WO1988000471A1 - Composition et procede d'immunisation contre des agents pathogenes viraux du sida et de l'arc - Google Patents

Composition et procede d'immunisation contre des agents pathogenes viraux du sida et de l'arc Download PDF

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Publication number
WO1988000471A1
WO1988000471A1 PCT/US1987/001733 US8701733W WO8800471A1 WO 1988000471 A1 WO1988000471 A1 WO 1988000471A1 US 8701733 W US8701733 W US 8701733W WO 8800471 A1 WO8800471 A1 WO 8800471A1
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peptide
aids
htlv
polyamide resin
iii
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PCT/US1987/001733
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English (en)
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Patrick Kanda
Ronald C. Kennedy
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Southwest Foundation For Biomedical Research
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Priority to DK149188A priority Critical patent/DK149188A/da

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A61K39/21—Retroviridae, e.g. equine infectious anemia virus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/58—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained by reactions only involving carbon-to-carbon unsaturated bonds, e.g. poly[meth]acrylate, polyacrylamide, polystyrene, polyvinylpyrrolidone, polyvinylalcohol or polystyrene sulfonic acid resin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6093—Synthetic polymers, e.g. polyethyleneglycol [PEG], Polymers or copolymers of (D) glutamate and (D) lysine
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16122—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2740/00—Reverse transcribing RNA viruses
    • C12N2740/00011—Details
    • C12N2740/10011—Retroviridae
    • C12N2740/16011—Human Immunodeficiency Virus, HIV
    • C12N2740/16111—Human Immunodeficiency Virus, HIV concerning HIV env
    • C12N2740/16134—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

  • the present invention relates to a composition and method for vaccination against acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC).
  • the present invention relates to a polyamide resin-synthetic peptide conjugate which can be used in a method of immunization against the viral causative agents of AIDS and ARC and in a diagnostic assay for AIDS and ARC.
  • AIDS was first discovered as a severe immune deficiency which resulted in reports of opportunistic infections occurring among male homosexuals (see Gottling, M.S., et al., 305 N. Engl. J. Med. 1425-1431 (1981) and Masur, H., et al., 305 N. Engl. J. Med. 1431-1438 (1981)).
  • the incidence of this new human disease, named "acquired immunodeficiency syndrome" (AIDS) is rapidly growing.
  • sexual transmission appears to be the primary mode of transfer, a number of cases in which the disorder was transferred by blood transfusion have been reported (see Gottlieb, et al., supra).
  • the etiologic agent of this disease has been shown to be a human retrovirus, known variously as human T lymphotropic virus type III (HTLV - III), lymphadenophathy-associated virus (LAV) (Barre-Sinoussi, F., et al., 220 Science 868-871 (1983)), or AIDS-associated retrovirus (ARV).
  • HTLV - III human T lymphotropic virus type III
  • LAV lymphadenophathy-associated virus
  • ARV AIDS-associated retrovirus
  • HTLV-III - specific antibodies in the serum of most patients with AIDS or ARC.
  • the predominant antigens recognized by antibodies in sera obtained from AIDS patients and from hemophiliacs are associated with the envelope glycoproteins.
  • the most immunogenic proteins of the human T lymphotrophic viruses, HTLV-I and HTLV-II are cell surface-expressed glycoproteins (Chen, I.S. et al., 305 Nature (London) 502 (1983)).
  • the envelope (env) gene product of HTLV-III is synthesized as a polyprotein precursor and is subsequently glycosylated within infected cells. That glycosylated polyprotein, with an estimated molecular weight of 160 Kd (gp 160), is processed into an amino terminus subunit gp 120 and a carboxyl transmembrane subunit, gp 41.
  • the gp 41 subunit is one of the predominant polypeptides in purified virus preparations.
  • Antibodies from AIDS and ARC patients contain viral neutralizing activity; however, infection presumably occurred in those patients prior to the development of neutralizing antibody, and whether the induction of neutralizing antibody prior to infection would result in protective immunity is unknown.
  • retroviruses The general notion with retroviruses is that the antigenic determinants or epitopes associated with the induction of neutralizing antibodies are associated with the glycoprotein envelope (see Holden, H.T. and T. Taniyama, 150 J. Exp. Med. 1367 (1979) and Flyer, D.C. et al., 305 Nature (London) 815 (1983)). Until the present invention, that association had not been established for the AIDS associated viruses. As will be described, it has now been demonstrated that the gp 41, gp 120 and gp 160 envelope glycoproteins are the most immunogenic epitopes in virus-exposed individuals.
  • the present invention is premised upon the assumption that the critical epitopes involved in the induction of protective virus neutralizing antibody are associated with the two viral envelope glycoprotein subunits, gp 120 and gp 41.
  • the present invention is also based on the use of polypeptide portions of those immunogenic subunits to induce an immunogenic response to the intact causative agents of AIDS and ARC when conjugated to the solid phase resin on which that portion of the immunogenic subunit was synthesized.
  • Those polypeptide subunits are conveniently synthesized on solid phase resins. Solid phase peptide synthesis is a valuable tool for investigating the structure and mechanism of action of proteins and peptides (proteins and peptides are collectively referred to herein as "protides").
  • polystyrene based resins are most commonly used as supports in solid phase protide synthesis, their relatively hydrophobic character in comparison to the polar organic media required to solubilize reactants can be problematic in protide chain assembly. Such media may freely solvate the growing protide, yet incompletely swell the polystyrene matrix. Within the polymer lattice, impaired diffusion of reagents and steric hindrance can contribute to lowered efficiency during coupling cycles, which, on a repeated basis, lowers final yields appreciably. During the early stages of assembly, when the resin to protide mass ratio is high and the physical properties of the support dominate, this lowered efficiency is particularly acute.
  • That polyamide resin as the amino methyl derivative, can accommodate synthetic schemes incorporating alternate protection strategies through selection of the appropriate linker molecule, which links the C-terminal residue to the support.
  • peptides synthesized on that resin must be separated from the resin after the synthesis is completed and then purified, both time-consuming steps which decrease the final yield of the protide.
  • a significant advantage of the composition and method of the present invention is that the protide can be used to induce an immunogenic response in an experimental animal without being separated from the resin on which it was synthesized and then purified.
  • the resin-protide conjugate thus synthesized can be used in a number of investigative applications.
  • Of particular interest to the present invention is the use of certain polyamide resin-peptide conjugates, specifically, conjugates including portions of the gp 120 and gp 41 subunits of the gp 160 envelope glycoprotein, as immunogens.
  • An object of the present invention is, therefore, to provide a composition of matter capable of inducing an immunogenic response to the viral causative agents of AIDS and ARC comprising a polyamide resin and a synthetic peptide, the synthetic peptide comprising a chain of amino acids having a sequence homologous to a portion of the amino acid sequence of the gp 120 or gp 41 envelope glycoprotein of HTLV-III, ARV or LAV and having a hydrophilic region therein.
  • a further object of the present invention is to provide a polyamide resin, and a method of preparing that polyamide resin, upon which those synthetic peptides are synthesized using solid phase synthetic methods to produce a conjugate which, when injected into an experimental animal, induces an immunogenic response to the viral causative agents of AIDS and ARC without separating the synthetic peptide from the resin before injection into the experimental animal.
  • Figure 1 is an artist's rendition of the plot of the hydrophilic averages for each residue against the amino acid sequence of the gp 160 precursor glycoprotein of the gp 120 and gp 41 env glycoproteins of HTLV-III, LAV and ARV generated by a computer program utilizing the Chou-Fasman predictive scheme for secondary structure.
  • Figure 2 is an actual computer plot of a segment of the amino acid sequence of the plot of Fig. 1.
  • Figure 3 is a schematic representation of the secondary structure of the amino acid sequence of the plot of Fig. 1 showing the differences between the secondary structure of the gp 160 precursor of HTLV-III, LAV and ARV.
  • Figure 4 is a graph of the optical density vs. the reciprocal dilution of the antiserum obtained from rabbits immunized with the gp 120 peptide 503-532 (Peptide 6 on Table II) showing the binding of the Peptide 6 by the rabbit antibodies by enzyme linked immunosorbent assay.
  • Fig. 4A shows binding of Peptide 6
  • Fig. 4B shows the binding of a control peptide (see Example 15).
  • Data from anti-peptide antisera from rabbit 1 is represented by a (•) and data from anti-peptide antisera from rabbit 2 is represented by a (0) .
  • Data from pre-immune sera from each rabbit is represented by a ( ⁇ ) and a ( ⁇ ) , respectively .
  • Figure 5 illustrates the growth of HTLV-III virus on the A3.01 cell line as determined by assaying reverse transcriptase (RT) activity (uptake of 3 H-TTP; see
  • FIG. 5A shows viral replication at a 10 -1 dilution
  • Fig. 5B was at a 10 dilution
  • Fig. 5C at
  • the present invention is based in part upon the assumption that it is the gp 120 and gp 41 subunits of the envelope glycoprotein which are the most immunogenic epitopes of the viral causative agents of AIDS and ARC. As will be described, the accuracy of that assumption has now been verified. It was next necessary to determine the sequence of the amino acids of the gp 120 and gp 41 subunits and to select the portions of those envelope glycoprotein subunits which represent the most likely antibody-binding sites. This selection was accomplished by means of computer modeling of the structure of the gp 120 and gp 41 subunits.
  • chains of amino acids were synthesized on a polyamide resin to duplicate the amino acid sequence at each of those sites.
  • the usual method of coupling a synthetic peptide to polystyrene based resins is through a benzyl ester derivative, and separation of the peptide from the resin is usually accomplished by either acidic or basic cleavage.
  • Benzyl esters are susceptible to several such methods of cleavage, but are also stable throughout the multiple deprotection, neutralization and coupling reactions which are characteristic of solid phase synthetic methods. Hydrazine has also been used to separate the protide from the resin (Kessler, W. and B.
  • the polyamide resin and method of the present invention requires no such separation and purification, thereby decreasing the amount of time required to accomplish the synthesis and raising the peptide yield.
  • the polyamide resin of the polyamide resin-peptide conjugate of the present invention is prepared by cross-linking a commercially available dimethylacrylamide monomer in aqueous solution using a diaminoalkane, preferably a diaminoalkane having alkenoyl groups at either end of the molecule, such as N,N'-bis-alkenoyl-diaminoalkane,
  • the cross-linker is either N,N'-bisacrylyl- 1,3-diaminopropane or
  • N,N'-bisacrylyl-1,3-diaminobutane prepared according to the method of Halpern and Sparrow (J.A. Halpern and J.T. Sparrow, "An Improved Procedure For the Synthesis of N,N'-bisaerylyldiaminoalkanes", 10 Synthetic Comm. 569 (1980)), hereby incorporated in its totality by this specific reference thereto.
  • the use of the propane analog is preferred because it yields a polymer of larger pore size and improved swelling properties during protide synthesis than the polymer obtained by use of the ethyl analog.
  • a functional monomer is included in the cross-linked resin.
  • the term "functional monomer” refers to those alkenyl amines which are used to anchor the C-terminal amino acid of a synthetic peptide to the resin.
  • the functional monomer when protected with the methylsulfonylethyloxycarbonyl (MSC) group (see Tesser, G.I. and I.C. Balvert-Geers, "The Methylsulfonylethyloxycarbonyl Group, A New And Versatile Amino Protective Function", 7 Int. J. Peptide Protein Res. 295 (1975)), is referred to as an MSC alkenyl amine.
  • MSC methylsulfonylethyloxycarbonyl
  • Those functional monomers are prepared by reaction of the commercially available chloride derivative with the alkenylamine, and the MSC protective group is subsequently removed with base. However, the MSC group is not required.
  • the polyamide resin is also prepared by simply adding an excess of the allylamine, followed by filtering or other method to remove the resulting fines.
  • the amount of functional monomer added is selected to yield a resin substitution of between about 0.1 mmol and about 0.5 mmol per gram of resin, and preferably in the range of about 0.2 mmol to about 0.4 mmol per gram of resin.
  • the initiator can be any of the initiators known to those skilled in the art such as a persulfate or riboflavin, and is preferably ammonium persulfate.
  • the aqueous phase refers to an organic phase which, when combined with the aqueous phase and stirred, results in a suspension from which the resin is obtained.
  • organic phase comprises a mixture of hexane and carbon tetrachloride.
  • the emulsifier is added during the stirring to allow for the formation of beads of uniform size.
  • the emulsifier can be any detergent known to those skilled in the art, and in a presently preferred embodiment, is either sorbitan sesquioleate, sorbitan monolaurate or sorbitan monodecanoate.
  • the amount of detergent added is adjusted to give a spherical resin of approximately uniform size.
  • a decrease in the amount of detergent results in an emulsion which yields increased amounts of larger, amorphous material, which could contribute to a reduction to the internal growing chains of amino acids.
  • An increase in the amount of detergent increases the amount of fine material, which is difficult to remove without the loss of significant amounts of the resin. Those fines clog the reaction vessels of the peptide synthesizer as well as the associated lines and valves.
  • a promoter is then added to promote the polymerization of the monomers in the suspension, resulting in the formation of beads of the polyamide resin of the present invention.
  • a number of promotors are known to those skilled in the art, but particular success in preparing the polyamide resin has been obtained with N,N,N',N'-tetramethylethylenediamine (TEMED).
  • TEMED N,N,N',N'-tetramethylethylenediamine
  • the resulting beads are then filtered and washed, the MSC group (if present) is removed with base, and the beads are dried.
  • the beads may then be sifted through a mesh sieve to insure relatively uniform size. Overall yields using the method of the present invention ranged from about 87% to about 94% from starting monomers.
  • the resulting aminomethyl, cross-linked polydimethylacrylamide resin when conjugated to the synthetic peptide, provides maximum exposure of the peptide in an aqueous solution, and the resin-polymer backbone does not restrict the peptide conformationally.
  • the exposure of the peptide is the result of the ability of the polyamide resin to swell to many times its dry bed volume when highly solvated by water.
  • the synthetic peptides are synthesized on the beads by coupling to a linker which is attached to the resin with an activator.
  • linker refers to a linking group which links the carboxyl group of the first amino acid of the synthetic peptide to the polymeric resin.
  • this linker is an oxyalkyl benzoic acid (OBA) to which an amino acid residue is coupled to serve as the first amino acid in the peptide chain. Because the OBA linker is used to attach the C-terminal amino acid to the polyamide resin, anhydrous hydrogen fluoride can be used to remove the side chain protecting groups from the peptide without significant loss of the synthesized peptide from the resin.
  • OBA oxyalkyl benzoic acid
  • the amino acid of choice is glycine, which is protected with the t-butyloxycarbonyl (t-BOC) protecting group, but it will be understood by those skilled in the art who have the benefit of this disclosure that the amino acid could be any amino acid, particularly, the amino acid which is the first amino acid in the peptide to be synthesized, and that other protecting groups are equally suitable.
  • the glycine residue serves the additional function of a spacer between the peptide and the resin-polymer backbone.
  • the BOC-glycyl-4- (oxymethyl) benzoic acid which is the presently preferred linker was prepared by a modification of the method described by Mitchell, et al. (Mitchell, A.R., S.B.H. Kent, M. Engelhard and R.B. Merrifield, "A New Synthetic Route to tert-butyloxycarbonylaminoacyl-4-(oxymethyl) phenylacetamidomethyl-resin, An Improved Support of Solid-phase Peptide Synthesis", 43 J. Org. Chem. 2845 (1978)), which is incorporated herein in its totality by this specific reference thereto.
  • the activator used to couple the linker to the polyamide resin prepared as described above is diisopropyl carbodiimide and 4-dimethylaminopyridine, but it will be understood by those skilled in the art that other activators such as dicyclohexylcarbodiimide and 4-methylpyrrolindinopyridine are equally suitable for such a purpose.
  • the polyamide resin-protide conjugates of the present invention are used for a number of purposes, including in vitro assays for the presence of antibodies to HTLV-III, ARV or LAV, inducing an immunogenic response to the viral causative agents of AIDS or ARC in experimental animals, or mapping antigenic determinants on the viral causative agents of AIDS or ARC.
  • an in vitro assay is conducted by crushing a beaded polyamide resin-synthetic peptide conjugate with a mortar and pestle and absorbing the crushed conjugate onto a solid phase such as a microtiter test plate with neutral pH buffer.
  • Serum or other body fluid suspected of containing an antibody against the viral causative agents of AIDS or ARC is then incubated with the absorbed conjugate, unbound antibodies are removed by washing, and the bound antibodies are detected by enzyme linked immunosorbent assay, biotin-avidin amplified assay or other detection methods such as are known in the art.
  • the polyamide resin-synthetic peptide conjugate can also be used to map antigenic determinants on the viral causative agents of AIDS or ARC by simply removing a portion of the polyamide resin at intervals during the synthesis of the peptide, deprotecting the peptide, and testing each removed portion in serial fashion to determine that point in the synthesis at which the peptide binds to an antibody specific for the viral causative agents of AIDS and ARC. This method is made possible by the elimination of the separation and purification steps required in other synthetic methods.
  • the conjugate can also be tested for its ability to bind antibody by crushing and absorbing to a solid support such as a microtiter test plate and assayed as described above. Separation of the peptide from the resin and purification of the peptide is not required for such an assay.
  • the polyamide resin-protide conjugates are also useful as an immunogen against the viral causative agents of AIDS or ARC.
  • the conjugate is used directly for immunization of experimental animals with or without an adjuvant.
  • the term "experimental animal”, as used herein, refers to any animal capable of an immune response.
  • the experimental animals of primary interest are mammals, but an immunogenic response can be induced in other experimental animals such as birds using the method of the present invention.
  • an immune response specific for the viral causative agents of AIDS and ARC was induced by immunization of rabbits using a conjugate comprised of a synthetic peptide with a sequence corresponding to the protein coat of the HTLV-III virus and the polyamide resin.
  • amino acid sequence of the gp 120 and gp 41 subunits was determined by prediction based upon the nucleotide sequence of HTLV-III and the verification of those sequences by analysis of the sequence of the
  • envelope glycoproteins (and their precursor, gp 160) was obtained by screening serum samples from AIDS and ARC patients to identify those with antibodies against HTLV-III by indirect cell membrane immunofluorescence (MIF) using the H9/HTLV-III cell line and by radioimmunoprecipitation and sodium dodecylsulfate-polyacrylamide gel electrophoresis
  • MIF indirect cell membrane immunofluorescence
  • the next step was to synthesize a polypeptide with the same amino acid sequence (or a sequence which is similar enough so as to be treated in the same manner by the antibody which binds with that epitope) as that region of the glycoprotein.
  • the synthesis was carried out by the solid-phase methodology described above. A total of ten synthetic peptides were synthesized, each selected on the basis of the above-described tests for predicted immunogenicity. The amino acid sequences of each of those synthetic peptides is given in Table II.
  • the ten synthetic peptides are then used to induce an immune response in rabbits by injecting the rabbits with the polyamide resin-synthetic peptide conjugate.
  • the rabbits were also injected with synthetic peptide separated from the resin on which it was synthesized and then coupled to a carrier.
  • the antibody titer of the rabbit sera was tested by the ability of the antibody to bind with the peptide conjugated to bovine serum albumin (BSA). Those results were confirmed by conducting inhibition studies in which the inhibition of the binding of the rabbit anti-peptide to the peptide-BSA was measured.
  • BSA bovine serum albumin
  • the rabbit anti-peptide antibodies were then examined for their ability to recognize the native proteins associated with HTLV-III.
  • An HTLV-III infected T-cell line labelled with 35 [S]-cystine was used for immunoprecipitation to determine whether the anti-peptide sera would bind any radioactively labelled
  • HTLV-III native proteins Autoradiography with SDS-PAGE confirmed that the rabbit anti-peptide antibodies specifically precipitated a single protein which corresponded to the gp 160 precursor envelope glycoprotein gp 160 of HTLV-III.
  • the precursor gp 160 product is cleaved to yield the major gp 120 envelope glycoprotein and gp 41, the transmembrane glycoprotein.
  • the gp 41 envelope subunit does not radioactively label to the same degree with 35 [S]-cystine as the amino end of the precursor gp 160 glycoprotein, and was not detected by immunoprecipitation. However, when when when
  • the anti-peptide antibodies thus generated were then tested to determine whether they were capable of neutralizing the viral causative agents of AIDS or ARC. That determination can be made in a number of ways.
  • the polyamide resin-peptide conjugate is crushed with a mortar and pestle, and a suspension of the resin is made in buffered saline. That emulsion of peptide is absorbed to the solid phase of microtiter plates, and nonspecific sites are blocked with 10% normal goat serum.
  • the binding of rabbit antibodies to the peptide is detected by using biotin-goat antibody to rabbit IgG and avidin conjugated horseradish peroxidase.
  • Peroxidase activity is determined using 1,2'-azino-di(3-ethyl-benzthiazoline-sulfonic acid) and H 2 O 2 as the substrate.
  • a resin bound peptide corresponding to a hepatitis B surface antigen sequence serves as a control.
  • the binding of the rabbit anti-peptide is quantified spectrophotometrically at 410 nm with a plate reader.
  • the neutralizing ability of the anti-peptide antibodies was tested by incubating purified virus and rabbit anti-peptide antibodies with infected T-helper cell lines, then examining the lysed cells by Western transfer and immunoprecipitation for the presence of the virus.
  • the neutralizing ability of the rabbit antibodies to the polyamide resin-synthetic peptide conjugate was assessed by measuring the reduction of reverse transcriptase (RT) activity. The results were verified by radioimmunoprecipitation.
  • the most immunogenic synthetic peptides is then used in a diagnostic assay for AIDS and ARC and as a vaccine.
  • the preferred method involves the detection of antibody against the viral causative agent of AIDS and/or ARC.
  • That assay is conducted, for instance, by coating an insoluble matrix such as a column of polystyrene beads or micro-well test plate with a synthetic peptide or a synthetic peptide coupled to a carrier protein (i.e., bovine serum albumin) containing the amino acid sequence associated with the epitope(s) of one of the viral causative agents of AIDS or ARC.
  • a carrier protein i.e., bovine serum albumin
  • the insoluble matrix is coated with a number of different synthetic peptides (a "cocktail") containing the amino acid sequence of several epitopes.
  • the polyamide resin-synthetic peptide conjugate is crushed with mortar and pestle and absorbed onto a solid phase as described above.
  • a sample of biological fluid from the suspected patient is incubated with the synthetic peptide-coated matrix to immunocapture the predetermined antibody.
  • the resultant matrix, separated from the uncaptured sample is then incubated with a quantity of biotin-labeled antibody directed to the species of the predetermined antibody (e.g., anti-human antibodies would be the predetermined antibody if the body fluid is taken from a human patient) sufficient to bind a measurable number of human antibodies, if present.
  • the resultant matrix is then incubated with a quantity of labeled avidin, preferably avidin labeled with an enzyme such as alkaline phosphatase, sufficient to bind a measurable number of antibodies, if present.
  • the resultant matrix is separated from uncaptured avidin and a label detected and/or preferably quantified by adding the substrate which is specific for that enzyme to thereby determine indirectly the presence of antibody to AIDS virus in the sample.
  • the antibody could also be labeled with an enzyme directly, in which case the matrix is incubated with an enzyme-reactive substrate, and the change in the substrate, e.g., a color change or fluorescence emission is detected.
  • the binding pair formed by the antigen and antibody or the enzyme and substrate will be referred to as the "ligand” and "anti-ligand” of the specific binding pair.
  • a diagnostic assay can also be conducted for detection of the antigen rather than the predetermined antibody.
  • the solid phase matrix is coated with antibodies to the viral causative agents of AIDS, i.e., the antibodies produced by immunization with a synthetic peptide or polyamide resin-synthetic peptide. conjugate (or, preferably, several peptides) such as the peptides of the present invention.
  • the sample of biological fluid from a patient suspected of having been infected with the AIDS virus is then added to the matrix, followed by the addition of biotin-labeled antibody, where the antibody is an antibody which binds to the AIDS virus produced in the same way as discussed above.
  • the avidin-labeled enzyme is then added, followed by the substrate specific for the enzyme, and the color change or fluorescence emission is detected. Either of these assays is also conducted as an inhibition assay where, instead of adding biotin-labeled antibody to the AIDS virus to the bound antigen, a biotin-synthetic peptide or biotinpolyamide resin-synthetic peptide conjugate is added.
  • polyamide resin-synthetic peptide conjugate of the present invention As a vaccine against the viral causative agents of AIDS, approximately 100 to 1000 micrograms of synthetic peptide, or several synthetic peptides, prepared as a polyamide resin-synthetic peptide conjugate according to the teachings of the present invention, is administered to an individual with an adjuvant.
  • the synthetic peptides were also administered, after separation from the resin on which they were synthesized, coupled to a carrier.
  • Appropriate carriers include the toxoid components, any one of several large protein-containing substances which are foreign to the animal to be injected, any of several small peptide preparations which have demonstrated adjuvant activity and which behave as a carrier, or liposomes.
  • the toxoid components can be tetanus toxoid or diptheria toxoid.
  • KLH Keyhole limpet hemocyanin
  • BSA BSA
  • the small peptide preparations with demonstrated adjuvant activity which also act as a carrier include muramyldipeptide, murabutidine, and the polyamino acids such as poly-L-glutamic acid or poly-L-lysine.
  • Approximately 10 to 100 molecules of synthetic peptide are complexed to each molecule of carrier using a heterobifunctional cross-linker such as m-maleimidobenzyl-N-hydroxysuccinimide ester (MBS) (Liu, F.T., et al., 18 Biochemistry 690 (1979), Green, N. et al., 28 Cell 477 (1982)), glutaraldehyde, a carbodiimide, succinyl anhydride or N-succinimidyl-3-[2-pyridyldithio]-propionate.
  • MBS m-maleimidobenzyl-N-hydroxysuccinimide ester
  • Suitable adjuvants include alum (aluminum hydroxide) and any of a number of additional adjuvants such as are known to those skilled in the art.
  • alum aluminum hydroxide
  • Both the polyamide resin-peptide conjugate and the carrier-synthetic peptide complex are administered in a pharmaceutically acceptable diluent such as distilled water, phosphate buffered saline, citrate buffer or any neutral pH buffer, i.e. a buffer with a pH of between about 6 and about 8.
  • the polyamide resin-synthetic peptide conjugate of the present invention is also used to screen putative vaccine candidates against AIDS and/or ARC. Such screening is best conducted by coating an insoluble matrix with crushed beads of the polyamide resin-synthetic peptide conjugate as described above. The vaccine candidate is then incubated with antibodies against the peptide (with or without biotin) such as a 1:1000 dilution of IgG-rabbit anti-peptide-biotin antibody.
  • biotin labeled antibody If biotin labeled antibody is used, the avidin-enzyme conjugate is added (if no biotin is used, add biotin-labeled anti-species (such as biotin-labeled goat anti-rabbit IgG) antibody, then add avidin-enzyme), the substrate is then added and the reaction detected.
  • biotin-labeled anti-species such as biotin-labeled goat anti-rabbit IgG
  • the polyamide resin-synthetic peptide conjugate of the present invention is also used to serotype viral isolates from AIDS or ARC patients. Serotyping is conducted in the same manner as described above for screening vaccine candidates, because in both cases, the anti-peptide antibody must bind with the intact AIDS viral causative agent. However, in the case of the serotyping of the viral isolate, a portion of the isolate is added, in serial fashion, to a number of bound anti-peptide antibodies, each antibody being specific for a different polyamide resin-synthetic peptide conjugate and having been bound to a separate insoluble matrix.
  • Example 1 Maintenance and Radioactive Labeling of HTLV-III Infected Cells
  • HTLV-III producing cell lines H-9 and MOLT-3
  • RPMI-1640 supplemented with 20% fetal bovine serum, 2 mM glutamine, non-essential amino acids and 0.1% NaHCO 3 (maintenance medium).
  • Cell cultures were labeled by transferring cells from maintenance medium to cystine and glucose deficient medium for 1 hheour before adding 35 [S]-cystine (150 ⁇ Ci/ml) and 3[H] -glucosamine (20 ⁇ Ci/ml for 24 hr).
  • Cells were separated from tissue culture supernatants by low speed centrifugation (1,000 x g for 10 minutes).
  • Serum samples taken from subjects who came to a community health clinic in a high-risk area for AIDS and ARC and to hospitals in that area during 1983 and 1984 were screened for antibodies to HTLV-III by indirect cell membrane immunofluorescence (MIF) using the H9/HTLV-III cell line as described by Essex, et al., 320 Science 859 (1983). Briefly, this method involves separating the cells from the media as described in Example
  • HTLV-III glycoproteins were incubated with lentil lectin Sepharose 4B for four hours and then eluted with 0.2 M methyl mannoside. The resulting proteins were then immunoprecipitated with HTLV-III reference serum, and the precipitates bound to protein A-Sepharose were dissociated from antibody by boiling for two minutes in the presence of 0.1% SDS and 0.15 M sodium citrate pH 5.5. Equal portions were then incubated for three hours at 37°C in the presence or absence of 0.25 ⁇ g of endoglycosidase H.
  • the reaction was terminated by the addition of five volumes of cold 95% ethanol, and the proteins were precipitated overnight at -20°C.
  • the samples were then centrifuged at 12000 x g for 15 minutes and the proteins were reconstituted with electrophoresis sample buffer, boiled for three minutes, and subjected to electrophoresis.
  • Samples from four antibody-positive AIDS patients precipitated proteins of about 120 kD, 160 kD and 41 kD. Similar results were obtained with two antibody-positive ARC patients, and with two antibody-positive healthy homosexual males. No proteins of related sizes were detected in sera from antibody-negative healthy homosexual males or with sera from apparently healthy laboratory workers.
  • the predicted amino acid sequences of the gp 160 precursor glycoprotein from the three viral isolates HTLV-III, LAV and ARV were run through a computer program which utilizes the parameters and hydrophilic values arrived at by Hopp, T.P., and K.R. Woods (20 Mol. Immunol. 483-489 (1983)).
  • the computer program was written in Apple BASIC.
  • the program was written with the ability to save the amino acid sequence to disk in a format which is compatible with the Chou-Fasman predictive scheme (Chou, P.Y. and E. D. Fasman, 13 Biochemistry 222 (1974)).
  • the hydrophilicity program calculates the hydrophilic averages over a hexapeptide length, thereby increasing the accuracy of the predictions.
  • Fig. 1 is actually an artist's rendition of the computer graphical output of the hydrophilicity plots from the three viral causative agents of AIDS/ARC which have been characterized.
  • the highest peak (most hydrophilic) is shown in a similar area for all three sequences, with the maximum hydrophilic index occuring at residues 739, 744, and 738 for HTLV-III, LAV and ARV respectively.
  • the second highest hydrophilic region centers around the amino acid residues 653-659 just to the amino terminal side of peak 1.
  • the third highest hydrophilic region was found to be in close proximity to peak 1, centered around amino acid residues 733-739 for each of the three glycoproteins.
  • FIG. 2 An actual computer graph output of a segment of the HTLV-III sequence is depicted in Fig. 2. Due to the length of the entire HTLV-III sequence, only a segment is shown. A proline residue is shown graphically as a "P". Two or more aromatic amino acids in a row within the sequence are depicted as an "O". The presence of aromatic amino acids within a given sequence is indicative of regions that possess a high degree of potential for hydrogen bonding. Thus, hydrogen bonds may act to influence the overall confirmation of the protein. These data indicate that these regions are likely to be exposed on the surface of the glycoprotein. The predicted secondary structure of the HTLV-III glycoprotein, as determined by the Chou-Fasman predictive scheme, is depicted in Fig. 3.
  • Butyloxycarbonyl-S-4-methylbenzyl-L-cystine coupled to polystyrene using dicyclohexylcarbodimide with a catalytic amount of 4-N,N-dimethylaminopyridine was used as the solid-phase support for the synthesis.
  • the four amino groups were protected with tert-butyloxycarbonyl (t-BOC) and the side chain protecting groups were as follows: benzyl ether for the hydroxyl of serine, dichlorobenzyl ether for the phenolic hydroxyl of tyrosine, and the ⁇ and ⁇ benzyl-esters were used for the carboxyl groups on glutamic acid and aspartic acid, respectively.
  • Trifluoroacetic acid (40% in CH 2 CL 2 ) was used to remove t-BOC and the resulting salt was neutralized with N,N-diisopropylethylamine (10% in CH 2 CL 2 ).
  • Diisopropylcarbodiimide was used to couple the t-BOC amino acids.
  • the specific steps of the synthesis are published in Sparrow, J.T., 41 J. Org. Chem. 1350 (1976), hereby incorporated in its entirety by this specific reference thereto.
  • the protecting groups were removed and the peptide was cleaved from the resin at 0°C with anhydrous hydrogen fluoride containing 10% anisole and 1% ethanedithiol as scavengers.
  • the hydrogen fluoride reagent was removed under vaccuum at 0°C and the peptide was then precipitated and washed with anhydrous ether.
  • the solvent was evaporated to 15°C and the peptide was again precipitated with ether.
  • the ether was decanted after centrifugation and the pellet was dissolved in 5% acetic acid with 6 M guanadine HCl.
  • That solution was desalted on a BioGel P2 column equilibrated in 5% acetic acid and the peptide containing fractions were pooled and lyophilized.
  • a cysteine residue was then added to the carboxyl terminus of the peptide to provide a functional -SH group for the coupling of the peptide to carrier proteins.
  • a glycine residue was added after the cysteine to provide a spacer amino acid between the coupled cysteine residue and the amino acid sequences analogous to gp 160.
  • a tyrosine residue was added to the amino terminus for radioactive labelling with 125Iodine to determine peptide-to-carrier protein coupling efficiency and to identify the peptide during purification by adsorbance at 278 nm.
  • the numbering system used throughout this specification is taken from the numbers assigned to the amino acid residues of HTLV-III as set out in Ratner, L., et al., "Complete nucleotide sequence of the AIDS virus, HTLV-III," 313 Nature 277-284 (1985), hereby incorporated in its totality by this specific reference thereto.
  • the sequence of the polyamide resin-Peptide 6 conjugate was as follows:
  • N,N'-bisacrylyl-1,3-diamino ⁇ ropane was prepared according to the method set out in Helpern and Sparrow, supra. Briefly, diaminopropane (Aldrich) was dissolved in acetonitrile and added dropwise to an acrylyl chloride-acetonitrile solution at 4°C, allowed to warm to room temperature and stirred. The diaiminopropane dihydrochloride was removed by filtration, washed with warm acetonitrile, and the combined filtrates were concentrated in vacuo. N,N'-bisacrylyl-1,3-diaminopropane was crystallized at 4°C overnight and the resulting plates filtered and dried in vacuo.
  • the suspended emulsion was stirred for two hours under nitrogen atmosphere.
  • the resultant beaded material was then filtered and washed sequentially with water (one liter) methanol (one liter), a mixture of dioxane:methanol:2 N NaOH (14:5:1, two liters, to remove MSC group), water (two liters), 1 N HCl (two liters), water (two liters), and then methanol (two liters).
  • the resin was defined by suspension in methanol and decanting (3x). After swelling in methylene chloride (Baker HPLC grade), the resin was shrunk in hexane and dried in vacuo. Large amorphous material was removed by sifting the resin through an 80 mesh (180 micron) sieve.
  • the degree of functionalization was checked by coupling BOC-alanine to 100 mg of the resin using diisopropylcarbodiimide as activator and 4-dimethylaminopyridine (recrystallized from ethyl acetate) as catalyst.
  • Amino acid analysis showed a substitution of 0.15 to 0.35 mmol/g resin, depending on the lot, and resins were prepared with as little as about 0.1 and as much as about 0.5 mmol/g resin depending upon the amount of allylamine added.
  • the loaded resin gave no detectable staining with picryl-sulfonic acid, indicating the absence of unreacted free amine.
  • the beads When swollen in methylene chloride, the beads occupied about 2.5 times their dry bed volume.
  • dimethylformamide or an aqueous solution the beads occupied approximately four and six times their dry bed volume, respectively.
  • the linker BOC-glycyl-4-(oxymethyl) benzoic acid was prepared by modification of the method of Mitchell, et al., supra. Briefly, the 4-(bromomethyl) benzoic acid phenylacylester was prepared by dissolving 10.3 ml redistilled diisopropylethylamine and 12.05 g (60.6 mmol) bromoacetophenone in 450 ml ethyl acetate. 4- (bromomethyl) benzoic acid (13.89 g, 60.6 mmol) was added in seven equal portions over a three hour period to the stirred solution at 40-50°C. Stirring was continued for two more hours at room temperature.
  • the 4-(bromomethyl) benzoic acid phenylacylester was converted to BOC-glycyl-4-(oxymethyl) benzoic acid by dissolving BOC-L-glycine (25 mmol, 4.38g) in 15 ml methanol and titrating to neutrality with tetramethylammonium hydroxide (25% in methanol). Solvent was removed azeotropically with chloroform in vacuo, and the salt dissolved in 150 ml acetonitrile. To the stirred solution was added 5.8 g (17.5 mmol) of the 4-(bromomethyl) benzoic acid phenacyl ester prepared as described.
  • Amino acid analysis was performed using either (1) a Beckman Model 119 amino acid analyzer following either a two hour hydrolysis (12 N HCl:propionic acid, 1:1, 135°C) or 24 hour hydrolysis (6 N HCl, 110°C) of resin bound peptides or (2) a Beckman Model 7300 amino acid analyzer following a two hour hydrolysis (12 N HCl:propionic acid, 1:1, containing 0.05% phenol at 135°C.
  • the results of the amino acid analysis are set out in Table III.
  • Peptides 1, 2, 7, and 10 were synthesized in the same manner on the resin to give the corresponding polyamide resin-peptide conjugates.
  • Synthetic peptide 4 (see Table II) was conjugated via the -SH group on the cysteine residue to the amino groups on Keyhole limpet hemacyanin (KLH) (for immunization of rabbits) and bovine serum albumin (BSA) (for assaying anti-peptide activity) using a heterobifunctional cross-linker, M-maleimidobenzyl-N- hydroxysuccininmide ester (MBS).
  • KLH Keyhole limpet hemacyanin
  • BSA bovine serum albumin
  • MVS M-maleimidobenzyl-N- hydroxysuccininmide ester
  • Two rabbits were each immunized with 100 ug per dose of Peptide 4-KLH complex, prepared as described above, emulsified in Freunds incomplete adjuvant.
  • the rabbits received one intramuscular injection every two weeks, for a total of three injections, and serum was obtained following each immunization.
  • a solid phase radioimmunoassay was used to titrate the rabbit anti-peptide antisera. Briefly, 200 ng of Peptide 4 coupled to BSA prepared as described in Example 11 was adsorbed to the wells of polyvinyl microtiter plates, and incubated overnight at 4°C. Following the addition of 10% normal goat serum (NGtS) to block nonspecific sites, the rabbit anti-peptide antisera diluted in 10% NGtS was added and incubated 2 hours at 37°C. Antisera was obtained 14 days after each immunization.
  • NtS normal goat serum
  • microtiter wells were washed with Tween 20 phosphate buffered saline (T-PBS) and 125 [I] goat-anti-rabbit gamma globulin (approximately 500,000 cmp in 50 ⁇ l) was added. Following incubation for 1 hour at 37°C, the wells were washed of excess radioactivity with T-PBS , and counted in a gamma counter. All volumes were 50 ⁇ l and the anti-peptide titers shown in Table IV are expressed as the reciprocal of the endpoint titer dilution (the highest dilution of antisera that gave cpm above the preimmune rabbit sera).
  • the end point titers were based on fivefold dilutions and represent the mean of triplicate values.
  • the specificity of the antibody response was shown by the inability of the anti-peptide sera to bind the control peptide conjugated to BSA.
  • the HTLV-III peptide 728-745 (Peptide 3) completely inhibited (100%) the binding of the rabbit anti-peptide to peptide-BSA.
  • the two rabbits also produced high antibody titers to KLH; however, rabbit anti-KLH did not bind peptide-BSA.
  • Example 11 or a hepatitis B surface antigen resin bound peptide produced by the method of Example 5.
  • HTLV-III was examined as follows. MOLT-3, an HTLV-III infected T-cell line, was labeled with 35 [S]-cystine and used for immunoprecipitation as described in Example 2, above, to determine whether the anti-peptide sera would bind any radioactively labeled HTLV-III native proteins.
  • the rabbit anti-peptide antibody from rabbits immunized with the carrier-peptide 4 conjugate prepared as described in Example 11 specifically precipitated a single protein of approximately 160,000 daltons as shown by autoradiographs of SDS-PAGE. This protein is the precursor envelope glycoprotein gp 160 of HTLV-III. No reactivity to HTLV-III proteins was demonstrated when preimmune rabbit sera was used in the immunoprecipitation experiments.
  • the rabbit anti-peptide failed to recognize the gp 120 envelope subunit that is detected with 35 [S]-cystine labeled
  • MOLT-3 cells when human antisera from AIDS patients is used in immunoprecipitation.
  • the gp 41 envelope subunit does not radioactively label to the same degree with
  • HTLV-III infected MOLT-3 cells were double llaalbeled by the addition of both 35 [S]-methionine and 35 [S]-cystine.
  • the glycoprotein populations present in those double cystine-methionine labeled lysates were then enriched by affinity chromotography on lentil-lectin columns as described in Example 2, above.
  • Both gp 160 and gp 41 glycoproteins was observed when the rabbit anti-peptide sera were reacted with those glycoprotein enriched fractions when analyzed by the radioimmunoprecipitation experiment described in Example 2, above.
  • the nitrocellulose sheets were incubated with 100 ml of 5% w/v non-fat dry milk rehydrated in PBS containing 0.001% w/v methiolate and 0.0001% v/v Antifoam A (Sigma) for 30 minutes at room temperature. Serial dilutions of sera obtained from the rabbits immunized with Peptide 4 were then incubated with the nitrocellulose sheets for 1 hour at 37°C. Nitrocellulose sheets were then washed with 100 ml of Tween 20 phosphate bufferred saline (T-PBS).
  • T-PBS Tween 20 phosphate bufferred saline
  • Biotinylated goat anti-human IgG (5 jug/ml) was then incubated with the nitrocellulose sheets for 1 hour at 37°C in order to detect the binding of the rabbit antipeptide antibodies.
  • Nitrocellulose sheets were washed again with T-PBS followed by the addition of 1 ⁇ g/ml of avidin-labeled horse radish peroxidase (Av-HRP) for 20 min at room temperature.
  • Av-HRP avidin-labeled horse radish peroxidase
  • 100 ml of a peroxidase chromagen: substrate solution (0.2 mg/ml of O-dianisdine in PBS plus 1 nl/ml of 30% H 2 O 2 ) was added to the nitrocellulose membranes until precipitates were observed on the membrane (10-15 min.).
  • the peroxidase catalyzed reaction terminated by washing the nitrocellulose sheets in 2% SDS in water.
  • Controls for the Western transfer assay include the use of normal human sera and a side by side comparison of the reactivity of the antisera with infected and uninfected cell lysates. Binding with the gp 41 protein was observed, as well as with the gp 120 subunit.
  • An enzyme-linked immunosorbent assay was used for detection of human antibodies against the viral causative agents of AIDS and ARC.
  • the polyamide resin-peptide 6 conjugate prepared as described in Example 5 was crushed with a mortar and pestle and a suspension of crushed conjugate was made in borate buffered saline (BBS), pH 8.0.
  • BBS borate buffered saline
  • One hundred microliters of that emulsion containing approximately 10 ⁇ g of peptide 6 (weight basis as calculated by amino acid composition) was absorbed to the solid phase of Dynatech Immunolon II microtiter plates in (BBS), pH 8.0, for eight hours at 4°C.
  • Nonspecific sites were blocked with 10% normal goat serum (NGtS) in Tween 20 phosphate buffered saline (T-PBS) and then washed with T-PBS.
  • NtS normal goat serum
  • T-PBS Tween 20 phosphate buffered saline
  • the resin-bound hepatitis B surface antigen described in Example 13 served as a control.
  • the results are shown in Fig. 4, in which the results for the polyamide resin-peptide 6 conjugate are shown in graph A and the results for the polyamide resin-hepatitis B peptide conjugate are shown in graph B.
  • Rabbit anti-peptide antisera were obtained from: rabbit no. 1 (•); rabbit no. 2 (o). All tests were performed in triplicate and the brackets refer to the range of values.
  • HTLV-III viral stock To prepare the various dilutions of the HTLV-III viral stock, one normal human serum and the 4 preimmune rabbit sera was heat inactivated at 56°C for 1 hour and filter sterilized through a 0.2 ⁇ m filter. One hundred fifty microliters of a 1:5 dilution was incubated with an equal volume of 10 -1 , 10 -2, 10 -3 , 10 -4 and 10 -5 dilutions of an HTLV-III isolate, termed NY-5 for 1 hour at 37°C.
  • the NY-5 isolate has an infectious titer of 10 -5 units as determined on the human T-cell line A3.01.
  • the antibody treated virus mixture was added to 10 6 A3.01 cells.
  • the mixture was incubated for 2 hours at 37°C in the presence of 1 ⁇ g/ml of POLYBRENE (Cal Biochem).
  • the infected A3.01 cells were washed and resuspended in 1 ml of RPMI media containing 10% heat inactivated fetal calf serum and dispersed into 24 well microtiter plates. Five hundred microliters of spent media supernatant was removed at days 5, 8, 10, 12 and 15 after infection and frozen at
  • ELISA and Western blot were obtained from Dr. Thomas Folks, Laboratory of Immunoregulation, NIAID, Bethesda, MD.
  • the human sera and rabbit anti-peptide antisera were treated as described above.
  • the preimmune sera of that particular rabbit served as the negative control indicative of no neutralization of HTLV-III infectivity as determined by RT activity cpm when compared to the individual rabbit anti-peptide preparation.
  • the preimmune and rabbit anti-peptide antisera were incubated with 10 -1 , 10 -2 , 10 -3 , 10 -4 , and 10 -5 dilutions of the NY-5 isolate as described above.
  • HTLV-III was observed at days 12 and 15, whereas little or no replication was demonstrated with 10 -4 dilution of virus even by day 15. Readings of greater than 2000 cpm of 3 H-uptake were selected as an indication of HTLV-III replication based on the fact that 11 out of 12 determinations (days 5 and 8 with all dilutions of virus, day 10 for 10 -3 and 10 -4 dilutions and days 12 and 15 for 10 -4 dilution) with less than 2000 cpm had standard deviations and standard errors of the mean greater than or equal to the mean cpm.
  • RT assay would be difficult. Antibodies may not efficiently neutralize virus if overwhelming quantities of infectious virions are present. Based on the kinetic studies and the nature of the HTLV-III viral stock, it was determined that neutralization of HTLV-III infectivity by human and rabbit anti-peptide antisera would be examined at days 10, 12, and 15 for the 10 -1 and 10 -2 dilution and days 12 and 15 for the 10 -3 dilution of HTLV-III.
  • the antiserum to the polyamide resin-peptide 6 conjugate from one rabbit efficiently reduced HTLV-III replication at day 10 when compared to pooled human sera from AIDS patients at both 10 -1 and 10 -2 dilutions of virus.
  • a second rabbit anti-serum to that peptide failed to reduce HTLV-III replication and served as a control antiserum throughout the RT assay.
  • No anti-HTLV-III activity was detected in this particular antiserum based on radioimmunoprecipitation even though the rabbit received a similar immunogen and produced a detectable anti-peptide response.
  • the antiserum that neutralized HTLV-III detected both the gp 120 and gp 160 envelope glycoproteins.
  • Rabbit no. 1 antiserum was found to be less efficient in neutralizing HTLV-III when compared to human AIDS serum on day 12 and 15.
  • the percent reduction of RT activity decreased by day 12 from greater than 90 percent (day 10) to 23 and 45 percent for a 10 -1 and 10 -2 dilution of virus, respectively.
  • An insoluble support matrix is coated with 5 ⁇ g each of the polyamide resin synthetic peptide conjugates prepared as described above in Example 5 in borate buffer saline (BBS), pH 8.0, for 8 hours at 4°C.
  • BBS borate buffer saline
  • the matrix may be coated for one hour at
  • a serum sample suspected of containing antibody to the viral causative agents of AIDS and/or ARC is added and incubated for one hour at 37°C.
  • the support matrix is washed three times with T-PBS, and biotin-labeled goat anti-human Ig (1:1000 of 5 mg/ml in 10% NGtS, Vector
  • the solid phase matrix is coated with antibodies produced by immunization of an experimental animal with one or more of the polyamide resin-peptide conjugates prepared as described in Example 5, and the antibodies blocked and washed as described above.
  • the biological fluid sample suspected of containing the AIDS or ARC viral causative agent is then added and washed.
  • the assay can be conducted either as a direct binding assay or as an inhibition assay. If a direct binding assay is conducted, biotin-labeled antibodies to the AIDS and/or ARC virus produced as described above are added and washed.
  • the avidin-labeled enzyme is then added as described above and washed, and the substrate is added as described above. The reaction is stopped and the optical density is read.
  • biotin-labeled antibody instead of adding biotin-labeled antibody to AIDS virus, the biotin-labeled synthetic peptide is added and the insoluble support matrix is washed. The avidin-labeled enzyme is then added and washed. The substrate is added, the reaction stopped and optical density is read.
  • the IgG from human or chimpanzee AIDS-containing serum is purified by ion exchange chromotography on a Whatman DE-52 anion exchange column.
  • IgG from rabbit anti-peptide is purified with a protein A-Sepharose 4B column (Pharmacia).
  • the IgG is biotinylated using biotin-N-hydroxysuccinamide ester (Boehinger Manheim).
  • the synthetic peptides 1-9 are synthesized on the polyamide resin as described in Example 5, above.
  • the polyamide resin-synthetic peptide conjugate, or a mixture of several conjugates, is injected into the animal in a bolus of between 100 to 1000 ⁇ g of synthetic peptide in alum as an adjuvant.
  • Three separate injections may be given, either intramuscularly or subcutaneously, on a biweekly basis until a measurable antibody response to the virus is detected. Other time intervals such as 0,
  • 1 and 6 months may also be used for the injection of the synthetic peptide.
  • the synthetic peptide of the present invention can also be used to screen potential AIDS vaccine candidates for their ability to induce an immunogenic response in an animal subject.
  • One or more of the polyamide resin-synthetic peptide conjugates are coated onto the insoluble matrix as described above in Example 15.
  • the vaccine candidate is then incubated with antibodies against the peptide (with or without biotin labelling). If biotin labeled, the avidin-enzyme is added, if not, a biotin anti-species antibody such as biotin goat anti-rabbit IgG is added, followed by the addition of the avidin-enzyme.
  • the substrate is added, the reaction stopped and optical density read to determine the ability of the vaccine candidate to block the binding of the peptide.

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Abstract

La composition et le procédé décrit permettent d'induire un anticorps de neutralisation ayant une action contre les agents pathogènes viraux du SIDA et de l'ARC (complexe lié au SIDA). Ladite composition est un conjugué d'une résine de polyamides et d'un peptide synthétique, la séquence d'acides aminés du peptide synthétique étant suffisamment homologue à la séquence d'acides aminés des sous-unités gp 41 et gp 120 de la glycoprotéine à enveloppe gp 160 de l'HTLV-III, du LAV ou de l'ARV pour produire une réponse immunogène chez l'animal faisant l'objet de l'expérimentation, une région hydrophile étant incluse dans ledit peptide. Ladite composition est additionnée ou non d'un adjuvant en quantité suffisamment efficace pour induire une réponse immunogène, protégeant ainsi l'animal faisant l'objet de l'expérimentation de toute exposition aux agents pathogènes de l'HTLV-III, du LAV, et/ou de l'ARV (rétrovirus associé au SIDA) à l'origine du SIDA et/ou de l'ARC.
PCT/US1987/001733 1986-07-21 1987-07-20 Composition et procede d'immunisation contre des agents pathogenes viraux du sida et de l'arc WO1988000471A1 (fr)

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EP0231914A2 (fr) * 1986-02-03 1987-08-12 F. Hoffmann-La Roche Ag Peptides d'enveloppe de HTLV-III
EP0328403A2 (fr) * 1988-02-12 1989-08-16 United Biomedical Inc. Peptides synthétiques relatifs à la protéine HIV-GP120-env. et leur application
EP0362909A2 (fr) * 1988-08-26 1990-04-11 Akzo N.V. Polypeptides synthétiques immuno-actifs avec des anticorps VIH
EP0362910A2 (fr) * 1988-08-27 1990-04-11 Akzo N.V. Polypeptides synthétiques immuno-actifs avec des anticorps VIH
WO1990003984A1 (fr) * 1988-10-03 1990-04-19 Repligen Corporation Proteines et peptides du vih utiles pour le diagnostic, la prophylaxie ou la therapie du sida
EP0402088A2 (fr) * 1989-06-06 1990-12-12 Merck & Co. Inc. Conjugés immunogènes contre le Sida
WO1990015627A1 (fr) * 1989-06-20 1990-12-27 Board Of Regents, The University Of Texas System Methodes et compositions pour la preparation et l'utilisation de reactifs immunologiques cibles
EP0448095A1 (fr) * 1990-03-21 1991-09-25 Wolf, Hans Joachim, Prof. Dr. Sous-région de la protéine ENV rétrovirale, séquences d'ADN codant pour celle-ci et compositions pour le diagnostic, la prévention ou la thérapie des infections rétrovirales
WO1991009872A3 (fr) * 1989-12-13 1992-04-02 Univax Biolog Inc Polypeptides selectivement reactifs avec des anticorps contre le virus d'immunodeficience humaine et vaccins comprenant les polypeptides
US5166050A (en) * 1986-08-20 1992-11-24 Bristol-Myers Squibb Company Monoclonal antibodies and peptides useful in treating and diagnosing HIV infections
WO1992022577A1 (fr) * 1991-06-17 1992-12-23 Neovacs Composes immunogenes a effet notamment anti-cytokine, procede de preparation, compositions pharmaceutiques et kits les renfermant
EP0525828A2 (fr) * 1986-08-01 1993-02-03 Repligen Corporation Polypeptides recombinants, et leur utilisation, y compris un diagnostic pour le virus du SIDA
US5274122A (en) * 1992-10-15 1993-12-28 Merck & Co., Inc. Acidic derivatives of homocysteine thiolactone
US5346989A (en) * 1990-08-22 1994-09-13 Syntello Vaccine Development Kb Peptides for use in induction of T cell activation against HIV-1
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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
US6602705B1 (en) 1998-12-31 2003-08-05 Chiron Corporation Expression of HIV polypeptides and production of virus-like particles
US6659681B1 (en) 1999-02-10 2003-12-09 Traf Fix Devices, Inc. Laterally stable vertical panel system
US6689879B2 (en) 1998-12-31 2004-02-10 Chiron Corporation Modified HIV Env polypeptides
US7211659B2 (en) 2001-07-05 2007-05-01 Chiron Corporation Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US7282364B2 (en) 2001-08-31 2007-10-16 Novartis Vaccines And Diagnostics, Inc. Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides and uses thereof
US7935805B1 (en) 1998-12-31 2011-05-03 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV Type C polypeptides, polypeptides and uses thereof
US7943375B2 (en) 1998-12-31 2011-05-17 Novartis Vaccines & Diagnostics, Inc Polynucleotides encoding antigenic HIV type C polypeptides, polypeptides and uses thereof
US8263394B2 (en) 1998-12-31 2012-09-11 Novartis Vaccines & Diagnostics Inc. Polynucleotides encoding antigenic HIV type B polypeptides, polypeptides, and uses thereof
WO2017128969A1 (fr) 2016-01-28 2017-08-03 中国科学院过程工程研究所 Système et procédé de production d'électrolyte au vanadium de valence 3,5 de pureté élevée

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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
US5562905A (en) * 1988-04-26 1996-10-08 E. I. Du Pont De Nemours And Company Human immunodeficiency virus (hiv) env-coded peptide capable of eliciting hiv-inhibiting antibodies in mammals
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EP0362910A2 (fr) * 1988-08-27 1990-04-11 Akzo N.V. Polypeptides synthétiques immuno-actifs avec des anticorps VIH
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EP0402088A2 (fr) * 1989-06-06 1990-12-12 Merck & Co. Inc. Conjugés immunogènes contre le Sida
WO1990015627A1 (fr) * 1989-06-20 1990-12-27 Board Of Regents, The University Of Texas System Methodes et compositions pour la preparation et l'utilisation de reactifs immunologiques cibles
WO1991009872A3 (fr) * 1989-12-13 1992-04-02 Univax Biolog Inc Polypeptides selectivement reactifs avec des anticorps contre le virus d'immunodeficience humaine et vaccins comprenant les polypeptides
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EP0448095A1 (fr) * 1990-03-21 1991-09-25 Wolf, Hans Joachim, Prof. Dr. Sous-région de la protéine ENV rétrovirale, séquences d'ADN codant pour celle-ci et compositions pour le diagnostic, la prévention ou la thérapie des infections rétrovirales
US5606030A (en) * 1990-07-19 1997-02-25 Merck & Co., Inc. Coconjugates of OMPC, HIV related peptides and anionic moieties
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JPH01500432A (ja) 1989-02-16
EP0276279A4 (fr) 1990-01-08
DK149188D0 (da) 1988-03-18
EP0276279A1 (fr) 1988-08-03
DK149188A (da) 1988-05-06
AU7786387A (en) 1988-02-10

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