DD283936A5 - METHOD FOR THE PRODUCTION OF PEPTIDES THAT ARE IMMUNOLOGICALLY TO REACH THE NEUTRALIZING AREAS OF HIV PROTEINS - Google Patents

Abstract

The invention relates to a process for the production of peptides capable of immunologically mimicking the neutralizing regions of HIV proteins. The peptides according to the invention are prepared by isolating them from an HIV extract or lysate or a cultured biological expression system with the aid of an immunocomplex with the monoclonal antibodies according to the invention. The present invention's products are useful in medicine for the prophylactic and therapeutic treatment of HIV infection for the diagnosis of HIV infection. {Method; manufacture; monoclonal antibodies; peptides; peptide sequence; neutralizing area; Immune complexes; immunoaffinity; Medicine; To HIV; HIV detection}

Description

Field of application of the invention

The invention relates to a process for the preparation of peptides. These may be formulated into pharmaceutical agents useful in the treatment, diagnosis, neutralization, and seeding associated with the treatment of human immunodeficiency virus (HIV) infections.

Characteristic of the known state of the art

The infectious agent responsible for Acquired Immunodeficiency Syndrome (AIDS) and its prodromal phases, AIDS-related complex (ARC) and lymphadenopathy syndrome (LAS), and novel lymphotropic retrovirus. This virus has been referred to in various ways, namely as LAV, HTLV-III, ARV and more recently as HIV.

Since the pandemic spread of HIV, treating infected individuals and preventing transmission to uninfected high-risk groups is of paramount importance. A variety of treatment strategies address different phases in the life cycle of the virus. They line described in Mitsuya and Broder, 1987, Nature 325: 773. One possibility involves the use of antibodies that bind to the virus and inhibit viral replication, either because they interfere with virus entry into the host sites or because of other mechanisms. Once the viral component (s) undergoing intervention by the antibody is identified, it is hoped to be capable of producing antibody titers sufficient to neutralize the infectivity of the virus by vaccination or, alternatively, by passive administration of immunoglobulins or monoclonal antibodies of the desired specificity.

It is believed that the envelope glycoproteins of most retroviruses react with receptor molecules on the surface of the virus-susceptible cells, thus determining viral infectivity to particular hosts. Antibodies that bind to the glycoproteins can block the interaction of the virus with the cell receptors and thus neutralize the infectivity of the virus, see Tue Molecular Biology of Tumor Viruses, 534 (J. Tooze, ed., 1973) and RNA Tumor Viruses, 226,236 (R. Weiss et al, ed., 1982) to both publications is hereby incorporated by reference. See also Gonzalez-Scarano et al., 1982, Virology 120: 42 (La Crosse Virus); Matsuno and Inouye, 1983, Infect. Immune. 39: 155 (Neonatal Calf Diarrhea Virus); and Mathews et al., 1982, J. Irnmunol., 129: 2763 (Encephalomyelitis virus).

The general structure of HIV consists of a ribonucleoprotein core surrounded by a lipid-containing envelope which, in the course of its development, forms the virus from the membrane of the infected host cell. The virally encoded glycoproteins are embedded in the envelope and protrude outward. The envelope glycoproteins of HIV are initially synthesized in the infected cell as a precursor molecule of 150,000 to 160,000 daltons (gp150 or gp160) and then in the cell into an N-torminal fragment of 110,000 to 120,000 daltons (gp110 or gp120) around the external glycoprotein and converted into a C-terminal fragment of 41,000 to 46,000 daltons (gp41), which is the transmembrane envelope glycoprotein. For the reasons given above, the gp 110 HIV glycoprotein has been the subject of numerous subspans; studies on a potential target to disrupt the life cycle of the virus. It has been shown that sera from MV-infected individuals neutralize HIV in vitro and that antibodies that bind to purified gp110 are present in the sera, Hobert-Guroff et al., 1985, Nature 316: 72 Weiss et al., 1985, Nature 316: 69 and Mathews et al., 1986, Proc. Natl. Acad. Be. USA, 83: 9709. Purified and recombinant gp 100 stimulated the production of serum neutralizing antibodies when administered to animals for immunization, Robey et al., 1986, Proc. Natl. Acad. Be. USA, 83: 7,023; Lasky et al., 1986, Science 233: 209; and Zagury et al., 1986, Nature 326: 249. Furthermore, it has been shown that the gp110 molecule binds to the CD4 (T4) receptor and that monoclonal antibodies that recognize certain epitopes of the CD4 receptor block HIV binding, syncytium formation and infectivity, McDougal et al., 1986, Science 231: 382. Putney et al. (1986, Science 234: 1392) demonstrated neutralizing serum antibodies in animals after immunization with a recombinant fusion protein containing the carboxyl-terminal half of the gp 110 molecule. They further demonstrated that glycosylation of the envelope protein is unnecessary for response to the neutralizing antibody.

It would therefore be desirable to have a subunit vaccine against AIDS using the HIV gp 110 molecule or portions thereof. Subunit vaccines are an alternative to vaccines made from inactivated or weakened viruses. Inactivated vaccines are problematic because not all virus particles may have been killed and their weakened viruses have the ability to mutate and regain their disease-causing effects. Subunit vaccines use only those parts of the virus to immunize the host, which contain the antigens or epitopes capable of eliciting the immune response, i. H. a response to neutralizing antibodies, ADCC and ch-cytotoxic T-cell response. The main advantage of subunit vaccines is that irrelevant viral material is excluded. is.

Viral subunits for application in a vaccine can be generated by various methods. For example, the envelope glycoprotein may be expressed and purified in a Baku host, although this molecule lacks most post-translational modifications (such as glycosylation) or other processing. Such a modification can be achieved by using a eukaryotic expression system, for example, yeast or cultured mammalian cells. Viral genes have been introduced into mammalian cells using vaccinia virus as a vector, see, for example, Mackett, M. et al, 1982, Proc. Nat. Acad. Be. USA 79: 7415; D. Panicali and E. Paoletti, 1982, Proc. Nat. Acad. Be. USA 79: 4927.

Recombinant vaccinia viruses can be constructed according to the methods of Hu et al., Nature 320: 537 (1986) or Chakrabarti et al., Nature 320: 535 (1986), incorporated herein by reference. In these systems, the viral glycoproteins produced by cells infected with recombinant vaccinia are suitably glycosylated. You can too. Extrusion and final isolation are transported to the cell surface.

An important step in the production of a subunit vaccine is adequate purification of the desired glycoprotein from the complex mixture of the expression system. For this purpose, several methods are suitable. These include, but are not limited to, preparative polyacrylamide gel electrophoresis, golpermeation chromatography, various methods of chromatography (ie, ion exchange, reverse phase, immunoaffinity, and hydrophobic intact chromatography), etc. The majority of these methods are used in various combinations See, for example, DG Kleid et al., 1981, Science 214: 1125, Cabradilla CD et al., 1986, Biotechnology 4: 128, DJ Donowko, 1985, Proc Nat.Acad., See, USA 82: 7748, incorporated herein by reference, methods are needed to produce subunit vaccines that reduce the number of steps required to achieve the best purification of a particular viral antigen from a complex expression mixture Separation of antigens from foreign components may be accomplished using immunoaffinity chromatography, also known as immunoadsorption In principle, othode consists in selectively adsorbing an antigen to a solid support to which a specific antibody has been covalently linked. The selectively absorbed antigen is then eluted from such an antibody-affinity adsorbent by, for example, changing the pH and / or ionic strength of the buffer.

Polygonal antibodies obtained from animals immunized with the desired antigen or from naturally infected individuals (see, for example, Laksy et al., Supra) were frequently used as immunoadsorbents. However, these reagents generally have significant disadvantages, for example that (i) not all of the antibodies which bind to the insoluble support are specific to the molecule of interest, thus requiring further purification; (ii) the yields of the desired antigen are often low; and (iii) the antibody affinities often vary from one preparation to another, requiring modifications to the elution method. By using monoclonal antibodies specific for the desired viral antigen in subunit vaccine preparations instead of polyclonal antibodies, one could circumvent these difficulties.

Monoclonal mouse antibodies that bind HIV antigens have already been described. Several groups have reported monoclonal antibodies specific for the core protein p25 (see, for example, Marzo Veronese et al., 1985, Proc. Nat. Acad., U.S.A.82: 5199, and J.Chassagne et al., 1986, J. Immunol.

136: 1442). Monoclonal antibodies specific for the membrane glycoprotein gp41 have previously been described (see, for example, Marzo Veronese et al., 1985, Science 229: 1402).

Object of the invention

There is a need for peptides capable of mimicking the neutralizing regions of HIV proteins. Monoclonal antibodies specific for epitopes in well defined regions of the major envelope glycoprotein gp 110 are required for their production. These monoclonal antibodies can be used to purify the desired region of gp 110, for example for use in vaccines from disrupted viruses or recombinant expression systems. Further, the region containing (s) the epitope (s) recognized by the monoclonal antibodies could be chemically synthesized, resulting in the purification and administration of larger fragments of the gp HO molecule associated difficulties could be avoided. The present invention fulfills these and other needs.

Explanation of the essence of the invention

According to the invention peptides, which are able to mimic di i neutralizing epitopes of HIV proteins immunologically as well as other peptides which interfere with the HIV-lnfektivit it, provided. These novel materials find use, for example, in diagnostic assays for the detection of HIV infection and in the therapeutic treatment of or vaccination against HIV infection.

The invention relates to new agents and methods for neutralizing HIV infection, d. H. for preventing or substantially inhibiting the formation or cellulase transmission of infectious HIV in a host. In particular, peptides that mimic a neutralizing region of HIV, as well as monoclonal antibodies that react with such a region, are used for the diagnosis and treatment and vaccination against HIV infections. The term "neutralizing region" in this context refers to those parts of HIV, in particular HIV proteins which contain amino acid segments which define one or more epitopes which react with antibodies which are able either alone or in combination with other antibodies according to the invention Appropriate neutralization assays are known, including, for example, the reduction of HIV infections in T cell lines, the reduction of plaque-forming moieties of VSV (HIV) pseudotypes carrying the envelope glycoproteins of HIV, Syncytium Inhibition Assays and Virion Receptor Binding Assays As desired, the neutralizing activity is comparable to antibody reactivity in immunoreactive assays, such as immunofluorescence, immunoblot, and radioimmunoprecipitation assays.

The novel peptides, typically less than about 50 amino acids, contain 5 or more contiguous amino acids that form epitopes substantially similar to those epitopes found in neutralizing regions of HIV gp 110 or p.25, which are expressed by the env and gag Regions of the HIV genome are encoded. Of particular interest are those regions extending from about amino acid residue 301 to about 336 from gp 110 and from about 278 to about 319 and from about 315 to about 363 of ρ 25 (all from the HIV strain designated LAV _B ru). The name of the amino acid residues is derived from the Los Alamos database (AIDS virus sequence database, Los Alamos National Laboratory, Theoretical Division, Los Alamos, NM 87545).

It will be appreciated by those skilled in the art that other analogous regions ("homologues") of other HIV isolates can be identified based on their location in related proteins from different isolates In practice, such homologs can be identified by reference to LAVeAir sequence data as follows:

a) the amino acid sequences of HIV isolates and LAV _B nu can be aligned so that there is maximum homology between the two sequences.

b) one can identify peptides comprising those amino acid sequences of HIV isolates corresponding to the location of the LAVoRu peptides which immunologically mimic the LAV _B Ru proteins. Peptides comprising the thus identified amino acid sequences of HIV isolates typically mimic the corresponding proteins of the HIV isolate immunologically.

This method can also be applied to HIV strains v. ^ iden, which are still to be found. For example, when new HIV strains are identified, their envelope and core amino acid sequences can be aligned with those of LAV ₆ Ru for maximum homology. The methods by which the sequences are aligned are known to those skilled in the art. When the sequences are aligned, it is desirable to keep the homology between the cysteine residues as large as possible. The amino acid sequence of the new HIV strain or HIV species corresponding to the location of the peptides disclosed herein can be synthesized and used in the present invention.

Another method for determining sequences of a homologous region in other HIV strains has been described by Scharf et al., Science (1986) 233: 1076. Two oligonucleotide primers are used which bind to conserved sequences outside of the sequence region of interest, with different restrictive sites present in each primer. The DNA of HIV strains can then be multiplied in vitro and the resulting oligonucleotides can be cloned into vectors for sequence analysis and incorporated into a vaccine cassette representing a particular epitope of the HIV strain.

It is not required in the present invention that the epitopes present within such sequences cross-react with antibodies of all HIV strains or species. Peptides comprising immunological epitopes that distinguish one species or serogroup from another are used to identify particular species or serogroups and may be used to identify individuals infected with one or more HIV species or serogroups. They are also useful in therapeutic agents in combination with other peptides derived from either a homologous region or another neutralizing region.

The peptides of interest here are preferably derived from the gp 110 region of the virus. Of particular interest in this area are peptides encoded in the open env reading frame extending from about base pair (bp) 6667 to about the base pair 6774 of the LAV _BRU isolate. Different homologous regions of other HIV isolates thus include, as listed in Table I, the homologous sequences obtained from the Los Alamos database (except LAV 2). Other peptides useful for generating and screening for monoclonal antibodies are those encoded in the open env reading frame from about bp7246 to about 7317 of LAV _B ru. Such antibodies and reactive peptides are particularly useful in immunoassay.

In the gag region of the LAV _BRU isolate, the p25 amino acid sequences of about 278 to 319 and 315 to 363 represent further neutralizing regions of HIV.

It will be apparent to those skilled in the art that further neutralizing regions of HIV can be identified based on the teachings of the present invention. In particular, combinations of monoclonal antibodies that react with various HIV epitopes show neutralizing activity.

TABLE 1 HXB2 TGTACAACACCCAACAACAATACAAGAAAACA ATCCGTATC CytThrArg ProAenAenAenThrArgLyeArg IleArf XIt 309

BH102 Ser 309

BH8 --------- · .---- «.. ·· -» ----- ·. · »---- Lyi ~ ----» - · .-- ·. »---------- 309

HXB3 Lye 309

H9M -Ser 309

BRU - · .-- * --.----. ·· »- ·· -» --- ·· »« «- -« * --- ^ SCT "" * "**" ** "". "-". "- ·. ·. ··· - jIm MAL Gly ArgCly -KtePhe 314 ELI -le TyrGln GIn ThrPro 310 ARV2 - Ser Tyr-312 WMJ2 Tyr V * l-ArgSer LeuSer 306 RFENV -Ser ThrLys 322 Z6 TyrLye ClnSer- ThrPro-311

23 GlySerAspLysLysIle GlnSer 306

WY5 Lye GIy Ala 304 CDC42 Here's ValThrLeu 320 LAV2 GIy-Lys - VaI Gin HetLeu 302 HXB 2 CAGAGA CGACCAGGGAGAGCATTTGTTACAATAGGAAAATAGG AAATATG ClnArg ClyProClyArgAlaPheValThrlleClyLyelleGlyAenHet 326

BH102 326

BH8 - - 326

HXB3 326

H9M - -

BRU

HAL Gin-UuTyr Thr IleVal-Asplle ELI GlyLeu .. .GlnSerLeuTyrThr Arg IleValSerArgSer ARV2 His Thr ArgealGlyAsp WMJ2 Arg Argu ... IleGlylle RFENV VallleTyrAlaThr GIn IleGlyAep

26 ClyLeu.,. ClnAlaLeuTyrThr Arg - ArgThrLyellelle

Z3 Arglle LyeVal-TyrAlaLy · -Gy NY5 ClyPro ... ThrLeuTyrAlaArgClu A'plli CDC42 ValTrpTyr-Thr GIu LeuGlyAsn LAV2 MetSer HisVal-HieSerHieTyrClnProIle-Lys

11XB2 ... AGA ... CAAGCACATTGT

... Arg ... GlnAlaHieCyr 331

BH102 331

BH8 331

HXB3 - 331

H9M 331

BRU 336

MAL Arg-Tyr-334

fcLl IlelleGly- 330

ARV2 lets Lye ... 333

WMJ2 Ue 326

RFtNV He Lys ... 343

Z6 GIy ... 334

Z3 IleThrCly 326

NY5 325

CDC42 - 341

LAV2 ArgProArg Met- 330

Peptide I, also referred to as peptide 29, is encoded in the open env reading frame approximately from amino acid residues 308 through and has the following amino acid sequence in which the oligopeptides within this sequence comprise linear epitopes of this sequence:

1 (29) Y-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Y ' .

in the Y and Y ', if present, each represent sequences of biszuotwa 20 amino acids. For example, if Y and / or "are present, they may include one or more amino acids from sequences flanking amino acid residues 308 to 328 of the HIV envelope sequence, or any part of these flanking sequences. comprise, in whole or in part, the LAVeRu envelope amino acid sequence approximately from residues 301 to 307, and Y 'may comprise in whole or in part the LAVeRu envelope amino acid sequence approximately from the 329 to 336 residues, as follows:

Cys-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-Lys-Ile-Gly-Asn-Met-Arg-Gln-Ala-His-Cys.

Alternatively, one can prepare truncated sequences of the peptides of the invention. In this regard, the following sequences of peptide 29 are particularly useful:

III (29b) Y-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-Gly-Y '^

wherein Y and / or Y ', if present, each represents sequences of up to about 20 amino acid residues;

IV (29c) Y-Ile-Gln-Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Y ^

wherein Y and Y ', if present, each represent sequences of up to about 20 amino acid residues.

In another embodiment, particularly interesting homologous regions of the ARV-2 isolate are encoded in the open env reading frame of about amino acid residues Nos. 306 to about 323. They typically have the following amino acid sequence in which oligopeptides within this amino acid sequence comprise linear epitopes within such sequence.

V (177) Y-Thr-Arg-Lys-Ser-Ile-Tyr-Ile-Gly-Pro-Gly-Arg-Ala-Phe-His-Thr-Thr-Gly-Arg-Ile-Y ',

wherein Y and Y ', when present, each comprise 1 to about 20 or more amino acid residues. When Y and / or Y 'are present, they may comprise one or more amino acid residues of sequences flanking amino acid residues 306 to 323 of the ARV-2 envelope sequence, or a portion of these flanking sequences. In particular, Y may comprise all or part of the HIV envelope inosäuresequenz from residues 299 to 306; Y 'may comprise all or part of the HI V envelope amino acid sequence, for example from residues 324 to 333.

Alternatively, one can produce truncated sequences of peptides V. In this regard, the following sequences are particularly useful:

VI (177a) Y-Thr-Arg-Lys-Ser-Ile-Tyr-Ile-Gly-Pro-Gly-Y ';

VII Y-Asp-Cys-Lys-Thr-Ile-Leu-Lys-Ala-Leu-Gly-Pro-Ala-Ala-Thr-Leu-Glu-Glu-Norleu-Norleu-Thr-Ala-Cys-Yj

wherein Y and / or Y ', if present, each represent sequences of up to 20 or more amino acid residues.

Another example includes homologous regions of the LAV-2 isolate, such as encoded in the open env reading frame of about amino acid residues 311 to 330, for example. They typically have the following sequence:

VIII (110-2-2) Y-Lys-Thr-Val-Lys-Ile-Nor-Leu-Nor-Ser-Gly-His-Val-Phe-His-Ser-His-Tyr-Gln-Pro-Y ' .

wherein Y and / or Y ', if present, each represents sequences of up to 20 or more amino acid residues (see Nature 326: 662 (1987), which is hereby incorporated by reference).

The invention further relates to novel cell lines capable of producing monoclonal antibodies and agents containing these antibodies. The antibodies are capable of producing extremely high titers (from 10 ² to 10 ⁴ to about 10 ⁷ or more) neutralizing regions contained in a predetermined sequence of the envelope glycoprotein gp 110 or ρ 25, their protein precursors, biologically expressed recombinant fusion proteins and to selectively recognize synthetic peptides containing one or more epitopes within the predetermined sequence range of gp 110 or ρ 25. The hybrid cells have an identifiable chromosome in which the germline DNA has rearranged to encode an antibody that has a binding site for an epitope on gp 110 or ρ 25, which have some or all of the clinical HIV isolates. These monoclonal antibodies can be used in a variety of ways. These include the applications in diagnosis and therapy as well as the application to identify other cross-reactive antibodies, such as blocking antibodies. Peptides or polypeptides containing the epitope (s) with which they react find separate application as immunogens for vaccines or as therapeutic agents.

Blocking peptides

Another embodiment of the invention, primarily for use with the above peptides or neutralizing monoclonal antibodies, is directed to other peptides or antibodies that interfere with HIV binding to receptors to further attenuate HIV infectivity. Preferably, the so-called "blocking peptides" capable of inhibiting viral proliferation, as well as monoclonal antibodies specific for epitopes contained in such blocking peptides, can be used to enhance the efficacy of treating HIV infections HIV-blocking peptides typically correspond to that HIV amino acid sequence which is believed to be essential for attachment of the virus to a host cell, for example, the env-encoded amino acid residues about 190 to about 197 of LAVbru and about 185 to about 192 of ARV-2 and HTLV-IIKBH-10), including the peptide T-octapeptide (Ala-Ser-Thr-Thr-THr-Asn-Tyr-Thr) and its various derivatives (e.g., IX below) and Analogs (eg XI, bottom) described by Pert et al (1986, Proc Natl, Acad., USA 83: 9254-9258, which is hereby incorporated by reference) and which are located on the envelope glycoprotein (gp 110 or 120).

Of particular interest are, for example, blocking peptides having the following sequences, wherein preferably the NH 2 -terminus is acetylated and the COOH-terminus is amidated:

IX (173D) Y-oAla-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-Y ';

X (186) Y-Thr-Thr-Asn-Tyr-Thr-Y;

XI (187) Y-Thr-Thr-Ser-Tyr-Thr-Y ';

XII (188) Y-Thr-Asp-Asn-Tyr-Thr-Y ';

XIII (189) Y-Asn-Thr-Ser-Tyr-Gly-Y ';

Y-Asp-Thr-Asn-Tyr-Ser-Y '; XV (191) Y-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Y ';

wherein each peptide Y and Y ', if present, each represents an amino acid sequence of up to about 20 amino acids. Epitopes or antigenic determinants within these peptides are typically defined by at least 5 contiguous amino acids and are used, for example, to mimic naturally occurring HIV antigen sites in the production of HIV-reactive antibodies and vaccines.

Obtaining monoclonal antibodies

The preparation of monoclonal antibodies that specifically react with one or more neutralizing regions of HIV is accomplished by administering to a host an immunogenically effective amount of an HIV protein-enriched antigen preparation; monitors the production of the specifically reactive antibodies in the immunized host; recovering and immortalizing the antibody-producing cells from the host; selecting the immortalized cells producing antibodies to HIV; the immortalized cells cloned for the production of cell lines; and the cell lines capable of producing antibodies that react with one or more HIV neutralizing regions, cultured and the antibodies recovered.

Recombinant fusion proteins expressed by a eukaryotic or bacterial host or HIV proteins from an HIV extract or lysate are preferably used as HIV proteins.

Preferably, an immortalized control line is also produced which produces monoclonal antibodies capable of reacting with an envelope glycoprotein gp 110 antigenic determinant contained within the neutralizing or blocking peptide of HIV.

Thus, the production of monoclonal antibodies can be accomplished by immortalizing the expression of nucleic acid sequences encoding antibodies specific for HIV by introducing such sequences, typically cDNA encoding the antibody, into a culturable host. The immortalized cell line may be such a mammalian cell line transformed by oncogenesis, transfection, mutation or the like. Such cells include myeloma lines, lymphoma lines or other cell lines capable of inducing expression and secretion of the antibody in vitro. The antibody may be a naturally occurring immunoglobulin of a mammal produced by transformation of a lymphocyte, particularly a splenocyte, by means of a virus or by fusion of the lymphocyte with a neoplastic cell, for example a myeloma, to form a hybrid cell line. Typically, the splenocytes are obtained from an animal immunized against the HIV virus or a fragment having an epitopic site thereof.

Immunization methods are known, they can vary considerably and yet remain effective, see Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, 2 supra. (1986), which is incorporated herein by reference. Destroyed viruses, synthetic peptides and bacterial fusion proteins containing the antigenic fragments of the gp 110 or ρ 25 molecule can be used as immunogens. Preferably, the immunogen of disrupted viruses, peptides or recombinant proteins is enriched by proteins or fragments thereof containing the epitopes for which antibody-producing B cells or splenocytes are desired. In particular, solutions containing lysates or extracts of disrupted viruses, or supernatants of biologically expressed recombinant proteins or disrupted expression vectors by glycoproteins may be accumulated as desired using purification methods such as polyacrylamide gel electrophoresis. Lectin affinity purification is a preferred convenient method for purifying gp 110 and other glycoproteins, for example, affinity purification using lentil lectin. The extent to which the glycoproteins are purified from solutions for use as an immunogen can vary widely, i. H. from less than 50% to usually at least 75 to 95%, desirably 95 to 99% and most desirably to absolute homogeneity.

Having purified the proteins to the desired extent, they may be suspended or dissolved in a physiological carrier suitable for immunization, or they may be coupled to an adjuvant. For example, one preferred technique involves adsorption of the proteins and fragments thereof to lectin lectin agarose or another macromolecular carrier for injection. Immunogenic amounts of antigenic preparations enriched in HIV proteins, including the gp 110 glycoprotein and the ρ 25 core protein, or the antigenic portions thereof, are generally injected in concentrations ranging from 1 pg to 20 rrfg / kg of the host , The administration may be, for example, by intramuscular, peritoneal, subcutaneous, intravenous, etc. injection. Administration may be 1 or more times and usually at 1 to 4 week intervals. The immunized animals are controlled for the production of antibodies against the desired antigens, then the spleen is removed, the spleen B lymphocytes isolated and fused with a myeloma cell line or transformed. The transformation or fusion can be carried out in a conventional manner, the fusion method is described in numerous patents, for example in US Pat. Nos. 4,172,124, 4,350,683, 4,364,799, 4,338,292 and 4,423,147; compare also, Kennett et al. Monoclonal Antibodies (1980) and those cited therein, as well as Godin as mentioned above; All of these publications are hereby incorporated by reference.

The immortalized cell lines can be screened and screened according to conventional procedures. In the cell supernatants, one can detect the antibodies capable of binding to the desired gp 110 or ρ 25 HIV proteins, the recombinant fusion proteins, or the synthetic peptides containing the desired epitope region. Suitable immortalized cell lines may then be grown in vitro or injected into the peritoneal cavity of a suitable host for ascites fluid extraction. Because antibodies of the present invention are known to be specific for epitopes present, for example, within the regions defined by the LAv e _RU gene range from about bp6688 to about bp6750 (coding for peptide 29) or from about bp7246 to about 7317 ( coding for peptide 36) (bp numbering according to Wain-Hobson et al., Cell 44: 9 1985, incorporated herein by reference), the supernatants can be subjected to a competitive assay with the monoclonal antibodies. Thus, other immortalized hybridoma cell lines with the desired binding properties can readily produce from a variety of sources due to the availability of the antibody of the invention specific for a particular antigen. Alternatively, one may fuse these cell lines with other neoplastic B cells, which may serve other B cells as receivers for the genomic DNA encoding the antibodies.

Neoplastic B cells from rodents, especially mice or rats, are preferred. However, they may also be derived from other mammalian species, such as Lagomorpha, cattle, sheep, horses, pigs, birds (Avian) or the like. It is easy to immunize these animals and obtain their lymphocytes, especially the splenocytes, for fusion.

The monoclonal antibodies secreted by the transformed or hybrid cell lines may be from one of the classes or subclasses of the immunoglobulins, such as IgM, IgD, IgA, IgG ₁ , or IgE. IgG is preferred because it is the most common isotype used in diagnostic assays. The monoclonal antibodies can be used intact or as fragments such as Fv, Fab, F (ab ') 2. However, they are usually used intact. To circumvent a possible antigenicity of a non-human monoclonal antibody in a human host, one can construct chimeric antibodies in which the antigen-binding fragment of an immunoglobulin molecule (variable region) is bound by a peptide linkage of at least a portion of another protein that is not recognized as foreign in humans, such as the "cast out portion" of a human immunoglobulin molecule, by fusing the animal's variable region exons to human kappa or gamma exons of the constant region Several methods are known to those skilled in the art, for example those described in PCT 86/01533, EP-A-171496 and EP-A-173494, which are hereby incorporated by reference.

: harmless formulations and applications

The neutralizing-action monoclonal antibodies of the present invention, for example, those which react with an epitopic site on gp 110 or ρ 25 or with a blocking peptide, can be incorporated into pharmaceutical agents as a component to alleviate HIV infection. The agent should contain a therapeutic or prophylactic amount of at least one of the monoclonal antibodies of the invention together with a pharmaceutically active carrier. The pharmaceutical carrier should be a compatible, non-toxic substance capable of mediating the monoclonal antibodies to the patient. Sterile water, alcohol, fats, waxes and inert solids can be used as carriers.

Pharmaceutically acceptable adjuvants (buffers, dispersants) may also be included in the compositions of the invention. These agents may contain a single monoclonal antibody, for example, to be specific for HIV strains having envelope glycoproteins containing an epitopic site within a range encoded by bp6688 to bp 6750. Alternatively, a pharmaceutical agent may contain one or more monoclonal antibodies and form a "cocktail." For example, a cocktail containing monoclonal antibodies to various strains of HIV provides a universally applicable product with therapeutic or prophylactic activity, most of the clinical HIV / AIDS. The cocktail may contain monoclonal antibodies that bind to HIV proteins or glycoproteins that are different from gp 110 or ρ25, such as gp41 glycoprotein or p34 nuclease / integrase The molar ratio of the various monoclonal antibody components differs in the generally not more than a factor of 10, in particular not more than a factor of 5, and is generally about 1: 1-2 per each other antibody component.

The monoclonal antibodies of the invention can be used as a separate agent to be given together with other anti-retroviral agents, including blocking peptides. The current state of development of anti-retroviral agents and anti-HIV agents is described in detail in Mitsuya et al., Nature 325: 773-778, 1987, which is incorporated herein by reference.

The monoclonal antibodies, peptides and pharmaceutical agents of the invention are especially useful for oral and parenteral administration. Preferably, the pharmaceutical agents are administered parenterally, i. H. subcutaneously, intramuscularly or intravenously. The present invention therefore also provides means for parenteral administration comprising a solution of the monoclonal antibodies, peptides or a cocktail thereof in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, for example, water, buffered water, 0.4% saline, 0.3% glycine, and the like. These solutions are sterile and generally free of particles. The agents can be sterilized by conventional and known methods. The agents (may contain pharmaceutically acceptable excipients as required to adjust to near physiological levels, such as pH adjusters and buffers, toxicity adjusting agents, and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like

-9- Ü83 936

Antibody in these formulations can vary widely, i. H. less than about 0.5 wt.%, usually about or at least about 1C: iw.% up to 15 or 20 wt.%. This amount will be mainly in terms of liquid volumes, viscosities, and the like, and preferably chosen on the mode of administration. A typical pharmaceutical agent for intramuscular injection may thus contain 1 ml of sterile buffered water and 50 mg of monoclonal antibody. A typical intravenous infusion may contain 250 ml of sterile Ringer's solution and 150 mg of monoclonal antibody. Methods for the preparation of parenterally administrable agents are well known in the art, nt or Föchman and are described in detail, for example, in Remington's Pharmaceutical Science, 15th Ed., Mack Publishing Company, Easton, Pennsylvania (1980), which is hereby incorporated by reference.

The monoclonal antibodies and peptides of the invention may be lyophilized for storage and reconstituted in a suitable carrier before use. This method has proven to be effective in conventional immunoglobulins. In doing so, known lyophilization and reconstitution techniques can be used. It will be apparent to those skilled in the art that lyophilization and reconstitution may result in a different degree of antibody activity loss (in conventional immunoglobulins, qM antibodies are prone to greater loss of activity than IgG antibodies) and, therefore, the amount applied will be adjusted must to compensate for the loss. The agents containing the monoclonal antibodies, peptides or cocktails thereof according to the invention can be administered for the prophylactic and / or therapeutic treatment of HIV infections. In therapeutic administration, the agents are administered to a patient already infected with HIV in an amount sufficient to cure or at least partially arrest the infection and associated complications. The dose needed to achieve this is defined as the "therapeutically effective dose." The effective amount of η in this application depends on the severity of the infection and the general condition of the patient's immune system, and is generally within the range from about 1 to 200mg of antibody per kg of body weight, with dosages of 5 to 25mg per kg commonly used, since the products of the present invention will generally be used in severe illnesses, ie life threatening or potentially life threatening situations, it is daily and can If administered prophylactically, the agents containing the peptides, antibodies or cocktail thereof of the present invention may be administered to a patient not yet infected with HIV recently received such an infection was or had suspected that he was exposed or at risk of exposure to the virus. Prophylactic administration serves to enhance the patient's resistance to a potential infection or to vaccinate the patient against the virus. An amount suitable for this purpose is defined as a "prophylactically effective dose." Also in this application, the exact amounts to be administered depend on the health and general condition of the patient's immune system, generally ranging from 0.1 mg to 25 mg per kg, in particular 0.5 mg to 2.5 mg per kg.

Single or multiple administrations of the agents may be made, with the dose and regimen chosen by the attending physician. The pharmaceutical formulations should in any case provide an amount of antibodies of the invention sufficient to effectively treat the patient. In addition, the monoclonal antibodies according to the invention are used as target-specific carrier molecule. An antibody may be linked to a toxin to form an immunotoxin or to a radioactive material or drug agent to form a radiopharmaceutical or drug. Methods for the production of immunotoxins and radiopharmaceuticals are known (see, for example, Cancer Treatment Reports 68: 317 [1984]).

It is also possible that heteroaggregates of monoclonal antibodies of the invention and human T cell activators, such as monoclonal antibodies against the CD3 antigen or against the F _c -y receptor on T cells, human T cells or F _c -yielding Enable cells (such as K cells or neutrophils) to kill HIV-infected cells via antibody-dependent cell-mediated cytolysis (ADCC). Such heteroaggregates may be formed, for example, by covalently crosslinking the anti-HIV antibodies with the anti-CD3 antibodies using the heterobifunctional reagent N-succinimidyl-3- (2-pyridyldithio-propionate as described by Karpowsky et al., J. Chem. Exp. Med. 160: 1686 (1984), which is hereby incorporated by reference.

The peptide-based compositions of the invention alone can find therapeutic use, the administration resulting in the reduction or elimination of HIV in an infected host. These agents, such as peptide 29, the blocking peptides and peptide 126 described in USSN 930785, which is incorporated herein by reference, may be administered in suitable physiological carriers intravenously, subcutaneously, intramuscularly, intraperitoneally, etc. Various carriers are suitable, including phosphate buffered saline, saline, water, potassium chloride, sodium lactate, or the like. The peptide co-concentration may vary widely depending on the final application, activity and mode of administration. Preferably, the peptides have an amidation of the COOH-Terrninus, a formylation of NHrTerminus or other pharmaceutically acceptable derivatization. The addition of blocking peptides to the peptides that mimic a neutralizing HIV region and / or the specifically reactive antibodies of the invention result in significantly increased therapeutic efficacy. The formulations may also contain other anti-HIV agents (other than the monoclonal antibodies that bind the peptides), such as 3'-azido-3'-deoxythymidine, 2 ', 3'-dideoxycytidine, 2', 3'- Dideoxy-2 ', 3'-didehydrocytidine, etc.

Application of monoclonal antibodies in immunoaffinity purification

The monoclonal antibodies specific for polypeptides containing gp 110 or other antigenic determinants, especially those antigenic determinants obtained from biologically expressed recombinant fusion proteins or lysates or extracts of cultured HIV, are particularly useful in purification procedures. In general, the antibodies have affinity association constants in the range of 10 ⁸ to

10 ' ² M. Such antibodies can be used to purify recombinant fusion proteins from the culture medium of the recombinant expression system when the expressed protein is secreted or from the components of the disrupted biological expression system when the protein is not secreted. The monoclonal antibodies capable of reacting with gp 110 or other antigenic determinants are generally immobilized to a substrate or support or immobilized. The solution containing the HIV antigenic determinants is then contacted with the immobilized antigen under conditions suitable for the formation of immune complexes between the antibody and the polypeptides containing the gp 100 antigenic determinants. Unbound material is separated from the bound immune complexes, these complexes or the gp 110 antigen fragments are then separated from the carrier.

The monoclonal antibodies are typically roughly purified of ascites fluid or cell culture supernatants before being bound to the carrier. Such methods of purification are known to the person skilled in the art, including fractionation with neutral salts at high concentration.

Other methods such as DEAE chromatography, gel filtration chromatography, preparative gel electrophoresis or protein A affinity chromatography can also be used to purify the monoclonal antibodies before they are used as immunoadsorbent

 The carrier to which the monoclonal antibodies are immobilized should have the following general characteristics:

(a) generally weak interactions with proteins to minimize non-specific binding,

(b) good flow characteristics allowing flow of high molecular weight materials,

(c) presence of chemical groups which can be activated or modified to allow chemical binding of the monoclonal antibody to b, possibly

(d) physical and chemical stability under the conditions applicable to monoclonal antibody binding and

(e) Stability to the conditions and constituents of the buffers required for the absorption and elution of the antigen. Some commonly used carriers are agarose, derivatized polystyrenes, polysaccharides, polyacrylamide beads, activated cellulose, glass and the like. There are several chemical methods for attaching the antibodies to the substrate supports, see generally Cuatrecasas P., Advances in Enzymology 36:29 (1972). The antibodies according to the invention can be attached directly or alternatively to the support via a link or spacer (left, spacer).

The general conditions required for the immobilization of monoclonal antibodies to chromatographic supports are known, see, for example, P. Tijissen, 1985, Practice and Theory of Enzyme Immunoassay, incorporated herein by reference. The actual coupling method used depends to a small extent on the property and the type of antibody to be coupled. Monoclonal antibodies have properties that are usually consistent from lot to lot, so that the conditions mentioned can be optimized. The linkage is typically via covalent bonds.

To the separation matrix is then added a suspension of extracts or lysates of HIV virus, the supernatant from a cultured biological expression system or a suspension of the disrupted cells. The mixture is incubated under conditions and for a Zoitdauer sufficient to form the immune complex, usually at least 30 minutes, more preferably 2 to 24 hours. The immune complexes, which contain polypeptides with antigenic parts of gp110, are separated from the reaction mixture, for example by elution, and the bound immune complexes are extensively washed with adsorption buffer, which can then be eluted from the separation matrix used an eluent, which is compatible with the next to the application support. Such eluent are known in the art. the polypeptides comprising gp 110 or other antigenic parts can be selectively removed. for example, one can peptides containing an epitope ' that is recognized by the antibody to be used to compete with the antibody binding sites. This "lev el illustrates an alternative elution procedure that can be carried out under mild elution conditions. the selectively adsorbed polypeptide that contains the gp 110 antigen can be from a Elute antibody affinity adsorbent by changing the pH and / or the ionic strength of the buffer. C'iaotropic agents are also used to remove the bound antigen. The choice of a chaotropic agent, concentration, and elution conditions will depend on the characteristics of the antibody-antigen interaction, but once established, they should not be subjected to changes commonly required in polyclonal affinity separation systems.

It may be necessary to adjust the pH of the eiuated material to physiological pH if a high or low pH or ionic strength buffer was used to separate the bound gp 110 antigens from the separation matrix. Also, a dialysis or a Geifiltrations chromatography, excess removal, used in the eluent salts are required to contain the reconstitution of gp 110 or ν jn polypeptides antigen fragments of gp 110 to enable ra native conformations. For example, the methods of the present invention yield substantially pure gp 110 or substantially pure polypeptides containing antigenic fragments thereof, either naturally produced by infected cell cultures or recombinant expression systems of bacteria, yeasts or cultured insect or mammalian cells, gp 110, the fragments thereof or other purified proteins typically have a purity of greater than 50%, usually at least 75%, and often greater than 95 to 99%. These products have many uses.

The HIV gp 110 proteins, the polypeptides containing antigenic fragments thereof, or other proteins that have been substantially purified by the method of the present invention have many uses. These include AIDS subunit vaccine formulations in which the immunogen comprises an effective dose of antigenic determinants of, for example, gp 110 or a neutralizing region thereof. Other components of the formulation may include those antigenic portions of HIV proteins or glycoproteins which stimulate the production of antibodies (preferably neutralizing antibodies) in an immunized host, which antibodies are capable of providing protection against a subsequent immunoassay HIV infection? szuüben.

-11- 283 936 Use of Monoclonal Antibody in Diagnosis

The monoclonal antibodies of the invention are also useful for diagnostic purposes. You can mark or unmarked it for this purpose. Typically, a diagnostic assay involves the detection of complex formation by the binding of a monoclonal antibody to an HIV antigen. Unlabeled antibodies are commonly used in agglutination assays. In addition, unlabeled antibodies may be used in combination with other labeled antibodies (second antibodies) reactive with the monoclonal antibodies, such as antibodies specific for immunoglobulin. Alternatively, the monoclonal antibodies can be labeled directly.

A variety of labels can be used, for example, radionuclides, fluorescent substances, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (especially haptens), etc. Numerous types of immunoassays are available, including, for example, those described in US Pat. Nos. 3,817,827 3850752, 3901654, 3935074, 3954533, 3936345, 403407A and 4098876, which are hereby incorporated by reference. Usually, the monoclonal antibodies and peptids according to the invention are used in enzyme immunoassays, whereby, for example, the antibodies according to the invention are optionally conjugated to the second antibodies of another species to an enzyme. When combining a biological sample with HIV antigens such as human blood serum, saliva, semen, vaginal secretions or a virus-infected cell culture suspension with the antibodies, a binding occurs between the antibodies and those molecules having the desired epitope. One can then separate such proteins or viral particles from the unbound reagents and add a second antibody (labeled with an enzyme.) Then the presence of the antibody-enzyconjugate which is specifically bound to the antigen is determined Use methods known to those skilled in the art.

To detect HIV infection or the presence of HIV antigens, one can also provide kits using the antibodies of the invention. The monoclonal antibody compositions of the present invention may be provided, usually in lyophilized form either alone or in conjunction with other antibodies specific for other HIV epitopes. The antibodies, which may be conjugated to a label or may be unconjugated, are included in the kits along with buffers such as tris, phosphate, carbonate buffers, etc., stabilizers, biocides, inert proteins such as bovine serum albumin or the like. In general, these materials are present in an amount of less than about 5% by weight, based on the amount of active antibody, and usually in a total amount of at least about 0.001% by weight, based on the antibody concentration. It is often desirable to add an inert extender or excipient to dilute the active ingredients, which excipient may be present in an amount of about 1 to 99% by weight of the total composition. When a second antibody capable of binding to the monoclonal antibodies is used, it is usually present in a separate vial. The second antibody is typically conjugated to a label and formulated in an analogous manner as the antibody formulations described above.

The detection of gp 110 or p25 antigens or whole virus in biological samples is used in the diagnosis of HIV viral infection. Biological samples include, but are not limited to, blood serum, saliva, semen, tissue biopsy samples (brain, skin, lymph nodes, spleen, etc.), cell culture supernatants, disrupted eukaryotic and bacterial expression systems, and the like. The presence of the virus is tested by incubating the monoclonal antibodies with the biological sample under conditions which result in immune complex formation and subsequent detection of complex formation. In one embodiment, the complexation is accomplished by the use of a second antibody which is capable of binding to a monoclonal antibody, which is typically conjugated to a leaning agent and formulated in a manner analogous to the antibody formulations described above. In another embodiment, the monoclonal antibody is bound to a solid phase support which is then contacted with the biological sample. After incubation, the labeled monoclonal antibody is added to detect the bound antigen.

Production and application of synthetic peptides

The invention also provides novel peptides which inter alia immunologically mimic the protein epitopes encoded by the HIV Ketrovirus, in particular the epitopes encoded within the env or gag domain of the virus genome encoding gp 110 or encode ρ 25. In order to adapt to the different ratios of strain to strain in the respective isolates, one can make an adjustment for conservative substitutions and a choice among alternatives with non-conservative substitutions. These peptides may be used to inhibit or eliminate HIV antigen production in vitro or in vivo as immunogens for the detection of the virus or antibodies to the virus in a physiological sample. Depending on the nature of the scheme, one may conjugate the peptides to a carrier or to other compounds which are labeled or unlabelled, bound to a solid surface, and so on. In one embodiment, peptides of the invention from the gp 'x 10 area dr, y originate virus. Of particular interest is the region within the open env reading frame which extends from about the base pair (bpj6688 to about bp6750 and about from bp7246 to about 7317. The peptides of interest here, including blocky end peptides, comprise at least 5, sometimes 6, sometimes 8, sometimes 12, sometimes 21, often less than about 50, more preferably less than about 35 and preferably less than about 25 amino acids in a sequence encoded by an HIV retrovirus. Desirably, the peptides are as small as possible However, they contain substantially all of the immunoreactivity or antiviral activity of a larger peptide, and in some instances it may be desirable to join two or more non-overlapping oligopeptides to biode a single peptide structure, or to deliver them as single peptides simultaneously where these peptides have separately or together an equivalent sensitivity of the starting material.

The peptides can be modified by introducing conservative or non-conservative substitutions, with generally less than 2% points, in particular less than 10%, points of the amino acids being exchanged. If polymorphic regions are present, it may be desirable to alter one or more particular amino acids to more efficiently mimic the different epitopes of the various retrovirus strains. Frequently methionine will be replaced by norleucine (Nor) to achieve chemical stability.

It should be understood that the peptide of the present invention is not required to be identical to a particular HIV polypeptide sequence as long as the subject compound is capable of conferring immunological competition with the proteins of at least one strain of HIV retrovirus. The subject peptides can therefore be altered in a variety of ways, such as by insertions, deletions, and substitutions in a conservative or non-conservative manner, if such changes entail application benefits. By conservative substitution is meant substitutions within the groups, such as gly, ala; val, ile leu; asp, glu; asn, gin; ser, thr; lys, arg; phe, tyr; and nor, met.

Typically, the sequence will not differ by more than 20% from the sequence of at least one strain of an HIV retrovirus, unless additional amino acids can be added at the termini to provide an "arm" The strains are usually at least one amino acid long and may be 50 or more amino acids, more usually 1 to 10 amino acids long.

The peptide whose amino acid sequence is modified by substitution, addition or deletion of amino acid residues should retain substantially all of the immunoreactivity or antiviral activity of the unmodified peptides. This can be readily determined by various assays described herein. If desired, one may use the α-isomer of one or more amino acids to modify biological properties such as activity, degradation rate, and the like.

Moreover, one, two or more amino acids may be added to the termini of an oligopeptide or peptide to facilitate coupling of the peptides to one another for coupling to a carrier or larger peptide, for the reasons discussed below, to modify the physical or chemical properties of the peptide Peptides or oligopeptides or the like.

Amino acids such as tyrosine, cysteine, lysine, glutamic acid or aspartic acid or the like can be introduced at the C- or N-terminus of the peptide or oligopeptide to provide suitable linkage functionality.

Cysteine is particularly preferred for facilitating kovac binding to other peptides, or for formation of polymers by oxidation.

In addition, the peptide or oligopeptide sequences may differ from the natural sequence by a sequence modified by terminal NH ₂ acylation, for example acetylation or amidation with thioglycolic acid, terminal carboxyamidation, for example with ammonia or methylamine, for better stability, To achieve increased hydrophobicity for a connection with or binding to a carrier or other molecule or for a polymerization.

A preferred embodiment of the above-described peptides I to VIII and IX to XV when Y or Y 'is present is, for example, when Y or Y' comprises one or more cysteine residues or a combination of one or more spacer amino acid cysteine residues , Glycine is a particularly preferred spacer. Preferred peptides for use in oxidative polymerization are those in which Y or Y 'represents at least two cysteine residues.

When two cysteine residues 9 are present at the same end of the peptide, a preferred embodiment is when the cysteine residues are separated by 1 to 3 spacer amino acid residues, preferably glycine. The presence of cysteine residues may allow the formation of peptide dimers and / or increase the hydrophobicity of the resulting peptide, facilitating immobilization of the peptide to solid phase or immobilization assay systems.

Of particular interest is the use of the mercaptan groups of the cysteines or thioglycolic acids or the like used to acylate the terminal amino groups to link two peptides or oligopeptides or combinations thereof via a disulfide bridge or a longer bridge to form polymers comprising a series of epitopes contain. Such polymers have the advantage of an enhanced immunological reaction. In addition, when different peptides are used to make the polymer, they have the ability to induce antibodies that immunoreact with some antigenic determinants of various HIV isolates.

For the formation of antigenic polymers (synthetic multimers) it is possible to use compounds having bis-haloacetyl groups, nitroarylhaloganides or the like, the reagents being specific for thio groups. The linkage between two mercapto groups of different peptides or oligopeptides may thus be a single bond or a linker of at least 2, usually at least 4 and not more than about 16, usually not more than about 14 carbon atoms.

The peptides here in question can mar. bound to a soluble macromolecular carrier (for example not less than 5kDal). Conveniently, the carrier is a naturally occurring or synthetic poly (amino acid) against which antibodies in human serum are unlikely. Examples of such carriers are poly-L-lysine, keyhole limpet hemocyanin, thyroglobulin, albumins such as bovine mor- omalbumin, tetanus toxoid, etc. The choice of carrier is primarily dependent on the intended use for the antigen and is dependent on the convenience and availability of the carrier. Such conjugates present at least 1 molecule of at least one peptide per macromolecule and not more than 1 proO, 5kDal, usually not more than about 1 per 2kDal of the macromolecule. One or more different peptides can bind to the same macromolecule.

The compound is usually carried out using reagents such as p-maleimidobenzoic acid, p-methyldithiobenzoic acid, maleic anhydride, succinic anhydride, glutaraldehyde, etc. The binding may be at the N-terminus, C-terminus or between the ends of the molecule.

The peptide can be oerivatized by linkage, linked while attached to a support, or the like.

In order to detect the presence of antibodies against retroviral proteins or retroviral proteins themselves, one can proceed according to various, familiar to the expert assay method. Of particular interest is to use the peptide as a labeled reagent, wherein the label provides a detectable signal, or to bind the peptide directly or indirectly to a surface, wherein the antibody binds to the peptide in the sample at the surface of the peptide becomes. The presence of a human antibody bound to the peptide can then be achieved using a xenogenic antibody specific for human immunoglobulin, usually both human IgM and IgG, or a labeled protein specific for immune complexes, for example the Rf factor of S. aureus protein A.

An example of an assay method is the use of a sample container, for example wells of microwell plates, wherein the polypeptide or conjugates thereof are covalently or non-covalently adsorbed on the bottom and / or on the walls of the container. The sample, usually human blood or serum diluted with a suitable buffer medium, is placed in the container and allowed sufficient time for complex formation to take place between the polypeptide (s) and a corresponding antibody in the sample. Remove the supernatant and wash the container to remove non-specifically bound proteins. For detection, use is made of a labeled specific binding protein that specifically binds to the complex, such as xenogeneic human immunoglobulin antiserum.

The peptide can be prepared in a variety of ways. The peptide can be synthesized because of its relatively short length in solution or on a solid support according to conventional methods. Various automatic synthesizers are commercially available and can be used in a known manner, 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) 10ί x 6442. Alternatively, one can make use of the hybrid DNA technology which produces a synthetic gene using single strands encoding the polypeptide or substantially complementary strands thereof, with the single strands overlapping and in an annealing medium can be brought together to hybridize. The hybridized strands can then be ligated to a complete gene and the gene inserted into readily available expression vectors by choice of suitable termini, see, for example, Maniatis et al., Molecular Cloning, A Laboratory Manual, CSH, Cold Spring Harbor Laboratory, 1982. Or one can clone and expose the region of the viral genome encoding the peptide by conventional recombinant DNA techniques (see above Maniatis et al.). DNA code sequences of 'AV _B nu and ARV 2 isolates of HIV that can be used to express the peptides include the following:

LAV _BRU TGT ACA AGA CCC AAC AAC AAT ACA AGA AAA AGT ATC CGT ATC CAG AGG GGA CCA GGG AGA GCA TTT GTT ACA ATA GGA AAA ATA GGA AAT ATG AGA CAA GCA CAT TGT

ARV-2 TGT ACA AGA CCC AAC AAC AAT ACA AGA AAA AGT ATC TAT ATA GGA CCA GGG AGA GLA TTT CAT ACA ACA GGA AGA ATA ATA GGA GAT ATA AGA AAA GCA CAT TGT

For expression of peptide fragments one can use fragments of a sequence. One may make conservative base changes where the modified codon (s) encode (encode) the same amino acid (s), or perform non-conservative base changes in the coding sequence, wherein the resulting amino acid has a conservative or non-conservative change in amino acid sequence can be what was described above. The code sequence may be extended at the 5 'or 3' terminus, or both, to extend the peptide while retaining the epitope site (s). The extension may serve to provide an arm for attachment to, for example, a label product such as an enzyme, to join two or all peptides together in the same chain, to provide antigenic activity and appropriate restriction sites for cloning, or the like.

The DNA sequence itself, fragments thereof or large sequences, usually at least 15 bases, preferably less than 18 bases, can be used as nucleotide probes for detection of rotaviral RNA or proviral DNA or for identification of homologous regions for cloning or sequencing. Numerous methods are described, such as the foundation stone hogness method, Southern method, Northem method, dot blot, improvements thereof, as well as other methods such as those described in US Pat. No. 4,358,535, which is incorporated herein by reference are. The peptides of the invention, including the blocking peptides and their analogs, are used alone or in combination in vaccines. Similarly, vaccines can also use anti-idiotype antibodies, i. H. Antibodies that react with the idiotype of the antibodies of the invention containing epitopes that mimic the neutralizing HIV regions. The peptides or anti-idiotypic antibodies may be formulated in a conventional manner, generally in concentrations ranging from 1 pg to 20 mg / kg of the host.

Physiologically acceptable media may be used as the carrier, for example, sterile water, saline, phosphate buffered saline, and the like. Adjuvants such as aluminum hydroxide gel, interfacially active substances such as lysolecilin, pluric polyols, polyanions, peptides, proteins (e.g., diphtheria and cholera toxin) and oil emulsions can be used. The peptides can also be incorporated into vaccine formulations and liposomes or conjugated to polysaccharides, polypeptides or polymers. Administration may be by injection, for example

intramuscular, peritoneal, subcutaneous, intravenous, etc. Administration of an immunogenic effective dose may be one or more times, usually at intervals of 1 to 4 weeks. An "immunogenically effective dose" is that amount of a vaccine which is capable of eliciting an immune response in a host, the host exhibiting a gesoigerte infection.The following examples illustrate the invention without limiting it.

Example I Generation and characterization of the monoclonal antibodies

Example I describes the generation of hybrid cell lines producing monoclonal antibodies specific for HIV envelope glycoproteins. This method involves the use of lectin-purified extracts of LAV _B ru bound to lentil lectin agarose as an immunogen. The monoclonal antibodies subsequently produced by the hybrid cell lines are characterized by their ability to immunoblot gp 110 from purified LAV and to precipitate in a radioimmunoassay as well as a biologically expressed recombinant fusion protein. Dio monoclonal antibodies that bind to epitopes on gp 110 also react in ELISAs with disrupted whole viruses, fusion proteins, and synthetic peptides, and react with whole viruses in indirect fluorescence assays.

Generation of the monoclonal antibody-producing hybrid cell lines and characterization of the antibodies are as follows:

LAV viruses purified from infected CEM cells (ATCC No. CRL8904) were dissolved in 50 mM Tris, pH 7.4, 0, 15 M NaCl, 1.0% aprotinin, 2.0% Nonidet P-40® (NP-40) (octylphenoxypolyethoxyethanol) destroyed. The extract was clarified 2x by centrifugation and adjusted to 0.5% NP-40 by addition of 3 volumes of disruption buffer without NP-40. Lentil lectin-pharose (Pharmacia, Piscataway, NJ) was prewashed in disruption buffer without NP-40 and then in adsorption buffer (50 mM Tris, pH 7.4, 0, 15 M NaCl, 1.0% aprotinin, 0.5% NP-40). equilibrated. The clarified virus extract was adsorbed at 4 ° C. for 42 h with lenticular Lektinsepharose 42 h. Unadsorbed material was removed by washing with excess adsorption buffer. The elution of the adsorbed material was carried out with 0.2 M a-methyl mannoside in adsorption buffer. The eluate was dialyzed against PBS to remove the sugar and the material was re-adsorbed on lentil lectin-pharose. The glycoprotein-1-enylene-lectin-pharose complex was used to immunize BALB / c mice by means of three intraperitoneal injections without adjuvant, given at intervals of 2 to 3 weeks. The spleen of immunized mice was removed. Immunoblot RIP and / or ELISA reveals circulating antibodies to glycoproteins. The production of cell lines was generally carried out according to the instructions of Köhler and Milstein (Nature 256: 495 [1985]) with the modifications of LC Goldstein et al., (Infect. Immun. 38: 273 [1982]). The spleen B lymphocytes from the immunized mice were fused with NS-1 myeloma cells using 40% (w / v) polyethylene glycol. Following the fusion, the cell mixture was resuspended in HAT medium (RPMI-1640 supplemented with 15% fetal calf serum, 1 x 10 ^"4 M hypoxanthine, 4 x 10 ^-7 MAminopterinund 1.6 x 10" ⁵ M thymidine) suspended to select for the growth of hybrid cells, and then incubated on 96-well microculture plates at a concentration 1-3 χ 10 ^-6 cells / ml gegebon and at 37 ⁰ C in a humidified atmosphere containing 6% CO _2. The cultures were fed by replacing half of the supernatant with fresh HAT medium. The wells were observed using a plankton microscope for signs of cell proliferation, and as soon as the cells had sufficient density, the supernatants were assayed for anti-LAV antibody.

Those wells containing antibodies to LAV-producing hybrid cells were identified by ELISAs, determining binding to purified, disrupted whole viruses, or biologically expressed fusion proteins. The ELISA assays using disrupted viruses were performed on LAV EIA plates (Genetic Systems, Seattle, Washington). The plates were incubated with cell culture fluid at 37 ^° C. for 45 min and then washed 3x with 0.05% Tween 20 in phosphate buffered saline (PBS-Tween).

Peroxidase Zie'je anti-mouse IgG (1: 2000 dilution in PBS-Tween; Zymetfor oboratories, Inc., South San Francisco, California) was added (100 μl per well) and the plates were incubated at 37 min for 45 min Incubated ⁰ C and washed as above. Substrate was added (0.025M citric acid, 0.05M dibasic sodium phosphate, pH 5, containing 14mg o-phenylenediamine and 10μΙ 30% hydrogen peroxide per 50ml) and the mats became 30min. incubated at room temperature in the dark. The reaction was quenched with 3N sulfuric acid and the colorimetric reactions were quantitated with an automatic microplate reader. Those wells that gave positive results were subcloned by limiting dilution, re-tested for specificity, and then expanded.

The monoclonal antibodies secreted by the resulting hybrid cell lines were further characterized for specificity and reactivity by immunoblotting, immunoprecipitation and ELISA using disrupted LAV viruses, recombinant LAV fusion proteins and synthetic LAV peptides. It was found that other antibodies were of the IgG1 isotype. The cell lines HIV gp110-1, HIV gp 110-2 and HIV gp 110-3 have been deposited with the Amercian Type Culture Collection for filing this application and have accession nos. ATCC HB9175, HB9176 and HB9177. The recombinant fusion proteins tested for their reactivity were designated ENV 2, ENV3, ENV4 and ENV5. The ENV2 protein is expressed from pENV2 (ATCC No. 53071) and comprises the LAV region from base pair (bp) 6598 to bp 7178 (Wain-Hobson et al., Cell 44: 9 [1985].) ENV3 is derived from pENV3 ENV4 is expressed from pENV4 (ATCC No. 53073) and comprises bp7698 to bp8572 ENV5 is expressed from pENV5 (ATCC No. 53074) and comprises the LAV. (ATCC No. 53072) Range from bp5889 to bp7698 The production of the recombinant fusion proteins is described in detail in USSN721237, which is hereby incorporated by reference.

Assembly of synthetic peptides

Peptides I (29) and VIII (110-2-2) were assembled on a benzhydrylamine resin (polystyrene / divinylbenzene (Applied Biosystems, Inc., Foster City, California). Peptide V (177) was ligated to a t-butyloxycarbony KbocJ-ethylbenzylcysteine phenylacetamidomethyl Symmetric anhydride couplings were performed in an Applied Biosystems 430A synthesizer and cysteine was added as the first residue to the two peptides (PAM) polystyrene / divinylbenzene resin.

Couplings with dicyclohexylcarbodiimide in the presence of hydroxylbenzotriazole were used for asparagine and glutamine. As protecting groups came benzyl-based side chains and Boc-a-amingrupper. : ur application. Other routinely used side-chain protecting groups were Boc (formyl) tryptophan, Boc-methionine jlfoxide, Boc (tosyl) arginine, Boc (methylbenzyl) cysteine, Boc (tosyl) -histidine, bodchlorobenzyloxycarbonyl-lysine, and bocfbromobenzyloxycarbonylotyrosine.

The radiolabeling of the peptides was carried out by acetylation of the amino terminus with ³ H-acetic acid and an excess of dicyclohexylcarbodiimide.

Removal of the protecting groups and cleavage of the peptide from the resin was carried out according to the Tarn "low-high" HF method (Tarn et al., Supra) The extraction of the resin was carried out with 5% acetic acid and the extract was subjected to gel filtration chromatography in 5% acetic acid.

Synthetic HIV peptides tested for reactivity with the monoclonal antibodies included peptides 29, 36 and 39. Peptide 29 is encoded by the LAVenu genomic region from about bp6688 to bp 6750; peptide 36 is encoded by the region from about bp7246 to bp7317; and peptide 39 is encoded by the region from about bp7516 to bp7593. Peptides 36 and 39 are described in detail in U.S. Patent 4,629,783, which is incorporated herein by reference. The blocking peptides IX-XV were assembled essentially as described above on a methylbenzhydrylamine resin (polystyrene / divinylbenzene) (Applied Biosystems, Inc. Foster City, California). Symmetric anhydride couplings were performed on an Applied Biosystems 430A synthesizer. For asparagine, couplings with dicyclohexylcarbodiimide in the presence of hydroxybenzotriazole were used. As protective groups, benzyl group side chains and boc-a-amine protecting groups were used, whereas Boc (bromobenzyloxycaibonyl) was used specifically for tyrosine side chains. Acetylation was done, if any, using acetic anhydride or glacial acetic acid and dicyclohexylcarbodiimide. Deprotection and cleavage of the peptide from the resin were carried out according to the "high" standard HF procedure (Stewart et al., Supra) Extraction of the resin was done with 50% acetic acid and the extract was then subjected to gel filtration chromatography in If desired, high pressure liquid chromatography was performed on a Vydac C18 column (Rainin Instrument Co., Emeryville, CA) using a 0.1% trifluoroacetic acid-acetonitrile gradient.

Immunoblottind

Immunoblotting characterization was performed on clone supernatants or ascites fluid using purified LAV viruses and recombinant fusion proteins as antigens. The antigens were first separated by polyacrylamide gradient gel electrophoresis (7.0-15.0%) and transferred to a nitrocellulose membrane (NCM) by 4 hours of electrophoresis at 25 V in 25 mM sodium phosphate (pH 7.0). After transfer, the NCM was blocked by incubation at room temperature for 1 hour in PBS-Tween or Blotto (5% low-fat dry milk in PBS) to prevent nonspecific interactions. The NCM was incubated with cell culture supernatant or ascites fluid diluted in PBS-Tween for 1 h at room temperature and rinsed 3x with PBS-Tween. In the second step, the NCM was incubated with goat anti-mouse IgG horseradish peroxidase diluted in PBS-Tween for 1 h at room temperature. Following incubation, washing was carried out with PBS-Tween, followed by immersion for 20 minutes in horseradish peroxidase color developing solution (Bio-Rad Laboratories, Richmond, California). The reaction was stopped by immersion in deionized water. Monoclonal antibody activity was compared to a positive control serum that reacts with purified disrupted virus or expressed fusion protein. All antibodies were found to bind to gp 110 or its precursor molecule gp 150 using disrupted virus preparations. Antibodies 110-1 and 110-2 also recognized the fusion protein ENV3, whereas antibodies 110-3,110-4,110-5 and 110-6ENV2 convert to an immunocomplex.

Immunpräzipltatlon

Virus extracts for radioimmunoprecipitation were prepared from CEM cells infected with LdAVeRu HIV isolate, which was adapted to lytic growth by continuous passage. Once early cytopathic effects were demonstrated, the cells were transferred to labeling media containing ³⁵ S-methionine (0.05mCi / ml) or ³ [H] -glucosamine (0.025 mCi / ml), then incubated for 24 hours, until most of the cells had lysed and release the virus into the culture supernatant. The viruses were pelleted from the cell-free supernatant (1 h at 100,000xg) and detergent extracts were added to P-RIPA buffer (phosphate buffered saline containing 1.0% Triton X-100.1.0% deoxycholate, 0.1% SDS, and 1 % Aprotinin contains). Similar extracts were prepared from the supernatants of uninfected CEM cells.

Immunoprecipitation assays were performed with 100μΙ virus extract, which was incubated on ice with 100μΙ culture supernatant from the hybrid cell lines for 1 hr. 4μl rabbit anti-mouse Ig (Zymed Laboratories, So. San Francisco, California) was added to each sample and incubated for 30 min. To each sample was added immunoprecipitin (100 μΙ; Bethesda Research Laboratory, Bethesda, Maryland) resuspended in P-RIPA buffer containing 1% ovalbumin and incubated for an additional 30 min. The bound complexes were washed and separated by SDS-polyacrylamide gel-electrophoresis (15.0% acrylamide: DATD-GeI). Following electrophoresis, the gels were fixed, soaked in Enhance (New England Nuclear, Boston, MA), dried and contacted with a Kodak XR-5 film. A positive reference serum which immunoprecipitates with all HIV viral proteins was reacted as positive and negative controls with virus-infected and mock-infected CEM cell supernatants

 The results show that all six monoclonal antibodies specifically immunoprecipitated gp 110 and gp 150.

Enzyme-Llnked-Immunoadäorbant assay

To map the gp110 epitopes recognized by the monoclonal antibodies of the present invention, the culture supernatants from hybrid cell lines or ascites fluid were further characterized by reactivity in ELISA assays with biologically expressed fusion proteins and synthetic peptides. The procedures were the same as those described above except that the fusion proteins or synthetic peptides replaced the purified viruses as antigens adsorbed to the surface of the microtiter wells.

When using peptides as antigen, the procedure was as follows. The lyophilized peptides were in

6M guanidine HCl dissolved. Immediately before plating into the 96-well plates, the guanidine solution was diluted in 0.05 M carbonate / bicarbonate buffer (pH 9.6) to a final peptide concentration of up to 100 μg / ml. 50 μΙ of the diluted peptide was added to each microtiter well. The plates were then incubated over Nficht at 4 ° C. Excess peptide solution was "shaken out", the plates were blocked with blotto and the further ELISA assay was performed according to the procedure described above Similarly, recombinant protein was reduced to a final concentration of about 2 μg / ml in 0.05 M carbonate / bicarbonate Diluted buffer (pH 9.6) and the same procedure repeated.

The results are shown in Table II. The monoclonal antibodies produced by cell lines HIV-gp 110-1 and HIV-gp 110-2 reacted with ENV3, ENV5, peptide 36 and destroyed viruses. The antibodies from the cell lines HIV gp 110-3, HIV gp 110-4, HIV gp 110-5 and HIV gp 110-6 reacted with ENV2 and peptide 29 as well as with destroyed viruses.

TABLE Il

ELISA assays showing the reactivity of the monoclonal antibodies with recombinant proteins and synthetic peptides

Recombinant fusion protein ENV2 ENV3 ENV4 ENV5 Synthetic peptides 36 39 LAV CEM 0.077 " 3000 0113 3000 29 2421 0054 control control 110-1 -0,003 3000 0000 3000 ND 2305 -0,005 0908 0125 110-2 3000 0011 ND ND ND ND 0017 1214 0009 110-3 3000 0020 ND ND 3000 ND 0016 0363 0046 110-4 3000 0014 ND ND 3000 ND 0016 0383 0067 110-5 3000 0033 ND ND 3000 ND 0017 0368 0025 110-6 1937 0486 0032

The results summarized in Table II show that the monoclonal antibodies 110-1 and 110-2 recognize an antigenic determinant encoded by the DNA probe in the pENV3 region, in particular by the region of the HIV genome that has been produced by a Sequence of amino acids in peptide 36 is defined. That is, the monoclonal antibodies gp110-1 and 110-2 bind to a peptide region of gp 110 encoded in bp7246-7317, as shown by the formation of immune complexes with peptide 36 and ENV3. This area of the HIV genome was identified as conserved, i. H. with only minor changes in the DNA sequence in the region encoded by peptide 36 on different viral isolates of different geographical origin, see Starcich et al., Cell 46: 637 (1986). In contrast, the monoclonal antibodies gp 110-3, -4, -5 and -6 bind to HIV peptides defined by the region encoded by peptide 29 from bp6688 to about bp6750, the region in gp110, which is defined by peptide 29 has been identified as containing multiple nucleotide substitutions in different virus isolates The monoclonal antibodies that selectively bind gp 110 polypeptides containing conserved epitopes, such as antibodies 110-1 and 110-2, are common especially in affinity chromatography, etc. Also in an ELISA assay, peptide 110-2-2 reacts with sera from the subject from whom LAV-2 was isolated.

Indirect immunofluorescence assay

Indirect immunofluorescence assays using monoclonal antibodies to HIV gp 110 antigen were performed on acetone-fixed and live cells. Acetone-fixed slides were prepared from LAV-infected CEM cells were incubated with diluted culture supernatant or ascites fluid 1 h at 37 ⁰ C, whereas living cells with culture supernatant or ascites fluid 1 h at 4 ° C were incubated before the cells on the slides were placed and fixed with acetone. In both methods, fluorescein isothiocyanate-labeled anti-mouse IgG was used to detect those cells which have the reactive gp110 antigen. The monoclonal antibodies HIV-gp110-1 gave positive results both when using live or acetone-fixed LAV-infected cells.

Example Il

Neutralization of HIV Infectivity by Anti-gp 110 Monoclonal Antibodies This example describes and characterizes the neutralization of HIV infectivity using monoclonal antibodies that bind to gp 110 and peptides within gp 110. The results show that the antibodies gp 110-3, -4, -5 and -6 have neutralizing activity and that gp 110-3 and -4 have particularly high neutralizing activity.

Neutralization assay

A sensitive neutralization assay was developed to quantify the effect of monoclonal antibodies on HIV infectivity. A CD4 + cell line, CEM, which is particularly sensitive to HIV infection, was chosen as the target cell for infectivity comparisons. Ascites fluid prepared as described in Example I or the IgG fraction thereof purified using ammonium sulfate precipitation was inactivated for 30 min. At 56 ° C, then in the required extent in RPMI medium containing 10% fetal calf serum , diluted. A suspension of HIV strain LAVbhu was harvested from about 4 days old CEM cultures in the log growth phase, filtered through a 0.2 or 0.45 micron filter, aliquoted and frozen at -70 ⁰ C. An aliquot was thawed, titrated to determine TCIDg _0, and then assays were performed with freshly thawed aliquots diluted to a ratio of 1: 500 in culture medium to approximately 10X the concentration required by 50% CEM cells in the culture (10 TCID ₆₀ ) to infect. The virus suspension was mixed with the same volume (250 μΙ) of a monoclonal antibody preparation in 5-fold dilutions of 1: 5 to 1: 7 765625. The virus / antibody mixture was incubated for 45 min at 37 ⁰ C and then voin. 1: duplicated 9 765 625: 5 to 1. The virus / antibody mixture was then incubated at 37 ^° C. for 45 min. Subsequently, duplicate 200μΙ samples were used to inoculate wells containing 1 ml with approximately 2χ10 ⁵ -CEM cells per well. The cultures were incubated at 37 ° C in a humidified atmosphere with 5% CO _{2 for} 14 days. Cells were harvested, pelleted and lysed with 1% Triton-X-100 in PBS for about 10 min. The amount of virus (or virus antigen) present in the lysed cells was determined using a sensitive HIV antigen capture assay.

The titer of neutralizing activity, if any, was determined to be the reciprocal word of the dilution of monoclonal antibodies which inhibited more than 50% of the antigen production of virus control cultures lacking antibody or with a monoclonal antibody Antibodies of the same isotype which were previously shown to have no neutralizing activity were incubated In the above-mentioned HIV antigen capture assay, two monoclonal antibodies to p25 antigens were used as the capture reagent, and these hybridoma cell lines were determined of the methods described above with minor modifications, including the use of a purified gag recombinant fusion protein as immunogen and the characterization of the resulting monoclonal antibodies for specificity and reactivity using those referred to as GAG-1, GAG-2 and GAG-3 recombinant fusion proteins and the synthetic peptide 141. The protein GAG-1 is expressed from PGAG-1 (ATCC No. 53379), GAG-2 is expressed from pGAG-2 (ATCC No. 53111) and GAG-3 is isolated from pGAG- 3 (ATCC No. 53112). The production of the recombinant fusion proteins is described in detail in USSN 764460 and 828828, to which reference is hereby made. The synthetic peptide 141 is encoded by the LAV _BRU gene region corresponding to amino acid residues 198 to 242. The monoclonal antibodies produced by the hybridoma cell lines ρ 25-2 and ρ 25-3 react with the recombinant fusion proteins GAG-1, GAG-2 and GAG-3. The monoclonal antibodies obtained from ρ 25-3 also react with the synthetic peptide 141.

To perform the antigen capture assay, the capture reagents were first adsorbed to a solid support. Ascites fluid derived from hybridoma cell lines ρ25-2 and ρ25-3 was diluted 1: 5000 in 25mM Tris buffer pH8.5. 200μΙ were placed in the wells of microwell plates. The wells were sealed and left for about 16h at ·! ⁰ C incubated. The solution was aspirated from the plates before adding a blocking solution of 0.3% Blotto in PBS. The blocking was carried out at room temperature for 15 min. The blocking solution was aspirated and the sample was added. 200μl of the lysed cell suspension and 5μΙ detection conjugate, prepared as described below, were added to each well. The corrugated strips or plates were incubated for 2 h at 37 ⁰ C, then the suspension was aspirated and the wells were washed 4x with buffer (0.05% Tween 20 in PBS). The detection conjugate was prepared as follows. The monoclonal antibodies p25-6 and p26-7 were subjected to horseradish peroxidase (HRP) in a molar ratio of 3: 1 (ex: HRP) for 3 h using the periodate oxidation method (Nakane et al., J. Histüchem.Cytochem. 22: 1084 (FIG. 1984)). The conjugates wu. Dilute 1: 1500 in 2.5% (w / v) low-fat dry milk, 0.01 thimerosal and 0.005% Antifoam A in 2OmM sodium citrate. The remaining portion of the ELISA assay was performed as described in Example I. The determination of the neutralizing activity with the antibodies gp 110-3, -4, -5 and -6 gave the following results. The highest titers with retained neutralizing activity were: gp 110-3 = 15625; gp 110-4 '= 9765625; gp 110-5 = 125 and gp110-6 = 625.

Based on the predicted genetic variability of the range defined by peptide 29, the ability of monoclonal antibody gp 110-4 to recognize other HIV isolates was examined. The immunofluorescence assays were carried out as described above. The antibody gp 110-4 was able to detect antigen in cultures of at least three out of 16 HIV isolates.

The antibody gp 110-4 was able to neutralize viruses isolated over a period of 15 weeks from a chimpanzee inoculated into LAV _B as a control animal in an AIDS vaccine trial. The monoclonal antibody was able to neutralize the isolates, although the animal had serum antibodies that neutralized HIV in vitro and although the animal had developed a measurable, cell-mediated immune response to HIV infection. This demonstrates the lack of antigenic drift in vivo for the epitope recognized by the gp 110-4 antibody.

Example III

Neutralization of HIV Intactivity by a Cocktail of Anti-gp110 and Antl-p25 Monoclonal Antibodies This example describes the neutralization of HIV infectivity using monoclonal antibodies that bind to gp 110 and peptides within gp 110 in combination with monoclonal antibodies which bind to ρ25 and to peptides within p25 and which alone have little or no neutralizing activity. The results show that the monoclonal antibody gp 110-2 in combination with ρ 25-6 or ρ 25-7 has a particularly high neutralizing activity.

Hybridoma cell lines p25-6 and p25-7 were generated by the methods described above using the modifications which included the use of inactivated disrupted viruses or purified gag recombinant fusion proteins expressed as immunogen in E. coli as well as the characterization of the monoclonal antibodies specificity and reactivity, using the recombinant fusion proteins designated GAG-1, GAG-2 and GAG-3, and the synthetic peptides 15, 88, 150, 148 and 148. The synthetic peptides are encoded by the LAV _8RU gene region, which corresponds to the following amino acid residues: Peptide 15, amino acid residues 329 to 350; Peptide 88, amino acid residues 315 to 350; Peptide 150, amino acid residues 318 to 363; Peptide 147, amino acid residues 278 to 319; and peptide 148, amino acid residues 290 to 319. The monoclonal antibodies produced by the hybridoma cell lines p25-6 and ρ25-7 react with the recombinant fusion protein GAG-3; p25-6 reacts with synthetic peptides 147 and 148; and ρ25-7 with the synthetic peptides 15,88 and 150.

The neutralization assays were performed as described above, except that when using cocktails, the individual monoclonal antibodies were first diluted 1: 5 in culture medium and then mixed in equal proportions to give a final dilution of 1:10. The remaining part of the assay was performed as above.

The monoclonal antibodies gp 110-2, ρ 25-6 and ρ 26-7 show less than 50% neutralizing activity when used alone. When using a cocktail comprising the monoclonal antibodies gp 110-2 together with ρ 25-6 or gp 110-2 with p25-7 at a dilution of 1:10, complete neutralization takes place. A cocktail containing the monoclonal antibodies ρ 25-6 in combination with ρ 25-7 gave a neutralizing activity in the range of 60 to 90%.

Immunoefficacy of the peptide 29 and homologs thereof

The ability of peptide 29 and the homologous peptides, including peptide 177, to stimulate an immune response against η HIV was examined on two mouse strains. The preparation of the peptide immunogens, the immunization methods and the characterization of the triggered immune responses are described below.

Peptide 29 is prepared for immunization by conjugation to a purified thyroglobulin. Thyroglobulin can be derivatized for conjugation with N-succinimidyl 4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) according to the methods described in US Patent 4,629,783, col. 10, lines 28-51. As a second immunogen, thyroglobulin is derivatized with glutaraldehyde as follows. 27 mg porcine thyroglobulin are dissolved in 1 ml of 0.1 M sodium bicarbonate. For this purpose, 8.3 μΙ of a 25% glutaraldehyde solution is dried and the mixture is stirred overnight at room temperature. You give

1 ml of sodium carbonate / bicarbonate buffer of pH 9.3 to the solution and then dialyzed for 8 h against 2I of the same buffer at 4 ⁰ C, with a complete replacement of the dialysate after 4 h. The peptide 29 is then added to the derivatized thyroglobulin in about 100-fold molar excess and the mixture is stirred overnight at room temperature. M'.ii blocks unreacted G lataraldehyde with 200 μL of a 0.2 M lysine solution and stirs the mixture for several hours (or overnight) at room temperature. The peptide-thyroglobulin conjugate is then dialyzed extensively against PBS at 4 ° C.

Two mouse strains (C57 black and BALB / c) are inoculated with the peptides produced by the respective conjugation method. All animals were about 2 to 4 weeks old at the time of inoculation. Inoculation may be in the footpad, by cauterization, subcutaneously, intranasally or intraperitoneally. The inoculum consists of 25 μς of the conjugated peptide suspended in 0.5 ml Freund's complete adjuvant, with booster inoculations given after 2, 3 and 5 weeks with the same immunogen suspended in incomplete Freund's adjuvant. Serum samples are collected from each mouse before immunization, 4 days after booster immunization after 3 weeks, and 4 days after the last booster immunization at 5 weeks. The serum samples were analyzed for antibodies against homologous peptides or whole viruses by screening in an ELISA assay. Those sera showing antibody activity against LAV are subjected to further screening for neutralizing activity. This is followed by analysis of the sera in immunoblot assays against destroyed LAV antigen and in radioimmunoprecipitation assays with radiolabelled gp 110. The results of the immunizations show that the mice immunized with peptide 29 developed antibodies that interact with peptide 29 and destroyed LAV-1 viruses in ELISA assays. Conjugation of peptide 29 by maleimide results in the formation of anti-peptide-29 antibodies in some balb / c mice. Conjugation of peptide 29 by glutaraldehyde generally gives higher titres of antibodies to peptide 29 in balb / c mice. Mice immunized with peptide 177 will develop antibodies to the peptide. Conjugation of peptide 177 by glutaraldehyde was better in eliciting an immune response. C57 mice respond more strongly to peptide 177 immunostimulation than balb / c mice. Mice immunized with LAV-2 peptide 110-2-2 develop antibodies to 110-2-2 and LAV-2 viruses, as demonstrated by ELISA assays. The glutaraldehyde-conjugated peptide 110-2-2 is immunogenic in both C57 and balb / c mice, with titers to the 110-2-2 peptide generally higher in balb / c mice than in C57 subjects. Mice are, while the titers against the LAV-2 virus are generally higher in C57 mice than in balb / c mice.

Those serum samples from immunized mice showing (i) whole virus antibodies; (ii) being able to neutralize HIV, as in the assays described in Example II, for example, and (iii) reacting with peptide 29 in ELISA assays cumulatively demonstrate the efficacy of using peptide 29 in a vaccine formulation.

Example V Immunoaffinity separation of gp 110 using monoclonal antibodies

Monoclonal antibodies to the gp 110 HIV antigen can be used in immunoaffinity separation procedures to purify the bacterially expressed recombinant fusion proteins. When the expressed protein is secreted by the bacteria, the protein can be isolated from the culture supernatant. If the protein is not secreted, destruction of the bacterial cells may be required.

The construction of plasmid pENV-5 (ATCC No. 53074) is described in USSN721237, which is hereby incorporated by reference. The plasmid pENV-5 encodes the majority of the carboxyl end of gp 110 and a portion of the amino terminus of gp41 of LAV inserted into the trp expression vector. E.CoIi C600 transformed by this vector express the gp 110 fusion protein, but do not secrete it.

E.CoIi C600 containing the plasmid pENV-5, allowed to grow overnight at 37 ⁰ C with aeration in a medium containing tryptophan L2Q \ ig / m \) and (ampicillin lOOpg / ml contains). The overnight cultures are then inoculated to 1: 100 in fresh minimal medium containing ampicillin (100pg / ml), but not tryptophan. These cultures are allowed to aeration at 37 ⁰ C.

2 to 3h grow (until the early log phase). The inducer, 3-B indole-acrylic acid (Sigma Chemical Co., St. Louis, MO) is added from a freshly prepared stock solution of 20 mg / ml in 95% ethanol to a final concentration of 20 mg / ml. The induced cultures are allowed at 37 ⁰ C with aeration 4 to grow 5h, then pelletized and freezes optionally a. The protein yields of pENV-5 are typically less than 1 mg / l.

The pelleted bacterial cells are lysed using P-RIPA buffer (PBS containing 1% Triton X-100.1% deoxycholate, 0.1% sodium dodocyl sulfate, and 1% aprotinin) which lyses E. coli cells. The suspension is ultrasonicated for shearing DNA and RNA and then centrifuged to remove particles. Dilution or concentration may be required to standardize the protein concentration.

The monoclonal antibody HIV-gp 110-1 is originally precipitated from ascites fluid or cell culture supernatants at room temperature or in the cold with (NH ₄ I ₂ SO ₄ or Na ₂ SO ₄ solutions at pH 7.3 to a final saturation of 33 or The precipitated proteins are removed by centrifugation and redissolved in PBS and precipitated a second time with 33% (NH ₄ I ₂ SO ₄ or 12 to 15% Na ₂ SO _4. If necessary, this step can be repeated The pellet is redissolved in PBS and the excess sets are removed by gel filtration through a desalting matrix or by exhaustive dialysis against PBS.

The purified monoclonal antibody 110-1 can then be coupled to cyanogen bromide-activated sepharose. The

required amount of gel is allowed to swell in 1O- ³ M HCI solution on a glass filter (1 g of freeze-dried material gives a Gelendvolumen of about 3.5 ml), washed 15 min. With the same solution and are the antibody immediately thereafter. In general, the coupling reaction is most effective in a pH range of 8 to 10, but it is also possible to operate at a lower pH if required for reasons of antibody stability. The antibody should be dissolved in PBS or in a carbonate / bicarbonate or borate buffer of high ionic strength with 15OmM NaCl. The activated Sepharose and the antibody suspension are stirred gently at room temperature for 2 to 4 hours or overnight at 4 ° C. and then washed on a coarse glass filter frit with coupling buffer. As far as active groups remain, they are blocked by treatment with 1.0M ethanolamine at pH 8 for 2 hours. The final antibody-sepharose product is then washed alternatively 4 or 5x with high or low pH buffer solutions (borate buffer, 0.1 M, pH 8.5, 1 M NaCl, and acetate buffer, 0.1 M, pH 4.0 , 1 MNaCI). Washing removes traces of noncovalently absorbed materials. The final immunoaffinity separation matrix is stored below 8 ° C in the presence of a suitable bacteriostatic agent such as 0.01% azide.

The addition of the expressed protein suspension to the immunoaffinity separation matrix results in the selective removal of the gp110 antigen. The mixture is allowed to act for 2 to 24 hours, preferably 12 to 18 hours, with gentle stirring or shaking. One can also proceed by placing the immunoaffinity matrix in a column, equilibrating and slowly adding the expressed protein suspension to the column. Upon addition of the protein suspension, the flow should be stopped to best effect the formation of the immune complex.

Unbound material is washed or separated by extensive washing with adsorption buffer, by using a coarse glass filter frit under vacuum or by passage through the column The unbound material is then subjected to low or high pH buffers (acetate buffer , pH 4.0 or borate buffer, pH 8.56) or a chaotropic agent.

Example Vl

Immunoaffinity Purification of Recombinant gp110 from a Subliminal Expresslon System The monoclonal antibodies of the present invention find use in the immunoaffinity purification of recombinant fusion gp 110 as experimentally expressed by mammalian cells. The mammalian cells are infected with recombinant vaccinia (Mackett et al., J. Virol. 49: 857 [1984], incorporated herein by reference) containing sequences encoding at least the portion of gp 110 which is antigenic and which Formation of neutralizing antibodies dissolves. The recombinant vaccinia are constructed according to the method described in USSN 842,984, which is hereby incorporated by reference. The sequences coding for the envelope glycoprotein of HIV are inserted in a plasmid vector (pGS20) downstream from a vaccinia transcriptional control element. This chimera gene is flanked by sequences coding for the viral thymidine kinase (TK) gene.

The chimeric plasmid vectors containing vaccinia virus promoters ligated to the LAV envelope gene are used to transform the E. coli strain MC 1000. Insertion of the chimeric LAV env sequence into the vaccinia virus genome was performed by in vivo recombination, which was made possible by flanking the chimeric genes in plasmids pv-env 5 of vaccinia virus sequences encoding the TK gene. This plasmid is then introduced into cells previously infected with wild type vaccinia virus. One then allows recombination between the TK sequences on the plasmid and the homologous sequences in the vaccinia virus genome with insertion of the chimeric gene. African Green Monkey kidney cells (BSC-40 strain, a BS-1 derived cell line, ATCC No.CCL26) are used as host in the expression system.

Confluent BSC-40 cells are infected at a multiplicity of infection of 10 by recombinant vaccinia viruses. The infection is allowed to proceed for 12 hours, then the cells are harvested, washed once with PBS and centrifuged off. The cell pellets are suspended in lysis buffer (1.0% NP-40.2.4% sodium deoxycholate, 0.1 M NaCl, 0.01 M Tris-HCl, pH 7.4.1 mM EDTA) and the lysate is clarified by centrifugation , The immunoaffinity separation of the expressed recombinant fusion protein is performed as described above for the bacterial expression system using monoclonal antibody gp 110-1. The proteins produced by this expression system are more naturally produced by HIV-gp110 due to mammalian cell treatment and glycosylation.

Example VII

Inhibition of HIV infection with blocking peptides

The effectiveness of blocking peptides in inhibiting infection of tissue culture cells by the LAV _B ru HIV strain was examined using a modification of the method for the evaluation of peptide T published by Pert et al., Supra. The preferred HIV lnhibitions assays carried by equal volumes of blocking peptides and CEM cells (2.5 χ 10 ⁶⁾ in medium (RPMI, 10% FCS and 2 mg / ml polybrene) combined, and 45 min. At 37 ⁰ C incubated. The virus is then added in different dosages (10.50, 500 TCID50) and the mixture is incubated at 37 ° C for 14 days. The supernatant is then assayed for the production of viral antigen (eg ρ 25 core antigen).

Preincubation of the CEM cells with the peptides prior to virus addition to the cultures enhances the inhibitory effect in the assays. It has been shown that the inhibition is dependent on the virus dosage, with a high activity at low and medium doses, a lesser effect, however, was observed at the highest virus doses.

With peptide T, inhibition of virus antigen production of about 60% to 90% could be achieved in experiments with low virus doses. Further experiments with peptides X through XV, which were typically amidated at the COOH terminus and acetylated at the NH 2 terminus, gave similar results, with peptide Xl being particularly effective over a broad dose range. It can be seen that based on the monoclonal antibodies and peptides of the invention, including the blocking peptides, a better method of neutralizing and / or inhibiting HIV infection is available. This allows for the creation of prophylactic and therapeutic agents effective against infection by most, if not all, HIV strains. In addition, the new materials are used in diagnostic assays and other known methods.

Subscriber numbers of the microorganisms

The following microorganisms that are part of the present invention, v.urden with the American Type Culture Collection, 12301 Parkiawn Drive, Rockville, Maryland 20852, US \ deposited. The deposit data is as follows:

ATCC-No. Deposit

dates

HB9175 August 15,1986

HB9176 August 15,1986

HB 9177 August 15,1986

HB 9404 April 30,1987

HB 9405 April 30,1987

HB 9406 April 30,1987

HB 9407 "April 30,1987

HB 9408 April 30,1987

HB 9409 April 30.19E7

HB 9410 April 30,1087

Hybridomas HB9175, HB9176 and HB9177 were tested and were viable on August 26, 1986. The remaining hybridomas were also tested and were viable on May 4, 1987.

Scientific. designation designation of depositor Mouse hybridoma HIV-1 gpHO (BalbC / NS-1) Mouse hybridoma HIV gp 110-2 (BalbC / NS-1) Mouse hybridoma HIV gp110-3 (BalbC / NS-1) Mouse hybridoma HIV gp 110-6 (BalbC / NS-1) Mouse hybridoma HIV gp 110-4 (BalbC / NS-1) Mouse hybridoma HIV gp 110-5 (BalbC / NS-1) Mouse hybridoma HIV-p 25-2 (BalbC / NS-1) Mouse hybridoma HIV-p 25-3 (BalbC / NS-1) Mouse hybridoma HIV-p 25-6 (BalbC / NS-1) Mouse hybridoma HIV-p 25-7

Claims (9)

A process for the preparation of peptides capable of immunologically mimicking the neutralizing regions of HIV proteins, characterized in that monoclonal
Antibodies that specifically react with one or more neutralizing regions of HIV bind to or immobilize a substrate or carrier; the recombinant
Fusion proteins expressed from a eukaryotic or bacterial host or which brings HIV proteins from an HIV extract or lysate into contact with the immobilized antibodies to form an immune complex; bringing the immune complexes into contact; the
Separating immune complexes or antigenic fragments from the carrier; and the peptides wins. 2. The method according to claim 1, characterized in that one uses antibodies, the
react specifically with a neutralizing region comprising epitopes encoded in the env or gag regions of the HIV genome. 3. The method according to any one of the preceding claims, characterized in that one
using monoclonal antibodies specifically reactive with a neutralizing region comprising epitopes located on the LAV _BRU HIV proteins gp 110 or p25. 4. The method according to any one of the preceding claims, characterized in that one
used monoclonal antibodies that specifically react with a neutralizing region having the HIV gp 110 amino acid sequence of about 301 to 336 or 308 to 328 or homologues
of these sequences. 5. The method according to one of claims 1 to 3, characterized in that monoclonal
AntikO 'which are specific for a neutralizing region comprising the HIV p25 amino acid sequence of about 278 to 319 or about 315 to 363 or homologues of these sequences. 6. The method according to any one of claims 1 to 5, characterized in that one obtains peptides containing at least 5 contiguous amino acids of the following HIV gp 110 amino acid sequence
or the homologs thereof include: Cys-Thr-Arg-Pro-Asn-Asn-Asn-Thr-Arg-Lys-Ser-Ile-Arg-Me-Gln-Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile- Gly-Lys-Ile-Gly-Asn-Met-Arg-Gln-Ala-His-Cys. 7. The method according to claim 6, characterized in that the contiguous amino acids define at least one Amigen determinant, which is capable after immunization of a
It causes the formation of antibodies that are protective against HIV infection
have. 8. The method according to claim 7, characterized in that one obtains peptides which are one of
include the following HIV gp 110 amino acid sequences or the homologs thereof; I (29)
Y-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Y ' VI117)
YTHR-Arg-Lys-Ser-lle-Tyr-Ile-Gly-Pro-Gly-Arg-Ala-Phe-His-Thr-Thr-Gly-Arg-Ile-Y ' VIII (110-2-2)
Y-Lys-Thr-Val-Lys-Ile-Leu-Nor-Noi-Ser-Gly-His-Val-Phe-His-Ser-His-Tyr-Gln-Pro-Y ' where Y and Y ', if present, each have an amino acid sequence of up to about 20
Mean amino acids. 9. The method according to claim 8, characterized in that one obtains peptides of the sequence I, V or VIII, in which Y and / or Y 'comprise a binding residue which is selected from the group consisting essentially of glycine, tyrosine , Cysteine, lysine, glutamic acid or asparagine.