HU214439B - Method for the production of monoclonal antibodies and peptids useful in treating hiv infections and for the production of pharmaceutical compositions and vaccines - Google Patents

Abstract

FIELD OF THE INVENTION The present invention relates to a novel in vivo neutralizing epitope specific for HIV gp110 or gag p25, capable of combating HIV infection, and to a neutralizing epithelial antibody to a human germinating antibody to HIV gp110 or gag p25 . It also includes the invention of pharmaceutical compositions and vaccines for the treatment of HIV infections. ŕ

Description

The present invention relates to novel monoclonal antibodies specifically reactive with a neutralizing epitope of HVV gp 10 or gag p25 capable of combating HIV infection with amino acids LAV 301-336, and cell lines producing said monoclonal antibody and a HIV neutralizing epitope antigen. for the preparation of a major immunoreactive peptide. The invention also relates to pharmaceutical compositions and vaccines for the treatment of HIV infections.

The infectious agent responsible for Acquired Immunodeficiency Syndrome (AIDS), its introductory phases, AIDS-related complex (ARC), and Lymphoadenopathy Syndrome (LAS) is a new lymphotrophic retrovirus. The virus has been named in many ways, including LAV, HTLV-III, ARV, and most recently HIV.

As the spread of HIV reaches epidemic proportions, treatment of infected individuals and prevention of transmission are not contaminated, but are of paramount importance to individuals at risk of infection. Different therapeutic strategies target different phases of the viral life cycle, outlined by Mitsuya and Broder (Natúré 325, 773 (1987)). One approach involves the use of antibodies that bind to the virus and inhibit viral replication, either by interfering with virus entry into host cells or by other mechanisms. Once the viral component susceptible to antibody interference is identified, it is hoped that antibody titers capable of neutralizing the viral infectivity can be generated by vaccination or, alternatively, by passive administration of immunoglobulins or monoclonal antibodies of the desired specificity.

The envelope glycoproteins of most retroviruses are thought to react with receptor molecules on the surface of susceptible cells, thereby determining the infectivity of the virus to certain hosts. Antibodies that bind to glycoproteins prevent the virus from interacting with cellular receptors by neutralizing the infectivity of the virus. See, in this regard, The Molecular Biology of Tumor Viruses (1973) (ed. Tooze J.), page 534; and RNA Tumor Viruses (1982) (eds. Weiss, R. et al.), 226-236. side; both references are incorporated herein by reference in their entirety. Other literature on the subject is Gonzalez-Scarano et al., Virology 120: 42 (1982) (LaCrosse virus); Matsuno and Inouye, Infect. Immun. 39, 155 (1983) (Neonatal calf diarrhea virus); and Mathews et al., J. Immunol. 129, 2763 (1982) (Encephalomyelitis virus).

The general structure of HIV is a ribonucleoprotein core structure surrounded by a lipid-containing envelope that the virus obtains during germination from the membrane of an infected host cell. The virally encoded glycoproteins are encapsulated and protruded outward. HIV envelope glycoproteins are initially synthesized in the infected cell as a precursor (gp150 or gp160) of 150,000 to 160,000 daltons, which is then processed in the cell at a molecular weight of 110,000 to 120,000 daltons

N-terminal fragment (gp10 or gp120) to form the outer glycoprotein and a 41,000 46,000 dalton C-terminal fragment (gp41) representing the transmembrane envelope glycoprotein.

For the reasons discussed above, the gpl 10 glycoprotein of HIV has been the subject of much research as it is a potential target for interrupting the virus's life cycle. Serum obtained from HIV-infected individuals has been shown to neutralize HIV in vitro and antibodies are present in the serum that bind to purified gp10 (Robert-Guroff et al., Natura, 316, 72 (1985)). ; Weiss et al., Natur., 316, 69 (1985); and Mathews et al., Proc. Natl. Acad. Sci. USA, 83, 9709 (1986)]. Purified and recombinant gp10 stimulated the production of neutralizing serum antibodies when used for immunization of animals (Robey et al., Proc. Natl. Acad. Sci. USA, 83, 7023 (1986); Lasky et al., 1986, Science 233: 209; and a human result, Zagury et al., Natur. 326, 249 (1986)]. Binding of the gp110 molecule to the CD4 (T4) receptor has also been demonstrated, and monoclonal antibodies that recognize certain epitopes on the CD4 receptor have been shown to inhibit HIV binding, syncytia formation, and infectivity [McDougal et al., Science 23 382 (1986)]. Putney et al. (Science 234, 1392 (1986)) developed neutralizing serum antibodies in animals after immunization with a recombinant fusion protein containing the carboxyl-terminal half of the gp10 molecule, and demonstrated that glycosylation of the envelope protein does not require a neutralizing antibody. answer.

Thus, a subunit vaccine utilizing the HIV gp 110 molecule or portions would be desirable. Subunit vaccines are alternatives to vaccines prepared from inactivated or attenuated viruses. Inactivated vaccines may give cause for concern because of the potential error that not all virus particles will be killed, and attenuated viruses may have the ability to mutate and regain their disease-causing potential. For subunit vaccines, only portions of the virus that contain antigens or epitopes that are capable of eliciting an immune response, for example, are used to immunize the host. neutralizing antibodies, ADCC, and a cytotoxic T cell response. The great advantage of subunit vaccines is that viral material that is not there is excluded.

Viral subunits can be formed for use in vaccines by a variety of methods. so e.g. the envelope glycoprotein may be expressed and purified from a bacterial host, although this molecule may lack most post-translational modifications (e.g., glycosylation) or other processing steps. Such modifications may be obtained using a eukaryotic expression system, e.g. yeast or mammalian cell culture. Viral genes were introduced into mammalian cells by using Vaccinia virus as vector. See, e.g., Mackett, M., et al., Proc. Natl. Acad. Sci. USA, 79, 7415 (1982); Panicali D. and Paoletti E. Proc. Natl. Acad. Sci. USA, 79, 4927 (1982). Recombinant Vaccinia

HU 214,439 Β can be constructed according to the method of Hu et al. (Naturre, 320, 537 (1986)) or Chakrabarti et al. (Naturre, 320, 535 (1986)), both references are incorporated herein by reference. . In these systems, the viral glycoproteins produced by cells infected with recombinant Vaccinia are glycosylated and can be transferred to the cell surface for selection and final isolation.

An important step in the preparation of a subunit vaccine is the proper purification of the desired glycoprotein from a complex mixture of the expression system. There are several methods that can be used to carry out the cleaning. These include, but are not limited to, preparative polyacrylamide gel electrophoresis, gel permeation chromatography, various methods of chromatography (e.g., ion-exchange, reverse-phase, immuno-affinity, hydrophobic interaction chromatography, etc.). Most of these methods are used in various combinations to swallow essentially pure formulations. The following literature references, which are incorporated herein by reference, provide a description of the above methods: KleidD.G. et al., Science, 214, 1125 (1981); Cabradilla C.D. et al., Biotechnology 4: 128 (1986); Dowbenko, D.J., Proc. Natl. Acad. Sci. USA, 82, 7748 (1985).

Methods are required for the preparation of subunit vaccines which require a reduced number of steps to achieve the maximum purification of a given viral antigen from a complex expression mixture. Effective separation of antigens from foreign components can be accomplished using immunoaffinity chromatography. This technique, also known as immuno-adsorption, consists in principle of selective adsorption of an antigen to a solid support previously bound to a covalently specific antibody. The selectively adsorbed antigen is then eluted from such an antibody affinity adsorbent, e.g. by changing the pH and / or ionic strength of the buffer.

Polyclonal antibodies obtained from animals immunized with the desired antigen or naturally infected animals (see, e.g., Lasky et al., Cited above) are often used as immuno-adsorbents, but generally, these reagents cause significant disadvantages, e.g. (i) not all insoluble carriers are specific to the molecule in question, thus requiring further purification; (ii) the yield of the desired antigen is often low; and (iii) antibody affinities often vary from formulation to formulation, requiring modifications in elution procedures. The use of monoclonal antibodies specific for the desired viral antigen to be used in subunit vaccine formulations, in place of polyclonal antibodies, circumvents these difficulties.

Rodent monoclonal antibodies that bind to HIV antigens have already been described. Many research groups have already reported monoclonal antibodies specific for the p25 gag protein. so e.g. diMarzo Veronese et al., Proc. Natl. Acad. Sci. USA 82, 5199 (1985)] describe monoclonal antibodies to HVV p25, but do not emphasize specificity and neutralization characteristics. Chassagne et al., J. Immunol. 136, 1442 (1986)] also report monoclonal antibodies against the p25 protein, but do not mention neutralizing activity. Monoclonal antibodies specific for gp41 membrane glycoproteins have also been reported. so e.g. diMarzo Veronese et al., Science 229, 1402 (1985) disclose monoclonal antibodies to gp41, but do not mention neutralizing activity. EP 214,709 discloses synthetic peptides in gp41 but does not mention gp10 and does not mention that the peptides define a HIV neutralizing region. EP 219.106 also discloses gp41 polypeptides, but does not mention gp 110 coat protein and neutralizing activity.

There are other literature sites which, even if remotely, are relevant to the process of the present invention. Proc. Natl. Acad. Sci. USA 83, 9254-58 (1986) describes some proven blocking peptides, but there is no mention of combination with monoclonal antibodies binding to HIV gp 110. PCT Publication No. WO 86/06099, which is our prior patent, describes recombinant gag proteins, but does not disclose the possibility of monoclonal antibodies that neutralize HIV. EP 187,041 discloses recombinant HIV polypeptides but does not show neutralizing regions.

Thus, the literature cited above describes methods that are significantly different from those of the present invention.

There remains a need in the art for monoclonal antibodies specific for epitopes within the well-defined region of the major envelope glycoprotein, gp1. Monoclonal antibodies that bind to these regions and reduce or abolish replication and transmissibility of CHV may have important therapeutic and prophylactic uses. In addition, monoclonal antibodies may be used to purify the desired region of the gp1 from disrupted virus or recombinant expression systems, e.g. for use in vaccines. In addition, the region containing the epitope recognized by the monoclonal antibodies can be chemically synthesized, thereby avoiding the difficulty of purifying and administering the larger fpag fragments of the gp10 molecule. The present invention fulfills these and other similar needs.

The following is a brief summary of the invention.

We provide peptides capable of immunologically mimicking the epitopes of HIV proteins, nucleic acid probes encoding such peptides, monoclonal antibodies reactive with such peptides, and other peptides that interfere with HIV infectivity. These new materials will find applications eg. diagnostic methods for detecting HIV infections; and therapeutic methods for treating or vaccinating against such infections.

The specific embodiments are described in more detail below.

HU 214 439 Β

The present invention provides novel compositions and methods for neutralizing HIV infections, i.e., preventing or substantially inhibiting the formation or intracellular transmission of infectious HIV in a host. More specifically, peptides that mimic the HIV neutralization region and monoclonal antibodies reactive with such an area are used for the diagnosis, treatment and vaccination against HVV infections. In this context, the term "neutralizing region" refers to portions of the HVV protein, or more specifically, the HV protein, which contain one or more epitope-defining amino acid segments reactive with antibodies capable of neutralizing HV infections alone or in combination with other antibodies of the present invention. . Assays for neutralization are well known, including reduction of HIV infections in T-cell lines, reduction of plaque forming units of VSV (HIV) pseudotypes carrying HIV envelope glycoproteins, syncope inhibition assays, and virion receptor binding assays. If desired, neutralizing activity can be compared to antibody reactivity in immunochemical assays, e.g. immunofluorescence, immunoblotting, and radioimmunoassay assays.

In one aspect, the novel peptides, typically consisting of less than 50 amino acids, contain five or more contiguous amino acids, forming epitopes similar to epitopes located in the neutralizing regions of the HVV encoded by the env or gag regions of the HVV genome. Of particular interest are the regions ranging from about 301 to about 336 of gpl10 and from about 278 to about 319 of p25 and about 363 of about 315 and called LAV _BRU strain. The amino acid residue designations were all from the Los Alamos Data Bank (AIDS Virus Sequence Database, Los Alamos National Laboratory, Theoretical Division, Los Alamos, NM 87545).

Those skilled in the art will appreciate that additional analogous regions from other HIV isolates can be identified by their location within related proteins from different isolates. In practice, such homologues can be identified by reference to LAV _bru sequence data as follows.

(a) The HIV isolates and the LAV _BRU amino acid sequences can be sequenced to obtain the maximum homology between the two sequences.

(b) Peptides containing the amino acid sequences of HIV isolates can be identified which correspond to the location of LAV _BRU peptides that immunologically mimic the LAV _bru proteins. Peptides containing the amino acid sequences of the HIV isolate thus identified typically immunologically mimic the corresponding HIV isolate proteins.

This method can also be applied to HIV strains that are still to be discovered. so e.g. when a new strain of HIV is identified, the envelope and core amino acid sequences can be aligned with similar sequences of LAV _BRU to obtain maximum homology. Methods for sequencing sequences are known to those of skill in the art. When aligning the sequences, it is desirable to maintain as much homology between the cysteine residues as possible. The amino acid sequence of the novel HIV strain or species, corresponding to the location of the peptides specifically described herein, can be synthesized and used in accordance with the present invention.

Another method for determining the sequences of a homologous region in another HIV strain is described in Scharf et al., 1986, Science 233: 1076. This method employs two oligonucleotide primers which bind to conserved sequences outside of said sequence sites and contain different restriction sites in each primer. DNA from HIV strains can then be grown in vitro and the resulting oligonucleotides cloned into vectors for sequence analysis and incorporated into vaccines as a "cassette" representing a particular epitope from the HIV virus strain.

It is not necessary for the present invention that epitopes within such sequences be cross-reactive with antibodies against all strains or species of HIV. Peptides containing immunological epitopes that differentiate one species or serogroup from another will be useful in identifying particular species or serogroups and will actually help identify individuals infected with one or more species or serotypes of HIV. They may also be useful in combination with other peptides, or from a homologous region or other neutralization region in pharmaceutical compositions.

Most preferably, the peptides in question are derived from the gpl10 region of the virus. Of particular interest in this field are peptides encoded within the env open reading frame from about 6667 bp (bp) to about 6774 bp of the LAV _BRU isolate. Thus, the various homologous regions of additional HIV isolates include the homologous sequences obtained from Los Alamos Data Bank (except LAV2), which are listed in Table I.

Other peptides suitable for generating or screening for monoclonal antibodies include those encoded within the env open reading frame from about 7246 bp to about 7317 bp of the LAV _BRU . Such antibodies and reactive peptides are particularly useful in immunoassays.

In the gag region of the LAV _bru isolate, the p25 amino acid sequences from 278 to 319 and 315 to 363 are further neutralizing regions of HVV. Those skilled in the art will appreciate that additional neutralizing regions of the HIV can be identified based on the teachings herein - in particular combinations of monoclonal antibodies reactive with different HIV epitopes show neutralizing activity.

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Table I

HXB2

BH102

BH8

HXB3

H9M

BRU

MAL

ELI

ARV2

WMJ2

RFENV

Z6

Z3

NY5

CDC42

LAV2

HXB2

BH102

BH102

BH8

HXB3

H9M

BRU

MAL

ELI

ARV2

WMJ2

RFENV

Z6

Z3

NY5

CDC42

LAV2

HXB2

BH102

BH8

HXB3

TGTACAAGACCCAACAACAATACAAGAAAÁAGA ................ ATCCGTATC

CysThrArgProAsnAsnAsnThrArgLysArg .........'...... IleArglle

------------------- ------------------------------ Ser --------------- ---------------------------------- Lys -------------------------------------- ----------- LYG ------------------------------------------ g _er ------ ----------------------------------------------- g _er - ---------------------------------- Gly ------------ ArgGly-- ---------------- HisPhe

Falling TyrGln --- ------ --------- ---------- Thr Gin ------------------ ---------------------- S _er ------------------ Tyr ------- ------- ------ Val Tyr Leu --------------- --------------- --- AtgSer ----------------- S _er ------------------ ThrLys

------------ --------- TyrLys GlnSer --------------- ----------- Thr GlySerAspLysLysIle --- -------------------------- ------------------ GlnSer Lys --- -------------------- Falling ------------------ Gly His ------ ------ ValThrLeu ...............

------------ Lys Gly --- --- --- Gln Val ------------------ MetLeu

CAGAGA ......... GGACCAGGGAGAGCATTTGTTACAATAGGAAAAATAGGAAATATG

GlnArg ......... GlyProGlyArgAlaPheValThrlleGlyLysIleGlyAsnMet

Thr Gin ------------------------ --- --- --- IleVal leu --- Asplle

GlyLeu --------------- GlnSerLeuTyrThr --- Arg --- IleValSerArgSer

------------------------------. . Thr Arg --- --- --- .His IleGlyAsp

Arg --- --------------------------------- ArgGlu. . IleGlylle .---

Gin IleValTyrAlaThr -------------------------- --- --- IleGlyAsp

GlyLeu ------------ * · * GlnAlaLeuTyrThr --- Arg --- ArgThrLysIlelle

Arglle ------------------ Ly sVal --- TyrAlaLys --- Gly .............

GlyPro ------------ · · .------ ThrLeuTyrAlaArgGlu --------- Asplle

........................... YalTrpTyr --- --- Glu Thr --- LeuGlyAsn

MetSer HisVal ------------------ --- --- HisSerHisTyrGlnProIle Lys

AGA CAAGCACATTGT ... ...

... Arg ... GlnAlaHisCys

309

309

309

309

309

314

314

310

312

306

322

311

306

304

320

302

326

326

326

326

326

331

329

323

327

320

337

327

319

320

335

323

331

331

331

331

HU 214 439 Β

Table I (continued)

H9M --------------------- '331

BRU --------------------- 336

MAL --------- Arg --- Tyr --- 334

ELI IlelleGly ------------ 330

ARY2 Ile --- Lys ...--------- 333

WMJ2 Ile ------------------ 326

RFENV Ile --- Lys ...--------- 343

Z6 Gly ...--------------- 334

Z3 IleThrGly ------------ 326

NY5 --------------------- 325

CDC42 He ------------------ 341

LAV2 ArgProArg ------ Met - Peptide I, also called peptide 29, is encoded in the env open reading frame between amino acid residues 308 to about 328, the amino acid sequence of which is described below. The oligopeptides included in the sequence below include linear epitopes.

1 (29th)

Y-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-GlyPro-Gly-Arg-Ala-Phe-Val-Thr-Ile-Gly-thysylle-Y ', wherein Y and Y' , when present, each represents a sequence of up to 20 amino acids. When Y and / or Y 'are present, these are e.g. may comprise one or more of the amino acid sequences shown in pages 308 to 328 of the HTV envelope. or from any part of these flanking sequences. For example, and not by way of limitation, Y can be the amino acid sequence 301 -307 of the LAV _BRU envelope. or Y 'may be comprised of amino acids 329 to 336 of the LAV _BRU envelope amino acid sequence. all or part of the following amino acids:

II (29a)

Cys-Thr-Arg-Pro-Asn-Asn-Asn-Thr-Arg-Lys-Sei-IleArg-Ile-Gln-Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Gly-Lys-Mithril Ile-Gly-Asn-Met-Arg-Gln-Ala-His-Cys.

Alternatively, truncated sequences of the peptides of the present invention may be prepared. In this regard, the following sequences of peptide 29 may be particularly useful:

III (29b)

330

IV (29c)

Y-Ile-Gln-Arg-Gly-Pro-Arg-Ala-Phe-Val-ThrIle-Gly-Lys-Ile-Y ', wherein Y and / or Y', if present, each represent up to 20 amino acid sequences .

In another embodiment, homologous regions of a particularly significant ARV-2 isolate are encoded within the env open reading frame between about amino acids 306 to 323, typically having the amino acid sequence set forth below. The oligopeptides included in the sequence below include linear epitopes.

V (177)

Y-Thr-Arg-Lys-Ser-Ile-Tyr-Ile-Gly-Pro-Gly-ArgAla-Phe-His-Thr-Thr-Gly-Arg-Ile-Y ', wherein Y and Y' when is present, each having a sequence of up to 20 amino acids. When Y and / or Y 'is present, they may consist of one or more of the sequences set forth in ARV-2 envelope 306-323. or from any part of these flanking sequences. Y is mainly the amino acid sequence 299-306 of the HIV envelope. can consist of all or part of the part of the roots of number 1; Y 'is the coding sequence for amino acids 324 to 333 of the HIV envelope amino acid sequence; can be all or part of.

In another embodiment, truncated sequences of peptide V may be prepared. The following sequences may be particularly useful in this regard:

VI (177a)

Y-Thr-Arg-Lys-Ser-Ile-Tyr-Ile-Gly-Pro-Gly-Y *; and

VII

Y-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-Gly-Y *

Y-Asp-Cys-Lys-Thr-Ile-Leu-Lys-Ala-Leu-Gly-Proahol Y and / or Y ', when present, each represents a sequence of up to 20 amino acids.

Ala-Ala-Thr-Leu-Glu-Glu-Thr-norLeu-norLeu AlaCys Y>,

Wherein Y and / or Y ', when present, each represents a sequence of up to 20 amino acids.

Another example includes homologous regions of the LAV-2 isolate encoded by the env open reading frame at about 311-330. amino acids, and typically have the following sequence:

VIII (110-2-2)

Y-Lys-Thr-Yal-Lys-Ile-Nor-Leu-Nor-Ser-Gly-His-ValPhe-His-Ser-His-Tyr-Gta-Pro-Y ', where Y and / or Y' if is present, each having a sequence of up to 20 amino acids (see Naturre, 326,662 (1987)), which is incorporated herein by reference.

Provided a monoclonal antibody capable of producing a new Cell Lines and compositions comprising such antibodies in accordance with another aspect of the present invention, these antibodies recognize they are capable of selectively extremely high titers than those (10 ² from 10 ⁴ to 10 ⁷ by weight, or even more is) the neutralization regions contained within the predetermined sequences of the gp 110 or p25 envelope glycoproteins; protein precursors thereof, biologically expressed recombinant fusion proteins, and synthetic peptides containing one or more epitopes within the predetermined regions of gp110 or p2. The hybrid cells in question have an identifiable chromosome in which the DNA germ line has been rearranged to encode an antibody that binds to an epitope that is common to gpl10 or p25 in some or all of the HVV clinical isolates. These monoclonal antibodies can be used in a variety of ways, including diagnosis and treatment, as well as other cross-reactive antibodies, e.g. detection of blocking antibodies. Peptides or polypeptides containing the epitopes with which they react may have a variety of uses as immunogens for vaccines or as pharmaceutical agents.

Blocking peptides

In addition to its primary use with the peptides or neutralizing monoclonal antibodies described above, another embodiment of the present invention encompasses the use of additional peptides or antibodies that interfere with binding of HIV to receptors to further reduce the infectivity of HIV. The so-called "blocking peptides", which are capable of inhibiting viral proliferation as well as the monoclonal antibodies specific for epitopes within such blocking peptides, can be used to increase the effectiveness of treatments against HTV infections. Typically, HIV blocking peptides correspond to amino acid sequences of HIV that are believed to be essential for binding of the virus to the host cell, such as. the env coded amino acid residues of LAV _BRU between about 190 to about 197 amino acids and between about 185 to about 192 of ARV-2 and HTLV-III (BH-10). These include the octapeptide T (Al-Ser-Thr-Thr-Asn-Tyr-Thr) and its various derivatives (e.g., IX.-1 below) and analogues (e.g., XI-1 below), which are used by Pert and et al., Proc. Natl. Acad. Sci. USA 83, 9254-9258 (1986), which is incorporated herein by reference, which is located on the envelope glycoprotein (gp 110 or 120).

so e.g. blocking peptides having the following sequences are of particular importance, preferably acetylated by the

-NH2-terminal and amidated at -COOH-terminal: IX (173D.) Y-D-Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr-Y ';

X (186.) Y-Thr-Thr-Asn-Tyr-Thr-Y ¹ ;

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 *;

XIV (190.) Y-Asp-Thr-Asn-Tyr-Ser-Y ';

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

wherein Y and Y ', when present, each represent a sequence of up to 20 amino acids. Epitopes and antigenic determinants are typically defined by at least five contiguous amino acids and are, e.g. mimicking naturally occurring HIV antigenic sites are used in the production of HIV reactive antibodies and vaccines.

Development of monoclonal antibodies

The term monoclonal antibody specifically reactive with the neutralizing epitope of the HVV 301-336 corresponding to LAV 301-336 includes, as used herein, monoclonal antibodies capable of analogous amino acid sequences to other HIV strains (e.g., Table I above). bind. The same applies to peptides in this range.

The production of monoclonal antibodies can be accomplished by rendering the expression of nucleic acid sequences encoding HIV-specific antibodies non-destructive by introducing such sequences, typically cDNA encoding the antibody, into a host cell. , which can be cultured in cell culture. The non-killed cell line may be a mammalian cell line transformed by oncogenesis, transfection, mutation or the like. Such cell lines include myeloma cell lines, lymphoma cell lines, or other cell lines capable of supporting antibody expression and secretion in vitro. The antibody may be a naturally occurring immunoglobulin of a mammal produced by a virus-transformed lymphocyte, particularly a splenocyte, or a lymphocyte fused to a neoplasmic cell, such as myeloma, to form a hybrid cell line. Typically, the splenocyte is obtained from an animal immunized against the HIV virus or an epitope-containing fragment thereof.

The immunization protocol is well known and can be significantly modified to remain effective. See Goding: Monoclonal Antibodies:

HU 214 439 Β

Principles and Practice, 2nd Edition (1986), Academic Press; this literature is incorporated herein by reference. Crushed viruses, synthetic peptides, and bacterial fusion proteins containing antigenic fragments of the gp10 or p25 molecule may be used as immunogens. Preferably, the immunogen of the disrupted viruses, peptides, or recombinant proteins is rich in proteins or fragments thereof containing the appropriate epitopes. Specifically, solutions containing disrupted virus lysates or extracts or supernatants of biologically expressed recombinant proteins or disrupted expression vectors may be enriched in glycoproteins, if necessary, using purification techniques such as. polyacrylamide gel electrophoresis. Lectin affinity purification is a preferred and convenient method for purifying gp10 and other glycoproteins, e.g. affinity purification using lens lectin. The degree of purification to which glycoproteins are purified from solutions for use as immunogens may vary within wide limits, e.g. less than 50%, generally 75% -95%, preferably 95% -99%, but most desirably to complete homogeneity.

Once the proteins have been purified to the desired extent, they can be suspended or diluted in an appropriate physiological vehicle for immunization or adjuvanted. One preferred technique is e.g. involves adsorbing proteins and fragments thereof to lens lectin agarose or other macromolecular carriers for injection. Immunogenic amounts of antigen formulations enriched in HIV proteins (including gp10 glycoproteins and p25 core proteins, or antigenic portions thereof) are injected into the host, typically in the range of 1 pg to 20 mg / kg host. Administration may be by injection, e.g. intramuscular, peritoneal, subcutaneous, intravenous, etc. Administration can be done once or more times, usually over a period of one to four weeks. Immunized animals are observed for the production of antibodies to the desired antigens, and the spleen is harvested and spleen B-lymphocytes are isolated and fused or transformed with a myeloma cell line. The transformation or fusion can be carried out in a conventional manner, and the fusion technique is described in numerous patents, e.g. a 4,172,124; 4,350,683; 4,363,799; 4,381,292; and U.S. Patent Nos. 4,423,147; In addition, see also Keimet et al., Monoclonal Antibodies (1980). and the literature references therein (all incorporated herein by reference) and Goding's work cited above.

Immortalized cell lines can be cloned and screened in accordance with conventional techniques and demonstrated in the cell supernatants for binding to the desired gp10 or p25 HIV virus viral proteins, recombinant fusion proteins or synthetic peptides containing the desired epitope region. Appropriate non-killed cell lines can then be grown in vitro or injected into the peritoneal cavity of a suitable host to produce ascites fluid. Due to the availability of some of the antibodies of the present invention that are known to be specific, e.g. epitopes within the area encoded by 6668 bp of the LAV _B ru genomic region (which encodes peptide 29) or by about 7246 bp (encoding peptide 36), the supernatants can be screened by inhibiting the monoclonal antibodies in question in a competitive assay (the above numbers are to be understood according to the method of Wain-Habson et al., Cell, 44, 9 (1985); this literature is incorporated herein by reference]. Thus, additional non-killed hybridoma cell lines can be readily produced with the desired binding characteristics from a variety of sources, based on the availability of specific antibodies specific to the antigen. Alternatively, these cell lines may be fused to other neoplasmic B cells, where such other B cells receive the genomic DNA encoding the antibody.

Although rodent, especially murine neoplasmic B cells are preferred, other mammalian species, e.g. rabbits, cattle, sheep horses, pigs, birds, or similar species. Immunization of these animals can be easily accomplished and their lymphocytes, especially splenocytes, obtained for fusion.

The monoclonal antibodies selected by the transformed or hybrid cell lines may be of any class or subclass of immunoglobulins, e.g. can be IgM, IgD, IgA, IgG ₄ or IgE immunoglobulins. Because IgG is the most common isotype used in diagnostic tests, it is often beneficial. Monoclonal antibodies may be used in intact form or fragments thereof, e.g. Fv, Fab, F (ab ') ₂ , but generally in intact form.

To counteract the potential antigenicity of an animal-derived monoclonal antibody, chimeric antibodies can be constructed in which an antigen-binding fragment (variable region) of an immunoglobulin molecule is linked by peptide linkage to at least a portion of another protein not recognized by humans, e.g. an excised portion of a human immunoglobulin molecule. This can be accomplished by fusing the exon of the animal variable region with human kappa or gamma constant region exons. Various techniques are known to those of ordinary skill in the art, such as those described herein. PCT Publication Nos. PCT 86/01533, EP 171496 and EP 173494, the disclosure of which is incorporated herein by reference.

pharmaceutical formulation and application

The antibodies of the present invention which show neutralizing activity, e.g. those that react with epitope sites on gp10 or p25, or that react with a blocking peptide, may be incorporated as a component into a pharmaceutical composition for attenuating HIV infections. The composition should contain at least one therapeutic or prophylactic amount of the monoclonal antibody of the present invention.

EN 214 439, with a pharmaceutically effective carrier. The pharmaceutical carrier may be any compatible, non-toxic material that is capable of delivering the monoclonal antibody to the patient. Suitable carriers include sterile water, alcohol, fats, waxes and inert solids. Pharmaceutically acceptable adjuvants (buffering agents, dispersants) may also be incorporated into the pharmaceutical composition. Such compositions may contain a unique monoclonal antibody, e.g. specific for 6688. bp-6750. bp to HIV strains containing an epitope site encoded within an area encoded by bp. Alternatively, a pharmaceutical composition may comprise one or more monoclonal antibodies to form a "cocktail." so e.g. A cocktail containing monoclonal antibodies against various strains of HIV could be a universal product with therapeutic or preventive activity against the vast majority of clinical isolates of HIV. The cocktail may contain monoclonal antibodies that bind to HIV proteins or glycoproteins other than gp110 or p25, e.g. gp41 glycoprotein or p34 nuclease / integrase. The molar ratio of the various monoclonal antibody compositions generally does not differ by more than a factor of 10, more particularly by a factor of 5, and the molar ratio is preferably about 1: 1-2 to any of the other antibody components.

The monoclonal antibodies of the present invention may be used in the form of individually administered compositions in combination with other antiretroviral agents, including blocking peptides. The current state of the development of anti-retroviral agents, and in particular anti-HIV agents, is reviewed by Mitsuya et al. (Natúre, 325, 1987), which is incorporated herein by reference.

The monoclonal antibodies, peptides, and pharmaceutical compositions of the present invention are particularly useful for oral or parenteral administration. The pharmaceutical compositions are preferably administered parenterally, that is, subcutaneously, intramuscularly or intravenously. Thus, the present invention provides compositions for parenteral administration comprising a monoclonal antibody, peptide, or "cocktail" thereof in an acceptable carrier, preferably an aqueous carrier. A wide variety of aqueous carriers can be used, e.g. water, from the buffer! water, 0.4% saline, 0.3% glycine, etc. These solutions are sterile and generally free of particulate matter. These compositions may be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable excipients, if necessary under appropriate physiological conditions, e.g. pH adjusting and buffering agents, toxicity adjusting agents, e.g. sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like. The concentration of antibody in these formulations may vary within wide limits, e.g. less than 0.5%, usually at least 1% to 15-20% (by weight), and the concentration is primarily based on the volume, viscosity, etc. of the liquid. selected, with particular regard to the chosen method of administration.

Thus, a typical pharmaceutical composition can be prepared for intramuscular injection containing 1 ml of sterile buffered water and 50 mg of monoclonal antibody. A typical pharmaceutical formulation can be prepared for intravenous infusion containing 250 ml of sterile Ringer's solution and 150 mg of monoclonal antibody. Methods for preparing parenteral compositions are known or obvious to those skilled in the art, and are described in more detail, e.g. in Remington's Pharmaceutical Science, 15th Edition, Mack Publishing Company, Easton, Pennsylvania (1980), which is incorporated herein by reference.

The monoclonal antibodies and peptides of the present invention may be lyophilized for storage and reconstituted in a suitable carrier prior to use. This technique has already been shown to be effective against conventional immunoglobulins, so that lyophilization and recovery techniques known in the art can be used. Those skilled in the art are aware that lyophilization and recovery may lead to variable decreases in antibody activity (e.g., IgM antibodies show greater loss of activity than IgG antibodies in conventional immunoglobulins) and that levels of use may need to be adjusted, to compensate for this.

The present monoclonal antibodies, peptides or cocktails thereof may be administered for the prophylactic and / or therapeutic treatment of HIV infection. For therapeutic use, the composition is administered to a patient who has already been infected with HIV, in this case in an amount that will cure or at least partially arrest the infection and its complications. The amount suitable to accomplish this task is defined as a "therapeutically effective dose". For this application, the effective amount will depend on the severity of the infection and the condition of the patient's own immune system, but is most commonly used in the range of 1-200 mg antibody / kg body weight at doses of 5-25 mg / kg body weight. It will be appreciated that the agents of the present invention are generally used in the severe condition of the disease, that is, in patients at risk of, or at risk of, life threatening. In such cases, it is possible, and if the attending physician feels it is desirable, to administer a significant excess of these antibodies.

For prophylactic applications, compositions comprising the peptides, antibodies, or cocktail of the present invention are administered to patients who have not yet been infected with HIV but may have recently been exposed to or are thought to have been exposed to the virus; in this case we want to increase the patient's resistance to such possible infections or my vaccines against the virus. The amount required for this is defined as the "prophylactic effective dose". The exact amount for such use will again depend on the patient's state of health and the general level of immunity, but this dose will generally be in the range of 0.1 mg to 25 mg / kg, particularly 0.5 mg to 2.5 mg / kg.

Dosage levels and dosing schedules can be used to determine the individual or multiple administration of the compositions.

EN 214 439,, this will be selected by your doctor. In any event, the pharmaceutical formulations should provide an amount of antibodies of the present invention sufficient to effectively treat the patient. In addition, the monoclonal antibodies of the present invention may be useful as target-specific carrier molecules. An antibody may be bound to a toxin to produce an immunotoxin, or to a radioactive substance or drug to form a radiopharmaceutical or pharmaceutical composition. Methods for producing immunotoxins and radiopharmaceuticals are well known (see, for example, Cancer Treatment Reports 68, 317 (1984)).

It is also possible to monoklonábs antibodies and human T-cell activators (eg. Monoclonal antibodies against _Fc gamma in against CD3 antigen or T cells receptors) of this invention heteroaggregátuma render the T cells or _Fc gamma bearing cells present empower (eg K cells or neutrophils) to kill HIV-infected cells by antibody-dependent cell-mediated cytolysis (ADCC). Such heteroaggregates are e.g. can be covalently cross-linked to anti-CD3 antibodies using the N-succinimidyl-3- (2-pyridyldithio) propionate heterobifunctional reagent as described by Karpinsky et al. Exp. Med. 160, 1686 (1984)]; this literature is incorporated herein by reference.

The peptide compositions themselves can also find therapeutic use, where administration results in a decrease or disappearance of HIV in an infected host. These compositions, e.g. peptide 29, blocking peptides, and peptide 126 in suitable physiological vehicles, intravenously, subcutaneously, intramuscularly, intraperitoneally, and the like. can be injected. Various carriers include phosphate buffered saline, water, potassium chloride, sodium lactate, and the like. Concentrations of peptides may vary within wide limits, depending on their end use, activity, and route of administration. Preferably, the peptides are amidated at the -COOH terminal, formylated at the NH ₂ terminal, or in the form of other pharmaceutically acceptable derivatives. Addition of blocking peptides to peptides that mimic the neutralizing HIV region and / or the specific reactive antibodies of the present invention results in significantly increased therapeutic efficacy. Other anti-HIV agents may also be included in the formulations (agents other than the monoclonal antibodies that bind the peptides), e.g. 3'-azido-3'-deoxythymidine; 2 ', 3'-dideoxycytidine;2'3'-dideoxy-2'3'-didehydrocytidine and the like.

Use of Monoclonal Antibodies for Immuno-Affinity Purification Monoclonal antibodies specific for polypeptides containing gp10 or other antigenic determinants, particularly those derived from biologically expressed recombinant fusion proteins or cultured HIV lysates or extracts, are particularly preferred for purification. In general, antibodies have an affinity association constant of the order of 10 ⁸ 12 ¹² M. Such antibodies can be used to purify recombinant fusion proteins from the culture medium of a recombinant expression system when the expressed protein is secreted or to purify proteins from components of the disrupted biological expression system if the protein is not secreted. In general, monoclonal antibodies that are capable of reacting with gp10 or other antigenic determinants are fixed or immobilized on a substrate or support. The solution containing the HIV antigenic determinants is then contacted with the immobilized antibody under conditions suitable for the formation of immune complexes between the antibody and the gp10 antigenic determinant polypeptides. Unbound material is separated from the bound immune complexes, which complexes or gp10 antigen fragments are then isolated from the vehicle.

Typically, the monoclonal antibodies are roughly purified from ascites fluid or cell culture supernatant before attachment to a vehicle. Such procedures are well known to those skilled in the art and may involve fractionation with neutral salts at high concentrations. Other methods, e.g. DEAE chromatography, gel filtration chromatography, preparative gel electrophoresis, or protein A affinity chromatography may also be used to purify monoclonal antibodies before being used as immunosorbent.

The carrier to which the monoclonal antibodies are immobilized must have the following general characteristics: (a) weak interactions with proteins in general to minimize non-specific binding; (b) good flow characteristics allowing flow through high molecular weight materials; (c) has chemical moieties that can be activated or modified to allow chemical binding of monoclonal antibodies; (d) physical and chemical stability under the conditions used for coupling the monoclonal antibodies; and (e) stability under the conditions required for adsorption and elution of the antigen and buffer components. Commonly used carriers include agarose, polystyrene derivatives, polysaccharides, polyacrylamide beads, activated cellulose, glass and the like. There are various chemical methods for attaching antibodies to carrier substrates, see, for example, Cuatrecasas P .: Advances in Enzymology 36, 29, 1972. The antibodies of the present invention may be directly attached to the vehicle; or, alternatively, the attachment may be effected by means of a switch or spacer lever.

The general conditions required for immobilization of monoclonal antibodies on chromatographic supports are well known in the art. See e.g. Tijssen P .: Practice and Theory of Enzyme Immunoassay (1985); this reference is incorporated herein by reference. The actual coupling procedures depend somewhat on the characteristics and type of antibody to be coupled. Monoclonal antibodies have characteristics that are batch-to-batch, allowing

EN 214 439 Β to optimize these conditions. The attachment is typically made through covalent bonds.

A suspension of extracts or lysates of the HIV virus or a supernatant from a cultured biological expression system or a suspension of disrupted cells is then added to the separating matrix. The mixture is incubated under conditions and for a time appropriate to the formation of an immune complex, usually for at least 30 minutes, most preferably from 2 to 24 hours. Immune complexes containing polypeptides having parts of the gpl10 antigen are then separated from the mixture. Typically, the mixture is e.g. Elution is removed and the bound immune complexes are washed extensively with adsorption buffer. Immune complexes are then eluted from the separating matrix using an eluting agent compatible with the particular vehicle to be used; such eluting agents are well known to those skilled in the art. Polypeptides containing gp10 or other antigenic portions may be selectively removed, e.g. peptides containing an epitope recognized by the antibodies may be used to compete for the antibody binding site; this is an alternative elution technique that can be applied under mild elution conditions. The selectively adsorbed polypeptide containing the gp110 antigen can be eluted from an antibody affinity adsorbent by changing the pH and / or ionic strength of the buffer. Chaotropic agents may also be useful in the removal of bound antigen. The selection, concentration and further elution conditions of a chaotropic agent will depend on the characteristics of the antibody-antigen interaction, but once determined, they do not need to undergo further changes, e.g. are commonly required in polyclonal affinity separation systems.

The eluted material may require adjustment to physiological pH when low or high pH or ionic strength buffers are used to separate the bound gp10 antigens from the separation matrix. Dialysis or gel filtration chromatography may also be necessary to remove excess salts used in the eluent to allow the recovery of polypeptides containing gp 110 or gp 110 antigen fragments to native conformations.

The method of this invention is e.g. produces polypeptides containing substantially purified gp110 or antigenic fragments thereof, or naturally produced by infected cell cultures, or by recombinant expression systems of bacteria, yeast, or cultured insect or mammalian cells. Gp10 and its fragments or other purified proteins are typically more than 50% pure but preferably at least 75% pure, often more than 95% to 99% pure. These molecules can then be further used in a wide range of applications.

The HIV gp10 proteins, polypeptides containing antigenic fragments thereof, or other substantially different applications, including the AIDS subunit vaccine formulation, in which the immunogen, e.g. gp 110 or its neutralizing region contains an effective dose of antigenic determinants. Other components of the formulation may include antigenic portions of HIV proteins or glycoproteins that stimulate the production (preferably neutralizing antibodies) of an antibody in an immunized host that can protect against subsequent infections by HTV.

Diagnostic Use of Monoclonal Antibodies The monoclonal antibodies of the present invention are also useful for diagnostic purposes. They may be marked or unmarked for this purpose. Typically, diagnostic assays involve the detection of the formation of a complex formed by binding of a monoclonal antibody to an HIV virus. If the antibodies are unlabeled, e.g. can be used in agglutination tests. In addition, unlabeled antibodies may be used in combination with other labeled antibodies ("second antibodies") that are reactive with the monoclonal antibody, such as. antibodies specific for immunoglobulins. Alternatively, monoclonal antibodies may be directly labeled. A wide variety of markings can be used, e.g. radionuclides, fluorescent substances, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (mainly haptens), etc. There are many types of immunoassays available, but by way of example only those described in U.S. Patent Nos. 3,817,827; 3,850,752; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; and 4,098,876; all of these references are incorporated herein by reference.

In general, the monoclonal antibodies and peptides of the present invention are used in enzyme immunoassays, e.g. the antibodies in question, or second antibodies from other species, are conjugated to an enzyme. When a biological sample containing HIV antigens, e.g. human blood serum, saliva, semen, vaginal secretions, or virus-infected cell culture suspensions are combined with the antibodies in question, binding between the antibodies and the molecules showing the desired epitope. Such proteins or viral particles can then be separated from unbound reagents and a second antibody (labeled with an enzyme) can be added. The presence of antibody-specific antibody-enzyme conjugates is then determined. Other conventional techniques are well known to those skilled in the art.

Kits can also be provided using the antibodies in question for the detection of HIV infection or for the presence of HIV virus. Thus, the monoclonal antibody compositions of the present invention may be provided, usually in lyophilized form, either alone or in combination with additional antibodies specific for other epitopes of HIV. Antibodies, which may be conjugated to a label or may be unlabeled, are provided in kits with buffers, e.g. tris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins, e.g. bovine serum albumin or the like. These materials generally11

They are present in less than 5% by weight relative to the amount of active antibody and are generally present in less than 0.001% by weight relative to the antibody concentration. It is often desirable that an inert binder or filler be present in the kit to dilute the active ingredient, wherein the filler may be present in an amount of from about 1% to about 99% of the total composition. Where a second antibody capable of binding the monoclonal antibody is used, it is usually present in a separate vial. The second antibody is typically conjugated to a label and is formulated in a manner similar to the antibody formulations described above.

Detection of gpl10 or p25 antigens or whole virus in various biological samples may be used to diagnose ongoing infection with the HIV virus. Biological samples include, but are not limited to, serum, saliva, semen, tissue sections (brain, skin, lymph nodes, spleen, etc.), cell culture supernatants, disrupted eukaryotic and bacterial expression systems, and the like. The presence of the virus is assayed by incubating the monoclonal antibody with the biological sample under conditions that lead to the formation of an immune complex and then detecting the formation of the complex. In one embodiment, complex formation is detected by the use of a second antibody capable of binding to the monoclonal antibody, wherein the second antibody is typically conjugated to a label and is formulated in a manner similar to the antibody formulations described above. In another embodiment, the monoclonal antibody is bound to a solid phase fixative which is then contacted with the biological sample. Following the incubation step, labeled monoclonal antibody is added to detect bound antigen.

Preparation and Use of Synthetic Peptides The present invention provides novel peptides which, inter alia, immunologically mimic protein epitopes encoded by HIV retroviruses, particularly those encoded within the env or gag region of the viral genome encoding gpl105 or p25. To balance strain-to-strain variations between different isolates, adjustments can be made for conservative substitution and selection can be made from variants that do not include conservative substitution. These peptides can be used as immunogens to inhibit or abolish HIV antigen production in vitro or in vivo, and can be used to detect virus or antiviral antibodies in physiological samples. Depending on the nature of the session, the peptides may be linked to a carrier or other material, labeled or unlabelled, or attached to a solid support, etc.

In one embodiment, the peptides of interest are derived from the gp10 region of the virus. Of particular interest are the areas within the env open reading frame that range from about 6688 bp (bp) to about 6750 bp and from about 7246 bp to about 7317 bp.

Such peptides, including blocking peptides, include at least 5, sometimes 6, sometimes 8, sometimes 20, sometimes 21, generally less than 50, more generally less than 35, and preferably less than 25 amino acids, including one amino acid. HIV into a retroviral coded sequence. Desirably, the peptide should be as small as possible, but still retain the substantially complete immunoreactivity or antiviral activity of the larger peptide. In some cases, it may be desirable to combine two or more non-overlapping oligopeptides to form a unique peptide structure or to be used as single peptides simultaneously; Thus, individually or in combination, they provide the same sensitivity to parents.

The peptides may be modified by introducing conservative or non-conservative substitutions into the peptide; generally less than 20%, more generally less than 10%, of the amino acids are exchanged. In situations where the regions are found to be polymorphic, it may be desirable to alter one or more particular amino acids to more effectively mimic different epitopes of different retroviral strains. In many cases, methionine can be replaced by norleucine (Nor) to provide chemical stability.

It will be appreciated that the peptide used in the present invention need not be identical to any particular HTV polypeptide sequence provided that the material is capable of immunologically competing with the proteins of at least one strain of HIV retrovirus. Therefore, the peptides in question may be the subject of various changes, e.g. insertions, bypasses, and substitutions, whether conservative or non-conservative, where such changes provide certain benefits to the application. Conservative substitutions provide for substitutions within the following groups: gly, ala, val, ile, leu; asp, glu; asn, gin; ser, thr; lys, arg; phe, tyr; and nor (norleucine), met. Generally, the sequence will not differ by more than 20% from that of at least one strain of an HIV virus retrovirus, except that additional amino acids may be added at any end to provide an "arm" by which the peptide of the present invention may be immobilized in a conventional manner. The arms are generally at least one amino acid in length and may be 50 or more amino acids in length, but most preferably 1-10 amino acids in length.

The peptide in which the amino acid sequence is modified by substitution, addition or deletion of amino acids must retain substantially complete immunoreactivity or antiviral activity of the unmodified peptides; these can be measured in a conventional manner by various assay techniques described herein. The d-isomeric form of one or more amino acids may also be used, if desired, with biological properties, e.g. activity, rate of degradation, etc. .

In addition, one, two or more amino acids may be added to the ends of an oligopeptide or peptide to provide ease of attachment of one peptide to another peptide, or to a larger peptide or carrier.

EN 214,439 Β for the purpose of modifying the physical or chemical properties of the peptide or oligopeptide.

As the amino acid, tyrosine, cysteine, lysine, glutamic acid, or aspartic acid may be introduced at the C or N-terminus of the peptide or oligopeptide to provide useful functional groups for coupling. Cysteine is particularly preferred for facilitating covalent coupling to other peptides or for forming polymers by oxidation.

In addition, the peptide or oligopeptide sequences may differ from the natural sequence with a sequence which, by terminal NH 2 acylation, e.g. acetylation or amidation of thioglycolic acid, or e.g. modified by terminal carboxyl amidation with ammonia or methylamine to provide stability and increased hydrophobicity for coupling or bonding to a carrier or other molecule or for polymerization.

so e.g. in one of the above-described peptides I-VIII and IX XV, where Y and Y 'are present, Y or Y' contains one or more cysteine residues, or a combination of one or more cysteine residues with spacer amino acids . Glycine is a particularly preferred bulking agent. Preferred peptides for use in oxidative polymerization are those wherein Y or Y 'represent at least two cysteine residues. When two cysteine residues are present at the same end of the peptide, there is a preferred embodiment of separating the cysteine residues from one to three spacer residues, preferably glycine. The presence of cysteine residues allows dimers to be formed and / or the hydrophobicity of the resulting peptide to be increased, which facilitates immobilization of the peptide in solid phase or immobilized measuring systems.

Of particular interest is the use of mercaptan groups of cysteines or thioglycolic acids used to acylate terminal amino groups to link two or more peptides or oligopeptides or combinations thereof via disulfide bonds or longer bonds to form polymers containing multiple epitopes. Such polymers have the advantage that they have increased immunological reactivity. Where different peptides are used to construct the polymer, they will have the additional ability to induce antibodies that are immune to various antigenic determinants of different HIV isolates.

To achieve the formation of antigenic polymers (synthetic multimers), materials which have bis-haloacetyl groups, nitroaryl halides and the like can be used. where the reagents are specific for thio groups. Thus, the linkage between two mercapto groups of different peptides or oligopeptides may be a single bond or a binding group may be at least 2, generally at least 4, not more than about. 16, and generally no more than 14 carbon atoms.

The peptide of interest may be used bound to a soluble macromolecular support (e.g., not less than 5 kdalton). Conveniently, the carrier may be a poly (amino acid), either naturally occurring or synthetic, with which antibodies are unlikely to encounter in human serum. Examples of such carriers are poly-L-lysine, keyhole limpet hemocyanin, thyroglobulin, albumins, e.g. bovine serum albumin, tetanus toxoid, etc. The choice of carrier will primarily depend on the intended end use of the antigen, as well as convenient handling and availability.

Such conjugates must have at least one molecule of at least one peptide of interest in the macromolecule and no more than about 1 molecule per 0.5 kdalton of the macromolecule, generally no more than 1 molecule per 2 kdalton of the macromolecule. One or more different peptides can be linked to the same macromolecule.

The coupling method is conventional using reagents such as p-maleimido-benzoic acid, p-methyldithiobenzoic acid, maleic anhydride, tartaric anhydride, glutaraldehyde and the like. The linkage may be at the N-terminus, at the C-terminus, or at a site between the ends of the molecule. Derivatives of the peptide in question can be prepared by coupling or coupled to a carrier.

Various assay and measurement protocols familiar to those skilled in the art can be used to detect the presence of antibodies to retroviral proteins or to detect the retroviral proteins themselves. Of particular importance is the use of peptides as labeled reagents where the label provides a detectable signal or provides a direct or indirect binding of the peptide to a surface, whereby the antibody against the peptide becomes bound to the peptide on the surface. The presence of a human antibody bound to the peptide can then be detected by using a specific xenogeneic antibody to human immunoglobulin (normally both human IgM and human IgG), or to immunocomplexes, e.g. A labeled protein specific for the A ^f protein of S. aureus A protein was used.

Application of a sample container typical of the measurement-test technique, e.g. using cavities of microtiter plates, wherein said polypeptide or conjugates thereof are covalently or non-covalently adsorbed to the bottom and / or walls of the container. The sample, normally human blood or serum, is placed in a container in approximately buffered medium and allowed sufficient time to allow complex formation between the polypeptide (s) and any related antibody in the sample. The supernatant is removed and the container is washed to remove non-specifically bound proteins. A labeled specific binding protein that specifically binds to the complex, e.g. as a xenogeneic antiserum to human immunoglobulin, is used for detection.

The peptide can be prepared in many ways. Because of its relatively short size, the peptide can be synthesized in solution or on a solid support in accordance with conventional techniques. Various automatic synthesizers13

These devices are now commercially available and can be used in accordance with known procedures. See e.g. Stewart and Young, Solid Phase Peptide Synthesis, 2nd Edition, Pierce Chemical Co. (1984); and Tam et al., J. Am. Chem. Soc. 705, 6442 (1983).

Alternatively, hybridization DNA technology may be used, wherein a synthetic gene can be generated using single strands encoding the polypeptide, or substantially complementary strands thereof, where the individual strands overlap and can be assembled in a fusion medium to hybridize. The hybridized strands can then be ligated by selecting the appropriate ends to form the complete gene and inserted into the gene expression vectors, which are readily available today. See e.g. Maniatis et al., Molecular Cloning, Laboratory Manual, CSH, Cold Spring Harbor Laboratory (1982). The viral genomic region encoding the peptide can also be cloned by conventional recombinant DNA techniques and then expressed (see Maniatis, cited above).

DNA coding sequences derived from the HAV LAV _bru and ARV2 isolates which may be used to express the peptides include:

^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 GCA ACA CAT OVER THE TGT GGA AAA OVER THE GGA AAT ATG AGA CAA ARV-2 TGT ACA AGA CCC AAC AAC AAT ACA AGA AAA AGT ATC TAT OVER THE GGA CCA GGG AGA GLA TTT CAT ACA ACA CAT GGA TGT AGA OVER THE OVER THE GGA DAM OVER THE AGA AAA GCA

Fragments of a sequence may be used to express peptide fragments, conservative base exchanges where the modified codon (s) encode identical amino acids, or non-conservative changes in the coding sequence, whereby the resulting amino acid may represent a conservative or non-conservative amino acid sequence change; we will discuss this later.

The coding sequence may be extended at either the 5'- or 3'-terminus, or at both ends to increase the peptide while retaining the epitope site (s). The extension may provide a lever for switching, e.g. for some signaling, such as enzyme, fusion of two or all peptides in the same chain, providing antigenic activity, or restriction sites convenient for cloning, etc.

The DNA sequence itself, or fragments thereof, or larger sequences may generally be used as nucleotide probes with at least 15 bases, preferably at least 18 bases, to detect retroviral RNA or proviral RNA, or to identify homologous regions for cloning or sequence analysis. Many techniques have been described, e.g. Grunstein-Hagness technique, Southern technique, Northern technique, dot-blot technique, and other methods, e.g. that described in U.S. Patent No. 4,358,535; the latter is incorporated herein by reference.

The peptides of interest, including blocking peptides, and analogs thereof can be used alone or in combination in vaccines. Similarly, anti-idiotype antibodies, i.e., reactive with the idiotypes of the antibodies of the present invention and thus containing epitopes mimicking the neutralizing regions of the HVV, may also be used in vaccines. Peptides or anti-idiotype antibodies can be conveniently formulated, usually at a concentration of 1 pg-20 g / kg host. A physiologically acceptable medium may be used as a carrier, e.g. sterile water, physiological saline, phosphate buffered saline, and the like. Adjuvants may also be employed, e.g. aluminum hydroxide gel, surfactants such as lysolecithin, polyvalent polyols, polyanions, peptides, proteins (e.g., diphtheria or cholera toxin) and oil emulsions. The peptides may also be incorporated into liposomes or conjugated to polysaccharides, polypeptides, or polymers for use in vaccine formulations. Administration may be by injection, e.g. intramuscular, peritoneal, subcutaneous, intravenous, etc. The immunogenically effective dose may be administered simultaneously or over a period of time, usually at 1-4 week intervals. An "immunogenically effective dose" is the amount of vaccine sufficient to elicit an immune response in a host by which the host exhibits increased infection.

Other aspects and advantages of the present invention will be apparent from the following experimental descriptions which set forth the invention by way of example. The examples are provided for illustrative purposes only and are not intended to be limiting.

Example 7

Development and characterization of monoclonal antibodies

Example I describes the construction of hybrid cell lines that produce monoclonal antibodies specific for HIV envelope glycoproteins. The method involves the use of LAVbru lectin-purified, lens-lectin-agarose-bound extracts as immunogen. The monoclonal antibodies, which are subsequently generated by the hybrid cell lines, are characterized by their ability to form an immunoblot and radioimmune precipitate with gpl10 from purified LAV, which are biologically expressed recombinant fusion proteins. [Gpl 10 is a long-known and standardized glycoprotein of the HIV envelope; is described, inter alia, in Robey et al., Proc. Natl. Acad. Sci. USA 83, 7023 (1986)]. Monoclonal antibodies that bind to epitopes on gp1010 are also reactive in ELISA with whole virus, fusion proteins and synthetic peptides, and react with whole virus in indirect fluorescence assays.

HU 214 439 Β

The procedure for the formation of the hybrid cell lines producing the monoclonal antibody and the characteristics of the antibodies are as follows:

Purification of LAV virus from infected GME cells (ATCC CRI 8904) according to the procedure disclosed in British Patent No. 83,24,800 (details of purification are described below) is broken down into 50 mM Tris pH 7.4 / 0.15 mM. 1 in a solution containing NaCl, 1.0% aprotinin and 2% Nonidet (-40 (NP-40) (octylphenoxy polyethoxyethanol). The extract was purified twice by centrifugation and adjusted to 0.5% NP-40 by adding three volumes of disintegrating buffer without NP-40. Lens lectin Sepharose (Pharmacia, Piscataway, New Jersey, USA) was prewashed in disintegration buffer without NP-40 and then equilibrated in adsorption buffer [50 mM h, pH 7.4, 0.15 M NaCl, 1.0% aprotonin, 0.5% NP40]. Purified virus extracts were adsorbed with lens lectin Sepharose at 40 ° C for 42 hours. The non-adsorbed material is removed by washing with excess adsorption buffer. Elution of the adsorbed material was performed with 0.2 M alpha-methyl mannoside in adsorption buffer. The eluate is dialyzed against PBS (phosphate buffered saline) to remove the sugar and re-adsorbed onto the lens lectin Sepharose.

The glycoprotein-lens-lectin Sepharose complex was used to immunize BALB / c mice by three intraperitoneal injections without adjuvant given 2-3 weeks apart. The immunization session was performed by Godstein et al., Infect. Immun. 38: 273-281 (1982). Spleens are removed from immunized mice that demonstrate circulating antibodies to HIV glycoproteins by immunoblotting, RIP, and / or ELISA.

The procedures used to construct cell lines are generally those described by Kohler and Milstein (Naturre, 256, 495 (1985)), modified by Goldstein LC et al., Infect. Immun. 38: 277 (1982)]. Spleen B-lymphocytes obtained from immunized mice were fused with NS-1 myeloma cells (ATCC TIB 18) using 40% (w / v) polyethylene glycol. After fusion, the cell mixture was resuspended in HAT medium (RPMI-1640 medium, 15% fetal calf serum, 110 ^ * mol / 1 hypoxanthine, 4 × 10 ⁷ mole / 1 aminopterirmal and 1.6 to 10 ^-5 mol / 1 thymidine); to select for hybrid cell growth and then plated in 96-well microculture plates at 1-3T0 ⁶ cells / ml and incubated at 37 ° C in a humidified atmosphere containing 6% CO 2. The cultures are fed by replacing half of the supernatant with fresh HAT medium. The cavities are observed using a reverse microscope for signs of cell proliferation, and when the cells are of sufficient density, the supernatants are screened for anti-LAV antibody.

Wells containing hybrid cells that produce anti-LAV antibody are identified by ELISA, measuring binding to either purified whole viruses or biologically expressed fusion proteins. ELISAs using the disrupted virus were performed on LAV EIA plates (Genetic Systems, Seattle, Washington). Plates were incubated at 37 ° C for 45 minutes with cell culture fluids and then washed three times with phosphate buffered saline containing 0.05% Tween20 (PBS-Tween).

Peroxidase goat anti-mouse IgG (1: 2000 dilution in PBS-Tween; Zymed Laboratories, Inc., South San Francisco, California) (100 μΐ / well) was added and the plates were incubated for 45 minutes at 37 ° C. , and wash as above. A substrate (0.025 mol / L citric acid, 0.05 mol / L sodium dihydrogen phosphate, pH 5.0 containing 50 ml of 14 mg o-phenylenediamine and 10 ml of hydrogen peroxide) was added and plates were incubated for 30 minutes at room temperature in the dark. The reaction is stopped with 3N sulfuric acid and the calorimetric reaction is quantitatively evaluated using an automated microplate reader. Cavities that produce positive results are subcloned by limiting dilution, re-examined for specificity, and used,

The monoclonal antibodies secreted by the resulting hybrid cell lines are further characterized for specificity and reactivity by immunoblotting, immunoprecipitation, and ELISA using disrupted LAV virus, recombinant LAV fusion proteins, and synthetic LAV peptides. All antibodies are defined as IgG). The HIV-gpl 10-1, HIV-gpl 10-2, and HIV-gpl 10-3 cell lines were deposited with the American Type Culture Collection prior to the filing of this application, designated ATCC HB 9175 and HB 9176 and HB 9177, respectively. they got.

The recombinant fusion proteins tested for reactivity were previously designated as ENV2, ENV3, ENV4, and ENV5. The ENV2 protein is expressed from pENV2 (ATCC 53071), which is an LAV 6598 base pair (bp) 7178 bp (Wain-Hobson et al., Cell, 44.9 (1985)); ENV3 is expressed from pENV3 (ATCC 53072), which contains the LAV from 7178 bp to 7698 bp; ENV4 is expressed in pENV4 (ATCC 53073), which includes the bp 7698 to bp 8572; and ENV5 is expressed from pENV5 (ATCC 53074), which contains the LAV region from 5889 bp to 7698 bp. The preparation of recombinant fusion proteins is described in detail in European Patent Application EP 201 716.

Composition of synthetic peptides

Peptide I (29) and VIII. peptide (110-2-2) was prepared on a benzhydrylamine (polystyrene / divinylbenzene) resin (Applied Biosystems, Inc., Foster City, California). Peptide V (177) was prepared on t-butoxycarbonyl (Boc) ethylbenzylcysteine phenylacetamidomethyl (PAM) polystyrene / divinylbenzene resin. Reference is made to Stewart and Young in "Solid Phase Peptide Synthesis", Pierce Chem. Co., (ed.), Rockford, Illinois (1984).

Symmetric anhydride couplings were performed on an Applied Biosystems 430A synthesizer. Cysteine is added as the first residue on both peptides.

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Asparagine and glutamine use dicyclohexylcarbodiimide couplings in the presence of hydroxybenzotriazole. Benzyl-based side chain protection and Boc alpha amine protection are used.

Other routine side chain protectors include Boc (formyl) tryptophan, Boc methionine sulfoxide, Boc (tosyl) arginine, Boc (methylbenzyl) cysteine, Boc (tosyl) histidine, Boc (chlorobenzyloxycarbonyl) lysine, and Boc (bromobenzyloxycarbonyl) tyrosine.

When the peptides are radiolabeled, this is done by acetylation at the amino terminus with ³ H-acetic acid and excess dicyclohexylcarbodiimide. For the reactions in the last two paragraphs, reference is also made to Stewart's book.

Deprotection and cleavage of the peptide from the resin is accomplished by a Tam low-to-high HF session (Tam et al., Supra). The resin is extracted with 5% acetic acid and the extract is subjected to gel filtration chromatography in 5% acetic acid.

Synthetic HIV peptides tested for reactivity with monoclonal antibodies include peptides 29, 36, and 39. Peptide 29 is encoded by the LAV _BRU genomic region from about 6688 bp to 6750 bp; peptide 36 is encoded by a region of about 7246 bp to 7317 bp; and peptide 39 is encoded in the region of about 7516 bp to 7593 bp. Peptides 36 and 39 are described in detail in U.S. Patent 4,629,783.

The blocking peptides IX-XV are prepared essentially as described above on methyl benzylhydrylamine (polystyrene / divinylbenzene) resin (Applied Biosystems, Inc., Foster City, California). Symmetric anhydride couplings were performed on an Applied Biosystems 430A synthesizer. For asparagine, dicyclohexylcarbodiimide was used in the presence of hydroxybenzotriazole. Benzyl-based side chain and Boc alpha-amine protection are used for protection, while Boc (bromobenzyloxycarbonyl) is used specifically for tyrosine side chain. Acetylation, if any, is performed using acetic anhydride or glacial acetic acid and dicycloheximide. Deprotection and peptide cleavage from the resin is performed using a standard "high" HF procedure (Stewart et al., Supra). Extraction from the resin was performed with 50% acetic acid and the extract was then subjected to gel filtration chromatography in 20% acetic acid. If necessary, high performance liquid chromatography was performed on a Vydac C18 column (Rainin Instrument Co., Emeryville, CA) using a gradient of 0.1% trifluoroacetic acid / acetonitrile.

Immunoblotting

Immunostaining is performed on clonal supernatants or ascites fluids using purified LAV virus and recombinant fusion protein as antigen. Antigens were first separated by polyacrylamide gradient gel electrophoresis (7.0-15.0%) and transferred to nitrocellulose membrane (NCM) by electrophoresis (4 hours, 25 V, 25 mM sodium phosphate, pH 7.0). After transfer, NCM is blocked by incubation for one hour at room temperature in PBS-Tween or Blotto (5% nonfat milk powder in PBS) to prevent non-specific interactions. The NCM is incubated with cell culture supernatant or ascites fluid diluted in standard ratio with PBS-Tween for one hour at room temperature and rinsed three times with PBS-Tween. In the second step, NCM was incubated with goat anti-mouse IgG horseradish peroxidase diluted 1: 2000 in PBS-Tween for one hour at room temperature. This incubation is followed by washing with PBS-Tween and the membrane is immersed in horseradish peroxidase color development solution (Bio-Rad Laboratories, Richmond, CA) for 20 minutes. The reaction is stopped by immersing the membrane in deionized water. The reactivity of the monoclonal antibody is compared with a positive control serum reactivated with purified disrupted virus or an express fusion protein. The results show that all antibodies bind to gp110 and its precursor molecule, gp150, using a disrupted virus preparation. Antibodies 110-1 and 110-2 also recognize the ENV3 fusion protein, while antibodies 110-3,110-4,110-5 and 110-6 form an immune complex with ENV2.

Immune precipitation

For radioimmune precipitation, virus extracts were prepared from HEM LAV _B ru isolates (prepared according to Montagnier, GB 83 24 800) from infected CEM cells adapted for continuous passage of HIV by lytic growth. When the early cytopathic effects become apparent, the cells are transferred to a signaling aqueous medium containing ³⁵ [S] -methionine (0.05 mCi / ml) or ³ [H] -glucosamine (0.025 mCi / ml) and then for 24 hours. incubate until most of the cells are lysed, releasing virus into the culture supernatant. Virus was pelleted (1 hour at 100,000 g) from cell-free supernatant and detergent extract was prepared in P-RIPA buffer (phosphate buffered saline containing 1.0% Triton Χ-100, 1.0% deoxycholate, 1% SDS and 1% aprotinin). Similar extracts were prepared from supernatants of uninfected CEM cells.

Immunoprecipitation assays were performed by incubating 100 µl of virus extract with 100 µl of culture supernatant from a hybrid cell line for 1 hour on ice. 4 microliters of rabbit anti-mouse Ig (Zymed Laboratories, South San Francisco, California) was added to each sample and incubated for 30 minutes. Immunoprecipitin (100 µl; Bethesda Research Laboratory, Bethesda, Maryland), resuspended in P-RIPA buffer containing 1% ovalbumin, was added to each sample and incubated for an additional 30 minutes. The bound complexes were washed and separated by SDS-polyacrylamide gel electrophoresis (15.0% acrylamide; DATD gel). Following electrophoresis, the gels were fixed, soaked in Enhance (New England Nuclear, Boston, Massachussets), dried and placed on Kodak XR-5 film. A positive reference serum that elicits an immunoprecipitation with each of the HIV virus proteins is reacted with virus-infected and

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II. spreadsheet

Monoclonal antibodies reactive with recombinant proteins and synthetic peptides, as detected by ELISA

Recombinant fusion protein Synthetic uentides LAV control CEM control ENV 2 ENV3 EMV4 ENV 5 29th 36th 39th 110-1 0.077 3,000 0.113 3,000 ND 2.421 0,054 0,908 0,125 110-2 -0.003 3,000 0,000 3,000 ND 2,305 -0.005 1,214 0,009 110-3 3,000 0.011 ND ND 3,000 ND 0,017 0,363 0.046 110-4 3,000 0,020 ND ND 3,000 ND 0,016 0.383 '0.067 110-5 '3,000 0,014 ND ND 3,000 ND 0,016 .368 0,025 110-6 3,000 0,033 ND ND 1.937 ND 0,017 .486 0.032 ' * ND = not shown Who

spiked CEM cell supernatants as positive and negative controls.

The results show that all six monoclonal antibodies produce specific immunoprecipitation with gp10 and gp150.

Enzyme-linked Immunoadsorption Assay To screen for gp10 epitopes recognized by the monoclonal antibodies of the present invention, culture supernatants from hybrid cell lines or ascites fluid were further characterized by reactivity in ELISA with biologically expressed fusion proteins and synthetic peptides. The procedure is the same as described above except that fused proteins or synthetic peptides replace the purified virus as an antigen adsorbed on the surface of the microtiter wells.

When peptides are used as antigen, the plate application procedure is as follows. The lyophilized peptide was dissolved in 6M guanidine HCl. Immediately before application to the 96-well plate, the guanidine solution was diluted in 0.05 M carbonate / bicarbonate buffer, pH 9.6, to a final peptide concentration of 100 pg / ml. A volume of 50 ml of diluted peptide was added to each microtiter well and the plates were then incubated at 4 ° C overnight. The excess peptide solution is "shaken", the plates are blocked with Blotto, and the process described above is followed by a further ELISA session. Similarly, the recombinant protein was diluted to a final concentration of 2 pg / ml in 0.05 M carbonate / bicarbonate buffer, pH 9.6, before following the same procedure.

The results are shown in Table II. Table. The monoclonal antibodies produced by the HVV gpl 10-1 and HVV gpl 10-2 cell lines react with ENV3, ENV5, peptide 36 and the viruses that have been destroyed. Antibodies derived from the HIV-gp10-3, HIV-gp10-4, HIV-gp10-5 and HIV-gp1010 cell lines react with ENV2 and peptide 29, as well as with the shattered virus.

II. The results in Tables 1 to 4 show that monoclonal antibodies 110-1 and 110-2 recognize an antigenic determinant encoded by the DNA sequence within the pENV3 region, more specifically the antigenic determinant encoded by the HIV genome region defined by an amino acid sequence within peptide 36. . This means that monoclonal antibodies gp10-1 and gp 110-2 bind to gp 110 7246 bp. and 7317 bp. region, as evidenced by the formation of immune complexes formed with peptide 36 and ENV3. This region of the HVI genome has been identified as having been conserved in the past, i.e. there are only minor changes in the DNA sequences encoding peptide 36 between different virus isolates from different geographic locations (see Starcich et al., Cell, 46, 637 (1986)). In contrast, gpl 10-3, -4, -5, and -6 monoclonal antibodies were identified by peptide 29, bp 6688. and about 6750 bp. are bound to HIV peptides encoded by the region. The gp10 region defined by peptide 29 was identified as having numerous nucleotide substitution between different virus isolates. Monoclonal antibodies that selectively bind gp10 polypeptides containing conserved epitopes, e.g. antibodies 110-1 and 110-2 are increasingly useful in a variety of conditions, e.g. affinity chromatography, etc. In an ELISA, peptide 110-2-2 reacts with serum from an individual from whom LAV-2 has been isolated.

Indirect immunofluorescence measurement

Indirect immunofluorescence assays using acetone-immobilized and live cells using a monoclonal antibody directed against the HIV gpl 10 antigen. Acetone-fixed plates prepared from LAV-infected CEM cells were incubated with diluted culture supernatants or ascites fluid for one hour at 37 ° C, while live cells were incubated with culture supernatant or ascites fluid for one hour at 4 ° C. and fixed with acetone. Both methods were labeled with anti-mouse IgG fluorinated with Escein isothiocyanate (Zymed

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Laboratories, Inc., South San Francisco, California) uses cells to detect reactive gpl 10 antigen. HIV-gp 110-1 monoclonal antibody produces positive results, whether using live or acetone-fixed, LAV-infected cells.

II. example

Neutralization of HIV infections with artti-gpl10 monoclonal antibodies

This example describes and characterizes HVV infectivity neutralization using monoclonal antibodies that bind to gpl 10 and gpl 10 peptides. The results indicate that gp1 has a neutralizing activity of 10-3, -4, -5, and -6, and that gp1 has a particularly high level of neutralizing activity.

Examination of neutralization

A sensitive neutralization assay was developed to quantitatively investigate the effect of monoclonal antibodies on HIV infectivity. A highly susceptible CD4 + cell line (CEM), which is highly susceptible to HIV infection, is selected as the target cell to compare infectivity. Ascites fluids prepared as described in Example I, or their purified IgG fractions using ammonium sulfate precipitation, are heat-inactivated at 56 ° C for 30 minutes and then diluted as necessary in RPMI medium containing 10% fetal bovine serum. A suspension of the LAVbru HIV strain was recovered from a CEM culture in a log phase for about four days, filtered through a 0.2 or 0.45 micron filter and frozen at -70 ° C. An aliquot is thawed, titrated to determine TCID ₅₀ / TCID ₅₀ means "tissue culture infectious dose", i.e. the concentration at which 50% of tissue culture cells are infected with the virus / and subsequent measurements are made with freshly thawed aliquots which are Dilute 1: 500 with culture medium to obtain a concentration (10 TCID ₅₀ ) of approximately 10 times the amount required to infect 50% of CEM cells in culture. The virus suspension was mixed with an equal volume (250 μΐ) of a 5-fold dilution of a monoclonal antibody composition in a ratio of 1: 5 to 1: 9765625. The virus / antibody mixture was incubated for 45 minutes at 37 ° C and a copy of 1: 5-1: 9765625 was made. The virus / antibody mixture was incubated for 45 min at 37 ° C and 200 μl replicate wells were used to inoculate wells of approximately 210 ⁵ CEM cells per ml. Cultures were incubated at 37 ° C in a humidified atmosphere containing 5% CO ₂ for 14 days. Cells were harvested, pelleted, and lysed with PBS containing 1% Triton Χ-100 for approximately 10 minutes. The amount of virus (or viral antigen) present in the lysed cells is determined by using the "sandwich" enzyme immunoassay for sensitive HIV antigen described below. The titer of neutralizing activity, if any, is defined as the reciprocal of the monoclonal antibody dilution that inhibits antigen production in more than 50% of virus-control cultures incubated in the absence of antibody, or in several cultures of virus of the same isotype. 50% of which have previously been shown to have no neutralizing activity.

The HIV antigen capture assay, referred to above, uses two monoclonal antibodies directed against p25 antigens as capture reagent. These hybridoma cell lines were constructed essentially by the methods described above, including the use of a purified recombinant fusion protein as an immunogen and characterization of the resulting monoclonal antibodies so that they are specific and reactive with GAG-1, GAG-2 and Recombinant fusion proteins called GAG-3 and synthetic peptide 141. GAG-1 is expressed by pGAG-1 (ATCC 53379), GAG-2 is expressed by pGAG-2 (ATCC 53111), and GAG-3 is expressed by pGAG-3 (ATCC 53112). The production of recombinant fusion proteins is described in detail in PCT Publication No. WO 86/06099.

Synthetic peptide 141 is encoded by the LAV _B ru genomic region, pages 198-242. corresponding to amino acids. Monoclonal antibodies produced by the p25-2 and p25-3 hybridoma cell lines were found to be reactive with recombinant fusion proteins GAG-1, GAG-2 and GAG-3, and the monoclonal antibody p25-3 was also reactive with synthetic peptide 141.

To perform the antigen capture assay, the first capture reagents are adsorbed onto solid supports. The ascites fluid from the p25-2 and p25-3 hybridoma cell lines was diluted 1: 5000 in 25 mM Tris buffer, pH 8.5, and 200 μΐ was added to the wells of the microplates. The wells were sealed and incubated at 4 ° C for about 16 hours. The solution was removed by aspiration from the wells before the blocking solution (0.3% in Blotto PBS) was added. The blocking was carried out for 15 minutes at room temperature. The blocking solution is aspirated and the sample is added. Two hundred microliters of lysed cell suspension and 5.0 μΐ of detection conjugate prepared as described below are added to each well. The hollow strips or plates are incubated for 2 hours at 37 ° C, then the suspension is aspirated and the wells are washed four times with buffer (0.05% Tween 20 in PBS). The detection conjugate is prepared as follows. Monoclonal antibodies p25-6 and p25-7 are conjugated to horseradish peroxidase (HRP) in a 3: 1 molar ratio (Ab: HRP) for three hours using a periodic oxidation procedure (Nakone et al., J. Histochem. Cytochem. 22, 1084 (1974)]. The conjugates were diluted 1: 1500 in a 20 mM sodium citrate solution containing 2.5% (w / v) non-fat milk powder, 0.01% thimerosal and 0.005% Antifoam A. The remainder of the ELISA procedure was performed as described in Example I.

The results of the assay for neutralizing activity with gpl 10-3, -4, -5, and -6 are as follows. As the highest titers that retain neutralizing activity,

The following is defined as: gpl 10-3 = 15.625; gp10-4 = 9,765,625; gp 10-5 = 125; and gp10-6 = 625.

Due to the anticipated genetic variability of the region defined by peptide 29, the ability of the gpl 10-4 monoclonal antibody to detect other isolates of HIV is examined. Immunofluorescence assays are performed as described above. The gp10-4 antigen expresses antigen in culture of at least three of the 16 tested HIV isolates.

The gpl 10-4 antibody is capable of neutralizing viruses isolated from a LAV _BRU- inoculated chimpanzee as a control animal in an AIDS vaccination experiment for 15 weeks. The monoclonal antibody can neutralize isolates even when the animal receives antibodies that neutralize HIV in vitro and develop a measurable cell-mediated immune response to HIV infection. This indicates the absence of antigen drift in vivo at the epitope recognized by the gp10-4 antibody.

III. example

Neutralization of HIV infectivity with a cocktail of anti-gpl 10 and anti-p25 monoclonal antibodies This example describes neutralization of HIV infectivity using monoclonal antibodies that bind gpl 10 and peptides in gpl 10 when used with monoclonal antibodies, which bind p25 and the peptides in p25, which alone have little or no neutralizing activity. The results indicate that gpl 10-2 monoclonal antibody, when combined with either p25-6 or p25-7, has an extremely high level of neutralizing activity.

The p25-6 and p25-7 hybridoma cell lines are constructed by the methods described above, including modifications involving the use of an inactivated, disrupted virus or purified gag recombinant fusion protein expressed in E. coli as an immunogen (see WO 86/06093), and characterizing the resulting monoclonal antibodies in a manner that is specific and reactive to recombinant fusion proteins formerly known as GAG-1, GAG-2, and GAG-3 88, 150, 147, and 148 using synthetic peptides. The synthetic peptides are encoded by the LAV _BRU genomic region and correspond to the following amino acid residues: peptide 15 = 329-350. amino acid residues; 88. peptide = 315-350. amino acid residues; Peptide 150 = 318-363. amino acid residues; 147. pcptid = 278. 319. amino acid residues; and peptide 148 = 290-319. amino acid residues. The hybridoma cell lines produced by the hybridoma cell lines p25-6 and p25-7 are reactive with the recombinant GAG-3 fusion protein, p25-6 reactive with synthetic peptides 147 and 148, and p25-7 reactive with the 15, 88, and 150. with synthetic peptides.

Neutralization measurements are performed as described above, except that when using cocktails, each monoclonal antibody is first diluted 1: 5 in culture medium and then mixed in equal proportions to give a final dilution of 1:10. The remainder of the measurement is carried out as described above.

Monoclonal antibodies gp10-2, p25-6 and p25-7 show less than 50% neutralizing activity when used alone. When used in a cocktail containing gp10-2 with p25-6 or gp10-2 with p25-7 at 1:10 dilution, neutralization is complete. The cocktail containing p25-6 monoclonal antibody in combination with p25-7 exhibits 60-90% neutralizing activity.

ARC. example

Immunopotential of peptide 29 and homologues thereof

The ability of peptide 29 and its homologous peptide, including peptide 177, to stimulate an immune response against HIV was first tested in two mouse strains. Methods for producing peptide immunogens, immunization procedures, and methods for characterizing the resulting immune response are detailed below.

Peptide 29 is prepared by conjugation to purified thyroglobulin for immunization. Thyroglobulin can be derivatized with N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) for conjugation, outlined in U.S. Patent No. 4,629,783 (column 10, lines 28-51). procedure. As a second immunogen, thyroglobulin is introduced into the derivative with glutaraldehyde as follows. Porcine thyroglobulin (27 mg) was dissolved in 1 ml of 0.1 M sodium bicarbonate followed by dropwise addition of 8.3 µl of 25% glutaraldehyde solution and the mixture was stirred overnight at room temperature. 1 ml of sodium carbonate / bicarbonate buffer (pH 9.3) was added to the solution and dialyzed against 2 liters of the same buffer at 4 ° C for 8 hours with complete replacement of the dialysate.

At 4 o'clock. Peptide 29 is then added to the derivatized thyroglobulin in an approximately 100-fold molar excess and the mixture is stirred overnight at room temperature. Unreacted glutaraldehyde is blocked with 200 µl of a 0.2 M lysine solution, which is stirred for several hours (or overnight) at room temperature. The peptide-thyroglobulin conjugate is then vigorously dialyzed against PBS at 4 ° C.

Two mouse strains (C37 black and BALB / c) were inoculated with peptides prepared by each method of conjugation. Each animal is about 2 to 4 weeks old at the time of inoculation. The route of inoculation may be paw, tail incision, subcutaneous, intranasal, or intraperitoneal. The inoculum consisted of 25 mg of conjugated peptide suspended in 0.5 ml of complete Freund's adjuvant, with booster doses given in sections 2, 3 and

At the end of week 5, it is repeated with the same immunogen suspended in incomplete Freund's adjuvant. Serum samples were collected from each mouse prior to immunization, 4 days after Week 3 booster vaccination and 4 days after the last booster week 5. Serum samples are analyzed for antibodies against homologous peptides or whole virus

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By ELISA. Serums showing antibody activity to LAV are subjected to further screening for neutralizing activity, followed by analysis of sera by immunostaining for disrupted LAV antigens and radioimmunoprecipitation assays with radiolabeled gpl10.

Immunization results indicate that mice immunized with peptide 29 develop antibodies reactive with peptide 29 and LAV-1 according to ELISA. Conjugation of peptide 29 with glutaraldehyde generally results in higher titers of anti-peptide 29 antibodies in BALB / c mice, although conjugation with maleimide also successfully elutes anti-29. peptide antibodies in some BALB / c mice. Mice immunized with peptide 177 develop antibodies to the peptide, and peptide 177 conjugation with glutaraldehyde is better at eliciting an immune response. C57 mice are more responsive to peptide 177 stimulation than BALB / c mice. Mice immunized with the 110-2-2 LAV-2 peptide develop antibodies to 110-2-2 and the LAV-2 virus as detected by ELISA. Glutaraldehyde-conjugated peptide 110-2-2 is immunogenic in both C57 and BALB / c mice, although the titers of peptide 110-2-2 are generally higher in BALB / c mice than in C57 mice, whereas LAV-2 given sites are generally higher in C57 mice than in BALB / c mice.

Generally, serum samples from immunized mice show (i) antibodies to the whole virus; (ii) they are capable of neutralizing HIV eg. II. in the assay described in Example 1; and (iii) reactive with peptide 29 by ELISA, collectively indicating the efficacy of using peptide 29 in vaccine preparation.

Example K Immunoaffinity isolation of gp10 using a monoclonal antibody

Monoclonal antibodies against the HIV-10 gp1 antigen can be used in immunoaffinity separation techniques to significantly purify bacterially expressed recombinant fusion proteins. If the expressed protein is selected by bacteria, the proteins can be isolated from the culture supernatant; if the protein is not secreted, bacterial cells may need to be disrupted.

The construction of plasmid pENV-5 (ATCC 53074) is described in the present patent application filed by the present applicants under the number 721,237, which is incorporated herein by reference. PENV-5 encodes most of the 10 carboxyl terminus of gp1 and a portion of the amino terminus of gp41 of LAV, inserted into the trp expression vector. E. coli C600 transformed with this vector expresses but does not select for the gp10 fusion protein.

E. coli C600 containing plasmid pENV-5 was grown in medium containing tryptophan (20 pg / ml) and ampicillin (100 pg / ml) overnight at 37 ° C with aeration. Overnight cultures are then inoculated 1: 100 into fresh minimal medium containing ampicillin (100 pg / ml) but not tryptophan. These cultures are grown using aeration for 2-3 hours (up to early hanging phase) at 37 ° C. The inductor, 3-β-indole acrylic acid (Sigma Chemical Co., St. Louis, Missouri) was added to a final concentration of 20 pg / ml in a fresh stock solution of 20 mg / ml in 95% ethanol. The induced cultures were then grown at 37 ° C for 4-5 hours with aeration, and the cultures were pelleted and, if desired, frozen. The protein yield from pENV-5 is typically less than 1 mg / L.

The sedimented bacterial cells were lysed using P-RIPA buffer (PBS containing 1% Triton Χ-100, 1% deoxycholate, 0.1% sodium dodecyl sulfate, and 1% aprotinin), which lysed E. coli cells. The suspension may be subjected to sonication to fragment the DNA and RNA, followed by centrifugation to remove the solid particles. A dilution or concentration step may also be required to make the protein concentration standard.

Monoclonal antibody HTV-gp 110-1 is first precipitated from ascites fluid or cell culture at room temperature with (NH ₄ ) ₂ SO ₄ or pH 7.3 buffered Na ₂ SO ₄ solution to give 33% and 18% final saturation, respectively. let's live. The precipitated proteins are recovered by centrifugation and redissolved in PBS and precipitated a second time with 33% (NH ₄ ) ₂ SO ₄ or 12-15% Na ₂ SO ₄ . This step is repeated if necessary. The pellet was redissolved in PBS and excess salts were removed by gel filtration using a desalting matrix or by exhaustive dialysis against PBS.

Purified monoclonal antibody 110-1 can then be bound to Sepharose activated with cyanogen bromide. The required amount of gel was swelled in 10 ~ ³ M HCl solution on a glass filter (1 g of freeze-dried material yields a final volume of about 3.5 ml) and washed with the same solution for 15 minutes and the antibody was added immediately. The coupling reaction is generally most effective in the pH range of 8-10, but lower pH may be employed if required for antibody stability. The antibody should be dissolved in PBS or in high ionic strength carbonate / bicarbonate or borate buffer with 150 mM NaCl. The activated Sepharose and antibody suspension are gently stirred for 2 to 4 hours at room temperature or overnight at 4 ° C and then washed with a layer of sintered glass filter with a switching buffer. All remaining active groups are blocked by treatment with 1.0 M ethanolamine at pH 8 for 2 hours. The final antibody Sepharose product was then washed alternately with high and low pH buffer solutions (0.1 M borate buffer, pH 8.5; 1 M NaCl and 0.1 M acetate buffer, pH 4.0; and 1 M NaCl) four or five times.

This wash removes traces of non-covalently adsorbed material. The finished immunoaffinity separation matrix is stored below 8 ° C with a suitable bacteriostatic agent, e.g. 0.01% in the presence of azide.

Addition of the Expressed Protein Suspension to the Immunoaffinity Separator Matrix

EN 214 439 Β. The mixture is allowed to interact for 2-24 hours, preferably 12-18 hours, with slow stirring or shaking. It is also possible to use a column shape in which the immunoaffinity matrix is filled into a column, equilibrated, and a suspension of the expressed protein is slowly fed to the column. After the protein suspension has been added, the flow must be stopped to allow maximum immune complex formation.

Unbound material is washed or separated by vigorous washing with adsorption buffer. Laminated sintered glass filters can be applied under vacuum or through a column. The bound material is then eluted using low or high pH buffers (acetate buffer, pH 4.0 or borate buffer, pH 8.56) or chaotropic agents.

VI. example

Immunoaffinity purification of recombinant gpl10 from mammalian expression systems The monoclonal antibodies of the present invention can be used to immunoaffinity purify recombinant fusion gpl10 in mammalian cells. Mammalian cells are infected with recombinant Vaccinia virus [Mackett et al., J. Virol. 49,857 (1984); this is incorporated by reference in the present application, which contains a sequence encoding at least a portion of gp1010, wherein the sequence is antigenic and induces neutralizing antibodies.

The recombinant Vaccinia is prepared according to the method described in British Patent No. 2,181,435, the disclosure of which is incorporated herein by reference. Briefly, sequences encoding the HIV envelope glycoprotein are inserted into a plasmid vector (pGS20), downstream of a Vaccinia transcription control element. This chimeric portion is flanked by sequences encoding the viral thymidine kinase (TK) gene.

Chimeric plasmid vectors containing the Vaccinia virus promoter ligated to the LAV envelope gene were used to transform E. coli strain MC1000 (a derivative of E. coli MC1009 / ATCC 33760). Insertion of the chimeric LAVenv sequences into the Vaccinia virus genome is accomplished by in vivo recombination; this is made possible by the fact that the chimeric genes present in pv-env5 plasmids are flanked by Vaccinia sequences encoding the TK gene. This plasmid is then introduced into cells infected with wild-type Vaccinia virus and allowed to recombine between the TK sequences in the plasmid and the homologous sequences in the Vaccinia virus genome, thereby inserting the chimeric gene. African green monkey kidney cells (BSC-40 strain, a cell line derived from BSC-1 cells (ATCC CCL26)) are used as host in the expression system.

Pooled BSC-40 cells were infected 10 times with recombinant Vaccinia virus. The infection was allowed to proceed for 12 hours, after which the cells were harvested, washed once with PBS and collected by centrifugation. The cell pellet was resuspended in lysis buffer (1.0% NP 40, 2.5% sodium deoxycholate, 0.1 M NaCl,

0.01 M Tris-HCl, pH 7.4, 1 mM EDTA) and the lysate was clarified by centrifugation. Immunoaffinity separation of the expressed recombinant fusion protein is performed as described above for bacterial expression systems using the gp 110-1 monoclonal antibody. The protein produced by this expression system is more reminiscent of naturally produced HIV gp10 due to processing and glycosylation by mammalian cells.

VII. example

Inhibition of HIV infection by blocking peptides

The efficacy of the blocking peptides was investigated to infect tissue culture cells with the HAV LAV _BRU strain using a modified version of the T-peptide evaluation procedure (Pert et al., Supra). Preferred HIV inhibition assays involve combining an equal volume of blocking peptide and CEM cell (2,510 ⁵ ) in culture medium (RPMI, 10% fetal calf serum (FCS) and 2 mg / ml polybrene) and incubating for 45 minutes at 37 ° C. The virus is then added at various doses (10, 50, 500 TCID 50) and the supernatant assayed for viral antigen production (e.g., p25 internal).

Preincubation of CEM cells with peptides before adding the virus to the culture increases the inhibitory effect in the assays. Inhibition is found to be dependent on the dose of the virus, showing strong activity at low and medium doses, while having less effect at the highest doses of virus.

About 60% to 90% inhibition of viral antigen production is achieved in low viral dose T-peptide experiments. Further experiments X.-XV. peptides, which are typically amidated at the -COOH end and acetylated at the -NH ₂ end, show similar results. peptide is particularly effective over a wide dosage range. Neutralizing antibodies may be added either during the preincubation period or when the virus is added to induce additive or synergistic inhibition of viral antigen production.

It will be appreciated from the foregoing that the monoclonal antibodies and peptides of the present invention, including blocking peptides, provide an improved method for neutralizing and / or inhibiting HIV infection. This allows us to easily develop preventive and therapeutic compositions that are effective against infections with most, if not all, HIV strains. In addition, the new materials are used in diagnostic tests and other well-known methods.

While several details of the present invention have been described by way of illustration and examples, in order to clarify the understanding, it is understood that certain changes and modifications may be made in practice, but are within the scope of the appended claims.

Depositing Microorganisms The following microorganisms, which are part of the present invention, are deposited with the American Type Culture Collection (12301 Parklawn Drive,

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description number number date of issue Mouse Hybrid ma (BALBc / NS-1) HIV-gp 110-1 HB 9175 1986th August 15 HIV-gp 110-2 HB 9176 1986th August 15 _Π_ HIV-gp 110-3 HB 9177 1986th August 15 HIV-gp 110-6 HB 9404 1987th April 30 _ II __ HIV-gp 110-4 HB 9405 1987th April 30 HIV-gp 110-5 HB 9406 1987th April 30 HIV-p25-2 HB 9407 1987th April 30 HIV-p25-3 HB 9408 1987th April 30 HIV-p25-6 HB 9409 1987th April 30 _ ll_ HIV-p25-7 HB 9410 1987th April 30 Rockville, Maryland 20852, United States ATCC HB 9177, ATCC HB 9405, ATCC HB 9406,

Mok). The deposit details are as follows:

HB 9175, HB 9176 and HB 9177 hybridomas were found in 1986.

on August 26, we examined it and found it alive. Further hybridomas were examined on May 4, 1987 and found alive.

Claims (15)

PATENT CLAIMS 1. Procedure for HIV infection, LAV about 301-336. HIV gpl corresponding to its amino acid residues with a neutralizing epitope within a 10 region to produce a specifically reactive monoclonal antibody, culturing a cell line expressing said monoclonal antibody, and recovering the monoclonal antibody produced by the cell line. (Priority: August 20, 1986) The method of claim 1 for producing a monoclonal antibody reactive with the following peptide: Cys-Thr-Arg-Pro-Asn-Asn-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly Characterized in that a cell line expressing the appropriate monoclonal antibody is cultivated and the appropriate monoclonal antibody is cultured and monoclonal antibody. (Priority: August 20, 1986) 3. The method of claim 1, wherein the cell lines are cultured as follows: or ATCC HB 9404 (Priority 01/05/1987) 30 4. A method for producing monoclonal antibodies responsive to HIV-gag p25 protein, comprising culturing the HIV-p25-6 (ATCC HB 9409) or HIV-p25-7 (ATCC HB 9410) cell line and recovering the monoclonal antibody produced in the culture. (Priority 35: May 1, 1987) 5. Procedure for LAV 301-336. for the production of cell lines producing monoclonal antibodies reactive with HIV gpl 10 antigen determinants corresponding to - administering to an mammalian host an immunogenic amount of an antigenic composition comprising HIV gp10 proteins or fragments thereof; recovering antibody-producing cells from the host organism and immortalizing those cells; 45 - selecting the immortalized cells that are LAV 301-336. are capable of producing antibodies against HIV gpl10 antigenic determinants corresponding to its amino acids, and - cell lines by cloning immortalized cells We make 50. (Priority: August 20, 1986) 6. The method of claim 5, wherein the antigenic composition comprises delivery of proteins derived from an extract or lysate of HIV, or recombinant fusion proteins expressed by a eukaryotic or bak55 terial host. (Priority: 08/20/1986) 7. The method of claim 5, comprising HIV-gp-110-3, (ATCC HB 9177), HIV-gp-110-4 (ATCC HB 9405), 60 HIV-gp-110-5 (ATCC HB 9406) or HIV-gp-110-6 EN 214,439 Β (ATCC HB 9404) to produce antibody-producing cell lines having the same immunological properties, characterized in that the appropriate cells are cloned and transformed into a cell line. (Priority: 01.05.1987) 8. A method of producing an immunoreactive peptide comprising less than fifty amino acids defining the HIV antigen determinant comprising a HIV neutralizing epitope comprising at least six consecutive amino acids of the HIV gp10 301-336. characterized in that the amino acids which make up the peptide are chemically linked in a manner known per se, optionally using protecting groups and, if desired, removing the protecting groups. (Priority: August 20, 1986) 9. The method of claim 8, wherein at least six consecutive amino acids are selected from one of the following sequences: 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, or 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 GXn-Ala-His-Cys. (Priority: August 20, 1986) The method of claim 8, for preparing a synthetic peptide comprising one of the following HIV gp 110 amino acid sequences: Y-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Y ' Y-Thr-Arg-Lys-Ser-Ile-Tyr-Ile-Gly-Pro-Gly-Ala-Phe-Arg ~ Hi5-Thr-Thr-Gly-Arg-Ile-Gly-Y; obsession Y-Lys-Thr-Val-Lys-Ile-Nur-Leu-Wor-Ser-Gly-His-Val-Phs-His-Ssr-His-Tyr-Gln-Pro-Y ', wherein Y and Y' are independently represents the sequences of 0-20 consecutive amino acid residues of the HIV sequence, characterized by linking the corresponding amino acids. (Priority: 1986. 5 08.20.) The process for preparing a synthetic peptide according to claim 10, wherein the Y and / or Y 'moieties comprise a glycine, tyrosine, cysteine, lysine or glutamic acid linker, wherein the corresponding amino10 acids are coupled. (Priority: August 20, 1986) 12. A process for the preparation of a pharmaceutical composition for the treatment of HIV infections, characterized in that - a LAV 301-336. a monoclonal neutralizing antibody with a reactive epitope corresponding to its amino acid residues - mixing a pharmaceutically effective amount of the monoclonal antibody produced with a pharmaceutically acceptable carrier. (Priority: August 20, 1986) 13. A method of pre-treating a healing composition for treating HIV infections comprising: - producing two monoclonal antibodies, one of which is LAV 278-319. p25 neutralization epitope, or LAV 315-363. p25 neutralizing epitope, the other being LAV 25 301-336. HIV gp10 with its neutralizing epitope specific for its amino acids, mixing said monoclonal antibodies, and and a pharmaceutically effective amount of the mixed monoclonal antibodies in admixture with a pharmaceutically acceptable carrier. (Priority: 01.05.1987) 14. A process for the preparation of a vaccine against HIV infection, characterized in that one or more of the vaccines according to any one of claims 8-10. A peptide according to any one of claims 1 to 6, which is immunologically active 35 volumes are admixed with a physiologically acceptable carrier. (Priority: August 20, 1986) 15. The method of claim 14, wherein an effective amount of multiple peptides is mixed with a physiologically acceptable carrier. (Priority: August 20, 1986)