IE872219L - Treatment of hiv infections - 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 and prevention of viral infections. More particularly, the invention provides compositions and methods for 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-iu, ARV, and most recently HIV.

As the spread of HIV reaches pandemic proportions s the treatment of infected individual® 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 Sroder, 19874, Nature 325s7?3,„ One ap 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) susceptible 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 desired specificity.

The envelope glycoproteins of most retroviruses are thought.to react with receptor molecules on the surface of susceptible cells* thereby determining the virus8 infectivity for certain hosts. Antibodies that bind to the glycoproteins may block the interaction of the virus with the cell receptors,, neutralising the infectivity of the virus. See generally, The Molecular Biology of Tumor Viruses, 534 (J. Tooze, ed., 1973) and RNA Tumor Viruses, 226e 236 (R. Weiss et al., eds., 1982) , to both of" which reference should be made for details. Pee also P Gonzales- Scarano et a.l. , 1982, Virology 120:42 (La Crosse Virus); Matsuno and Inouye,, 1983 f Infect. Iminun , 3 9 s 15 5 (Neonatal Calf Diarrhea Virus)? and Mathews et al., 1982, J. Immunol. t, 129 :2763 fEncephalomyelitis Virus) .

The general structure of HIV is that of a ribonucleoprotein core surrounded by a lipid-contain ing 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 fgplSO or gpl6Q), which is then processed in the cell into an N-terminal fragment of 110,000-120,000 dsltons (gpllO or gpl20) 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 gpl 10 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 neutralise HIV in vitro, and antibodies that bind to purified gpl10 are present in the sera. Kobert-Guroff et _al-, 1985, Mature 316s72; Weiss et al. , 1985Nature 316: 69; and Ha thews et_ al. , 1986 , Proc■ Natl■ Acad. Sei. U.S.A., 33:9709. Purified and recombinant gpl10 stimulated the production of neutralizing serum antibodies when used to immunize animals, Robey et jil-, 1986, Proc. Natl. Acad. Sci. U.S.A., 3J:7023; Lasky et_al.» 19bb, Science 233:209; and a munan, Zaguryjat al.f 1986e Nature 326:249. Binding of the gpl10 molecule to the CD4 |T4) receptor has also been shown, and monoclonal antibodies which recognise certain epitopes of the CD4 receptor have been shown to block HIV binding, syncytia formation and infectivity. McDougal et al., 1986, Science 231:382. Putney et al. (1986, Science 234:1392) elicited neutralising serum antibodies in animals after immunizing with a recombinant fusion protein containing the carboxyl-terminal half of the gpl10 molecule and further demonstrated that glycosylation of the envelope protein is unnecessary tor a neutralizing antibody response.

A subunit vaccine for AIDS utilising 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 atten 5 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., neutralising antibodies, ADCC, and cytotoxic T-cell response, are used to immunize the most. A major advantage of subunit vaccines is tnat irrelevant viral material is excluded.

Viral subunits for use in a vaccine can be generated by several methods. By way oi 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, M. , et aJL„, 1382;. Proc. Nat. Acad. Sci. USA 79:7415; Panicali, D. and Paoletti, 'E., 1982, Proc. Hat. ,&cad. Sci. USA 79s4927. Recombinant vaccinia virus may be constructed according to the method of Hu et- al. , Mature 320;537 (1986) or Cbakrabarti etjal., Nature 320 z535 (1986)t 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 sub-unit 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. Tnesa include but are not limited to preparative polyacrylamide gel electrophoresis,, gel 6 persneation chromatography and various methods ot chromatography {i.e., ion exchange, reverse pnase, immune-affinity, hydrophobic interaction) and others™ Most of these methods are used in various combinations to 5 achieve substantially pur© preparations (Kleid, D.G., et al., 1981, Science 214:1125; Cabradilla, C.D., et ■al., 1986, Biotechnology 4:128, Dowbenko, D.J.«. 1985, Proc. Wat. Acad. Sex. USA 82:7748) to which reference should toe made for details* iq 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 irom extraneous ^5 components could be accomplished using immunoatfinity chromatography™ This technique,, also known as immuno-adsorption, consists in principle of the selective adsorption of an antigen to a solid support on which & specific antibody has been covalently attached., Tne 2o selectively adsorbed antigen is then eluted trom such an antibody affinity adsorbent by changing,, for example , tne pH and/or ionic strength of the buffer.

Polyclonal antiDodies, obtained from animals immunized with the desired antigen or from naturally 25 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 (ij not all of the antibodies bound to the insoluble support are specific tor the molecule 20 of interest, necessitating additional purification; (ii) yields of the desired antigen are frequently low; and liii) antibody affinities often vary trom one preparation to another, requiring modifications in elution procedures„ The use or monoclonal antibodies specific 35 for the desired viral antigen to be usea 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 Marso Veronese, et al., 198b, Proc. Mat, Acad. Sci. USA 82:5199 and Chassagne, J*, et al., 1986, J. ImmunoJ.. 136:1442) . Monoclonal antibodies specific tor the membrane glycoprotein gp41 have also been reported (see, for example, di Marzo Veronese, et al_. 1985, Science 22911402) .

There remains a need in the art for monoclonal antibodies specific for epitopes within well defined regions of the saajor envelope glycoproteins gpl10» Monoclonal antibodies which bind these regions and cause a reduction in or elimination ot the replication and transmissibility of HIV would have substantial therapeutic and prophylactic utility. Moreover, the monoclonal antibodies could also be used to purify the desired region or gpl10 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 ©voiding the difficulties inherent in purification and administration of larger fragments of the gpl10 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 therepeutic regimens tor the treatment of or vaccination against such infections- The present invention provides novel compositions and nethods for neutralising HIV infectionsr i.e., preventing or substantially inhibiting the formation ox cellular transmission of infectious HIV in a host. More specifically„ peptides mimicking a neutralizing region of gpllO or p25 of HP/ and monoclonal antibodies reactive vitii such a region axe utilized to diagnose, treat and vaccinate against 3IV infections. In this regard,, the term "neutralising region" indicates those portions of HIV, particularly wiv proteins, containing amino acid segments defining one or wore epitopes reactive with antibodies which, either individually or in combination vita other antibodies of the present invention, are capable of neutralizing HIV infections. Suitable assays for neutralisation are well Jen own and can include reduction of HIV infections in T-cell lines, reduction of plague forming units of VSV(HIV) pseudotypes bearing the envelope glycoproteins of HIV„ syncytial inhibition tests and virion-receptor binding tests. As desired,, the neutralizing activity can be compered to antibody reactivity in ixnraunochemical testsr such as immunofluorescence* imnjunoblot and radioimmunoprecipitation as-say* In one aspectf 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 or p2S, encoded by the env and gag regions„ respectively, of the 3IV genome. Of particular interest axe the regions extending from about ammo acid residue 301 to about 336 of gpHOs and from about 278 to about 319 and from about 315 to about 363 of p25, all frost the HIV strain desiqnated LAVas_,,„ The amino.

SHU acid residue designations are from the Los Alamos Data Bank {AIDS virus sequence database, Los Alamos National Laboratory, Theoretical Division, Los Alamos, NM 8754S&.

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 homo log s may be identified by reference to LAVfiRU sequence data as follows; (a) The amino acid sequences or HIV isolates and LAVfiRU may be aligned to obtain maximum homology between the two sequences; }b) Peptides comprising HIV isolates5 amino acid sequences corresponding to the location of LAV ^ peptides that immunological ly mimic LAvbr(j proteins may be identified. The peptides comprising HIV isolate amino acid sequences so identified will typically lmmu-nologica1ly mimic corresponding HIv 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 LAVgmj 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. 10 10 Anotner method for determining sequences of a homologous region in other 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 priimer-JDNA from HIV strains can then be atnplitxed 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 ail strains or spe-cies 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 znxected with one or more species 20 or serogroups of HIV. Tney saay 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 25 be derived from the gpl10 region of the virus. Of particular interest in this region are peptides encoded within the env open reading jrrasne extending from about base pair (bp) 6b57 to about 6774 of the LAVgRU isolate. Thus, various homologous regions of other HIV 3q isolates include the homologous sequences obtained from Los Alamos Data Bank (except LAV2), as listed in Table I.

Other peptides suitable for generating or screening for monoclonal antibodies include those en-2^ coded in the env open reading frame from about bp 7245 II TABLE i tlXES TGI AC AAGACCCAACAACAAT AC AAG AAAAACA............... ATCCGTATC CysThrArgProAsnAsnAsnThrArgLyeArg...............IleArglle 309 5 BH8 -- — 1.ye- — 309 MAL BisFhe 314 10 ELI -—-Ala-—■—TyrCln-- ——-Gin—-———-—-—'IhrPs®—- 310 ASV2 312 UK J 2 ———---.—Tyr —Val—-ArgSer—---LeuSer—-• 306 RFENV —————— —See— --ThrLys 322 26 ———-TyifLys— —-ClssSer ——■———--ThrPro— 311 15 13 ClySerAspLysLyslle-————————ClnSer— 306 HYS ———————lyc— In the gag region of the LAVgRU isolate, the p25 amino acid sequences from about 278 to 319 and 315 5 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 snonoclonal antibodies reactive with various different jq HIV epitopes will exhibit neutralizing activity.

Peptide I, also designated peptide 29, is encoded in the env open reading frasae trosn about amino acid residue numbers 308 to about 328 and will have the following amino acid sequence, where oligopeptides in-15 eluded within the following sequence will include linear epitopes within such sequence: I {23) V-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gin-Arg-Gly- Pro-Gly~Arg-Ala-Phe-Val-Thr-Ile-Gly-Lys~ 20 in which Y and ¥", when present, each represents sequences of up to shout twenty amino acids. fc'hen Y and/or Y' are present, these may comprise, for example,, one or more amino acids from sequences which flank 25 amino acid residues 308 through 328 ot the HIV envelope sequence or any portion or these flanking sequences. By way of example and not limitation, Y can comprise all or portions of the LAVSRU envelope amino acid sequence from about residue numbers 301 to 307; and Y* 30 can comprise all or portions of the LAVgRU envelope amino acid sequence from about residue numbers 329 to 336 as follows: II (29a) Cys-Thr-Arg-Pro-Asn-Asn~Asn~Thr-Arg-Lys~Ser-Ile-2^ Arg~Ile-Gln-Arg-Gly-Pro-Gly-Arg-Ala~Phe~Val-Thr- Ile-Gly-Lys-Ile-Gly-Asn-Met-Arg-Gln-Ala-His-Cys. i ^ I t> alternatively., truncated sequences of peptides of the present invention may be prepared. In this ssgard, the following sequences from peptide 29 may he particularly useful: 5 111 12»0) Y-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-Gly- in which ¥ and/or y°» when present, each represents sequences of up to about twenty ami.no acid residues. 10 IV (29c) y~Ile-Gln-Arg-Gly-Pro-Gly-Arg~Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Y9 in which Y and ¥'„ when present, eacn represents sequences of up to about twenty amino acid residues, .j g In another embodiments homologous regions of the ARV-2 isolate of particular interest are encoded in the env open reading frame from about amino acid residue numbers 306 to about 323 and will typically have the following amino acid sequence,, where oligopeptides 9q included within the following amino acid sequence will include linear epitopes within such sequence; V (177) Y-Thr-Arg-Lys-Ser-Ile-Tyr-lle-Gly-Pro-Gly-Arg-Ala-Phe-His-Thr-Thr-Gly-Arg-Xle-Y' 25 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 2q 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 amino acid sequence from aoout residue numbers 3 24 to 333. 14 Alternatively* truncated sequences of peptide v may be prepared. In this regard, the following sequences may b© particularly useful: VI J 177a) 5 Y~Thr-Arg~Lys-Ser~Ile-Tyr-Ile~Gly-Pro-Gly~Y5; and in which Y and/or Y*, 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 10 open reading from about aiaino acid residue numbers ill to 330, ana will typically have the following sequences ¥111 (110-2-2) Y-Lys~Thr-Val-Lys-Ile-Nor-Leu-Nor~Ser-Gly~His~Val-?he-His~Ser-His-Tvr-Gln-Pro-Y8 in which Y and/or Y', when present, each represents sequences of up to twenty or more aiaino acid residues. (See, Nature 326:662 C1987J , 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 recognising at extremely high 2 4 7 titers (from 10 , to 10 to about 10 or more; neutral- 15 20 25 30 izing regions contained within a predetermined sequence of envelope glycoprotein gpl10 or p25, their protein precursors,, oiologically-expressed recombinant fusion proteins and synthetic peptides that contain one or more epitopes within the predetermined sequence region of gpl10 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 vide 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 isnmunogens for vaccines, or as therapeutic agents.

Blocking Peptides Primarily for use in conjunction with the loregoing peptides or neutralising monoclonal antibodies e 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 utilised 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 & host cell# such as env-encoded amino acid residues about 190 to about 197 of LAV° «... and about 185 to about 192 of ARV2 and HTLV-BRU III(BH-10). These include the peptide T oetapeptide JAla-Ser-Thr-Thr-Thr~Asn-Tyr-Thr) and its various derivatives (e.g., IX below) and analogs (e.g. „ XI below) described by Pert jet 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 sollowing sequences are of particular interest, prefer- 1.6 ably with ^H^-terrninus acetylation and COOH terminus amidation : IX (173D) Y-^Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-Y' ? 5 " X <186) Y-Thr-Thr-Asn-Tyr-Thr-Y'; XI 1187) Y-Thr-Thr-Ser-Tyr-Thr-Y9 g XIX 1188) 1 o Y-Thr-Asp-Asn-Tyr-Thr-Y9 ; XIII (189) Y-Asn-Thr-Ser-Tyr-Gly-Y' ? XIV |190) Y-Asp-Thr~Asn~Tyr-Ser-Y5 y 15 XV (191) Y-Ala-v&l-Phe-Thr-Asp-Asn-Tyr-Thr-Y *; in vhich, for each peptide, Y and Y'» if present, each comprises an amino acid sequence of up to about 20 aiaino acids. Epitopes or antigenic determinants within 20 these peptides are typically defined by at least about five contiguous amino acids, and find use int, e.g. , mimicking naturally-occurring HIV antigenic sites for producing HIV reactive antibodies and vaccines™ Generation of Monoclonal Antibodies 25 The preparation of monoclonal antibodies can be accomplished by immortalising 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 cul-30 tivation in culture. The immortalised 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 35 and secretion of the antibody in vitro. The antibody snay he a naturally occurring immunoglobulin of a mamma I, produced toy transformation of a lymphocyte, particularly a splenocyte, by meens 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 splenocyte will be obtained from an animal immunised 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, Goding, Monoclonal Antibodies a Principles and Practice, Academic Press, 2rsd edition fiSQS) , to which reference should be ®>ade for details. Disrupted virus,, synthetic peptides ana bacterial fusion proteins which contain antigenic fragments of the gpl10 or p2S molecule may be used as iimmunogens... Preferably the inununogen of disrupted virus, peptides or recombinant proteins will be enriched for proteins or fragments thereof containing the epitopes to which antibody-produ.cj.ng B cells or splenocytes are desired. More particularly,, solutions containing disrupted virus lysates or extracts, or supernatants or Diologically-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 gpl10 and other glycoproteinsf e.g., affinity purification using lentil lectin. The extent t:o which the glycoproteins are purified from the solutions for use as an inununogen can vary widely,, i.e., from less than S0%{ usually or at least 75% to 95%, desirably 95% to 9.9% and, most desirably, to absolute homogeneity.

Once the proteins have been purified to the extent desired,, they may be suspended or diluted in an 18 appropriate physiological carrier for immunization, or may be coupled to an adjuvant. One preferred technique , for example, involves adsorbing the proteins and fragments thereof to lentil lectin agarose or other 2 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 by 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 technique being described in 20 an extensive number of patents., e.g., 4,172,124; 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 25 screened in accordance with conventional techniques, and antibodies in the cell supernatants detected that are capable of binding to the desired gpl10 or p25 HIV viral proteins, recombinant fusion proteins or synthetic peptides which contain the desired epitopic region. 30 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 contained, e.g., within the regions encoded by the LAV3RU genomic region from about bpS688 to about hp&750 (encoding peptide 29), or from about bp7246 to about 731/ (encoding peptide 36) Cop numbering according to Wain-Hobson et «al., Cell 44:9 1985, to which reference should be made for details)* tne supernatant® may be screened in competition with the subject monoclonal antibodies in a competitive assay- Thus, additional immortalised hybridoma ceil 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 S~ceils may serve as recipients for genomic DNA coding for the antibody.

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

The monoclonal antibody secreted by the transformed or hybrid cell lines may be of 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 assaysc it is often preferred- Tne monoclonal antibodies may be used intact, or as fragments, such as Fv, Fab, F {ab') 2' 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 at least part of another pro- 10 tein not recognised as foreign by humans, such as the cast out portion of a human immunoglobulin molecule. This can be accomplished toy 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 m PCT 86/01533, EP171496, and EP173494, to which reference should be made for details - Pharmaceutical Formulations and Use The monoclonal antibodies or this invention t.nat 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 attenu-<15 ate HIV infections. The composition should contain a therapeutic or prophylactic amount of at least one of the monoclonal antibodies of this invention with a pharmaceutical^ 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. Pharmaceutic cally acceptable adjuvants (burtering 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 bp5888~bp675Q. Alternatively, a pharmaceutical composition can contain one or more monoclonal antibodies to rorm 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 35 clinical isolates of HIV. The cocktail may contain 20 25 30 monoclonal antibodies which bind to proteins or glycoproteins o.t HIV other than gpllO or p25, such as, for example, to gp41 glycoprotein or p34 nuclease/inte-grase. The mole ratio ox the various monoclonal antibody components will usually not ditter by more than a factor of 10„ snore usually by not more than a factor of 5, and will usually be in a isole ratio of about 1:1-2 to each of 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 el.-/ Mature j2hs773~778„ 1987, to which reference should be made for details.

The monoclonal antiDodies, peptides and pharmaceutical 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. Thus, this invention provides compositions for parenteral administration which comprise a solution of the monoclonal antibody, peptide or a cocktail thereof dissolved xn an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and tne like. These solutions are sterile and generally free of particulate matter. These compositions sway 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 9n im & 10 15 20 25 30 35 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 15U 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 Ed™, Hack Publishing Company, Easton, Pennsylvania (1980) , to which reference should be made for details.

The monoclonal antibodies and peptides of this invention can be Ivophilised 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 lyophiligation and reconstitution techniques can be employed. It will be appreciated by those skilled in the art tnat lyo~ philization 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 9 9 treatment of HIV infections. In therapeutic application, compositions are administered to a patient al-ready 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 wilx depend apon the 1Q severity ot the infection and tine general state of the patient's own immune system, but generally range from about 1 to about 200 sag or antibody per kilogram ox body weight with dosages ot from 5 to 25 mg par kilogram being more commonly used. It must be kept in mind ^ that th® materials of this invention may generally be employed in serious disease states, that is life-threatening 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., 20 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 "prophyiacticaliy eftective dose.1™ In this usef 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 pre- 24 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 5 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 Treat-10 ment Reports 68:317 {19 84)) .

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 antiqen or to the F gamma reeeptor on T-15 cells, 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 cyto-lysis (ADCC). Such heteroaggregates may be assembled, for example, by covalently cross-liking the anti-HIV 20 antibodies to the anti-CD3 antibodies using the hetero-bifunctional reagent N~succinimidyl-3-O-pyridyldithi™ ol)propionate, as described in Karpowsky et al.( J. Exp. Med. 160: 1686 (1984) , to which reference should be made for details. 25 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 30 which is disclosed in commonly owned pending application U. S.S.N. 930,785, may be administered in appropriate physiological carriers intravenously, subcutaneouslyf intramuscularly, intrsoeritoneallyc, etc. Various carriers include 35 phosphate buffered saline, saline, water, potassium 10 15 20 25 30 35 9% ht V chloride, sodium lactate f or the like. The concentration of the peptides will vary widely depending on its ultimata use,, activity, and imode of administration. Preferably, the peptides will have COOH-terminus aroida-tion(, HHj-terminus formylation or other pharmaceutieal-ly 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 ©nti-BIV agents may also be included in the formulations (other than the monoclonal antibodies which bind the peptides) such as 3 ' -azido-3" -deoxythyjnidine „ 2 %3'-dideoxycyti-dine, 2° 33«dideoxv-29 ,3"-didehydrocytidine, etc.

Use of Monoclonal Antibodies in Iinemunoa f tin it y Purification Monoclonal antibodies specific for polypeptides containing gpl10 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 con- 8 i "> stants on the order of 10 to 10""M. 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. Generallythe 'monoclone 1 antibodies which are capable of reacting with gpl10 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 SJ if* lb the antibody and the polypeptides containing the gpl10 antigenic determinants„ Unbound material is separated from the bound immune complexes,, which complexes or gpl 10 antigenic fragments are then separated from the 5 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, 10 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 15 prior to its use as an immunoadsorbant.

The support to which the monoclonal antibodies are immobilized should have the following general characteristics: fa I weak interactions with proteins in general to minimize non-specific binding, Cb) good 20 flow characteristics which allow the flow through of high molecular weight materials, Cc) 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 25 the monoclonal antibody, and (e) steble 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, polyaerylamide beads,, activated ce 1 lu~ 30 lose,, glass and the like. Various chemical methods exist for the attachment of antibodies to substrate supports. See generally„ Cuatrecasas P P., Advances in Enzymology 36s29 (1972). The antibodies of the present invention may be attached directly to the support or, 35 alternatively, through a linker or spacer arm. 27 General conditions required for immobilisation of monoclonal antibodies to chromatographic supports are well known in the art. See, for example, Tijssen, P., 1985, Practice and Theory of Engym® Xmmu-5 noassay, to which reference should be made for details,.

Actual coupling procedures will depend slightly on the characteristics and type of the antibody to be coupled. Monoclonal antibodies possess characteristics which are usually consistent from batch to batch,, thereby allow-10 ing 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 15 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 us\>ally 2 to 24 hours. The immune complexes containing polypeptides with antigenic por-2o tions of gpl10 are then separated from the mixture.

Typically the mixture is removed,, a.g. ? by elutiont. and the bound immune complexes extensively washed with adsorption buffer. The immune complexes may then be eluted from the separation matrix using eluant cosnpati-25 ble with the particular support being used, which elu-ants are wall known by those skilled in the art., Also, the polypeptides containing the gpl10 or other antigenic portions may be selectively removed. For example, peptides that contain an epitope recognized by the 30 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 gpl10 antigen may be eluted from an antibody 35 affinity adsorbent by altering the pH and/or ionic 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 gpl10 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 gpl10 or polypeptides containing antigenic fragments of gpllO to native conformations.

The methods of this invention yield, e.g.,, substantially purified gpl10 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 gp110 and fragments or other purified proteins will.typically be greater than 501 pure, more usually at least "JSI pure, and frequently greater than 95% tc 991 pure. These molecules may then find subsequent use in a wide variety of applications.

Trie neutralising regions of HIV gpllO proteins, polypeptides containing the antigenic fragments thereof., or other proteins substantially purified according to the methods of the present invention, may find use in & wide variety of applications, including AIDS subunit vaccine formulations, in which the immunogen comprises an effective dose of antigenic determinants of, for example, gpl10 or a neutralising region thereof. Other components of the formulation would include those antigenic portions of HIV proteins or glycoproteins that stimulate the 9 Cj fv el production of antibody (preferably neutralising antibodies) in an immunized host, which antibodies are capable of protecting against subsequent infection by HIV. Diagnostic Uses of Monoclonal Antibodies 5 Monoclonal antibodies of the present inven tion 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 ©£ a complex through the binding of the mono-10 clonal 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 fsecond antibodies) that are reactive with the monoclonal antibody,, 15 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,. 20 ligands (particularly haptens), etc. Numerous types of immunoassays are available and, by way 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 25 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 30 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 oceurs between the antibodies and 35 those molecules exhibiting the desired epitope. Such 30 proteins or viral particles may then be separated from the unbound reagents, and a second antibody (labeled vith an enzyme) added. Thereafter,, the presence of the antibody-enzyme conjugate specifically bound to the 5 antigen is determined. Other conventional techniques well known to those skilled in the art roay also be utilised* Kits can also be supplied for use with the subject antibodies in the detection of HIV infection or 10 for the presence of HIV antigen™ Thus, the subject monoclonal antibody compositions of the present invention may be provided,, usually in a lyophilized form,, either alone or in conjunction with additional antibodies specific for other epitopes of HIV. The anti-15 bodies, which snay be conjugated to a label or unconjugs ted, are included in the kits with buffers, such as Tris, phosphate« carbonate, etc., stabilisers,, hio-ciaes, inert proteins, e.g., bovine serum albumin, or the like. Generally, these materials will be present 20 in less than about 5% wt. based on the amount of active antibody, and usually present in total amount of at least about 0.001% wt. based again on the antibody concentration „ Frequently, it will be desirable to include an inert extender or excipient to dilute the ac-25 tive ingredients, where the excipient may be present in from about li to 99% wt. of the total composition.

Where a second antibody capable of binding to the monoclonal antibody is employed, this will usually he present in a separate vial. The second antibody is typi-30 cally conjugated to a label and formulated in an analogous manner with the antibody formulations described above.

The detection of gpl 10 or p25 antigens t, or the whole virus, in various biological samples may find 35 use in diagnosing a current infection by the HIV virus. 31 Biological samples can include, but are not limited to, blood serum, saliva e ssmen , tissue biopsy samples {brain, skin, lymph nodes,? spleen? etc.), cell culture supernatants, disrupted eukaryotic and bacterial ex-5 pression systems ©nd the like. Presence of virus is tested for by incubating the monoclonal antibody with the biologieal sample under conditions conducive to iimnune complex formation, followed fov the detection of complex formation. In one embodiment, complex forfait) tion is detected through 'isse 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 13 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 20 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 gpl10 or p25, 25 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, may be made. These peptides can be used as 30 immunogens, to inhibit or eliminate HIV antigen production in_ vitro or ijn 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 32 or other compounds, labeled or unlabeled, bound to a solid surface, or the like.

Id one embodiment,, peptides of interest will be derived from the gpl10 region of the virus™ Of par-5 ticular interest is the region within the env open reading frame extending from about base pair IbpS 6688 to about fop6750 and front about bp7246 to about 731/.

The peptides of interest,, including blocking peptides, will include at least five, sometimes six, 10 sometimes eight, sometimes twelve, sometimes 21, usually fewer than about 50, snore 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 15 possible while still maintaining substantially all of the immunoreactivity or antiviral activity of the larger peptide. In some instances it snay be desirable to join two or snore oligopeptides which are non-overlapping to form a single peptide structure or 20 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 25 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 30 mimic the differing epitopes of the different retroviral strains. In many instances to provide chemical stabilityf methionine may be replaced by norleucine (Nor) .

It should be understood that the peptide 35 employed in the subject invention need not be identical 33 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 5 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 with-10 in groups such as gly, al®? val, ile, leu; asp, glu; asa, gin; eets thr? lys, arg; phe, tyr; and nor, met. Usually,, the sequence will sot differ by siore than 20% from the sequence of at least one strain of an HIV retrovirus except where additional aiaino acids may be 15 added at either terminus for th© purpose of providing an "arm" by which the peptide of this invention may 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 acids, in length. 20 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 25 by various assay techniques disclosed herein „ The d~ icomer form of one or more amino acids may be used, as desired, to modify biologic properties , such as activity „ rate of breakdown, etc.

In addition,, one, two, or more amino acids 30 may be added to the 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 phys ical or chemical properties of the peptide or 35 oligopeptide, or the like. 9 A d> & Amino acids such as tyrosine, cysteine,, lysine, glutamic or aspsrtic 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 5 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 se~ 10 quence being modified by terminal- NH^ acylation, e.g.„ acetylation, or thioglycolic acid arnidation, terminal-c&rboxy arnidation, e.g., with ammonia or methylamine, to provide stability,, increased hydrophobicity for linking or binding to a support or other molecule, or 15 for polymerization.

Thus, for example, in the peptides I-VIII and IX-XV disclosed above, when Y or Y' are present, a preferred embodiment exists when Y or y" comprises one or snore cysteine residues or a combination of one or 20 snore 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 YB represents at least two cysteine residues. When two cysteine residues are present at 25 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 30 the hydrophobicity of the resulting peptide which facilitates immob i ligation of the peptide in solid phase or immobilized assay systems.

Of particular interest is the use of the mer-captan group of cysteines or thioglycolic acids used 35 for acylating terminal amino groups or the like for linking two of the peptides or oligopeptides or combinations thereof by £ 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 the polymer,, they possess the additional ability to induce antibodies that immunore&ct with several antigenic determinants of different HIV isolates .

To achieve the formation of antigenic polymers (synthetic mult inters) , 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 foe 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 poIy-L-Xysine, 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 vill be at least one molecule of at least one subject peptide per macro-molecule and not more than about 1 per 0.SkDal, usually not more than about 1 per 2kDal of the macromolecule. One or more different peptides may be linked to the same macromolecule. 36 The manner of linking is conventional, employing such reagents as p-maleimidobenzoic acid, p-methyldithiobenzoic acid, maleic acid anhydride, succinic acid anhydride, glutaraldehyde, etc. The linkage S may occur at the N-terminus, C-terroinus or at a site intermediate to the ends of the molectsle- 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 15 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 hu~ 20 inan immunoglobulin,, normally both human IgM and IgGP or a labeled protein specific for immune complexes, e.g., Hf factor of S. aureus protein A.

Illustrative of an assay technique is the use of sample container f e.g., wells of microwe11 plates, 25 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 30 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 37 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, stay be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesisers 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 Tarn et_ al., J. Am. Chem„ Soc. (1983) 105:8442.

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 en annealing -medium so as to hybridise., The hybridised strands may then be ligated to form the complete gene, and, by choice of appropriate termini, the gene snay be inserted into expression vectors, which are readily available today. See, for example, Maniatis et al. , Molecular Cloning, A Laboratory Manual, CSK, Cold Spring Harbor Laboratory,, 1982. Or, the region of the viral genome coding for the peptide may be cloned by conventional recombinant DNA techniques and expressed (see, Maniatis, supra).

DNA coding sequences from the LAV and ARV 2 isolates of HIV which may be used for expressing the peptides include the following^ LAV TGT ACA AGA CCC AAC AAC AAT ACA hGh AAA AGT FKU 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 5 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 10 expression of peptide fragments, conservative base changes can be made, where the modified codon ts) code for the same amino acid(s) , or non-conservative changes in the coding sequence may be made, where the resulting amino acid may be a conservative or non-conservative 15 change in the amino acid sequence, which ^as discussed previously.

The coding sequence may be extended at either the 59 - or 3'-terminus or both termini to extend the peptide, while retaining its epitopic site (s). The ex- 20 tension 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. 23 The DMA sequence by itself, fragments there of, or larger sequences, usually at least 15 bases, preferably at least 13 bases, snay be used as nucleotide probes for detection of retroviral RNA or proviral DNA„ or for identifying homologous regions for cloning or 30 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 Ho. 4,358,535, to which reference should be made 35 for details.

The subject peptides,, including blocking peptides, and their analogs find use by themselves or in combination in vaccines. Similarly, antiidiotype antibodies., i.e., reactive with the idiotypes of the antibodies of the present invention and thereby containing 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 formulation . Administration may be injections, e.g., intramuscularly , peritoneallye subcutaneously, intravenously, etc. Administration of an immunogenics 1ly effective dose may be one or a plurality of times,, usually at one to four week intervals. An "inununogenically effective dose® is that amount of a vaccine suitable to elicit immune response in a host,, whereby the host demonstrates increased infection.

The words NONIDET, WEEN, TRITON and KODAK are registered Trade Marks and the words SEPHAROSE and POLYBREMS are Trade Marke.

Other features and advantages of the present invention will become apparent from the following experimental descriptionsa which describe the invention by way of example. The examples are offered by way of illustration and not by way of limitation. ie EXAMPLE I Generation and Characterisation of B'onoclonal Antibodies Example I describes the generation of hybrid 5 cell lines vhich produce monoclonal antibodies specific for the envelope glycoproteins of HIV. This method involves the use of lectin purified extracts of l*AvgRU attached to lentil lectin agarose as the iramunogen. The monoclonal antibodies subsequently generated hy the 10 hybrid cell lines are characterised by their ability to immunoblot and radioimmune precipitate gpl 10 from purified LAV and as biologically-expressed recombinant fusion protein. The monoclonal antibodies that bind to epitopes on gpl10 are also reactive in ELISAs with dis-15 rupted 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 20 characterization of the antibodies were as follows.

LAV virus purified from infected CEM cells JA.T.T.C. ^o» CRL8904) was disrupted in 50 mK Tris, pH 7.4P OolS.K NaCl, 1.0* Aprotinin, 2.0% Nonidet P~40(R) (MP-4 0) (octvlphenoxypolyethoxyethanol). The extract 25 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, K.J.) was prewashed in disrupt tion buffer without KP-40 and then equilibrated in ad-30 sorption buffer (50 mK Tris, pH 7.4, 0.15 K NaCl, 1.0% Aprotinin, 0.51 NP-40). Clarified viral extract »es adsorbed with lentil lectin Sepharose for 42h at 4°C. Unadsorbed material was removed by washing with excess adsorption buffer. Elution of adsorbed materiel was 35 carried out with 0.2 M alpha methyl mannoside in adsorption buffer. The eluent was dialyzed against PBS to remove the sugar arid the material was readsorbed to the lentil lectin Sepharose.

The glycoprotein-lentil lectin Sepharose com-S plex was used to iimmunize BALB/c mice by three intraperitoneal injections without adjuvant given 2-3 weeks apart. Spleens were removed from immunized mice which demonstrated circulating antibody to glycoproteins of HIV by immunoblot» RIP and/or ELISA. 10 Protocols used for the generation of cell lines were generally those of Xohler and Milstein (Nature 256:495 (1985)5 with the modifications of Goldstein, L.C.* et al., finfeet. Immun. 38s273 (1982)). Splenic 3-lymphocytes from the immunized mice were 15 fused with NS-1 myeloma cells using 40% fw/v ) polyethylene glycol. Following fusion the cell mixture was resuspended in HA? medium (RPMI - 1640 medium supple- -4 mented with 15% fetal calf serum, 1x10 ' M hypoxan-thine, 4x10 7 H aminopterin and 1.6x10 ^ M thymidine) 20 to select for the growth of hybrid cells, and then dispensed into 96-well microculture trays at a concentre- * tion of 1 to 3x100 cells/ml and incubated at 37°C in a humidified atmosphere containing 6% CO^. Cultures were fed by replacement of one-half the supernatant with 25 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 super-natants were tested for anti-LAV antibody.

Wells containing hybrid cells producing anti-30 body to LAV were identified by ELISAs 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 35 were incubated with cell culture fluids at 37°C for 4S &2 minutes and then washed three times with 0,05% Tveer. 20 in phosphate buffered saline (PBS-Tween).

Peroxidase-goat anti-mouse JgG 11t2.000 dilution in P3S-Tween; 2ymed Laboratories, Inc.,, South Sen 5 Francisco, California) was added flOO ul per well), and the pi re incubated for 45 minutes at 37°C and washed as above. Substrate 10.025 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 10 per 50 ml) was added and the plates were incubated for 30 lain at room temperature in the dark. The reaction was stopped with 3N sulfuric acid,, and coloriinetric reactions were quantitated with an automated microplate reader. Wells that gave positive results were subclon-15 ed 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, immu-20 noprecipitation 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-gpl10-1, HIV-gpl10-2, and HIV—gpl10-3 have been deposited with the American Type 25 Culture Collection prior to the filing of this application and designated A.T.C.C. Kos- BB 9175, KB 9176, and HB 9177 , respectively.

Recombinant fusion proteins tested for reactivity have previously been designated ENV2, ENV3, ENV4 30 and ENV5. Protein ENV2 is expressed from pENV2 (A.T.C.C. No. 53071), which is a region of LAV from base pair (bp) SS98 through bp 7178 (numbering according to Wa in-Hobson et a1., Cell 44 :9 (1985)) , ENV3 is expressed from pENV3 (A.T.C.C. No, 53072), which com-35 prises the LAV region from bp "178 through bp 7698; 43 SNV4 is expressed from pENV4 JA.T.C.C. No- 53073)f and 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 1 129) and VIII <110-2-2) were assembled on a benzhydrylamine Ipolystyrene/divinylben-zene) resin (Applied Biosystems, Inc., Foster City, California) . Peptide V 11775 was assembled on a t~ butyloxycarbonyl(Boc)-ethylbenzylcysteine-phenylace-tamidomethyl (PAM) polystyrene/divinylkenzene resin. Symmetrical anhydride couplings were carried out in an Applied Biosystems 430A synthesiser. Cysteine was added as the first residue in both peptides.

Dicyclohexlycarbodiimidc couplings in the presence of hydroxylbenzotria2ole were used for aspara™ gine and glutamine„ Benzyl-based side chain protection and Boc alpha-amine protection vera used. Other side chain protection routinely used was Boc (forrnvl) tryptophan „ Boc methionine sulfoxide, Boe (tosyl) arginine, Boc (methylbenzyl) cysteine Boc Jtosvl) histidine, Boc (chlorobenzyloxycarbonyl) lysine and Boc Jbromobenzyl-oxycarbonyl) tyrosine.

When the peptides sere radiolabeledf it was by acetylating the amino terminus with "H-acetic acid and an excess of dicyclohexylcarbodiitnide.

Deprotection and cleavage of the peptide from the resin was by the Tarn "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. 44 Synthetic HIV peptides tested for reactivity with the monoclonal antibodies included peptides 29, 36, and 39 - Peptide 29 is encoded by the LAVgRU genomic region from about bp 6688 through bp 6750j peptide 5 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. 10 The blocking peptides, IX-XV, were assembled essentially as described above on a methyl-benzhydryla-mine (polystyrene/divinzlbenzene) resin (Applied Biosystems , Inc., Foster City, California). Symmetrical anhrydride couplings were performed on an. Applied Bio-15 systems 430A synthesizer. Dicyclohexylcarbodiimide couplings in the presence of hydroxyIbenzotriazole were used for asparagine. For protection, benzyl-based side chain and Boc alpha-amine protection were used, while Boc (brontobenzyloxycarbonyl) was used specifically for 20 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 "high68 HF protocol (Stewart et ajL , 2 5 supra). Extraction from the resin was performed with 501 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 Vydac CIS column (Hainin Instru-30 ment Co., Emeryville, CAl using a 0.1% 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 polv-acrylaroide gradient gel electrophoresis {7„0-15„0%) and transferred to nitrocellulose membrane ?NCM) by electrophoresis for four hours at 25 v in 25 mM sodium phosphate fpH 7.0). After transferf the NCM was blocked to prevent nonspecific interactions by incubation in PBS^Tween or Blotto fS't non-fat dry milk in PBS) for one hour at room temperature. The PCM was incubated with cell culture supernatant or ascites fluid' diluted in PBS-Tveen for one hour at room temperature and was rinsed with three changes of PBS-Tween. In the second step the PCM was incubated with goat anti-mouse igG-horseradish peroxidase diluted in ?BS-Tween for one hovar 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 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 gpl10 and its precursor molecule, gpl50, using disrupted virus preparations . Antibodies 110-1 and 110-2 also recognised the fusion protein ENV3, whereas antibodies 110-3,, 110-4, 110-5, and 110-6 immunocomplexed ENV2.

Immu n oprec i p i t a tion Viral extracts for radioimmune precipitation were prepared from CEK cells infer feed with the LAV^,, isolate of HIV adapted to lytic growth by continuous passage. When early cvtopathic effects were evident, the cells were transferred to labeling media containing 3 5 3 (S)-methionine (0.05 mCi/m1) or [H]-glucosamine (0.025 mCi/ml), then incubated for 24h until most of fl iff* the cells had lysed, releasing virus into the culture supernatant. Virus was palleted {one hour at 100,000 >tg) from the cell-free supernatant,, anril detergent extracts were prepared in P-RIPA buffer (phosphate 5 buffered saline containing 1.0% Triton X-100, 1.0* de-oxycholate, 0.1% SDS, and 1% Aprotinin) . Similar extracts vers prepared from the supernatants of wain-fee ted CEM cells.

Immunoprecipitation 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. Immuno-15 precipitin (100 ul; Sethesda Research Laboratory, Sethesda, 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 20 gel electrophoresis C15.0% 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 re-25 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 gpl10 and gpl50. 30 Enzyme-Linked Imrunoadsorbant Assay To map the gpl10 epitopes being recognised by the monoclonal antibodies of the present invention f culture supernatants from hybrid cell lines or ascites fluid were further characterized by reactivity in 35 ELISAs with biologically-expressed fusion proteins and 47 synthetic peptides. Procedures were the same as those described above with the exception that fusion proteins or synthetic peptides replaced purified virus es the antigen adsorbed to the surface of the microliter wells.

When peptides were used as antigen the plating protocol was as follows - Lyophilized peptide was dissolved in BM guanidine HC1. Just prior to plating in the 96 well plates, the guanidine solution was diluted into 0,05 M carbonste/bicarbonate buffer (pH 9.6) to a final peptide concentration of up to 100 ug/ml. A 50 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 the rest of the ELISA„ Similarly# recombinant protein was diluted to a final concentration of about 2 ug/ml in 0.05 H carbonate/bicarbonate buffer fpH 9.6) before the same procedure was followed.

The results are shown in fable II- Monoclonal antibodies produced by cell lines HIV gpl10-1 and HIV gpll0-2 reacted with SNTV3, ENV5» peptide 36 and disrupted virus. Antibodies from cell lines HIV«gpll0-3, HIV-gp~l10-4 , HIV-gpl.tO-5 and HIV-gpllO-6 reacted with ENV2 and peptide 29 as well as disrupted virus. 48 TABLE II ELlSAs Showing Reactivity of Monoclonal Antibodies with Recombinant ?roteir>s and Synthetic Peptides Becorablnent Fusion fToteiti Synth? >tlc ?«otide LAV Control CEM Contti ENV 2 ENV 3 ENV A ENV5 29 36 39 110-1 0.077 3.000 0.113 3»000 ND 2.421 0.054 0.908 0.125 110-2 -0.003 o o o o © o 3.000 HD 2.305 -0.005 1.214 0.009 110-3 3.000 0.031 ND HD 3.000 ND 0.017 0.363 0.046 110-4 3.000 0.020 B'O ND 3.000 m> 0.016 0.383 0.067 110-5 3.000 0.014 MB ND 3.000 HD 0.016 0.368 0.025 no-6 3.000 0.033 ND ND 1.937 HD 0.017 0.486 0.032 The results in Table II demonstrated that monoclonal antibodies 110-1 ©nd 110-2 recognised an antigenic determinant encoded by a DNA sequence within the pENV3 region,, snore particularly by the region of the HIV genome defined by a sequence of amino acids within peptide 36. That is, monoclonal antibodies gpl 10-1 and 11-0-2 bind to a peptide region of gpl 10 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 the DNA sequence in the region encoded by peptide 36 among different viral isolates from diverse geographical locations. See Starcich et al., Cell 4€sS37 (1986). In contrast, monoclonal antibodies gpl 10-3,, -4, -5, and -6 biad to peptides of HXV defined by the region encoded by peptide 29 from bp €688 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 gpl10 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 chromatographyP etc. Also„ in an ELISA assay, the peptide 110--2-2 reacted with sera frorr the individual from whom LAV-2 was isolated. Indirect Inununofluorescent Assay Indirect immunofluorescent assays using laono-clonal antibodies directed against the gpl10 antigen of HIV were carried out on acetone-fixed and live cells. Acetone-fixed slides prepared from LAV-infeeted 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 placed on slides and acetone fixed. Both snethods used fluorescein isothiocyanate-labeled anti mouse IgG to detect cells bearing the reactive gpl 10 antigen. Monoclonal antibody HIV-gpllO-1 gave positive results using either live or acetone-fixed LAV~in£ected cells.

EXAMPLE II Neutralisation of HIV Infectivit y by Anti-gpllO Monoclonal Antibodies This example describes and characterises the neutralization of HIV infectivity -using monoclonal antibodies vhich bind to gpl 10 and peptides within gpl10. The results indicate that monoclonal antibodies gpl10™ 3,, -4, -5 and -6 possess neutralising activity,' and that gpl10-3 and -4 possess particularly high levels of neutralising activity™ Neutralization Assay A sensitive neutralisation assay was developed to quantitate the effect of the monoclonal antibodies on HIV infectivity. A CD4+ cell line highly susceptible to HIV infection, CEM, was 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 10% 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 TCID^q f and subsequent assays performed with freshly thawed sliquots, 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 TCID^). The virus r* -II 31 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 fixture was incubated for 45 minutes at 37"C and then duplicate 5 from 1:5 to 1:9,765,625- The virus/antibody mixture was incubated for 45 minutes at 37°C and then duplicate samoles of 200 ul s«sed to inoculate veils containing 5 1.0 ml of approximately 2x10 CEM cells per veil. The cultures were incubated 37*c in a humidified, 5% COj 10 atmosphere for 14 days. The cells were harvested * pelleted, and lysed with 1% Triton X-100 in PBS for about 10 minutes. The amount of virus Cor viral anti~ gen) present in lysed cells was quantitated using a sensitive HIV antigen capture "sandwich" enzyme immuno-15 assa-y described below. The titer of neutralising activity, if any,, was determined as the reciprocal of the dilution of monoclonal antibody vhich inhibited antigen production by greater than 501 of ifirws control cultures incubated without antibody, or with a mono-20 clonal antibody of the same.isotype which had previously been shown to lack neutralising activity.

The HIV antigenic capture assay referred to above employed two monoclonal antibodies directed against p25 antigens as capture reagents. These hybri-* 25 doma 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 fu~ 30 sion 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-35 duction of the recombinant fusion proteins is described d u in detail in commonly owned pending patent application numbers U.S.S.N. 764,460 and 828,828, to uhich reference sJxxild be made for cSetai3s® Synthetic peptide 141 is encoded by the LAV3R0 9enon,ic region corresponding to S the amino acid residues 198-242. Monoclonal antibody produced by hybridoma cell lines p25-2 and p2S-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, 10 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 8.5 and 200 ul was- placed into wells of 15 microwell plates. Wells were sealed and incubated for about 16 hours at 4'5'C,., The solution v.»s 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 solu- 20 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 37°C, after which the 25 suspension was aspirated and the wells washed four times with buffer 10.05% Tween 20 in PBS). The detection conjugate was prepared as follows. Monoclonal antibodies p25-6 and p25-7 were conjugated to horseradish peroxidase (HRP) at a 3:1 molar ratio (AbsHRP) for 30 three hours using a periodate oxidation procedure CNakane, et al., J. Histochem. Cytochem. 22;1084 (1974)). Conjugates were diluted Is 1500 in 2.5% (w/v) non-fat dry milk, 0„01% thimerosal, and 0.0051 Anti-foam A in 20mM sodium citrate. The remainder of the 35 ELISA procedure was performed as described in Example 1.

Th® results of the assay of neutralizing activity with antibodies gpl10-3, -4, -5, and -6 are as follows. The highest titers which retained neutralizing activity were determined to be: gpll0-3 « 15,625; gpl 10-4 «= 9,765,625; gpll0-5 * 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 recognize other isolates of HIV was examined. The immunofluorescence assays were performed as described above. Antibody gpll0-4 detected antigen in cultures of at least 3 of 16 HIV isolates tested.

Antibody gpll0~4 was able to neutralise viruses isolated over fifteen weeks from a chimpanzee inoculated with LAV^y as a control animal in an AIDS vaccine trial. The monoclonal antibody was able to neutralise the isolates even though the animal had serum antibodies which neutralised HIV in vitro, and had developed a measurable cell mediated immune response to the HIV infection. This indicates a lack of antigenic drift _in vivo for the epitope recognized by the gpll0-4 antibody.

EXAMPLE III Neutralisation of HIV Infectivity by a Coctail of Anti-gpl10 and hntx~v25 Monoclonal Antibodies This example describes the neutralization of HIV infectivity using monoclonal antibodies which bind to gpl10 and peptides within gpl10 in combination with monoclonal antibodies which bind to p2S and peptides within p25, which alone show little or no neutralizing activity. The results indicate that monoclonal antibody gpl10-2 in combination with either p25-6 or p25~7 possesses particularly high levels of neutralizing activity.

Hybridoma cell lines p25-6 and p25-7 were generated by the methods described above with modifi 54 cations which include using inactivated disrupted virus or a purified crag recombinant fusion protein expressed in E. coli as the isnmunogen „ and characterising the resultant monoclonal- antibodies as to specificity and re-5 activity 'osing the recombinant fusion proteins previously designated GAG-1, GAG-*2? and GAG-3 and the synthetic peptides 15, 38, 150, 147r and 148. The synthetic peptides are encoded by the LAVBRy genomic region corresponding to amino acid residues as follows: 10 peptide 15, amino acid residues 329 through 350; peptide 88, amino acid residues 315 through 350? peptide 150* aiaino acid residues 310 through 363; peptide 147, amino acid residues 278 through 319; and peptide 148, aiaino acid residues 290 through 319. Monoclonal anti-15 bodies 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. 20 Neutralization assays ware carried out as de scribed 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 25 was carried out as described above.

Monoclonal antibodies gpll0-2, p25-6 and 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 gpll0-2 30 with p25-7 at a Is 10 dilution, there was total neutralisation. A cocktail including the monoclonal antibodies p25-6 in combination with p25-7 gave neutralizing activity range of 60-90%. 55 EXAMPLE XV Xmmunopotency of Peptide 29 and Homoloqs The ability of peptide 29 and homologous peptides, including peptide 177, to stimulate an immune 5 response against HIV was examined in two strains of mice. The procedures for the preparation of the peptide immunogens, immuniaation protocols, and for the characterisation of the immune response generated are detailed below. 10 Peptide 29 was prepared for immunization by conjugation to a purified thyroglobulin. Thyroglobulin may be derivatised with N-succinimidyl-4-(N-maleimido-methyl)eyelohexane-l-carboxylate 5SMCC) for conjugation according to the procedure outlined in U.S. 4 629 ?7 8 3 e 15 col. 10lines 28-51. As a second inununogen, thyro globulin %?as derivatized with glutaraldehyde as follows,, Porcine thyroglobulin,, 27 mg, was dissolved in 1 ml of 0.1 M sodium bicarbonate, to which 8,3 pi of a 25% glutaraldehyde solution was added dropwise„ and the 20 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 eC, • a complete change of dialysate at 4 hours,. Peptide 29 was then 25 added to the derivatized thyroglobulin at approximately 100 molar excess, and the mixture stirred overnight at room temperature. Unreacted g3.utaraldehyde was blocked with 200 pi of a 0.2 M lysine solution, which mixture was stirred for several hours {or overnight) at room 30 temperature. Th© pepticl©-thyroglobu 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 35 at the time of inoculation. The routes of inoculation 5G 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 5 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 et 5 10 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 analysing the sera in immunoblots to dis-15 rupted LAV antigen and radioimmunoprecipitation assays with radiolabeled gpl10.

The results of immunizations indicated that mice immunized with peptide 29 developed antibodies reactive with peptide 29 and disrupted LAV-l virus in 20 ELISA*s. Conjugation of peptide 29 through glutaraldehvde generally elicited higher titer of antibodies to peptide 29 in Balb/c mice f, although conjugation through maleimide did successfully elicit anti-peptide 29 antibodies in some Balb/c mice. Mice 25 immunised with peptide 177 developed antibodies to the peptide, and conjugation of peptide 177 through glutaraldehvde was better at eliciting an immunological response. C57 mice were more responsive to immune stimulation with peptide 111 than Balb/c mice. Mice 30 immunized with the LAV-2 peptide 110-2-2 developed antibodies to 110-2-2 and LAV-2 virus as shown by ELISA5s. Peptide 110-2-2 conjugated through 6 glutaraldehvde was immunogenic in both C57 and Balb/c mice? although titers to the 110-2-2 peptide were 57 generally higher in Balb/c than C57 mice, while titers to hhV-2 virus were generally higher in C5? mice than Balb/c jnice.

Generally, serum samples from immunized mice 5 which (i) demonstrate antibodies to whole virus? ?ii) are able to neutralize HIV, such ss in the assays described in Example II; and IiJ.i> are reactive with peptide 29 in ELISAs» cumulatively indicate the efficacy of the use of peptide 29 in a vaccine 10 formulation.

EXAMPLE V Iramunoaffinity Separation of GPl10 Using Monoclonal Antibody Monoclonal antibodies to the gpl10 antigen of 15 HIV can be used in imrnunoaffinity 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 secretedc, disruption of the bacterial cells may be nec essary. 58 Construction of plasmid pENV-5 (A.T.C.C. Mo. 53074) is described in copending commonly-owned patent application U.S.S.N. 721,237, to which reference should be made for details. Plasmid pENV-5 encodes a major 5 portion of the carboxvl end of gpllO and a portion of the amino terminus of gp41 of LAV inserted into the trp expression vector. coli C600 transformed by this vector expresses but does not secrete the gpl10 fusion protein. 1Q E. coli C600 containing the plamid pENV-5 are grown in medium containing tryptophan J20 ug/ml) and aropicillin (100 ug/ml) overnight at 37°C with aeration. The overnight cultures are then inoculated at Is 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 inducere 3-B-indole~acrylic acid (Sigma Chemical Co. , St. Louis, MO), is added to © final concentration of 20 ug/ml from freshly made stocks of 20 20 mg/ml in 95% ethanol. Induced cultures are then grown at 37°C 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 25 P-RIPA buffer (PBS containing 11 Triton 1-100,, 1% de-oxycholate, 0.1% sodium dodecyl sulfate, and 1% Aprotinin ) which will lyse EL coli cells. The suspension can be sonicated to shear DNA and RNA, followed by cen-trifugation to remove particulates, A dilution or con-3o centration step may then be needed to standardize the protein concentration™ Monoclonal antibody Hiv-gpl10-1 is precipitated initially from ascites fluid or cell culture supernatants at room temperature or in the cold with (NHkJ-SO, or Na,SO, solutions buffered at pH 7.3 to 35 424 2 4 59 final saturation of 33 or 181, respectivelv. Precipitated proteins are removed by contrifugation and redis-solved in PBS and precipitated a second time with 33% (NH^),SO^ or 2,2-15% NajSO^ - This step can be repeated 5 as necessary. The pellet is again dissolved in PBS and the excess salts are removed by gel filtration through a desalting matrix or toy exhaustive dialysis against PBS.

Purified 110-1 monoclonal antibody can then 10 be coupled to the cyanogen bromide-activated Sepharose- -3 The necessary amount of gel is swollen in 10 M HC1 solution on a glass filter 11 g of freeze-dried material yields a final gel volume of approximately 3„5 snl) and washed for 15 minutes with the same solution, and 15 the antibody added immediately thereafter. In general,, the coupling reaction proceeds most efficiently in a pH range 8-10, but a lower pH can be used if needed for antibody stability. The antibody should foe dissolved in PBSf or a high ionic strength carbonate/bicarbonate 20 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 25 blocked by treatment with 1.0 M ethanolarnine 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) 30 four or five times. This washing removes traces of non-covalently adsorbed materials. The finished inunu-noaffinity separation matrix is stored below 8°C in the presence of a suitable bacteriostatic agent, such as 0.01% aside. 60 The addition of the expressed protein suspension to the immunoaffinity separation matrix results in the selective removal of the gpl10 antigen. The mixture is allowed to interact for 2-24 hoursf preferably 5 12-18 hours,, with slow stirring or rocking. A column format can also foe 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 10 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 8.5S) or a chaotropic agent.

EXAMPLE VI Immunoaffinity Purification of Recombinant GP110 From a Mammalian Expression Svstem 20 - The monoclonal antibodies of the present invention find use in the immunoaffinity purification of recombinant fusion gpllO expressed by mammalian cells. Mammalian calls are infected with recombinant vaccinia (Mackett et al., J. Virol. 49:857 U984) , to which reference 25 slxxild be msde 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 & vaccinia transcriptional control element. This 6i chimeric gene is flanked by sequences encoding the viral thymidine kinase JTK) gene.

Chimeric plasmid vectors containing vaccinia virus promoter ligated to the bKV envelope gene are S used to transform E. coli strain NCI000. Insertion of the chimeric LAV-env sequences into the vaccinia virus genome was achieved by _in vivo recombinations made possible by the fact that the chimeric genes in plasmids pv-env5 are flanked fey vaccinia virus sequences coding 10 for the TK 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 chi~ 15 meric gene. African Green Monkey kidney cells (strain 3SC-40, a line derived from 3SC-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 of 10 by recombinant vaccinia 20 virus. Infection is allowed to proceed for 12 hours, at which time the cells are harvested, washed once with PBS„ and collected by eentrifugation. Cell pellets are resuspended in lysis buffer 0.0% NP 40, 2.5% sodium deoxycholate, 0.1 M NaCl, 0.01 M K Tris-HCl, pH 7.4, 1 25 mM EDTA) , and the lysc.te is then cleared by eentrifugation . Immunoaffinity separation of the expressed recombinant fusion protein is performed as described above for the bacterial expression system, employing the monoclonal antibody gpl10-1. The proteins produced 30 by this expression system more closely resemble naturally produced HIV gpl1Q because of the processing and clycosylation afforded by the mammalian cells. * 62 EXAMPLE VII Inhibition of HIV Infection with Blocking Peptides The effectiveness of blocking peptides in inhibiting the infection of tissue culture cells by the 5 LAV strain of HIV was studied utilizing a modifies- bKU tion of the protocol for peptide T evaluation published by Pert et al.r supra. The preferred HIV inhibition assays comprise combining equal volumes of blocking peptide and CEM cells |2„5 X 10S} in medium (RPMI, 10% 10 FCS and 2 rog/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. 15 Preincubation of the CEM cells with the pep tides prior to virus addition into the cultures enhances the inhibiting effect in the assays. The inhibition was found to be dependent on the virus dose, showing strong activity at low and medium doses, but less ef-20 feet 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 NH,-terminus 25 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 at the time the virus is added to produce an additive or synergistic inhibition of 30 viral antigen production.

From the foregoing,, it will be appreciated that the monoclonal antibodies and peptides, including blocking peptides, of the present invention provide U el 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 ehanges and modifications may be practiced within the scope of the appended claims.

MICROORGANISM DEPOSIT DATA The following microorganisms which form a part of the present invention ware deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852,, U.S.A. Data regarding the deposits are as follows; Scientific Description Mouse hvbridOMa (boslb c/NS-1) W m m is |fiB m w 09 99 P®EfieitO£S_Mf_2. HIV-gp 110-1 HIV-gp 110-2 HIV-gp 110-3 HIV-gp H0-6 HIV-gp 110-4 HIV-gp HO-5 HIV-p 25-2 HIV-p 25-3 HIV-p 25-6 HIV-p 25-7 IB 9X75 HB 9176 HB 9177 HB 9404 HB §405 HB 94OS HB 9407 HB 9408 HB 9409 HB §410 Deoosit Data August 15,» 1986 August 15, 1985 August 15, 1986 April 30t, 1987 April 30, 1987 April 30, 1987 April 30, 1987 April 30, 1987 April 30, 1937 April 30, 1967 Hybridomas HB 9175, HB 9176 and HB 9177 were tested and found viable on August 26, 1986. The remaining hybridonas were tested and found viable on Hay 4, 1987.

Claims (34)

1. CLAIMS; 84 1. A composition for use in the treatment of HIV infections comprising a therapeutically effective dose of at least one monoclonal antibody, specifically reactive with 5 one or more neutralising regions (as hereinbefore defined) of gpllO or p25 of HIV, and a pharmaceutical!]/ effective carrier.

2. A composition according to claim 1, wherein the neutralising regions comprise epitopes encoded within the 10 env or gag 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 IjAVbbu P25 ainino acid sequence 278 to 319 and/or homologs thereof, and a second monoclonal antibody is reactive with at least one epitope in LAVBRU p25 amino acid sequence 315 to 363 and/or homologs thereof.

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

7. A composition according to claim 6, wherein the p2 5 epitope is located within LAVBR(J amino acid sequence 278 to 30 3 3.9 or 315 to 363, or homologs of said sequences. 25 V Vj» Si'

8. A composition according to claim 6, wherein the gpl10 eoitooe is located within LAV..,,, amino acid seauence 3 08 to «. p. BRU •*;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 gpl10 or p25 of HIV.;

10. A composition according to claim 9, wherein the neutralising region is located on HIV gpl10.;

11. A composition according to claim 10, wherein said lOregion comprises HIV gpl10 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.;15

13. A composition according to claim 9, wherein the neutralising region is located on HIV p25„;

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

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.;30

16. A peptide comprising one of the following HIV gpl10 amino acid sequences or homologs thereof;;till;I (29);Y-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-Gly-Arg~Ala-Phe-Val~Thr-Ila~Gly~Lys-Ile-Y8;;V (177);Y-Thr-Arg-Lys-Ser-Ile-Tvr-Xle-Gly-Pro-Gly-Arg-Ala-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-Val-Phe-His-Ser-His~Tyr-Gln-Pro-Y0;;in which Y and Y', if present, each comprises an amino acid sequence of up to about 20 amino acids.;

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

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

19. A composition comprising a nucleic acid segment, said segroent having from 15 to 150 nucleotides encoding a peptide having from 5 to 50 amino acids from a neutralising region (as hereinbefore defined) of gpl10 or p25 of HIV.;

20. A nucleic acid segment according to claim 19, further encoding a blocking peptide of HIV.;

21. A nucleic acid segment according to claim 19, wherein said peptide comprises at least about five amino acids from LAVqru gpl 10 amino acids 301 to 336 or from LAV°BRU p25 amino acid sequences 278 to 319 or 315 to 363, and homologs of the sequences.;

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

23. A vaccine against HIV infection comprising an immunologically effective dose of one or more peptides of less than about 50 amino acids containing neutralising regions from gpl10 and/or p25, wherein said peptides are;5 admixed with a physiologically acceptable carrier„;

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

25. An immortalized cell line that produces a monoclonal antibody capable of reacting with an envelope glycoprotein;10 gpl10 antigenic determinant contained within a neutralizing region of HIV.;

26. The cell lines HIV-gpllO-1, HIV-gpllO-2, Hiv-gpiio-3, HIV-gpllO-4, HIV-gpllQ-5, HIV-gpllO-6, HIV-p25-6, and HIV-p25-7.;15

27. A monoclonal antibody produced by a cell line of claim 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;20 cell lines of claim 26„;

29. A monoclonal antibody capable of reacting with an antigenic determinant of envelope glycoprotein gpl10 of HIV,;

30. A method for generating cell lines which produce antibodies reactive with antigenic determinants of HIV;25 gpl10, which method comprises:;administering to a host an immunogenic amount of an antigenic preparation enriched for HIV proteins;;monitoring the immunized host for the production of antibodies reactive with gpl 10 antigenic determinants; 30 obtaining antibody producing cells from the host and immortalising said cells;;68;selecting the immortalised cells which produce antibodies to HIV gpllO| and cloning the immortaliaed cells to produce the cell lines.;5

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.;

32. A method for diagnosing the presence of HIV in a 10 biological sample, comprising:;incubating a monoclonal antibody capable of reacting with HIV gpl10 with said biological sample; and detecting the presence of immune complexes formed between the monoclonal antibody and the antigenic 15 determinant in the biological sample, and therefrom determining the presence or absence of HIV in the sample.;

33. A method according to claim 32, wherein the monoclonal antibody is capable of reacting with a neutralising region or blocking peptide of gpllO,;20

34. A method for diagnosing the presence of antibodies to HIV 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 2 5 between the peptide and antibodies in the sample reactive with the peptide, and therefrom determining the presence or \ * absence of HIV in the sample. v 3 5. A method of diagnosing for the presence of HIV in a biological sample comprisings 30 incubating under hybridisation 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. 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. 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. 38. A monoclonal antibody specifically reactive with a neutralising region (as hereinbefore defined) of gpl10 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. 39. An antidiotypic monoclonal antibody, wherein the antibody is specifically reactive with an idiotvpe of a second monoclonal antibody capable of binding to a neutralising region (as hereinbefore defined) of gpllO or p25 of HIV for us® in an immunologically effective dose for vaccinating a patient against HIV infections. 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 gpl10 amino acid sequences or homologs thereof: IX (173) Y~Ala-Ser~Thr~Thr~Thr-Asn-Tyr-Thr-Y9; or XV (191) Y-Ala-Val-Phe~Thr-Asp-Asn-Tyr-Thr-Y'j in which Y and if present, each comprises an amino acid sequence of up to about 20 amino acids. 41. A composition comprising (a) a monoclonal antibody specific for a neutralising region of HIV and (to) a peptide comprising at least one of the following amino acid sequences or homologs thereof; XI (187) Y-Thr-Thr~Ser-Tyr-Thr-Yu; or XII (188) Y-Thr-Asp-Asn-Tyr-Thr-Y5; or XIII (189) Y-Asn-Thr-Ser-Tyr-Gly-Y5; or XIV (190) Y-Asp-Thr-Asn-Tyr-Ser-Y0 -; in which Y and Y', if present, each comprises an amino acid sequence of up to about 20 amino acids. 42. A therapeutically or prophy1actica1ly 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. 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. P. R» KELLY & CO., AGENTS FOR THE APPLICANTS.