PL161520B1 - Method of obtaining a novel peptide - Google Patents

Abstract

1. A method of obtaining a new peptide consisting of at least 5 adjacent amino acids of the amino-acid sequence HIV gp110 or homologues of this sequence of the formula II (29a):Cys-Thr-Arg-Pro-Asn-Asn-Asn-Thr-Arg-Lys-Ser-Ile-Arg-IleGln-Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile-Gly-Lys-IleGly-Asn-Met-Arg-Gln-Ala-His-Cys,characterised in that an expression is caused of the sequence of nucleic acids of the HIV virus coding the amino acids from 301 to 336 of the isolate LAVBRU gp 110 of homologues of this sequence.3. A method of obtaining of a new peptide having one of below given amino acid sequences HIV gp 110 or a homologue of these sequences:I (29)Y-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Y',V (177)Y-Thr-Arg-Lys-Ser-Ile-Tyr-Ile-Gly-Pro-Gly-Arg-Ala-Phe-His-Thr-Thr-Gly-Arg-Ile-Gly-Y', orVIII (110-2-2)Y-Lys-Thr-Val-Lys-Ile-Nor-Leu-Nor-Ser-Gly-His-Val-Phe-His-Ser-His-Tyr-Gln-Pro-Y'in which Y and Y', if present, then independently indicate amino acid sequences of length up to 20 amino acids, characterised in that an expression is caused of env regions of nucleic acids sequence of the virus HIV coding respectively amino acids 308 to 328 of the isolate LAVBRU, amino acids 306 to 323 of the isolate ARV-2 and amino acids 311 to 330 of the isolate LAV-2.

Description

The subject of the invention is a method of producing a new peptide useful for the detection, neutralization and for the purpose of protective vaccination against HIV (virus that causes immune defect in humans).

The causative agent of Acquired Immunodeficiency Syndrome (AIDS) and its precursor infections, AIDS-related syndrome (ARC) and lymphocytic leukemia (LAS) infections is a novel retrovirus growing in lymphocytes. Various names have been adopted to denote this virus, namely LAV, HTLV-III, ARV and more recently HIV.

Faced with the pandemic spread of HIV, treating infected patients and preventing the disease from spreading to uninfected individuals at risk of this virus is a fundamental task. The various treatment strategies proposed so far are based on an attack targeting different development stages of this virus. They are described by Mitsuya and Broder in Nature, 325,773 (1987). One such strategy is the use of antibodies that bind to the virus and inhibit its replication either by transforming the virus into the host cell or by some other mechanism. With the identification of an antibody sensitive viral component (or component), the hope was raised that by vaccination or passive immunization by administering immunoglobulins or monoclonal antibodies with the desired specificity

To neutralize the contagiousness of the virus, it is possible to obtain sufficient antibody titers in the body to neutralize it.

The coat glycoproteins of most retroviruses are believed to react with receptor molecules on the surface of sensitive cells, which determines viral contagiousness towards certain hosts. Antibodies that bind to glycoproteins can block the action of the virus and the receptor cells, neutralizing its infectivity. See (The Molecular Biology of Tumor Viruses, 534, ed. J. Tooze, 1973) and RNA Tumor Viruses, 226, 236 (R. Wiss et al., 1982). See also: Gonzalez-Scarano et al., Virology 120, 42 (1982), La Crosse virus; Matsuno and Inouye, Infect. Immun., 39, 155 (1983) - Neonatal Calf Diarrhea Virus, and Mathews et al., J. Imminol., 129, 2763 (1982) - Encephalitis virus.

The general structure of HIV has a ribonucleoprotein core surrounded by a lipid-containing coat which the virus acquires from the membrane of the infected host cell during its development. The glycoproteins encoded by the virus are embedded in the coat and protrude outside. HIV coat glycoproteins are initially synthesized in an infected cell in the form of a precursor molecule of 15,000 (0-160,000 daltons (gpl 50 or gpl 60) and then processed in the cell into an N-terminal fragment of 110,000-120000 daltons (gpl 10 or gpl20) with the simultaneous generation of an outer glycoprotein and a C-terminal fragment of 41,000-46,000 daltons (gp41) representing the transmembrane mantle glycoprotein.

For the reasons discussed above, HIV gpl 10 glycoproteins have been the subject of much research, serving as a potential target by which the virus development cycle can be broken. The raw materials of HIV infected patients neutralized HIV in vitro. Antibodies binding to purified gpl10 were present in these sera (Robert Guroff et al., Nature, 316, 72, 1985; Weiss et al., Nature 316, 69 (1985) and Mathews et al., Proc. Natl. Aced. Sci. USA, 83, 9709 (1986) Purified and recombinant gpl10 used to immunize animals stimulated the production of neutralizing antibodies in serum [Robey et al., Proc. Natl. Acad, Sci. USA, 83, 7023 (1986) ; Lasky et al., Science, 233, 209 (1986). Zagury et al., Nature, 326, 249 (1986)] reported the same immunization experience in humans. Binding of gpl10 to the CD4 (T4) receptor has also been demonstrated. Furthermore, monoclonal antibodies that recognize certain CD4 receptor epitopes have been shown to block HIV binding, syncytia formation and infectivity [(McDougal et al., Science, 231,382 (1986)]. Putney et al., [Science 234,1392]. (1986)] demonstrated the presence of neutralizing antibodies in serum in animals after immunization with recombinant fur protein containing C the terminal half of the gpl10 molecule and then proved that the glycosylation of the coat protein is necessary for obtaining an immune response in the form of neutralizing antibodies.

Thus, a subunit vaccine for AIDS in which an HIV gpl molecule or part thereof is used may prove useful. Vaccines containing antigenic subunits are an alternative to vaccines prepared from inactivated or non-virulent (attenuated) viruses. In the case of inactivated vaccines, it should be borne in mind that not all viral particles were killed during the preparation of the vaccine, while attenuated viruses may be able to mutate and recover disease-causing activity. In the case of subunit vaccines, only those parts of the virus which contain antigens or epitopes capable of eliciting an immune response are used to immunize the host, i.e. antibody neutralization, ADCC and lymphocyte T cell cytotoxic responses. The main advantage of subunit vaccines is that they exclude irrelevant viral material.

The viral subunits for vaccine preparation can be generated by several methods. For example, one may express a coat glycoprotein in a bacterial host and then purify the glycoprotein, although post-translational modification (such as glycosylation) or other such processes are unlikely to occur in that host. Such modifications can be achieved using a eukaryotic expression system such as yeast or mammalian cell culture. Viral genes are introduced into cells

161,520 mammals using vaccinia virus as vectors (Mackett M. et al., Proc. Nat. Acad. Sci. USA, 79, 7415, 1982; Panicali D. and Paoletti E., Proc. Nat. Acad. Sci. USA, 79, 4927, 1982).

Recombinant vaccinia virus can be obtained by the method of Hu et al., Nature, 320, 537 (1986) or Chakrabarti et al., Nature, 320, 535 (1986). Both of these methods are given in this specification. In such systems, the viral glycoproteins produced by cells infected with recombinant vaccinia virus are suitably glycosylated and can be transported to the cell surface, removed outside the cell and isolated.

An important step in the production of subunit vaccines is the appropriate purification of the desired glycoprotein from the complex mixture of the expression system. For purification purposes, a number of methods can be used, such as preparative polyacrylamide gel electrophoresis, gel permeation chromatography, as well as various other chromatographic methods (e.g., ion exchange, reverse phase, immunological affinity, hydrophobic interaction) and others. To obtain truly pure formulations, most of these methods are used in various combinations (Kleid DG et al., Science, 214, 1125 (1981), Cabradilla CD, et al., Biotechnology, 4, 128 (1986), Dowbenko DJ, Proc Nat. Acad. Sci. USA. 82, 7748 (1985)) reported herein. For the production of subunit vaccines, methods are needed to limit the number of steps required to maximize purification of a particular viral antigen isolated from a complex expression mixture. The efficiency of separating antigens from foreign components is achieved by using affinity chromatography. This technique, also known as immunoadsorption, basically relies on the selective adsorption of an antigen onto a solid support previously covalently bound to a specific antibody. The antigen, selectively adsorbed on the antibody-a <d, orbent system, is then eluted by changing, for example, the pH and / or ionic strength of the buffer.

Often used as immunoadsorbents are polyclonal antibodies obtained from animals immunized with the desired antigen or from naturally infected individuals (e.g., Lasky et al., Supra). However, such reagents generally suffer from major disadvantages, namely, (i) not all antibodies bound to the insoluble carrier are specific to the molecule, requiring additional purification, (ii) the yields of the desired antigen are generally low, and (iii) the affinity of an antibody often differs from preparation to preparation, necessitating modification of the elution process. The use of monoclonal antibodies specific for the desired viral antigen to be used in the subunit vaccine formulation in place of polyclonal antibodies overcomes these disadvantages.

Murine monoclonal antibodies binding to HIV antigens have been described in the literature. There have been reports of monoclonal antibodies specific for the p25 core protein [e.g., di Marżo Veronese et al., Proc. Nat. Acad. Sci. USA, 82, 5199 (1985) and Chassagne J., et al., J. Immunol. 136, 1442 (1986)]. Monoclonal antibodies specific for the membrane glycoprotein gp41 have also been described [di Marżo Veronese et al., Acience, 229, 1402 (1985)].

It remains to obtain epitope-specific monoclonal antibodies within the well-known regions of the larger gpl 10 coat glycoprotein. Monoclonal antibodies that bind to these regions and reduce or completely eliminate HIV replication and transmission could have significant therapeutic and prophylactic applications. Such monoclonal antibodies could also be used to purify the desired gpl10 region into cleaved parts of the virus or to isolate it from recombinant expression systems, for example as vaccines.

Furthermore, the region containing the epitope or epitopes recognized by monoclonal antibodies could be chemically synthesized, thereby avoiding the difficulties associated with purifying and administering larger fragments of the gpl10 molecule. The present invention fulfills these and other objectives.

The subject of the invention is a method of obtaining peptides capable of immunologically mimicking (mimicking) neutralizing epitopes of HIV proteins and nucleic acid probes containing the code of these peptides, as well as other peptides reducing the infectivity of HIV. The obtained new materials are used, for example, in diagnostics, for the detection of HIV infections and in therapy, for the treatment of these infections or for the purpose of vaccination against them.

161 520

The obtained peptides allow for the preparation of new compositions for the neutralization of HIV infection, that is, to prevent or significantly inhibit the emergence or cellular spread of HIV infections in the host. More particularly, peptides mimicking the HVV neutralizing region and monoclonal antibodies reactive with this region are used for diagnosis, treatment and vaccination against HIV infection. In this aspect, the term "neutralizing region" means those parts of the HIV virus, especially HIV proteins containing amino acid segments defining one or more antibody-reactive epitopes that, either singly or in combination with other antibodies, are capable of neutralizing HIV infection. Appropriate neutralization tests are known. These include: HIV reduction tests on T cell lines, reduction of plaque-forming units in VSV pseudotypes (HIV) containing HIV coat glycoproteins, syncytia inhibition tests, and virion receptor binding tests. If necessary, the neutralizing activity is compared with the reactivity of the antibodies in immunochemical tests such as immunofluorescence, colored immune reaction (immunoblot) and radioimmunoprecipitation.

The novel peptides of the invention of less than 50 amino acids contain five or more contiguous amino acids forming epitopes substantially similar to epitopes located in the HIV gpl10 or p25 neutralizing regions encoded by the env and gag regions of the HIV genome, respectively. Regions of particular interest are those extending from amino acid residues 301 to 336 in gpl10 and in p25 peptide, residues 278 to 319 and from residue 315 to residue 363. All these regions are derived from the HIV strain designated LA Vbru. The designation of the amino acid residues is derived from Data Bank of Los Alamos (AIDS virus sequence database, Los Alamos National Laboratory, Theoretical Division, Los Alamos, NM 87545).

It is clear that additional analogous regions ("homologues) from other HIV isolates can be identified based on their localization in related proteins from different isolates. In practice, these homologues can be identified by comparison with the LAVbru sequence data as follows:

a / the amino acid sequences of the LAVbru isolates can be aligned so as to obtain the maximum homology of these two sequences, b / the peptides containing the amino acid sequences of the HIV isolates corresponding to the location of the LAVbru peptides can be identified, immunologically mimicking the LAVbru proteins. appropriate proteins from HIV isolates.

This method can be applied to strains of HIV that have not yet been detected. For example, when a new HIV strain is detected, its coat and core amino acid sequences are compared to LAVbru so as to obtain maximum homology.

Methods for serial comparison of sequences are known. In carrying out this work, the greatest possible homology should be maintained between the cysteine molecules. An amino acid sequence of a new HIV strain or species that corresponds to the location of the peptides disclosed herein may be synthesized and used in accordance with the invention.

Scharf et al. (Science, 233, 1076 (1986)) describe another method for determining the sequence of the homologous region in other HIV strains. This method uses two oligonucleotide primers, each having different restriction sites in each of them, binding to conserved sequences outside the region of interest.

DNA from HIV strains can then be amplified in vitro and the resulting oligonucleotides cloned into vectors for sequence analysis and inserted into a vaccine as a cassette representing a specific epitope from that HIV strain.

The present invention does not require epitopes contained in such sequences to cross-react with antibodies against all HIV strains or species. Peptides incorporating immune epitopes that distinguish between a particular species or serogroup will find use in identifying those particular species or serogroups and may be helpful in detecting individuals infected with one or more HIV species or serogroups. In combination with other peptides derived from the homologous region or another neutralizing region, they may also be useful in the preparation of therapeutic compositions.

161 520

ABOUT about What CA WHAT M. ABOUT CM CD Cj -"and What re about cm WHAT V0 CC) What ABOUT i — t about about about ABOUT about ri <H. i — I ABOUT CM about about CM about CM C \ l CM CM WHAT this this this rO t <O (ABOUT 1 * 0 t <O this r-r, this "this\ ro ί'Ό ro ;about this ►O this this

o cj F A <M A EH hO o Ci

O <4 sts

EH A <and A >>

A ¢ 2) P <i

Pi

AND

El a

Pi 03 in Pi Pi about > ł >> ABOUT ABOUT :about AND AND What ABOUT

ω 1 | 1 03 1 1 • 2 Cj -p and i <- 1 1 | >> 1 1 • 23 Ei 03 1 1 AND 1 1 1 AND | | 1 • AND <4 -A ^- 1 1 03 about Pł Ph Pi ABOUT Pi customs duty • -p Eh 1 1 AND Pi > » 03 AND P. C. rH • G3 <4 00 1 1 3 AND Eh AND Eh AND AND • £ AND 1 i 1 Pi and WITH 1 PI β | • 1 -4 > 5 and 1 1 /AND ""AND and about and AND AND 1 • 1 CJ AND 1 1 1 EH 1 AND 1 EH 1 • 1 Cj. CI 1 1

AND

Table I

I '

C o P A ω Pi <>

Pi A EH 03> »

ABOUT

I and

I I I I

I I I I I I I I

And Cj

J $

I EH ί 3 ł 3

I Eh I EH ά 8

A&H? 3 i <5 73 * i e>! o and o o o <a>> O

A Q

I <t I Cs J Cj

From > OHM ABOUT this OI CJ 3 Έ- A C 3 ES AND B S. about 3 AND AND AND § Ai Si SO K \ IN 3 3 in ►A m s « 5 3 CS3 this 3 about

A >>

£ ·

A oU c

Pi A EH> c A

S.

3 r "H

O I δ%

r—

About • · I ti) I $ 11!

this

M ca pa ³ 3 A.

161 520

cn rc t- ABOUT t— σ> ABOUT lh KO KO MH U0 «Η M. CM rc CM rc CM KO CM «Η rc tM CM HO AND KO CM (this fK \ KO CM this M this KO KO CM KO CM KM KM tM this

Fri.

SO • ai

AND

AND

I t>

ω

P.

Φ

Cl hO a

P.

Φ

Cl t>

φ tlO • Η

Ο rO

Ν ω

rt

Ό rt ο

• Η •

P.

Λ

Eh

AND

AND

J u

Eh β

Φ

AND

AND

AND

And β

r — ł

AND

And bO

P.

AND

AND

AND

P.

AND

Eh

Eh β

Φ

A β

φ by β

And this·

AND

AND

And ω

ρ ι

t β

Α

ΕΗ

AND

And η

τΗ

Κ

AND

AND

AND

AND

AND

AND

AND

AND

AND

AND

And β

Φ

Α >>

• this

A Α rt «ί Η S

Ι-Η

Α

Α

AND

AND

AND

AND

AND

AND

CM

Φ AND Φ • Φ β CM i 1 1 1 1 I i i i i t i III III AND Ml rH • and- yeah >> 1 l 1 1 1 + P AND «: AND • AND < H. 1 1 1 1 1 Φ > » >> Φ • H. 1 1 1 1 1 ε ^φ AND r — I AND • CM hH 1 ω 1 1 1 1 1 AND rb cb H. • «< this ωτ 1 1 1 1 1 AND Φ Φ What • 1 β φ 1 1 1 1 1 1 rH rH > 5 • 1 Φ rH Ο • 1 1 1 1 1 AND I-H H. • 1 H. AND • 1 1 1 1 AND 1 1 β • 1 J. AND ABOUT >> • 1 1 1 1 AND 1 AND Λ • AND P. tr this 1 > 5 1 1 hO 1 EH • 1 AND Ed m t » 1 rH 1 1 Fi • c bM β AND cH AND 1 £ 1 1 • rH β r— 1 H. rt 1 • ^£ 1 1 ABOUT • ? -and! this this this x> | • AND 1 1 P. β AND Ύ β Ύ H. AND • Eh 1 1 hH rH AND AND AND rH AND > 0 Φ Φ 1 Φ bD & AND β AND ω C. & AND P. Eh What AND rH l-H M. rH l-H 1 1 rH IN P. < β AND ρ > 1 P. H. Ή · < 1 * 3 1 7§ H. AND fs rt about 1 rH AND Hf hH AND Φ CM 1 -at AND <4 Cl CM because β £ β AND fc fc P. > 0 What H. AND rt HW kO KO ILO Jh 8 > <ί AND Eh Φ AND EH 1 1 EH AND AND EH E- ΓΊ. CC 1 1 Hi Pi N N and about AND 1 ^AT Φ & 1 AND EH AND H. EH AND 1 73 β 73 P. 73 73 and Φ H. 1 t> AND > EH > > j 1 ro ra 1 1 What rH AND ^ H. rH rH MO about K0 MO 1 1 AND >> 1 1 AND KH η Kr KO KO KO rc rc rc CM 1 1 c; H. 1 1 AND KO KO KO M. o. o KO KO tc rc ABOUT 1 1 β 1 1 1 1 1 1 1 1 1 1 1 1 this 1 1 1 1 1 1 1 1 • 1 • 1 1 EH yeah 1 1 1 1 1 1 1 1 • 1 • 1 1 ABOUT 1 1 1 4 1 AND and 1 1 1 1 1 • 1 • 1 1 H. ro 1 1 1 1 1 1 1 1 1 1 1 1 AND 1 H. yeah 1 1 1 1 1 £ 1 1 1 1 1 1 1 1 Ł 1 1 1 1 1 rO) 1 1 1 1 1 1 and 1 1 1 1 1 • 1 1 M. 1 1 1 1 1 1 1 1 1 1 AND AND 1 AND 1 1 1 1 1 1 Ł 1 1 • 1 M. AND 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 • AND M. «0 1 1 1 1 1 AND 1 1 1 1 1 1 f 0 1 c 1 1 1 1 1 ω 1 • » 1 1 1 1 1 • 1 < H. 1 1 1 1 1 P. AND • 1 1 1 1 1 1 • 1 about M. 1 1 1 1 AND V • 1 1 | • 1 • • 1 1 1 1 1 1 > 0 (0 1 1 1 1 • vol • • 1 1 1 1 1 AND hH >> 1 1 1 1 ¹ and • J. « • 1 1 1 1 ! AND AND 1 • • β Φ p • β < bD 1 1 1 and 1 Φ AND 1 • • Φ rH β • φ tJ5 P. 1 1 1 1 1 1 rH AND AND ! • • £ AND % • What 1 1 1 1 1 4 and • • r * ł bD • -P • ABOUT 1 1 1 1 1 1 1 φ Φ Φ • • hH P. • Φ about • 1 Ł Ł 1 1 rH AND rH • 9 C3 about • ε 0 • 1 1 1 1 AND AND AND > Cm ' CM and CM CM about KO CM CM ► “3 AND LM about > IN AND What AND ε AND AND AND ε AND MO KO AND AND AND in σο & c AND Kj ε s = this t <] N S. M. H. AND IN H. tc in ε in «Ί 2 =

161 520

The peptides of interest most preferably should be derived from the gpl 10 region of the virus, and within this region they should be encoded in the env open reading frame region extending from 6667 bp to 6774 bp of the LAVbru isolate, and thus different homologous regions of other HIV isolates contain sequences homologues obtained from the Los Alamos Data Bank (except LAV2) as shown in Table I.

Other peptides suitable for generating or screening monoclonal antibodies include peptides encoded in the open reading frame env from 7246 bp to 7317 bp LAVbru. Such antibodies and reactive peptides are useful, especially for immunoassays.

In the gag region of the L AVbru p25 isolate, amino acid sequences 278 to 319 and 315 to 363 are additional neutralizing regions for HIV. It will be appreciated by those skilled in the art that additional HIV neutralizing regions can be identified based on the teachings provided herein. In particular, neutralizing activity will be exhibited by a combination of monoclonal antibodies which react with many different HIV epitopes.

Peptide I, also designated peptide 29, is encoded in the open reading frame env from 308 to 328 amino acid residues. It will have the following amino acid sequence (the oligopeptides contained in this sequence will contain linear epitopes):

1 (29)

Y-Thr-Arg-Lys-Ser-ne-Arg-ne-Gln-Alg-Gly-iPO-Gly-Arg-Ala-Phe-VaI-Thr-ne-Gly-Lys-ne-Y 'where Y and Y ', if present, are sequences up to about 20 amino acids in length. If present, Y and / or Y 'may include, for example, one or more amino acids derived from the flanking sequences, amino acid residues 308 to 328 of the HIV coat sequences, or any portion of these flanking sequences. For example, Y may contain the entire amino acid sequence of the LA Vbru coat from 301 to 307 or a portion thereof, and Y 'may contain all or a portion of the LAVbru amino acid sequence from residues 329 to 336 as shown below:

II (29a)

Cys-Th - Arg-Pro-Asn-Asn-Asn-'T ^ Γ-Arg-Lys-Ser-ele-Ag-ele-Gln-AΓg-Gly-Pro-Gly-Ag-Ala-PheVal-Th- -Ile-Gly-Lys-Ile-Gly-Asn-Me - AΓg-Gln-Ala-His-Cys.

Alternatively, truncated ("truncated) peptide sequences can be obtained, of which sequences derived from peptide 29 are particularly useful with the following structure:

III (29b)

Y-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-Gly-Y 'where Y and / or Y', if present, are sequences of up to 20 amino acid residues, and

IV (29c)

Y-IleIGlnIA-g-Gly-PΓOIGly-Arg-Ala-PheIValITh - Ile-Gjy-Lys-ele-Y 'wherein Y and Y', if present, are sequences of up to 20 amino acid residues.

In a further aspect, the homologous regions of the ARV-2 isolate of interest are encoded in the open reading frame env from 306 to 323 amino acid residues. They will have the V amino acid sequence, and the oligopeptides contained in this sequence will contain linear epitopes:

V (177)

YI'T'hr-Arg-Lys-Ser-Πe-IT ^ r-Ile-Gly-Pro-GlyIA-gIAlaIPhe-HisITh-IGly-A-gIeleIY 'where Y and Y', if present, are from 1 to 20 amino acid residues. Y and / or Y ', if present, may contain one or more amino acid residues derived from the flanking sequences, amino acid residues 306 to 323 of the ARV-2 coat, or any portion of these flanking sequences. More particularly, Y may contain all or part of the HIV coat amino acid sequence from residue 299 to 306 amino acid residues, Y 'may contain all or part of the HIV coat amino acid sequence from residue 324 to residue 333.

Alternatively, truncated ("truncated) V peptide sequences are prepared. In this case, sequences VI and VII are particularly useful."

VI (177a)

YITh - A-gILys-Ser-Ile-Tyj-Ile-Gly-Pro-GlyIY 'and

161 520

VII

Y-Asp-Cys-Lys-Thr-IIe-Leu-Lys-Ala-Leu-GIy-Pro-AIa-Ala-Thr-Leu-Glu-Glu-Nor-Leu-Nor-LeuThr-Ala-Cys-Y ' wherein Y and / or Y ', if present, are sequences of up to 20 or even more amino acids.

Another example are the homologous regions of the LAV-2 isolate, encoded, for example, in the open reading frame env from 311 to 320 amino acid residues with the following sequence:

VIII (110-2-2)

Y-Ly - ThΓIVal-Lys-IIe-Nor-Leu-No - Ser-GlyIHis-Val-Phe-His-SeΓ-His-Tyr-Gln-PoY 'where Y and / or Y', if present , represent sequences of up to 20 and even more amino acids (Nature, 326, 662, 1987).

A further aspect of the invention provides novel cell lines capable of producing monoclonal antibodies and compositions containing these antibodies. These antibodies, with extremely high titers (from 10-10 ⁴ to about 107 and even higher), are able to recognize the neutralizing regions contained in a specific sequence of the gpl10 or p25 coat glycoprotein in the protein precursors of these glycoproteins, in fusion proteins produced by biological expression and in synthetic proteins. peptides that contain one or more epitopes in that particular region of gp110 or p25. These hybrid cells have an identifiable chromosome in which the DNA has rearranged in the newly emerging lines such that it encodes an antibody having a binding site with an epitope contained in gpl10 or p22 common to some or all clinical HIV isolates. These monoclonal antibodies have a wide variety of uses, including diagnosis and therapy, as well as the identification of other cross-reactive antibodies, such as blocking antibodies. Peptides or polypeptides containing the epitope with which they react may find separate use as immunogens for vaccines or as therapeutic agents.

Production and use of synthetic peptides.

According to the invention, new peptides are produced which, inter alia, immunologically mimic epitopes encoded by the HIV retrovirus, especially epitopes encoded in the env or gag regions of the viral genome, encoding gpl10 and p25, respectively.

To compensate for differences between strains derived from different isolates, adjustment of conservative substitutions and selection of those with non-conservative substitutions can be performed. These peptides can be used as immunogens to inhibit or eliminate the production of HIV antigen in vitro or in vivo, to detect viruses or antibodies against these viruses in samples of biological material.

Depending on the procedure, the peptides can be conjugated to the support or other compounds, labeled or unlabeled, or attached to a solid surface.

The peptides produced according to the invention are derived from the gpl10 region of the virus, in particular the open reading frame of the env region, ranging from 6688 bp to 6750 bp and from 7246 bp to 7317 bp. These peptides, including blocking peptides, contain at least 5, sometimes 6, sometimes 8, sometimes 12, sometimes 21, generally less than 50, usually less than 35, and preferably less than 25 amino acids in the HIV retrovirus coding sequence. It is preferred that these peptides are as small as possible while retaining substantially complete immunological or antiviral reactivity of the larger peptide. In some cases, it is preferable to combine two or more non-overlapping oligopeptides to form a single peptide structure, or to use them as separate peptides. Together or separately, they constitute the immune equivalent of the parent peptide.

These peptides can be modified with conservative or non-conservative substitutions, generally being less than 20 percent, and usually less than 10 percent, of the number of amino acids replaced. In those cases where the regions show polymorphism, it is preferable to exchange one or more amino acids in order to more effectively mimic the variable epitopes of the different retrovirus strains. In many cases, methionine is replaced with norleucine (Nor) for chemical stability.

161 520

It is noted that the peptides used need not be identical to any HVV polypeptide sequence as long as the compound is immunologically competitive with at least one HIV strain.

Thus, these peptides may be subject to various changes, such as insertions, deletions, and substitutions, whether conservative or non-conservative, if these changes provide advantages in their use. Conservative substitutions are considered to be substitutions within groups such as gly, ala; val, ile, leu, asp, glu; asn, gln; cheese, thr; lys, arg; phe, tyr; and burrows, met. The altered sequence should not differ by more than 20 percent from that of at least one HIV retrovirus strain, unless additional amino acids have been added at either end to serve as the "arm 1 by which the peptide is immobilized on the support. Such an arm may consist of 1 to 50 or even more amino acids, most commonly arms of 1 to 10 amino acids in length are constructed.

A peptide in which the amino acid sequence has been modified by substituting, adding or deleting amino acid residues should retain substantially complete immunological or antiviral reactivity of the unmodified peptide, which can be readily determined using the assay techniques disclosed herein. One or more amino acids in D-isomeric form can also be used to modify biological properties such as activity, rate of disintegration and the like.

In addition, to modify the physical or chemical properties of a peptide or oligopeptide, one, two, or more amino acids may be added to the ends of the peptide or oligopeptide for the purposes discussed below to facilitate binding of the peptides to each other and binding to a carrier or to a larger peptide .

In order to introduce functional groups needed for bonding, amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid are placed at the carboxy or amino terminus of the peptide or oligopeptide. Cysteine is especially preferred to facilitate covalent bonding to other peptides or to make polymers by oxidation.

The peptide or oligopeptide sequences may also differ from the sequences of natural origin in that they have modified sequences, for example by acylation of the amino terminal group, or amine terminal blocking, or thioglycolic acid amidation, ammonia or methylamine terminal blocking, thereby obtaining stability and increased hydrophobicity - qualities needed for bonding to a carrier or to another molecule or for polymerization. Thus, in the disclosed peptides I-VIII and IX-XV, if Y and Y 'are present, they preferably represent one or more cysteine residues or a combination of one or more cysteine groups with an amino acid residue as an arm for further processes.

A particularly preferred arm amino acid is glycine. Preferred peptides for use in oxidative polymerization are those wherein Y and Y 'are at least two cysteine residues. If there are two cysteine residues at the same terminus of the peptide, it is preferred that they are separated by one to three shoulder amino acid residues, preferably glycine. The presence of cistern residues allows the formation of dimers of the given peptide and / or increases the hydrophobicity of the obtained peptide, which facilitates its immobilization in a solid phase or facilitates immobilization tests.

Of particular importance is the use of the mercaptan group of cysteine or glycolic acid used to acylate amine or similar end groups to link two peptides or oligopeptides or combinations thereof with disulfide or longer bonds to form polymers having multiple epitopes. Such polymers are marked by increased immunological reactivity. When different peptides are used to make the polymer, they have the additional ability to induce antibodies which react immunologically with several antigens and determinants of different HIV isolates.

For the preparation of antigenic polymers (synthetic multimers), compounds having bis-haloacetyl groups, nitroaryl halides and the like are used where the reagents are thiol-specific. Thus, a combination of two mercaptan groups

The different peptides or oligopeptides can be a single bond or a bridging group of at least 2, generally at least 4, but not more than 16, usually no more than 14 carbon atoms.

These peptides may be used bound to a soluble macromolecular carrier (not less than 5 kilodaltons). Such a carrier may be a poly (amino acid), either naturally occurring or synthetic in which no antibodies are present in human serum.

Examples of such carriers are poly-L-lysine, leech haemocyanin, thyroglobulin, albumin such as bovine serum albumin, tetanus toxoid and the like. The choice of carrier primarily depends on the intended use of the antigen and the ease of surgery and availability of that carrier.

In such conjugates there should be at least one peptide molecule in the macromolecule, but no more than one in 0.5 kilodalton, typically no more than one in every 2 kilodalton macromolecules, one or more different peptides may be attached. A conventional bonding method is used, using such reagents as p-methyldithiobenzoic acid, p-methyldithiobenzoic acid, maleic anhydride, succinic anhydride, glutaraldehyde and the like. The linkage may be at the amino terminus, carboxyl terminus, or intermediate to the ends of the molecule. The peptide can be derivatized by binding, it can also be coupled in a carrier-bound state.

Various tests, similar to the known ones, are used to detect the presence of antibodies to the retroviral proteins or the retroviral proteins themselves. It is especially useful to use a peptide as a labeled reagent in which the labeling produces a detectable signal or the binding of the peptide directly or indirectly on the surface, whereby the antibody against the peptide in the test sample will be bound to the peptide on that surface. The presence of the peptide-bound human antibody is then detected using a xenogeneic anti-human immunoglobulin antibody, generally both human IgM and IgG, or a labeled protein specific for immune complexes, e.g. protein A Rf factor from S. aureus.

An illustration of the testing technique is the use of a sample container, that is, cavities in a plastic microtest plate in which cavities the polypeptide or its conjugates adsorb to the bottom and / or walls of the cavity covalently or non-covalently. Human blood or serum, diluted with an appropriate buffer, is then added to the wells and allowed to complex until a complex is formed between the polypeptide and related antibodies in the sample. The supernatant is then removed, and the wells washed to remove unspecifically bound proteins. For detection, a labeled, specific binding protein is used which binds to the complex formed, such as an anti-human immunoglobulin xenogeneic antiserum.

These peptides are produced using various synthetic routes. As the peptides are relatively short, they are synthesized in solution or on a solid support in accordance with the techniques currently used. Various automatic synthesizers currently commercially available can be used in this work. See Stewart and Young, "Slid Phase Peptide Synthesis, 2nd ed. Pierce Chemical Co., 1984 and Tam et al., J. Am. Chem. Soc., 105, 6442 (1983).

Alternatively, hybrid DNA technology can be used. In this technology, a synthetic gene is prepared using single strands encoding that polypeptide or complementary strands overlapping the single strands and joined together in a suitable hybridization solution. The hybridized strands are ligated to form the complete gene by selecting the appropriate ends. Such a gene is inserted into expression vectors which are readily available today. (Maniatis, Molecular Cloning, A Laboratory Manual, CSH, Cold Sprong Harbor Laboratory, 1982).

Another technique is to clone the region of the viral genome encoding the peptide by known recombinant DNA methods (see Maniatis, supra). DNA coding sequences from isolates can be used to express the peptides

161 520

LAV

LAV

BRU

BRU and ARV-2 of HIV with the following structure:

ARV-2

TGT AGA AGA Γ1 Γ »·! "at AAG AAG AAT AC A AGA AAA AGT ATC GGT ATG GAG AGG GGA GGA GGG AGA GGA TTT GIT AGA ATA GGA AAA ATA GGA AAT ATG AGA GAA GGA GAT TGT TGT AGA AGA GGG AAG AAC AAT AGA AGA AAA AGT a ron TAT ATA GGA CCA GGG AGA GLA TIT GAT AGA AGA GGA AGA ATA ATA GGA GAT ATA AGA AAA GGA

CAT TGT

A fragment of these sequences is used to express peptide fragments. Conservative base changes may also be made where the modified codon encodes the same amino acid, or non-conservative changes in the coding sequence, whereby the resulting amino acid may be a conservative or non-conservative change in the amino acid sequence, as discussed above.

The coding sequence can be extended at the 5 'or 3' end, or both ends, thereby extending the produced peptide and preserving its epitope site. The purpose of the extension is to add an "arm for binding to, for example, a labeling agent such as an enzyme, to link two or all of the peptides into a single chain to obtain antigenic activity, or to obtain convenient restriction sites for cloning.

As such, fragments or larger sequences of these DNA sequences of at least 15 bases, preferably at least 18 bases, are used as nucleotide probes to detect retroviral RNA or proviral RNA or proviral DNA or to identify regions homologous for cloning or sequencing. Various techniques are available for this purpose, such as the Grunstein-Hogness techniques, the Southern technique, the Northern technique, the dot-blot technique and improvements thereof, and other methodologies such as that disclosed in US Patent No. 4,358,535.

The peptides obtained by the method of the invention are used in vaccines as such or in combinations. Anti-idiotype antibodies, that is, reactive with the idiotypes of the antibodies of the invention, and therefore having epitopes mimicking HIV neutralizing regions, can also be used as vaccines. These peptides and anti-idiotype antibodies are formulated into pharmaceutical forms in a known manner, generally in doses of 1 µg to 20 mg / kg of host weight. As carriers, physiologically acceptable agents such as sterilized water, body fluid, phosphate buffered body fluid, and the like are used. Adjuvants such as aluminum hydroxide gel, surface-active substances such as lyso-lecithin, pluoron polyols, polyanions, peptides, proteins (for example diphtheria or cholera toxins and oil emulsions) can also be used. These peptides can also be incorporated into liposomes or conjugated to polysaccharides, polypeptides or polymers and used in vaccines. These vaccines are administered parenterally, that is, intramuscularly, intraperitoneally, subcutaneously and intravenously. The immunogenically effective dose is administered one or more times, generally at intervals of one to four weeks. "An immunogenically effective dose is the amount of vaccine needed to elicit an immune response in the host with evidence of increased infection.

Other advantages and details of the invention are set forth in the following examples, which are included in the description for the purpose of illustration only, and should not be construed as limiting its scope.

161 520

Example I. Production and characterization of monoclonal antibodies.

This example describes a method for generating hybrid cell lines producing monoclonal antibodies specific for HIV coat glycoproteins. In this method, lectin-purified LAVbru extracts attached to lentil lentin lectin are used as immunogen. Subsequently, monoclonal antibodies generated by the hybrid cell lines are characterized by an "immunoblot" assay and for the ability to radioimmunologically precipitate gpl10 from purified LAV, and as a recombinant fusion protein produced by biological expression. Monoclonal antibodies that bind to epitopes on gpl10 are also reactive in whole virus disrupted ELISAs with fusion proteins and synthetic peptides, and react with whole virus in intermediate fluorescence samples.

The method of producing hybrid monoclonal antibody producing cell lines and characterizing these antibodies is as follows:

LAV virus isolated from infected CEM cells (ATCC No. CRL8904) is disrupted in a solution composed of: 50 mM Tris (pH 7.4), 0.15 M NaCl, 1.0 percent aprotinin, 2.0 percent "Nonidet P" reagent -40 * R (NP-40, octylphenoxypolyethoxyethanol). The extract is clarified twice by centrifugation and brought to a concentration of 0.5% NP-40 by adding three volumes of the above buffer but without NP-40. Lentil lectin agarose (Pharmacia, Piscataway, NJ) is pre-washed with disruption buffer without NP-40 and equilibrated in an adsorption buffer composed of: 50 mM Tris (pH 7.4), 0.15 M NaCl, 1.0% aprotinin, 0.5% NP-40. The clarified viral extract is adsorbed to lentil lectin agarose for 42 hours at 4 ° C. Unadsorbed material is removed by washing with an excess of adsorption buffer. The adsorbed material is eluted with a 0.2 M solution of? -Methylmannoside in an adsorption buffer. The eluent is dialyzed against PBS to remove the sugar and the resulting material is re-adsorbed onto lectin sepharose.

Complex: glycoprotein - lentil lectin sepharose, is used to immunize BALB / c mice by administering three intraperitoneal injections without adjuvant at 2-3 week intervals. Spleens are collected from mice that show circulating antibodies to HIV glycoproteins in immunoblot, RIP and / or ELISA assays.

The generation of the cell lines is performed essentially as described by Kohler and Milstein (Nature, 256, 496 (1985) with modifications by Goldstein LC et al. (Infect. Immunol. 38, 273 (1982). B cells from the spleen of immunized mice are fused. with myeloma cells NS-1 to 40% (w.), polyethylene glycol. After the fusion the cell mixture was resuspended in HAT medium (RPMI-1640 medium enriched with 15% fetal calf serum which more ^{LE 1} XM ^{-4 M high} poksantyny ⁴ XK) ⁷ M am ⁱ no ^p ter ^y n ^y l, ⁶ XW ^{"5 M} tymtóyn ^ dV generate conditions of selection allowing the growth of only the hybrid cells, and then the mixture is poured into depressions in the plates microculture 96 well at a concentration of 1 to 3 X 10 ⁶ cells / ml and incubated at 37 ° C in a humidified atmosphere with 6% CO2. Cultures are fed by replacing half of the supernant with fresh HAT medium. The wells are observed under an inversion microscope for signs of cell proliferation k and after a sufficient cell density is reached, the supemants are tested for the presence of anti-LAV antibodies.

The wells containing the LAV antibody producing hybrid cells are identified by ELISA by their binding either to the whole purified disrupted virus or to fusion proteins produced by biological expression. The disrupted virus ELISA is performed on LAV RIA plates (genetic Systems, Seattle, Washington). The cell culture fluid plates are incubated at 37 ° C for 45 minutes and then washed three times with a solution of 0.05% Tween 20 in phosphate buffered saline (PBS-Tween).

Peroxidase - goat anti-mouse IgG (1: 200 dilution in PBS-Tween, Zymed Laboratories, Inc., South San Francisco, California) is added at 100 µl per well and the plates are incubated for 45 minutes at 37 ° C and rinses as above. Then it is added

161 520 substrate (0.025 M citric acid, 0.05 M dibasic sodium phosphate (pH 5.0) and 14 mg o-phenylenediamine and 10 µl 30% hydrogen peroxide in 50 ml) and incubate the plates for 30 minutes at room temperature in the dark. The reaction is stopped with 3N sulfuric acid and readings of the colorimetric reaction are taken in an automatic microplate reader. Positive wells are further subcloned using limiting dilution, specificity determined again and expanded.

Monoclonal antibodies secreted by the resulting hybrid cell lines are then characterized by determining their specificity and reactivity in "immunobloC, immunoprecipitation and ELISA assays using disrupted LAV viruses, recombinant LAV fusion proteins, and synthetic LAV peptides." The determinations show that all the antibodies are of the IgG-ι isotype. Prior to this application, the HIV-gpl 10-1, HIV-gpl 10-2, and HIV-gpl 10-3 cell lines were deposited with the American Type Culture Collection under ATCC Nos. HB 9175, HB 9176, and HB 9177, respectively.

Recombinant fusion proteins tested for reactivity were previously designated ENV2, ENV3, ENV4 and ENV5. The ENV2 protein is derived from the expression of pENV2 (ATCC 53071) which is the LAV region from 6598 bp to 7178 bp (base pair numbering according to Wain-Hobson et al., Cell, 44, 9 (1985), ENV3 is derived from the expression of pENV3 (ATCC 53072) containing the LAV region from 7178 bp to 7698 bp ENV4 is derived from the expression of pENV4 (ATCC No. 53073) containing the sequence from 7698 to 8572 bp and ENV5 is derived from the expression of pENV5 (ATCC 53074) which contains the LAV region from 5889 to 7698 bp. Generation of recombinant fusion proteins is described in detail in pending United States Patent Application No. 721,237.

Preparation of synthetic peptides.

Peptides I (29) and VIII (10-2-2) are synthesized from benzhydrylamine resin (polystyrene / divinylbenzene) (Applied Biosystems, Inc., Foster City, California). Peptide V (177) is synthesized on t-butyloxycarbonyl (Boc} -ethylbenzylcysteine-phenylacetamidomethyl (PAM) polystyrene / divinylbenzene resin Symmetric anhydride coupling is performed in Applied Biosystems 430 A synthesizer. Both peptides use cysteine as the first residue.

For asparagine and glutamine, dicyclohexylcarbodiimide coupling is used in the presence of hydroxybenzotriazole. Side chain benzylation and BOC blocking of the σ-amino group are used. In addition, the following blockade of reactive groups in the side chains is routinely used: Boc (formyl) tryptophan, Boc methionine sulfoxide, Boc (toluenesulfonyl) arginine, Boc (methylbenzyl) cysteine, Boc (toluenesulfonyl) histidine, Boc (chlorobenzyloxycarbonyloxycarbonyl). tyrosine.

Labeling of the peptides with the radioactive element is carried out by acetylating the amino terminus with ³ H-acetic acid using an excess of dicyclohexylcarbodiimide.

Deblocking and cleavage of the peptide from the resin was performed by the Tania "low-high" method using HF (Tam et al., Supra). The peptide is extracted from the resin 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 LAVbru genomic region from 6688 bp to 6750 bp, peptide 36 is encoded by a region ranging from 7246 bp to 7317 bp, and peptide 39 is encoded by a region from 7516 bp to 7593 bp. Peptides 36 and 39 are disclosed in US Patent No. 4,629,783.

Blocking peptides IX through XV are constructed as described above on a methylbenzhydralamine (polystyrene / divinylbenzene) resin (Applied Biosystems Inc., Foster City, California). Symmetric anhydride coupling is performed on an Applied Biosystems 430 A synthesizer. For asparagine, dicyclohexylcarbodiimide coupling is used in the presence of hydroxybenzotriazole. Side chain benzylation and Boc (amino) blocking are used, while BOC (bromobenzyloxycarbonyl) blocking is used for tyrosine side chains. Acetylation, if necessary, is performed with acetic anhydride.

161 520 or glacial acetic acid in dicyclohexylcarbodiimide. Deblocking and cleavage of the peptide from the resin was carried out by the standard technique "high using HF (Stewart et al., Supra). The peptide is extracted from the resin with 50% acetic acid and the extract is subjected to gel filtration chromatography in 20% acetic acid. Purification by high performance liquid chromatography on a Vydac C18 column (Rainin Instrument Co., Emeryville-CA) using a 0.1% gradient of trifluoroacetic acid in acetonitrile is performed where necessary.

Color immune reaction ("Immunoblotting).

In this assay, the characterization is performed on clone supernatants or on the exudate using purified LAV and recombinant fusion proteins as antigens. Antigens are separated by gradient electrophoresis of polyacrylamide gel (7.0-15.0%) and transferred to a nitrocellulose (NMC) membrane by electrophoresis for 4 hours at 25 V in 25 mM sodium phosphate (pH 7.0). After transfer, non-cellulose membranes are blocked to avoid non-specific reactions by incubating in PBS-Tween or Blotto (5% non-fat dry milk in PBS) for one hour at room temperature.

Then, these non-cellulose membranes are incubated with the cell culture supernatant or with the effusion fluid diluted in PBS-Tween for one hour at room temperature and required replacing three times with PBS-Tween. In the next step, nitrocellulose membranes are incubated for one hour at room temperature with a solution of goat IgG anti-mouse horseradish peroxidase diluted in PBS-Tween. After incubation, elution is carried out with PBS-Tween solution and the membranes are immersed in a solution giving a color reaction with horseradish peroxidase (Bio-Rad Laboratories, Richmond, California) for 20 minutes. The reaction is stopped by immersion in deionized water. The reactivity of the monoclonal antibodies is compared to a positive control serum reactive with the purified disrupted virus or expression fusion protein. The results of the assay indicate that when using the disrupted virus preparations, all antibodies bind to gp110 and its precursor molecule, gpl50. Antibodies 110-1 and 110-2 also recognize the ENV3 fusion protein, while antibodies 110-3, 110-4, 110-5, and 110-6 immunologically compensate for ENV2.

Immunoprecipitation.

Viral extracts for radioimmune precipitation are prepared from CEM cells infected with HIV LAVbru isolate adapted to lytic growth by continuous passaging. When observing early cytopathic changes in the cell is transferred to the fluid ^D incl znakowama ^and devouring lying ^± "^ j ^ medoni ^(0, 05mCi / ml) afoo ^{3 (H)} -glukozamm ^E (0.025 mCi / ml), then incubated 24 hours. During this time, most cells are lysed and the virus is released into the culture supernatant. The virus is centrifuged from the cell-free supernatant (centrifugation for one hour at 100,000 g) and detergent extracts are made in P-RlP buffer (phosphate buffered saline containing 1.0% Triton Χ-100,

1.0% deoxycholate, 0.1% SDS and 1% aprotinin). Similar extracts are prepared from the supernatants of uninfected CEM cells.

For immunoprecipitation assays, 100 µl of viral extract are incubated with 100 µl of hybrid cell line culture supernatant for one hour on ice. 4 µl of rabbit anti-mouse Ig (Zymed Laboratories, San Francisco, California) was added to each sample and incubated for 30 minutes. Immunoprecipitin (100 [mu] l), Bethesda Research Laboratory, Bethesda, Maryland) suspended in P-RIPA buffer containing 1.0% ovalbumin is then added to each sample and incubated for an additional 30 minutes. The bound complexes are washed and separated by SDS polyacrylamide gel electrophoresis (15.0% acrylamide: DATD gel). After electrophoresis, the gels are fixed, immersed in an Enhance bath (New England Nuclear, Boston, MA), dried, and filmed onto Kodak XR-5 film. Serum is used as comparative positive and negative controls, which immunoprecipitates all HIV proteins by reacting with supernatants of virus-infected and mock-infected CEM cells.

The results of the assay show that all 6 monoclonal antibodies are specifically precipitated by gplO and gpl50.

161 520

Solid phase enzyme immunoassay.

For the determination of gpl10 epitopes recognized by the monoclonal antibodies of the invention, culture supernatants from hybrid cell lines or from the exudate are characterized by determining their reactivity with biologically expressed fusion proteins and synthetic peptides in ELISA assays. The procedure is the same as described above except that fusion proteins or synthetic peptides are used instead of the virus as the antigen adsorbed on the surface of a well in a microtiter plate.

When using peptides as an antigen, the procedure is as follows: The lyophilized peptide is dissolved in 6 M guanidine HCl. Immediately before dispensing into the wells on 96-well plates, the guanidine solution is diluted with 0.05 M carbonate / bicarbonate buffer (pH 9.6) to a final peptide concentration to K0) prg / ml. 50 µL of the diluted peptide is placed in each well and the plates are incubated overnight at 4 ° C. The excess peptide solution is "shaken", the plates are blocked with a "Blotto solution" and the procedure is continued as described above for the ELISA assay. Similarly, the recombinant protein is diluted to a final concentration of 2 µg, µmL with 0.05M carbonate / bicarbonate buffer (pH 9.6) and the same procedure is followed.

The results are shown in Table 2. Monoclonal antibodies produced by the HIV gpl 10-1 and HIV gpl 10-2 cell lines react with ENV3, ENV5, peptide 36 and the disrupted virus. Antibodies from the HIV-gp-110-3, HIV gp-11 OM, HIV gp-110-5 and HIV-gp110-6 cell lines react with ENV2 and peptide 29 and with disrupted virus.

Table 2 Results of the ELISA test showing the reactivity of monoclonal antibodies against recombinant proteins and synthetic peptides

Recombinant fusion protein Synthetic peptide . LAV virus control CEM control cells ENV2 ENV3 ENV4 ENV5 29 36 39 110-1 0.077 3,000 0.113 3,000 ND 2.421 0.054 0.908 0.125 110-2 -0.003 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 0.368 0.025 110-6 3,000 0.033 ND ND 1.937 ND 0.017 0.486 0.032

The results presented in Table 2 show that monoclonal antibodies 110-1 and 110-2 recognize an antigenic determinant encoded by a DNA sequence in the pENV3 region, in particular by the region of the HIV genome defined by the amino acid sequence of peptide 36. Thus, monoclonal antibodies gpl 10-1 and 110-2 bind to the gpl10 peptide region encoded in the 7246 bp to 7317 bp segment, as evidenced by the formation of immune complexes with peptide 36 and ENV3.

This region of the HIV genome was previously identified as a conserved region, that is, there were only minor changes in the DNA sequence of the peptide 36 region derived from different virus isolates from different geographic regions. See Starcich et al., Cell, 46.637 (1986). In contrast, monoclonal antibodies gpl 10-3, -4, -5 and -6 bind to HIV peptides as defined by a peptide 29 coding region ranging from 6688 bp to 6750 bp. This region in gpl10, defined by peptide 29, however, contains a number of nucleotide substitutions in different viral isolates. Monoclonal antibodies that selectively bind to gpl10 polypeptides containing conserved epitopes, such as antibodies 110-1 and 110-2, have greater utility in many circumstances, for example in affinity chromatography. Also in the ELISA assay, peptide 110 2-2 reacts with the serum of the individual from which LAV-2 was isolated.

161 520

Indirect immunofluorescence test.

Indirect immunofluorescence tests with monoclonal antibodies directed against the HIV gpl antigen are performed on acetone-fixed and live cells. Acetone-fixed samples prepared from LAV-infected CEM cells are incubated with diluted culture supernatant or effusion fluid for 1 hour at 37 ° C, while viable cells are incubated with culture supernatant or effusion fluid for 1 hour at 4 ° C, whereby cell preparations fixed with acetone are prepared. In both methods, fluorescein isothiocyanate-labeled anti-mouse IgG is used to detect cells containing reactive gpl10 antigen. The HIV-gpl 10-1 monoclonal antibody gives positive results with both live and acetone-fixed LAV infected cells.

An example. HIV contagiousness neutralization by monoclonal antibodies against gpl 10.

In the present example, the neutralization of HIV infectivity by the use of monoclonal antibodies that bind to gpl10 and peptides within gpl10 is described and characterized. The results of the trials indicate that monoclonal antibodies gpl10-3, -4, -5 and -6 have the activity of neutralizing activity and that gpll0n3 and -4 have extremely high levels of neutralizing activity.

An attempt to neutralize. A sensitive neutralization assay has been developed to quantify the effect of monoclonal antibodies on HIV infectivity. CD4 + (CEM) cell line very sensitive to HIV infection was selected as the target cell for the infectivity comparisons. The exudate, prepared as described in Example 1, or its IgG fraction is purified by ammonium sulfate precipitation and inactivated by heating at 56 ° C for 30 minutes, then diluted in RPMI medium containing 10% fetal calf serum. HIV virus suspension, strain LA Vbru, is harvested from 4-day log-phase CEM cultures, filtered through 0.2 or 0.45 micron filters, aliquoted and frozen at -70 ° C. One aliquot is thawed, titrated to determine TCID50, and subsequent trials are run with freshly thawed aliquots diluted 1: 500 in culture medium to a concentration of about 1.0 times the concentration needed to infect 50% of the CEM cells in the culture (10 TCID50). The viral suspension is mixed with an equal volume (250 µl) of five-fold dilutions of the monoclonal antibody preparation 1: 5 to 1: 9, 765, 625. The virus / antibody mixture is incubated for 45 minutes at 37 ° C then diluted 1: 5 to 1: 9, 765, 625. This virus / antibody mixture is incubated for 45 minutes at 37 ° C, then 200 μl aliquots of this mixture are used to inoculate wells containing 1.0 ml of a solution of approximately 2 × 10 ⁵ cells CEM / cavity. The cultures are incubated at 37 ° C in a humidified atmosphere with 5% CO2 for 14 days.

Cells are then harvested, paletted and lysed in 1% Triton Χ-100 in PBS for 10 minutes. The amount of virus (or viral antigen) present in lysed cells is quantified using a sensitive HIV antigen capture sandwich immunoassay described below. The titer of neutralizing activity is determined as the reciprocal of the dilution of the monoclonal antibody that inhibits the production of antigen by more than 50% compared to control viral cultures incubated without the antibody or with a monoclonal antibody of the same isotope that has not shown neutralizing activity in previous tests. In the above HIV antigen capture assay, two monoclonal antibodies directed against p25 antigens are used as capture (hood) reagents. These hybrid cell lines are generated as described above, with minor modifications, which include the use of the purified recombinant gag protein as an immunogen and the characterization of the produced monoclonal antibodies for specificity and reactivity, using recombinant fusion proteins designated GAG-1, GAG-2 and GAG-3 and synthetic peptide 141. The GaG-1 protein is the expression product of the pGAG-1 plasmid (ATCC No. 53379), GAG-2 is the expression product of pGAG-2 (ATCC 53111) and the GAG-3 protein is the expression product of pGAG-3 (ATCC No. 53112). The production of recombinant fusion proteins is described in detail in application 18

In US Patent Nos. 764,460 and 828,828. The synthetic peptide 141 is encoded by the genomic LAVbru region corresponding to amino acid residues 198-242. It turned out that the monoclonal antibody produced by the p25-2 and p25-3 hybrid cell lines reacts with the recombinant fusion proteins GAG-1, GAG-2 and GAG-3, and the monoclonal antibody with p25-3 also reacts with the synthetic peptide 141.

To carry out the antigen capture assay, the capture reagents are adsorbed onto a solid support. The exudative fluid from the p25-2 and p25-3 hybrid cell lines is diluted 1: 5000 in 25 mM Tris buffer (pH 8.5) and 200 μ amounts are placed in the wells of the microplate. These wells are sealed and incubated for 16 hours at 4 ° C. The solution is removed from the wells by aspiration and 0.3% Blotto in PBS blocking solution is added. Blocking is performed for 15 minutes at room temperature. Blocking solution is aspirated and the sample is added. 0.02 µl of cell suspension after lysis was added to each well and 5.0 µl of the detection conjugate was prepared as described below. The strips or well plates are incubated for 2 hours at 37 ° C, after which the suspension is aspirated and the wells are washed 4 times with buffer (0.05% Tween 20 in PBS). The detection conjugate is prepared as follows: The monoclonal antibodies p25-6 and p25-7 are conjugated to horseradish peroxidase (HPR) at a molar ratio of 3: 1 (Ab: HRP) for 3 hours using the periodate oxidation procedure (Nakane et al., J. Histochem Cytochem. 22, 1084 (1974) These conjugates are diluted 1: 1500 in 2.5% (w / v) skimmed dry milk with 0.01% thimerosal and 0.005% "Antifoam" A "in 20 mM sodium citrate. The rest of the ELISA procedure is carried out as in Example I. The following results are obtained in the test of the neutralizing activity of the gpl 1 antibody (0-3, -4, -5 and -6. the neutralizing activity was: gpl 10-3 = 15.625, gpl 104 = 9.765.625, gpl 1 (05 = 125 and gpl 10-6 = 625.

Due to the predicted genetic variation in the region defined by peptide 29, the ability of the gpl 10-4 monoclonal antibody to recognize other HIV isolates was tested. Immunofluorescence assays were performed as described above. The gpl 10-4 antibody detects antigen in cultures of at least 3 out of 16 HIV isolates tested.

In AIDS vaccination trials, the gp 11 (0-4 antibody is able to neutralize viruses isolated after 15 weeks from LAVbru infected chimpanzees as control animals. The monocloned antibody is able to neutralize isolates despite the animal possessing HIV neutralizing antibodies in vitro and in serum). that a measurable cell-mediated immune response to this infection has already developed, indicating no in vivo antigenic shift to the epitope recognized by the gpl 110-4 antibody.

Example III. HIV contagiousness neutralization by a mixture of monoclonal anti-gpl 10 and anti-p25 antibodies.

This example describes the neutralization of HIV infectivity by using monoclonal antibodies that bind to gpl10 and peptides within gpl10 in combination with monoclonal antibodies that bind to p25 and peptides within p25 and, when used alone, do not have neutralizing activity. or very little activity. The results indicate that the monoclonal antibody gpl 1 (0-2 in combination with either p25-6 or p25-7 has particularly high levels of neutralizing activity.

The p25-6 and p25-7 hybrid cell lines are generated by the methods described above, with modifications involving the use of inactivated, split viruses or purified recombinant gag fusion protein secreted by E. coli as an immunogen, and characterization of the obtained monoclonal antibodies for specificity using recombinant fusion proteins previously labeled GAG-1, GAG-2 and GAG-3, and synthetic peptides 15, 88, 150, µ7, and 148. These synthetic peptides are encoded in the LAVbru genomic region corresponding to the following amino acid residues: peptide 15: amino acid residues 329 to 350, peptide 88; amino acid residues 315 to 350, peptide 150: amino acid residues 318 to 363, peptide 147: amino acid residues 278 to 319 and peptide 148: amino acid residues 290 to 319. Monoclonal antibodies produced by hybrid lines

Cellular p25-6 and p25-7 react with the recombinant GAG-3 fusion protein p25-6 and react with synthetic peptides 147 and 148, and p25-7 react with synthetic peptides 15, 88 and 150.

The neutralization assays are carried out as described above, except that if the mixture (cocktail) is used, the individual monoclonal antibodies are first diluted 1: 5 in the culture medium and then equilibrated to give a final 1:10 dilution. The rest of the test is carried out as described above.

Monoclonal antibodies gp 110-2, p25-6, and p25-7 show less than 50% neutralizing activity when used alone. When used in a mixture that contains the monoclonal antibodies gpl10-2 together with p25-6 or p25-7 at a 1:10 dilution, complete neutralization is achieved. The mixture containing monoclonal antibodies p25-6 in combination with p25-7 gives a neutralizing activity of 60 to 90%.

Example IV. Strength of the immune peptide 29.

The ability of peptide 29 and the homologous peptides including peptide 177 to stimulate an immune response against HIV was determined in two mouse strains.

The method of preparing the peptide immunogens, the method of immunizing and determining the immune response is given below.

Peptide 29 preparation for immunization purposes is prepared by conjugation with purified thyroglobulin. Tyroglobulin is derivatized with N-succinimidyl-4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (SMCC) in accordance with the disclosure in US Patent No. 4,529,783, column 10, lines 28-51. As a second immunogen, thyroglobulin derivatized with glutaraldehyde is prepared as follows: 27 mg of porcine thyroglobulin is dissolved in 1 ml of 0.1 M sodium bicarbonate to which 8.3 μ \ 25% glutaraldehyde solution is added dropwise and the mixture is stirred overnight. in room temperature. 1 ml of buffer: sodium carbonate / sodium bicarbonate, pH 9.3, was added to the solution and dialyzed for 8 hours against 2 liters of the same buffer at 4 ° C, with complete dialysate exchange after 4 hours. Peptide 29 is then added to the derivatized thyroglobulin in a 100 molar excess, and the mixture is stirred overnight at room temperature. The unreacted glutaraldehyde was blocked with 200 µl of 0.2 M lysine solution and the mixture was stirred for several hours (24 hours) at room temperature. The peptide-thyroglobulin conjugate is then dialyzed extensively against PBS at 4 ° C.

Peptides prepared by either of these conjugation methods are administered to two mouse strains (black C57 and BALB / c). At the time of inoculation, all mice were 2-4 weeks old. The preparations were administered to the footpads, by scarification of the tail, subcutaneously, intranasally or intraperitoneally. The inoculum contains 25 µg of the conjugate peptide suspended in 0.5 ml of complete Freund's adjuvant. In addition, supportive inoculations repeated at weeks 2,3 and 5 with the same immunogen in incomplete Freund's adjuvant are used. Serum samples are collected from individual mice prior to immunization, 4 days after the support incubation at week 3, and 4 days after the last incubation at week 5. Antibodies to analogous peptides or to whole viruses are detected in serum samples by screening by ELISA. Sera showing antibody activity against LAV are further screened for neutralizing activity, then these sera are analyzed by colored immunoassays ("immunoblots) against disrupted LAV antigen and by radioimmunoprecipitation against labeled gpl 10".

The immunization results show that mice immunized with peptide 29 had antibodies reactive with peptide 29 and the disrupted LAV-1 virus by ELISA. Conjugation of peptide 29 with glutaraldehyde generally resulted in higher titers of anti-peptide 29 antibodies in BALB / c mice, although maleimide conjugation gave satisfactory results in the production of anti-peptide 29 antibodies in some BALB / c mice. Mice immunized with peptide 177 developed antibodies against this peptide and conjugation of this peptide with glutaraldehyde to a greater extent induced an immune response. C57 mice were more sensitive to immune stimulation with peptide 177 than BALB / c mice. As shown by ELISA assays, mice immunized with peptide 110-2-2 from LAV-2 possessed antibodies against 110-2-2 and against LAV-2 virus. Peptide 110-2-2 conjugated through glutaraldehyde was immuno20

161 520 genes versus C57 and BALB / c mice, although titers for peptide 110-2-2 were generally higher in BALB / c mice than in C57 mice, while titers for LAV-2 were generally higher higher in C57 mice than in BALB / c mice.

In summary, it can be concluded that serum samples from immunized mice that (i) have antibodies to whole virus particles, (ii) neutralize HIV, as shown in the tests described in Example 2, and (iii) react with peptide 29 in an ELISA assay. collectively demonstrate the efficacy of peptide 29 in vaccine formulations.

Example 5 Resolution of gpl10 by immunoaffinity method using monoclonal antibodies.

Monoclonal antibodies against the HIV gpl10 entigen can be used in the immunoaffinity separation process to completely purify the bacterially expressed recombinant fusion proteins. If the protein produced by the bacteria is secreted, this protein can be isolated from the culture supernatant. If, on the other hand, it is not secreted from the bacterial cell, it is necessary to break the cell. The construction of the plasmid pENV-5 (ATCC No. 53074) is given in pending U.S. Patent Application No. 721,237. The plasmid pENV-5 encodes most of the carboxyl terminus of gpl10 and the amino terminus portion of LAV gp41 which is an insertion in the expression vector trp. E. coli C600 transformed with this vector produces the gpl10 fusion protein but does not secrete it outside the cell.

E. coli C600 containing the pENV-5 plasmid is grown in tryptophan (20 µg / ml) and ampicillin (100 µg / ml) medium overnight at 37 ° C under aeration conditions. The one-day-old cultures are then inoculated at a 1: 100 ratio into fresh minimal medium containing ampicillin (/ g / ml) but not tryptophan. These cultures are grown under aeration for 2-3 hours (until the early log phase of growth) at 37 ° C. The inducer, 3J-Inoleacrylic acid (Sigma Chemical Co., St. Louis, MO) is then added as a freshly prepared solution at 20mg / ml in 95% ethanol (to a final culture concentration of 20pg / ml). The induced colonies are grown at 37 ° C with aeration, then pelleted for 4 to 5 hours and optionally frozen. The protein yield from pENV-5 is generally below 1 mg / liter.

The bacterial cell pellet is lysed in P-RIPA buffer (PBS containing 1% Triton Χ-1Ο0.1% deoxycholate, 0.1% sodium dodecyl sulfate and 1% aprotinin) inducing lysis of E. coli cells. The suspension is sonicated to shear the DNA and RNA then centrifuged to remove undissolved particles. A dilution or concentration step may be required to standardize the protein concentration.

HIV-gpl 10-1 monoclonal antibodies are initially precipitated from the exudate or from cell culture supernatants at room temperature or below with (NHi) zSO4 or Na2SO <buffered (pH 7.3) solutions added in the amount needed to obtain final concentrations, respectively 33 and 18%. The precipitated proteins are centrifuged and redissolved in PBS and precipitated a second time with 33% (NH4) 2SO4 or with 12-15% Na2SC4. This step may be repeated. The pellet formed is re-dissolved in PBS and excess salt is removed by gel filtration through a desalting matrix or by exhaustive dialysis against PBS.

The purified monoclonal antibody 110-1 is coupled to cyanogen bromide sepharose. pow ^ ^l ^ Ma axis ^C us ^EP Including t ^o in solution ^{1: 0} ~ ³ M ^HC1 on a glass filter (g freeze-dried material gives a final gel volume approximately 3.5 ml), and washed for 15 minutes with the same solution, and the antibody is then added immediately. In general, the coupling reaction is most effective at a pH of 8-10, but lower pH may be used if necessary to maintain the stability of the antibody. The antibody should be dissolved in PBS or in a high ionic strength carbonate / bicarbonate or borate buffer containing 150 mM NaCl. The activated Sepharose and antibody suspension is gently stirred for 2-4 hours at room temperature or overnight at 4 ° C, then on a sintered glass filter with a coupling buffer. All remaining active groups were blocked by the 1.0M treatment

161 520 with ethanolamine for pH 8 for 2 hours. Final product - antibody - sepharose is washed 4 to 5 times in turns with high and low pH buffer solutions (borate buffer, 0.1 M, pH 8.5, 1 M NaCl and acetate buffer, 0.1 M, pH 4 0.1 M NaCl). This washing removes residual covalently non-adsorbed materials. The resulting immunoaffinity separation matrix is stored below 8 ° C in the presence of a suitable bacteriostatic agent, such as 0.01% azide.

The addition of a suspension of the produced protein to such a matrix selectively removes the gpl 10 antigen. The mixture is allowed to stand for 2 to 24 hours, preferably 12-18 hours with slow stirring or shaking. A column may also be used, in which case the immunoaffinity matrix is loaded onto the column, equilibrated, and the protein suspension is slowly fed. After the protein suspension is administered, the flow through the column is stopped to obtain maximum immune complex formation.

Unbound material is either washed off or separated by exhaustive elution with an adsorption buffer. A sintered glass filter with a vacuum or column flow-through can be used. Unbound material is then eluted using both low and high pH buffers (acetate buffer pH 4.0 or borate buffer pH 8.56) or a chaotropic agent.

Example VI. Purification of recombinant gpl10 obtained from a mammalian expression system by immunoaffinity method.

Monoclonal antibodies find use in the immunoaffinity purification of recombinant gpl10 protein expressed from mammalian cells. Mammalian cells are infected with recombinant vaccinia (Mackett et al., J. Virol. 49, 857 (1984), containing sequences encoding at least a portion of the gp 110 protein which is an antigen and causes the production of neutralizing antibodies.

Recombinant vaccinia is prepared as described in U.S. Patent No. 842,984. In this method, sequences encoding the HIV coat glycoprotein are inserted into a plasmid vector (pGS20) after the vaccinia transcription control element. This chimeric gene is flanked by sequences encoding the thymidine kinase (TK) gene.

Chimeric plasmid vectors containing a vaccinia virus promoter ligated to the LAV coat gene are used to transform the E. coli MC1000 strain. The insertion of the chimeric LAV-env sequences into the vaccinia virus genome was accomplished by in vivo recombination, made possible by the fact that the chimeric genes in the pv-env5 plasmids are flanked by vaccinia virus sequences encoding the TK gene. The plasmid is then introduced into cells previously infected with wild type vaccinia virus, whereby recombination occurs between the TK sequences in the plasmid and the homologous sequences in the vaccinia viral genome, resulting in the insertion of the chimeric gene. African green monkey kidney cells (BSC-40 strain BSC-1.ATCC cell line No. CCL26) were used as the host in the expression system.

BSC-40 cells are infected with a multiplicity of recombinant vaccinia virus. 12 hours after infection, cells are harvested, washed once in PBS and centrifuged. Cell pellets are resuspended in cell 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 lysate cell is clarified by centrifugation. The immunoaffinity separation of the expressed recombinant fusion protein is performed as described above for a bacterial expression system using the gpl 110-1 monoclonal antibodies. Proteins produced in this expression system are more closely related to the naturally produced HIV gpl10 proteins due to the fact that glycosylation processes take place in mammalian cells.

Example VII. Inhibition of HIV infection with blocking peptides.

The effectiveness of the blocking peptides in inhibiting infection of a tissue cell culture with the HIV strain LAVbru was tested using a modified T-peptide evaluation method published by Petr et al., Supra. A preferred attempt to inhibit HIV infection is to combine equal volumes of blocking peptide and CEM cells (2.5 X 10 ⁵ ) in media (RPMI, 10% FSC and 2mg / ml polybrene) and incubation for 45 minutes at 37 ° C.

161 520

The virus is then added at various doses (10, 50, 500 TCID 50), the mixture is incubated for 14 days at 37 ° C, and the supernatant is then analyzed for viral antigen production (e.g. p25 core).

Pre-incubation of CEM cells with the peptides before adding the virus to the culture enhanced the inhibitory effect seen in the tests performed. This inhibition appears to be dose-dependent: at low and medium doses, high inhibitory activity is observed, but at the highest doses of virus, this activity is lower.

In the T peptide assays, about 60 to 90% inhibition of the production of viral antigens was achieved at low viral doses. Additional experiments with X-XV peptides generally after amidation of the free carboxyl terminus and after acylation of the free, terminal amino group gave similar results. On the other hand, peptide XI was particularly effective over a wide dose range. To achieve additional or synergistic inhibition of the production of viral antigens, neutralizing antibodies can be added either during preincubation or during virus addition.

From the above examples, it can be seen that the monoclonal antibodies and peptides, including the blocking peptides of the invention, allow for improved methods of neutralizing and / or inhibiting HIV infection. They enable the development of prophylactic and therapeutic compositions effective against infections with most, if not all, strains of HIV. In addition, the new materials find use in diagnostic samples and other known procedures.

The present invention has been described in detail for illustration and the examples are provided to aid understanding thereof, and it is obvious that changes and modifications may be made within the scope of the appended claims. Data on the deposit of microorganisms. The following microorganisms produced and used in the present invention are deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockwille, Maryland 20852, uSa.

Data on deposits are given below:

Scientific name Name given by the depositor. ATCC Number Date of deposit

Hybrid mouse cells

HIV-gp 110-1 HIV-gp 110-2 HB9175 HB 9176 August 30, 1986 at HIV-gp 110-3 HB9177 at at HIV-gp 110-6 HB 9404 April 30, 1987 at HIV-gp 110-4 HB 9405 at HIV-gp 110-5 HB 9406 at HIV-p 25-2 HB 9407 at at HIV-p 25-3 HB9408 at HIV-p 25-6 HB 9409 at at HIV-p 25-7 HB1M10 at

Hybrid HB 9175, HB 9176 and HB 9177 cells were tested and found viable on August 26, 1986. The remaining cells were tested and found viable on May 4, 1987.

Publishing Department of the UP RP. Circulation of 90 copies. Price: PLN 10,000

Claims (4)

Patent claims A method of producing a new peptide having at least 5 contiguous amino acids from the amino acid sequence of HIV gpl10 or homologues of this sequence of formula II (29a): 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, characterized by expressing HIV nucleic acid sequences encoding amino acids 301 to 336 of LAVbru gp 110 isolate or homologues thereof. 2. The method according to p. The method of claim 1, wherein in the produced peptide, the sequence of contiguous amino acids defines at least one antigenic determinant which causes the production of antibodies against HIV infection in the host after immunization of the host. 3. A method of producing a new peptide having one of the following amino acid sequences of HIV gp 110 or homologues of these sequences: 1 (29) Y-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Y ', V (177) Y-Thr-Arg-Lys-Ser-Ile-Tyr-Ile-Gly-Pro-Gly-Arg-Ala-Phe-His-Thr-Thr-Gly-Arg-Ile-Gly-Y ', or VIII (110- 2-2) Y-Lys-Thr-VallLys-IIe-Nor-Leu-Nor-Ser-Gly-His-Val-Phe-His-Ser-His-Tyr-Gln-Pro-Y 'where Y and Y', if present , these independently mean amino acid sequences up to 20 amino acids long, characterized by expressing the env regions of the HIV nucleic acid sequence encoding amino acids 308 to 328, respectively, of the LAVbru isolate, amino acids 306 to 323 of the ARV-2 isolate and amino acids 311 to 330 of the LAV isolate -2. 4. The method according to p. The process of claim 3, wherein the peptide is prepared in which Y and Y 'are independently a bonding moiety consisting mainly of glycine, tyrosine, cysteine, lysine, glutamic acid or asparagine.