BE1000811A4 - Monoclonal Antibodies, PEPTIDES AND COMPOSITIONS CONTAINER FOR THE DIAGNOSIS AND TREATMENT OF HIV INFECTION BY VIRUSES. - Google Patents

Abstract

Monoclonal antibodies, peptides and compositions useful for the treatment of HIV virus infections. The compositions according to the present invention comprise a therapeutically effective dose of at least one monoclonal antibody reacting with one or more neutralizing regions of HIV, optionally one or more blocking peptides capable of attenuating the infectious power of HIV and / or reactive monoclonal antibodies. with epitopes of these peptides, as well as a pharmaceutically acceptable excipient.

Description

       

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  Monoclonal antibodies, peptides and compositions containing them, intended for the diagnosis and treatment of infections by the HIV virus
The present invention relates generally to the diagnosis, treatment and prevention of viral infections.



   It relates more particularly to compositions and methods for the production of monoclonal antibodies as well as peptides useful in the diagnosis, neutralization and vaccination against infections with Human Immunodeficiency Virus (Human Immunodeficiency Virus).



   The infectious agent responsible for acquired immunodeficiency syndrome (AIDS) and its prodromal phases, the AIDS-related complex (ARC), and lymphadenopathy syndrome (LAS), is a new lymphotrophic retrovirus. . The virus has been variously called LAV, HTLV-III, ARV, and more recently HIV.



  Since the HIV virus is reaching pandemic proportions, the treatment of infected individuals and the prevention of transmission to exposed and uninfected individuals is of fundamental interest. Various therapeutic strategies have targeted different stages in the life cycle of the virus and are emphasized by Mitsuya and Broder, 1987, Nature 325: 773.



   One approach is to use antibodies that bind to the virus and inhibit viral replication, either by interfering with entry of the virus into host cells, or by other mechanisms. Once the component (s) of the virus susceptible or sensitive to the intervention of the antibody are identified, it is hoped that antibody titers sufficient to neutralize the infectious power of the virus can be generated by vaccination or, alternatively,

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 by passive administration of immunoglobulins or monoclonal antibodies of desired specificity.



   The envelope glycoproteins of most retroviruses are believed to react with receptor molecules on the surface of susceptible cells, allowing the infectivity of the virus to be determined for certain hosts. Antibodies that bind to glycoproteins can block the virus’s interaction with cell receptors, neutralizing the virus’s infection power. See generally, The Molecular Biology of Tumor Viruses. 534 (J. Tooze, ed., 1973) and RNA Tumor Viruses, 226, 236 (R. Weiss et al., Eds., 1982) these two documents being cited here in their entirety for reference. See also,
Gonzalez-Scarano et al., 1982, Virology 120: 42 (La Crosse Virus); Matsuno and Inouye, 1983, Infect.

   Immun. 39: 155 (Neonatal Calf Diarrhea Virus); and Mathews et al. 1982, J. Immuno1., 129: 2763 (Encephalomyelitis Virus).



   The general structure of an HIV virus is that of a ribonucleoprotein nucleus surrounded by an envelope containing lipids which the virus acquires during budding from the membrane of the infected extractor cell. Housed inside and projecting from the envelope are the virus encoded glycoproteins.



  The HIV envelope glycoproteins are initially synthesized in the infected cell in the form of a 150,000-160 precursor molecule. 000 daltons (gpl50 or
 EMI2.1
 gp160), which is then processed in the cell to form an N-terminal fragment of 110,000-120. 000 daltons (gpl10 or gpl20) in order to generate the external glycoprotein, and a C-terminal fragment of 41,000-46. 000 daltons (gp 41), which represents the transmembrane envelope glycoprotein.



   For the reasons given above, the glycoprotein
 EMI2.2
 HIV virus gpl10 has been the subject of important research as a potential target for interrupting the life cycle of the virus. It has been shown that sera from infected individuals

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 EMI3.1
 by HIV neutralized HIV in vitro, and that antibodies which bind to purified gp110 are present in the sera.



  See Robert-Guroff et al., 1985, Nature 316 Weiss et al., 1985, Nature 316 and Mathews et al., 1986, Proc. Natl. Acad. Sei. U. The recombinant and purified gap110 molecule can stimulate the production of neutralizing serum antibodies when used to immunize animals, see Robey et al., 1986, Proc. Natl.



  Acad. Sei. U. others, 1986, Science 233 and to immunize a human being, see Zagury et al., 1986, Nature 326 The binding of the gp110 molecule to the (T4) CD4 receptor has also been described, and of monoclonal antibodies which recognize certain epitopes of the CD4 receptors have been described as blocking HIV binding, syncytial formation and infectious power. See McDougal et al., 1986, Science 231 Putney et al. (1986, Science 234 have found antibodies to neutralizing sera in animals after immunization with a recombinant fusion protein containing half of the carboxyl-terminal of the gpl10 molecule and in addition have demonstrated that glycosylation of the envelope protein is not necessary for a neutralizing antibody response.



  An AIDS subunit vaccine using the HIV gp110 molecule or parts thereof may thus be desirable. Vaccines forming subunits are an alternative to vaccines prepared from attenuated or inactive viruses. Inactivated vaccines are of concern given the possible defect that kills all viral particles, and may have the power to mutate and regain their ability to cause disease. With subunit vaccines, only those parts of the virus that contain antigens or epitopes that are capable of causing immune responses, i.e. neutralizing antibodies, ADCC, and a cytoxic T cell response , are used

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 to immunize the host.

   A major advantage of subunit vaccines is that uninteresting viral material is excluded
Viral subunits for use in a vaccine can be produced by several methods. For example, the envelope glycoprotein can be expressed and purified from a bacterial host, although this molecule may be deficient in most post-translational modifications (such as glycosylation), or from other methods. Such a modification can be achieved using a eukaryotic expression system, such as yeast or cultured mammalian cells.



  Virus genes have been introduced into mammalian cells using the vaccine virus as a vector.



  See, for example, Mackett M. et al., 1982, Proc. Nat. Acad.
 EMI4.1
 Sei. USA 79; Panicali, 0. and Paoletti, E.,
Nat. Acad. Sci USA 79: 4927. A recombinant vaccine virus can be constructed according to the method of Hu et al., Nature 320: 537 (1986) or Chakrabarti et al., Nature 320: 535 (1986), both of which are given here as reference. In these systems, viral glycoproteins produced by cells infected with recombinant vaccines are appropriately glycosylated and can be transported to the "cell surface" for extrusion and ultimate isolation.



   An important phase in the production of a subunit vaccine is the adequate purification of the desired glycoprotein from the complex mixture of the expression system.



  Several methods can be used to accomplish the
 EMI4.2
 purification. These include, but are not limited to, preparative polyacrylamide gel electrophoresis, gel permeation chromatography, and various chromatography methods (i.e., ion exchange, reverse phase, immunoaffinity, interaction hydrophobic), as well as other methods. Most of these methods are used according to

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 EMI5.1
 various combinations for substantially pure preparations (Kleid, O. et al., 1981, Science 214 Cabradilla, C. et al., 1986, Biotechnology 4 Dowbenko, D. Proc. Nat. Acad. Sci. USA 82 these articles being given here at reference title.



  Methods that reduce the number of phases required to achieve maximum purification of a particular viral from a complex expression mixture are needed to make subunit vaccines. Efficient separation of antigens from foreign components could be achieved using chromatography of innunoaffinite. This technique, legally known under the name in principle to selectively adsorb an antigen on a solid support on which a specific antibody has been covalently attached. The antigen selectively adsorbs is then eluted from such an antibody affinity adsorbent, for example by changing the pH and / or ionic strength of the buffer.



  Polyclonal antibodies, obtained from animals immunized with the desired antigen or from naturally infected or contaminated individuals (see for example Lasky et al., Supra), have been used frequently as immunoadsorbents, but, in general, these reagents with substantial drawbacks, such as (i) the antibodies bound to the insoluble support are not all specific for the molecule of interest, which requires additional purification (ii) the yields of the desired antigen are frequently low and ( (ii) affinities for the antibody often vary from preparation to preparation, which requires modifications in the elution processes.

   The use of monoclonal antibodies specific for the desired viral antigen and intended for use in the preparation of subunit vaccines, rather than polyclonal antibodies, should overcome these difficulties. Murine monoclonal antibodies which bind to

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    HIV antigens have been described. Several research groups have described monoclonal antibodies specific for the p25 nucleus protein (see for example di Marzo Veronese et al., 1985, Proc. Nat. Acad. Sci. USA 82: 5199 and
Chassagne, J., et al., 1986, J. Immunol. 136: 1442). Monoclonal antibodies specific for the membrane glycoprotein gp41 have also been described (see for example di Marzo Veronese et al., 1985, Science 229: 1402).



   It remains that there is a need in the art concerned for specific monoclonal antibodies for fear of epitopes in well defined regions of the important envelope glycoprotein, gpl10. Monoclonal antibodies which bind to these regions and cause a reduction or elimination of the replication and transmissibility of the HIV virus would present substantial prophylactic and therapeutic utility. Furthermore, monoclonal antibodies could also be used to purify the desired region of gpl10 from disrupted or broken viruses, or from recombinant expression systems for use in vaccines, for example.

   Furthermore, the region containing the epitope or epitopes recognized by the monoclonal antibodies could be chemically synthesized, which avoids the difficulties inherent in the purification and administration of the larger fragments of the gp110 molecule. The present invention meets the above needs as well as others.



   The present invention provides peptides capable of imitating or mimicking immunologically epitopes immunologically.
 EMI6.1
 HIV protein binders, nucleic acid coding probes of such peptides and monoclonal antibodies reacting with such peptides, as well as other peptides interfering with the infectious power of HIV. These new materials find a use for example in diagnostic tests for the detection of HIV infections and in therapeutic actions for the treatment of or vaccination against

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 such infections.



   The present invention relates to new compositions and new methods for neutralizing HIV infections, that is to say for the prevention or substantial inhibition of the formation or trans-
 EMI7.1
 cellular mission of HIV infections in a hood. More particularly, peptides mimicking an HIV neutralizing region and monoclonal antibodies reacting with such a region are used to diagnose, treat and vaccinate against HIV infections.

   In this regard, the expression "neutralizing region" indicates the parts of HIV, in particular the HIV proteins, containing amino acid segments defining one or more epitopes reacting with antibodies which, either individually or in combination with other antibodies of the present invention are capable of neutralizing HIV infections. Tests suitable for neutralization are well known and may include reducing HIV infection in T cell lines, reducing VSV (HIV) pseudotype plaque-forming units carrying HIV envelope glycoproteins, assays syncytial inhibition, and virion receptor binding assays.

   If desired, neutralizing activity can be compared to antibody reactivity
 EMI7.2
 in immunochemical tests such as the immunofluorescence test, radioimmunoprecipitation immunoblot.



  According to one aspect of the invention, the new peptides, which typically have less than about 50 amino acids, contain five or more contiguous amino acids forming epitopes substantially similar to epitopes located on the neutralizing regions of gpl10 or HIV p25, encoded by the env and gag regions, respectively, of the HIV genome.

   The regions of particular interest are those which extend from about 301 amino acid residues to about 336 gp110, and from about 278 to about 319 and from about 315 to about 363 of p25, all

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 EMI8.1
 from the HIV strain designated LAV-- .. of acid residues from the Data Designations Bank of Los Alamos (Sequence virus database
AIDS. Los Alamos National Laboratory, Theoretical Division,
Los Alamos, NM 87545).



   Those familiar with the art will appreciate that additional analogous regions ("homologs") from other HIV isolates can be identified based on their location within the corresponding proteins from various isolates. In practice, such homologs can be identified by reference to LAVBRU sequence data, as follows: (a) The amino acid sequences of HIV isolates and LAVBRU can be aligned to obtain maximum homology between the two sequences; (b) Peptides comprising amino acid sequences of HIV isolates corresponding to the site of the peptides of
 EMI8.2
 LAV immunologically mimic the LAV oHU DrtU proteins can be identified.

   Peptides comprising amino acid sequences of HIV isolates so identified typically mimic or mimic immunologically, etc. this, the corresponding HIV isolate proteins.



   This method can be applied to HIV strains which are still to be discovered. For example, when new strains of HIV are identified, their core and envelope amino acid sequences can be aligned with that of LAVBRU to achieve maximum homology. The methods, by which the sequences are aligned, are known to those familiar with the art. By aligning the sequences, it is desirable to maintain as much homology as possible between the cysteine residues. The amino acid sequence of the new HIV species or strain, which corresponds to the situation or site of the peptides specifically described herein, can be synthesized and used according to the present invention.

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   Another method for determining the sequences of a homologous region in other HIV strains is described by
Scharf et al., Science (1986) 233: 1076. It uses two oligonucleotide primers which bind to sequences conserved outside the region of sequence of interest and contain different restrictive sites in each primer. AON from HIV strains can then be amplified in vitro thereafter, and the resulting oligonucleotides can be cloned into vectors for sequence analysis, and incorporated into a vaccine in the form of a cassette representing a particular epitope. of the HIV strain.



   It is not necessary for the present invention that the epitopes contained within such sequences be reactive by cross-reaction with antibodies for all strains or species of HIV. Peptides encompassing immunological epitopes which distinguish one species or serogroup from another, find utility in the identification of particular species or serogroups, and may in fact assist in the identification of individuals infected with one or more species or serogroups of HIV.



  They may also be useful in combination with other peptides from either a homologous region or another neutralizing region, in therapeutic compositions.
 EMI9.1
 



  The peptides of interest very preferably come from the g110 region of the virus. Peptides of particular interest in this region are the peptides encoded in the open reading frame env extending from about the base pair (bp) 6667 to about 6774 of
 EMI9.2
 isolate LAV., others o HU o ,,. Thus, various homologous regions of HIV isolates include the homologous sequences obtained from the Los Alamos Database (except LAV2), as set out in Table I.

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   TABLE 1.
 EMI10.1
 
 <tb>
 <tb>



  MXB2 <SEP> TCTACAACACCCAACAACAATACAACAAAAACA ............... <SEP> ATCCGTATC <SEP>
 <tb> CysThrArgProAsnAsnAsnThrArgLysArg ............... IleArgIle <SEP> 309
 <tb> BH <SEP> 102-Serr --------------- 309 <SEP>
 <tb> BH8 <SEP> ------------------------------ Lys ---------------- ------- <SEP> 309
 <tb> HSB3 <SEP> ------------------------------ Lys ---------------- ------- <SEP> 309
 <tb> H9M <SEP> ------------------------------ Ser ---------------- ------- <SEP> 309
 <tb> BRU <SEP> ------------------------------ Ser ---------------- ------- <SEP> 314
 <tb> MAL <SEP> ----------- Gly ------------- ArgGly ----------------- HisPhe <SEP> 314
 <tb> ELI <SEP> --- Als ------ TyrGly --------- Gly ----------------- ThrPro --- <SEP> 310
 <tb> ARV2 <SEP> ------------------------------ Ser ---------------- -Try --- <SEP> 312
 <tb> WMJ2 <SEP> ------------ Tyr ------ Val --- ArgSer -------------- LeuSer --- <SEP> 306
 <tb> RFENV <SEP>

  ------------------------------ Ser ----------------- ThrLys <SEP> 322
 <tb> Z6 <SEP> ------------ TyrLys --------- GlySer ----------------------- <SEP> 311
 <tb> Z3 <SEP> ------------ GlySerAspLysLysIle ----------------- GlySer --- <SEP> 306
 <tb> NY5 <SEP>
 <tb> ----- CDC42 <SEP> ------------------ His ------------ ValThrLeu ..............

    <SEP> 320
 <tb> LAV2 <SEP> ------------ Gly --- Lys --- Val --- Gln ----------------- MetLeu <SEP> 302
 <tb> HXB2 <SEP> CAGAGA ......... GGACCAGGGAGAGCATTTGTTACAATAGGAAAAATAGGAAATATG
 <tb> GlnArg ......... GlyProGlyArgAlaPheValThrIleGlyLysIleGlyAsnMet <SEP> 326
 <tb> BH102 <SEP> ----------------------------------------------- ----------- <SEP> 326
 <tb> BH8 <SEP> ----------------------------------------------- ----------- <SEP> 326
 <tb> HXB3 <SEP> ----------------------------------------------- ----------- <SEP> 326
 <tb> H9M <SEP> ----------------------------------------------- ----------- <SEP> 326
 <tb> BRU <SEP> ----------------------------------------------- ----------- <SEP> 331
 <tb> MAL <SEP> ......---------------- Gln --- LeuTyr --- Thr --- IlaVal --- AspIle <SEP> 329
 <tb> ELI <SEP> ClyLet ----------... GlnSerLeuTyrThr --- Arg --- IleValSerArgSer <SEP> 323
 <tb> ARV2 <SEP>

  ......------------------------- His --- Thr --- Arg --- IleGlyAsp <SEP> 327
 <tb> WMJ2 <SEP> ......------------------------- Arg --- ArgGlu ...--- IleGlyIle <SEP> 320
 <tb> RFENV <SEP> ......------------------- ValIleTyrAlaThr --- Gln --- IleGlyAsp <SEP> 337
 <tb> Z6 <SEP> GlyLeu ------------... GlnAlaLeuTyrThr --- Arg --- ArgThrLysIleIle <SEP> 327
 <tb> Z3 <SEP> ArgIle ------------------ LysVal --- TyrAlaLys --- Gly ............. <SEP> 319
 <tb> NY5 <SEP> GlyPro ------------...------ thrLeuTyrAlaArgGlu --------- AspIle <SEP> 320
 <tb> CDC42 <SEP> ...............------------ ValTrpTyr --- Thr --- Glu --- LeuGlyAsn <SEP> 335
 <tb> LAV2 <SEP> MetSer ------------------ HisVal --- HisSerHisTyrGlnProIle --- Lys <SEP> 323
 <tb> HXB2 <SEP> ... AGA ... CAAGCACATTGT
 <tb> ... <SEP> Arg ...

    <SEP> GlnAlaHisCys <SEP> 331
 <tb> SH102 ------------ 331
 <tb> BH8 <SEP> ----------------------- <SEP> 331
 <tb> HXB3 ------------ 33t
 <tb> H9M <SEP> ----------------------- <SEP> 331
 <tb> BRU ------------- 330
 <tb> --------- Arg --- Tyr - 33 <SEP>
 <tb> Ml <SEP> IlelleGly ------- <SEP> 330 <SEP>
 <tb> ARV2 <SEP> Ile --- Lys ...----- 333
 <tb> WJ2 <SEP> Ile ---------- 326
 <tb> RFENV <SEP> Ile --- Lys ...------ 33
 <tb> Z6 <SEP> Cly ...--------- 334 <SEP>
 <tb> Z3 <SEP> IleThrGly --------------- <SEP> 326
 <tb> NY5 <SEP> ----------------------- <SEP> 325
 <tb> CDC42 <SEP> Ile -------------------- <SEP> 341
 <tb> LAV2 <SEP> ArgProArg --- Mec - 330 <SEP>
 <tb>
 

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   Other peptides suitable for generating or obtaining per "screening" of monoclonal antibodies,

   include these codes in the open reading frame approx from around bp 7246 to around 7317 of LAVBRU. Such reactive antibodies and peptides are particularly useful in immunization tests.



   In region g of the LAV- isolate, the amino acid sequences p25 from about 278 to 319 and from 315 to 363 are additional neutralizing regions of HIV. Those familiar with this technique may understand that additional HIV neutralizing regions can be identified based on the teachings given here; in particular, combinations of monoclonal antibodies reacting with various different HIV epitopes exhibit neutralization activity.



   Peptide I, also called peptide 29, is encoded in the reading frame open approx from a number of
 EMI11.1
 in acids and will present the following sequence of amino acids, where the following included oligopeptides will include (29) Y-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-GlyPro-Gly-Arg- Ala-Phe-Val-Thr-Ile-Gly-LysIle-Y 'in each When Y amines from enviran 308 to about 328, for example, one or more amino acids from sequences which are available alongside amino acid residues 308 to 328 of the sequence of the HIV envelope or of any part of these lateral or arranged sequences.

   AT
 EMI11.2
 as an example parts of BRU

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 from about a number of residues 301 to 307; and it can comprise all or parts of the LAV BRU envelope amino acid sequence from a number of residues of approximately 329 to 336 as follows:
II (29a)
 EMI12.1
 Cys-Thr-Arg-Pro-Asn-Asn-Asn-Thr-Arg-Lys-Ser-IleArg-Ile-Gln-Arg-Gly-Pro-Gly-Arg-Ala-Phe-Val-ThrIle-Gly-Lys- Ile-Gly-Asn-Met-Arg-Gln-Ala-His-Cys. Alternatively, truncated sequences of peptides according to the present invention can be prepared.

   In this regard, the following sequences of peptide 29 may be particularly useful:
III (29b)
Y-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-Pro-Gly-
 EMI12.2
 Y 'in which Y and / or Y', if present, each represent sequences of up to about twenty acid residues IV (29c) Y-Ile-Gln-Arg-Gly-Pro-Gly-Arg -Ala-Phe-Val-Thr- amines. Ile-Gly-Lys-Ile-Y 'in which Y and Y', if present, each represent sequences of up to about twenty residues
 EMI12.3
 acids
In another embodiment, homologous regions of the ARV-2 isolate of particular interest are encoded in the open reading frame env from numbers of amino acid residues from about 306 to about 323 and typically have the following amino acid sequences,

   where the oligopeptides included in the following amino acid sequence include linear epitopes within such a sequence:
V (177)
Y-Thr-Arg-Lys-Ser-Ile-Tyr-Ile-Gly-Pro-Gly-Arg-
Ala-Phe-His-Thr-Thr-Gly-Arg-Ile-Y '

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 wherein Y and Y ', if present, each represent from one to about twenty or more amino acid residues. If Y and / or Y 'are present, they may comprise one or more amino acid residues of sequence which are arranged next to amino acid residues 306 to 323 of the ARV-2 envelope sequence or any part of these lateral or adjacent sequences.



  In particular, Y may include all or parts of the HIV envelope amino acid sequence from about numbers of residues 299 to 306; It can include all or parts of the HIV envelope amino acid sequence from residue numbers 324 to 333.



   Alternatively, truncated peptide V sequences can be prepared. In this regard, the following sequences can be particularly useful:
VI (177a)
 EMI13.1
 Y-Thr-Arg-Lys-Ser-Ile-Tyr-Ile-Gly-Pro-Gly-Y 'and VII
Y-Asp-Cys-Lys-Thr-Ile-Leu-Lys-Ala-Leu-Gly-Pro-
Ala-Ala-Thr-Leu-Glu-Glu-Norleu-Norleu-Thr-Ala-
Cys-Y 'in which Y and / or Y', if present, each represents sequences of up to twenty or more residues
 EMI13.2
 acids
Another example includes homologous regions of the LAV-2 isolate, as encoded in the open reading frame env from about a number of amino acid residues 311 to 330. and typically have the following sequence:

  
VIII (110-2-2) Y-Lys-Thr-Val-Lys-Ile-Nor-Leu-Nor-Ser-Gly-His-Val-
Phe-His-Ser-His-Tyr-Gln-Pro-Y 'in which Y and / or Y', if present, each represents sequences of up to twenty or more amino acid residues. (See article in Nature 326: 662 (1987),

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   which is given here for reference).



  According to another aspect of the present invention, new cell lines capable of producing monoclonal antibodies and compositions comprising such
 EMI14.1
 antibodies are to selectively recognize 24 (from 10, 10 to about 10 or more) neutralizing regions contained in a predetermined sequence of proposed, which antibodies are capable of gpl10 or p25 envelope glycoproteins, their protein precursors, proteins biologically expressed recombinant fusion, and synthetic peptides which contain one or more epitopes in the region of predetermined sequence of gpl10 or p25.

   The hybrid cells which are the subject of the invention have an identifiable chromosome in which the germ line DNA has rearranged to encode an antibody having a binding site for an epitope on gpl10 or p25 common to some or all of the clinical HIV isolates. These monoclonal antibodies can be used in a wide variety of ways including diagnostics and therapy, as well as to identify other cross-reactive antibodies, such as blocking antibodies. Peptides or polypeptides containing the epitope or epitopes with which they react can find separate uses as immunogens for vaccines, or as therapeutic agents.



   Blocking peptides
According to another embodiment of the present invention, there are used, for joint use with the neutralizing monoclonal antibodies or the preceding peptides, additional peptides or antibodies which interfere with the HIV bond to the receptors in addition to moderate the infectious power of HIV. Oe preference. peptides called "blocking peptides" capable of inhibiting virus profiling, as well as monoclonal antibodies specific for the epitopes contained in such

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 blocking peptides, can be used to increase the effectiveness of treatments against HIV infections.



  The HIV blocking peptides typically correspond to the amino acid sequences of HIV which are believed to be essential for the attachment of the virus to a host cell, such as the amino acid residues encoded approximately 190 about 197 from LAV BRU and about 185 to about 192 from ARV-2 and HTLV-III (BH-10). These peptides include octapeptide T (Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr) and its various derivatives (see IX below) and analogs (for
 EMI15.1
 Example XI below) described by Pert et al. (1986, Proc. Natl. Acad. Sci. USA 83 is given here for reference) located on the envelope glycoprotein (gp110 or 120).



   For example, blocking peptides having the following sequences are of particular interest, preferably with acetylation of the NH2-terminal and amidation of the COOH-terminal:
IX (173D)
 EMI15.2
 X (186) Y-Thr-Thr-Asn-Tyr-Thr-Y '; XI (187) Y-Thr-Thr-Ser-Tyr-Thr-Y '; XII (188) Y-Thr-Asp-Asn-Tyr-Thr-Y '; XIII (189) Y-Asn-Thr-Ser-Tyr-Gly-Y '; XIV (190) Y-Asp-Thr-Asn-Tyr-Ser-Y '; XV (191) Y-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Y 'in which, for each peptide, Y and Y', if present, each comprise an amino acid sequence from up to about 20 amino acids.

   Epitopes or determinants

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   Antigenics in these peptides are typically defined by at least about five contiguous amino acids, and find use in, for example, mimicking naturally occurring HIV antigen sites to produce HIV reactive antibodies and vaccines.



   Monoclonal antibody production
The preparation of the monoclonal antibodies can be carried out by immortalizing the expression of the nucleic acid sequences which code antibodies specific for HIV, by introducing such sequences, typically cDNA coding for the antibody, in a host capable of being cultivated in a culture. The immortalized cell line can be a mammalian cell line which has been transformed by oncogenesis, by transfection, mutation or the like. Such cells include myeloma lines, lymphoma lines or other cell lines capable of supporting the expression and secretion of the antibody in vitro.

   The antibody may be a naturally occurring immunoglobulin from a mammal, produced by transformation of a lymphocyte, particularly a splenocyte, by means of a virus or by fusion of the lymphocyte with a neoplastic cell, for example a myeloma, to produce a hybrid cell line. Typically, the splenocyte will be obtained from an animal immunized against the HIV virus or a fragment thereof containing an epitopic site.



   Immunization protocols are well known and can vary widely to remain effective, however. See Goding, Monoclonal Antibodies: Principles and Practice, Academic Presse, 2nd edition (1986), which is given here for reference. A ruptured or broken virus, synthetic peptides, and bacterial fusion proteins which contain antigenic fragments of the gpl10 or p25 molecule can be used as immunogens. Preferably, the immunogen of the broken virus, peptides or

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   Recombinant proteins will be enriched in proteins or in its fragments containing the epitopes for which the cells 8 producing the antibody or the splenocytes are desired.



   More particularly, solutions containing extracts or lysates of broken or disrupted virus, or supernatants of biologically expressed recombinant proteins or disrupted or separated expression vectors, can be enriched in glycoproteins, if desired, using purification, such as, for example, polyacrylamide gel electrophoresis. Affinity purification by lectin is a practical and preferred method for purifying gpl10 and other glycoproteins, for example affinity purification using lens lectin.

   The extent to which glycoproteins are purified from solutions for use as immunogens can vary widely, i.e., less than 50%, usually or at least 75% to 95%. %, desirably from 95% to 99% and, more desirably, to absolute homogeneity.



   Once the proteins have been purified to the desired degree, they can be suspended or diluted in a physiological carrier suitable for immunization, or can be combined with an adjuvant. A preferred technique, for example, is the adsorption of proteins and their fragments onto lens lectin agarose or other macromolecular support for injection. Immunogenic amounts of antigen preparations enriched in HIV proteins, including glycoprotein gpl10 and p25 nucleus protein or antigenic fractions thereof, are injected, generally at concentrations in the range l ug to 20 mg / kg d 'host.

   Administration can be by injection, for example intramuscular, peritoneal, subcutaneous, intravenous etc. Administration can be done in one or more steps, usually at intervals of

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 one to four weeks. The immunized animals are checked for production of the antibody into the desired antigens, then the spleens are removed and the splenic B cells are isolated and combined with a cell line of myelomas or transformed.

   The transformation or the fusion can be carried out according to conventional manners, the fusion technique or combination being described in a large number of patents, for example the American patents NO 4,172,124;
NO 4,350,683; NO 4,363,799; NO 4,381,292; and NO 4,423,147.



   See also, Kennett et al., Monoclonal Antibodies (1980), and references cited in this article which are cited by reference, and still see Goding, supra.



   Immortalized cell lines can be cloned and sorted according to conventional techniques, and antibodies are detected in the supernatants which are capable of binding to the desired HIV viral proteins of gpl10 or p25, recombinant fusion proteins or synthetic peptides which contain the desired epitopic region.

   The appropriate immortalized cell lines can then be grown in vitro or injected into the peritoneal cavity of a suitable host for production of ascites fluid. By virtue of the availability of certain antibodies of the present invention which are known to be specific for epitopes contained for example in the regions coded by the genomic region
 EMI18.1
 LAV from about 6688 bp to about 6750 bp (peptide coding for HU 29), or from about 7246 bp to about 7317 (peptide coding for 36) (bp numbering according to Wain-Hobson et al., Cell 44: 9 1985, which is given here for reference). the supernatants can be selected in competition with the monoclonal antibodies object of the invention in a competition test.



  Thus, additional immortalized hybridoma cell lines with the desired binding characteristics can be readily produced from a variety of sources depending on the availability of antibodies.

  <Desc / Clms Page number 19>

 presents specific for the particular antigen. Alternatively, these cell lines can be combined with other neoplastic cells 8, where such other 8 cells can serve as recipients for the DNA of the genome encoding the antibody.



   Although neoplastic rodent, particularly murine B cells are preferred, other mammalian species can be used such as lagomorph, bovine, sheep, equine, porcine, avian or the like. Immunization of these animals can be easily accomplished and their lymphocytes, particularly splenocytes, can be obtained for fusion.



   The monoclonal antibody secreted by the hybrid or transformed cell lines can be any of the classes or subclasses of immunoglobulins, beautiful as
IgM, IgD, IgA, IgG., Or IgE. Since IgG is the most common isotype used in diagnostic tests, it is often preferred. Monoclonal antibodies can be used intact, or in the form of fragments, such as Fv, Fab, F (ab '), but usually intact.



   To attenuate the possible antigenicity in a human hole of a monoclonal antibody, derived from an animal other than a human being, chimeric antibodies can be constructed, in which the antigen-binding fragment of a molecule d immunoglobulin (variable region), is linked by a peptide bond to at least part of another protein not recognized as foreign by humans, such as the evacuated part of a human immunoglobulin molecule. This can be achieved by merging or combining regio exons. svariables of animal with exons of gamma or kapa constant regions of a human being.

   Various techniques are known by those familiar with the technique, such as those described in international patent application NO 86/01533, and in European patent applications 171496 and 173494, which are given here as

  <Desc / Clms Page number 20>

 reference.



  Pharmaceutical formulations and use
The monoclonal antibodies of this invention which
 EMI20.1
 exhibit neutralizing activity, such as those which react with an epitopic site on gpl10 or p25 or which react with a blocking peptide, can also be incorporated as constituents of a pharmaceutical composition to attenuate the infections by HIV. The composition must contain a prophylactic or therapeutic amount of at least one of the monoclonal antibodies of this invention with a pharmaceutically effective carrier. The pharmaceutical support must be constituted by any compatible, non-toxic and suitable substance to allow the administration of monoclonal antibodies to the patient.

   Sterile water, alcohol, fatty products, waxes and inert solid products can be used as a carrier. Pharmaceutically acceptable adjuvants (buffering agents, dispersing agents) can also be incorporated into the pharmaceutical composition. Such compositions may contain a single monoclonal antibody so as to be, for example, specific for HIV strains with envelope glycoproteins containing an epitopic site in a region encoded by 6688 bp-6750 bp.



  Alternatively, the pharmaceutical composition can contain one or more monoclonal antibodies to form a "cocktail". For example, a cocktail containing monoclonal antibodies against various strains of HIV would constitute
 EMI20.2
 a universal product with prophylactic or therapeutic activity against the vast majority of clinical HIV isolates.

   The cocktail may contain monoclonal antibodies which bind to HIV proteins or glycoproteins other than gpl10 or p25, such as, for example, glycoprotein gp41 or integrase / p34 molar ratio of the various constituents of monoclonal antibodies will usually not differ by more than a factor of 10 plus

  <Desc / Clms Page number 21>

 usually not more than a factor of 5, and will usually be between about 1: 1-2 for each of the other antibody constituents.



   The monoclonal antibodies of the present invention can be used in the form of compositions administered separately and given in conjunction with other anti-retroviral agents, including blocking peptides.



   The current state of development of anti-retroviral agents, and anti-HIV agents in particular, is described in the publication by Mitsuya et al., Nature 32b: 773-778, 1987, which is given here for reference.



   Monoclonal antibodies, peptides and pharmaceutical compositions thereof according to the present invention are particularly useful for oral or parenteral administration.



   Oe preferably, the pharmaceutical compositions can be administered parenterally, that is to say by subcutaneous, intramuscular or intravenous.



   Thus, this invention provides compositions for parenteral administration, which comprise a solution of the monoclonal antibody, a peptide or a cocktail thereof, dissolved in an acceptable carrier, preferably an aqueous carrier. A wide variety of aqueous carriers can be used, for example water, buffered water, 0.4% saline, 0.3% glycine or the like. These solutions are sterile and generally free of particles. These compositions can be sterilized by conventional and well known sterilization techniques.



  The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximately satisfy physiological conditions, such as buffering and pH adjusting agents, toxicity adjusting agents or the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate etc. The concentration of

  <Desc / Clms Page number 22>

 the antibody in these formulations can vary widely, i.e., less than about 0.5%, usually
 EMI22.1
 ment at or at least about 1% up to as much as 15 or 20 by weight, and the concentration will be chosen essentially according to the volumes of fluid, viscosities, etc., preferably according to the particular mode of administration chosen.



   Thus, a typical pharmaceutical composition for intramuscular injection can be made up to contain 1 ml of sterile buffered water, at 50 mg of monoclonal antibody. A typical composition for intravenous injection could be made up to contain
250 ml of sterile Ringer's solution, and 150 mg of monoclonal antibody. Methods for preparing parenterally administrable compositions are well known to those familiar with the art and are described in more detail in, for example, the publication Remington's Pharmaceutical Science, 15th Ed. Mack Publishing Company, Easton,
Pennsylvania (1980), which is shown here for reference.



   The monoclonal antibodies and peptides of this invention can be lyophilized for storage and reconstituted in a suitable carrier or vehicle before use.



  This technique has been shown to be effective with conventional immune globulins and reconstitution and lyophilization techniques known in the art can be used. It will be appreciated by those familiar with the art that lyophilization and reconstitution can lead to various degrees of loss of activity of the antibody (eg with conventional immunoglobulins, IgM antibodies tend to have greater loss of activity than IgG antibodies) and that levels or proportions of use may require adjustment for compensation.



   The compositions containing the monoclonal antibodies

  <Desc / Clms Page number 23>

 of the present invention, the peptides or their cocktails can be administered for therapeutic treatment and / or
 EMI23.1
 prophylactic of HIV infections. In therapeutic applications, the compositions are administered to a patient already infected with HIV, in an amount sufficient to or at least partially stop the infection and its complications. An adequate proportion to achieve this is defined as a "therapeutically effective dose".

   Amounts effective for this use depend on the severity of the infection and the general condition of the patient's own immune system, but are generally between about 1 and about 200 mg of antibodies per kilogram of body weight, doses between 5 and
25 mg per kilogram being more commonly used. It should be borne in mind that the materials of this invention can generally be used in serious disease states, that is, in situations where life is threatened or potentially threatened. In such cases, it is possible and may be considered desirable by the treating physician to administer these antibodies in appreciable excess.



   In prophylactic applications, the compositions containing the present peptides, antibodies or their cocktails are administered to a patient who is not already infected with the HIV virus, but who may have been recently exposed or who is believed to have been exposed to a risk of infection with the virus or with the virus itself, so as to reinforce the patient's resistance to such a potential infection or so as to vaccinate him against the virus. The amount administered is defined by a "prophylactically effective dose".



  In this use, the precise amounts again depend on the patient's state of health and on his general immune state, but are generally between 0, 1 mg and 25 mg per kilogram, especially between 0, 5 mg and 2,5 mg per kilogram.



   Single or multiple administrations of

  <Desc / Clms Page number 24>

 compositions can be performed with doses and a protocol chosen by the attending physician. In all cases, the pharmaceutical formulations should supply an amount of the antibody or antibodies of this invention which are sufficient to effectively treat the patient.



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



   It is also possible that heteroagregates of monoclonal antibodies of the present invention and human T cell activators, such as monoclonal antibodies for CD3 antigen or the gamma F receptor on T cells, may allow human T cells or cells carrying Fc-gama (such as K cells or neutrophils) to kill HIV-infected cells via cytolysis via antibody dependent cells (antibody dependent cell-mediated cytolysis " : 'ADCC "). Such heteroagregates can be assembled, for example, by covalent binding of anti-HIV antibodies to anti-CD3 antibodies using the hetero-bifunctional reagent N-succinimidyl-3- (2-pyridyldithiol) propionate, as described by Karpowsky et al., J. Exp.

   Med. 160: 1686 (1984), this publication being given here for reference.



   The peptide compositions themselves which are the subject of this invention can also find therapeutic use, when the administration produces the reduction or elimination of the HIV virus in an infected host. These compositions. such as peptide 29, blocking peptides and peptide 126 can be administered in carriers

  <Desc / Clms Page number 25>

 or suitable physiological vehicles, intravenously, subcutaneously, intramuscularly, intraperitoneal, etc. Various vehicles include phosphate buffered saline, saline, water, potassium chloride, sodium lactate or the like.



   The concentration of peptides varies widely depending on its ultimate use, activity and method of administration.



   Preferably, the peptides comprise an amidation of the COOH-terminal, a formylation of the NH2-terminal or other pharmaceutically acceptable derivatives. Addition of blocking peptides to peptides mimicking a neutralizing HIV region and / or the specifically reactive antibodies of the present invention will provide significantly increased therapeutic efficacy. Other anti-HIV agents can also be included in the formulations (other than the monoclonal antibodies which bind the peptides) such as 3'-azido-3'-deoxythymidine, 2 ', 3'-dideoxycytidine, 2' , 3'-dideoxy-2 ', 3'-didehydrocytidine, etc.



  Use of monoclonal antibodies in the purification of immunoaffinity
Monoclonal antibodies specific for polypeptides containing gpl10 or other antigenic determinants, particularly the antigenic determinants obtained from biologically expressed recombinant fusion proteins or cultured HIV lysates or extracts, are particularly advantageous for use in the protocols of purification.

   Generally, antibodies have association affinities of the order
 EMI25.1
 8 12 from to Such antibodies can inter alia be used to purify recombinant proteins from the culture medium of the recombinant expression system if the expressed protein is secreted or from the constituents of the disrupted biological expression system if it is not secreted. Generally, monoclonal antibodies that are able to react with gpl10 or other antigenic determinants are attached

  <Desc / Clms Page number 26>

 or immobilized on a substrate or support.

   The solution containing the HIV antigenic determinants is then brought into contact with the immobilized antibody under conditions suitable for the formation of immune complexes between the antibody and the polypeptides containing the antigenic determinants gpl10. The unbound material is separated from the bound immune complexes, which antigenic complexes or fragments of gpl10 are then separated from the support.



   Typically, the monoclonal antibodies are crude purified from ascites fluid or cell culture supernatants before attachment to a support. Such processes are well known to those familiar with the art, and may include fractionation with high concentration neutral salts. Other methods. such as DEAE chromatography, gel filtration chromatography, preparatie gel electrophoresis, or protein A affinity chromatography, can also be used to purify the monoclonal antibody before its use as an immunoadsorbent.



   The support on which the monoclonal antibodies are immobilized must have the following general characteristics: (a) weak interactions with proteins in general to minimize non-specific bonds, (b) good flow characteristics which allow flow through of high molecular weight materials, (c) possession of chemical groups which may be active or modified to allow chemical binding of the monoclonal antibody, (d) physical and chemical stability under the conditions used to bind the monoclonal antibody and ( e) stability to the conditions and components of the buffers required for the adsorption and elution of the antigen.

   Some supports commonly used are agarose, polystyrenes derived. polysaccharides, polyacrylamide grains, activated cellulose, glass and the like. Various chemical methods exist for the attachment of antibodies to

  <Desc / Clms Page number 27>

 supports forming substrates. See generally Cuatrecasas, P.,
Advances in Enzymology 36:29 (1972). The antibodies of the present invention can be fixed directly to the support or, alternatively, via a link or spacer arm.



   The general conditions required for the immobilization of the monoclonal antibodies on the chromatographic supports are well known in the art. See for example
Tijssen, P. 1985, Practice and Theory of Enzyme Immunoassay, this publication is given here for reference. Current coupling processes depend slightly on the characteristics and type of the antibody to be coupled. Monoclonal antibodies have characteristics which are usually constant from mixture to mixture, which allows such conditions or characteristics to be optimized. Fixation typically occurs through covalent bonds.



   A suspension of extracts or lysates of HIV virus, the supernatant of a cultured biological expression system, or a suspension of ruptured or broken cells is then added to the separation matrix. The mixture is incubated under conditions and for a time sufficient for the formation of the immune complex, usually for at least 30 minutes, more usually for a period of 2 to 24 hours. Immune complexes containing
 EMI27.1
 polypeptides with antigenic parts of gpl10 are then separated from the mixture. Typically, the mixture is removed, for example by elution, and the bound immune complexes are washed thoroughly with adsorption buffer.

   The immune complexes can then be flowed from the Separation matrix using an eluent compatible with the particular support that is used, the-
 EMI27.2
 which eluents are well known to those familiar with the art. Also, polypeptides containing gpl10 or other antigenic fractions can be eliminated

  <Desc / Clms Page number 28>

 selectively. For example, peptides that contain an epitope recognized by antibodies can be used to compete with the binding site of the antibody, which is an alternative elution technique that can be performed under mild elution conditions. .

   The selectively adsorbed polypeptide containing the gpl10 antigen can be eluted from an antibody affinity adsorbent by altering the pH and / or ionic strength of the buffer. Chaotropic agents may also find use in the elimination of bound antigen. The selection of the chaotropic agent, its concentration and other elution conditions depend on the characteristics of the antibody-antigen interaction, but once determined, they should not usually be changed. required in polyclonal affinity separation systems.



   The selected material may require adjustment to physiological pH if low or high ionic strength or pH buffers are used to separate bound gpl10 antigens from the Separation matrix. Dialysis or gel filtration chromatography may also be required to remove excess salts used in the eluent so as to allow reconstitution of gpl10 or polypeptides containing antigenic fractions of gpl10 on nascent conformations.



   The methods of this invention provide, for example, substantially purified gpl10 or polypeptides containing antigenic fragments thereof, either produced naturally by cultures of infected cells or by
 EMI28.1
 expression systems for bacteria, yeast, or mammalian or cultured insect cells. The recombinants gp110 and the fragments or other purified proteins will typically be greater than 50% purity, more usually at least 75%, and frequently greater than 95-99%. These molecules can then find a use

  <Desc / Clms Page number 29>

 later in a wide variety of applications.



   The gpl10 HIV proteins, polypeptides containing antigenic fragments thereof, or other proteins substantially purified according to the methods of the present invention, can find use in a wide variety of applications, including subunit formulations of AIDS vaccine, in which the immunogen comprises an effective dose of antigenic determinants of, for example, gp110 or a neutralizing region of gpl10.

   Other constituents of the formulation may include
 EMI29.1
 antigenic fractions of HIV proteins or glycoproteins which stimulate the production of antibodies (preferably neutralizing antibodies) in an immunized hole, which antibodies are capable of providing protection against subsequent HIV infection.



   Uses for diagnosis of monoclonal antibodies
The monoclonal antibodies of the present invention are also useful for diagnostic purposes. They may be known or unmarked for this purpose. Typically, diagnostic tests allow the detection of the formation of a complex by means of the binding of the monoclonal antibody to an HIV antigen. If they are unmarked, antibodies find use, for example, in agglutination tests. In addition, unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) which react with the monoclonal antibody, such as antibodies specific for immunoglobulin. Alternatively, the monoclonal antibodies can be labeled directly.

   A wide variety of markers can be used, such as radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (especially hapten5) etc. Various types of immunoassays are available, and, by way of example, those described in
 EMI29.2
 U.S. patents 3,817,827 3,850,752 3,901,654

  <Desc / Clms Page number 30>

    ; 3935074; 3,984,533; 3,996,345; 4034074; and 4,098,876 which are given here for reference.



   Usually, the monoclonal antibodies and the peptides of the present invention are used in enzyme immunoassays where, for example, the antibodies object of the invention, or the second antibodies of a different species, are conjugated to an enzyme. When a biological sample containing HIV antigens, such as human blood serum, saliva, semen, vaginal secretions or a culture suspension of virus-infected cells, is combined with the antibodies, a bond occurs between the antibodies and molecules presenting the desired epitope.



   Such proteins or virus particles can then be separated from unbound reagents, and a second antibody (labeled with an enzyme) added. Subsequently, the presence of the antibody-enzyme conjugate specifically linked to the antigen is determined. Other conventional techniques well known to those familiar with the art can legally be used.



   Kits can also be produced for use with the antibodies in the detection of HIV infection or for the presence of the HIV antigen. Thus, the monoclonal antibody compositions according to the present invention can be provided, usually in lyophilized form, either alone or in conjunction with additional antibodies specific for other HIV epitopes.



  Antibodies, which can be conjugated to a marker or unconjugated, are included in the kits with buffers, such as Tris, phosphate, carbonate etc ..., stabilizers, biocides, inert proteins, for example 1 ' bovine serum albumin, or the like. Generally speaking, these materials will be present in a proportion of less than about 5K by weight based on the amount of active antibodies, and will usually be present in a total amount of at least about 0.001% by weight based on the antibody concentration.

   Frequently it is desirable

  <Desc / Clms Page number 31>

 including an inert diluent or excipient to dilute the active ingredients, where the excipient may be present in an amount of from about 1% to 99% by weight of the total composition. If a second antibody capable of binding to the monoclonal antibody is used, it will usually be present in a separate container. The second antibody is typically conjugated to a label and formulated in a manner analogous to the antibody formulations described above.



   The detection of p25 or gpl10 antigens, or of the whole virus, in various biological samples may find use in the diagnosis of a common infection with the HIV virus. Biological samples can include, but are not limited to, blood serum, saliva, semen, tissue samples obtained by biopsy (brain, skin, lymph nodes, spleen, etc.), culture culture supernatants disrupted or broken cells, bacterial and eukaryotic expression systems, and the like. The presence of the virus is tested by incubation of the monoclonal antibody with the biological sample under conditions contributing to the formation
 EMI31.1
 of an immune complex, after which the formation of the complex is detected.

   According to one embodiment, the formation of complex is detected by the use of a second antibody capable of binding to the monoclonal antibody which is typically conjugated to a marker and formulated in a manner analogous to the antibody formulations described above. above. In another embodiment, the monoclonal antibody is fixed on a solid phase support which is then contacted with a biological sample. After an incubation phase, the labeled monoclonal antibody is added to detect the bound antigen.



  Preparation and use of synthetic peptides
The subject of the present invention is new peptides which, inter alia, imitate immunologically epitopes of protein coded by the HIV retrovirus,

  <Desc / Clms Page number 32>

 in particular epitopes coded in the gag or env regions of the viral genome coding respectively for gpl10 or p25. to satisfy strain-to-strain variations among different isolates, adjustments for conservative substitutions, and selection among alternatives if non-conservative substitutions are required, may be made.

   These peptides can be used as immunogens, to inhibit or eliminate the production of anti
 EMI32.1
 HIV gene in vitro or in vivo, for detecting the virus or antibodies to the virus in a physiological sample.



   Depending on the nature of the protocol, the peptides can be conjugated to a carrier or other labeled or unlabeled compounds bound to a solid surface, or the like.



   According to one embodiment, interest peptides can be derived from the gpl10 region of the virus. The region of particular interest is that in the open reading frame env extending from about the base pair 6688 bp to about 6750 bp and from about 7246 bp to about 7317.



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



   The peptide can be modified by introducing conservative or non-conservative substitutions in the

  <Desc / Clms Page number 33>

 peptide, usually less than 20%, more usually less than 10% of the amino acids being exchanged. In situations where regions are found to be polymorphic, it may be desirable to vary one or more particular amino acids to more effectively mimic the different epitopes of the different retroviral strains. In many cases to obtain chemical stability, methionine can be replaced by norleucine (Nor).



   It should be understood that the peptide used in the present invention need not be identical to a particular HIV polypeptide sequence, so long as the compound is capable of providing immunological competition with proteins at least one of the strains of the HIV retrovirus. Therefore, the peptide of the invention may be subject to various changes, such as insertions, deletions and substitutions, either conservative or non-conservative, if such changes can provide certain advantages in use. By conservative substitutions is meant substitutions in groups such as gly, ala; val, ile, leu; asp, glue; asn, gln; ser, Ihr; lily. arg; phe, tyr; and nor, met.

   Usually, the sequence does not differ by more than 20% from the sequence of at least one strain of an HIV retrovirus except where additional amino acids can be added to either terminal to provide an "arm" by which the peptide of this invention can be suitably immobilized. The arms can have a length which is usually at least 1 amino acid and can be 50 or more amino acids, more often from 1 to 10 amino acids.



   The peptide in which the amino acid sequence is modified by the substitution, addition or deletion of amino acid residues, must retain substantially all of the antiviral or immunoreactivity activity of unmodified peptides, which may be conveniently measured by various

  <Desc / Clms Page number 34>

 test techniques currently described. The isomeric form of one or more amino acids can be used, if desired, to modify biological properties, such as activity, breakdown rate, etc.



   In addition, one, two or more amino acids may be added to the terminal portions of an oligopeptide or peptide to facilitate bonding of the peptides to each other, for coupling to a carrier or a more peptide. large, for reasons which will be discussed later, to modify the physical or chemical properties of the peptide or oligopeptide, or the like.



   Amino acids such as tyrosine, cysteine, lysine, aspartic or glutamic acid. or the like, can be introduced to the C or N terminal of the peptide or oligopeptide to provide useful functionality in binding. Cysteine is particularly preferred to facilitate covalent coupling to other peptides or to form polymers by oxidation.



   In addition, the peptide or oligopeptide sequences can differ from the natural sequence by the fact that the sequence is modified by acylation of the MH - terminal, for example acetylation or amidation by thioglycolic acid, amidation of the carboxy-terminal, for example , with ammonia or methylamine, to provide stability, increased hydrophobicity for binding to a support or other molecule, or for polymerization.



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

   If two

  <Desc / Clms Page number 35>

 
 EMI35.1
 Cysteine residues are present at the same end of the peptide, a preferred embodiment exists when the cysteine residues are separated by means of one to three spacer amino acid residues, preferably glycine. The presence of cysteine residues can allow the formation
 EMI35.2
 of peptide dimers and / or increase the hydrophobicity of the resulting peptide which facilitates immobilization of the peptide in immobilized test systems or solid phase systems.



   One use of particular interest is that of the mercaptan group of cysteines or thioglycolic acids used for amino terminal acylation groups or the like to link two of the peptides or oligopeptides or their combinations by a disulfide bond or a larger bond to form polymbers that contain a number of epitopes. Such polymers have the advantage of an increased immunological reaction. If different peptides are used to make the polymer, they have the additional possibility of inducing antibodies which immunoreact with several antigenic determinants of
 EMI35.3
 different HIV isolates.



   In order to obtain the formation of antigenic polymers (synthetic multimeters), it is possible to use compounds having bis-haloacetyl groups, nitroarylhalides, or the like, when the reagents are specific for thio groups. Thus, the bond between the two mercapto groups of the different peptides or oligopeptides can be a single bond or a linking group of at least 2, usually at least 4, and not more than about 16, usually not more than about 14 carbon atoms.



   The peptide object of the invention can be used while being linked to a soluble macromolecular support (for example not less than 5 kDal). Conveniently, the support can be a poly (amino acid), either natural or synthetic, against which antibodies are unlikely to be encountered.

  <Desc / Clms Page number 36>

 
 EMI36.1
 in human serum. Examples of such supports are poly-L-lysine, hemocyanin limpet keyhole, thyroglobulin, albumin, such as bovine serum albumin, tetanus toxoid, etc. The choice of support is essentially dependent the ultimate use of the antigen, and is dependent on amenities and availability.



   With such conjugates, there will be at least one molecule of at least one peptide of the invention per macro-molecule, and no more than about 1 per 0.5kOal, usually not more than about 1 per 2kDal of the macromolecule. One or more different peptides can be linked to the same macromolecule.



   The way of making the connection is conventional, and uses reagents such as p-maleimidobenzoic acid, p-methyldithiobenzoic acid, maleic acid anhydride, succinic acid anhydride, glutaraldehyd etc. Binding can occur on the N-terminal, the C-terminal or on an intermediate site between the ends of the molecule.



   The peptide object of the invention can be derived by binding, can be linked while being linked to a support, or the like.



   Various assay protocols known to those familiar with the art can be used to detect the presence of either the antibodies on the retroviral proteins or the retroviral proteins themselves. There is a particular interest in using the peptide in the form of the labeled reagent. if the marker allows a detectable signal, or to bind the peptide, either directly or indirectly on a surface, if the antibody on the peptide in the sample 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 xenogenic antibody specific for human immunoglobulin, normally both human IgM and IgG, or a labeled protein specific for immune complexes, for example factor Rf protein A S. aureus.



   As an illustration of a test technique, we

  <Desc / Clms Page number 37>

 will cite the use of a sample container, for example wells of plates comprising microwells, where the polypeptide object of the invention or the conjugated products thereof are adsorbed on the bottom of the container and / or the walls either covalently or non-covalently.



   The sample, which is normally human blood or serum diluted in an appropriately buffered medium, is placed in the container and sufficient time is left for the complex to form between the polypeptide (s) and all of the parent antibodies in it. 'sample. The supernatant is discarded and the container washed to remove non-specifically bound proteins. A specific labeled binding protein which specifically binds to the complex, such as the xenogenic antiserum for human immunoglobulin, is used for detection.



   The peptide can be prepared in a variety of ways. The peptide, due to its relatively short dimensions. can be synthesized in solution or on a solid support according to conventional techniques. Various automatic synthesis devices are commercially available today and can be used with known protocols. See, for example, Stewart and Young, Solid Phase Peptide Synthesis, 2nd ed., Pierce Chemical Co., 1984; and Tarn et al., J. Am. Chem. Soc. (1983) 105: 6442.



   Alternatively, hybrid AON technology can be used if a synthetic gene can be prepared using single strands that code for the polypeptide or strands substantially complementary to it, if the single strands overlap and can be put together in a medium annealing so as to allow hybridization. The hybrid strands or filaments can then be ligated to form the complete gene. and, by choosing appropriate terminations, the gene can be inserted into expression vectors, which are readily available today.



  See for example Maniatis and others, Molecular Cloning,

  <Desc / Clms Page number 38>

   A Laboratory Manual. CSU. Cold Spring Harbor Laboratory, 1982.



  Alternatively, the region of the viral genome encoding the peptide can be cloned by conventional recombinant DNA techniques and expressed (see Maniatis, supra).
 EMI38.1
 Gold LAVSRU isolates <u of HIV which can be used to express the peptides, are the following: LAV BRU TGT ACA AGA CCC AAC AAC AAT ACA AGA AAA AGT
ATC CGT ATC CAG AGG GGA CCA GGG AGA GCA TTT
GTT ACA ATA GGA AAA ATA GGA AAT ATG AGA CAA
GCA CAT TGT
ARV-2 TGT ACA AGA CCC AAC AAC AAT ACA AGA AAA AGT
ATC TAT ATA GGA CCA GGG AGA GLA TTT CAT ACA
ACA GGA AGA ATA ATA GGA GAT ATA AGA AAA GCA
CAT TGT
Fragments of a sequence can be used for the expression of peptide fragments, conservative basic changes can be made, if the codon (s) code for the same amino acid (s),

   or non-conservative changes in the coding sequence can be made, if 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 either to
5 'or 3' terminal or at both terminals to lengthen the peptide, while retaining its epitopic site (s).



  The extension can provide a link for binding, for example to a marker, such as an enzyme, to join two or all of the peptides together in the same chain, so as to provide antigenic activity, restriction sites suitable for cloning, or the like.



   The DNA sequence itself, its fragments, or larger sequences, usually at least 15 bases, preferably at least 18 bases, can be used as nucleotide probes for the detection of retroviral RNA

  <Desc / Clms Page number 39>

 or proviral DNA, or to identify homologous regions for cloning or sequence formation. Oe many techniques are described, such as for example the technique
Grunstein-Hogness, the Southern technique, the technique
Northern, dot-blot, their improvements, as well as other methodologies, such as those described in US Patent No. 4,358,535 which is given here for reference.



   The peptides object of the invention, including blocking peptides, and their analogs find use in themselves or in combination in vaccines. Similarly, anti-idiotype antibodies, that is to say reacting with the idiotypes of the antibodies of the present invention and therefore containing epitopes mimicking the neutralizing regions of HIV, can also be used in vaccines. The anti-idiotypic peptides or antibodies can be formulated in a suitable manner, generally at concentrations in the range of 1 yg to 20 mg / kg of host. Physiologically acceptable media can be used as carriers, such as sterile water, saline, phosphate buffered saline or the like.

   Adjuvants can be used such as aluminum hydroxide gel, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, proteins (eg cholera toxin or diphtheria) and emulsions oil.



  The peptides can also be incorporated into liposomes, or conjugated to polysaccharides, polypeptides or polymers for use in a vaccine formula. Administration can be by injection, for example intramuscularly. peritoneal, subcutaneous, intravenous etc. Administration of an immunogenically effective dose may be done in one or more doses, usually in the range of one to four weeks. An "immunogenically effective dose" corresponds to

  <Desc / Clms Page number 40>

 a quantity of vaccine suitable for obtaining an immune response in a hole, which demonstrates in the host an increased infection.



   Other characteristics and advantages of the present invention will appear better in the experiments which follow, and which describe the invention by way of example.



  These examples should not be considered as limiting the present invention.
 EMI40.1
 



  EXAMPLE 1 Production and characterization of monoclonal anti-microps
Example I describes the production of hybrid cell lines which produce anti-monoclonal bodies specific for HIV envelope glycoproteins. This method involves the use of purified lectin extracts of LAvn., Fixed to the lens lectin agarose such as the immunogen. The monoclonal antibodies subsequently produced by the hybrid cell lines are characterized by their ability to precipitate radio-immune and in "immunoblot" gp110 from purified LAV and as a biologically expressed recombinant fusion protein.

   The monoclonal antibodies which bind to the epitopes on gpl10 are also reactive in the ELISA test with the whole ruptured virus, fusion proteins and synthetic peptides, and react with the whole virus in indirect fluorescence tests.



   The protocols for the production of hybrid cell lines producing a monoclonal antibody and the characterization of the antibodies are as follows.



   LAV virus purified from infected CEM cells (ATTC NO CRL8904), was broken or broken in 50 mM Tris, pH 7.4, 0.15 M NaCl, 1% aprotinin, Nonidet P-40 (R) (NP -40) (octylphenoxypolyethoxyethanol) 2%. The extract was clarified twice by centrifugation and adjusted to 0.5% NP-40 with the addition of three volumes of dislocation buffer

  <Desc / Clms Page number 41>

 
 EMI41.1
 100 NP-40. Lens lectin Sepharosis (Pharmacia, Piscataway, N. J.) was prewashed in the dislocation buffer
100 NP-40 and then equilibrated in the adsorption buffer (Tris 50 mM, pH 7.4, NaC1 0.15 M, aprotinin 1%, NP-40 0.5%).



   The clarified viral extract was adsorbed with lens lectin Sepharose for 42 hours at 4OC. The non-adsorbed material was removed by washing with excess adsorption buffer. The elution of the adsorbed material was carried out with 0.2 M alpha mannoside in adsorption buffer.



   The eluent was dialyzed against PBS to remove the sugar and the material was rgadsorbed on the lectin lectin Sepharose.



   The lens lectin-glycoprotein Sepharose complex was used to immunize SALB / c mice by three intraperitoneal injections without adjuvant and given separately over a period of 2-3 weeks. Spleens were eliminated from the immunized mice, which demonstrated the presence of an antibody circulating on the glycoproteins of
HIV by immunoblot, RIP and / or ELISA.



   The protocols used for the production of cell lines were generally those of Kohler and Milstein (Nature 256: 495 (1985)) with modifications by Goldstein L. C. and others (Infect. Immun. 38: 273 (1982)). The splenic B cells of the immunized mice were fused with NS-1 myeloma cells using 40% (weight / volume) of polyethylene glycol.

   After the fusion, the mixture of cells was resuspended in HAT medium (RPMI medium - 1640 supplemented with 15% fetal calf serum, hypoxanthine lx 10-4M, aminopterin 4 x 10-7M and thymidine 1, 6 x 10-5M) to choose the growth of hybrid cells, and then the mixture was delivered in 96-well microculture trays at a concentration of 1 to 3 x 10 6 cells / ml and incubated at 370C in a humid atmosphere containing 6% of culture cells was fed by replacing half of the supernatant with

  <Desc / Clms Page number 42>

 fresh HAT medium.

   The wells were observed using an inverted microscope for indications of cell proliferation and when the cells were of sufficient density the supernatants were tested for the anti-LAV antibody.



   Wells containing hybrid cells producing antibody to LAV were identified by test
ELISA by measuring the binding to either the purified whole ruptured virus or the biologically expressed fusion proteins.



   ELISA tests using disrupted virus were performed on LAV EIA plates (Genetic Systems, Seattle, Washington).



   The plates were incubated with cell culture fluids at 37 ° C for 45 minutes and then washed three times with 0.5% Tween 20 in phosphate buffered saline (PBS-Tween).



   Goat anti-mouse peroxidase IgG (dilution
1: 2,000 in PBS-Tween; Zymed Laboratories, Inc., San
Francisco Sud, California) was added (100 null per well), and the plates were incubated for 45 minutes at 370C and washed as above. A substrate (0.025 M citric acid, 0.05 M dibasic sodium phosphate, pH 5 containing 14 mg of o-phenylenediamine and 10 μl of 30% hydrogen peroxide per
50 ml) was added and the plates were incubated for 30 minutes at room temperature in the dark. The reaction was stopped with 3N sulfuric acid, and colorimetric reactions were quantified with an automatic microplate reader.

   The wells which gave positive results were subcloned by limited dilution, retested for specificity, and then extended.



   The monoclonal antibodies secreted by the resulting hybrid cell lines were then characterized with regard to specificity and reactivity by immunoblot, immunoprecipitation and ELISA test using disrupted LAV virus, recombinant LAV fusion proteins and synthetic LAV peptides. All antibodies were determined

  <Desc / Clms Page number 43>

 as being of the IgGl isotype. Cell lines HIV-gpl10-1, HIV-gpl10-2 and HIV-gpl10-3 were deposited with (the American Type Culture Collection) before the filing of this request and are referenced under NOs ATCC HB 9175 , HB 9176 and HB 9177 respectively.



   The recombinant fusion proteins tested for reactivity were previously called ENV2, ENV3, ENV4 and ENV5. The ENV2 protein is expressed from pENV2 (ATCC NO 53071), which is an LAV region of the base pair bp 6598 to bp 7178 (numbered according to Wain-Hobson and others Cell 44: 9 (1985)). ENV3 is expressed from pENV3 (ATCC NO 53072) which includes the LAV region of bp 7178 to bp 7698; ENV4 is expressed from pENV4 (ATCC NO 53073),
 EMI43.1
 and includes bp 7698 to bp 8572 and ENV5 is expressed from pENV5 (ATCC NI 53074) which includes the LAV region bp 5889 to bp 7698.



   Assembly of synthetic peptides
Peptides I (29) and VIII (110-2-2) were assembled on a benzhydrylamine (polystyrene / divinylbenzene) resin (Applied Biosystems, Inc., Foster City, California). Peptide V (177) was assembled on a p-butyloxycarbonyl (Boc) -ethylbenzylcysteine-phenylacetamidomethyl (PAM) polystyrene / divinylbenzene resin. Symmetrical anhydride couplings were carried out in a 430A synthesizer (Applied Biosystems). Cysteine was added as a first residue in the two peptides.



   Dicyclohexylcarbodiimide couplings in the presence of hydroxylbenzotriazole were used for asparagine and glutamine. A side or branched chain protection based on benzyl and an alpha-amine protection Boc were used. Other side chain protections commonly used were tryptophan (formyl) Boc, methionine sulfoxide Boc, arginine (tosyl) Boc, cysteine (methylbenzyl) Boc, histidine (tosyl) Boc, lysine (chlorobenzyloxycarbonyl) Boc and tyrosine (bromobenzyl-

  <Desc / Clms Page number 44>

 oxycarbonyl) Boc.



   When the peptides were radiolabelled, it was by acetylation of the amino terminal with acetic acid-H and an excess of dicyclohexylecarbodiimide.



   Deprotect. ion and cleavage of the peptide from the resin were performed by the HP "low-high" Tarn protocol (Tam et al., supra). The resin extraction was carried out with 5% acetic acid and the extract was subjected to gel filtration chromatography in acid.
 EMI44.1
 acetic 5%.



   HIV synthetic peptides tested for reactivity with monoclonal antibodies included peptides 29,36 and 39. Peptide 29 is encoded by the genome region
 EMI44.2
 LAVBRU from 6688 bp to 6750 bp the peptide 36 D HU is coded by the region from approximately 7246 bp to 7317 bp; and peptide 39 is encoded by the region from about 7516 bp to 7593 bp. Peptides 36 and 39 are described in detail in US Patent No. 4,629,783 which is given here for reference.



   The blocking peptides, IX-XV were assembled essentially as described above on a methyl-benzhydrylamine (polystyrene / divinylbenzene) resin (Applied Biosystems, Inc., Foster City, California). The symmetrical anhydride couplings were realistic on a 430A synthesis device (Applied Biosystems). Dicyclohexylcarbodiimide couplings in the presence of hydroxylbenzotriazole were used for asparagine. For protection, protection by Boc alpha-amine and by side chain based
 EMI44.3
 Benzyl was used, while Boc (bromobenzyloxycarbonyl) was used specifically for the branched chains of tyrosine. Acetylation, if present, was performed using acetic anhydride or iced acetic acid and dicyclohexylcarbodiimide.

   The deprotection and cleavage of the resin peptide were accomplished by the standard "high" HF protocol (Stewart et al., See above).

  <Desc / Clms Page number 45>

 



  The resin extraction was carried out with 50% acetic acid, and the extract was then subjected to gel filtration chromatography in 20% acetic acid. As desired, high performance liquid chromatography was performed on a Vydac C18 column (Rainin Instrument Co., Emeryville, CA) using 0.1% trifluoacetic acid, acetonitrile gradient.



  Immunoblot
The immunoblot characterization was performed on clone supernatants or ascites fluid using purified LAV virus and recombinant fusion proteins as antigens. The antigens were first separated by polyacrylamide gradient gel electrophoresis (7-15%) and transferred to a nitrocellulose membrane (NCM) by electrophoresis for 4 hours at 25 V in 25 mM sodium phosphate (pH 7). After transfer, the NCM was blocked to prevent non-specific interactions by incubation in PBS-Tween or Blotto (5% non-fat dry milk in PBS) for one hour at room temperature.

   The NCM was incubated with cell culture supernatant or ascites fluid diluted in PSS-Tween for one hour at room temperature and was rinsed with three changes of PBS-Tween.



  In the second phase, the NCM was incubated with a goat anti-goat mouse IgG black radish peroxidase, diluted in PBS-Tween for one hour at room temperature. This incubation was followed by washing with PSS-Tween and then immersion in a color development solution of black radish peroxidase (Bio-Rad Laboratories, Richmond, California) for 20 minutes. The reaction was stopped by immersion in deionized water. The reactivity of the monoclonal antibody was compared to a control positive serum reacting with the purified disrupted virus or an expressed fusion protein.



  The results showed that all antibodies bind to gpl10 and its precursor molecule, gpl50, using ruptured or separated virus preparations. Antibodies 110-1

  <Desc / Clms Page number 46>

 and 110-2 also recognized the ENV3 fusion protein, while antibodies 110-3,110-4, 110-5 and 110-6 formed an ENV2 immunocomplex.



    Immunoprecipitation
Virus extracts for radioimmune precipitation were prepared from CEM cells infected with the LAVgp .. isolate of HIV capable of lytic growth by continuous passage. When the early cytopathic effects were evident, the cells were transferred to a
 EMI46.1
 labeling medium containing methionine rs 3 r i (0, then incubated for 24 hours until most of the cells have been lysed, which releases the virus into the culture supernatant.

   The virus was transformed into pills or tablets (one hour at 100,000 xg) from the cell-free supernatant, and detergent extracts were prepared in P-RIPA buffer (buffered saline
 EMI46.2
 phosphate containing 1% Triton X-100, 1% deoxycholate, 0, 1% SOS and 1% aprotinin). Similar extracts were prepared from the supernatants of uninfected CEM cells.



   The immunoprecipitation tests were carried out with 100 μl of virus extract incubated with 100 μl of culture supernatant from hybrid cell lines for one hour on ice. Four microliters of rabbit anti-mouse Ig (Zymed Laboratories, So. San Francisco, California) was added to each sample and incubated for 30 minutes. The 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 linked complexes were washed and separated by electrophoresis on polyacrylamide-SOS gel (15% acrylamide; OATD gel). Following the electrophoresis, the gels were fixed, soaked in the product "Enhance" (New England Nuclear, Boston, MA),

  <Desc / Clms Page number 47>

 dry and exposed to Kodak XR-5 film. A positive reference serum which immunoprecipitates all of the HIV viral proteins was reacted with supernatants of falsely infected and virus infected CEM cells as negative and positive controls.



   The results showed that all six monoclonal antibodies specifically immunoprecipitate gp110 and gpl50.



   Enzyme-linked immunoadsorbent test
To establish that the gpl10 epitopes were recognized by the anti-monoclonal bodies of the present invention, culture supernatants from hybrid cell lines or ascites fluids were further characterized by reactivity in the ELISA test with biologically expressed fusion proteins and synthetic peptides.



   The processes were the same as those described above except that the fusion proteins or the synthetic peptides replaced the purified virus such as the antigen adsorbed on the surface of the microtiter wells.



   When peptides were used as the antigen, the plate-setting protocol was as follows. The lyophilized peptide was dissolved in guanidine 6M HC1. Just before plate-mounting in 96-well plates, the guanidine solution was diluted in a 0.05 M bicarbonate / carbonate buffer (pH 9.6) to a final peptide concentration of up to 100 p9 / ml. A volume of 50 µl of the diluted peptide was placed in each microtiter well and the plates were then incubated overnight at 4 ° C.



  The excess peptide solution was "shaken", the plates closed or blocked Blotto, and the process described above was followed for the remainder of the ELISA test. Similarly, a reconstituting protein was diluted to a final concentration of about 2 V9 / ml in a bicarbonate / carbonate buffer.
 EMI47.1
 0, M (pH 9, before the same process is followed.



  The results are given in Table II. The

  <Desc / Clms Page number 48>

 monoclonal antibodies produced by the HIV-gpl10-1 and HIV-gpl10-2 cell lines reacted with ENV3, ENV5, peptide 36 and the disrupted virus. Antibodies from HIV-gpl10-3, HIV-gpl10-4, HIV-gpl10-5 and HIV-gpl10-6 cell lines reacted with ENV2 and peptide 29 as well as disrupted or disrupted virus.



   TABLE II
ELISA test showing the reactivity of monoclonal antibodies with recombinant proteins and synthetic peptides
 EMI48.1
 
 <tb>
 <tb> Protein <SEP> from <SEP> fusion <SEP> recombinant <SEP> Peptide <SEP> from <SEP> summary <SEP> LAV <SEP> CEM
 <tb> te- <SEP> tes- <SEP>
 <tb> ENV2 <SEP> ENV3 <SEP> ENV4 <SEP> ENV5 <SEP> 29 <SEP> 36 <SEP> 39 <SEP> months <SEP> my <SEP>
 <tb> 110-1 <SEP> 0.077 <SEP> 3,000 <SEP> 0.113 <SEP> 3,000 <SEP> ND <SEP> 2,421 <SEP> 0.054 <SEP> 0.908 <SEP> 0.125
 <tb> 110-2 <SEP> -0.003 <SEP> 3,000 <SEP> 0,000 <SEP> 3,000 <SEP> ND <SEP> 2.305 <SEP> -0.005 <SEP> 1,214 <SEP> 0.009
 <tb> 110-3 <SEP> 3,000 <SEP> 0.011 <SEP> ND <SEP> ND <SEP> 3,000 <SEP> ND <SEP> 0.017 <SEP> 0.363 <SEP> 0.046
 <tb> 110-4 <SEP> 3,000 <SEP> 0.020 <SEP> ND <SEP> ND <SEP> 3,000 <SEP> ND <SEP> 0.016 <SEP> 0.383 <SEP> 0.067
 <tb> 110-5 <SEP> 3, <SEP> 000 <SEP> 0,

    <SEP> 014 <SEP> NO <SEP> NO <SEP> 3, <SEP> 000 <SEP> ND <SEP> 0, <SEP> 016 <SEP> 0, <SEP> 368 <SEP> 0, <SEP> 025 <SEP>
 <tb> 110-6 <SEP> 3, <SEP> 000 <SEP> 0, <SEP> 033 <SEP> NO <SEP> NO <SEP> 1, <SEP> 937 <SEP> ND <SEP> 0, <SEP> 017 <SEP> 0, <SEP> 486 <SEP> 0, <SEP> 032 <SEP>
 <tb>
 
 EMI48.2
 The results in Table II show that the monoclonal antibodies 110-1 and 110-2 recognized an antigenic determinant coded by a DNA sequence in the pENV3 region, more particularly by the region of the HIV genome defined by an amino acid sequence in peptide 36.



  In other words, the monoclonal antibodies gpl10-1 and 110-2 bind to a peptide region of gpl10 encoded in 7246 bp at 7317, as evidenced by the formation of immune complexes with peptide 36 and ENV3. This region of the HIV genome has previously been identified as conserved, that is, little change in the DNA sequence in the region encoded by peptide 36 among different viral isolates from various geographic locations. See Starcich et al., Cell 46 (1986). In contrast, the monoclonal antibodies gpl10-3, -4, -5 bind to peptides of

  <Desc / Clms Page number 49>

 
HIV defined by the region coded by peptide 39 from 6688 bp to approximately 6750 bp.

   The region in gpl10 defined by peptide 29 has been identified as containing several nucleotide substitutions among different virus isolates. Monoclonal antibodies which selectively bind to gpl10 peptides which contain conserved epitopes, such as antibodies 110-1 and 110-2 may have enhanced utility in different circumstances, such as in affinity chromatography etc. Also, in a test
ELISA, peptide 110-2-2 reacted with sera from the individual from whom LAV-2 was isolated.



   Indirect immunofluorescent test
Indirect immunofluorescent tests using monoclonal antibodies directed against the HIV gp110 antigen were carried out on living cells and fixed with acetone. Frames or Chassis fixed by acetone, prepared from CEM cells infected with LAV were incubated with a diluted culture supernatant or an ascites fluid for one hour at 370C, while live cells were incubated with a culture supernatant or a fluid ascites for one hour at 4 Å before cells are placed on frames or the like and fixed by acetone.



  Both methods used anti-mouse IgG labeled with fluorescein isothiocyanate to detect cells carrying the reactive gp110 antigen. The monoclonal antibody HIV-gp110-1 gave positive results using either
 EMI49.1
 living cells or cells infected with LAV fixed by acetone.



  EXAMPLE 11 Neutralization of the infectious power of HIV by anti-gpl10 monoclonal antibodies
This example describes and characterizes the neutralization of the infectious power of HIV using monoclonal antibodies which bind to gpl10 and peptides in gpl10. The results indicate that the monoclonal antibodies Gpl10-3,

  <Desc / Clms Page number 50>

   -4, -5 and -6 have neutralizing activity, and that gp110-3 and -4 have particularly high levels of neutralizing activity.



  Neutralization test A sensitive neutralization test was developed to quantify the effect of monoclonal antibodies on the infectious power of HIV. A CD4 + cell line highly susceptible or sensitive to HIV infection, CEM was chosen as target for comparisons of infectivity. An ascites fluid prepared as described in Example I, or its IgG fraction purified using ammonium sulfate precipitation, was heat quenched at 560C for 30 minutes, then diluted as required in RPMI medium containing 10% fetal calf serum. A suspension of
 EMI50.1
 LAVSRU was harvested from crops of around D HU
4 days of CEM in a logarithmic growth phase, filtered through filters of 0, 2 or 0. 45 micron, put in the form of aliquots and frozen at -70 C.

   An aliquot was thawed, titrated to determine the TCID50, and subsequent tests were performed with freshly thawed aliquots, diluted 1: 500 in culture medium to a concentration of approximately ten times the amount required to infect 50 % of CEM cells in the culture (10 TCID50).

   The virus suspension was mixed with an equal volume (250 µl) of a monoclonal antibody preparation in five dilutions from 1: 5 to 1: 9, 765, 625. The virus / antibody mixture was incubated for 45 minutes at 37 C and then duplicated from 1: 5 to 1: 9, 765, 625. The virus / antibody mixture was incubated for 45 minutes at 37 C and then duplicate samples of 200 pI were used to inoculate wells containing approximately 1 ml 2 x 105 CEM cells per well. The cultures were incubated at 37 ° C in a 5% CO 2 atmosphere humidified for 14 days. The cells were harvested, compressed, and lysed with 1% Triton X-100 in PBS for about 10 minutes.

   The

  <Desc / Clms Page number 51>

 
 EMI51.1
 The amount of virus (or viral antigen) present in the lysed cells was quantified using a sandwich enzyme immunoassay to capture sensitive HIV antigen. The titration of the neutralizing activity, if there is one, was determined as the reciprocal of the dilution of monoclonal antibody which inhibited the production of antigen on more than 50% of virus control cultures incubated without antibody, or with a monoclonal antibody of the same isotype which had previously
 EMI51.2
 been shown to lack activity The HIV antigen capture test described above used two monoclonal antibodies directed against neutralizing p25 antigens as capture reagents.

   These hybridoma cell lines were produced by the methods described above with minor modifications, including using a purified recombinant gag fusion protein as the immunogen and characterizing the resulting monoclonal antibodies with respect to specificity and reactivity using the recombinant fusion proteins previously called GAG-1, GAG-2 and GAG-3, and the peptide synthesis 141.



  The GAG-1 protein is expressed from pGAG-1 (ATCC NO 53379), GAG-2 is expressed from pGAG-2 (ATCC NO 53111) and GAG-3 is expressed from pGAG-3 (ATCC NO 53112).



  Synthetic peptide 141 is encoded by the genome region
 EMI51.3
 LAV corresponding to amino acid residues 198-242. B HU
BRUD Monoclonal antibodies produced by hybridoma cell lines p25-2 and p25-3 have been found to react with recombinant fusion proteins GAG-1, GAG-2 and GAG-3 and the monoclonal for p25-3 is also reactive with synthetic peptide 141.



   To carry out the antigen capture test, the capture reagents were first adsorbed on a solid support. An ascites fluid derived from hybridoma cell lines p25-2 and p25-3 was diluted 1: 5,000 in 25 mM Tris buffer, pH 8, 5 and 200 µl were placed in wells of microwell plates. The wells were closed in a manner

  <Desc / Clms Page number 52>

   sealed and incubated for approximately 16 hours at 4 ° C. The solution was removed from the wells by suction before adding a blocking solution of 0.3% Blotto in BBS. The blocking was carried out for 15 minutes at room temperature.



  The blocking solution was aspirated and the sample was added. Two hundred microliters of the lysed cell suspension and 5 l of detection conjugate, prepared as described below, were added to each well. The plates or strips of wells were incubated for 2 hours at 37 ° C., after which the suspension was aspirated and the wells washed four times with buffer (0.05% Tween 20 in PBS). The conjugate detection product was prepared as follows. The monoclonal antibodies p25-6 and p25-7 were conjugated with black radish peroxidase (HRP) in a 3: 1 molar ratio (Ab: HRP) for three hours using a periodate oxidation method (Nakane et al., J. Histochem.



  Cytochem. 22: 1084 (1974). The conjugates were diluted 1: 1500 in 2.5% (weight / volume) of non-fat dry milk, 0.01% thimerosal and 0.05% antifoam A in 20 mM sodium citrate. The remainder of the ELISA test was performed as described in Example I.



  The results of the neutralizing activity test with the antibodies gpl10-3, -4, -5 and -6 are as follows.



  The highest titers which retained neutralizing activity were determined to be: gp110-3 = 15. 625; gpl10-4 = 9,765,625; gpl10-5 = 125; and gp110-6 = 625.



  Given the predicted genetic variability of the region defined by peptide 29, the power of the monoclonal antibody gp110-4 to recognize other HIV isolates was examined. The immunofluorescence tests were carried out as described above. The antibody gpl10-4 detected an antigen in cultures of at least 3 to 16 HIV isolates tested.



  The antibody gp110-4 was able to neutralize viruses isolated over fifteen weeks from a chimpanze

  <Desc / Clms Page number 53>

 inoculated with LAV BRU as a control animal in an SIOA vaccine trial. The monoclonal antibody was able to neutralize isolates even if the animal had serum antibodies that neutralized HIV in vitro, and had developed a measurable immune response generated by cells to HIV infection. This indicates a defect in antigenic movement in vivo for the epitope recognized by the antibody gpl10-4.



   EXAMPLE III
Neutralization of the infectious power of HIV with a cocktail of anti-gpl10 and anti-p25 monoclonal antibodies
This example describes the neutralization of HIV infectivity using monoclonal antibodies which bind to gpl10 and the peptides in gpl10 in combination with monoclonal antibodies which bind to p25 and peptides in p25, which alone show little or no d 'neutralizing activity. The results indicate that the gpl10-2 monoclonal antibody in combination with either p25-6 or p25-7 has particularly high levels of neutralizing activity.



   Hybridoma cell lines p25-6 and p25-7 were produced by the methods described above with modifications which include the use of inactivated disrupted or disrupted virus or or a purified recombinant gag fusion protein expressed in E. Coli, as an immunogen. and characterizing the resulting monoclonal antibodies with regard to specificity and reactivity, using the recombinant fusion proteins previously called GAG-1, GAG-2 and GAG-3 and the synthetic peptides 15,88, 150, 147 and 148.

   Synthetic peptides are encoded by the LAV-p genome region. corresponding to the amino acid residues as follows: peptide 15, amino acid residues 329 to 350; peptide 88, amino acid residues 315-350; peptide 150, amino acid residues 318 to 363; peptide 147, amino acid residues 278 to 319; and peptide 148, residues

  <Desc / Clms Page number 54>

 amino acids 290 to 319. The monoclonal antibodies produced by the hybridoma cell lines p25-6 and p25-7 react with the recombinant protein GAG-3, p25-6 reacts with the synthetic peptides 147 and 148, and p25-7 reacts with the synthetic peptides 15,88, and 150.



   Neutralization tests were carried out as described above, except when Cocktails were used; monoclonal antibodies from individuals were first diluted 1: 5 in culture medium and then mixed in equal ratios to give a final dilution of 1: 10.



  The remainder of the test was carried out as described above.



   The monoclonal antibodies gpl10-2, p25-6 and p25-7 show less than 50% of neutralizing activity when used alone. When used in a cocktail which includes the monoclonal antibodies gpl10-2 with p25-6 or gpl10-2 with p25-7 at a 1:10 dilution, complete neutralization occurs. A cocktail comprising the monoclonal antibodies p25-6 in combination with p25-7 gave
 EMI54.1
 a neutralizing activity rate of 60-90%.



   EXAMPLE IV Immunopotentiality of Peptide 29 and Its Counterparts
The power of peptide 29 and homologous peptides, including peptide 177, to stimulate an immune response against HIV was first examined in two strains of mice.



  The procedures for the preparation of peptide immunogens, the immunization protocols, and for the characterization of the generated immune response are given in detail below.



   Peptide 29 was prepared for immunization by conjugation with a purified thyroglobulin. Thyroglobulin can be derived with N-succinimidyl-4- (N-maleimidomethyl) cyclohexane-l-carboxylate (SMCC) for conjugation according to the process underlined in US Patent No. 4,629,783, column 10, lines 28 to 51 As the second immunogen, thyroglobulin was derived with glutaraldehyde as follows.

  <Desc / Clms Page number 55>

 



   Swine thyroglobulin, 27 mg, was dissolved in 1 ml of 0.1 M sodium bicarbonate, to which 8.3 µl of 25% glutaraldehyde solution was added dropwise, and the mixture was stirred overnight. Room temperature.



   1 ml of bicarbonate / sodium carbonate buffer, pH 9.3, was added to the solution and then dialyzed for 8 hours on 2 liters of the same buffer at 4 ° C., with a complete change of the dialysate after 4 hours. Peptide 29 was then added to the derived thyroglobulin in approximately a molar excess of 100, and the mixture was stirred overnight at room temperature. The unreacted glutaraldehyde was blocked with 200 µl of 0.2 M lysine solution, which mixture was stirred for several hours (or overnight) at room temperature. The thyroglobulin-peptide conjugate was then dialyzed extensively on 4OC PBS.



   Two mouse strains (black C57 and BALB / c) were inoculated with peptides prepared by each method of conjugation. All animals were around 2-4 weeks of age at the time of inoculation. The routes of inoculation include the tip of the leg, scarification of the tail, subcutaneous, intranasal, or intraperitoneal administration. The inoculum consisted of 25 μg of conjugated peptide suspended in 0.5 ml of complete Freund's adjuvant, with reinforced and repeated inoculations at weeks 2, 3 and 5, with the same immunogen suspended in an incomplete Freund's adjuvant. . Serum samples from individual mice were collected before immunization, 4 days after the enhanced immunization at 3 weeks, and 4 days after the enhanced inoculation at 5 weeks.

   The serum samples were analyzed for antibodies against the homologous peptides or the whole virus by "screening" in the ELISA test. Sera with antibody activity for LAV are further sorted for neutralization activity, after which the

  <Desc / Clms Page number 56>

 sera in immunoblots for ruptured LAV antigen and radioimmunoprecipitation tests with radiolabelled gpl10.



   Immunization results indicated that mice immunized with peptide 29 developed antibodies reactive with peptide 29 and the ruptured or broken LAV-1 virus in ELISA tests. Conjugation of peptide 29 through glutaraldehyde generally revealed a higher antibody titer for peptide 29 in Balb / c mice, although conjugation through maleimide successfully produced anti-peptide 29 antibodies in some 8alb / c mice.



   Mice immunized with peptide 177 developed antibodies to the peptide, and conjugation of peptide 177 through glutaraldehyde was better to elicit an immunological response. C57 mice were more sensitive to immune stimulation with peptide 177 than Balb / c mice. Mice immunized with the peptide
110-2-2 of LAV-2 developed antibodies against 110-2-2 and the LAV-2 virus as shown by ELISA tests.

   A 110-2-2 peptide conjugates through glutaraldehyde being immunogenic in both Balb / c and C57 mice, although the titers for peptide 110-2-2 are generally higher in Balb / e mice than C57 , while LAV-2 titers were generally higher in C57 mice than in Balb / c mice.



   Usually. serum samples from immunized mice which (i) exhibit antibodies to the whole virus; (ii) are capable of neutralizing HIV, as in the tests described in Example II; and (iii) react with peptide 29 in the ELISA test, cumulatively indicate the effectiveness of the use of peptide 29 in a vaccine formulation.

  <Desc / Clms Page number 57>

 
 EMI57.1
 



  EXAMPLE V Separation of a Monoclonal Antibody Using Gallo The anti of the gpl10 HIV antigen can be used in immunoaffinity separation procedures to substantially purify the recombinant proteins of bacterial expression. If the expressed protein is secreted by bacteria. the protein may be isolated from the culture supernatant if the protein is not secreted, bacterial cell breakdown may be necessary.



  The plasmid pENV-5 (ATCC NO encodes a major fraction of the carboxyl end of gpl10 and a fraction of the amino terminal of LAV inserted into the expression vector trp. C600 transformed by this vector expresses but does not secrete the protein gpl1D.



  E. Coli C 600 containing the plasmid pENV-5 are subjected to growth in a medium containing tryptophan (20 μg / ml) and ampicillin overnight overnight at 370C with aeration. The overnight cultures are then inoculated according to 1 in a fresh minimal medium containing ampicillin (100 μg / ml) but no tryptophan. These cultures grow with aeration for 2-3 hours (up to the early log phase) at 37OC. The inducer, 3-B-indole-acrylic acid (Sigma Chemical Co., Saint-Louis, MO) is added to a final concentration of 20 Mg / ml from freshly obtained stocks of 20 mg / ml in 95 % ethanol. Induced cultures are then grown at 370C with aeration for 4 hours, then tableted and, optionally, frozen.

   Protein yields from pENV-5 are typically less than 1 mg / liter.



  The bacterial cells in granules or pills are lysed using P-RIPA buffer (PBS containing 1% Triton X-100, 1% desoxycholate, 0, sodium sulfate

  <Desc / Clms Page number 58>

 immunoaffinitedodecyl, and 1% aprotinin) which will cause lysis of E. cells - Coli. The suspension can be subjected to sound waves to shear DNA and RNA, after which centrifugation is carried out to remove the particles.



   A dilution or concentration phase may be necessary to standardize the protein concentration.



   The HIV-gpl10-1 monoclonal antibody is initially precipitated from ascites fluids or cell culture supernatants at room temperature or in the cold with solutions of (NH4) 2SO4 or Na2SO4 buffered to pH 7.3 at final saturation of 33 or 18% respectively. The precipitated proteins are eliminated by centrifugation and redissolved in PBS and precipitated a second time with 33% of (NH4) 2SO4 or 12-15% of Na2SO4. This phase can be repeated if necessary. The tablet is again dissolved in PBS and the excess salts are removed by gel filtration through a desalting matrix or by exhaustive dialysis on PBS.



   The purified monoclonal antibody 110-1 can then be paired with cyanogen bromide activated Sepharosis. The required amount of gel is swollen in
 EMI58.1
 a solution of HC1 on a glass filter (l of
10Material dried by freezing gives a final gel volume of approximately 3.5 ml) and washed for 15 minutes with the same solution, and the antibody is added immediately thereafter. In general, the coupling reaction takes place more efficiently in a pH range of 8-10, but a lower pH can be used if necessary for the stability of the antibody. The antibody should be dissolved in PBS or a high ionic strength borate or bicarbonate / carbonate buffer with 150 mM NaCl.

   The suspension of antibodies and
 EMI58.2
 Active sepharose is stirred gently for 2-4 hours at room temperature, or overnight at 4QC, and then washed on a sintered glass filter with coupling pad. All remaining active groups are blocked by treatment

  <Desc / Clms Page number 59>

 with 1 M ethanolamine at pH 8 for 2 hours. The final Sepharose-antibody product is then washed alternately with high and low pH buffer solutions (borate buffer, 0.1 μM, pH 8.5, acetate buffer and 1M NaCl, 0.1 μM, pH 4, 1 M NaC1, respectively ) four or five times. This washing removes traces of non-covalently adsorbed materials.



  The finished immunoaffinity separation matrix is stored below 8 "C in the presence of a suitable bacteriostatic agent, such as 0.01% azide.
 EMI59.1
 



  Addition of the expressed protein suspension to the immunoaffinity separation matrix causes selective elimination of the gpl10 antigen. The mixture is allowed to interact for 2 to 24 hours, preferably 12-18 hours, with oscillation or slow agitation. A column system can also be used in which the immunoaffinity matrix is poured onto a column, equilibrated and the suspension of
 EMI59.2
 expressed protein added slowly to the column. After the protein suspension has been added, the flow can be stopped to allow formation of maximum immune complex.



   The unbound material is washed or removed by separation by extensive washing with an adsorption pad. A vacuum sintered glass filter can be used or a flow through the column is used. The bound material is then eluted using buffers of low or high pH (acetate buffer, pH 4 or borate buffer, pH 8.6) or else a chaotropic agent.



  * EXAMPLE VI
 EMI59.3
 immunoaffinity
Purification from a mammalian expression system
The monoclonal antibodies of the present invention find use in the purification of immunoaffinity of recombinant gpl10 expressed by mammalian cells.



  Mammalian cells are infected with recombinant vaccines (Mackett et al., J. Virol. 49: 857 (1984) which

  <Desc / Clms Page number 60>

 contain sequences encoding at least the portion of gpl10 which is antigenic and updates neutralizing antibodies.



   Recombinant vaccines are constructed according to the method described in American patent application NO 842 984 which is given here for reference. Briefly speaking, sequences encoding the HIV envelope glycoprotdin are inserted into a plasmid vector (pGS20) downstream of a control element of vaccine transcription. This chimeric gene is flanked by sequences encoding the viral thymidine kinase (TK) gene.



   Chimeric plasmid vectors containing a vaccine virus promoter ligated to the LAV envelope gene are used to transform the E. Coli MC1000 strain.



  The insertion of chimeric LAV-env sequences into the genome of vaccine viruses was carried out by in vivo recombination, which is made possible by the fact that the chimeric genes in the pv-env5 plasmids are flanked or arranged
 EMI60.1
 Next to vaccine virus sequences encoding the TK gene.



   This plasmid is then introduced into cells previously infected with a wild type vaccine virus and the recombination between the TK sequences on the plasmid and the homologous sequences in the viral vaccine genome is allowed to occur, which ensures the insertion of the chemical gene.



  Kidney cells of the African Green Monkey (strain BSC-40, line derived from BSC-1 cells. ATCC NQ CCl26) are used as host cell in the expression system.



   The confluent BSC-40 cells are infected following an infection multiple of 10 with recombinant vaccine virus. The infection is allowed to proceed for 12 hours, at which time the cells are harvested, washed once with PBS, and collected by centrifugation. The cell grains are resuspended in a lysis buffer (1% NP 40, 2.5% sodium deoxycholate, 0.1 M NaCl, 0.01 M Tris-HCl, pH 7.4, 1 mM EDTA) , and the lysate is then clarified

  <Desc / Clms Page number 61>

 by centrifugation.

   An immunoaffinity separation of the expressed recombinant protein is carried out as described above for the bacterial expression system, using the monoclonal antibody gpl10-1. Proteins produced
 EMI61.1
 by this expression system resemble the 9pilla HIV produced naturally by the processing and glycosylation provided by mammalian cells.



   EXAMPLE VII
Inhibition of HIV infection with blocking peptides The efficacy of blocking peptides in the inhibition of infection of tissue cells by the LAVBRU strain of HIV was studied using a modification of the protocol for the evaluation of T peptide , as published by Pert et al., see above. Preferred HIV inhibition tests
 EMI61.2
 consist of combining equal volumes of blocking peptide and CEM cells (2.5 x 105) in medium (RPMI, 10% FCS and 2 mg / ml polyethylene), and incubate for 45 minutes at 37 ° C.



  Virus is then added at different doses (10, 50, 500 TCID 50) the mixture is incubated for 14 days at 370C and then the supernatant tested for the production of anti-virus) (eg nucleus p25) .



   Preincubation of CEM cells with peptides before the addition of virus to the cultures reinforces the inhibition effect in the tests. The inhibition was found to be dependent on the dose of virus, and exhibits strong activity at medium and low doses, but less effect for very high doses of virus.



   About 60% to 90% inhibition of antigen production in experiments with low virus doses was obtained with peptide T. Additional experiments with X-XV peptides, typically COAH-terminal
 EMI61.3
 amide and NHL-terminal gave similar acetylated results, while peptide XI was particularly effective over a wide dose range. Antibodies

  <Desc / Clms Page number 62>

 Neutralizers can be added either during the preincubation period or when the virus is added to produce synergistic or additive inhibition of viral antigen production.



   It will be understood from the above that the monoclonal antibodies and peptides, including the blocking peptides, of the present invention constitute improved methods for neutralizing and / or inhibiting infections by
HIV. This allows therapeutic and prophylactic compositions to be more easily developed and which can be effective against infections caused for the most, if not all, of HIV strains. In addition, the new materials of the invention find uses in diagnostic tests and other well-known processes.



   Below is a table showing the occurrence of a number of microorganisms which constitute part of the present invention and which have been deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, USA.
 EMI62.1
 
 <tb>
 <tb>



  Description
 <tb> scientific <SEP> Ref. <SEP> Applicants <SEP> Ref. <SEP> ATCC <SEP> Date <SEP> departure
 <tb> Hybridoma
 <tb> mouse
 <tb> (balb <SEP> c / NS-1) <SEP> HIV-gp <SEP> 110-1 <SEP> HB <SEP> 9175 <SEP> 15 <SEP> August <SEP> 1986
 <tb> " <SEP> HIV-gp <SEP> 110-2 <SEP> HB <SEP> 9176 <SEP> 15 <SEP> August <SEP> 1986
 <tb> It <SEP> HIV-gp <SEP> 110-3 <SEP> Ha <SEP> 9177 <SEP> 15 <SEP> August <SEP> 1986
 <tb> HIV-gp <SEP> 110-6 <SEP> HS <SEP> 9404 <SEP> 30 <SEP> April <SEP> 1987
 <tb> " <SEP> HI <SEP> V- <SEP> gp <SEP> 110- <SEP> 4 <SEP> HB <SEP> 9405 <SEP> 30 <SEP> April <SEP> 1987
 <tb> HIV-gp <SEP> 110-5 <SEP> HB <SEP> 9406 <SEP> 30 <SEP> April <SEP> 1987
 <tb> HIV-p <SEP> 25-2 <SEP> HS <SEP> 9407 <SEP> 30 <SEP> April <SEP> 1987
 <tb> HIV-p <SEP> 25-3 <SEP> HB <SEP> 9408 <SEP> 30 <SEP> April <SEP> 1987
 <tb> n <SEP> HIV-p <SEP> 25-6 <SEP> HB <SEP> 9409 <SEP> 30 <SEP> April <SEP> 1987
 <tb>

  HIV-p <SEP> 25-7 <SEP> HB <SEP> 9410 <SEP> 30 <SEP> April <SEP> 1987
 <tb>
 

  <Desc / Clms Page number 63>

 
 EMI63.1
 It will be noted that the hybridomas HB 9175, HB 9176 and HB 9177 were tested and found viable on August 26, 1986.



  The remaining hybridomas were tested and found viable on May 4, 1987.



   Of course, the present invention is in no way limited to the examples given which must be considered as clarifying the invention but not limiting it.
 EMI63.2
 



  In other words, the invention includes all the technical equivalents of the means described, as well as their combinations if these are carried out according to the spirit.


    

Claims (54)

 EMI64.1   R E V E N D I C A T 1 0 N S CLAIMS 1.- Composition to be used for the treatment of infections by HIV, characterized in that it comprises a therapeutically effective dose of at least one monoclonal antibody specifically reactive with one or more neutralizing regions of HIV, and a pharmaceutically effective excipient.  2. Composition according to Claim 1, characterized in that the neutralizing regions contain epitopes encoded by the env or gag regions of the HIV genome.  3. Composition according to any one of claims 1 and 2, characterized in that the epitopes are located on the proteins gp110 or p25 of HIV.  4. Composition according to any one of the preceding claims, characterized in that it also comprises one or more blocking peptides capable of attenuating the infectious power of HIV and / or monoclonal antibodies reactive with epitopes of these peptides.  5. Composition according to Claim 4, characterized in that the blocking peptides comprise at least approximately 5 contiguous amino acids originating from a protein gpl10 of HIV.  6. Composition according to Claim 5, characterized in that the blocking peptides comprise peptides originating from the amino acid sequence 190 to 197 of LAVBRU, or homologues of this sequence.  7. Composition according to any one of the preceding claims, characterized in that a monoclonal antibody is reactive with at least one epitope of the amino acid sequence 278 to 319 of p25  <Desc / Clms Page number 65>  of LAVBRU and / or its counterparts, and a second monoclonal antibody is reactive with at least one epi tope of the amino acid sequence 315 to 363 of p25 of LAVBRU and / or its counterparts.  8. Composition according to any one of Claims 1 to 5, characterized in that a monoclonal antibody is reactive with at least one epitope of HIV p25 and a second monoclonal antibody is reactive with at least one epitope of gpl10 of HIV.    9. - Composition according to claim 8, characterized in that the epitope of p25 is located in the amino acid sequence 278 to 319 or 315 to 363 of LAVBRU, or in the homologues of these sequences.  10.-Composition according to any one of claims 8 and 9, characterized in that the epitop of gpl10 is located in the amino acid sequences of the envelope 190 to 197,301 to 336 or 308 to 328 of LAVBRTj, or in homologs of these sequences.  11. Composition according to any one of claims 7 to 10, characterized in that it further comprises a cocktail of monoclonal antibodies reactive with these sequences or different homologs of these sequences.  12.-Composition of monoclonal antibodies, characterized in that it is specifically reactive with a neutralizing region of HIV.  13. A composition according to claim 12, characterized in that it further comprises a monoclonal antibody reactive with an HIV blocking peptide.  14. - Composition according to any one of claims 12 and 13, characterized in that the neutralizing region is located on gpl10 of HIV.  15.-Composition according to Claim 14, characterized in that the region comprises the sequence  <Desc / Clms Page number 66>    EMI66.1  of acids 301 to 336 or 308 of amines of environgpl10 of HIV or of the homologs of these sequences.  16. Composition according to Claim 14, characterized in that the neutralizing region and the blocking peptide are located on HIV gp101.  17. Composition according to Claim 16, characterized in that the blocking peptide comprises the amino acid sequence of approximately 190 to 197 of HIV gpl10, or of homologues of this sequence.  18. Composition according to any one of claims 12 and 13; characterized in that the neutralizing region is located on p25 of HIV.  EMI66.2   19.- in that it comprises (a) at least one antibody Composition, characterized with an HIV neutralizing region and, (b) a blocking peptide containing at least about 5 contiguous amino acids from the following amino acid sequences of HIV gpl10, or their counterparts IX (173) Y-Ala-Ser-Tr-Thr-Thr-Asn-Tyr-Thr-Y ', or XV (191) Y-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Thr-Y 'where Y and Y', if present, each include an amino acid sequence of up to about 20 amino acids.  20.-Composition, characterized in that it comprises (a) a monoclonal antibody specific for an HIV neutralizing region, and (b) a blocking peptide containing at least one of the following amino acid sequences or their counterparts:  <Desc / Clms Page number 67>   XI (187) Y-Thr-Thr-Ser-Tyr-Thr-Y ', or XII (188) Y-Thr-Asp-Asn-Tyr-Thr-Y ', or XIII (189) Y-Asn-Thr-Ser-Tyr-Gly-Y ', or XIV (190) Y-Asp-Thr-Asn-Tyr-Ser-Y 'where Y and Y', if present, each include an amino acid sequence of up to about 20 amino acids.  21. Composition according to either of Claims 19 and 20, characterized in that the blocking peptide defines at least one antigenic determinant capable of causing antibodies to appear by immunization of a host, the antibodies thus appeared being protective against infections by HIV.  22. Composition according to any one of Claims 19 to 21, characterized in that Y and / or Y ′ comprise a binding residue chosen from the class formed essentially by glycine, tyrosine, cysteine, lysine, l glutamic acid and asparagine.  23. - Composition according to any one ,. es claims 19 to 22, characterized in that the peptide comprises an acetylated amino terminal and / or an amidated carboxy terminal and / or an amino acid d.    24. - Peptide, characterized in that it comprises at least about 5 contiguous amino acids originating from the amino acid sequence of HIV gpl10 below, or from its counterparts:  <Desc / Clms Page number 68>   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.  25. Peptide according to claim 24, characterized in that the contiguous amino acids define at least one antigenic determinant capable of producing antibodies by immunization of a host, these antibodies being protective against infections by HIV.    26. - Peptide, characterized in that it comprises one of the amino acid sequences of gpl10 of HIV below or of their counterparts: 1 (29)  EMI68.1  Y-Thr-Arg-Lys-Ser-Ile-Arg-Ile-Gln-Arg-Gly-ProGly-Arg-Ala-Phe-Val-Thr-Ile-Gly-Lys-Ile-Y ', V (177) Y -Thr-Arg-Lys-Ser-Ile-Tyr-Ile-Gly-Pro-Gly-ArgAla-Phe-His-Thr-Thr-Gly-Arg-Ile-Gly-Y ', or VIII (110-2-2) Y-Lys-Thr-Val-Lys-Ile-Nor-Leu-Nor-Ser-Gly-His-Val- Phe-His-Ser-His-Tyr-Gln-Pro-Y ', where Y and Y', if present, each comprise an amino acid sequence of up to about 20 amino acids.  27. Peptide according to claim 26, characterized in that Y and / or Y ′ comprise a binding residue chosen from the class formed essentially by glycine, tyrosine, cysteine, lysine, glutamic acid and asparagine.  EMI68.2   28. laughed at in that they encode the following peptides  <Desc / Clms Page number 69>    EMI69.1  any one of claims 24 to 27. 29.-Composition, in that it comprises a segment of nucleic acids of approximately 150 nucleotides or less coding for an HIV blocking peptide segment.  30. A composition according to claim 29, characterized in that the nucleic acid segment also codes for a peptide from a neutralizing region of HIV.     31. - Segment of nucleic acids of approximately 150 nucleotides or less, characterized in that it codes for a peptide of a neutralizing region of HIV.    32.-A nucleic acid segment according to claim 31, characterized in that it also codes for a HIV blocking peptide.    33.-nucleic acid segment according to any one of claims 31 and 32, characterized in that the peptide of a neutralizing region comprises at least about 5 amino acids originating from amino acids 310 to 336 of gpl10 of LAVBRU, or amino acid sequences 278 to 319 or 315 to 363 of p25 of LAVBRU, and homologs of these sequences.   34.-Set of probes, to be used for diagnostic tests by hybridization, characterized in that it comprises at least two nucleic acid sequences coding for the peptides mentioned in Table I.    35.-Vaccine against an HIV infection, characterized in that it comprises an immunologically effective dose of one or more peptides of less than about 50 amino acids containing neutralizing regions of gpl10 and / or p25, these peptides being mixed with a physiologically acceptable excipient.    36.-Vaccine according to claim 35, characterized in that it further comprises at least  <Desc / Clms Page number 70>  about 5 amino acids of a blocking peptide.    37.-Vaccine according to any one of claims 35 and 36, characterized in that the peptides are conjugated with an immunogenic support.    38. - Immortalized cell line, characterized in that it produces a monoclonal antibody capable of reacting with an antigenic determinant of the envelope glycoprotein gpl10 contained in a neutralizing region or an HIV blocking peptide.  39.-The cell lines HIV-gpl10-1, HIV-gpl10-2, HIV-gpl10-3, HIV-gpl10-4, HIV-gpl10-5,  EMI70.1  HIV-gpl10-6, HIV-p25-6 and HIV-p25-7. 40. en ce - Monoclonal antibody:, characterized that it is produced by a cell line according to any one of claims 38 and 39.  41.- Monoclonal antibody, characterized in that it is capable of reacting with an antigenic determinant of the glycoprotein gpl10 of the HIV envelope.  42. - Monoclonal antibody, capable of reacting specifically with an antigenic determinant of HIV, characterized in that it blocks the binding of an antibody produced by the cell lines according to any one of claims 38 and 39.    43. "Method for generating cell lines which produce antibodies reactive with antigenic determinants of HIV gpl10, charac-  EMI70.2  terized in that it comprises: the administration to a host of an immunogenic amount of an antigenic preparation enriched in HIV proteins, the observation, in the host, immunized of  EMI70.3  production of antibodies reactive with gpl10 antigenic determinants, obtaining, from the host, cells  <Desc / Clms Page number 71>  producing antibodies and immortalizing these cells, 1a selection of immortalized cells which  EMI71.1  produce antibodies to HIV gpl10, and cloning immortalized cells to produce cell lines.    44. - process according to claim 43, characterized in that the HIV proteins are recombinant fusion proteins expressed by a eukaryotic or bacterial host, or else the HIV proteins are obtained from an HIV extract or lysate.    45. - method for diagnosing the presence of HIV in a biological sample, characterized in that it comprises: the incubation of a monoclonal antibody capable of reacting with gpl10 of HIV contained in the said biological sample, and the detection the presence of immune complexes formed between the monoclonal antibody and the antigenic determinant in the biological sample, and therefore the determination of the presence or absence of HIV in the sample.    46.- Method according to claim 45, characterized in that the monoclonal antibody is capable of reacting with a neutralizing region or a gpl10 blocking peptide.    47. - method for diagnosing the presence of antibodies against HIV in a biological sample, characterized in that it comprises: the incubation of a peptide originating from a neutralizing region of gpl10 or p25 of HIV with said biological sample , and detecting the presence of immune complexes formed between the peptide and the antibodies in the sample reactive with the peptide, and therefore the  <Desc / Clms Page number 72>  determination of the presence or absence of HIV in the sample.    48. - A method for diagnosing the presence of HIV in a biological sample, characterized in that it comprises: the incubation, under hybridization conditions, of a nucleic acid segment coding at least for part of an HIV neutralizing region with the nucleic acid contained in the biological sample, and the detection of the presence of hybrid complexes formed between the nucleic acid segment and the nucleic acid of the sample, and therefore the determination of the presence or the absence of HIV in the sample.    49. - method for determining a strain of HIV  EMI72.1  in an infected host, characterized in that it comprises: the incubation of a biological sample taken from the hole with a peptide originating from a neutralizing region of HIV, and the detection of the presence of immune complexes formed between the peptide and antibodies from the sample, and hence the determination of the HIV strain infecting the host.  50.-Composition to be used for the treatment of a patient suspected of having been exposed to HIV, characterized in that it comprises a therapeutically or immunologically effective dose of at least one monoclonal antibody reactive with a neutralizing region of HIV.    51. - Kit to be used for the treatment of a patient suspected of having been exposed to HIV, characterized in that it comprises a therapeutically or prophylactically effective dose of one or more  <Desc / Clms Page number 73>  several monoclonal antibodies reactive with an HIV neutralizing region, blocking peptides capable of attenuating the infectious power of HIV and / or monoclonal antibodies reactive with the blocking peptides.    52. Kit according to claim 51, characterized in that the monoclonal antibody reactive with the neutralizing region of HIV is produced by a cell line bearing the designation ATCC HB 9405 and a blocking peptide is the T peptide or a derivative, or the like of it. - 53. - Composition to be used for the treatment of a patient infected with HIV after determination of the HIV strain by the method according to claim 49, characterized in that it comprises a monoclonal antibody specifically reactive with a neutralizing region of the strain of HIV.    54. - Composition to be used for vaccinating a patient against HIV infections, characterized in that it comprises an immunologically effective dose of an anti-idiotypic monoclonal antibody, the antibody being specifically reactive with an idiotype of a second antibody monoclonal capable of binding to a neutralizing region of HIV.