WO1989005820A1 - 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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WO1989005820A1
WO1989005820A1 PCT/US1988/004552 US8804552W WO8905820A1 WO 1989005820 A1 WO1989005820 A1 WO 1989005820A1 US 8804552 W US8804552 W US 8804552W WO 8905820 A1 WO8905820 A1 WO 8905820A1
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hiv
peptide
antibody
sequence
antibodies
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PCT/US1988/004552
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Mark E. Gurney
David D. Ho
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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
    • 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 ho ology with proteins of Human Immunodeficiency Virus type 1 ("HIV-1") and with the human neurotrophic factor, neuro- leukin (“N K”), 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
  • N K neuro- leukin
  • Human Immunodeficiency Virus Type 1 has been designated as the viral causative agent of acquired immunodeficiency syndrome ("AIDS") and AlDS-related complex ("ARC").
  • AIDS acquired immunodeficiency syndrome
  • ARC AlDS-related complex
  • Numerous review publications address the etiology of the disease state, the p thogenesis 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), 8 , 9278-9283 (1986) and Ho et al.. New Eng. Jour. Med., 317, 278-286 (1987).
  • vaccinia-based strategies envision vaccination with recombinant vaccinia strains engineered to encode the HIV-1 envelope glycoprotein, gpl ⁇ O, hopefully thereby presenting the glycoprotein as an immunogen to the indi- viduals vaccinated.
  • immunopurified preparations of a gpl ⁇ O 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 , 9709-9713 (1986).
  • Recombinant methods have been applied to secure relatively large scale production of various forms of HIV-l.gpl60, as well as its lower molecular weight subunits, gp41 and gpl20 [see, e.g., asky et al.. Science, 233, 209-212 (1986); Weiss et al.. Nature, 324, 572-578 (1986); and Lasky et al.. Cell, 50, 975-985 (1987)] and even "sub- subunit" polypeptides such as the fusion protein com ⁇ prising a 180 amino acid fragment of gpl20 known as "PB1" [see. Putney et al.. Science, 234, 1392-1395 (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).
  • these publications disclose that the deduced amino acid sequence of neuroleukin is partially homologous to the amino acid sequence of the gpl20 env glycoprotein of HIV-1 in a region which is highly "conserved” among known HIV-1 subtypes. Also disclosed is the determination that gpl20 is, per se, capable of in vitro inhibition of the action of neuroleukin on neuroleukin-dependent cultured neuron cells, suggesting that the antigen may display neurotoxic effects in an infected host by directly suppressing neuronal responses to neurotrophic factors. This determination is noted to provide valuable insights into the pathogenesis of the AIDS dementia complex.
  • 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 in vitro inhibition of neuroleukin action on neuroleukin-dependent neurons.
  • the peptides are further characterized by sufficient amino acid sequence homology to residues 254 to 280 [according to the numbering system of Ratner et al..
  • glycoprotein gpl20 of HIV-IIIB to allow for the capacity to stimulate forma- tion of antibodies of the invention which, in turn, are characterized by immunoreactivity with gpl20 and/or gpl60 isolates of HIV-IIIB and at least one other HIV-1 subtype and by the capacity for neutralization of infec ⁇ tivity, _in vitro, of HIV-IIIB and at least one other HIV-1 subtype [e.g., HIV-IIIRF referred to as HAT-3 and sequenced by Starcich et al., Cell, 45, 637-648 (1986)].
  • Peptides of the invention are thus seen to include amino acids in sequence which correspond to at least one antigenic determinant shared by gpl20 of HIV-IIIB and at least one other HIV-1 subtype.
  • Preferred antibodies of the invention including mono ⁇ clonal and polyclonal antibodies, neutralize HIV-1 infectivity (with respect to, e.g., H9 cells in culture) without substantially diminishing the capacity for association (binding) of viral particles to host cell surfaces.
  • Antibodies of the invention also comprehend monoclonal anti-idiotypic antibodies raised, e.g., against variable, antigen-combining regions ("paratopes") of neutralizing monoclonal antibodies of the invention.
  • Chi eric antibodies, and fragments thereof, and especially bi-specific antibodies are also products within the contemplation of the present invention, as are antibody-related products produced in microbial hosts, (e.g., procaryotic and eucaryotic cells in culture) which hosts are transformed or transfected with DNA sequences encoding the desired polypeptide products.
  • microbial hosts e.g., procaryotic and eucaryotic cells in culture
  • procaryotic and eucaryotic hosts such as E. coli, yeast, insect, and mammalian cells in culture to produce useful antibody fragments (such as fab' and f(ab') 2 fragments).
  • chimeric antibodies e.g.
  • mouse/human antibodies may be prepared using transformed mouse myeloma cells or hybridoma cells (especially heavy chain deletion mutant cells) as production hosts. Hybrid hybridoma cell producing bi-specific antibodies having diagnostic and therapeutic uses are contemplated.
  • Presently preferred antibodies of the invention are monoclonal antibodies of the IgG 3 subtype and in particular that monoclonal antibody produced by cell line JK112, deposited August 19, 1988 with the American Type Culture Collection, Rockville, Maryland under accession No. A.T.C.C. HB9799.
  • novel binary immuno ogical compositions comprising HIV-1 viral par ⁇ ticles bound to antibodies of the invention, as well as ternary immunological compositions comprising antibodies " of the invention bound to HIV-1 viral particles which are, in turn, bound to susceptible host cell surfaces.
  • Presently preferred peptides of the invention include the peptide having the following sequence, -
  • CTHGIRPWSTQLLLNGSLAE which sequence possesses total homology with the con- tinuous sequence of amino acid residues spanning posi ⁇ tions 254 through 274 of gpl20 of both HIV-IIIB and HIV-IIIRF and differs from the corresponding sequence of residues of gpl20 of HIV-ARV-2 by a single residue. Also presently preferred is 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.
  • 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: S T Q L L L N G S L A E.
  • Vaccine compositions of the invention comprise immunologically effective amounts of one or more pep ⁇ tides or anti-idiotypic antibodies of the invention in combination with a diluent, adjuvant or carrier and vaccination methods of the invention involve administration of such compositions to a host susceptible to HIV-1 infection. Also within the contemplation of the invention are passive immunization compositions and vaccination methods based on ad ini- stration of antibodies of the invention.
  • 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
  • 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 ._
  • Example 7 relates to the mapping of the amino acid sequence recognized by the JK112 monoclonal antibody.
  • con ⁇ struction of peptide C19Q is based on homology to resi ⁇ dues 236 through 253 of gpl20 of HIV-IIIB; peptide T19V is based on homology to residues 243 through 261; pep- tide C21E is based on homology to residues 254 through 274; and peptide S19C is based on homology to residues 263 through 280 and includes an additional carboxy ter ⁇ minal cysteine residue. It is also apparent that pep ⁇ tide M20L is based on homology to residues 401 through 421 of neuroleukin and that peptide F20H preserved the amino acid composition, but not the sequence, of peptide M20L.
  • 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 - 12 -
  • the water soluble cross-linker is dissolved in 100 mM sodium phosphate (pH 7.4) at a weight ratio of 1 mg MSAC per mg BSA and incubated 10 min. at room temperature. Removal of un ⁇ reacted MSAC and buffer exchange is performed by chroma- tography as before. For coupling, varying molar ratios of the peptide are incubated with the activated carrier overnight at room temperature and then unreacted peptide is removed by dialysis. Coupling with 1% glutaraldehyde in phosphate buffered saline is performed for 1 h . at room temperature and the reaction is terminated by addi ⁇ tion of sodium borohydride.
  • Peptides T19V, C21E and S19C were conjugated to KLH through cysteine residues using m-maleimidoben- zoic acid-N-hydroxy-succinimide ester.
  • the amount of peptide bound to the carrier was determined by amino acid analysis and was generally 0.2-0.4 mg peptide bound per mg of KLH.
  • Female New Zealand White rabbits were immunized three times at two-week intervals with 500 ug of the conjugate emulsified in Freund's complete adju- vant for the primary immunization and in Freund's incom ⁇ plete adjuvant for the secondary immunizations. Titer and specificity of rabbit antiserum were assayed by ELISA.
  • 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).
  • Each of the rabbits developed a strong anti ⁇ body response to its respective immunizing peptide, but not to a control peptide, M20L.
  • the antiserum to C21E also reacted with the peptide that overlaps C21E at its N-terminus (T19V) , but not with the peptide that over ⁇ laps at its C-terminus (S19C).
  • the antisera to T19V and S19C showed little cross-reactivity with C21E.
  • the following example relates to characteriza ⁇ tion of antibodies according to the invention.
  • the rabbit preimmune and immune sera were tested for neutralization of HIV infectivity using an assay as described in Ho et al., J.Virol.. , 61(6) , 2024- 2028 (1986) .
  • 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- *-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.
  • Supernatant fluid from HIV-IIIB-infected Molt-III cells were pre- cleared by sequential centrifugation (300 g for 7 minutes, followed by 1500 g for 20 minutes) and then concentrated 1000-fold by ultracentrifugation (90,000 g for 90 minutes) onto a cushion of 15% Renografin-60 (Squibb, Princeton, N.J.) in a 0.01 M Tris, 0.15 M NaCl, 1 mM EDTA, pH 8.0.
  • Ten ul of this HIV preparation were pretreated with 10 ul of test sera for 30 minutes at room temperature prior to incubation with C8166 cells (5 X 10 5 , 30 minutes at 37°C) .
  • the cells were washed and resuspended in 25 ul of a 1:50 dilution of human anti-HIV conjugated to fluroescin. After 30 minutes at 4°C, the cells were then washed, fixed in 1% paraformaldehyde, and analyzed by flow cytometry. The results are summarized in Table III, below.
  • the preimmune sera had no neutralizing acti ⁇ vity.
  • the antiserum to peptide C21E strongly neutralized the homologous isolate, HIV-IIIB, at a titer of 1:128. More importantly, the antiserum was strongly neutralizing against the "heterologous" isolates tested, HIV-IIIRF and HIV-ARV-2.
  • the antisera to peptides T19V and S19C had weak, but detectable neutralizing titers of 1:8 and 1:16, respectively, against HIV-IIIB, and had similar titers against HIV-IIIRF and HIV-ARV-2.
  • Animal sera produced against the entire gpl20 envelope protein frequently show restricted neutralization of only the HIV-1 strain from which the gpl20 protein was pre- pared.
  • a goat antiserum to gpl20 prepared from HIV-IIIB neutralizes HIV-IIIB at a titer of -1:32, has a very low neutralizing titer against HIV-IIIRF and does not neutralize HIV-ARV-2.
  • the neutralizing titer of the antiserum to C21E is several fold higher than the mean titer of sera from H V-1 seropositive persons.
  • human serum P982 is more reactive with gpl20 and gpl ⁇ O 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.
  • 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.
  • T19V peptide con ⁇ jugate to be a potent inhibitor of neuroleukin in the culture assay.
  • Table IV below provides a summary of the results of screening of conjugates of peptide whose preparation is referred to in Example 1. In the Table, 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.
  • 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 I 50 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 ⁇ g of 2 pM (two experiments).
  • T19V-BSA conjugates with equivalent peptide loadings had an I 50 of approximately 100 pM.
  • the inhibition of NLK by M20L-BSA was sequence speci ⁇ fic.
  • 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 ug 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 Milstein, Methods Enzy ology, 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 ug/ml 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.
  • the antigen binding site of the JK112 monoclonal antibody was pre-saturated with C21E peptide (1 ug per immunoprecipitation). Subsequent attempts at immunoprecipitation of gpl ⁇ O and gpl20 from HIV IIIB isolate was blocked thereby indicating a specific reactivity of the JK112 monoclonal antibody with the target C21E sequence.
  • 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 C21E. One of each of these subclones was retained and designated JK112.6a, JK112.16a, and JK112.17a.
  • JK112 is an IgG3 subtype antibody. Because the 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 CL4B 1 ". 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 in 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 ug 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. Samples of the culture supernatant were harvested 4, 7, 10 and 14 days after infection for assay of p24 GAG antigen as an index of HIV-1 infection and replication.
  • 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 not 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 n vitro infection of T-lymphocytic cells by the IIIB, RF, and MN isolates of HIV-1. See Table III, supra. Thus, a minimum of two distinct epitopes are present in C21E, either of which will elicit antibodies that efficiently neutralize HIV-1 infection in the JLn vitro model.
  • 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.
  • hybrid hybridomas e.g., mouse/human
  • human/human hybridomas prepared, for example, in a manner consistent with Borrebaeck, IBTECH, June, 1986, pp. 147-153; Abrams et al.. Methods in Enzymology, 121, pp. 107-119 (1986); Kozbor et al.. Methods in
  • Immunological complexes of the invention are 0 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 in vivo upon practice of vaccination procedures of the invention and expected to preclude infectivity of the virus. 5
  • 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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  • Oncology (AREA)
  • Hematology (AREA)
  • AIDS & HIV (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

Des réactifs immunochimiques nouveaux, y compris des peptides non-neurotoxiques ayant des séquences d'acides aminés homologues aux résidus 254 à 280 de la glycoprotéine gp 120 du virus IIIB d'immunodéficience humaine, et des anticorps de ces derniers, sont capables d'inhiber in vitro l'infectiosité des virus I d'immunodéficience humaine. Ces peptides et ces anticorps sont utiles dans des dosages diagnostiques et en tant que constituants actifs de compositions de vaccination active et passive.
PCT/US1988/004552 1987-12-21 1988-12-20 Antigenes et anticorps associes au virus d'immunodeficience humaine WO1989005820A1 (fr)

Applications Claiming Priority (4)

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US13533787A 1987-12-21 1987-12-21
US135,337 1987-12-21
US23501188A 1988-08-22 1988-08-22
US235,011 1988-08-22

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WO1989005820A1 true WO1989005820A1 (fr) 1989-06-29

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991007493A1 (fr) * 1989-11-13 1991-05-30 Xoma Corporation Anticorps murins humains chimeriques a specificite aux antigenes du vih
WO1991007494A1 (fr) * 1989-11-13 1991-05-30 Xoma Corporation Anticorps murins humains chimeriques a specificite aux antigenes du vih
WO1992005800A1 (fr) * 1990-09-27 1992-04-16 Syntello Vaccin Development Kb Peptides destines a etre utilises dans la vaccination et dans l'induction d'anticorps neutralisants pour lutter contre le virus d'immunodeficience humaine
WO1992021377A1 (fr) * 1991-06-03 1992-12-10 Syntello Inc. Peptides a utiliser pour induire l'activation de lymphocytes t contre le vih-1
US5346989A (en) * 1990-08-22 1994-09-13 Syntello Vaccine Development Kb Peptides for use in induction of T cell activation against HIV-1

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
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

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HO, JOURNAL OF VIROLOGY, 61, 2024-2028, Published June 1987. *
LEE, SCIENCE, 237, 1047-1051, Published 28 August 1987. *
STARCICH, CELL, 45, 637-648, Published 6 June 1986. *
WILLEY, PROCEEDINGS NATIONAL ACADEMY OF SCI. USA; Volume 83, 5038-42, Published July 1986. *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991007493A1 (fr) * 1989-11-13 1991-05-30 Xoma Corporation Anticorps murins humains chimeriques a specificite aux antigenes du vih
WO1991007494A1 (fr) * 1989-11-13 1991-05-30 Xoma Corporation Anticorps murins humains chimeriques a specificite aux antigenes du vih
US5346989A (en) * 1990-08-22 1994-09-13 Syntello Vaccine Development Kb Peptides for use in induction of T cell activation against HIV-1
WO1992005800A1 (fr) * 1990-09-27 1992-04-16 Syntello Vaccin Development Kb Peptides destines a etre utilises dans la vaccination et dans l'induction d'anticorps neutralisants pour lutter contre le virus d'immunodeficience humaine
AU650911B2 (en) * 1990-09-27 1994-07-07 Syntello Vaccine Development Kb Peptides for use in vaccination and induction of neutralizing antibodies against human immunodeficiency virus
US5589175A (en) * 1990-09-27 1996-12-31 Syntello Vaccine Development Kb Peptides for induction of neutralizing antibodies against human immunodeficiency virus
WO1992021377A1 (fr) * 1991-06-03 1992-12-10 Syntello Inc. Peptides a utiliser pour induire l'activation de lymphocytes t contre le vih-1

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