IE60671B1 - Monoclonal antiobodies to HIV and related peptides - Google Patents

Abstract

Disclosed are monoclonal antibodies reactive with one or more neutralizing regions of HIV proteins, peptides or homologs of such neutralizing regions, and nucleic acid segments coding for said peptides. Neutralizing regions include portions of the env and gag genes from various HIV isolates. The monocional antibodies may be used to treat HIV infection or in diagnosis and the peptides may be used to elicit antibodies and to block HIV infection. [DE3727703A1]

Description

The present invention relates generally to the diagnosis« treatment end prevention of viral infections. More particularly, the invention provides compositions and methods fox the production of monoclonal antibodies and peptides useful in diagnosing, neutralizing and vaccinating against Human Immunodeficiency Virus (HIV) infections.

The infectious agent responsible for acquired immunodeficiency syndrome (AIDS) and its prodromal phases, AIDS-related complex (ARC) and lymphadenopathy syndrome (LAS), is a novel lymphotrophic retrovirus.

The virus has been variously termed LAV, HTLV-ΐϊϊ, ARV, and most recently HIV.

As the spread of HIV reaches pandemic proportions, the treatment of infected individuals and the preventing the transmission to uninfected individuals at risk of exposure is of paramount concern. A variety of therapeutic strategies have targeted different stages in the life cycle of the virus and are outlined in Mitsuya and Broder, 1987, Nature 325;773. One apΟ ti proach involves the use of antibodies which bind to the virus and inhibit viral replication, either by interfering with viral entry into host cells or by some other mechanism. Once the viral component(s) susceptiS ble to antibody intervention are identified, it is hoped that antibody titers sufficient to neutralize the infectivity of the virus could be engendered by vaccination or, alternatively, by the passive administration of immune globulins or monoclonal antibodies of the de10 sired specificity.

The envelope glycoproteins of most retro» viruses are thought.to react with receptor molecules on the surface of susceptible cells, thereby determining the virus' infectivity for certain hosts. Antibodies that bind to the glycoproteins may block the interaction of the virus with the cell receptors, neutralizing the infectivity of the virus. See generally, The Molecular Biology of Tumor Viruses, 534 (J. Tooze, ed., 1973) and RNA Tumor Viruses, 226, 236 (R. Weiss et al., eds., 1982), to both of which reference should be made for details. See. a^so p GonzalezScarano et al.,, 1982, Virology 120:42 (La Crosse Virus); Matsuno and Inouye, 1983, Infect. Immun. 39:155 (Neonatal Calf Diarrhea Virus); and Mathews et al., 1982, J. Immunol., 129:2763 (Encephalomyelitis Virus).

The general structure of HIV is that of a ribonucleoprotein core surrounded by a lipid-containing envelope which the virus acquires during the course of budding from the membrane of the infected host cell.

Embedded within the envelope and projecting outward are the viral encoded glycoproteins. The envelope glycoproteins of HIV are initially synthesized in the infected cell as a precursor molecule of 150,000-160,000 daltons (gplSO ©2’ gp!60) , which is then processed in the cell into an N-terminal fragment of 110,000-120,000 daltons (gpllO or gp!20) tc generate the external glycoprotein, and a C-terminal fragment of 41,000-46,OuO daltons (gp41), which represents the transmembrane envelope glycoprotein.

For the reasons discussed above, the gpllO glycoprotein of HIV has been the object of much investigation as a potential target for interrupting the virus’ life cycle. Sara from HIV infected individuals have been shown to neutralize HIV in vitro, and antibodies that bind to purified gpllO are present in the sera. Kobert-Gurofx e t a 1. , 1985, Nature 316:72? Weiss et al., 1985, Nature 316:69? and Mathews et_al., 1986, Proc. Natl- Acad. Sci. (J.S.A. , 33:9709. Purified and recombinant gpllO stimulated the production of neutralizing serum antibodies when used to immunize animals, Robey e t a 1. ,-, 1986, Proc. Natl. Acad. Sci. U.S.A., 83:7023; Lasky et al., 1986, Science 233:209; and a numan, gagury et _al., 1986, Nature 326:249. Binding of the gpllO molecule to the CD4 A subunit vaccine for AIDS utilizing the HIV gpllO molecule or portions thereof may thus be desirable. Subunit vaccines are an alternative to vaccines prepared from inactivated or attenuated viruses. Inactivated vaccines are worrisome due to the possible failure to kill all of the viral particles, and atten5 uated viruses may possess the ability to mutate and regain their disease-causing capability. With subunit vaccines, only those portions of the virus that contain the antigens or epitopes that are capable of eliciting immune responses, i.e., neutralizing antibodies, ADCC, and cytotoxic T-cell response, are used to immunize the post. A major advantage of subunit vaccines is that irrelevant viral material is excluded.

Viral subunits for us® in a vaccine can be generated by several methods. By way of example, the envelope glycoprotein can be expressed and purified from a bacterial host, although this molecule would lack most post-translational modifications (such as glycosylation) or other processing. Such modification may be obtained using a eukaryotic expression system, such as yeast or cultured mammalian cells. Viral genes have been introduced into mammalian cells using the vaccinia virus as a vector. See, for example, Mackett, Μ., et al., 1982, Proc.,Nat. Acad. Sci. USA 79:7415; Panicali, D. and Paoletti, Έ., 1982, Proc. Nat. Acad.

Sci. USA 79:4927. Recombinant vaccinia virus may be constructed according to the method of Hu et al., Nature 320:537 (1986) or Chakrabarti et al., Nature 320:535 (1986), to both of which reference should be made for details. ln these systems viral glycoproteins produced by cells infected with recombinant vaccinia are appropriately glycosylated and may be transported to the cell surface for extrusion and ultimate isolation.

An important step in the production or a subunit vaccine is adequate purification of the desired glycoprotein from the complex mixture of the expression system. Several methods can be used to accomplish the purification. These include but are not limited to preparative polyacrylamide gel electx’ophoresis, gel permeation chromatography and various methods ot chromatography (i.e., ion exchange, reverse phase, immuneaffinity, hydrophobic interaction) and others. Most of these methods are used in various combinations to achieve substantially pure preparations (Kleid, D.G., et al., 1981, Science 214;1125? Cabradilla, C.D., et al., 1986, Biotechnology 4:128, Dowbenko, D.J., 1985, Proc. Nat. Acad. Sex. USA 82:7748) to which reference should be made for details.

Methods which would reduce the number of steps required to achieve maximum purification of a particular viral antigen from a complex expression mixture are needed to manufacture subunit vaccines. The efficient separation of the antigens from extraneous components could be accomplished using immunoazfinity chromatography. This technique, also known as immunoadsorption, consists in principle of the selective adsorption of an antigen to a solid support on which a specific antibody has been covalently attached. The selectively adsorbed antigen is then eluted xrom such an antibody affinity adsorbent by changing, for example, the pH and/or ionic strength of the buffer.

Polyclonal antiDodies, obtained from animals immunized with the desired antigen or from naturally infected individuals (see, for example, Lasky et al. , supra), have frequently been used as immunoadsorbants, but, in general these reagents present substantial disadvantages, such as (ii not all of the antibodies bound to the insoluble support are specific tor the molecule of interest, necessitating additional purification; (ii) yields of the desired antigen are frequently low? and (iii) antibody affinities often vary from one preparation to another, requiring modifications xn elution procedures. The use or monoclonal antibodies specific for the desired viral antigen to be used in the subunit vaccine preparation, rather than polyclonal antibodies, would circumvent these difficulties.

Murine monoclonal antibodies that bind HIV antigens have been described. Several groups have reported monoclonal antibodies specific for the core protein p25 (see, tor example, di Marzo Veronese, et al., 1905, proc. Nat. Acad, Sci. USA 82:5X99 and Chassagne, 3., et al., 1985, J. Immunol. 136:1442). Monoclonal antibodies specific tor the membrane glycoprotein gp41 have also been reported (seeg for example, di Marzo Veronese, et' al. 1985, Science 229:1402).

Ther© remains a need in the art for monoclonal antibodies specific for epitopes within well defined regions of the major envelope glycoprotein, gpllO. Monoclonal antibodies which bind these regions and cause a reduction in or elimination ot the replication and transmissibility of BIV would have substantial therapeutic and prophylactic utility. Moreover, the monoclonal antibodies could also be used to purify the desired region or gpllO from disrupted virus or recombinant expression systems for use in vaccines, for example» Additionally, the region containing the epitope (s) recognized by the monoclonal antibodies could be chemically synthesized, thereby avoiding the difficulties inherent in purification and administration of larger fragments of the gpllO molecule. The present invention fulfills these and other related needs.

Peptides capable of immunologically mimicking neutralizing epitopes of HIV proteins, nucleic acid probes encoding such peptides and monoclonal antibodies reactive with such peptides, as well as other peptides interfering with HIV infectivity, are provided. These novel materials find use in, for example, diagnostics assays for the detection of HIV infections and in therapeutic regimens tor the -treatment of ox vaccination against such infections.

The present invention provides novel compositions and methods for neutralizing HIV infections, i.e.,, preventing or substantially inhibiting the formation ox cellular transmission of infectious HXV in a host. More specifically., peptides mimicking a neutralizing region of gpUOor p25 of HIV and monoclonal antibodies reactive with such a region axe utilized to diagnose, treat and vaccinate against HIV infections. Xn this regard, the term neutralizing region indicates those portions of HIV, particularly HIV proteins, containing amino raeid segments defining one or more epitopes reactive with antibodies which, either individually or in combination witn other antibodies of the present invention, axe capable of neutralizing HIV infections. Suitable assays for neutralization axe well known and can include reduction of HIV infections in T-cell lines, reduction of plaque forming units of VSV(firv) pseudotypes bearing the envelope glycoproteins of HIV, syncytial inhibition tests and virion-receptor binding tests. As desired, the neutralizing activity can be compared to antibody reactivity in immunochemical tests, such ss immunofluorescence, imraunoblot and radioimmunoprecipitation assay. 3n one aspect, the novel peptides, typically less than about 50 amino acids, contain five or more contiguous amino acids forming epitopes substantially similar to epitopes located on neutralizing regions of HIV gpllO ox p25, encoded by the env and gag regions„ respectively, of fche HIV genome. Of particular interest axe the regions extending from about amino acid residue 301 to about 336 of gpllO, to about 319 and from about 315 to all from the HIV strain designated acid residue designations are from Bank (AIDS virus sequence database Laboratory, Theoretical Division, 8?545>. and from about 278 about 363 of p25, ^AVBRU° Tne ®m*no the Los Alamos Data , Los Alamos National Los Alamos, NM to LAV„„„ sequence data as follows Those skilled in the art will appreciate that additional analogous regions ("homologs) from other HIV isolates may be identified based upon their location within related proteins from various isolates. In practice, such homologs may be identified by reference BRU (a) The amino acid sequences ot HIV isolates and bAV3RU may be aligned to obtain maximum homology between the two sequences; (b) peptides comprising HIV isolates' amino acid sequences corresponding to the location of LAV peptides that immunologically mimic LAV^^y proteins may be identified. The peptides comprising HIV isolate amino acid sequences so identified will typically immunologically mxmic corresponding Hlv isolate proteins.

This method can be applied to HIV strains in general and their envelope and core amino acid sequences may be aligned with that of LAV^^ to obtain maximum homology. The methods by which the sequences are aligned are known to those skilled in the art. In aligning the sequences it is desired to maintain as much homology between cysteine residues as possible. The amino acid sequence of the new HIV strain or species which corresponds to the location of the peptides specifically disclosed herein can be synthesized and used in accordance with the invention.

Anctner method for determining sequences of a homologous region in otner HIV strains is described by Scharf et al. , Science (1986) 233:107b. It employs two oligonucleotide primers which bind to conserved sequences outside the sequence region of interest, and contain different restrictive sites in each primer.

DNA from HIV strains can then be amplified in vitro thereafter, the resulting oligonucleotides may be cloned in vectors for sequence analysis and incorporated into a vaccine as a cassette representing a particular epitope from the HIV strain.

It is not necessary to the present invention that the epitopes contained within such sequences be cross-reactive with antibodies to all strains or species of HIV. Peptides encompassing immunological epitopes which distinguish one species or serogroup over another will find utility in identifying particular species or serogroups, and may in fact assist in identifying individuals xnrected with one or more species or serogroups of HIV. Tney may also be useful in combination with other peptides, from either a homologous region or another neutralizing region, in therapeutic compositions .

The peptides of interest will most preferably be derived from the gpllO region of the virus. Of particular interest in this region are peptides encoded within the env open reading xrame extending from about base pair (bp, 6b67 to about 6774 of the LAVgRU isolate. Thus, various homologous regions of other HIV isolates include the homologous sequences obtained from bos Alamos Data Bank (except LAV2), as listed in Table 1.

Other peptides suitable for generating or screening for monoclonal antibodies include those encoded in the env open reading frame from about bp 7246 TABLE A KLXa2 TGTACAAGACCCAACAACAATACAAGAAAAAGA............... ATCCGTATC GyeThrArgFroAsnAsnAsnThrArgLyeArg...............TleArglle BHI 02 ———-----------—~——Ser---—-----—«·------. BH8 — ——— —----—·——l,y®«---------— HAL ———----Gly-—-------ArgGly-----—————.—Β 1 @?he ELI —-ala—~-~TyrGXm~---»—-—-GITS-—-—---—--ASV2 w—w~^R—w—Se WHJ2 ———Tyr--—Val——ArgSer—-—————LeuSe?'-’FJFEEW ----—Ser—————------ThrLys 23 ————GlySerAspLysLyslle———————SInSer— KY 5 —J,y(— <23042--------—gie— ---—ValThrLeu. ..............

LAV2 Gly-—-Lys—'-Val-'—Sla—Me eL«eu 309 309 309 309 309 314 314 310 312 305 322 311 305 304 320 302 HXB2 CAOAGA.........GGACCAGGGAGAGCATTTGTTACAATAi6GAAAAATAGGAAATATG GlftArg.........GXy?roGlyArgAl«?heValThrXleClyLysUeGlyAsnMet 326 BHI02 326 25 mAL ————CIb—UuTyt—Thr—XleVal—Asp lie 329 ELI GlyLeu——-—-.. .GlnSerLeuTyrThr—Arg—IleVelSerArgSer 323 ARV2 ......-—·----------—-----------Sis—Thr—Arg— HeClyAsp 327 WMJ2 ——————— ----———-Arg—ArgGlu..ZleGlylle 320 30 RFEKV ......---------------------VallleTyrAlsThr Gin—HeClyAsp 337 Z6 GlyLeu-------—---.. .GlnAlaLeuTyrThr——Arg—-ArgThrLysllelle 327 Arglle———----————LysVal-—TyrAlaLys—-Cly............ 319 ΜΪ5 ClyPro-—— ——...-——-ThrLeuTyrAlsArgGJu———-—Asplle 320 COC4 2 ..............—ValTrpTyr—Thr—Glu—LeuGlyAsn 335 LAV2 MecSer— -----————HisVal—HisSerHisTyrGlnProIle—Lys 323 HXB2 ... AGA...GAAGCACATTGT ...Arg...GlnAlsKisCys SH102 ——---------BH8 --------------------HXB3 ——---------H9M ---------------------BRU ---------------------MAL -----——Arg—Tyr-— ELI IlelleCly.......—AFW2 Ile—Lys...·—-----WMJ2 lie-----------------RFEftV lie—Lys...-------— Gly... —------— JlsThrGly—---------NY5 —..............— CDC42 He·----LAV 2 ArgProArg——Met— 331 331 33’ 33’ 331 336 334 330 333 326 343 334 326 325 341 330 tc about 7317 of LAV . Such antibodies and reactive s Ku peptides are particularly useful in immunoassays.

In the gag region of the LAV^y isolate, the p25 amino acid sequences from about 278 to 319 and 315 to 363 are additional neutralising regions of HIV.

Those skilled in the art will appreciate that additional neutralizing regions of HIV can be identified based on the teachings herein—in particular, combinations of monoclonal antibodies reactive with various different HIV epitopes will exhibit neutralizing activity.

Peptide I, also designated peptide 29, is encoded in the env open reading frame from about amino acid residue numbers 308 to about 328 and will have the following amino acid sequence, where oligopeptides included within the following sequence will include linear epitopes within such sequences I (29) Y-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gin-Arg-GlyPro-Gly-Arg-Ala-Phe-Val-Thr-Ile-Gly-LysIle-Y' in which ¥ and Y e, when present, each represents sequences of up to about twenty amino acids. When Y and/or ¥’ are present, these may comprise, for example, one or more amino acids from sequences which flank amino acid residues 308 through 328 ot the HIV envelope sequence or any portion ox these flanking sequences.

By way of example and not limitation, ¥ can comprise all or portions of the LA'V^y envelope amino acid sequence from about residue numbers 301 to 307; and Y' can comprise all or portions of the LAVSRU envelope amino acid sequence from about residue numbers 329 to 335 as follows: II (29a) Cys-Thr-Arg-Pro-Asn-Asn-Asn-Thr-Arg-Lys-Ser-XleArg~Ile-Gln-Arg-Gly-Pro~Gly-Arg-Ala~Phe~Vai~ThrIie-Gly-Lys-Ile-Gly-Asn-Met-Arg-Gln-Ala-His-Cys» Alternatively, truncated sequences of peptides of the present invention may be prepared. In this regard, the following sequences from peptide 29 may be particularly useful: III (2SO) Y-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-GlyY’ in which Y and/or Y’, when present, each represents sequences of up to about twenty amino acid residues.

IV (29c) Y~XXe-Gln-Arg-Gly-Pro-Gly-Arg~Ala-Phe-Val-ThrIXe-Gly-Lys-Ile-Y’ in which Y and Y’, when present, eacn represents sequences of up to about twenty amino acid residues.

In another embodiment, homologous regions of the ARV-2 isolate of particular interest are encoded in the env open reading frame from about amino acid residue numbers 30» to about 323 and will typically have the following amino acid sequenceL, where oligopeptides included within the following ammo acid sequence will include linear epitopes within such sequence.· V (177) Y-Thr’Arg-Lys-Ser-Ile-Tyr-lle-Gly-Pro-Gly-ArgAla-Phe-His-Thr-Thr-Gly-Arg-Ile-Y* in which Y and Y’, when present, each represents one up to about twenty or more amino acid residues. When Y and/or Y' are present, these may comprise one or more amino acid residues from sequences which flank amino acid residues 306 through 323 of the ARV-2 envelope sequence or any portion of these flanking sequences.

In particular, Y can comprise all or portions of the HIV envelope amino acid sequence from about residue numbers 299 to 306; Y’ can comprise all or portions of HIV envelope ammo acid sequence from aoout residue numbers 324 to 333.

Alternatively, truncated sequences of peptide V may be prepared. In. this regard, the following sequences may be particularly useful: VI (177a) Y-Thr-Arg-Lys-Ser-Ile-Tyr-Ile-Gly-Pro-Gly-y«- and in which ¥ and/or ¥', when present, each represents sequences of up to twenty or more amino acid residues.

A further example comprises homologous regions of the LAV-2 isolate, such as encoded in the env open reading from about amino acid residue numbers Jll to 330, ana will typically have the following sequence: VIII (110-2-2) ¥-Lys-Thr-Vai-Lys-Ile-Nor-Leu-Nor-Ser-Gly-His-Val?he-His~5er-His-Tyr-Gln~Pro-¥8 in which ¥ and/or ¥’, when present, each represents sequences of up to twenty or more amino acid residues.

(See, Nature 326:662 (1987), to which reference should be made for details).

In accordance with another aspect of the present invention, novel cell lines capable of producing monoclonal antibodies and compositions comprising such antibodies are provided, which antibodies are capable of selectively recognizing at extremely high 2 4 7 titers (from 10 , to 10 to about 10 or more) neutralizing regions contained within a predetermined sequence of envelope glycoprotein gpllO or p25, their protein precursors, biologically-expressed recombinant fusion proteins and synthetic peptides that contain one or more epitopes within the predetermined sequence region of gpllO or p25. The subject hybrid cells have an identifiable chromosome, in which the germ line DNA has rearranged to encode an antibody having a binding site for an epitope on gpllO or p25 common to some or all HIV clinical isolates. These monoclonal antibodies may be used in a wide variety of ways including diagnosis and therapy, as well as to identify other cross-reactive antibodies, such as blocking antibodies. Peptides or polypeptides containing the epitope(s) with which they react may find separate uses as immunogens for vaccines, or as therapeutic agents.

Slocking Peptides Primarily for use in conjunction with the roregoing peptides or neutralizing monoclonal antibodies, another embodiment of the present invention comprises utilizing additional peptides or antibodies that interfere with HIV binding to receptors to further mitigate HIV infectivity. Preferably, so-called blocking peptides· capable of inhibiting virus proliferation, as well as monoclonal antibodies specific for epitopes contained within such blocking peptides, can be utilized to increase the effectiveness of treatments against HIV infections. HIV blocking peptides typically correspond to amino acid sequences of HIV thought to be essential tor virus attachment to a host cell, such as env-eneoded amino acid residues about X90 to about 197 ox LAVbru and about 185 to about 192 of ARV2 and HTLVZII(BH-IO). These include the peptide T octapeptide (Ma-Ser-Thr-Thr-Thr-Asn-Tyr-Thr) and its various derivatives (e.g., IX below) and analogs (e.g., XI below) described by Pert et al. (1986, Proc. Natl.

Acad.’ Sci. USA 83:9254-9258, to which reference should be made for details)located on the envelope glycoprotein (gpllO or 120).

For example, blocking peptides having the rollowing sequences are of particular interest, preferίδ ably with HHj-terminus acetylation and COOH terminus amidation : IX ¢1730) Y-^Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-Y9 ; X U86) Y-Thr-Thr-Asn-Tyr-Thr-Y' ; XI (187) Y~Thr-Thr-Ser*Tyr-Thr-Y9 ? XII (188) Y-Thr-Asp-Asn-Tyr-Thr-Y5 ,· XIII (189) Y-Asn-Thr-Ser-Tyr-Gly-Y' s XIV (190) Y-Asp-Thr-Asn-Tyr-Ser-Y5 ? XV (191) Y-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Y9 j in which, for each peptide, Y and Y*, if present, each comprises an amino acid sequence of up fco about 20 amino acids. Epitopes or antigenic determinants within these peptides are typically defined by at least about five contiguous amino acids, and find use in,, e.g. , mimicking naturally-occurring HIV antigenic sites for producing HIV reactive antibodies and vaccines. Generation of Monoclonal Antibodies The preparation of monoclonal antibodies ean be accomplished by immortalizing the expression of nucleic acid sequences that code for antibodies specific for HIV, by introducing such sequences, typically cDNA encoding for the antibody, into a host capable of cultivation in culture. The immortalized cell line may be a mammalian cell line that has been transformed through oncogenesis, by transfection, mutation, or the like. Such cells include myeloma lines, lymphoma lines, or other cell lines capable of supporting the expression and secretion of the antibody in vitro. The antibody may be a naturally occurring immunoglobulin of a mammal, produced by transformation of a lymphocyte, particularly a splenocyte, by means of a virus or by fusion of the lymphocyte with a neoplastic cell, e.g., a myeloma, to produce a hybrid cell line. Typically, the spienocyte will be obtained from an animal immunized against the HIV virus or a fragment thereof containing an epitopic site.

Immunization protocols are well known and can vary considerably yet remain effective. See, Coding, Monoclonal Antibodies: Principles and Practice, Academic Press, 2nd edition <1986}, to which reference should be made for details. Disrupted virus, synthetic peptides and bacterial fusion proteins which contain antigenic fragments of the gpllO or p25 molecule may be used as immunogens... Preferably the immunogen of disrupted virus, peptides or recombinant proteins will be enriched for proteins or fragments thereof containing the epitopes to which antibody-producing B cells or splenocytes are desired. More particularly, solutions containing disrupted virus lysates or extracts, or supernatants or biologically-expressed recombinant proteins or disrupted expression vectors, may be enriched for glycoproteins, as desired, using purification methods, such as, for example, polyacrylamide gel electrophoresis. Lectin affinity purification is a preferred and convenient method for purification or gpllO and other glycoproteins, e.g., affinity purification using lentil lectin. The extent to which the glycoproteins are purified from the solutions for use as an immunogen can vary widely, i.e., from less than SOS, usually or at least 75% to 95%, desirably 95% to 99% and, most desirably, to absolute homogeneity.

Once the proteins have been purified to the extent desired, they may be suspended or diluted in an appropriate physiological carrier for immunization, or may he coupled to an adjuvant. One preferred technique p for example, involves adsorbing the proteins and fragments thereof to lentil lectin agarose or other macromolecular carrier for injection- Immunogenic amounts of antigenic preparations enriched in HIV proteins, including gpllO glycoprotein and p25 core protein, or antigenic portions thereof, are injected, generally at concentrations in the range of 1 ug to 20 mg/kg of host- Administration may be hy injection, e.g., intramuscularly, peritonealiy, subcutaneously, intravenously, etc. Administration may be one or a plurality of times, usually at one to four week intervals. Immunized animals are monitored for production of antibody to the desired antigens, then the spleens are removed and splenic B-lymphocytes isolated and fused with a myeloma cell line or transformed. The. transformation or fusion can be carried out in conventional ways, the fusion tecnnique being described in an extensive number of patents, e.g., «,172,124; 4,350,683; 4,363,799? 4,381,292; and 4,423,147. See also, Kennett et al., Monoclonal Antibodies (1980), and references therein, and Ceding, supra.

The immortalized cell lines may be cloned and screened in accordance with conventional techniques, and antibodies In the cell supernatants detected that are capable of binding to the desired gpllO or p25 HIV viral proteins, recombinant fusion proteins or synthetic peptides which contain the desired epitopic region. The appropriate immortalized cell lines may then be grown in vitro or injeeted into the peritoneal cavity of an appropriate host for production of ascites fluid, By virtue of having some antibodies of the present invention, which are known to be specific tor epitopes IS contained, e.g., within the regions encoded by the IAVBRy genomic region from about boS688 to about bp6750 (encoding peptide 29), or from about bp7246 to about 7317 {encoding peptide 36) {bp numbering according to t^ain-Hobson et al., Cell 44:9 1985, to which reference should be made for details), the supernatants may be screened in competition with the subject monoclonal antibodies in a competitive assay» Thus, additional immortalized hybridoma cell lines with the desired binding characteristics can be readily produced from a variety of sources based on the availability of the present antibodies specific for the particular antigen. Alternatively, these cell lines may be fused with other neoplastic 3-cells, where such other 3-cells may serve as recipients for genomic DNA coding for th© antibody.

While rodent, particularly murine, neoplastic 3-cells are preferred, other mammalian species may be employed, such as lagomorpha, bovine, ovine, equine, porcine, avian or the like. Immunization ox these animals can be readily performed and their lymphocytes, particularly splenocytes, may be obtained ror fusions.

The monoclonal antibody secreted by the transformed or hyorid cell lines may be o£ any of the classes or subclasses of immunoglobulins, such as IgM, IgD, IgA, IgG^^, or IgE. As IgG is the most common isotype utilized in diagnostic assays, it is often preferred. Tne monoclonal antibodies may be used intact, or as fragments, such as Fv, Fab, Fiab’ij, but usually intact.

To circumvent the possible antigenicity in a human host of a monoclonal antibody, derived from an animal other than human, chimeric antibodies may be constructed wherein the antigen binding fragment of an immunoglobulin molecule (variable region) is connected by a peptide linkage to af least part of another pro35 tein not recognized as foreign by humans, such as the cast out portion of a human immunoglobulin molecule. This can be accomplished by tusing the animal variable region exons with human kappa or gamma constant region exons. Various techniques are known to the skilled artisan, such as those described in PCT 86/01533, EP171496, and EP173494, to which reference should be made for details.

Pharmaceutical Formulations and Use The monoclonal antibodies or this invention tnat exhibit neutralizing activity, such as those which react with an epitopic site on gpllO or p25 or which react with a blocking peptide, can also be incorporated as components of pharmaceutical compositions to attenuate HIV infections. The composition should contain a therapeutic or prophylactic amount of afc least one of the monoclonal antibodies of this invention with a pharmaceutically effective carrier. A pharmaceutical carrier should be any compatible, non-toxic substance suitable to deliver the monoclonal antibodies to the patient. Sterile water, alcohol, fats, waxes, and inert solids may be used as the carrier. Pharmaceutically acceptable adjuvants (buflering agents, dispersing agents) may also be incorporated into the pharmaceutical composition. Such compositions can contain a single monoclonal antibody so as to be, for example, specific for strains of HIV with envelope glycoproteins containing an epitopic site within a region encoded by bp6S88~bp675Q. Alternatively, a pharmaceutical composition can contain one or more monoclonal antibodies to lorm a cocktail. For example, a cocktail containing monoclonal antibodies against the various strains of HIV would be a universal product with therapeutic or prophylactic activity against the great majority of the clinical isolates of HXV. The cocktail may contain monoclonal antibodies which bind to proteins or glycoproteins ot HIV other than gpllO or p25, such as, for example, to gp41 glycoprotein, or p34 nuclease/integrase. The mole ratio ox the various monoclonal antibody components will usually not differ by more than a factor of 10, more usually by not more than a factor of 5, and will usually be in a mole ratio of about 1:1-2 to each ox the other antibody components.

The monoclonal antibodies of the present invention can be used as separately administered compositions given in conjunction with other anti-retroviral agents, including blocking peptides. Tne current status of the development of anti-retroviral agents, and of anti-HIV agents in particular, is reviewed in Mitsuya et tl.·/ Nature j2bs773-778, 1987, to which reference should be made for details.

The monoclonal antibodies, peptides and pnarmaceutical compositions tnereof of this invention are particularly useful for oral or parenteral administration. Preferably, the pharmaceutical compositions may be administered parenterally, i.e., subcutaneously, intramuscularly or intravenously. Tnus, this invention provides compositions for parenteral administration which comprise a solution of the monoclonal antibody, peptide or a cocktail thereof dissolved m an acceptable carrier, preferably an agueous carrier. A variety of agueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and tne like. These solutions axe sterile and generally free of particulate matter. These compositions may be sterilized by conventional, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for fe L example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of antibody in these formulations can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., preferably for the particular mode or administration selected.

Thus, a typical pharmaceutical composition for intramuscular injection could foe made up to contain 1 ml sterile buffered water, and 50 mg of monoclonal antibody. A typical composition tor intravenous infusion could be made up to contain 250 ml of sterile Ringer’s solution, and 150 mg of monoclonal antibody. Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science, 15th Sd.„ Mack Publishing Company, Saston, Pennsylvania (1980), to which reference should be made for details.

The monoclonal antibodies and peptides of this invention can be lyophilised for storage and reconstituted in a suitable carrier prior to use. This technique has been shown to be effective with conventional immune globulins and art-known lyophilisation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art tnat lyophilization and reconstitution can lead to varying degrees of antibody activity loss (e.g., with conventional immune globulins, IgM antibodies tend to have greater activity loss than IgG antibodies) and that use levels may have to be adjusted to compensate.

The compositions containing the present monoclonal antibodies, peptides or cocktails thereof can be administered for the prophylactic and/or therapeutic treatment of HIV infections. In therapeutic application, compositions are administered to a patient already infected with HIV, in an amount sufficient to cure or at least partially arrest the infection and its complications. An amount adequate to accomplish this is defined as a therapeutically effective dose. Amounts effective for this use will depend upon the severity ox the infection and the general state of the patient’s own immune system, but generally range from about 1 to about 200 mg ot antibody per kilogram or body weight with dosages ox from 5 to 25 mg per kilogram being more commonly used. It must be kept in mind that the materials of this invention may generally be employed in serious disease states, that is lifethreatening or potentially life-threatening situations. In such cases, it is possible and may be felt desirable by the treating physician to administer substantial excesses of these antibodies., In prophylactic applications, compositions containing the present peptides, antibodies or a cocktail thereof are administered to a patient not already infected by HIV, but perhaps recently exposed to or thought to have been exposed to, or at risk of being exposed to the virus, to enhance the patient’s resistance to such potential infection or to vaccinate against the virus. An amount is defined to be a prophylactically effective dose. In this use, the precise amounts again depend upon the patient’s state of health and general level of immunity, but generally range from 0.1 mg to 25 mg per kilogram, especially 0.5 mg to 2.5 mg per kilogram.

Single or multiple administrations of the compositions can be carried out with dose levels and pattern being selected by the treating physician. In any event, the pharmaceutical formulations should pre35 vide a quantity of the antibody(ies) of this invention sufficient to effectively treat the patient.

In addition, the monoclonal antibodies of the present invention· may find use as a target-specific carrier molecule. An antibody may be bound to a toxin to form an immunotoxin or a radioactive material or drug to form a radiopharmaceutical or pharmaceutical. Methods for producing immunotoxins and radiopharmaceuticals are well known (see, for example, Cancer Treatment Reports 68:317 (1984)).

It is also possible that heteroaggregates of monoclonal antibodies of the present invention and human T-cell activators, such as monoclonal, antibodies to the CD3 antigen or to the F'c gamma receptor on Tcells, may enable human T-cells or Fc~gamma bearing cells (such as K cells or neutrophils) to kill HIV infected cells via antibody dependent cell-mediated cytolysis (ADCC). Such heteroaggregates may be assembled, for example, by covalently cross-liking the anti-HIV antibodies to fhe anti-CD3 antibodies using the heterobifunctional reagent N~succinimidyl-3-(2-pyridyldithiol)propionate, as described in Karpowsky et al., J.

Bxp. Med. 160: 1686 (1984), to which reference should be made for details.

The subject peptide compositions themselves may also find use therapeutically, where administration results in the reduction or elimination of HIV in an infected host. These compositions, such as peptide 29, the blocking peptides, and peptide 126, the latter of which is disclosed in commonly owned pending application U.S.S.N. 930,785, may be administered in appropriate physiological carriers intravenously, subcutaneously, intramuscularly, intraperitoneally, etc. various carriers include phosphate buffered saline, saline, water, potassium chloride, sodium lactate, or the like. The concentre·» tion of the peptides will vary widely depending on its ultimate use, activity, and mode of administration. Preferably, the peptides will have COOH-terminus amidation, NKj-terminus formvlation or other pharmaceutically accetable derivatives. The addition of blocking peptides to the peptides mimicking a neutralising HIV region and/or the specifically reactive antibodies of the present invention will result in significantly increased therapeutic effectiveness. Other anti-HIV agents may also be included in the formulations (other than the monoclonal antibodies which bind the peptides) such as 3 '-arido-3 -deoxythymidine, 2 ’ ,3'-dideoxycyti-* dine? 2’, 3 ’-dideoxy-2’, 3B-dldehydrocytidine, etc.

Use of Monoclonal Antibodies in Iwnunoaff inity Purification Monoclonal antibodies specific for polypeptides containing gpllO or other antigenic determinants, particularly those antigenic determinants obtained from biologically-expressed recombinant fusion proteins or lysates or extracts of cultured HIV, are particularly advantageous for use in purification protocols. Generally the antibodies will have affinity association con8 τ stants on the order of 10 to 10~"Μ. Such antibodies may be used to purify the recombinant fusion proteins from the culture medium of the recombinant expression system if the expressed protein is secreted, or from the components of the disrupted biological expression system if it is not secreted. Generally, the monoclonal antibodies which ©re capable of reacting with gpllO or other antigenic determinants are attached to or immobilized on a substrate or- support. The solution containing the HIV antigenic determinants is then contacted with the immobilized antibody under conditions suitable for the formation of immune complexes between the antibody and the polypeptides containing the gpllO antigenic determinants. Unbound material is separated from the bound immune complexes, which complexes or gpllO antigenic fragments are then separated from the support.

Typically, the monoclonal antibodies will be crudely purified from ascites fluid or cell culture supernatants prior to attachment to a support. Such procedures are well known by those skilled in the art, and may include fractionation with neutral salts at high concentration. Other methods, such as DEAE chromatography, gel filtration chromatography, preparative gel electrophoresis, or Protein A affinity chromatography, may also be used to purify the monoclonal antibody prior to its use as an immunoadsorhant.

The support to which the monoclonal antibodies are immobilized should have the following general characteristics: (a) weak interactions with proteins in general to minimise non-specific binding, (b) good flow characteristics which allow the flow through of high molecular weight materials, ic) possess chemical groups that can be activated or modified to allow chemical linkage of the monoclonal antibody, (d) physically and chemically stable in the conditions used to link the monoclonal antibody, and (e, stable to the conditions and constituents of the buffers required for adsorbtion and elution of the antigen. Some supports commonly used are agarose, derivatized polystyrenes, polysaccharides, polyacrylamide beads, activated cellulose, glass and the like. Various chemical methods exist for the attachment of antibodies to substrate supports. See generally, Cuatrecasas, P., Advances in Enzymology 36:29 (1972). The antibodies of the present invention may be attached directly to the support or, alternatively, through a linker or spacer arm.

General conditions required for immobilization of monoclonal antibodies to chromatographic supports are well known in the art. See, for example, Tijssen, Ρ., 1985, Practice and Theory of Bnsyme Immunoassay, to which, reference should be made for details. Actual coupling procedures will depend slightly on the characteristics and type of th© antibody to be coupled. Monoclonal antibodies possess characteristics which are usually consistent from batch to batch, thereby allowing such conditions to be optimised. Attachment typically occurs through covalent bonds.

A suspension of extracts or lysates of HIV virus, the supernatant from a cultured biological expression system, or a suspension of the disrupted cells is then added to the separation matrix. The mixture is · incubated under conditions and for a time sufficient for immune complex formation to occur, usually at least 30 minutes, more usually 2 to 24 hours. The immune complexes containing polypeptides with antigenic portions of gpllO are then separated from the mixture. Typically the mixture is removed, e.g., by elution, and the bound immune complexes extensively washed with adsorption buffer. The immune complexes may then be eluted from the separation matrix using eluant compatible with the particular support being used, whieh eluants are well known by those skilled in the art. Also, the polypeptides containing the gpllO or other antigenic portions may be selectively removed. For example, peptides that contain an epitope recognized by the antibodies can be used to compete for the antibody binding site, which presents an alternative elution technique that can be performed under mild elution conditions, The selectively adsorbed polypeptide containing the gpllO antigen may be eluted from an antibody affinity adsorbent by altering th® pH and/or ionic 0 strength of the buffer. Chaotropic agents may also find use in removing the bound antigen. The selection of a chaotropic agent, its concentration and other eluting conditions are dependent on the characteristics of the antibody-antigen interaction, but once determined should not be subject to changes usually necessary in polyclonal affinity separation systems.

The eluted material may require adjustment to a physiologic pH if low or high pH or ionic strength buffers are used to separate the bound gpllO antigens from the separation matrix. Dialysis or gel filtration chromatography may also be required to remove excess salts used in the eluent to permit reconstitution of gpllO or polypeptides containing antigenic fragments of gpllO to native conformations.

The methods of this invention yield, e.g., substantially purified gpllO or polypeptides containing antigenic fragments thereof, either naturally produced by infected cell cultures or by recombinant expression systems of bacteria, yeast, or cultured insect or mammalian cells. The gpllO and fragments or other purified proteins will. typically be greater than 50¾ pure, more usually at least 75% pure, and frequently greater than 95¾ tc 99% pure. These molecules may then find subsequent use in a wide variety of applications.

The neutralising regions of HIV gpllO proteins, polypeptides containing the antigenic fragments thereof» or other proteins substantially purified according to the methods of die present invention, may find use in a wide variety of applications, including AIDS subunit vaccine formulations, in which the immunogen comprises an effective dose of antigenic determinants of, for example, gpllO or a neutralizing region thereof. Other components of the formulation would include those antigenic portions of HIV proteins or glycoproteins that stimulate the 9(] production of antibody (preferably neutralizing antibodies) in an inenunized host, which antibodies are capable of protecting against subsequent infection by HIV. Diagnostic Uses of Monoclonal Antibodies Monoclonal antibodies of the present invention are also useful for diagnostic purposes. They can be either labeled or unlabeled for this purpose. Typically, diagnostic assays entail the detection of the formation of a complex through the binding of the monoclonal antibody to an HIV antigen. When unlabeled, the antibodies find use, for example, in agglutination assays. In addition, unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) that are reactive with the monoclonal antibody, such as antibodies specific for immunoglobulin.· Alternatively, the monoclonal antibodies can be directly labeled. A wide variety of labels may be employed, such as radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens), etc. Numerous types of immunoassays are available and, by wav of example, some include those described in U.S. Patent Nos. 3,817,827; 3,850,752; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876, to all of which reference should be made for details.

Commonly, the monoclonal antibodies and peptides of the present invention are utilized in enzyme immunoassays, where, for example, the subject antibodies, or second antibodies from a different species, are conjugated to an enzyme. When a biological sample containing HIV antigens, such as human blood serum, saliva, semen, vaginal secretions or viral infected cell culture suspension, is combined with the subject antibodies, binding occurs between the antibodies and those molecules exhibiting the desired epitope. Such proteins or viral particles may then be separated from the unbound reagents, and a second antibody (labeled with an enzyme) added. Thereafter, the presence of the antibody-enzyme conjugate specifically bound to the antigen is determined. Other conventional techniques wall known to those skilled in the art may also be utilized.

Kits can also be supplied for use with the subject antibodies in the detection of HIV infection or for the presence of HIV antigen. Thus, the subject monoclonal antibody compositions of the present invention may be provided, usually in a lyophilised form, either alone or in conjunction with additional antibodies specific for other epitopes of HIV. Th© antibodies, which may be conjugated to a label or unconjugated, are included in the kits with buffers, such as Tris, phosphate, carbonate, etc.., stabilizers, biocides, inert proteins, e.g», bovine serum albumin, or the like. Generally, these materials will be present in less than about 5% wt. based on the amount of active antibody, and usually present in total amount of at least about 0.001¾ wt. based again on the antibody concentration. Frequently, it will be desirable to include an inert extender or excipient to dilute the active ingredients, where the excipient may be present in from about 1¾ to 99% wt. of the total composition.

Where a second antibody capable of binding to the monoclonal antibody is employed, this will usually be present in a separate vial. The second antibody is typically conjugated to a label and formulated in an analogous manner with the antibody formulations described above .

The detection of gpllO or ρ25 antigens, or the whole virus, in various biological samples may find use in diagnosing a current infection by the HIV virus.

Biological samples can include, but are not limited to, blood serum, saliva, semen, tissue biopsy samples (brain, skin, lymph nodes, spleen, etc.), cell culture supernatants, disrupted eukaryotic and bacterial expression systems and the like. Presence of virus is tested for by incubating the monoclonal antibody with the biological sample under conditions conducive to immune complex formation, followed by the detection of complex formation. Xn one embodiment, complex formation is detected through use of a second antibody capable of binding to the monoclonal antibody which is typically conjugated to a label and formulated in an analogous manner with the antibody formulations described above. In another embodiment, the monoclonal antibody is attached to a solid phase support which is then contacted with a biological sample. Following an incubation step labeled monoclonal antibody is added to detect .the bound antigen.

Preparation and Use of Synthetic Peptides Novel peptides are provided in the present invention which, inter alia, immunologically mimic protein epitopes encoded by the HIV retrovirus, particularly epitopes encoded within the env or gag regions of the viral genome encoding the gpllO or ρ25, respectively. To accommodate strain-to-strain variations among different isolates, adjustments for conservative substitutions, and selection among the alternatives where non-conservative substitutions are involved, mav be made. These peptides can be used as immunogens, to inhibit or eliminate HIV antigen production in vitro or in vivo, for the detection of the virus or of antibodies to the virus in a physiological sample. Depending upon the nature of the protocol, the peptides may be conjugated to a carrier or other compounds, labeled or unlabeled, bound to a solid surface, or the like.

In one embodiment, peptides of interest will be derived from the gpllO region of the virus. Of particular interest is the region within the env open reading frame extending from about base pair (bp) 6688 to about bp6750 and from about bp7246 to about 7317.

The peptides of interest, including blocking peptides, will include at least five, sometimes six, sometimes eight, sometimes twelve, sometimes 21, usually fewer than about 50, more usually fewer than about 35, and preferably fewer than about 25 amino acids included within a sequence coded for by an HIV retrovirus. Desirably, the peptide will be as small as possible while still maintaining substantially all of the immunoreactivity or antiviral activity of the larger peptide. In some instances it may be desirable to join two or more oligopeptides which are non-overlapping to form a single peptide structure or to use them as individual peptides at the same time, which separately or together provide equivalent sensitivity to the parent.

The peptide may be modified by introducing conservative or non-conservative substitutions in the peptide, usually fewer than 20 number percent, more usually fewer than 10 number percent of the amino acids being exchanged. In those situations where regions are found to be polymorphic, it may be desirable to vary one or more particular amino acids to more effectively mimic the differing epitopes of the different retroviral strains. In many instances to provide chemical stability, methionine may be replaced by norleucine (Ncr) .

It should be understood that the peptide employed in the subject invention need not be identical to any particular HIV polypeptide sequence, so long as the subject compound is able to provide for immunological competition with proteins of at least one of the strains of the HIV retrovirus. Therefore, the subject peptide may be subject to various changes, such as insertions, deletions, and substitutions, either conservative or non-conservative, where such changes might provide for certain advantages in their use. By conservative substitutions is intended substitutions with10 in groups such as gly, ala? val, ile, leu? asp, glu; asn, gin; ser, thr? lys, arg; phe, tyr; and nor, met. Usually, the sequence will not differ by more than 20% from the sequence of at least one strain of an HIV retrovirus except where additional amino acids may be added at either terminus for the purpose of providing an arm" by which the peptide of this invention aay be conveniently immobilized. The arms will usually be at least 1 amino acid and may be 50 or more amino acids, more often 1 to 10 amino acids9 in length.

The peptide in which the amino acid sequence is modified by the substitution, addition or deletion of amino acid residues should retain substantially all of the immunoreactivity or antiviral activity of the unmodified peptides, which may be conveniently measured by various assay techniques disclosed herein. The d'isomer form of one or more amino acids may be used, as desired, to modify biologic properties, such as activity „ rat© of breakdown, etc.

In addition, one, two, or more amino acids may be added to th© termini of an oligopeptide or peptide to provide for ease of linking peptides one to another, for coupling to a support or larger peptide, for reasons to be discussed subsequently, for modifying the physical or chemical properties of the peptide or oligopeptide, or the like.

Amino acids such es tyrosine, cysteine, lysine, glutamic or aspartic acid, or the like may be introduced at the C-or N-terminus of the peptide or oligopeptide to provide for a useful functionality for linking. Cysteine is particularly preferred to facilitate covalent coupling to other peptides or to form polymers by oxidation.

Additionally, the peptide or oligopeptide sequences may differ from the natural sequence by the sequence being modified by terminal- NH2 acylation, e.g., acetylation, or thioglycolic acid amidation, terminalcarboxy amidation, e.g., with ammonia or methylamine, to provide stability, increased hydrophobicity for linking or binding to a support or other molecule, or for polymerization.

Thus, for example, in the peptides I-VIII and IX-XV disclosed above, when Y or ¥’ are present, a preferred embodiment exists when ¥ or Y comprises one or more cysteine residues or a combination of one or more cysteine residues with spacer amino acids.

Glycine is a particularly preferred spacer. Preferred peptides for use in oxidative polymerisation are those in which Y or Y' represents at least two cysteine residues. When two cysteine residues are present at the same end of the peptide, a preferred embodiment exists when the cysteine residues are separated by from one to three spacer amino acid residues, preferably glycine. The presence of cysteine residues may allow the formation of dimers of the peptide and/or increase the hydrophobicity of the resulting peptide which facilitates immobilisation of the peptide in solid phase or immobilized assay systems.

Of particular interest is the use of the mercaptan group of cysteines or thioglycolic acids used for acylating terminal amino groups or the like for linking two of the peptides or oligopeptides or combinations thereof by ε disulfide linkage or a longer linkage to form polymers that contain a number of epitopes. Such polymers have the advantage of increased immunological reaction. Where different peptides are used to make up the polymer, they possess the additional ability to indue® antibodies that immunoreact with several antigenic determinants of different HIV isolates .

To achieve the formation of antigenic polymers (synthetic multimers, i9 compounds may be employed having bis-haloacetyl groups, nitroarylhalides, or the like, where the reagents are specific for thio groups. Thus, the linking between the two mercapto groups of the different peptides or oligopeptides may be a single bond or a linking group of at least 2, usually at least 4, and not more than about 16, usually not more than about 14 carbon atoms.

The subject peptide may be employed linked to a soluble macromolecular (e.g., not less than SkDal) carrier. Conveniently, the carrier may be a poly(amino acid), either naturally occurring or synthetic, to which antibodies are unlikely to be encountered in human serum. Exemplary of such carriers are poly-Llysine, keyhole limpet hemocyanin, thyroglobulin, albumins, such as bovine serum albumin,, tetanus toxoid, etc. The choice of the carrier is primarily dependent upon the ultimate use intended for the antigen, and one of convenience and availability.

With such conjugates, there will be at least one molecule of at least one subject peptide per macromolecule and not more than about 1 per 0„5kDal, usually not more than about 1 per 2kDal of the macromolecule. One or more different peptides may be linked to the same macromolecule.

The manner of linking is conventional, employing such reagents as p-maleimidobenzoic acid, pmethyldithiobenzoic acid, maleic acid anhydride, succinic acid anhydride, glutaraldehyde, etc. The linkage S may occur at the N-terminus, C-terminus or at a nite intermediate to the ends of the molecule. The subject peptide may be derivatized by linking, may be linked while bound to a support, or the like.

Various assay protocols familiar to those 10 skilled in the art may be employed for detecting the presence of either antibodies to retroviral proteins or retroviral proteins themselves, of particular interest is using the peptide as the labeled reagent, where the label allows for a detectable signal, or binding the peptide, either directly or indirectly to a surface, where antibody to the peptide in the sample will become bound to the peptide on the surface. The presence of human antibody bound to the peptide can then be detected by employing a xenogeneic antibody specific for hu20 man immunoglobulin, normally both human IgM and IgG, ox' a labeled protein specific for immune complexes, e.g., Rf factor of S. aureus protein A.

Illustrative of an assay technique is the use of sample container, e.g., wells of microwell plates, where the subject polypeptide or conjugates thereof are adsorbed to the container bottom and/or walls either covalently or non-covalently. The sample, normally human blood or serum diluted in appropriately buffered medium, is added to the container and a sufficient time allowed for complex formation between the polypeptide (s) and any cognate antibodies in the sample. The supernatant is removed and the container washed to remove non-specifically bound proteins. A labeled specific binding protein which specifically binds to the complex, such as xenogeneic antiserum to human immunoglobulin, is employed for detection.

The peptide can be prepared in a wide variety of ways. The peptide# because of its relatively short size, may be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers are commercially available today and can be used in accordance with known protocols, See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., 1984? and Tam et al.» J. Am. Chem. Soc. (1983) 105:6442.

Alternatively, hybrid DNA technology may be employed where a synthetic gene may be prepared by employing single strands which code for the polypeptide or substantially complementary strands thereof, where the single strands overlap and. can be brought together in an annealing-medium so as to hybridize. The hybridized strands may then be ligated to form the complete gene, and, by choice of appropriate termini, the gene may be inserted into expression vectors, which are readily available today. See, for example, Haniatis et a1.. Molecular Cloning, A Laboratory Manual, CSH, Cold Spring Harbor Laboratory, 1982. Or, the region of the viral genome coding for the peptide may be cloned by conventional recombinant DMA techniques and expressed (see, Haniatis, supra).

DNA coding sequences from the LAV and ARV 2 isolates of HIV which may be used for expressing the peptides include the following: ^BRU TGT ACA AGA CCC AAC AAC AAT ACA AGA AAA AGT ATC CGT ATC CAG AGG GGA CCA GGG AGA GCA TTT GTT ACA ATA GGA AAA ATA GGA AAT ATG AGA CAA GCA CAT TGT ARV-2 TGT ACA AGA CCC AAC AAC AAT ACA AGA AAA AGT ATC TAT ATA GGA CCA GGG AGA GLA TTT CAT ACA ACA GGA AGA ATA ATA GGA GAT ATA AGA AAA GCA CAT TGT Fragments from a sequence may be employed for expression of peptide fragments, conservative base changes can be made, where the modified codon is) code for the same amino acid is), or non-conservative changes in the coding sequence may be made, where the resulting amino acid may be a conservative or non-conservative change in the amino acid sequence, which was discussed previously.

The coding sequence may be extended at either the 58- or 3’-terminus or both termini to extend the peptide, while retaining its epitopic site(s). The extension may provide for an arm for' linking, e.g., to a label, such as an enzyme, for joining two or all of the peptides together in the same chain, for providing antigenic activity, convenient restriction sites for cloning, or the like.

The DNA sequence by itself, fragments thereof, or larger sequences, usually at least 15 bases, preferably at least 18 bases, may be used as nucleotide probes for detection of retroviral RNA or proviral DNA, or for identifying homologous regions for cloning or sequencing. Numerous techniques are described, such as the Grunstein-Hogness technique. Southern technique, Northern technique, dot-blot, improvements thereon, as well as other methodology, such as disclosed in U.S. Patent No. 4,358,535, to which reference should be made for details.

The subject peptides, including blocking peptides, and their analogs find use by themselves or in combination in vaccines. Similarly, anti-idiotype antibodies, i.e., reactive with the idiotypes of the antibodies of th® present invention and therebycontaining epitopes mimicking neutralizing regions of HIV, may also be used in vaccines. The peptides or anti-idiotype antibodies may be formulated in a convenient manner, generally at concentrations in the range of 1 ug to 20 mg/kg of host. Physiologically acceptable media may be used as carriers, such as sterile water, saline, 'phosphate buffered saline, and the like. Adjuvants may be employed, such as aluminum hydroxide gel, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, proteins (e.g., diptheria or cholera toxin) and oil emulsions. The peptides may also be incorporated into liposomes, or conjugated to polysaccharides, polypeptides or polymers for use in vaccine formula20 tion. Administration may be injection, e.g., intramuscularly, peritoneally, subcutaneously, intravenously, etc. Administration of an immunogenically effective dose may be one or a plurality of times, usually at one to four week intervals. An Bimmunogenically effective dose is that amount of a vaccine suitable to elicit ®n immune response in a host, whereby the host demonstrates increased infection.

The words NQMIDET, TWEEN, TRITON and KODAK are registered Trade Marks and the words SEPHAROSE and POLYBRENS are Trade Marks.

Other features and advantages of the present invention will become apparent from the following experimental descriptions, which describe the invention by way of example. The examples are offered by wav of illustration and not by way of limitation.

EXAMPLE I Generation and Characterization of Monoclonal Antibodies Example I describes the generation of hybrid cell lines which produce monoclonal antibodies specific for the envelope glycoproteins of HIV. This method involves the use of lectin purified extracts of attached to lentil lectin agarose as the immunogen.

The monoclonal antibodies subsequently generated by the hybrid cell lines are characterized by their ability to immunoblot and radioimmune precipitate gpllO from purified LAV and as biologically-expressed recombinant fusion protein. The monoclonal antibodies that bind to epitopes on gpllO are also reactive in ELISAs with disrupted whole virus, fusion proteins and synthetic peptides, and react with whole virus in indirect fluorescent assays.

The protocols for the generation of the hybrid cell lines producing monoclonal antibody and the characterization of the antibodies were as follows.

LAV virus purified from infected CEM cells JA.T.T.C. No. CHL8904) was disrupted in 50 mM Tris, pH (R) 7.4, 0.15 H NaCl, 1.0% Aprotinin, 2.0% Nonidet P-40 (NP-4 0) (octvIphenoxypolyethoxyethanol). The extract was clarified twice by centrifugation and adjusted to 0.5% NP-40 with the addition of three volumes of disruption buffer without NP-40. Lentil lectin Sepharose (Pharmacia, Piscataway, N.J.) was prewashed in disruption buffer without NP-40 and then equilibrated in adsorption buffer (50 mM Tris, pH 7.4, 0,15 M NaCl, 1.0% Aprotinin, 0.5% NP-40). Clarified viral extract was adsorbed with lentil lectin Sepharose for 42h at 4eC. Unadsorbed material was removed by washing with excess adsorption buffer. Elution of adsorbed material was carried out with 0.2 M alpha methyl mannoside in adsorption buffer. The eluent was dialyzed against PBS to remove the sugar and the material was readsorbed to the lentil lectin Sepharose.

The glycoprotein-lentil lectin Sepharose com5 plex was used to immunise BALB/c mice by three intraperitioneal injections without adjuvant given 2-3 weeks apart. Spleens were removed from immunised mice which demonstrated circulating antibody to glycoproteins of HIV by immunoblot, RIP and/or ELISA.

Protocols used for the generation of cell lines were generally those of Kohler and Milstein (Nature 256s495 (1985)5 with the modifications of Goldstein, I».C. * et al., (Infect. Immun,. 38s273 (1982)). Splenic 3-lymphocytes from the immunized mice were fused with NS-1 myeloma cells using 40% (w/v) polyethylene glycol. Following fusion fche cell mixture was resuspended in HAT medium (RPMI - 1640 medium supple-4 merited with 15% fetal calf serum, 1x10 ' M hypoxanthine,, 4x10 M aminopterin and 1.6x10 M thymidine) to select for the growth of hybrid cells, and then dispensed into 96-well microculture trays at a concentra6 tion of 1 to 3x10 cells/ml and incubated at 37°C in a humidified atmosphere containing 6% Cultures were fed by replacement of one-half the supernatant with fresh HAT medium. The wells were observed using an inverted microscope for signs of cell proliferation and when the cells were of sufficient density the supernatants were tested for anti-LAV antibody.

Wells containing hybrid cells producing anti30 body to LAV were identified by ELlSAs measuring the binding to either purified whole disrupted virus or biologically-expressed fusion proteins. ELISA assays using disrupted virus were carried out on LAV EIA plates (Genetic Systems, Seattle, Washington,. Plates were incubated with cell culture fluids at 37°C for 45 minutes and then washed three times with 0.05% Tween 20 in phosphate buffered saline (PBS-Tween).

Peroxidase-goat anti-mouse IgG ¢1:2,000 dilution in PBS-Tween? Zymed Laboratories, Inc., South San Francisco, California) was added ¢100 ul per well), and the plates were incubated for 45 minutes at 37°C and washed as above. Substrate (0.025 M citric acid, 0.05 H dibasic sodium phosphate, pH 5.0 containing 14 mg of o-phenylenediamine and 10 ul of 30% hydrogen peroxide per 50 ml) was added and the plates were incubated for 30 min at room temperature in the dark. The reaction was stopped with 3N sulfuric acid, and colorimetric reactions were quantitated with an automated microplate reader. Wells that gave positive results were subcloned by limiting dilution, retested for specificity, then expanded.

Th© monoclonal antibodies secreted by the resulting hybrid cell lines were further characterized as to specificity and reactivity by immunoblotting, immunoprecipitation and ELISA using disrupted LAV virus, recombinant LAV fusion proteins and synthetic LAV peptides. All antibodies were determined to be of the IgG^ isotype. Cell lines HIV-gpllO-l, HIV-gpl10-2, and HIV-gpHO-3 have been deposited with the American Type Culture Collection prior to the filing of this application and designated A.T.C.C. Nos. HB 9175, H3 9176, and HB 9177, respectively.

Recombinant fusion proteins tested for reactivity have previously been designated ENV2, ENV3, ΕΝΎ4 and ENV5. .Protein ENV2 is expressed from pENV2 (A.T.C.C. No. 53071), which is a region of LAV from base pair (bp) 6596 through bp 7178 (numbering according to Wain-Hobson et al», Cell 44:9 (1985)), ENV3 is expressed from pENV3 (A.T.C.C. No. 53072), which comprises the LAV region from bp 7178 through bp 7696; SNV4 is expressed from pENV4 (A.T.C.C. No. 53073,, end comprises bp 7698 through bp 8572,- and ENV5 is expressed from pENV5 (A.T.C.C. 'No. 53074) , which comprises the LAV region bp 5889 through bp 7698. The production of the recombinant fusion proteins is described in detail i commonly-owned pending U.S. patent application, Serial No.. 721.,237, to which reference should be made for details.

Assembly of Synthetic Peptides Peptides I (29, and VIII (110-2-2, were assembled on a benzhydrylamine (polystyrene/divinylbenzene) resin (Applied Biosystems, Inc,^ Foster City, California, . Peptide V (177, was assembled on a tbutyloxycarbonyl (@oe, -ethylbenzylcysfceine-phenylacetamidomethyl (PAH, polystyrene/divinylkenzene resin. Symmetrical anhydride couplings were carried out in an Applied Biosystems 430A synthesizer. Cysteine was added as the first residue in both peptides.

Dicyclohexlycarbodiimide couplings in the presence of hydroxylbenzotriazole were used for asparagine and glutamine. Benzyl-based side chain protection and Boc alpha-amine protection were used. Other side chain protection routinely used was Boc (formyl, tryptophan, Boc methionine sulfoxide, Boc (tosyl, arginine, Boc (methyIbenzyl) cysteine Boc (tosyl) histidine, Boc (chlorobenzyloxycarbooyl) lysine and Boc (bromobenzyloxycarbonyl, tyrosine. >Jhen the peptides were radiolabeled, it was . by acetvlating the amino terminus with Ή-acetxc acid and an excess of dicyclohexylcarbodiimide.

Deprotection and cleavage of the peptide from the resin was by the Tam "low-high" HF protocol (Tam et al., supra). Extraction from the resin was with 5% acetic acid and the extract was subjected to gel filtration chromatography in 5% acetic acid.

Synthetic HIV peptides tested for reactivity with the monoclonal antibodies included peptides 29, 36, and 39. Peptide 29 is encoded by the LAVg^^ genomic region from about bp 6688 through bp 6750? peptide 36 is encoded by the region from about bp 7246 through bp 7317? and peptide 39 is encoded by the region from about bp 7516 through bp 7593. Peptides 36 and 39 are described in detail In U.S. Patent 4,629,783, to which reference should be made.

The blocking peptides, IX-XV, were assembled essentially as described above on a methyl-benzhydrylamine (polystyrene/divinzlbenzene) resin {Applied 3io~ systems, Inc., Foster City, California). Symmetrical anhrydride couplings were performed on an. Applied Biosystems 430A synthesizer. Dicyclohexylcarbodiimide couplings in the presence of hydroxyIbenzotriazole were used for asparagine. For protection, benzyl-based side chain and Soc alpha-amine protection were used, while Boc (bromobenzyloxycarbonyl) was used specifically for tyrosine side chains. Acetylation, when present, was carried out using acetic anhydride or glacial acetic acid and dicyclohexylcarbodiimide. Deprotection and cleavage of the peptide from the resin was accomplished by the standard high" HF protocol (Stewart ef al., supra). Extraction from the resin was performed with 50% acetic acid, and the extract was subsequently subjected to gel filtration chromatography in 20% acetic acid. As desired, high performance liquid chromatography was performed on a Vyaac CIS column (Rainin Instrument Co., Emeryville, CA) using a 0.11 trifluoracetic acid, acetonitrile gradient™ Immunoblotting Characterization by immunoblotting was carried out on clone supernatants or ascites fluid using purified LAV virus and recombinant fusion proteins as antigens. The antigens were first separated by polyacrylamide gradient gel electrophoresis (7.0-15.0%) and transferred to nitrocellulose membrane ?NCM) by elec5 trophoresis for four hours at 25 V in 25 mM sodium phosphate (pH 7.0). After transfer® the NCM was blocked to prevent nonspecific interactions by incubation in PBS-Tween or Blotto <5t non-fat dry milk in PBS) for one hour at room temperature. The NCM was incubated with cell culture supernatant ox ascites fluid' diluted in PBS-Tween for one hour at room temperature and was rinsed with three changes of PBS-Tween. In the second step the NCM was incubated with goat anti-mouse IgGhorseradish peroxidase diluted in PBS-Tween for one IS hour at room temperature. This incubation was followed by washing with PBS-Tween and then immersion in horseradish peroxidase color development solution (Bio-Rad Laboratories, Richmond, California) for 20' minutes.

The reaction was stopped by immersion in deionized 2C water. Monoclonal antibody reactivity was compared to a positive control serum reactive with purified disrupted virus or expressed fusion protein. The results showed that all antibodies bound to gpllO and its precursor molecule, gpl50, using disrupted virus prepara*· tions. Antibodies 110-1 and 110-2 also recognized the fusion protein ENV3, whereas antibodies 110-3, 110-4, 110-5, and 110-6 immunocomplexed ENV2.

Immunoprecipitation Viral extracts for radioimmune precipitation were prepared from CEM cells infected with the LAVg^r, isolate of HIV adapted to lytic growth by continuous passage. When early eytopathie effects were evident, the cells were transferred to labeling media containing 3 · (Sj-methionine (0.05 mCi/ml) or (H)-glucosamine (0.025 mCi/ml), then incubated for 24h until most of the cells had lysed, releasing virus into the culture supernatant. Virus was pelleted (one hour at 100,000 xg) from the cell-free supernatant, and deter» gent extracts were prepared in P-RIPA buffer (phosphate buffered saline containing 1.0% Triton X-100, 1.01 deoxycholate, 0.1% SDS, and It Aprotinin). Similar extracts were prepared from the supernatants of uninfected CEM cellsImmunoprecipitation assays were performed 10 with 100 ul of virus extract incubated with 100 ul culture supernatant from the hybrid cell lines for one hour on ice. Four microliters of rabbit anti-mouse Ig (Zymed Laboratories, So. San Francisco, California) was added to each sample and incubated 30 minutes. Immuno15 precipitin (100 ul; Bethesda Research Laboratory, Bethesda, Maryland) resuspended in P-RIPA buffer containing 1.0% ovalbumin was added to each sample and incubated for an additional 30 minutes. The bound complexes were washed and separated by SDS-polyacrylamide gel electrophoresis (15.0% acrylamide: DATD gel).

Following electrophoresis the gels were fixed, soaked in Enhance (New England Nuclear, Boston, MA), dried and exposed to Kodak XR-5 film. A reference positive serum which immunoprecipitated all HIV viral proteins was re25 acted with viral-infected and mock-infected CEM cell supernatants as positive and negative controls.

The results showed that all six monoclonal antibodies specifically immunoprecipitated gpllO and gplSO.

Enzyme-Linked Immunoadsorbant Assay To map the gpllO epitopes being recognized by the monoclonal antibodies of the present invention, culture supernatants from hybrid cell lines or ascites fluid were further characterized by reactivity in ELISAs with biologically-expressed fusion proteins and 4? synthetic peptides, Procedures were the same as those described above with the exception that fusion proteins or synthetic peptides replaced purified virus as the antigen adsorbed to the surface of the microtiter weIls.

When peptides were psed as antigen the plating protocol was as follows» Lyophilised peptide was dissolved in 6M guanidine HCI. dust prior to plating in the 96 well plates, the guanidine solution was diluted into 0.05 M carbonate/bicarbonate buffer (pH 9.6) fco a final peptide concentration of up to 100 ug/ml. A SO ul volume of the diluted peptide was placed in each snicrotiter well and the plates were then incubated overnight at 4°C. Excess peptide solution was shaken out, plates blocked Blotto, and the procedure described above was followed for fche rest of the ELISA- Similarly, recombinant protein was diluted to a final concentration of about 2 ug/ml in 0.05 M carbonate/bicarbonate buffer (pH 9.6) before the same procedure was followed.

‘The results are shown in Table II- Monoclonal antibodies produced by cell lines HIV gpll0-l and HIV gpllO-2 reacted with ENV3, ENV5, peptide 36 and disrupted virus. Antibodies from cell lines HIV~gpll03, HIV-gp-110-4? HIV~gpll0-5 and HIV-gpllO»6 reacted with ENV2 and peptide 29 as well as. disrupted virus. <8 TABLE II ELISAe Shoving Reactivity of Monoclonal Antibodies With Racorabinant Proteins and Synthetic Peptides Recombinant Fusion Protein Synthetic P< aptide 39 LAV Control CEH Control ENV 2 ENV 3 EMV4 EMV5 29 35 110-1 0.077 3.000 0.113 3.000 MD <5 A *> 1 β» β ai, £ 0.054 0.908 0.125 no-2 -0.003 3.000 0.000 3.000 MD 2.305 -0.005 1.214 0.009 110-3 3.000 o.on HD MD 3.000 HD 0.0Π 0.363 0.046 110-4 3.000 0.020 MD MD 3.000 MD 0.016 0.383 0.067 Π0-5 3.000 0.014 MD MD 3.000 HD 0.016 0.368 0.025 no-6 3.000 0.033 MD MD 1.937 MD 0.017 0.486 0.032 The results in Table II demonstrated that monoclonal antibodies 110-1 and 110-2 recognized an antigenic determinant encoded by a DNA sequence within the pENV3 region, more particularly by the region of the HIV genome defined by a sequence of amino acids within peptide 36. That is, monoclonal antibodies gpllO-l and 110-2 bind to a peptide region of gpllO encoded within bp7246 through 7317, as evidenced by the formation of immune complexes with peptide 36 and ENV3. This region of the HIV genome has previously been identified as conserved, i.e.# little change in fche DNA sequence in the region encoded by peptide 36 among different viral isolates from diverse geographical locations. See Starcich et al. „ Cell 46:637 (1986),, In contrast, monoclonal antibodies gpllO-3» -4# -5, and -6 bind to peptides of sIV defined by the region encoded by peptide 29 from bp 6688 through about bp 6750. The region in gpllO defined by peptide 29 has been identified as containing several nucleotide substitutions among different viral isolates. Monoclonal antibodies that selectively bind gpllO polypeptides which contain conserved epitopes, such as antibodies 110-1 and 110-2, may have enhanced utility in a variety of circumstances, such as in affinity chromatography, etc. Also, in an ELISA assay, the peptide 110--2-2 reacted with sera from the individual from whom LAV-2 was isolated. Indirect Immunofluorescent Assay Indirect immunofluorescent assays using monoclonal antibodies directed against the gpllO antigen of HIV were carried out on acetone-fixed and live cells. Acetone-fixed slides prepared from LAV-infected CEM cells were incubated with diluted culture supernatant or ascites fluid for one hour at 37°C, while live cells were incubated with culture supernatant or ascites fluid for one hour at 4®C before the cells were plaged SO on slides and acetone fixed. Both methods used fluorescein isothiocyanate-labeled anti nouse IgG to detect cells bearing the reactive gpllO antigen. Monoclonal antibody Hiv-gplio~l gave positive results using either live or acetone-fixed LAV-infected cells.

EXAMPLE II Neutralization of HIV Infectivity by Anti-gpllO Monoclonal Antibodies This example describes and characterizes the neutralization of HIV infectivity using monoclonal antibodies which bind to gpllO and peptides within gpllO.

The results indicate that monoclonal antibodies gpllO3,, -4, -5 and -6 possess neutralising activity, and that gpllO-3 and -4 possess particularly high levels of neutralizing activity.

Neutralization Assay A sensitive neutralization assay was developed to quantitate the effect of the monoclonal antibodies on HIV infectivity. A CD4+ cell line highly susceptible to HIV infection, GEM, vas chosen as a target cell for infectivity comparisons. Ascites fluid prepared as described in Example I, or the IgG fraction thereof purified using ammonium sulfate precipitation, was heat inactivated at 56°C for 30 minutes, then diluted as required in RPMI medium containing 201 fetal calf serum. A suspension of HIV strain LAV-0RU was harvested from about four-day cultures of CEM in log growth phase, filtered through 0.2 or 0.45 micron filters, aliquoted, and frozen at ~70®C. One aliquot was thawed, titrated to determine the ΤϋϊΟ^θ, and subsequent assays performed with freshly thawed aliquots, diluted Is 500 in culture medium to a concentration of approximately ten times the amount required to infect 50% of CEM cells in culture <10 ΤΟΟ^θ). The virus suspension was mixed with an equal volume (250 ul) of monoclonal antibody preparation of five-fold dilutions from 1:5 to 1:9,765,625. The virus/antibody mixture was incubated for 45 minutes at 37C and then duplicate from 1:5 to 1:9,765,625. The virus/antibody mixture was incubated for 45 minutes at 37eC and then duplicate samoles of 200 ul wsed to inoculate wells containing 5 1.0 ml of approximately 2x10 CSM cells per well. The cultures were incubated 37®c xn a humidified, 5¾ C02 atmosphere for 14 days. The cells were harvested, pelleted, and lysed with 1¾ Triton X-100 in PBS for about 10 minutes. The amount of virus ior viral antigen) present in lysed cells was quantitated using a sensitive HIV antigen capture sandwich enzyme immuno15 assay described below. The titer of neutralizing activity, if any, was determined as the reciprocal of the dilution of monoclonal antibody which inhibited antigen production by greater than 501 of virus control cultures incubated without antibody, or with a mono20 clonal antibody of the sameisotype which had previously been shown to lack neutralizing activity.

The HIV antigenic capture assay referred to above employed two monoclonal antibodies directed against p25 antigens as capture reagents. These hybri25 dome cell lines were generated by the methods described above with minor modifications, including using a purified gag recombinant fusion protein as the immunogen and characterizing the resultant «monoclonal antibodies as to specificity and reactivity using recombinant fu30 ©ion proteins previously designated GAG-1, GAG-2, and GAG-3, and the synthetic peptide 141. Protein GAG-1 is expressed from pGAG-1 (A.T.C.C. No. 53379), GAG-2 is expressed from pGAG-2 (A.T.C.C. No. 53111) and GAG-3 is expressed from pGAG-3 (A.T.C.C. No. 53112). The pro- duction of the recombinant fusion proteins is described >2 in detail in commonly owned pending patent application numbers U.S.S.N. 764,460 and 828,828, to uhich reference should be mode for details. Synthetic peptide 141 is encoded by the LAV^^ genomic region corresponding to the amino acid residues 198-242. Monoclonal antibody produced by hybridoma cell lines p25-2 and p25-3 are found to be reactive with recombinant fusion proteins GAG-1, GAG-2, and GAG-3, and the monoclonal of p25-3 also is reactive with synthetic peptide 141.

To perform the antigen capture assay, the capture reagents which were first adsorbed to a solid support. Ascites fluid derived from hybridoma cell lines p25-2 and p25-3 were diluted 1:5000 in 25 mM Tris buffer, pH 3.5 and 200 ul was- placed into wells of microwell plates. Wells were sealed and incubated for about IS hours at 4®c„ The solution was removed from the wells by aspiration before a blocking solution of 0.3% Blotto in PBS was added. Blocking was carried out for 15 minutes at room temperature. The blocking solu20 tion was aspirated and the sample was added. Two hundred microliters of the lysed cellular suspension and 5.0 ul of detection conjugate, prepared as described below, were added to each well. The well strips or plates were incubated for 2 h at 37eC, after which the suspension was aspirated and the wells washed four times with buffer (0.05% Tween 20 in P3S) . The detection conjugate was prepared as follows. Monoclonal antibodies p25-6 and p25-7 were conjugated to horseradish peroxidase CHRP) at a 3:1 molar ratio (AbsHRP) for three hours using a periodate oxidation procedure (Nakane, et al., J. Bistochem. Cytochem. 22:1084 (1974)). Conjugates were diluted 1:1500 in 2.5% (w/v) non-fat dry milk, 0.01% thimerosal, and 0.005% Antifoam A in 20mM sodium citrate. The remainder of the ELISA procedure was performed as described in Example I.

Th® results of the assay of neutralizing activity with antibodies gpllO-3, -4, -5, and -6 are as follows. Th© highest titers which retained neutralizing activity were determined to be; gpllO-3 * 15,625; gpllO-4 «= 9,765,625; gpllO-5 « 125; and gpll0-6 « 625.

Due to the predicted genetic variability of the region defined by peptide 29, the ability -of monoclonal antibody gpllO-4 to recognise other isolates of HIV was examined. The immunofluorescence assays were performed as described above- Antibody gpllO-4 detected antigen in cultures of at least 3 of 16 HIV isolates tested.

Antibody gplio~4 was able to neutralize viruses isolated over fifteen weeks from a chimpanzee inoculated with LAVQRU as a control animal in an AIDS vaccine trial. The monoclonal antibody was able to neutralize the isolates even though the animal had serum antibodies which neutralized HJV in vitro, and had developed a measurable cell mediated immune response to the KIV infection. This indicates a lack of antigenic drift in vivo for the epitope recognized by the gpllO-4 antibody.

EXAMPLE III Neutralization of HIV Infectivity by a Coctail of Anti-gpllO and Anti-p25 Monoclonal Antibodies This example descx'ibes the neutralization of HIV infectivity using monoclonal antibodies which bind to gpllO and peptides within gpllO in combination with monoclonal antibodies which bind to p25 and peptides within p25, which alone show little or no neutralizing activity. The results indicate that monoclonal antibody gpllO-2 in combination with either p25-5 or p25~7 possesses particularly high levels of neutralizing activity.

Hybridoma cell lines p25-S and p25-7 were generated by the methods described above with modifi54 cations which include using inactivated disrupted virus or a purified gag recombinant fusion protein expressed in E. coli as the immunogen, and characterizing the resultant monoclonal antibodies as to specificity and reactivity using the recombinant fusion proteins previously designated GAG-1, GAG-2, and GAG-3 and the synthetic peptides 15, 38, 150, 147, and 148. The synthetic peptides are encoded by the I*AVSRy genomic region corresponding to amino acid residues as follows: peptide 15, amino acid residues 329 through 350; peptide 88, amino acid residues 315 through 350; peptide 150, amino acid residues 318 through 363; peptide 147, amino acid residues 278 through 319; and peptide 148, amino acid residues 290 through 319» Monoclonal antibodies produced by hybridoma cell lines p25-6 and p25-7 are reactive with the recombinant fusion protein GAG-3, p25-6 is reactive with synthetic peptides 147 and 148, and p25-7 is reactive with synthetic peptides 15, 88, and 150.

Neutralization assays were carried out as described above, except when cocktails were used, individual monoclonal antibodies were first diluted 1;5 in culture medium and then mixed in equal ratios to yield a final dilution of 1:10. The remainder of the assay was carried out as described above.

Monoclonal antibodies gpllO-2, p25-6 end p25-7 show less than 50% neutralizing activity when used alone. When used in a cocktail which includes monoclonal antibodies gpll0~2 with p25-6 or gpllO-2 with p25-7 at a 1:10 dilution, there was total neutralization. A cocktail including the monoclonal antibodies p25-5 in combination with p25-7 gave neutralizing activity range of 60-90%.

EXAMPLE IV Immunopotency of Peptide 29 end Homoloqs The ability of peptide 29 and homologous peptides, including peptide 177, to stimulate an immune response against HIV was examined in two strains of mice. The procedures for the preparation of the peptide immunogens, immunization protocols, and for the characterization of the immune response generated are detailed below.

Peptide 29 was prepared for immunization by conjugation to a purified thyroglobulin. Thyroglobulin may be derivatized with N-succinimidyl-4-(N-maleimidomethy1)cyclohexane-l-carboxylate (SMCC) for conjugation according to the procedure outlined in U.S. 4,629,783, col. 10, lines 28-51. As a second immunogen, thyroglobulin was derivatized with glutaraldehyde as follows. Porcine thyroglobulin, 27 mg, was dissolved in 1 ml of 0.1 M sodium bicarbonate, to which 8.3 jil of a 25% glutaraldehyde solution was added dropwise, and the mixture stirred overnight at room temperature. 1 ml of sodium carbonate/bicarbonate buffer, pH 9.3, was added to the solution and then dialysed for 8 hours against 2 liters of the same buffer at 4°C,-with a complete change of dialysate at 4 hours. Peptide 29 was then added to the derivatized thyroglobulin at approximately 100 molar excess, and the mixture stirred overnight at room temperature. Unreacted glutaraldehyde was blocked with 200 μϊ of a 0.2 M lysine solution, which mixture was stirred for several hours {or overnight) at room temperature. Tha peptide-thyrcglobu 1 in conjugate was then dialyzed extensively against PBS at 4°C.

Two strains of mice, (C57 black and BALB/c) were inoculated with peptides prepared by each method of conjugation. All animals were about 2-4 weeks old at the time of inoculation. The routes of inoculation £ include footpad, tail scarification, subcutaneous, intranasal, or intraperitioneal. The inoculum consisted of 25 pg of conjugated peptide suspended in 0„5 ml of complete Freund’s adjuvant, with booster inoculations repeated at weeks 2, 3 and 5 with the same immunogen suspended in incomplete Freund’s adjuvant. Serum samples from individual mice were collected prior to immunisation, 4 days after the booster immunization at 3 weeks, and 4 days after the final booster at 5 weeks. The serum samples were analyzed for antibodies against homologous peptides or whole virus by screening in ELISAs. The sera demonstrating antibody activity to LAV are further screened for neutralizing activity, followed by analyzing the sera in immunoblots to disrupted LAV antigen and radioimmunoprecipitation assays with radiolabeled gpllO.

The results of immunizations indicated that mice immunized with peptide 29 developed antibodies reactive with peptide 29 and disrupted LAV-l virus in ELISA’s. Conjugation of peptide 29 through glutaraldehyde generally elicited higher titer of antibodies to peptide 29 in Balb/c mice, although conjugation through maleimide did successfully elicit anti-peptide 29 antibodies in some Balb/c mice. Mice immunized with peptide 277 developed antibodies to the peptide, and conjugation of peptide 177 through glutaraldehyde was better at eliciting an immunological response. C57 mice were more responsive to immune stimulation with peptide 177 than Balb/c mice. Mice immunized with the LAV-2 peptide 110-2-2 developed antibodies to 110-2-2 and LAV-2 virus as shown by ELISA’s. Peptide 110-2-2 conjugated through glutaraldehyde was immunogenic in both C57 and Balb/c mice, although titers to the 110-2-2 peptide were generally higher in Salb/c than C57 mice, while titers to LAV-2 virus were generally higher in C57 mice than Salb/c mice, Generally, serum samples from immunized mice 5 which ii) demonstrate antibodies to whole virus; (ii) are able to neutralize HIV, such as in the assays described in Example II;; and (iii) are reactive with peptide 29 in ELISAs, cumulatively indicate the efficacy of the use of peptide 29 in a vaccine 1° formulation.

EXAMPLE V Immunoaffinity Separation of GP110 Using Monoclonal Antibody Monoclonal antibodies to the gpllO antigen of HIV can be used in immunoaffinity separation procedures to substantially purify bacterially-expressed recombinant fusion proteins. If the expressed protein is secreted by the bacteria, the protein may be isolated from the culture supernatant? if the protein is not 2o secreted, disruption of the bacterial cells may be necessary.

Construction of plasmid pEWV-5 (A.T.C.C. No. 53074) is described in copending commonIv-owned patent application U.S.S.N. 721,237, to which reference should be made for details. Plasmid pENV-5 encodes a major portion of the carboxyl end of gpllO and a portion of the amino terminus of gp4i of LAV inserted into the trp expression vector. coli C600 transformed by this vector expresses but does not secrete the gpllO fusion protein.

E. coli C600 containing the plamid pENV-5 are grown in medium containing tryptophan (20 ug/ml) and ampicillin (100 ug/ml) overnight at 3?®C with aeration. The overnight cultures are then inoculated at 1:100 into fresh minimal medium containing ampicillin (100 ug/ml) but not tryptophan. These cultures are grown with aeration for 2-3 hours (up to early log phase) at 37"C. The inducer, 3-B-indole-acrylic acid (Sigma Chemical Co., St. Louis, MO) f is added to a final concentration of 20 ug/ml from freshly made stocks of 20 mg/ml in 95% ethanol. Induced cultures are then grown at 37ΰΟ with aeration for 4 to 5 hours and then pelleted and, optionally, frozen. Protein yields from pENV-5 are typically less than 1 mg/liter.

The pelleted bacterial cells are lysed using P-RIPA buffer (PBS containing 1% Triton X-100? 1% deoxycholate, 0.11 sodium dodecyl sulfate? and 1% Aprotinin) which will lyse E. coli cells. The suspension can be sonicated to shear DNA end RNA, followed by centrifugation to remove particulates, A dilution or concentration step may then be needed to standardize the protein concentration.

Monoclonal antibody Hlv-gpllO-l is precipitated initially from ascites fluid or cell culture supernatants at room temperature or in the cold with iNH,)_SO, or Na-SO. solutions buffered at oH 7.3 to 4 2 4 2« final saturation of 33 or 18%, respectively. Precipitated proteins are removed by contrifugation and redissolved in PBS and precipitated a second time with 33% (NH^),SO4 or 12-15% This step can be repeated as necessary. The pellet is again dissolved in PBS and the excess salts are removed by gel filtration through a desalting matrix or by exhaustive dialysis against PBS.

Purified 110-1 monoclonal antibody can then be coupled to the cyanogen bromide-activated Sepharose.

The necessary amount of gel is swollen in 10 M HCl solution on a glass filter (1 g of freeze-dried material yields a final gel volume of approximately 3.5 ml) and washed for 15 minutes with the same solution, and the antibody added immediately thereafter.. In general, the coupling reaction proceeds most efficiently in a pH range 8-10, but a lover pH can be used if needed for antibody stability. The antibody should be dissolved in PBS, or a high ionic strength carbonate/bicarbonate or borate buffer with 150 mM NaCl. The activated Sepharose and antibody suspension is stirred gently for 2-4 hours at room temperature, or overnight at 4°C, and then washed on a course fritted glass filter with coupling buffer. Any remaining active groups are blocked by treatment with 1.0 M ethanolamine at pH 8 for 2 hours, The final antibody-Sepharose product is then washed alternately with high and low pH buffer solutions (borate buffer, 0.1M, pH 8.5, 1 M NaCl and acetate buffer, 0.1M, pH 4.0, 1 M NaCl, respectively) four or five times. This washing removes traces of non-covalently adsorbed materials. The finished immunoaffinity separation matrix is stored below 8°C in the presence cf a suitable bacteriostatic agent, such as 0.01% azide.

The addition of the expressed protein suspension to the immunoaffinity separation matrix results in the selective removal of the gpllO antigen. The mixture is allowed to interact for 2-,24 hours, preferably 12-18 hours, with slow stirring or rocking. A column format can also be used in which the immunoaffinity matrix is poured into a column, equilibrated, and the expressed protein suspension added slowly to the column. After the protein suspension has been added the flow should be stopped to allow maximum immune complex formation.

Unbound material is washed or separated away by extensive washing with adsorption buffer. A course fritted glass filter with vacuum can be used or column flow-through. The bound material is then eluted using low or high pH buffers (acetate buffer, pH 4.0 or borate buffer, pH 8.56) or a chaotropie agent.

EXAMPLE VI Immunoaffinity Purification of Recombinant GPliO From a Mammalian Expression System The monoclonal antibodies of the present invention find use in the immunoaffinity purification of recombinant fusion gpllO expressed by mammalian cells. Mammalian cells are infected with recombinant vaccinia (Mackett et al.e J. Virol. 49;857 (1984)e to which reference should be mede for details) that contain sequences encoding at least the portion of gpllO which is antigentic and elicits neutralizing antibodies.

The recombinant vaccinia is constructed according to the method described in U.S.S.N. 842,984, to which reference should be made for details. Briefly, sequences coding for the envelope glycoprotein of HIV are inserted into a plasmid vector (pGS20) downstream from a vaccinia transcriptional control element. This chimeric gene is flanked by sequences encoding the viral thymidine kinase (TK) gene.

Chimeric plssmid vectors containing vaccinia virus promoter ligated to the LAV envelope gene are used to transform E. colj, strain NC1O0O. Insertion of the chimeric LAV-env sequences into the vaccinia virus genome was achieved hy in vivo recombination, made possible hy the fact that the chimeric genes in plasmids pv-env5 are flanked by vaccinia virus sequences coding for the TS gene. This plasmid is then introduced into cells previously infected by wild-type vaccinia virus and recombination allowed to occur between the TK sequences on the plasmid and the homologous sequences in the vaccinia viral genome, thereby inserting the chimeric gene. African Green Monkey kidney cells (strain SSC-40, a line derived from BSC-1 cells, A.T.C.C. No.

CCL26) are used as the host in the expression system.

Confluent BSC-40 cells are infected at a multiplicity of infection ox 10 by recombinant vaccinia virus, infection is allowed to proceed for 12 hours, at which time the cells are harvested, washed once with PBS, and collected by centrifugation. Cell pellets are resuspended in lysis buffer (1.0% NP 40, 2.5% sodium deoxycholate, 0.2 M NaCl, 0.01 K M Tris-HCl, pH 7.4, 1 mM EDTA), and the lysate is then cleared by centrifugation. Immunoaffinity separation of the expressed recombinant fusion protein is performed as described above for the bacterial expression system, employing the monoclonal antibody gpllO-Ι. The proteins produced by this expression system more closely resemble natux’ally produced HIV gpllO because of the processing and glycosylation afforded by the mammalian cells. » fc EXAMPLE VII Inhibition of HIV Infeetlon_ with Blocking Peptides The effectiveness of blocking peptides in inhibiting the infection of tissue culture cells by the LAV stre^n HIV was studied utilizing a modification of the protocol for peptide T evaluation published by Pert et al., supra. The preferred HIV inhibition assays comprise combining equal volumes of blocking peptide and CEM cells <2.5 X 105) in medium (RPMI# 10% PCS and 2 mg/ml polybrene), and incubating for 45 minutes at 37®C. Virus is then added at various doses ¢10,. 50, 500 TCID 50) , the mixture incubated for 14 'days at 37°C# and then the supernatant assayed for virus antigen (e.g., p25 core) production.

Preincubation of the CEM cells with the peptides prior to virus addition into the cultures enhances the inhibiting effect in the assays. The inhibition «as found to be dependent on the virus dose# showing strong activity et low and medium doses, but less effect at the highest virus doses.

About 60% to 90% inhibition of virus antigen production in low virus dose experiments was achieved with Peptide T. Additional experiments with peptides X-XV, typically COOH-terminus amidated and ΝΗ,-terminus acetylated, produced similar results, while peptide XI was particularly effective over a broad dose range. Neutralizing antibodies may be added either during the preincubation period or ©t the time the virus is added to produce an additive or synergistic inhibition of viral antigen production.

From the foregoing, it will be appreciated that the monoclonal antibodies and peptides, including blocking peptides, of the present invention provide improved methods for neutralising and/or inhibiting HIV infections. This allows prophylactic and therapeutic compositions to be more easily developed that can be effective against Infections due to most, if not all, HIV strains. In addition, the novel materials find uses In diagnostic assays and other well-known procedures.

Although the present invention has been described In some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent that certain changes and modifications may be practiced within the· scope of the appended claims.

MKB2OSGms>L£Emi£J2m The following microorganisms which fora a part of the present invention were deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, IE S.A. Data regarding the deposits are as follows; Scientific Beposit Data Mouse (balb hvbridona c/NS-1) HIV-gp 110-1 HS 9175 August 15,, 1986 ¢10 HIV-gp 110-2 BS 9176 August 15, 1986 HIV-gp 110-3 KB 9177 August 15, 1986 HIV-gp 110-6 HB 9404 April 30, 1987 HIV-gp 110-4 KB 9405 April 30, 1987 IfiB HIV-gp 110-5 HS 9406 April 30, 1987 w HIV-p ; 25-2 KB 9407 April 30, 1987 a 09 HIV-p ; 25-3 Ξ3 94 08 April 30, 1987 HIV-p : 25-6 HB 9409 April 30, 1987 03 Hiv-p : 25-7 HB 9410 April 30, 1987 Hybridomas KB 9175, HB 9176 and KB 9177 were tested and found viable on August 26, 1986. The remaining hybridomas were tested and found viable on Hay 4, 1987.

Claims (41)

CLAIMS:

1.I. Ai composition for use in the treatment of HIV infections comprising a therapeutically effective dose of at least one monoclonal antibody, specifically reactive with one or more neutralising regions (as hereinbefore defined) of gpllO or p25 of HIV, and a pharmaceutically effective carrier.

2. A composition according to claim 1, wherein the neutralising regions comprise epitopes encoded within the 10 env or regions of the HIV genome.

3. A composition according to claim 1, further comprising one or more blocking peptides capable of attenuating HIV infectivity and/or monoclonal antibodies reactive with epitopes of said peptides. 15

4. A composition according to claim 3, further comprising a cocktail of monoclonal antibodies reactive with different homologs of said peptides.

5. A composition according to claim 1, wherein one monoclonal antibody is reactive with at least one epitope of 20 LAV BRIJ P 25 amino acid sequence 278 to 319 and/or homologs thereof, and a second monoclonal antibody is reactive with at least one epitope in LAV BRU p25 amino acid sequence 315 to 363 and/or homologs thereof.

6. A composition according to claim 1, wherein one * 25 monoclonal antibody is reactive with at least one epitope of HIV p25 and a second monoclonal antibody is reactive with at s , least one epitope of HIV gpllO.

7. A composition according to claim 6, wherein the p25 epitope is located within LAV BRU amino acid sequence 278 to 30 319 or 315 to 363, or homologs of said sequences.

8. A composition according to claim 6, wherein the gpllO epitope is located within LAV 0RU amino acid sequence 308 to 328 or homologs thereof.

9. A composition comprising a monoclonal antibody, said 5 composition being specifically reactive with a neutralising region (as hereinbefore defined) of gpllO or p25 of HIV.

10. A composition according to claim 9, wherein the neutralising region is located on HIV gpllO.

11. A composition according to claim 10, wherein said lOregion comprises HIV gpllO amino acid sequence from about 301 to 336 or 308 to 328, or homologs of said sequences.

12. A composition according to claim 10, further comprising a monoclonal antibody reactive with a blocking peptide of HIV. 1513. A composition according to claim 9, wherein the neutralising region is located on HIV p25.

13. 14. A peptide comprising at least five contiguous amino acids from the following HIV gpllO amino acid sequence or homologs thereof: 20 II (29a) Cvs-Thr-Arg-Pro-Asn-Asn-Asn-Thr-Arg-Lys-Ser-IleArg-Ile-Gln-Arg-Gly-Pro-Glv-Arg-Ala-Phe-Val-ThrIle-Gly-Lus-Ile-Gly-Asn-Met-Arg-Gln-Ala-His-Cys.

14. 15. A peptide according to claim 14, wherein said 25contiguous amino acids define at least one antigenic determinant capable of eliciting antibodies upon immunisation into a host, wherein said antibodies are protective against HIV infections. 3016. A peptide comprising one of the following HIV gpllO amino acid sequences or homologs thereof: 66' I (29) Y-Thr-Arg-Lys-Ser-Ile-Arg-1le-Gln-Arg-Gly-ProGly-Arg-Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Y·; V (177) 5 Y-Thr-Arg-Lys-Ser-lle-Tyr-Ile-Gly-Pro-Gly-ArgAla-Phe-His-Thr-Thr-Gly-Arg-Ile-Y; or VIII (110-2-2) Y-Lys-Thr-Val-Lys-Ile-Nor-Leu-Nor-'Ser-Gly-His-ValPhe-His-Ser-His-Tyr-Gln-Pro-Y; 10 in which Y and Y', if present, each comprises an amino acid sequence of up to about 20 amino acids.

15. 17. A peptide according to claim 16, wherein Y and/or Y s comprises a linking residue selected from the group consisting essentially of glycine, tyrosine, cysteine, 15 lysine, glutamic acid or aspartic acid.

16. 18. Nucleic acid sequences encoding the peptides of claims 14 or 16.

17. 19. A composition comprising a nucleic acid segment, said segment having from 15 to 150 nucleotides encoding a peptide

18. 20 having from 5 to 50 amino acids from a neutralising region (as hereinbefore defined) of gpllO or p25 of HIV. 20. A nucleic acid segment according to claim 19, further encoding a blocking peptide of HIV.

19. 21. A nucleic acid segment according to claim 19, wherein 25 said peptide comprises at least about five amino acids from LAV bru gpllO amino acids 301 to 336 or from LAV gRU p25 amino acid sequences 278 to 319 or 315 to 363, and homologs of the sequences.

20. 22. For use in diagnostic hybridisation assays, a panel 3q of probes comprising at least two nucleic acid sequences encoding the peptides in Table I.

21. 23. A vaccine against HIV infection comprising an immunologicallv effective dose of one or more peptides of less than about 50 amino acids containing neutralising regions from gpllO and/or p25, wherein said peptides are admixed with a physiologically acceptable carrier.

22. 24. A vaccine according to claim 23, further comprising at least five amino acids from a blocking peptide.

23. 25. An immortalised cell line that produces a monoclonal antibody capable of reacting with an envelope glycoprotein gpllO antigenic determinant contained within a neutralising region of HIV.

24. 26. The cell lines HlV-gpllO-l, HIV-gpllO-2, Hlv-gpllO3, HIV-gpllO-4, HIv-gpiio-5, BIV-gpllO-6, HIV~p25~6, and HIV-p25-7.

25. 27. A monoclonal antibody produced by a cell line of claim 26.

26. 28. A monoclonal antibody capable of specifically reacting with an antigenic determinant of HIV, wherein the monoclonal antibody blocks the binding of an antibody produced by the cell lines of claim 26.

27. 29. A monoclonal antibody capable of reacting with an antigenic determinant of envelope glycoprotein gpllO of HIV.

28. 30. A method for generating cell lines which produce antibodies reactive with antigenic determinants of HIV gpllO, which method comprises; administering to a host an immunogenic amount of an antigenic preparation enriched for HIV proteins; monitoring the immunised host for the production of antibodies reactive with gpllO antigenic determinants; obtaining antibody producing cells from the host and immortalising said cells; produce selecting the immortalized cells which antibodies to HIV gpllO; and cloning the immortalized cells to produce the cell lines.

29. 31. A method according to claim 30, wherein the HIV proteins are recombinant fusion proteins expressed by a eukaryotic or bacterial host or the HIV proteins are obtained from an extract or lystate of HIV.

30. 32. A method for diagnosing the presence of HIV in a biological sample, comprising: incubating a monoclonal antibody capable of reacting with HIV gpllO with said biological sample; and detecting the presence of immune complexes formed between the monoclonal antibody and the antigenic determinant in the biological sample, and therefrom determining the presence or absence of HIV in the sample.

31. 33. A method according to claim 32, wherein the monoclonal antibody is capable of reacting with a neutralizing region or blocking peptide of gpllO.

32. 34. A method for diagnosing the presence of antibodies to KIV in a biological sample comprising: incubating a peptide from a neutralizing region of HIV gpllO or p25 with the biological sample; and detecting the presence of immune complexes formed between the peptide and antibodies in the sample reactive with the peptide, and therefrom determining the presence or absence of HIV in the sample.

33. 35. A method of diagnosing for the presence of HIV in a biological sample comprising: incubating under hybridization conditions a nucleic acid segment encoding at least a portion of a neutralising region of HIV with nucleic acid in the biological sample; and detecting the presence of hybrid complexes formed between the nucleic acid segment and the nucleic acid in the sample, and therefrom determining the presence or absence of HIV in the sample.

34. 36. One or more monoclonal antibodies reactive with a neutralising region (as hereinbefore defined) of gpllO or p25 of HIV for use in a therapeutically or immunologically effective dose for treating a patient suspected of exposure to HIV.

35. 37. A method for determining a strain of HIV in an infected host comprising: incubating a biological sample from said host with a peptide from a neutralising region of HIV; and detecting the presence of immune complexes formed between the peptide and antibodies in the sample, and therefrom determining the strain of HIV infecting the host.

36. 38. A monoclonal antibody specifically reactive with a neutralising region (as hereinbefore defined) of gpllO or p25 of the HIV strain for use in treating a patient infected with HIV in which treatment: a biological sample from said patient is incubated with a peptide from a neutralising region (as hereinbefore defined) of gpllO or p25 of HIV; the presence of immune complexes formed between the peptide and antibodies in the sample is detected and therefrom the strain of HIV infecting the patient is determined.

37. 39. An antidiotypic monoclonal antibody, wherein the antibody is specifically reactive with an idiotype of a second monoclonal antibody capable of binding to a neutralising region (as hereinbefore defined) of gpllO or p25 of HIV for use in an immunologically effective dose for vaccinating a patient against HIV infections.

38. 40. A composition comprising (a) at least one monoclonal antibody reactive with a neutralising region of HIV and (b) a blocking peptide comprising at least five contiguous amino acids from the following HIV gpllO amino acid sequences or homologs thereof: IX (173) Y-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-Y 9 ? or XV (191) Y-AXa-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Y 9 ; in which Y and ?*, if present, each comprises an amino acid sequence of up to about 20 amino acids.

39. 41. A composition comprising (a) a monoclonal antibody specific for a neutralising region of HIV and (b) a peptide comprising at least one of the following amino acid sequences or homologs thereof: XI (187) Y-Thr-Thr-Ser-Tyr-Thr-Y“; or XII (188) Y-Thr-Asp-Asn-Tyr-Thr-Y 5 ; or XIII (189) Y-Asn-Thr-Ser~Tyr~Gly~Y e ; or XIV (190) Y-Asp-?hr-Asn~Tyr~Ser-Y’-; in which Y and Y, if present, each comprises an amino acid sequence of up to about 20 amino acids.

40. 42. A therapeutically or prophylactically effective dose of one or more monoclonal antibodies reactive with a neutralising region (as hereinbefore defined) of gpllO or p25 of HIV, and of blocking peptides capable of attenuating HIV infectivity and/or monoclonal antibodies reactive with the blocking peptides for use treating a patient suspected of exposure to HIV.

41. 43. A composition according to claim 1 or 9, wherein the one or more monoclonal antibodies present are substantially as described in any of the foregoing Examples.