WO1997014436A9 - Vaccin de synthese pour la protection contre l'infection par le virus de l'immunodeficience humaine - Google Patents

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

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WO1997014436A9
WO1997014436A9 PCT/US1996/016911 US9616911W WO9714436A9 WO 1997014436 A9 WO1997014436 A9 WO 1997014436A9 US 9616911 W US9616911 W US 9616911W WO 9714436 A9 WO9714436 A9 WO 9714436A9
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hiv
peptide
peptides
region
amino acid
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PCT/US1996/016911
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English (en)
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WO1997014436A1 (fr
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Priority claimed from US08/546,515 external-priority patent/US5993819A/en
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Priority to EP96936830A priority Critical patent/EP0868196A4/fr
Priority to AU74656/96A priority patent/AU7465696A/en
Priority to JP9516093A priority patent/JPH11515006A/ja
Publication of WO1997014436A1 publication Critical patent/WO1997014436A1/fr
Publication of WO1997014436A9 publication Critical patent/WO1997014436A9/fr

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  • the present invention relates, in general, to immunogenic preparations and, in particular, to peptides comprising amino acid sequences
  • HIV immunodeficiency virus
  • 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.
  • the human retrovirus HIV has been demonstrated to be the causative agent of acquired
  • AIDS immunodeficiency syndrome
  • saliva, semen, whole blood and plasma from individuals in high risk categories, including male homosexuals, intravenous drug users, patients
  • 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 cellss After infecting the cell, the virus subverts the ability of the T cell to fend off the virus.
  • gp120 external envelope glycoprotein
  • 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.
  • the HIV external envelope glycoprotein gp120 has been shown to be capable of inducing neutralizing antibodies in goats and in man (Robey et al, Proc. 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.
  • the recombinant protein PB1 (Putney et al., Science.
  • sequence mutation rate is a major obstacle to
  • HIV isolates share a consensus of V3 sequences that is similar to the HIV MN isolate, and that approximately 80% of HIV
  • 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.
  • an effective vaccine against HIV will induce protective immune responses in primates and in man, that is, will prevent subsequent HIV infection from occurring.
  • aggregates is capable of inducing the product ion in mammals of high titers of neutralizing antibodies against HIV, which peptides does not distrub normal immune function.
  • the invention relates to immunogenic
  • 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.
  • the present invention in one embodiment, the present invention
  • peptide 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-III B isolate (Ratner et al., Nature 313:277, 1985), or any portion thereof.
  • the present invention comprises an immunogenic conjugate capable of
  • conjugate comprising: (i) a carrier molecule covalently attached to (ii) a peptide comprising an amino acid sequence
  • the present invention comprises a method of producing immunity to HIV comprising administering the above-described
  • the present invention comprises a method of detecting the presence of anti-gp120 antibodies in biological test samples.
  • Figure 1 Recombinant proteins and relation to synthetic peptides.
  • Figure 2. Reactivity of AIDS patient antibodies to synthetic peptides.
  • Figure 3. Reactivity of gp120 of antibodies from HIV+ patient purified over synthetic peptide affinity columns.
  • FIG. 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 mice as determined by ELISA assay in 96 well plates using the peptide
  • 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.
  • FIG. 8 Comparison of the ability of various T1-SP10 peptides from the envelope of HIV MN to induce antibodies in Balb/c mice that neutralize HIV MN in syncytium inhibitium assays in vi tro .
  • 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
  • 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
  • Figure 12 shows goats immunized with the same batch of peptides used to immunize chimpanzees 884, 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
  • Figure 14 shows antibody to immunizing peptide in Rhesus monkeys immunized with T1-SP10MN(A) peptide.
  • Figure 15 shows neutralizing antibody levels in syncytium inhibition assay in serum of Rhesus monkeys immunized with F-T1-SP10MN(A) peptide.
  • Figure 16 shows serum antibody titers
  • Figure 17 shows the absorption of cross
  • 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 SPIOMN(A) peptides and partial absorption by DP2 peptide.
  • 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.
  • 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)).
  • FIG. 27 Sequence of T1-SP10(A) Th-B-Tc peptides for human immunization.
  • FIG. 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 ⁇ g/well) as described in detail in Haynes et al (J. Immunol.
  • Figure 28 shows that mab 48d bound to the T1-SP10CANO (A) peptide, and the 17b antibody did not. This plate was stripped with 8 molar urea (a treatment
  • T1-SP10CANO (A) peptide is required for maximal peptide binding to mab 48d.
  • [SP10CANO(A)] peptide were incubated on an ELISA plate with the total concentration of 2 ⁇ g/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.
  • FIG. 31 Schematic representation of the possible interaction of functional native HIV-1 envelope regions
  • Figure 31 shows that HIV-1 envelope protein gp41 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
  • Figure 32 Western blot analysis of guinea pig antisera against HIV gp120 and proteins.
  • Recombinant gp120, gp41-MBP fusion proteins (0.1 ⁇ g/lane), cell lysate of HIVLAI/IIIB infected- or mock-infected CEM cells (0.5 ⁇ 10 6 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.
  • 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 6 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 immunogenic epitopes of HIV and synthetic vaccines made therefrom. These novel immunogenic agents are prepared by chemically
  • 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
  • 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
  • the peptides of the instant invention correspond to, or are homologous with, B-cell epitopes present within the central region of the HIV isolate HTLV-III B 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.
  • the peptides themselves are not 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
  • peptides of the instant invention capable of inducing anti-HIV neutralizing antibodies, do not inhibit antigen-specific normal T cell
  • Peptides of the instant invention can have, for example, the sequence CTRPNNNTRKSIRIQRGPG (designated SP-10), corresponding to amino acids 303-321 of the HTLV-III B envelope glycoprotein gp120 (Ratner 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 HTLV-III B , or portions thereof, these sequences being designated "SP-10-like" (see, for example, sequences in Table I).
  • 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
  • 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 more 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 SP-10-like sequences, or portions thereof, to a carrier
  • 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
  • Conjugation of the carrier molecule to the peptide is accomplished using a coupling agent.
  • heterofunctional coupling agent M-maleimidobenzoyl-N-hydroxysuccinimide ester MES
  • sulfo-MES water soluble compound m-maleimidobenzoylsulfosuccinimide ester
  • 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.
  • the SP-10 sequence of HTLV-III B (see Table I) can be conjugated to or synthesized with either the HTLV-III B gp120 envelope T cell epitope T1 (amino acids 428-443 of gp120),
  • KQIINMWQEVGKAMYA or to the T2 epitope (amino acids 112-124 of HTLV-III B 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 HTLV-III B isolate of HIV,
  • T1 or T2 sequences from other HIV isolates can be linked to synthetic peptides derived from the SP-10 region of the corresponding isolates (see 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.
  • 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 cell epitopes, and used in a vaccine.
  • carrier molecules including gp120 T cell epitopes
  • 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 322-333, Ratner et al, Nature 313:277, 1985), 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,
  • Hypervariable sequences are linked advantageously C-terminal to the SP-10-like peptide. Linkage N-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 gp4l transmembrane region that is involved in viral-induced cell fusion, FLGFLG, (Gallagher, Cell 50:327, 1987) .
  • 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
  • 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
  • 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 polyvalent 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).
  • SP-10-like peptides linked to hypervariable sequences described above (see, for example, Table II).
  • a mixture of such polypeptides, coupled to appropriate carrier are described above (see, for example, Table II).
  • 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) 84:2479, 1987) 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.
  • the peptide 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.
  • 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
  • Neutralizing antibodies produced by T1-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, 1989). Similarly, neutralizing antibodies raised against the T1-SP10-like peptides containing
  • sequences from the MN or RF HIV isolate neutralize the homologous isolate but do not neutralize any of the other two HIV isolates.
  • anti-T1-SP10IIIB serum was observed to neutralize 1 of 9 HIV isolates, anti-T1-SP10RF serum neutralized 3 of 9 HIV isolates, and anti-T1-SP10MN serum neutralized 6 of 9 HIV isolates (Haynes et al AIDS Res. Retrol. 6:38, 1990) (see Table V) .
  • 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 et al reported that the T1-SP10-like peptide was advantageous over synthetic peptides coupled to carrier molecules such as KLH or tetanus toxoid.
  • carrier coupled peptides only induced large amounts of antibody against the carrier in polyvalent mixtures of peptides
  • T1 sequence of HIVIIIB env amino acids 429-443
  • 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.
  • Hart et al J.
  • 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.
  • 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.
  • CTL induced cytotoxic T cell
  • 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 other HIV-1, HIV-2 and simian immunodeficiency virus (SIV) isolates) has sequence homology to the FI (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.
  • 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 cell activity, anti-HIV neutralizing antibodies, and memory anti-HIV CD8+, HLA-Class I restricted cytotoxic T cell responses.
  • the present invention relates, in a preferred embodiment, to peptides of the general formulae:
  • F sequences are from the putative fusogenic domain of HIV env gp41 (for example, amino acids 519-530 in HIV isolate BH10/IIIB or homologous regions in other HIV-1, HIV-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 (below) 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 from 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
  • F sequences can be, for example, C-terminal to Th-SP10 (X) sequences.
  • Th, SP10 and (X) sequences can be arranged in any order in the peptide construct.
  • embodiment of the invention is capable of inducing anti-HIV neutralizing antibodies, anti-HIV helper T cells, and anti-HIV cytotoxic (killer) T cells.
  • immunogen which is a fusion protein
  • immunogen can either be synthesized chemically or by recombinant means known in the art.
  • the immunogen can have, for example, the
  • 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 those shown in Table VII.
  • WMJ1 sequence is from Brasseur et al. AIDS Res. Hum. Retrovirol. 4:83-90, 1988.
  • the (A) region-like sequences can be selected from (A)-homologous sequences from any HIV isolate, including those shown in Tables II and VIII.
  • 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) N 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.
  • 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
  • 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/IIIB (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 MHC Class I molecules and generates the development of CD8+ cytotoxic T cells in vivo.
  • the 519-530 amino acid region of HIV gp41 consisting of the 12 amino acids: AVGIGALFLGFL (F) or
  • F-derivatized hybrid HIV peptides can be constructed comprising the F amino acid sequence (see, for example, Table VII) coupled N or C terminal to an amino acid sequence of HIV proteins that are capable of inducing cytotoxic T cells in vivo .
  • F amino acid sequence see, for example, Table VII
  • Examples of described HIV peptides that are capable of being recognized by HLA Class I cytotoxic T cells are shown in Table IX.
  • 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 those 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
  • cytotoxic T cells 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.
  • 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
  • cytotoxic T cell epitopes and their HLA rest ⁇ cting elements that are the types of peptides that can be derivatized by F-sequences and used as a mixture with F-T1-SP10 (A) peptides.
  • 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 other cytotoxic T cell-inducing sequence such as are listed in Tables IX) .
  • T helper epitopes 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 cell response to the immunogen, sufficient T helper cell epitopes will need to be present in order to have available
  • 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 .
  • Sequences from the first 20 peptides above are from Schrier et al (J. Immunol. 142:1166-1176, 1989) and sequences T1, T2, Th4 and p18 are from Clerici et al (Nature 339:383-385, 1989).
  • T helper cell epitope may be recognized by T cells in the context of multiple HLA Class II specificities and,
  • T2 or T1 T helper cell epitopes see Table X.
  • Schrier et al J. Immunol. 142:1166-1176, 1989 have identified a number of helper T cell 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).
  • the AIDS vaccine of the present invention has the general structure and composition of mixtures of peptides of the formulation:
  • F sequences are from the putative fusogenic domain of HIV env gp41 (for example, amino acids 519-530 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
  • T helper cell epitopes listed in Table X 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, 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 MHC 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 F(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 cell responses in the majority of subjects in a given population cohort.
  • cytotoxic T cell (X) and Th epitopes needed to induce cytotoxic T cells and T helper cell responses in the majority of subjects in a given population cohort.
  • the order of F, Th, SP10 and (X) can vary as long as the above- indicated function of each is retained.
  • the specific SP10-like sequences can vary as long as the above- indicated function of each is retained.
  • F-Th-SP10 (X) peptides will 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.
  • 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
  • T1-SP10CANO A
  • Table XXIII T1-SP10CANO
  • 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.
  • 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 lmmunogens that need to be included in the F-Th-SP10 (X), Th-SP10 (X), Th-SP10 and F(X)
  • the peptides given to the subject are those necessary for eliciting the desired anti-HIV B and T cell responses.
  • the ability to conjugate the F-region from the HIV g41 envelope protein to any sequence capable of being recognized by cytotoxic T cells provides a simple and effective method of inducing MHC Class I restricted cytotoxic T cells to any peptide bearing cytotoxic T cell
  • F-derivatized peptides that include sequences from host proteins
  • F-derivatized peptides can be used comprising HIV gp41 F sequences (for example, amino acids 519-530 from the BH10/IIIB HIV-1 isolate or from homologous regions of other HIV-1, HIV-2 or SIV isolates, or sequences functionally equivalent thereto) conjugated either N- or C-terminal to peptides capable of being recognized by cytotoxic T cells 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.
  • HIV gp41 F sequences for example, amino acids 519-530 from the BH10/IIIB HIV-1 isolate or from homologous regions of other HIV-1, HIV-2 or SIV isolates, or sequences functionally equivalent thereto
  • TCR T cell receptor
  • the present invention provides a simple and effective method for inducing MHC-restricted Class I or Class II cytotoxic T cells to peptides of host antigens and thus represents a major advance in the development of vaccines for autoimmune disease.
  • sequences can be obtained from unique regions of the TCR molecules (Barns et al J. Exp. Med. 169:27, 1989).
  • F-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 cell-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.
  • a 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.
  • sequence of peptides from the region of the B cell immunoglobulin molecule that binds antigen can be identified using, for example,
  • 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 cell types for the treatment of clonal B and T cell malignancies that express on their surface clonal immunoglobulin or TCR molecules.
  • immunoglobulin molecules expressed on 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 kill the tumor cells.
  • F-derivatized non-HIV proteins A fourth example of the use of F-derivatized non-HIV proteins is the creation of an immunogen that kills pathogen-infected cells and thus facilitates the elimination of pathogen-infected cells from the host.
  • Hepatitis C Non-A, non-B
  • hepatitis is a disease that is caused by the
  • cytotoxic T cell epitope sequences of the Hepatitis C virus protein and injecting such sequences into individuals, memory anti-Hepatitis C specific
  • cytotoxic T cell 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.
  • 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).
  • 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)
  • 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
  • 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 promotes the neutralization of HIV primary isolates by these antibodies.
  • the peptides listed in Table XXVIII are against certain HIV strains (HIV MN or BAL). For effective neutralization of HIV primary isolates, it is
  • Peptides were conjugated to carrier molecules such as bovine serum albumin (BSA) or tetanus toxoid (TT) with MBS, as described by Green et al. (Cell, 28:477, 1982; Palker et al, Proc. Natl. Acad. Sci. (USA) 84:2479, 1987).
  • BSA bovine serum albumin
  • TT tetanus toxoid
  • Tetanus toxoid treated with MBS was then subjected to sieving chromatography on a PD-10
  • TT-MBS was then incubated with rocking at 23°C 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 4°C against PBS or again desalted on a PD-10 column and were used as an immunogen.
  • Decreased cpm values obtained in RT assays reflect decreased levels of HIV after cocultivation of virus and cells for 10 days.
  • 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, those 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 (Maddon 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 cell formation is death of the infected cell (Zagary et al., Science 231:850, 1986).
  • SP-10-TT alone was as good an antigen-specific T cell activator as TT alone.
  • SP-10-TT and SP-10-BSA when added to TT alone did not inhibit TT induced proliferation by normal T cells.
  • anti-SP-10 goat serum did not bind to peripheral blood lymphocytes or monocytes in indirect immunofluorescence assay using flow cytofluorometry.
  • vaccines comprising the small synthetic SP-10-like peptides (less than or equal to about 35 amino acids in length) have distinct advantages over HIV vaccines comprising recombinant gp120, or large subunits thereof, as the latter may interfere with normal immune function.
  • Synthetic peptide SP-10 has an amino acid sequence derived from and unique to the gp120 envelope protein of HIV isolates HTLV-III B 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-III B ) from the HTLV-III B isolate of HIV was coupled to tetanus toxoid and used to raise antibodies 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).
  • Fig. 5A HTLV-III B
  • Fig. 5B HTLV-III RF
  • Fig. 5C HTLV-IIIr ⁇
  • Fig. 5D HTLV-III SC
  • Goat anti-SP-10 antiserum ⁇ , pre-immune goat serum (O) and AIDS patient serum ( ⁇ ) 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 H-9 T cells by cocultivation of virus and cells for 10 days in vitro. Levels of HIV present in cell culture
  • Fig. 5A goat anti-SP-10 antiserum inhibited (i.e. neutralized) HTLV-III B infection of H-9 cells at a virus dilution of 10 -2 .
  • Pre-immune goat serum did not inhibit HTLV-III B infection at the same dilution of virus.
  • goat anti-SP-10 antiserum did not 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 HTLV-III B isolate that contains in its gp120 envelope protein the amino acid sequence present in SP-10.
  • 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.
  • FYTTK amino acids 322-326
  • T1-SP10 variant peptide made was the T1-SP10MN(A) peptide with the (A) signifying that amino acids 322-326 were added to the existing MN SP10 region of amino acids 303-321 (see Table III) .
  • 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 fu ⁇ ogenic region) (see Tables III and IV). Bosch et al
  • 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 ⁇ /ml of tne 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-pept_.de antibodies to the T1-SP10MN peptide in ELISA assay
  • 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(A) 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).
  • Table XIV shows that the cytotoxic T cells induced in Balb/c mice by the soluble F-T1-SP10MN (A) peptide in vivo were Thyl+, Ly2 (CD8)+. CD8+ anti-T1-SP10(A) cytotoxic T cells killed only H2 ' targets and did not kill H2' 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.
  • 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.
  • 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 memory T helper cells and B cell immunogen for anti-HIV neutralizing antibodies in vivo (Palker et al., PNAS (USA).
  • T1SP10(A) peptide induces anti-HIV neutralizing antibodies in mice, goats and rhesus monkeys (Palker et al., PNAS (USA). 85:1932-1936, 1983; Palker et al., J. Immunol., 142:3612-3619, 1989; Hart et al., J . Tmmunol., 145:2677-2685, 1990 and Hart et al., PNAS (USA), 88:9448-9452,
  • mice induces anti-HIV MHC Class I-restricted CTL in mice (Hart et al., PNAS (USA), 88:9448-9452, 1991), and induces anti-HIV T helper cell responses in mice, goats, rhesus monkeys, and chimpanzees (Palker et al., PNAS (USA). 85:1932-1936, 1988; Palker et al., J. Immunol., 142:3612-3619, 1989; Hart et al., J.
  • T1SP10IIIB peptides these mouse strains make no neutralizing antibodies to the HIVIIIB V3 loop neutralizing determinants
  • C57BL6 and Balb/c mice make good anti-HIV neutralizing antibodies when immunized with T1SP10 peptides containing sequences from the HIVMN V3 loop.
  • T1-SP10 peptides were excellent immunogens in animals 884 and 1028, 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.
  • F HIV gp41 fusion
  • animals 1045 and 1070 were challenged at month 16 with the immunogen T1-SP10IIIB(A) that induced good antibody titers in animals 884 and 1028.
  • Animals 1045 and 1028 did not respond to T1-SP10IIIB(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.
  • 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 1028 was with no adjuvant, but rather only PBS.
  • peripheral blood mononuclear cell (PBMC) proliferative response to the immunizing peptides was also studied (see Figure 10).
  • mice 1045 and 1070 that were immunized with F-T1-SP10IIIB(A) peptide, did not proliferate to T1-SP10IIIB(A) peptide.
  • T1-SP10IIIB(A) peptide that was a good immunogen in 884 and 1028
  • 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.
  • animals 884 and 1028 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 HIV gp120, PBMC PHA responses in these animals throughout the immunization period were normal.
  • 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) .
  • T1-SP10 peptides were superb immunogens with IFA in
  • T1-SP10IIIB(A) peptides did not induce anti-HIVIIIB neutralizing antibodies in animals 884 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 was to determine A) if the T1-SP10MN(A) peptide could break tolerance in animals 1045 and 1070, and B) 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
  • FIG. 18 shows that after immunization of all 4 chimpanzees with 0.1mg/kg of T1-SP10MN(A) peptide, three of the 4 animals (884, 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
  • IRIQRGPGR was used in ELISA assay with serum from chimpanzee 1070 End-point ELISA titers against this peptide were 1:800 or less from 10-23-90 through 12-3-91 (months 3 through 17 of study) Following the first immunization of animal 1070 with T1-SP10MN(A) on 12-3-92, the titers of antibody to SP10D peptide rose from 1 800 to 1:3200 on 1-7-92 and 1:12,800 on 2-4-92 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-SP10IIIB peptide rose from 1 3200 to 1:25,600. Absorption studies to absorb out the neutralizing antibodies in animal 1070 serum demonstrated that all of the anti-HIV MN neutralizing activity could be absorbed out with the SP10MN(A) peptide, and part of the HIV MN
  • IGPGRAIGPGRAIGPFRAC 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 studies described.
  • HIV seronegative subjects will be immunized with a polyvalent mixture of T1SP10 (A) peptides (see Tables XXII and XXIII) designed to generate neutralizing antibodies to about 80% of the current HIV isolates in the United States.
  • T helper and/or class I-restricted anti-HIV CTL will be measured.
  • the immunogens to be used will be T1SP10 (A) peptides which are expected to give rise to
  • Each patient will receive as immunogen dose about 0.05mg/kg/peptide or lmg 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.
  • IFA Incomplete Freund's adjuvant
  • the immunogens will be administered 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
  • 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 20cc 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 HTLV-IIIB, HTLV-IIMN and field HIV isolates in reverse transcriptase and/or syncytium inhibition assays. Routine serum chemistries for toxicity
  • Peripheral blood cells 60 ml blood will be used to study T cell proliferative responses to PHA, TT candidate TISP10 and SP10 peptides, gp120 and OKT3 (about 30 ml heparinized blood).
  • T cell, B cell, NK cell, CD4 and CD8 cell numbers will also be measured (about 5 ml heparinized blood).
  • CTL assays will be performed on autologous or HLA- identical EBV- transformed B cell lines or autologous EBV-transformed B cell lines using vaccinia gp160
  • 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
  • CTL cytotoxic T lymphocytes
  • Th l...n - B l...n includes the construct Th-SP10 and, by example, C4-V3.
  • Th-B Th epitopes of HIV proteins N-terminal to the gp120 envelope V3 loop neutralizing domain
  • Prototype synthetic peptide immunogens comprised of Th-B-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 (1988), Palker et al, J. Immunol. 142:3612 (1989), Hart et al, J. Immunol. 145:2677 (1990), Haynes et al, J.
  • Th-B-CTL epitopes reflective of 4 common HIV isolate motifs in Clave E, HIVMN, HIVRF, HIVEV91, and HIVCANO ( Figures 26 and 27).
  • Th-B-CTL epitopes reflective of 4 common HIV isolate motifs in Clave E, HIVMN, HIVRF, HIVEV91, and HIVCANO ( Figures 26 and 27).
  • In each of these prototype peptides resides at least two Th determinants, two Class I-restricted CTL
  • the 17b and 48d human anti-gp120 mabs were isolated from human PBMC B eel Is from patients infected with HIV (Thali et al, J. Virol. 67:3978-3988 (1993); Moore et al, AIDS Res. Human.
  • T1-SP10CANO(A) hybrid C4-V3 peptide 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 other 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.
  • HIV V3 loop by itself induces primarily type-specific anti-HIV neutralizing
  • 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 48d 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)).
  • C4-V3 peptide designs can be identified that reflect native gp120 C4 -V3
  • 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
  • An HLA-based vaccine for induction of anti-HIV T cell immunity is a
  • 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.
  • 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
  • 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
  • HIV CTL epitopes 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
  • HIV B cell immunogens that induce broadly neutralizing
  • antibodies for HIV primary isolates can be added to a multivalent HIV T cell immunogen.
  • HLA-based HIV vaccine formulated based on the
  • 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 gp120 (SP410-BAL), the AVERY region of gp41
  • T1-SP10(A) or GTH1-SP10(A) peptides were included in the mixture of HIV env peptide designs
  • T1-SP10(A) and GTHI-SP10(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-BAL, SP410-BAL, and the Th-B design peptides, GTH1-SP61, GTHI-SP10MN(A), and
  • T1-SP10(A)-BAL T1-SP10(A)-BAL.
  • GTHI-SP10MN(A) peptides induced antibodies that bound to recombinant gp120IIIB or to recombinant gp41 in Western bloc assays ( Figure 32A, B and C), and bound to the surface of HIVIIIB/LAI-infected CEM T cells
  • Table XXX The peptide designs listed in Table XXVI I 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.

Abstract

Préparations immunogènes de peptides comportant des séquences d'acides aminés correspondant aux déterminants antigéniques de la glycoprotéine d'enveloppe du VIH, couplées de manière covalente, et directement ou par l'intermédiaire d'une molécule intercalaire, à des molécules porteuses utilisables dans la vaccination des mammifères.
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US7611712B2 (en) * 2003-10-23 2009-11-03 Nelson M. Karp Immunogenic compositions capable of eliciting Th1 immune responses comprising an HIV-1 MA myristate binding site polypeptide
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