitope from the gp120 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 gp120 or gp41.
The present embodiment results, at least in part, from the realization that immunogenic peptides reflective of points of contact between HIV gp120 and gp41 (termed "gp120/gp41
touchpoints") together provide antibodies against multiple sites on native gp120 and gp41, and thereby facilitate the dissociation of gp41 and gp120. Dissociation of gp41 and gp120 promotes the neutralization of HIV primary isolates by these antibodies.
A number of sites have been identified on gp120 or gp4l that: are involved in, or regulate, the interactions of gp120 with gp41 (see Figure 31, Table XXVIII). These sites include: the gp120 V3 loop region (Willey, R.L. and Martin, M.A., J. Virol. 67:3639- 3643 (1993)), the gp120 C2 region cencered around asparagine ac aa 267 (Willey, R.L. and Martin, M.A. , J. Virol. 67:3639 -3643 (1993)), the C-terminus of gp120 (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 Res. 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 gp120 (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)) and A. Beretta has reported that antibodies against the C5 gp120 region can "loosen" the
gp41/gp120 interaction and thereby inactivate HIV (Fust et al, Immunol. Today 16:167-169 (1995)). Whether the C-terminus of gp120 by itself induces anti-HIV neutralizing antibodies is controversial. Some investigators have found anti-gp120
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 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.
The following nonlimiting examples
illustrate the invention in more detail.
EXAMPLE 1
Synthesis of Peptides and Preparation of Conjucates
Synthetic essantially pure peptices containing hydrophilic acinc acid sequences from the HTLV-IIIB envelope glycoprotein gp120 (Ratner, et
al. Nature, 313:277, 1985) were synthesited on an Applied Biosystems 430A peptide synthesiser using chemical and program cycles supplied by the
manufacturer. Sequences of synthetic peptides are given in Table XI .
The relationship of the peptides synthesized to known reccmbinant proteins PE3, PBI and PENV9 is shown in Figure 1 (Putney et al.,
Science, 234:1392, 1986; Petteway et al., Viruses and Human Cancer: UCLA Symposia on Molecular and Cellular Biology 1987).
Peptides were cenjugatad to carrier molecules such as bevine serum albumin (BSA) or tetanus toxoid (TT) with MBS, as described by Green et al. (Cell, 28:477, 1982; Palker et al, Proc. Katl. Acad. Sci. (USA) 84:2479, 1387). For the ccupling procedure, 24 eg of tetanus toxoid (for example] in 0.5 al of phosphate buffered saline, pH 7.2, vas incubated with 1 ag of HBS dissolved in 100μl of dimethylformamide for 1 hr. at 23ºC.
Tetanus toxoid treated with MES (TT-MBS) vas then subjected to sieving chromatography on a PD-10
(Pharmacia) column to remove unreacted MBS from TT-MBS, and fractions containing TT-MBS were recovered in the void volume of the column as determined by spectrophotometric assay at an optical density of 280 nm. TT-MBS was then incubated with recking at 23ºC for 3 hr. with 6-9 mg of synthetic peptide (molar ratio 30:1, peptide carrier protein) in PBS containing reduced cysteine at either the carboxyl or amino terminus. TT-peptide conjugates were dialyzed overnight at 4ºC against PSS or again desalted on a PD-10 column and were used as an immunogen.
Conjugation of peptides to BSA or tetanus toxoid was monitored by subjecting conjugates to sodium dodecylsulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) under non-reducing conditions and by measuring the increase in apparent molecular weights over that of ESA and TT treated with MBS. Coupling efficiencies also monitored by trace iodination of peptides varied from 10-30* depending on the peptide.
EXAMPLE 2
Reactivi ty of AIDS Pat ient Antibod ies
To Synthe tic Pept ides
Synthe tic peptides derived f rom hydrophilic reg ions o f gp120 coupled to BSA were us ed as ant igens in a radio immunoassay ( RIA ) w i th HIV+ patient sera (N=12) and noraal serum control (N=4), to evaluate the AIDS patient antibody response to epitopes on gp120 (Figure 2) (Palker et al., J.
Immunol . 136:2393, 1986; ibid, Proc. Hat'l. Acad. Sci (USA), 84:2479, 1987). The majority of EIV+ patient sera reacted with two synthetic peptides, SP-10 (9/12, 75%) and SP-22 (8/12, 671).
Results are expressed as a ratio (E/C) of duplicate cpm values obtained with experimental (E) AIDS sera and control (C) serum samples. E/C > 3.0 = positive.
EXAMPLE 3
Reactivity of gp120 to Antibodies from HIV+ Patient
Purified Over Synthetic Peptide Affinity Columns
For preparation of affinity columns, synthetic peptides containing aaino acid sequences from HTLV-IIIB gp120 (SP-10, 10A, 11, 14, 15, 22, see Figure 1) were coupled to BSA and then
covalently linked to CNBr-activated Sepharose.
Serum aliquots (2ml) from an HIV seropositive patient were then passed over each column and the antibodies that bound to the affinity columns were then tested for reactivity to purified 12SI-labelled ETLV-IIIB gp120 in RIP assay (Figure 3A) and for reactivity to the surface of E-5 cells infected with ETLV-IIIB in indirect immunofluorescence assays (Figure 3B).
A) In RIP assay (Palker et al., Proc.
Nat'l. Acad. Sci (USA) 84:2479, 1987; ibid, J.
Immunol. 136:2393, 1986), bound antibodies frca the SP-10 (lane 1), SP-10A (lane 2), SP-11 (lane 3), and SP-22 (lane 6) affinity columns reacted with gp120-IIIa in RIP assay, with antibodies from the SP-10 column showing the greatest reactivity to gp120- IIIB.
B) When tested in FACS analysis (Shapiro, Practiced Flow Cytometry, Alan R. Liss Pub., NY, NY, 1985), antibodies reactive with synthetic peptide SP-10 bound to the surface of HIV-infected cells while binding of affinity purified antibodies to SP- 14 or to SP-10A, 11, 15 or 22 (not shown) vas not detected. These data suggest that the antigenic site(s) defined by SP-10 are accessible to antibody binding when gp120 is present on the surface of HIV+ cells.
EXAMPLE 4
Neutralisation of HIV by Goat Anti-SP-10 Antisera
Goats were immunized subcutaneously with 28mg of tetanus toxoid SP-10 conjugates (SP-10-TT) in Freunds complete adjuvant (days 0) followed by biweekly inoculations in incomplete Freunds adjuvant (days 14 and 28). Serum samples were collected after the second immunization and tested for the ability to inhibit (i.e. neutralise) HIV infection of E-9 T cells in vitro as measured by the presence of reverse transcriptase (RT) activity in ceil culture supernatants (Figure 4). Decreased cpm values obtained in RT assays reflect decreased levels of HIV after cocultivation of virus and cells for 10 days.
When pre-incubated with 100 infectious units of ETLV-IIIB, goat anti-SP-10 antisarum neutralized the ability of HIV isolate HTLV-IIIB to infect E-9 T cells (● - ●, 50% neutralization titer = 1/145). In contrast, serve collected from the same goat prior to immunization did not appreciably neutralize HTLV-IIIB (O-O, 50% neutralization titer = 1/16).
The original animal injected with SP-10-TT (whose serum neutralized ETLV-IIIa in reverse transcriptase assay) vas subsequently injected with additional doses of SP-10-TT (0.5 mg/kg body
weight). The 50% neutralization titer rose to
1:1600 after two injections. Neutralization data from these and other experiments with SP-10-like peptides are shown in Table XII as the serum
dilutions that result in 80% rather than 50%
neutralizations of HIV.
In addition, a second goat vas injected twice with 0.5 mg/kg doses of SP-10-TT. Serum from the second goat neutralized ETLV-IIIB at a titer of 1:100. Importantly, both sera against SP-10-TT raised in goats also inhibited ETLV-III2 infectivity of T cells in the syncytium-inhibition assay (Table XII).
The syncytium-inhibition assay (Lifson et al., Nature 323:725, 1986) measures the ability of antibodies to inhibit the fusion of HIV-infected T cells, these expressing HIV gp120 envelope protein . on the cell surface, with CD4 (T4)+ uninfected T cells. The CD4 (T4) molecule serves as the receptor for the AIDS virus (Haddon et al., Cell 47:333, 1986). The result of fusion of these two cell types is the formation of giant cells that are infected with HIV. In many instances, the result of HIV infection of cells and giant ceil formation is death of the infected cell (Zagary et al., Science
231:850, 1986).
Therefore, the above-described ability of goat anti-SP-10 sera to inhibit ETLV-IIIB
infectivity in the syncytium inhibition assay and in the reverse transcriptase assay, indicated that anti-SP-10 antibodies are capable of blocking the binding of HIV gp120 protein to T cell CD4 (T4) molecules. In addition, goat antiserum raised to a peptide [SP-10 RF(A)] containing SP-10-like sequence from HIV isolate ETLV-IIIRF inhibited syncytium formation by ETLV-IIIRF but net by ETLV-IIIB, indicating that type-specific antigens contained in SP-10 RF(A) are suitable as vaccine components to raise antibodies that inhibit the interaction of ETLV-IIIRF gp120 and T cell CD4 (T4) molecules.
EXAMPLE 5
Induction of Antibodies Capable of
Inhibiting HIV gp120-CD4 (T4) Interactions
A series of studies were undertaken to determine 1) whether 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-SP-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). As seen in Table XIII, SP-10-TT alone was as good an antigen-specific T cell activator as TT alone. Moreover SP-10-TT and SP-10-BSA when added to TT alone did not inhibit TT induced proliferation by normal T cells. In addition, anti-SP-10 goat serum did not bind to peripheral blood lymphocytes or monocytes in indirect immunofluorescence assay using flow cytofluorometry.
These data indicate that the SP-10 peptide does not perturb normal human T cell function that is dependent on a functional CD4 (T4) molecule but does induce antibodies that will inhibit HIV ep120- CD4 (T4) interactions and neutralize HIV in reverse transcriptase inhibition assays.
Thus, vaccines comprising the small synthetic SP-10-like peptides (less than or equal to about 35 amino acids in length) have distinct
advantages ever HIV vaccines comprising recombinant gp120, or large subunits thereof, as the latter may interfere with normal immune function.
EXAMPLE 6
Isolate specific neutralization of HIV
Synthetic peptide SP-10 has an amino acid sequence derived from and unique to the gp120
envelope protein of HIV isolates ETLV-IIIB and LAV, while other HIV isolates have varying degrees of differing amino acid sequences in their SP-10-like gp120 envelope proteins. Synthetic peptide SP-10 (that is, SP-10-IIIB) from the HTLV-IIIB isolate of HIV vas coupled to tetanus toxoid and used to raise anybodies in goats (0.5 mg of conjugate per kg goat body weight) as described by Palker et al. (Proc. Nat'l. Acad. Sci. (USA) 84:2479, 1987). Coat antibodies raised to synthetic peptide SP-10 were tested for the ability to neutralize four different HIV isolates (Fig. 5A: HTLV-IIIB, Fig. 5B: HTLV-IIIRF, Fig. 5C: ETLV-IIIMN, Fig. 5D: HTLV-IIISC). Goat anti-SP-10 antiserum (#, pre-immune goat serum (O) and AIDS patient serum (S) all at a 1/10 dilution were first incubated with dilutions (10-1, 10-2, 10-3) of each virus isolate. Next, these virus isolates were tested for the ability to infect E-9 T cells by cocultivation of virus and cells for 10 days in vitro. Levels of HIV present in cell culture supernatants after 10 days in culture were 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 of HIV in culture.
As shown in Fig. SA, goat anti-SP-10 antiserum inhibited (i.e. neutralized) ETLV-IIIB infection of E-9 cells at a virus dilution of 10-2. Pre-immune goat serum did not inhibit HTLV-IIIB infection at the same dilution of virus, In contrast, gcat anti-SP-10 antiserum did net neutralize other isolates of HIV (Figs. 5 B-D). AIDS patient antibodies neutralized all four isolates of HIV (Figs. 5 A-D). The data indicate that goat antiserum to synthetic peptide SP-10 neutralizes the ETLV-IIIB isolate that contains in its gp120 envelope protein the amino acid sequence present in SP-10. These data, along with data in Table XII,indicate that a vaccine comprising SP-10-like amino acid sequences from a variety of HIV isolates will be effective against a wide spectrum of HIV isolates.
EXAMPLE 7 Binding of Goat Anti-SP-10 Serum To
ETLV-IIIB- But Not To ETLV-IIIPF-
Infected E9 T Cells
The reactivity of goat anti-SP-10 serum and autologous prebleed control serum were compared on either uninfected E9 T cells, E9 T cells infected with HIV isolate ETLV-IIIB, or E9 T cells infected with HIV isolate ETLV-IIIRF using flow
cytofluorometry and a Coulter EPICS V
cytofluorograph (Eaynes, Immunol. Rev. 57: 127, 1981; Eaynes et al., New Eng. J. Med. 304:319, 1911).
Goat anti-SP-10 serum (1:200) reacted with 40. of ETLV-IIIB-infected E9 T cells coopered to ETLV-IIIB-infected E9 cells incubated with control (prebleed) goat serum (1:200) (Figure 6A). Neither gcat anti-SP-10 nor control (prebleed) serum (1:50) reacted with noninfected H9 T cells (Figure 63). Neither control (prebleed) nor anti-SP-10 serum (1:50) bound to H9 T cells infected with the HTLV- IIIRF isolate of HIV (figure 6C). EXAMPLE 8
Development of a Synthetic Immunogen Comprising
Multiple Regions of the Human Immunodeficiency
Virus Envelope that Induces T Helper Cell.
CD8+ Cytotoxic T Cell and B Cell
Neutralizing Responses In 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 were prepared reflective of regions of the KIV EH isolate that have included therein a defined cytotoxic T cell epitope (see Table III). These studies were performed with the HN HIV isolate since it appears that this is the Best common prototypic virus in the US at present (La Rosa et al Science 249:932, 1990).
Takahashi et al. (Science 246:118, 1939) have defined a cytotoxic T cell (CTL) epitope that includes amino acids 322-326 (FYTTX) from the MN HIV isolate and includes amino acids 323-329 of the KIVIII3 isolates (see Table IV) (Taλahashi et al J. Eys. Ked. 170:2023, 1989). Thus, one T1- SP10 variant peptide made vas the T1-SP10MN(A) peptide with the (A) signifying that amino acids 322-326 were added to the existing KH SP10 region of amino acids 303-321 (see Table III). Secondly, to make a synthetic peptide that might insert into the cell membrane of antigen-presenting cells and therefore potentially be processed and expressed via KHC Class I molecules and therefore be recognized by CD8+ CTL, the first 12 amino acids of the gp41 HIV envelope protein (amino acids 519- 530 AVGIGALFLGFL in HIV isolate BH10/IIIB) were covalently linked N-terminal to T1-SP10 peptides. These amino acids (519-530) of HIV gp41 are highly hydrophobic. They have been postulated to be the primary amino acids that are capable of inserting into lipid membranes and to play a role in the ability of HIV to induce cell fusion (Brasseur et al AIDS Res, Hum, Retrovirol, 4:83, 1988).
Peptides with this 12 amino acid gp41 sequence have the prefix F- before the name of the peptide (F for fusogenic region) (see Tables III and IV). Bosch et al (Science 244:694, 1989) have
demonstrated that the homologous region in SIV (GV7VLGFLGFLATAG) to the P region in HIV
(AVGIGALFLGFL) is indeed the SIV fusion envelope peptide. Thus, it vas postulated that F-derivatized peptides eight also insert into antigen presenting cell membranes, the F- derivatized peptides would be internalized and CD8+ MHC Class I restricted CTL would be generated in vivo following immunization with F-derivatized peptides. (Deres et al (Nature 342:561, 1989) have shown the conjugation of a fatty acid tripalmitoyl-S-glycerylcysteinyl-seryl-serine moiety to synthetic peptides can promote synthetic peptide processing and presentation in the context of MHC Class I molecules and lead to generation of CD8+ CTL in vivo.)
A series of studies were performed in Balb/c mice with the MN series of T1-SP10 peptides (Table III) to compare their ability to induce anti-peptide antibodies (see Figure 7), to compare their ability to induce anti-HIV neutralizing antibodies (Figure 8), and to determine if any of these peptides could induce MHC Class I restricted CD8 CTL when injected in nice in vivo (Tables XIV and XV).
Comparison of the ability of various T1-SP10 peptides to induce anti-peptide antibodies in Balb/c mice:
Figure 7 shows a comparison of the levels of anti-peptide antibody generated in the serum of
Balb/c mice after 1, 2 and 3 immunizations with 10 μ/ml of the various peptides. Figure 7 shows that after the second immunization, addition of either the (A) region or the F region increased the level of anti-peptide antibodies to the T1-SP10MN peptide in ELISA assay.
Comparison of the ability of various T1-SP10 peptides to Induce anti-HIV neutralizing
antibodies in Balb/c mice:
Figure 8 shows the percent inhibition of
HIV syncytium formation in vitro when antisera from bleed 3 were added to a HIV syncytium
inhibition assay. Whereas only 1 animal in the
T1-SP10-injected group and none of the animals in the F-T1-SP10 and the T1-SP10(λ) groups had serum antibodies that inhibited syncytium formation by greater than 50%, 3 of 5 animals in the group of animals injected with F-T1-SP10MN(A) had antibody levels that neutralized syncytium formation by greater than 50% (Figure 8).
Ability of various T1-SP10 peptides to induce CP8+ CTL in vivo:
When injected into Balb/c mice, neither T1-SP10MN peptide nor F-T1-SP10MN peptide induced measurable CTL in Balb/c nice. However, soluble. F-T1-SP10MN(A) peptide (Table XIV) and T1- SP10MN(A) peptide in liposomes (Table XV) when injected in vivo were capable of inducing anti- HIV CTL in Balb/c mice that killed T1-S?10KX(A) coated D' target cells in vivo. Table XIV shows that the cytotoxic T cells induced in Balb/c mice by the soluble F-T1-5P10MN(A) peptide in vivo were Thyl+, Ly2 (CD8)+. CD8+ anti-T1-SP10(A) cytotoxic T cells killed only B2* targets and did not kill H2b targets. Table XV shows that the anti-T1- SP10(A) cytotoxic T cells induced by T1-SP10(A) peptide in liposomes were CD8+ and MHC Class I restricted. Thus , by addition of the F sequence and the (A) sequence (Table III) to the T1-SP10MN sequence, it was possible to construct a 52 amino acid peptide [F-T1-SP 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-HIV cytotoxic T cells have been induced in vivo by a 40 amino acid peptide, T1-SP10MN(A) incorporated into liposomes .
EXAMPLE 9
As has been shown, the construction of a synthetic peptide T1-SP10(A) containing aa303-327 of HIV gp120 V3 loop [SP10(A)] and aa 428-43 of HIV gp120 (T1) serves as a potent T cell immunogen for induction of activation of anti-HIV meaory T helper cells and B cell immunogen fcr anti-HIV neutralizing antibodies in vivo (Palker et al., PHAS (USA), 85:1932-1936, 1533; Palker et al., J . Immunol., 142:3612-3619, 1539; Hart et al., J. Immunol., 145:2677-2635, 1590 and Hart et al., PNAS (USA). 83:9443-9452, 1551). The TlSP10(λ) peptide induces anti-HIV neutralizing antibodies in mice, goats and rhesus monkeys (Palker et al., PNAS (USA), 85:1932- 1936, 1933; Palker et al., J. Immunol., 142:3612- 3619, 1985; Hart et al., J. Immunol. , 145:2677-2635, 1950 and Hart et al., PSAS (USA), 88:5443-9452, 1991), induces anti-HIV MHC Class I-restricted CTL in mice (Hart et al., PKAS (USA), 88:9443-5452, 1991), and induces anti-HIV T helper cell responses in mice, goats, rhesus monkeys, and chimpanzees (Palker et al., FKAS (USA). 85:1932-1936, 1938;
Palker et al., J. Immunol., 142:3612-3615, 1989; Hart et al., J. Immunol., 145:2677-2635, 1550 and Hart et al., PHAS (USA), 88:9448-9452, 1591). In a recently completad study in aice, it was found that while CS7SL/6 and Balb/c mica make high titers of anti-peptide antibodies to T1SP10IIIB peptides, these mouse strains make no neutralizing antibodies to the HIVIIIB V3 loop neutralizing determinants. In contrast, C57BL6 and Balb/c mice make good anti-HIV neutralizing antibodies when immunized with TISP10 peptides containing sequences from "the HIVMN V3 loop.
Immunization trials in chimpanzees were conducted to test the synthetic peptides. The results discussed below show that the chimpanzees at Holloman AFT, New Mexico, made good antibody and good helper T cell responses to T1SP10IIIB(A) peptides, but like Ealb/c mice, did not make
antibodies against the neutralizing antibody
determinants on the HIVIIIB V3 loop.
In Figure S the chimpanzees were immunized with HIV env synthetic peptides and their antibody titers tested using an ELISA assay.
Animals 834 and 1023 were immunized with peptide T1-SP10IIIB which was also used in the ELISA assay.
Peptide F-T1-SP10IIIB(λ) was used in the
immunization and ELISA assays for animals 1045 and 1070. All immunizations were in IFA + PBS (1:1) except for animal 1023 that developed IM abscesses after the third immunization and had one
immunization held. Subsequent immunizations were given in PBS only.
As can be seen, T1-SP10 peptides were excellent immunogens in animals 834 and 1023, while T1-SP10 peptides with the HIV gp41 fusion (F) domain synthesized N-terminal to the T1-SP10 peptide did not induce antibody titers as high or as of long duration as did peptides without the F domain.
It should be noted that animals 1045 and 1070 were challenged at month 16 with the immunogen T1-SP10IIIB (A) that induced good antibody titers in animals 834 and 1023. Animals 1045 and 1023 did not respond to T1-SP10III3(A) in IFA, thus demonstrating that they were tolerant to the T1-SP10(A) peptide from their prior immunizations with F-T1-SP10IIIB(A) peptide. It is also important to note that while boost of animal 884 at week 14 gave a rise in titer to T1-SP10IIIB(A) peptide, boost of animal 1028 at the same time did not. Animal 884 was boosted with IFA, while boost of 1023 was with no adjuvant, but rather only PBS.
The peripheral blood mononuclear cell (PBMC) proliferative response to the immunizing peptides was also studied (see Figure 10). Peptides T1-SP10IIIB and T1-SP10IIIB(A) induced high levels of proliferation of circulating PBMC in animals 884 and 1028. These levels fell to non-detectable levels after a 6 month rest (month 14) but rose again in animals 834 and 1028. Proliferative responses in animal 1023 rose with each boost after the 6 month rest even though the immunizaticns were in PBS alone with no adjuvant.
As with E cell responses, animals 1045 and 1070, that were immunized with F-T1-SP10IIIB(A) peptide, did net proliferate to T1-SP10IIIB(A) peptide. When these latter two animals were
immunized with the T1-SP10IIIB(A) peptide that was a coed immunogen in 834 and 102S, neither of the animals 1045, 1070 developed a proliferative
response to T1-SP10IIIB(A) which proves that the addition of the F-domain N-terminal to the T1-SP10 peptide created a toleragen that tolerized animals 1045 and 1070 to the T1 and SP10 regions of gp120. As shown in Table XVI, while animals 884 and 1023 both responded in proliferative assays to native gp120, animals 1045 and 1070 were tolerant to native gp120 as well as to immunizing peptides.
The PBMC proliferative responses of chimpanzees immunized with the synthetic peptides to PHA was also studied (see Figure 11). The data show that while animals 1045 and 1070 were tolerant to T1 and SP10 regions of EIV gp120, PBMC PHA responses in these animals throughout the immunization period were normal.
The same T1-SP10IIIB peptide batches used in the chimpanzee study, were also used as immunogens in goats, and good anti-HIV
neutralization titers in goats were obtained (See Figure 12). Thus, T1-SP10 peptides were superb iaaunogens with IFA in chimpanzees, with remarkable anti-peptide serum antibody titers of >1:102,40, (See Figure 9), and induction of T cell response to T1-SP10 and to native KIV gp120 (See Figure 10 and Table XVI below).
The high neutralizing antibody responses of coats to the same T1-SP10 peptide batches used in chimpanzees demonstrated that chimpanzees
selectively did not recognize the neutralizing V3 sequences as immunogenic, while other non- neutralization T1-SP10IIIB peptide sequences were immunogenic in chimpanzees. Thus, it is possible that selective proteolysis of the HIVIIIB V3 loop occurs by chimpanzee and mouse mononuclear cells in vivo, or more likely, that genetic restriction of antibody responses to neutralizing determinants of the V3 loop exist in chimpanzees and nice.
In rhesus monkeys, the injection of 500 ug of purified T1SP10MN(A) peptides vas shown to yield very high levels of anti-HIVMN neutralizing
antibodies in 4/4 animals (see Figure 13-15). In addition, in 1 cut of 4 monkeys, immunization yielded cross-reactive anti-HIV neutralizing antibodies that neutralized the HIVIII3 and HIVMN viruses (see Table XVIII below). Thus, if 25% chimpanzees and humans respond to the T1-SP10MN(λ) peptide and make cross-neutralizing anti-HIV env antibodies, then an additional 5% of subjects challenged with an otherwise non-MN-like HIV isolate could be protected from HIV challenge. See figures
16 and 17 for additional monkey data.
Because T1-SP10IIIB(A) peptides did not induce anti-HIVIIIB neutralizing antibodies in animals 834 and 1028, and because F-T1-SP10III(B)A peptides induced tolerance in animals 1045 and 1070, all of the chimpanzees were immunized at either month 16 (animals 884, 1028) or month 17 (animals 1045, 1070) with T1-SP10MN(A) peptide. The
rationale here vas to determine A) if the T1-SP10MN(A) peptide could break tolerance in animals 1045 and 1070, and E) if any of the animals could genetically see the V3 neutralizing determinants of HIV MN V3 loop, since it appeared that none of the animals could see the V3 determinants of HIV IIIB as presented by T1-SP10IIIB peptides. Figure 13 shows that after immunization of all 4 chimpanzees with 0. lag/kg of T1-SP10MN(A) peptide, three of the 4 animals (334, 1028 and 1045 showed the appearance of weak serum anti-HIV MN neutralizing antibodies
(dotted lines), while animal 1070 developed high levels of anti-HIV MN neutralizing antibodies that titered to >30% neutralization at 1:20, and also cross-neutralized HIV III3 (Table XIX, solid lines, Figure 13). This break in tolerance can also be seen in the rise in titer to T1-SP10MN(A) peptide in the serum of animals 834, 1045 and 1070 (Figure 19). Animal 1023 had an early abscess associated with the immunizations and did not receive IFA after month 4 of the study, and never had an antibody rise to peptide of HIV after the initial immunization of peptide with IFA.
The observation that neutralizing antibodies in chimpanzee 1070 neutralized both HIV HN and IIIB isolates could be cue to the presence of either type-specific neutralizing antibodies induced by both HIV MN and HIV III3 peptides (Rusche et al Proc. Natl. Acad. Sci. USA 95:3198 (1983)), or be due to the induction of cross-neutralizing anti-GPGRA antibodies by the T1-SP10MN(A) peptides.
Antibody titers against a truncated SP10IIIB
peptide, SP10D, IRIQRGPGR, vas used in ELISA assay with serum from chimpanzee 1070. End-point ELISA titers against this peptide were 1:800 or less froa 10-23-50 through 12-3-51 (months 3 through 17 of study). Following the first immunization of animal 1070 with T1-SP10MN (A) on 12-3-52, the titers of antibody to SP10D peptide rose from 1:800 to 1:3200 on 1-7-92 and 1:12,800 on 2-4-52. During the same time period, antibody titers of 1070 to T1-SP10MN (A) peptide rose from 1:12,800 to 1:102,400, while titers to the T1-5P10IIIB peptide rose from 1:3200 to 1:25,600. Absorption studies to absorb cut the neutralizing antibodies in animal 1070 serum demonstrated that all of the anti-HIV MN
neutralizing activity could be absorbed cut with the SP10KN(A) peptide, and part of the HIV KN
neutralizing activity could be absorbed cut with a peptide with the sequence IGPGRAIGPGRAIGPGRAC (DP2) (Janaverian et al Science, 250:1590 (1950)) that only contains sequences from the tip of the V3 loop that are common to both HIV KN and HIV IIIB
(Figure 20). Thus, a portion of the chimpanzee antibody response induced by T1-SP10MN(λ) peptides cross-neutralize HIV KN and HIV III3 and are directed against the conserved sequences at the tip of the HIV gp 120 V3 loop .
Importantly, in rhesus monkeys it was demonstrated that injection of 500ug of purified T1SP10KN(A) peptides yielded very high levels of anti-HIVMN neutralizing antibodies in 4/4 animals (Figures 20-24) and in 1 of 4 rhesus monkeys, yielded cross-reactive anti-HIV neutralizing antibodies that neutralized the HIVIIIB and HIVMN viruses (Tables XVIII and XX). Figure 25 shows that the DP2 (IGPGRAIGPGEAIGPFRAC) peptide absorbs the anti-HIVIIIB neutralizing activity in serum from rhesus monkey 18987. Table XXI shows the sequences of peptides used in the chimpanzee and rhesus monkey
Experimental Protocol
Human patients, both KLA 2A- and HLA 2A-, will be studied for up to two years. During
treatment, the generation of neutralizing antibodies against HIVMN and other HIV isolates as veil as the generation of T helper and/or Class I-restricted anti-HIV CTL will be measured.
The immunogens to be used will be TISP10(A) peptides which are expected to give rise to antibodies against 80% of the HIV isolates in the Los Alamos Data Set (Myers et al., Human
Retroviruses and AIDS 1991). 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 0.05mg/kg/peptide or 1mg of each peptide. If no responses to the original dose schedule result, the dose will be doubled and the regimen repeated after a three month rest.
Incomplete Freund's adjuvant (IFA) will be nixed with the immunogen in a 1 : 1 v/v mixture (Hart al Immunol . , 145 : 2677-2S35, 1990) Total volume for each immunization should be 2cc.
The immunogens will be administsrad by IM. The immunogens will be mixed in a total volume of 2c 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 HIV 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 TISP10 and SP10 peptide binding in RIA and HIV gp120 binding in RIP/Western blot assays. Serum will also be used to determine neutralization titers of ETLV-IIIB, HTLV-IIMN and field HIV 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 (10cc heparinized bleed) will be performed.
Peripheral blood cells (60 ml blood) will be used to study 7 cell proliferative responses to PEA, TT candidate TISP10 and SP10 peptides, gp120 and OKT3 (about 30 ml heparinized blood). T cell, B cell, NK cell, CD4 and CD8 call numbers will also be measured (about 5 ml heparinized blood). Finally, CTL assays will be performed on autologous or HLA- identical EBV-transformed B cell lines or autologous E3V-transformed B cell lines using vaccinia gp160 infected targets and peptide coated targets. 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 HIV native proteins or HIV 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), Haynes et al, AIDS Res. & Human
Retroviruses 6:35 (1990), Haynes et al, J. Immunol.
151:1646 (1993), Haynes et al, J. Exp. Med. 177:717 (1993), Haynes 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. Th1...n- B1...n includes the construct Th-SP10 and, by example, C4-V3. Thl...n- Tcl...n is equivalent to Th-CTL (CTL = X)).
A general immunogen design for induction of neutralizing antibodies requires the synthesis of one or more Th epitopes of HIV proteins N-terminal to the gp120 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 HIVIIIB, MN or RF env gp120 have: a) induced Th responses to native gp120 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), Haynes et al, J. Exp. Med. 177:717 (1993)) and b) induced in goats, rhesus monkeys and chimpanzees B cell neutralizing antibody responses that have neutralized laboratory HIV 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), Haynes et al, J. Exp. Med. 177:717 (1993), Haynes et al, Trans. Amer. Assoc. 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 HIV or SIV proteins (Hart et al, Proc. Natl. Acad. Sci. USA 88:5443 (1951), Yasutomi et al, J. Immunol. 151:5096 (1953)). In rhesus monkeys, it has been demonstrated that the T1-SP10MN (A) peptide induced in select animals antibodies that reacted primarily with the IGPGRAF sequence at the tip of the V3 loop, and cross-neutralized HIVIIIB, HIVMN, HIVRF as well as HIV primary isolates grown in CEM cells (Haynes et al , J . Immunol . 151 : 1646 (1993)).
Design of A Prototype Polyvalent HIV Immunogen
Because of the extreme variability that exists in HIV isolates both in geographic locations and among patients, a multivalent HIV immunogen design tailored to HIV isolates in specific geographic locations, will likely be required for successful preventive and therapeutic HIV immunogens (Falker et al, J. Immunol. 142:3612 (1585), Haynes et al, Trans. Amer. Asscc. Physician 106:31 (1993)). To this end, a prototype polyvalent HIV immunogen has been designed containing Th-B-CTL epitopes reflective of 4 common HIV isolate motifs in Clave E, HIVMN, HIVRF, HIVEV91, and HIVCANO (Figures 2S and 27). In each of these
prototype peptides resides at least two Th
determinants, two Class I-restricted CTL determinants, one restricted by ELA A2 and A3 (Clerici et al, Nature 339:383 (1939)) and another restricted by E7 (Safrit et al, Characterization of ELA-37-Restricted cytotoxic T lymphocyte clones specific for the third variable region HIV gp120, 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-HIV 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 26 and 27). Preclinial studies of this prototype peptide mixture in mice have demonstrated that two of the components CTI-SP10RF(A) and T1- SP10EV91(A)] induced type-specific anti-V3 peptide
[anti-SP10 (A)] responses (Table XXIV), while two of the components [T1-SP10MN(A) and T1-SP10CANO(A)] induced broadly cross-reactive anti-V3 peptide antibody response (Table XXIV).
Eliza method are described in Haynes et al, J. Immunol . 151:1646 (1593), Haynes et al, J. Exp. Med. 177:717 (1953), Haynes et al, Trans. Amer. Assoc. Physician 106:31 (1993). Ability of HIV Env Peptides to Induce Cross-Reactive Peptide Responses to African (Clave A) and Thailand (Clave E) HIV Isolate V3 Loop Peptides
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 T1-SP10(A) E. con. peptide (a Th-3-CTL peptide reflective of the consensus V3 loop sequence of Clave E in Thailand (Table XXV). When sera from mice immunized with only one of each of the 4 components of the polyvalent mixture were tested for their ability to bind to the African T1-SP10(A)A.con. peptide, it was found that the T1-SP10CANO(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 HIV env V3 sequences (Figure 27), secondary and possibly higher order structures of the V3 loop of the HIVCANO isolate appear to have the ability to induce cross-reactive anti-V3 antibodies against many different HIV V3 motifs.
Neutralizing Antibody Responses Generated By The
Polyvalent HIV Env Immunogen
Regarding neutralizing antibody responses, sera from animals immunized with the polyvalent
immunogen (Figure 27) bind to HIV gp120IIIB and
gp120SF2 in either radioimmunoprecipitation assay or in ELISA assay. Sera from these animals have neutralized HIVMN and RF isolates in syncytium inhibition assays.
Demonstration of a Neutralizing CD4-V3 Conformational Determinant in HIV gp120
The 17b and 48c human anti-gp120 mabs were isolated from human PBMC B cells from patients infected with HIV (Thali et al, J. Virol. 67:3978-3988 (1993); Moore et al, AIDS Res. Human. Retroviral. 9:1185
(1993)). The 17b and 48c mabs cross-block mouse mabs that block CD4 binding to gp120, broadly neutralize disparate HIV isolates, but do not in and of themselves block gp120-CD4 binding (Moore et al, personal
communication, 1994; Thali et al, J. Virol. 67:3978 (1993)). Rather, binding of the 48d mab is unregulated to native gp120 following ligation of gp120 by CD4. It has been found that one peptide, T1-SP10CANO(A), binds to the 48c mab (Figure 28), and the optimal binding of mab 43d to HIV env hybrid peptide T1-SP10CANO(A) depended on the presense of the CD4 peptide, T1 N- terminal to the SPIOCANO(A) peptide (Figure 29). Thus, the T1-SP10CANO (A) hybrid C4-V3 petide mirrors a conformational determinant of HIV gp120 recognized by a potent broadly neutralizing human mab. It is interest that Wyatt et al, J. Virol. 66:6997 (1992) and Moore et al, J. Virol. 67:4785 (1993) have suggested that the V3 loop [SP10(A)] and the C4, T1 region are in close physical proximity to each ether in native gp120.
Thus, the present data directly demonstrate that the T1-SP10CANO(A) synthetic peptide can mimic broadly neutralizing C4-V3 conformational determinants of native gp120.
General strategy for identification of multiple CD4-V3 [T1-SP10(A)] peptides that mirror other HIV isolate C4-V3 conformational determinants
Whereas the HIV V3 loop by itself induces primarily type-specific anti-HIV neutralizing
antibodies, the C4-V3 determinant as defined by the T1- SP10CANO(A) peptide will induce more broadly, cross-reactive neutralizing antibodies. This is known from the fact that the 43d human monoclonal antibody derived from a HIV seropositive patient binds to a complex conformational determinant on the surface of gp120, binds to a wide spectrum of HIV isolates and
neutralizes disparate HIV isolates such as HIVIIIB and HIVMN (Thali et al, J. Virol. 67:3978 (1993); Moore, J. personal communication (1994)). Thus, a general strategy for identifying multiple C4-V3 peptides would be to construct a large number of C4-V3 peptides derived from C4 sequences (for example, from amino acids 419 to 428 from the HIVMN isolate and from homologous regions in other HIV isolates) linked
N-terminal to SP10 or SP10(A) regions (such as amino acids 301-327 of HIVMN and from homologous regions in other HIV isolates) from sequences listed in the Los Alamos database (Human 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 Eiolocgy 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 to 100 of these different C4-V3 peptides against a combinatorial library of heavy and light chain immunoglobulin variable genes expressed on the surface of phage (Borbas et al, Proc. Natl.
Acad. Sci. USA 88:7978 (1991)). The work of Borbas et al provides a method for screening a large number (107 to 108) of human monoclonal antibodies derived from a patient with HIV 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 heterodimer
expressed in the combinatorial library on the surface of phage. These Fab monoclonals can be isolated and cloned (Borbas et al, Proc. Natl. Acad. Sci. USA,
88:7978 (1991)). Most importantly, C4-V3 peptide designs can be identified that reflect native gp120 C4-V3 conformational determinants of a wide variety of HIV strains. When this type of protein based selection is performed with combinatorial libraries derived from a large number of HIV-infected individuals from many different geographic sites of HIV infection around the world, a wide selection of C4-V3 peptides that mimic broadly reactive neutralizing determinants from the C4-V3 region of native gp120 can be identified and, for example, combined together with the T1-SP10CANO(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 multiple HIV strains.
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 of 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 epitopes which also can be derived from the available literature, 3) a compilation of the HIV 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 HIV 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)).
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 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 HIV 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-Verlag, 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) Immunopublishing 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 of 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 immunogenic for all four populations. These results are presented in Table XXVII.
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 class 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 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.
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 gp120 (SP410-BAL), the AVERY region of gp41 (SP400-BAL), the ELDKWAS region of gp41 (GTH1-SP61), and the C2 region of gp120 (T1-SP420-BAL). In addition, T1-SP10(A) or GTHI-SPIO(A) peptides were included in the mixture of HIV env peptide designs (the T1-SP10(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 gp120IIIB 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.
* * * * *
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 HIV isolates, without departing from the scope of the invention.

Claims

WHAT IS CLAIMED IS;
1. An essentially pure form of a
hydrophilic peptide consisting essentially of 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 HIV recognized by 3 lymphocytes, said peptide being capable, when covalently linked to a carrier
molecule, of inducing in a mammal the production of high titers of protective, type-specific, antibodies against HIV.
2. The peptide according to claim 1, wherein said amino acid sequence corresponds to the SP-10 region of the envelope glycoprotein of HIV, or some portion thereof.
3. The peptide according to claim 2, wherein said amino acid sequence consists
essentially of CTRPNNNTRKS IRIQRGPG, or some portion thereof.
4. An immunogenic conjugate capable of inducing in a mammal the production of high titers of protective, type-specific, antibodies against HIV, said conjugate comprising:
(i) a carrier molecule covalently attached to (ii) a hydrophilic peptide consisting essentially of 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 HIV recognized by B lymphocytes.
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 HIV, 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 T1 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.
9. The conjugate according to claim 8, wherein said spacer molecule consists of the dipeptide glycine-glycine.
10. A conjugate according to claim 4 wherein said amine acid sequence corresponds to the SP-10 region of the envelope glycoprotein of HIV, or some portion thereof.
11. A conjugate according to claim.10 wherein said amino acid sequence consists
essentially of CTRPNNNTRXSIRIQRGPG, or some portion thereof.
12. The conjugate according to claim 4, further comprising the amino acid sequence FLGFLG covalently linked to the SP-10-like peptide, or portion of the SP-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 HIV isolates located C terminal .to the SP-10 region.
14. A conjugate according to claim 13 wherein said sequence corresponding to said
hypervariable region is RAFVTIGKIGN and is directly linked to the C terminus of SP-10.
15. A method of producing immunity to HIV in a mammal comprising administering at least one conjugate according to claim 4 to said mammal.
16. A method of producing immunity to HIV in a mammal comprising administering to said mammal at least one covalently linked aggregate of at least two conjugates, each of said conjugates having (i) a carrier molecule covalently attached to (ii) at least one SP-10-like peptide, or portion thereof.
17. The method according to claim 16 wherein said aggregate comprises at least two SP-10-like peptides, or portions thereof, each of which corresponds to a different isolate of HIV.
18. The method according to claim 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 of 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 HIV recognized by B lymphocytes, said conjugates being linked to each other through at least one disulfide bend.
20. A method of determining the presence and titers in mammalian serum of neutralizing antibodies against a specific strain of HIV comprising the steps of: (i) contacting an SP-10-like peptide, or portion thereof, with antibodies from mammalian serum; and (ii) measuring the formation of SP-10-like peptide-antibody complexes by radioimmunoassay, or enzyme linked immunosorbent assay.
21. A peptide of the general formula:
F-TH-SP10 (X)
Th-SP 10 (X)
Th-SP10
or
F(X)
wherein:
F represents an amino acid sequence derivable from the putative fusogeni0 domain
of HIV env gp41, or sequence functionally
equivalent thereto;
Th represents an amino acid sequence comprising a T helper epitope;
SP10 represents a peptide as defined in claim 1; and
(X) represents an amino acid sequence corresponding to a HIV protein sequence
recognized by MHC Class I or Class II
restricted cytotoxic T cells .
22 . A method of inducing, in a primate, antibodies that neutralize human immunodeficiency virus (HIV) comprising administering to said primate a mimeotope of a conformational determinant of the native HIV gp120 C4 -V3 region.
23 . The method according to claim 22 wherein said mimetope is T1-SP10CANO (A) .
24 . A method of screening said peptides according to claim 21 for the ability to produce neutralizing antibodies against more than 1 HIV isolate comprising:
i) contacting one of said peptides with a monoclonal antibody that recognizes a conformational determinant of the native HIV gp120 C4 -V3 region under conditions such, that binding can occur, and
ii ) determining whether said peptide binds to said monoclonal antibody .
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 gp120/gp41 interactions .
26 . A composition comprising human
immunodeficiency virus envelope peptides that disrupt gp120/gp41 interactions , together with a
pharmaceutically acceptable carrier .
EP96936830A 1995-10-20 1996-10-18 SYNTHESIS VACCINE FOR PROTECTION AGAINST HUMAN IMMUNODEFICIENCY VIRUS INFECTION Withdrawn EP0868196A4 (en)

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PCT/US1996/016911 WO1997014436A1 (en) 1995-10-20 1996-10-18 Synthetic vaccine for protection against human immunodeficiency virus infection

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DE19814925C2 (en) * 1998-04-03 2000-10-05 Thomas Harrer Medicinal products for induction of cytotoxic T cells
CN1635876A (en) 2000-02-04 2005-07-06 杜克大学 Human immunodeficiency virus vaccine
US7033593B2 (en) 2000-09-22 2006-04-25 Duke University Immunogen comprising an HIV envelope protein, a ligand and H2 peptide
CA2423127A1 (en) 2000-09-22 2002-03-28 Duke University Immunogen comprising ligand bound hiv envelope protein
US7172761B2 (en) 2001-11-07 2007-02-06 Duke University Polyvalent immunogen
CA2466050A1 (en) 2001-11-07 2003-05-15 Duke University Polyvalent immunogen
US7195768B2 (en) 2001-11-07 2007-03-27 Duke University Polyvalent immunogen
US20050112139A1 (en) * 2003-10-23 2005-05-26 Nmk Research, Llc Immunogenic composition and method of developing a vaccine based on factor H binding sites
JP2010509340A (en) * 2006-11-09 2010-03-25 ポール スディール Binary epitope antibody and B cell superantigen immunostimulator
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
EP4070815A1 (en) 2016-10-03 2022-10-12 The U.S.A. as represented by the Secretary, Department of Health and Human Services Hiv-1 env fusion peptide immunogens and their use
CN118126132B (en) * 2024-03-06 2024-08-16 广东医保药业有限公司 Polypeptides and combinations thereof with cefotaxime sulbactam

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EP0201540B2 (en) * 1984-10-18 2001-10-31 Institut Pasteur Envelope antigens of lymphadenopathy associated virus and their applications
ATE87103T1 (en) * 1985-12-17 1993-04-15 Akzo Nv IMMUNOCHEMICAL REAGENT.
WO1987007616A1 (en) * 1986-06-12 1987-12-17 Biogen N.V. Peptides involved in the pathogenesis of hiv infection
US5019387A (en) * 1987-09-08 1991-05-28 Duke University Production of antibodies to HIV
US5013548A (en) * 1987-09-08 1991-05-07 Duke University Production of antibodies to HIV
PT762895E (en) * 1994-04-29 2002-11-29 Univ Duke SYNTHETIC VACCINE FOR PROTECTION AGAINST INFECTION OF HUMAN IMMUNODEFICIENCY VIRUSES

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EP0868196A4 (en) 2004-11-24
JPH11515006A (en) 1999-12-21
WO1997014436A1 (en) 1997-04-24
AU7465696A (en) 1997-05-07
CA2235168A1 (en) 1997-04-24

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