WO1989005821A1 - Antigenes et anticorps associes au virus d'immunodeficience humaine - Google Patents

Antigenes et anticorps associes au virus d'immunodeficience humaine Download PDF

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WO1989005821A1
WO1989005821A1 PCT/US1988/004553 US8804553W WO8905821A1 WO 1989005821 A1 WO1989005821 A1 WO 1989005821A1 US 8804553 W US8804553 W US 8804553W WO 8905821 A1 WO8905821 A1 WO 8905821A1
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hiv
peptide
gpl20
antibodies
beta
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PCT/US1988/004553
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Mark E. Gurney
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Arch Development Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C07K16/1063—Lentiviridae, e.g. HIV, FIV, SIV env, e.g. gp41, gp110/120, gp160, V3, PND, CD4 binding site
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes

Definitions

  • the present invention relates generally to immunological methods and materials and more particu- larly to peptides sharing amino acid sequence homology with proteins of Human Immunodeficiency Virus type 1 ("HIV-1”) and with the human neurotrophic factor, neuro- leu in (“NLK”), to antibodies specific for and related to such peptides, and to immunotherapeutic and diagnostic procedures involving such peptides and anti ⁇ bodies.
  • HIV-1 Human Immunodeficiency Virus type 1
  • NLK neuro- leu in
  • Human Immunodeficiency Virus Type 1 has been designated as the viral causative agent of acquired immunodeficiency syndrome ("AIDS") and AIDS-related complex ("ARC").
  • AIDS acquired immunodeficiency syndrome
  • ARC AIDS-related complex
  • Numerous review publications address the etiology of the disease state, the pathogenesis of. the viral causative agent, and the prospects for rapid development of AIDS and ARC diagnostic and/or thera ⁇ plastic agents. See, e.g., Fauci, Proc. Nat'l. Acad. Sci. (USA), 3, 9278-9283 (1986) and Ho et al.. New Eng. Jour. Med., 317, 278-286 (1987). _.
  • immunopurified preparations of a gpl60 subunit, gpl20 have been suggested as protective vaccine components. See, Robey et al., Proc. Nat'l. Acad. Sci (USA), 83, 7023-7027 (1986); and Matthews et al., Proc. Nat'l. Acad. Sci (USA), E$3_, 9709-9713 (1986).
  • protective subunit vaccines based on, e.g., HIV-1 env glycoproteins or fragments thereof is the existence of significant intertypic heterogeneity in amino acid sequence and the corresponding hetero ⁇ geneity,of immune responses to administration of pro ⁇ teins derived from differing HIV-1 isolates.
  • monoclonal antibodies raised against or speci- fically immunoreactive with gpl20 derived from one HIV-1 isolate may be immunologically significant in terms of in vitro neutralization capacity with respect to the specific isolate, but may not at all recognize gpl20 of other subtypes. See, e.g., Fung et al., Bio/Technology, j>, 940-946 (1987).
  • Natural isolates and recombinant products should be free of foreign DNA and contaminating foreign proteins. Finally, because little is known concerning the effects of HIV-1 proteins per se in infected hosts, substantial attention must be taken to avoid, e.g., adverse non-immunological effects which might attend administration of viral subunits.
  • these publications and published patent applications relate to immunochemical reagents projected for use in diagnosis and vaccination for AIDS and ARC and, more specifically, describe synthetic peptides modeled after, i.e., sharing amino acid sequence homology with, con- tinuousgsequences of amino acids within the envelope glyccJl libems of HIV-1.
  • Ho et al., J.Virolfl 61(6) , 2024-2028 (1986) describes the prepara ⁇ tion and testing of anti-sera to 87 "overlapping" pep- tides spanning the env glycoprotein, gpl60.
  • peptides corresponding to amino acids 298-314, 458-484 and 503-532 (within the gpl20 portion of the gpl60 sequence) as well as amino acids 728-752 and 616-632 (within the gp41 portion of the sequence) were highlighted for further study. All five synthetic peptides correspond to extremely hydrophilic regions of the HIV-1 envelope and the latter three correspond to regions which are reported to be highly conserved in HTLV-III and LAV strains of HIV-1.
  • synthetic "sub-subunit" peptides provide the basis for such reagents, they should be readily assembled and admini ⁇ stered (e.g., in combination with adjuvants and "carrier” proteins), should be based on homology to "constant” rather than “variable” regions or domains in HIV-1 proteins, should be non-neurotoxic so as to avoid deleterious neurological side effects upon administra ⁇ tion, and should be capable of provoking immune res ⁇ ponses operative in neutralizing viral infectivity.
  • novel immunochemical reagents including non-neurotoxic pep- tides and antibodies specifically immunoreactive there ⁇ with.
  • Peptides of the invention are characterized by the absence of capacity for ⁇ n vitro inhibition of neuroleukin action on neuroleukin-dependent neurons.
  • chimeric antibodies e.g. mouse/human antibodies
  • hybridoma cells especially heavy chain deletion mutant cells
  • HIV-IIIRF differs from the corresponding sequence of residues of gpl20 of HIV-ARV-2 by a single residue.
  • the peptide having the sequence, STQLLLNGSLAEEEWIRC which possesses total homology to residues 263 through 280 within gpl20 of all three of the above-mentioned HIV-1 isolates and includes a carboxy terminal cysteine residue.
  • Peptides of the invention may optionally be provided with a variety of additional amino acid residues at their amino or carboxy terminal or at inter ⁇ mediate positions within the sequence in order to enhance their immunogenicity and immunochemical reagent potential.
  • one or more tyrosine resi- dues may be provided for association with a radiolabel substance such as ⁇ 125 with the peptide.
  • a radiolabel substance such as ⁇ 125 with the peptide.
  • one or more -reactive terminal or intermediate cysteine resi ⁇ dues may be provided to facilitate association of the peptide with carrier proteins commonly employed in the development of peptide vaccine ⁇ compositions.
  • polyamino acids such as poly-L-glutamic acid may be linked to peptides of the invention to enhance anti- genicity. In general, however, care should be exercised to avoid incorporation of residues adversely affecting the immunogenicity of antigenic sites within the pep- tides.
  • Monoclonal antibodies presently preferred are those reactive with a peptide having the sequence:
  • Peptides and corresponding antibodies of the invention are useful for immunodiagnostic procedures (e.g., ELISA*s, RIA's, and the like) for the detection of HIV-1 infection and/or for monitoring progress of vaccination treatment.
  • immunodiagnostic procedures e.g., ELISA*s, RIA's, and the like
  • the present invention provides, as novel immunochemical reagents, peptides (and antibodies related thereto) whose sequences are based on those regions of amino acids within residues 234-300 of HIV-III B, which presumptively form loops separating (i.e., intermediate between) beta-sheet forming residues within the normal secondary and tertiary conformation of that region.
  • Figure 1 shows the predicted beta-sheet secondary structure for HLA-A A-2 domain, HCMV-H301 gene, HLA-DR B-2 domain, and HIV gpl20.
  • Figure 2 shematically illustrates a typical anti-parallel arrangement of the beta-sheets of a constant Ig domain.
  • Figure 3 illustrates predicted strands of beta-sheets, B-2 through B-6, for the second conserved region of HIV-III B.
  • Figure 4 shows the alignment of contact residues between the HLA-A A-3 domain strands beta-4 and beta-5 and B2"-m with the homologous domains of HCMV- H301, HLA-DR, and gpl20.
  • Example 1 relates to preparation of immunologically active pep ⁇ tides and reagents of the invention.
  • Example 2 relates to generation of antibodies specific for peptides of the invention.
  • Example 3 relates to screening procedures permitting characterization of immunological properties of antibodies of the invention.
  • Example 4 relates to screening of peptides of the invention for potential neurotoxic effects as determined according to the cul ⁇ tured chick sensory neuron survival assay_as described in Lee et al., supra.
  • Example 5 relates to the preparation of monoclonal antibodies.
  • Example 6 relates to m vitro neutralization of HIV-1 using JK112 monoclonal antibody.
  • Example 7 relates to the mapping of the amino acid sequence recognized by the JK112 monoclonal antibody.
  • Example 8 relates to the predicted three-dimensional conformation of the antigenic epitopes of HIV-III B gpl20 and the peptides derived therefrom.
  • Table I below sets out partial amino acid sequence of glycoprotein gpl20 of HIV-IIIB, HIV-IIIRF and HIV-ARV-2 isolates in the so-called "second con ⁇ served region" as reported in Ratner et al., supra; Wain-Hobson, Cell, 40, 9-17 (1985); Sanchez-Pescador et al., Science, 227, 484-492 (1985); and Starcich et al., supra, using the numbering system of Ratner et al., supra. A dash indicates identity with the first-listed residue.
  • Set out immediately following the HIV-1 sequences is the sequence of a "corresponding" sequence of amino acids in human neuroleukin. Asterisks desig ⁇ nate sequence homology with the HIV-1 proteins.
  • Table I sets out the sequence of six synthetic peptides — three 19-mers and a 21-mers based on the HIV-1 sequences and two 20-mers based on NLK sequences.
  • Conjugates of the peptides with a carrier protein are readily prepared by suitable means well known in the art.
  • Suitable carriers include keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) .
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • Coupling of cysteine residue-containing peptides may be accomplished through use of m-maleimidobenzoic acid N-hydroxy succinimide ester (MBS) or the
  • N-maleimido-6-aminocaproyl ester of l-hydroxy-2-nitro-4- benzenesulfonic acid (MSAC) .
  • MSAC N-maleimido-6-aminocaproyl ester of l-hydroxy-2-nitro-4- benzenesulfonic acid
  • coupling to primary amino groups through glutaraldehyde may be employed.
  • the carrier is first reacted with MBS (25 mg/ml in for ⁇ mamide) .at a ratio of 0.125 mg MBS per mg BSA in 50 mM sodium phosphate buffer (pH 7.5) for 30 min. at room temperature. Removal of unreacted MBS and buffer exchange is accomplished by chromatography over Sephadex G-50 equilibrated with 50 mM sodium phosphate buffer (pH 7.0).
  • Bound rabbit immunoglobulin was detected with a Vectastain kit (Vector Labs.) utilizing biotinylated goat antibody to rabbit immunoglobulin, avidin, and biotinylated horse radish peroxidase. Color development was achieved with o-phenylenediamine and hydrogen peroxide and the ELISA was read on a dual wavelength microplate spectrophotometer at 450 nm. - The titers in Table II below are the dilution of rabbit antiserum that gave one-half maximal color development (usually optical density of 0.5-0.6).
  • the following example relates to characteriza ⁇ tion of antibodies according to the invention.
  • Each virus inoculum (100 ul, fifty 50% tissue culture infective dose) was preincubated with the test serum (100 ul, serial two-fold dilutions) for 1 hr. at 37°C before inoculation onto 2.0 X 10 6 H9 cells in 5 ml of RPMI 1640 medium supplemented with fetal calf serum (20%) , N-2-hydroxyethylpiperazine-N'-2-ethane-sulfonic acid (10 mM) , penicillin (250. U/ml), streptomycin (250 ug/ml), and L-glutamine (2 mM) .
  • each culture was examined for characteristic cytopathic effects with syncytia formation and for p24 antigen in- the supernatant fluid by an immunoassay (Abbott Laboratories, North Chicago). Neutralization was defined as >90% reduction in both syncytia formation and supernatant p24 antigen compared to control cultures, which were similarly established except that the virus inoculum was preincubated with culture medium or normal rabbit serum. HIV binding inhibition studies were per ⁇ formed using the protocol of McDougal et al., J.Immunol. , 137, 2937-2944 (1986). The virus stock used in the assays was prepared as follows.
  • the neutralizing titer of the antiserum to C21E is several fold higher than the mean titer of sera from HIV-1 seropositive persons.
  • human serum P982 is more reactive with gpl20 and gpl60 by radioimmune precipitation
  • the antiserum to C21E is four-fold more neutralizing against HIV-IIIB and HIV-ARV-2.
  • the antiserum to C21E is also more neutral ⁇ izing than the goat antiserum to gpl20.
  • HIV-1 seropositive individuals show restricted neutralization of different HIV-1 strains, as do animal antisera prepared against gpl20.
  • the immune response to gpl20 is directed against antigenically variable domains as opposed to regions of conserved sequence.
  • HIV-1 seroposi- tive human sera were largely negative in an ELISA with the T19V, C21E, and S19C peptides, and those few scoring as positive were not strongly reactive.
  • sera from animals immunized with gpl20 also did not react significantly with the peptides as determined by ELISA.
  • the following example relates to characteriza- tion of peptides of the invention.
  • Tests were performed to determine whether synthetic peptides having homology to gpl20 would inhibit the biological activity of neuroleukin as reported in Lee et al., supra.
  • initial procedures involving peptides T19V, C21E and S19C prepared accord ⁇ ing to Example 1 up to 10 ug per ml (from 4-5 uM) of each of the unconjugated peptides was added to culture medium containing 4-5 biological units of recombinant mouse NLK. That concentration of NLK maintains maximum survival and growth of sensory neurons cultured from 10 day chick embryos. No inhibition of sensory neuron growth was obtained with any of the peptides.
  • Table IV provides a summary of the results of screening of conjugates of peptide whose preparation is referred to in Example 1.
  • concentrations of stock solutions were calculated from amino acid analysis. Molecular weights employed for the calculations were: C19Q, 1985; T19V, 1970; C21E, 2208; S19C, 2074; M20L, 2306; F20H, 2306; BSA, 67,000.
  • fifty percent inhibition of sensory neuron survival in culture medium containing 4-5 bio ⁇ logical units of NLK (defined as the I 50 for the inhi ⁇ bitor) was obtained with the T19V-BSA(C) conjugate added at 5 ng per ml of culture medium (94 pM) .
  • the C19Q peptide which overlaps the amino terminus of T19V was also an inhibitor of NLK, while the C21E peptide which overlaps the carboxyl terminus of T19V was not inhibi ⁇ tory.
  • the C19Q-BSA conjugate even though it had a slightly greater loading of peptide, was less inhibitory than the T19V-BSA conjugate and had an IC Q of 13 ng per ml (278.pM).
  • BSA conjugates prepared with C21E or S19C did not inhibit sensory neuron growth in NLK at up to 1 ug per ml (> 100 nM) .
  • the set of peptides define a sequence within the midsection of the T19V peptide that is a potent inhibitor of NLK.
  • the homologous NLK peptide is also an antagonist, and even more potent than T19V.
  • An M20L-BSA conjugate with a loading of 10 nmol M20L peptide per nmol BSA was found to have an I 50 of 2 pM (two experiments).
  • T19V-BSA conjugates with equivalent peptide loadings had an Igg of approximately 100 pM.
  • the inhibition of NLK by M20L-BSA was sequence speci ⁇ fic.
  • # - determined by amino acid analysis mean ⁇ SD where n is the number of amino acids in the peptide that were analyzed.
  • Antibodies of the present invention include polyclonal and monoclonal antibodies of diverse mam ⁇ malian origins. Monoclonal antibodies derived from murine hybridoma cell sources and chimeric (e.g., mouse/human) and anti-idiotypic antibodies are currently undergoing development.
  • C21E peptide was coupled to keyhole limpet hemocyanin (KLH) with m-maleimidobenzoic acid-N-hydroxy- succinimide ester (MBS) as described in Table I and Example 1, supra. Approximately 0.2 - 0.4 mg of peptide was bound per mg of KLH. Eight 12-week old female BALB/c mice were immunized with 50 yg C21E-KLH conjugate emulsified in Freund's complete adjuvant at multiple intradermal sites (day 0).
  • mice were immunized a second time with C21E-KLH emulsified in Freund's incomplete adjuvant on day 14, and then were boosted on day 28 with C21E-KLH in saline delivered intraperitonealy.
  • the spleens were harvested on day 31 for fusion with mouse SP2/0 cells.
  • Hybridoma fusions of immunized mouse spleen cells with SP2/0 cells followed the protocol of Galfre and Mils ⁇ ein, Methods Enzymology, 73, 1-45 (1981). Hybridomas were selected for growth in hypoxanthine-aminopterin-thymidine.
  • Monoclonal antibodies reactive with C21E were detected using ELISA.
  • the C21E peptide was used to coat the wells of 96-well polyvinylchloride microtiter plates at a concentration of 5 yg/ l in 0.1 M sodium bicarbonate (pH 9.6).
  • gpl60 ELISA recombinant gpl60 coated ELISA plates were used that were purchased from MicroGeneSys (West Haven, CT) .
  • Supernatants from hybridoma microcultures were incubated in the wells for 2 hr at 37°C.
  • the assay was then developed with a Vector Laboratories ABC kit using biotinylated antibody to mouse immunoglobulin and a biotinylated horseradish peroxidase, avidin complex. Color was developed with o- phenylenediamine/hydrogen peroxide and quantitated at 450 nm.
  • radioimmunoprecipitation analysis as described Example 3, supra, was performed on sixteen of the 55 monoclonal antibodies.
  • the HTLV-IIIB isolate of HIV-1 was used to infect H9 T- lymphocytic cells.
  • the infected cell cultures were fed S-35 labeled methionine and cysteine to incorporate S-35 label into viral proteins.
  • Detergent lysates were prepared from the infected cells.
  • mouse monoclonal antibodies from the culture supernatants were attached to SepharoseTM beads through goat anti-mouse immunoglobulin which previously was covalently bound to the surface of the beads.
  • the beads were then washed and subsequently incubated with S-35 labeled, HIV- infected H9 cell lysates. After incubation for 2 hr at room temperature, the beads were then washed with RIPA buffer containing 0.5 M sodium chloride, and bound antigen was released by boiling in sample ⁇ buffer containing sodium dodecyl sulfate and B- mercaptoethanol. The released antigen was analyzed by electrophoresis on 7.5% SDS-polyacrylamide gels and autoradiography against X-ray film to determine whether the mouse monoclonal antibodies had reacted with any HIV-1 glycoproteins.
  • JK1112 monoclonal antibody with HIV-1 envelope glycoproteins additional isolates of HIV-1 were radiolabeled, including isolates designated RF, MN, COSTA, Z34, Z84, and AL.
  • the RF isolated has been described in Starcich et al.. Cell, 4_5, 637-648 (1986); the MN isolate has been described in Gurgo et al., Virol. , 164, 531-536 (1988); the remaining isolates are clinical isolates of one of the co-inventors.
  • Monoclonal antibody JK112 reacted with the gpl60 and/or gpl20 envelope glycoprotein of every isolate of HIV-1 tested.
  • JK112 hybridoma To demonstrate the genetic stability of the JK112 hybridoma, it was subcloned by limiting dilution ' using feeder cells prepared from un-immunized BALB/c mouse spleens. In the first subcloning, 36 of 36 subclones secreted antibody reactive with C21E. Three subclones were chosen (JK112.6, JK112.16, and JK112.17) and each was subcloned a second time by limiting dilution. In the second subcloning, 28 of 28 JK112.6 subclones, 36 of 36 JK112.16 subclones, and 22 ' of 22 JK112.17 subclones secreted antibody reactive with
  • JK112 monoclonal antibody binds to protein A, it can be purified from culture supernatants in one step.
  • Supernatants were harvested from JK112 hybridoma cultures, adjusted to pH 8.0 by addition of sodium phosphate buffer to a final concentration of 20 mM, and then were passed over a column of protein A covalently bound to Sepharose CL4BTM. The optical absorbance of the effluent was washed to baseline with pH 8 buffer containing 20 mM sodium phosphate and 150 mM sodium chloride.
  • JK112 monoclonal antibody was eluted from the column using pH 4.5 buffer containing 100 mM sodium citrate. The JK112 antibody was dialyzed into phosphate-buffered saline, concentrated to 0.5 - 0.25 mg protein per ml, sterilized by filtration through a 0.22 micron filter, and stored frozen until use.
  • An HIV-1 Ln vitro neutralization assay of the JK112 monoclonal antibody was performed as follows. A titered stock of HIV-1 containing 50 tissue culture infectious doses was incubated with varying amounts (from 10 ⁇ g to 10 pg) of JK112 monoclonal antibody for 30 minutes at 37°C before dilution twenty-five fold for inoculation onto H9 cells, as per Example 3, supra.
  • peptides Three overlapping peptides were used to map the amino acid sequence recognized by JK112 monoclonal antibody.
  • the peptides are designated T19V, C21E, and S19C, as described in Table I, supra.
  • JK112 reacted with C21E and S19C, but notr-with T19V.
  • JK112 reacts with the minimum sequence S-T-Q-L-L- L-N-G-S-L-A-E comprising the carboxy-terminus of the C21E peptide.
  • the rabbit antiserum to C21E reacted with T19V and C21E peptides, but not with S19C.
  • the rabbit antiserum reacted with the minimum sequence C-T-H-G-I-R-P-V comprising the amino-terminus of the C21E peptide.
  • the rabbit antiserum produced by immunizing with the C21E peptide also efficiently neutralized in vitro infection of T-lymphocytic cells by the IIIB, RF, and MN isolates of HIV-1. See Table III, supra.
  • a minimum of two distinct epitopes are present in C21E, either of which will elicit antibodies that efficiently neutralize HIV-1 infection in the i ⁇ ⁇ vitro model.
  • the HLA class II molecule is a dimer consist ⁇ ing of an alpha and a beta chain [Kappes et al., Ann. Rev. Biochem., 57, 991-1028 (1988)]. Each chain con ⁇ sists of an external T-cell recognition domain (A-l and B-l, respectively) and a membrane-proximal domain (A-2 and B-2).
  • the B-l domain of HLA-DR is highly polymor ⁇ phic and is important for presenting processed antigen to HLA class II-restricted T-cells.
  • the A-2 and B-2 domains probably have a structural function and have considerable homology to immunoglobulin constant domains (the C-l Ig homology set defined by Williams et al.. Ann. Rev.
  • Class II- restrictjf .-cells express CD4 and monoclonal antibodies to specifi ⁇
  • the same antibodies which block CD4 function also block binding of HIV-1 [Sattentau et al.. Science, 234, 1120-1123 (1986)].
  • CD4 binds to class II molecules and probably helps stabilize the contact between class II molecules and the T-cell antigen receptor [Doyle et al.. Nature, 330, 256-259 (1987)].
  • the HLA class II molecule is structurally homologous to the HLA class I molecule. Instead of two polymorphic chains, the class I molecule has an epoly- morphic chain (with 3 domains designated A-l through A-3) which is associated with an invariant protein, B2-microglobulin (B 2 -m).
  • the A-l and A-2 chains form a groove or saddle which has contact residues for T-cell recognition and a binding site for foreign antigen
  • the A-3 domain and B2 ⁇ provide structural support for the T-cell recognition domains, and like the membrane proximal domains of class II molecules, are also members of the C-l Ig homology set [Williams et al., Ann. Rev. Immunol. , 6 , 381-405 (1988)].
  • the H2 HLA-DR, A-3 HLA-A and B2 ⁇ m sequences possess considerable homology and readily align.
  • the HLA class I A-3 domain contains two anti- parallel beta-pleated sheets. One sheet contains four beta-strands; the other contains three beta-strands, and the two sheets are connected by a disulfide which is a hallmark of Ig domains.
  • the association of B 2 ⁇ m with the class I molecule is primarily through contacts on the A-3 domain. These contacts cluster mainly over a 12 amino acid stretch (residues 231, 233, 234, 237 and 238) comprising the beta 4-strand and the loop between strands beta-4 and beta-5 [Bjorkman et al.. Nature, 329, 506-512 (1987)].
  • the A-3 beta-4 strand of the class I molecule contacts B2 ⁇ m perpendicularly to the B2 ⁇ m anti- parallel beta-sheet along a shallow groove across the inner surface of the protein.
  • the secondary structure of the A-3 domain is schematized in Figure 1 with the corresponding pre ⁇ diction of beta-strands plotted immediately underneath (Chou-Fasman algorithm. University of Wisconsin Computing Genetics Group) .
  • Figure 1 The secondary structure of the A-3 domain is schematized in Figure 1 with the corresponding pre ⁇ diction of beta-strands plotted immediately underneath (Chou-Fasman algorithm. University of Wisconsin Computing Genetics Group) .
  • Figure 1 are represented predicted structures for the HLA-A A-2 (residues 203-259), the HCMV-H301 gene (residues 224- 281) r the HLA-DR B-2 (residues 117-173) and HIV gpl20 (residues 235-295).
  • the class I and class II molecules are aligned with respect to conserved cysteine residues in the Ig homology domain and the gpl20 sequence has been aligned with the region of homology in the class II B-2 domain (residues 142 to 151). Note that each sequence, including that of HIV-1, predicts a pattern of five alternating, anti-parallel beta- strands. The five beta-strands predicted for the gpl20 sequence (numbered 2-6 following the numbering ' of the A-3 class I structure) fall at residues 238-242,
  • FIG. 2 provides a schematic representation of a constant Ig domain showing the anti-parallel arrangement of beta- sheets. Strands of the beta-sheets are indicated by the ribbon arrows. Loops of polypeptide chain which are believed to be antigenic determinants link adjacent strands of the beta-sheets. Linear epitopes are frequently found to comprise loops of polypeptide chain exposed on the surface of proteins [Tainer et al.. Nature, 312, 127-134 (1984)].
  • the model of anti-parallel beta-strands for gpl20 residues 235-300 is in good agreement with the existing mutational analysis and the mapping of epitopes within that region.
  • the epitope apparently recognized by both anti-C21E and anti-1-110, for example (CTHGIRPV) maps precisely to the proposed loop between strands beta-3 and beta-4.
  • the epitopes recognized by JK112 monoclonal antibody (STQLLLNGSLAE) and anti-S19C (LAEEEWIR) overlap the proposed loop between strands beta-4 and beta-5.
  • the antiserum to T19V was poorly reactive with gpl20, perhaps because it was directed against a sequence (TGPCTNVSTVQ) located in the beta-3 strand rather than a loop region.
  • Figure 3 diagrammatically represents a structure for residues 234-300 of gpl20 which is consistent with the above.
  • B-2 through B6 are the predicted strands of beta-sheet. The residues beginning and ending the inter-strand acid sequences of the loops below, correlates projected the gpl20 region with their location in the predicted beta-sandwich structure.
  • the region of homology between gpl20 and HLA-DR aligns with the contact residues (underlined) between the HLA-A class I beta 4-strand and B 2 -m. Figure . 4.
  • the contact residues are located at the end of the beta-4 strand (V 231 , ⁇ 233 , and R 234 ); within the inter-strand loop (G 237 and D 238 ); and at the beginning of the beta-5 strand (Q 242 and W 244 ).
  • the class I molecule encoded by HCMV aligns with this sequence and is believed to bind B 2 -m [Beck et al., Nature, 331, 269- 272 (1988)], but as can be seen only two contact residues are conserved.
  • the gpl20 and HLA-DR sequences also align with the class I sequences over the contact region.
  • Four contact residues are conserved between HLA-DR and HLA-A, and three of these are ⁇ also present in the gpl20 sequence.
  • the alignment between HLA-DR and gpl20 is strongest over the length of the predicted contact region in the beta-4 strand and the loop between beta-strands 4 and 5. Mutations in the inter-strand beta-4/-5 loop destroy HIV-1 infectivity.
  • Three mutations have been made in the predicted beta-3 strand of gpl20, one in the loop between strands beta-3 and beta-4, and three in the predicted loop between strands beta-4 and beta-5 which align with the contact residues in the HLA-A sequence.
  • Non-random association of HIV-1 virions is seen with two cellular antigens, both of which are members .of the Ig superfamily, B2 ⁇ m and HLA-DR [Hoxie et al.. Hum. Immunol., 18, 39-52 (1987)].
  • B2 ⁇ m members .of the Ig superfamily
  • HLA-DR HLA-DR
  • the B 2 ⁇ m detected in HIV-1 virion preparations is not associated with HLA class I molecules.
  • l60 versus gpl20 also provides some evidence that th ⁇ l3econd conserved domain of gpl20 may be complexed with an additional protein.
  • JK112 binds strongly to gpl60, but has little reactivity with gpl20.
  • peptides having sequence homology to the "second conserved region" of HIV-1 gpl20 glycoprotein can provide valuable immuno- chemical reagents. Not all such peptides are valuable and, indeed, some display a substantial potential for generating neurotoxic effects.
  • peptides of the invention are prepared based on homology to an antigenic determinant or epitope within the region spanning gpl20 residues 254-280 and preferably 254-274, neurotoxic potential is not observed.
  • preferred peptides of the inven ⁇ tion include an epitope homologous to the gpl20 sequences spanning residues 254 through 262.
  • peptides of the invention as portions of larger polypeptides such as poly-L-glutamic acid or poly-L-lysine, to subject the peptides to cationization and thereby enhance immunogenicity as in Muckerheide et al., J.Immunol., 138, 833-837 (1987), to couple peptides of the invention with antibodies known to facilitate strong serological responses to protein antigens as in Carayanniotis et al., Nature, 327, 59-61 (1987), to attach peptides to foreign helper T-cell epitopes according to the procedures of Francis et al., Nature, 330, 168-170 (1987), or to use anti-idiotypic antibodies of the invention as in Chanh et al., Proc. Nat'l. Acad. Sci. (USA), 84, 3891-3895
  • hybrid hybridomas e.g., mouse/human
  • human/human hybridomas prepared, for example, in a manner consistent with Borrebaeck, TIBTECH, June, 1986, pp. 147-153; Abrams et al.. Methods in Enzymology, 121, pp. 107-119 (1986); Kozbor et al.. Methods in Enzymology, 121, pp. 120-140 (1986); Suresh et al. , Methods in Enzymology, 121, pp. 210-228 (1986); and Masuho et al., Biochem. & Biophys. Res. Comm. , 135(2) , pp. 495-500 (1986). See, also, Klausner, '"Single
  • Immunological complexes of the invention are formed ⁇ n vitro upon contact between antibodies of the invention with HIV-1 virus and virus particles and host cells. Such complexes are expected to be formed iji vivo upon practice of vaccination procedures of the invention and expected to preclude infectivity of the virus. Numerous modifications and variations in prac ⁇ tice of the invention will occur to those skilled in the art upon consideration of the foregoing illustrative examples and consequently only such limitations as appear in the appended claims should be placed thereon.

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Abstract

Des réactifs immunochimiques nouveaux, y compris des peptides non-neurotoxiques ayant des séquences d'acides aminés homoloques aux séquences en boucle contenues dans les résidus 230 à 300 de la glucoprotéine gp 120 du virus IIIB d'immunodéficience humaine et des anticorps de ces derniers, sont capables d'inhiber in vitro l'infectivité des virus I d'immunodéficience humaine. Ces peptides et ces anticorps sont utiles dans des essais de diagnostic et en tant que composants actifs de compositions de vaccination active et passive.
PCT/US1988/004553 1987-12-21 1988-12-20 Antigenes et anticorps associes au virus d'immunodeficience humaine WO1989005821A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991015512A2 (fr) * 1990-04-03 1991-10-17 Genentech, Inc. Polypeptides d'enveloppe du vih
EP0459779A1 (fr) * 1990-05-29 1991-12-04 Cedars-Sinai Medical Center Réactifs immunologiques réagissant avec un épitope constant du virus immunodéficient humain du type I (HIV-I) GP120 et méthodes d'utilisation
FR2677364A1 (fr) * 1991-06-05 1992-12-11 Pasteur Institut Sequences peptidiques de la glycoproteine externe d'enveloppe du retrovirus hiv-1.
GB2313376A (en) * 1996-05-22 1997-11-26 Diapharm Limited Polypeptide immunologically cross-reactive with NTM peptide and with vasoactive intestinal peptide (VIP)
EP0808846A2 (fr) * 1996-05-22 1997-11-26 Diapharm Limited Peptides réactifs avec des anticorps servant de révélateurs de maladie liée au VIH
US5763160A (en) * 1988-02-12 1998-06-09 United Biomedical, Inc. Synthetic peptides and process of using same for the detection of antibodies to human immunodeficiency virus (HIV) gp120 envelope protein, diagnosis of AIDS and pre-AIDS conditions and as vaccines
WO2019092703A1 (fr) * 2017-11-07 2019-05-16 Memed Diagnostics Ltd. Anticorps anti-trail et procédés d'utilisation

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US4629783A (en) * 1985-04-29 1986-12-16 Genetic Systems Corporation Synthetic antigen for the detection of AIDS-related disease
WO1987002775A1 (fr) * 1985-10-24 1987-05-07 Southwest Foundation For Biomedical Research Peptides synthetiques et leur utilisation pour le diagnostic et la vaccination contre le sida et son complexe associe

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US4629783A (en) * 1985-04-29 1986-12-16 Genetic Systems Corporation Synthetic antigen for the detection of AIDS-related disease
WO1987002775A1 (fr) * 1985-10-24 1987-05-07 Southwest Foundation For Biomedical Research Peptides synthetiques et leur utilisation pour le diagnostic et la vaccination contre le sida et son complexe associe

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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5763160A (en) * 1988-02-12 1998-06-09 United Biomedical, Inc. Synthetic peptides and process of using same for the detection of antibodies to human immunodeficiency virus (HIV) gp120 envelope protein, diagnosis of AIDS and pre-AIDS conditions and as vaccines
WO1991015512A2 (fr) * 1990-04-03 1991-10-17 Genentech, Inc. Polypeptides d'enveloppe du vih
WO1991015512A3 (fr) * 1990-04-03 1991-12-12 Genentech Inc Polypeptides d'enveloppe du vih
EP0459779A1 (fr) * 1990-05-29 1991-12-04 Cedars-Sinai Medical Center Réactifs immunologiques réagissant avec un épitope constant du virus immunodéficient humain du type I (HIV-I) GP120 et méthodes d'utilisation
EP0614985A1 (fr) * 1990-05-29 1994-09-14 Cedars-Sinai Medical Center Réactifs immunologiques réagissant avec l'épitope du virus immunodéficient humain type 1 (VIH-1) gp120 et méthodes d'utilisation
US6054284A (en) * 1990-05-29 2000-04-25 Cedars-Sinai Medical Center Immunoreagents reactive with a conserved epitope of human immunodeficiency virus type I (HIV-1) GP120 and methods of use
FR2677364A1 (fr) * 1991-06-05 1992-12-11 Pasteur Institut Sequences peptidiques de la glycoproteine externe d'enveloppe du retrovirus hiv-1.
GB2313376A (en) * 1996-05-22 1997-11-26 Diapharm Limited Polypeptide immunologically cross-reactive with NTM peptide and with vasoactive intestinal peptide (VIP)
EP0808846A2 (fr) * 1996-05-22 1997-11-26 Diapharm Limited Peptides réactifs avec des anticorps servant de révélateurs de maladie liée au VIH
EP0808846A3 (fr) * 1996-05-22 1998-08-26 Diapharm Limited Peptides réactifs avec des anticorps servant de révélateurs de maladie liée au VIH
WO2019092703A1 (fr) * 2017-11-07 2019-05-16 Memed Diagnostics Ltd. Anticorps anti-trail et procédés d'utilisation

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