Useful compositions, vaccine and monoclonal antibodies for the treatment of the affections by virus HIV and processes of production of these antibodies and diagnosis of the presence of this virus.

The present invention relates generally to^- at diagnosis, treatment and prevention of viral infections.

It relates more particularly to compositions and methods for the production of monoclonal antibodies and peptides useful in the diagnosis, neutralizing and vaccination by human immunodeficiency virus (human immuno-deficiency virus) HTV.

The infecting agent syndrome of immuno-to-10 deficiency (aids) and its acquired prodromal phase, the corresponding complex AIDS bow ("sida by extreme complex derivatives"), and the syndrome Lymphadenopathy (SAL), is a novel retrovirus lymphotrophic. The virus has been variously called LAV which, to HTLV-III, ARV1, HTV and more recently.

Etant.donné than HIV reached pandemic proportions, the treatment of infected individuals and the prevention of transmission to uninfected individuals exposed and is a fundamental interest. Various therapeutic strategies. have target various stages in the life cycle of the virus and 20 are underlined by Babb JR and embroidering, 1987, In nature 325:773.

One approach is to use antibodies that bind virus and inhibit viral replication, either by interfering ' with virus entry into cells hfltes, or by other mechanisms. After the one or more constituents of the viruses that may or sensitive to the intervention of the antibody are identified, one can expect that protective levels of antibody to neutralize the infectivity of the virus can be generated by vaccination or, alternatively,

2

0∩O* f. V HAS i) ' L * b C.

by the passive administration of immunoglobulins or monoclonal antibodies having a desired specificity.

It is believed that forming the envelope glycoproteins of most retroviruses react with receptor molecules on the surface of susceptible cells, thereby determining the infectivity of the virus for some hosts. Antibodies that bind to the glycoproteins can block the interaction of the virus with cellular receptors, in ' neutralizing the infectivity of the virus. See generally, Tea Mineralogical d'Tumor Microbiology, 534 (J Tooze who, ed, 1973) and ANN Tumor Microbiology, 226, 236 (R Weiss and other, EDS, 1982) these two documents being cited herein in their entirety as a reference. See also, González-to-Scarano and others, 1982, Virology 1 20:42 (The stock viruses); Matsuno and Inouye, 1983, Disgusting. Immune. 39:155 (Neonatal nursing the Calf Diarrhea viruses); and Mathews and others, 1982, J., Immunol. , 129:2763 (Encephalomyelitis viruses).

The general structure of a virus is the that of a ribonucleoprotein core surrounded by a shell containing lipids that the virus acquires during a budding from the membrane of the infected host cell. Housed within the casing and projecting therefrom, the glycoproteins found encoded virus.

The glycoproteins of the envelope of HIV are initially synthesized in-the infected cell in the form of a precursor molecule of 150,000 - 160,000 dalton (gp150 or gp 60!), which is then processed in the cell to form an n-terminal fragment of 110,000 - 120,000 dalton (gpllO or gp! 20) to generate the external glycoprotein, and a c-terminal fragment of 41,000 - 46,000 dalton (the GP 41), which represents the envelope glycoprotein transmembrane.

For the reasons given above, the glycoprotein gpllG HIV has been the subject of significant research as a potential target for the life cycle of the virus. It has been shown that sera of infected individuals

hIV neutralizing HTV i_nin vitro , and that antibodies that bind to purified gpllO are present in sera.

See Robert Guroff and other, 1985, In nature 316:72; Weiss and other, 1985, . In nature 316:89; and Mathews and other, 1986, Process. Proc. CDAA. IBS. PAN., 83:9709. The purified recombinant gpllO molecule and can enhance the production of serum neutralizing antibodies when used to immunize animals, see Robey and other, 1986, Process. Proc.

CDAA. IBS. PAN., 83:7023; Lasky and other, 1986, Forensic science 233:209; and for immunizing a human, see Zagury and other, 1986, In nature 326:249. The binding of the molecule to the receiver gpllO (Τ 4) has also been described cd4, and monoclonal antibodies that recognize certain epitopes cd4 receptor have been described as blocking the binding HTV, syncytial formation and infectivity. See McDougal and other, 1986, Forensic science 231:382. Putney and other (1986, Forensic science 234:1392), have discovered antibodies neutralizing sera in animals following immunization with a recombinant fusion protein containing the carboxyl-terminal half of the molecule gpllO and have further proved ' that glycosylation of the envelope protein is not required for a neutralizing antibody response.

A vaccine for AIDS English using the molecule gpllO HIV or portions thereof may thus be desirable. Forming subunit vaccines provide an alternative th vaccines prepared from attenuated or inactivated virus. Killed vaccines are concern given the possible defect that they have to kill all viral particles, and attenuated vaccines may have more mutation and recover their capacity th causing disease. With subunit vaccines, only the parts of the virus that contain antigens or epitopes that are capable reveal immune responses, it is to say neutralizing antibodies, the ADCC, and a cytotoxic T cell response, are used to immunize the host. A major advantage of subunit vaccines is that the material is excluded from the viral uninteresting.

Viral subunits in usable. a vaccine can be produced by several methods. By way of example, the envelope glycoprotein can be expressed and purified from a bacterial host, although this molecule can be deficient at most of the post-translational modifications (such as glycosylation), or other methods. Such modification can be achieved using a eukaryotic expression system, such as a yeast or mammalian cells cultured.

Virus genes have been introduced into mammalian cells using virus as vector vaccines.

See e.g. Mackett m and other, 1982, Process. The nat. CDAA, IBS. REVENUE 79: 741b; Panicali, D. and Paûletti, e, 1982, Process, the nat. CDAA. Revenue IBS 79:4927. A recombinant vaccine virus can be constructed according to the method hu and others, In nature (1986) or 320:537 Chakrabarti and others, In nature (1986) 320:535, these two documents being given herein for reference. In these systems, the viral glycoproteins produced by infected cells with recombinant vaccines are glycosylated in a suitable manner and can be transported to the cell surface for extrusion and ultimate insulation.

An important step in the manufacture of a vaccine

of subunit is adequate purification of glycoprotein desired from the complex mixture of the expression system.

Several methods can be used to accomplish•purification. These include, but are not limited to, preparative polyacrylamide gel electrophoresis, the gel permeation chromatography and various chromatography methods (it is to say ion exchange, reverse phase, immunoaffinity, hydrophobic interaction), as well as other methods. Most of these methods are used in various combinations to provide preparations sensitive * telling a pure (Kleid, D.G., and others, 1981, Forensic science 214:1125; Cabradilla, CD and other, 1986, At Toronto 4:128, Oowbenko, Mrs. O.J., 1985, Process. The nat. CDAA. IBS. REVENUE 82:7748), said items being given herein for reference.

Methods that reduce the number of phases required for the purification most viral antigen in particular from a mixture expression complex, are required to manufacture subunit vaccines. Effective separation of the extraneous components antigens could be effected by utilizing chromatography

d.' immunoaffinity. This technique, also known as immunoadsorption, consists in its principle to selectively adsorbing an antigen on a solid support to which a specific antibody was covalently attached. Selectively adsorbed antigen is subsequently eluted from, such an adsorbent to affinity of antibodies, for example by changing the pH and/or the ionic strength of the buffer.

Polyclonal antibodies, obtained from animals immunized with the desired antigen or th from individuals naturally infected or contaminated (see e.g. Lasky and other, above), have been used frequently as immunosorbent, but, in general, these reagents have disadvantages substantial, such as (the I) the antibodies bound to the insoluble carrier are not all specific for the molecule of interest, thereby requiring further purification; (ll) yields the desired antigen are frequently low; and (II) the affinities for the antibody often vary from preparation to the other, which requires changes in the eluting process. The use of monoclonal antibodies specific for the viral antigen desired for use in the preparation of subunit, rather than polyclonal antibodies, should circumvent these difficulties.

Murine monoclonal antibodies which bind to the hiv antigens have been described. Several groups of investigators have described monoclonal antibodies specific for the protein core p25 (see e.g. di-mars Veronese and other, 1985, Process. The nat. CDAA. IBS. REVENUE¹ 82:5199 and prefer to, J, and other, 1986, J Immunol. 136:1442). Monoclonal antibodies specific for the membrane glycoprotein gp41 have also been described (see for example the di mars Veronese and other, 1985, Forensic science 229:1402).

It remains that there is a need in the technique concerned for monoclonal antibodies specific for epitopes in regions well ' defined envelope glycoprotein important, gpilO. Monoclonal antibodies that bind to these regions and cause a reduction or elimination of the replication and the transmissibility of HIV prevention and utility would be substantial. In addition, the monoclonal antibodies could also be used to purify the region, desired gpilO from broken or shattered viruses, or recombinant expression systems for use in vaccines, for example. Further, the region containing the epitope or epitopes recognized by the monoclonal antibodies could be chemically synthesized, thus avoiding the difficulties inherent in the purification and administering larger fragments of the molecule gpilO. The present invention addresses the needs above as well as other.

The present invention provides peptides capable of mimicking or immunologically neutralizing epitopes of proteins HTV, nucleic acid probes encoding such peptides and monoclonal antibodies reactive with such peptides, as well as other peptides interfering with the infectivity of HIV. These new materials find use e.g. in the diagnostic tests for the detection of infections by HIV and in the treatment actions for treating or vaccination against 0 ∩ Ρ Π. K ?

such infections.

The present invention provides novel compositions and methods for neutralizing the infectiqns HIV, it is to say for the prevention or inhibition of the formation or substantial cellular transmission HIV infections in an hflte. More particularly, peptides which mimic a region HIV and neutralizing monoclonal antibodies reactive with such region are used for diagnosing, treating and vaccinating against HIV infections. In this connection, the expression "neutralizing region" indicates those portions of HIV, particularly proteins HTV, 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 the HIV infections. Assays to the neutralization are well known and may include the reduction in HIV infections in the T cell lines, the reduction of plaque-forming units of pseudotyped VSVs (HTV) bearing the envelope glycoproteins of HIV, syncytia inhibition assays, receptor binding assays and a virion. If desired, the neutralizing activity can be compared to the reactivity of antibodies in the immunochemical tests such as testing the immunofluorescence, 'd ' blotting and radioimmunoprecipitation.

According to one aspect of the invention, the novel peptides, which typically have less than about 50 amino acids, contain five or more contiguous amino acid forming epitopes substantially similar to epitopes located on the regions-neutralizing gpllO p25 or HIV, encoded by the regions clade and the gag respectively, of the HIV genome. The regions that are of particular interest include those which extend from about 301 amino acid residues up to about 336 of gpllO, and from about 278 up th about 319 and from about 315 to about 363 of p25, all from the strain designated the LAV HTV_gRU . Designations of dibasic amino acid residues originate from the database of the ICRP (database sequence viruses aids, ICRP U.S. National Laboratory, fill the dividing, ICRP, with nM-87545).

The familiar art appreciate that regions like additional ("homologous") from other isolates of HIV can be identified based on their location within corresponding proteins from various isolates. In practice, such homologs can be identified by reference to the sequence data LAVgpjj, as follows:

(has) the amino acid sequences of isolates hiv and LAVgRy may be aligned to obtain a highest homology between the two sequences;

(b.) peptides comprising amino acid sequences corresponding to the site of HIV isolates peptides

THE LAV

LND

the LAV proteins immunologically mimic

Daughter-in-law can be identified. Peptides comprising amino acid sequences of isolates HTV thus identified immunologically mimic or imitate, and typically, the corresponding proteins isolate HTV.

This method can be applied to the HTV strains

which are still to be discovered. For example, when new strains of HIV are identified, their amino acid sequences of core and skin may be aligned with that of LAVgRU to obtain a highest homology. The means by which the sequences are aligned, are known to those familiar with the technique. By aligning the sequences, it is desirable to maintain as much as possible homology between cysteine residues. The amino acid sequence of the new species or strain HTV, which corresponds to the situation or site peptides specifically disclosed herein, can be synthesized and used according to the present invention.

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

It is not necessary for the present invention that the epitopes contained within such sequences are reactive by cross-reacting with antibodies for all species or strains of HIV. The peptides including immunological epitopes that distinguish species or serogroup of another, find use in the identification of particular species or serogroups, th_T- may in fact to help identify 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 other region neutralizing, in therapeutic compositions.

The peptides of interest very preferably come from the region gllO virus. The peptides of particular interest in this region are peptides encoded in the open reading frame clade extending from about the base pair (LP) 6667 to about 6774 isolate the LAV_mDD . Thus, various homologous regions of other isolates HTV include homologous sequences obtained from the database of the ICRP (except LAV2), as set forth in Table I.

10

1 TA8LSAU.

008652

309,309 309,309 309 3U 314,310 312,306 322,311 306,304 320,302

2 ΗΧΒ CACAGA

GlnArg

BH102

BH3

HXB3

K9M

LND

HÂL!••"•■

In Eli GlyLeu

ARV2

KMJ2

RFENV

GlyLeu z6

23 Clay

Nï5GlyPro

.42

LAV2MetSer

UXB2... AGM... CAAGCACATTGT

... Αarg... GlnAlaHisCys 331

BH102 331

BH8 331

HXB3 331

H9M 331

Diagonalizing of Arg-Tyr 334

fcLIIlelleGly 330

Lys-lle ARV2... 333

2 326 W m j-lle

Lys-lle RFtNV... 34, 3

Gly-z6... 334

Z3 IleThrGIy 326

NY5 325

Lle .42 341

330 .Ggaccagggagagcatttgttacaataggaaaaataggaaatatg LAV2ArgProArg puts

Gln. glyproglyargalaphevalthrlleglylysileglyasnmec 326,326 326,326 326,331 329,323 327,320 337,327 319,320 335,323 LeuTyr Thr-IleVal-to-Asplle

... GlnSerLeuTyrThr-Arg IleValSerArgSer His MC - - - - - the R Arg IleGlyAsp - Arg ArgGlu...-to-UeGlylleVallleTyrAlaThr Gln-IleGlyAsp

. -. GlnAlaLeuTyrThr-Arg ArgThrLysIlelle - Lys-Val TyrAlaLys-to-Gly

... - ThrLeuTyrAlaArgGluAsplle. r 'ValTrpTyr Thr-Glu-LeuGlyAsn~- - HisVal-to-HisSerHisTyrGlnProIle Lys hx32 TGTACAAGACCCAACAACAATACAAGAAAAAGAATCCGTATCCysThrArgProAsnAsnAsnThrArgLysArgIleArglle

- - - - H9m■RCS

Poorly-Gly - ArgGly■HisPhe LIS - Ala-to-TyrG Lnis■of Thr - Gln - proinsulin

J is 2 W m - - - - - Tyr-Val ArgSer-to-a-a-LeuSer-to-RFENV RCS - -: - - - ThrLys z6 TyrLysGlnSer - - - of Thr Gly-pro z3 rAspLy th e e - Lys-I the a-a-a-a-a-a-GlnSe Gly-Lys-R ny5 Ala yeastCDC4 ValThrLeu 2 HSI

Val-Lys-Gly-Gln LAV2MetLeu other peptides suitable for generating or obtaining by "Mutation" monoclonal antibodies, include those encoded in the open reading frame clade from about LP 7246 up to about 7317 ^ ^ L-Vavg. Such antibodies and peptides are especially useful in testing of immunization.

In the region the gag the isolate LAVg ^, the amino acid sequences p25 from about 278 to and from 315 to 319, 363 are additional neutralizing regions of HIV. Those familiar of this technique will include that regions of additional neutralizing HIV can be identified based on the teachings given herein; in particular, combinations of monoclonal antibodies reactive with various epitopes of HIV different have neutralizing activity.

The peptide-I, also called a peptide 29 is encoded in the open reading frame clade from a number of amino acid residues from about 30b up to about 328, and will have the following amino acid sequence, wherein the oligopeptides are included in the following sequence will include linear epitopes within any such sequence:

(29) The I-Y-Thr-Arg-Lys-Ser-Ile-Arg-Ile Gln Gly - Pro rGly Arg-Ala-Phe-Val-Thr-Ile-Gly-Lys -

Ile there '

wherein Y and Y ', if present, each represent sequences up to about twenty amino acids.

When Y and/or Y ' are present, they may include, for example, one or more amino acids from sequences that are arranged adjacent to amino acid residues 328 to 308 of the envelope sequence HIV or any part.ie side of these sequences or arranged to c3té. By way of non-limiting example, one or more Y may comprise

portions of the amino acid sequence of Rj ' the LAV envelope

LND

12 ο λ-U-R-Y-κ 9 υ ΰ 4 from about a number of residues 301 to 307; and Y ' can include any at portions of the amino acid sequence of envelope LAVg ^ Y from a number of residues from about 323 to 336 as follows:

The II (29a)-cys-thr-Lys Asn-Asn-Asn-Thr-Arg-Lys-Ser-Ile - Arg-Ile-arg-Gly-Pro Arg-Ala-Phe-Val-Thr - Ile-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 the peptide 29 may be particularly useful:

Lll (29b) therein Thr-Lys-Ser-Ile-Arg-Ile-arg-Gly-Pro -

THERE '

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

VI (29c) Y-Ile-Gln-Gly-Gly-Pro Arg-Ala-Phe-Val-Thr - Ile-Gly-Lys-Ile there '

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

According to another embodiment, the homologous regions of the isolate ARV1 and 2, interest particuliersont encoded in the open reading frame clade from numbers of amino acid residues from about 306 to about 323 and have typically, the following amino acid sequences, wherein the oligopeptides are included in the following amino acid sequence comprise linear epitopes within any such sequence:

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

wherein Y and Y ', if present, are each one to about twenty or more amino acid residues. If Y and/or Y ' are present, they may include one or more amino acid residues of the sequences being arranged adjacent to amino acid residues 306 to 323 of the envelope sequence ARV1-a 2 or any part of these flanking sequences or disposed beside.

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

Alternatively, sequences truncated peptide V may be prepared. In this regard, the following sequences can be particularly useful:

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

VII. Y-Asp-Cys-Lys-thr-Ile-Leu-Lys-Ala-Leu-Gly-Pro - Ala-Thr - Leu - Glu-to-Lew norLeu-to-Lew norLeu Thr-Ala-Cys-Y '

in which Y and/or Y ', if present, each represent sequences of up to twenty or more amino acid residues.

Another example includes the homologous regions of the isolate the LAV-to-2, such as encoded in the open reading frame clade from about a number of amino acid residues 311 to 330, and typically have the following sequence:

Viii (110 - 2 - 2) the Y-Lys-thr-val-Ile-nor-Leu-NORs Ser-Gly-his-val--

H e - h I-s-I-s - t - r - h r - g1 n - p therein is R-o - y '

wherein Y and/or Y ', if present, each represent sequences of up to twenty or more amino acid residues. (See item in Nature 326:662 (1987),

which is used herein as reference).

In another aspect of the present invention, new cell lines capable of producing monoclonal antibodies and compositions comprising such antibodies are provided, wherein the antibodies are capable of recognizing selectively to extremely high titers

2, 4 7

(of 10, to 10 up to about 10 or more) neutralizing regions in a predetermined sequence of envelope glycoproteins or p25 gpllO, their protein precursors, the recombinant fusion proteins biologically expressed, and synthetic peptides which contain one or more epitopes in the region of predetermined sequence of gpllO or p25. The hybrid cells object of the present invention have a chromosome identifiable in which the DNA of germline was rearranged to encode an antibody having a binding site for an epitope on gpllO p25 or common to several or all HIV clinical isolates. Monoclonal antibodies can be used in a variety of ways including the diagnostics and therapy, as well as to identify other antibodies reactive cross, such as blocking antibodies. The peptides or polypeptides containing the epitope or epitopes with which they react can find use as immunogens for vaccines separate, or as therapeutic agents.

According to another embodiment of the present invention, there is used, for use in conjunction with the monoclonal antibodies neutralizing or the foregoing peptides, additional peptides or antibodies that interfere with the binding of HIV receptors for further moderating the infectivity of HIV. 0th preferably, peptides called "blocking peptides" capable of inhibiting the proliferation treaty virus, as well as monoclonal antibodies specific for epitopes contained in such blocking peptides, can be used to increase the effectiveness of treatments against HIV infections.

HTV blocking peptides typically correspond to the amino acid sequence of HIV that is judged to be essential for the binding of virus to a host cell, such that the encoded amino acid residues clade of envirort 190 to about 197 of LAVgpy and from about 185 to about 192 of ARV1 and 2' and to HTLV-III(the BH and 10). Peptides include 1'T-octapeptide sequence (Ala-Thr Thr-Asn-Tyr-Thr) and its various derivatives (see IX below) and the analogs (. example XI below) described by PERT and other (1986, Process. Proc. CDAA. IBS. REVENUE 83:9254 - 9258, which publication is incorporated herein as reference) located on the envelope glycoprotein (gp110 or 120).

For example, blocking peptides having the

•following sequences are of particular interest, preferably with an acetylation nf ^ - terminal and an amidation of the carboxy-terminal:

IX. (173d) ^ - V-Ala-Thr Thr-Asn-Tyr-thr thereto ';

THE X (186)

The Y-Thr-Thr-Asn-Tyr-thr thereto ';

(187) XL

The Y-Thr-Thr-Ser-Tyr-thr there ' ;

(188) XII AS

There Thr Asp-Asn-Tyr-thr thereto ';

(189) XIII.

Y-Asn-Thr-Ser-Tyr thereto ';

(190)-XIV

There Asp Thr-Asn-Tyr-Ser-there ';

(191) Xv frames Y-Ala-val-Phe-Thr-Asp-Asn-Tyr-thr thereto ';

in which, for each peptide, Y and Y ', if present, each comprise an amino acid sequence of up to about 20 amino acids. The epitopes or antigenic determinants in the peptides are typically defined by at least about five contiguous amino acids, and find use in e.g. the simulated sites naturally occurring HIV antigens to generate antibodies reactive HIV and vaccines.

The preparation of monoclonal antibodies may be made immortalizing the expression of nucleic acid sequences that encode antibodies specific for hiv, by introducing such sequences, typically AONc encoding the antibody, in a host capable of being grown in culture. The immortalized cell line may be a mammalian cell line that has been transformed by oncogenesis, by transfection, mutation or the like. 0th such cells include myeloma cell lines, the lymphoma cell line or other cell lines capable of supporting the expression and secretion of the antibody the in vitro. The antibody can be an immunoglobulin naturally occurring mammalian, produced by transforming a cell, particularly a splenocyte, by using a virus or by melting of the lymphocyte with a neoplastic cell, such as a myeloma, to produce a hybrid cell line. Typically, the splenocyte shall be obtained from an animal immunized against the HIV virus or a fragment thereof containing an epitopic site.

The immunization protocols are well known and can vary greatly for remain however effective. See Goding, The monoclonal Mitochondrial Antibodies: Health Organization, When Academic press, (1986) 2è editing " which is used herein as reference. A virus broken or shattered, synthetic peptides, and bacterial fusion proteins which contain antigenic fragments of the molecule gpllO p25 or, can be used as immunogens. 0th preferably, the immunogenic virus broken, peptides or recombinant proteins will be enriched proteins or fragments thereof containing the epitopes for which the neighbour producing B antibody in the splenocytes are desired.

More particularly, solutions containing extracts or lysates of viruses broken or ruptured, or supernatants of recombinant proteins biologically expressed or expression vectors is broken or separated, may be enriched in glycoproteins, if desired, using purification methods, such as e-xemple, polyacrylamide gel electrophoresis. The affinity purification by lectin is a convenient method and preferred to purify gpllO and other glycoproteins, for example the affinity purification using the lectin. The extent to which the glycoproteins are purified from solutions for use as immunogens, may vary significantly, it is to say less than 50%, typically 75% or at least 95% to, a manner of 95% to 99% and desirable, more desirably, absolute to homogeneity.

Once the proteins were purified to the desired degree, they may be suspended or diluted in a suitable physiological carrier for immunization, or may be coupled to an adjuvant. A preferred technique, for example, involves the adsorption of proteins and fragments on agarose of lentil lectin or other macromolecular carrier for injection. Immunogenic amounts of antigenic preparations enriched for proteins HTV, comprising the glycoprotein gpllO p25 and the core protein or antigenic moieties thereof, are injected, generally at concentrations in the range from 1 to 20 mg/kg of GP host. Administration may be by injection, e.g. intramuscular, intraperitoneal, subcutaneous, intravenous administration may be like in one or more stages, usually at intervals of one to four weeks. Immunized animals are monitored for production of the antibody in the desired antigens, then the spleens are removed and splenic lymphocytes 8 isolated and combined with a myeloma cell line or transformed. The transformation or fusion can be carried out according to conventional ways, the fusion technique or combination being described in a large number of patents, for example patents Americans no. 4,172 124.;

No. 4,350 683; № 4,363 799; no. 4,381 292; and no. 4,423 147.

See yet, Kennett and others, The monoclonal Mitochondrial Antibodies (1980), and the references cited article that are illustrative, and see still Goding, super.

The immortalized cell lines may be cloned and sorted, according to conventional techniques, and detecting the antibodies in the supernatants products which are capable of binding to the desired HIV viral proteins or p25 gpllO, the recombinant fusion proteins or synthetic peptides which contain the desired epitope region. The appropriate immortalized cell lines may then be subjected to a growth in in vitro or injected into the peritoneal cavity of a suitable host for production of ascites fluid. By virtue of the fact that one of certain antibodies of the present invention what is known as the specific for epitopes contained e.g. in the regions encoded by the genomic region LAVBRU about 6688 bp to about 6750 LP (29 encoding a peptide), or about 7246 bp to about 7317 (encoding a peptide 36) (numbering LP as tourists for a-Hobson and other, Glial cell 4 4:9 1985, which is used herein as reference), supernatants may be selected in competition with the monoclonal antibodies of the invention in a competition assay, thus, hybridoma cell lines immortalized with the additional the characteristics desired binding can be produced easily in th from a variety of sources depending on the availability of specific antibodies present for the particular antigen. Alternately, these cell lines can be combined with other neoplastic b cells, where such other B cells can be used as containers to genomic DNA encoding the antibody.

Although rodent neoplastic b cells, particularly murine is preferred, other mammalian species can be employed such as lagomorph, bovine, ovine, equine, porcine, avian or analogs. Immunization of these animals can be readily performed and their lymphocytes, particularly splenocytes, may be obtained for fusions.

The monoclonal antibody secreted by the transformed or hybrid cell lines may be any one of the classes or subclasses of immunoglobulins, such as IgMs, DDI, an IgA, _ ^ ^ IgGs, or IgE. Since IgG is the isotype most common used in diagnostic testing, it is often preferred. The monoclonal antibodies may be used intact, or as fragments, such as Fv-, Fabs, F. (Aβ ')₂ , but usually intact.

For attenuating the antigenicity possible in a human host of a monoclonal antibody, derived from an animal other than a human, chimeric antibodies can be constructed, in which the antigen binding fragment of an immunoglobulin molecule (variable region), is connected by a peptide bond to at least part of another protein not recognized as foreign by humans, such that the removed part of a human immunoglobulin molecule. This may be done by merging or combining of the exons of variable regions of animal with exons of alphaS2 gamma or constant regions of a human. Various techniques are known by those familiar technology, such as those described in Patent no. 86/01533 multinational, and in requests 171496 173494 and European patents, which are given here as

reference.

The monoclonal antibodies of the invention that exhibit neutralizing activity,, such as those that react with an epitopic site on gpllO or p25 or reactive with a blocking peptide, may also be incorporated as components of a pharmaceutical composition for ameliorating HIV infections. The composition should contain a prophylactic or therapeutic amount of at least one of the monoclonal antibodies of the present invention with a pharmaceutically effective carrier. The pharmaceutical carrier: must be constituted by any compatibility material, non-toxic and suitable for allowing the administration of monoclonal antibodies to the patient. Sterile water, alcohol, fatty products, waxes and inert solid products can be used as support.. pharmaceutically acceptable adjuvants (buffering agents, dispersing agents) may also be incorporated into the pharmaceutical composition. Such compositions may contain a single monoclonal antibody so as to be for example specific for strains of HIV with envelope glycoproteins containing an epitopic site in a region encoded by 6688 BP 6750 LP.

Alternatively, the pharmaceutical composition may contain one or more monoclonal antibodies to form a "nuclease". For example, a cocktail containing monoclonal antibodies against various strains of HIV would be a universal product with a prophylactic or therapeutic activity against the vast majority of clinical isolates of HIV. The a-cocktail which may contain monoclonal antibodies that bind to proteins or glycoproteins of HIV other than gpllO or p25, such as, for example, to glycoprotein gp41 or 1' integrase p34/nuclease. The molar ratio of individual constituents of the monoclonal antibodies do not typically either more than a factor of 10, more usually not more than a factor of 5, and will usually be between about 1:1 -2 for each of the other components of antibodies.

The monoclonal antibodies of the present invention peùvent be used in the form of compositions administered separately and data in conjunction with other anti-retroviral agents, including blocking peptides. The current state of the development of anti-retroviral agents, agents and especially hiv, is described in Publication Babb JR and other, In nature 32b:773 - 778, 1987, which is incorporated herein as a reference.

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

Preferably, the pharmaceutical compositions may be administered parenterally, it is i.e. subcutaneously, intramuscularly or intravenously.

Thus, the present invention provides compositions for parenteral administration, which comprise a solution of 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 may be sterilized by conventional techniques well known and sterilization.

The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions to meet, such as buffering agents and pH adjustment, the adjusting agents of the toxicity or the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and so on the concentration of the antibody in the formulations can vary significantly, it is L-e. least about 0.5%, usually at least about 1% or to as much as 15 or 20% by weight, and the concentration will be chosen primarily upon the volume formulas. fluid, viscosities, and so forth, preferably depending on the particular mode of administration chosen.

Thus, a typical pharmaceutical composition for intramuscular injection could be made up to contain 1 ml sterile buffered water, and 50 mg of monoclonal antibody. A typical composition for intravenous infusion 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 technique and is described in more detail in example publishing Remington-Peppers pharmaceutique forensic science, 15è DE. By Mack Publishing, Easton in, Pennsylvania (1980), which is indicated herein as a reference.

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

This technique have shown to be as effective with conventional immune globulins and restoration procedures for and freeze drying known in the technique can be used. It will be appreciated by those familiar with the technical lyophilization and reconstitution can lead to varying degrees of loss of activity of the antibody (e.g. immunoglobulins or conventional, IgM antibodies tend to have greater loss of activity that the igg antibodies) and that levels or concentrations of use may require adjustment for compensation.

Compositions comprising the monoclonal antibodies of the present invention, the peptides or their preparer can administered for the therapeutic and/or prophylactic treatment of HIV infections. For therapeutic applications, the compositions are administered to a patient already infected with HIV, in an amount sufficient to cure or at least partially arrest the infection and its complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose". Amounts effective for this use depend on the severity of the infection and the general status of the patient's own immune system, but are generally between about 1 to about 200 mg of antibody per kilogram of body weight, doses between 5 and 25 mg per kilogram being more commonly used. You have to keep in mind that the materials of this invention can be typically utilized in serious disease conditions, it is to say in situations where the life is threatened or potentially at risk. In such cases, it is possible and may be estimated desirable by the treating physician to administer these antibody excess sensitive.

In prophylactic applications, compositions containing the present peptides, antibodies or their cocktail are administered to a patient not already infected with HIV, but that has possibly been recently exposed or which is deemed to have been exposed to a risk of infection by the virus or the virus itself, to enhance the patient's resistance to such a potential infection or to the vaccinate against the virus. Defining the amount administered by a "prophylactically effective amount".

In this use, the precise amounts again depend on the state of health of the patient and condition of general immune, but are generally between 0.1 mg and 25 mg per kilogram, particularly between 0.5 mg and 2.5 mg per kilogram.

Single or multiple administrations of the compositions can be formed with doses and protocol selected by the treating physician. In any event, the pharmaceutical formulations should provide a quantity of the antibody or antibodies of the present invention sufficient to effectively treat the patient.

Further, the monoclonal antibodies of the present invention may find use as a carrier molecule or vehicle specific for a target. An antibody may be linked to a toxin to form an immunotoxin or a radioactive material or drug to form a pharmaceutical or radiopharmaceutical. Methods to produce immunotoxins and radiopharmaceuticals are well known (see e.g., The carries her dimerization inhibitor of cancer 68:317 (1984)).

It is also possible that data hétéroagrégats of monoclonal antibodies of the present invention and activators of human T-cells, such as monoclonal antibodies for the antigen or receptor gamma F. cd3_C. on T cells, can cause to human T cells or to cells bearing F._C. ~the gamma (such as K cells or neutrophils) kill cells infected with HIV via a cytolysis via antibody-dependent cell ("mediated cytotoxic Glial cell loop-mediated cytolysis depend" ' "THE ADCC"). Such hétéroagrégats may be joined, e.g., by covalent bonding of the anti-HIV antibodies with anti-cd3 using the hetero-bifunctional reagent-n-succinimidyl 3 - (2 a-pyridyldithiol) propionate moiety, as described by Karpowsky and others, J with exp. The MED. (1984) 160:1686, where the publication is given herein th for reference.

The peptide compositions themselves herein may also find therapeutic use, when administering product reduction, or elimination of the HIV virus in an infected host. These compositions, such as the peptide 29, blocking peptides and peptide 126 may be administered in brackets () 25 C. Rr 5?

suitable physiological or carriers, intravenous, subcutaneous, intramuscular, intraperitoneal and so on various vehicles include a phosphate buffered saline, saline, water, potassium chloride, sodium lactate or the like.

The peptide concentration varies greatly with its last use, activity and the mode of administration.

Preferably, the peptides have an amidation of the terminal■c00h - x *, a formylating Ni L-terminal or other derivatives Pharma S. ceutiquement acceptable. The addition of blocking peptides to peptides that mimic a region of HIV neutralizing and/or antibodies specifically reactive of the present invention will provide an enhanced therapeutic efficacy significantly. Other anti-HIV agents can also be included in the formulations (other than the monoclonal antibodies that bind the peptides) such as the 3 'a-azido 3' - deoxythymidine, the 2 ', 3' a-dideoxycytidine, the 2 ', 3'-dideoxy ', 2 3' a-didehydrocytidine, andc.

Use in the purification of monoclonal antibodies d.' immunoaffinity

Monoclonal antibodies specific for the polypeptides containing gpllO or other antigenic determinants, particularly epitopes obtained from recombinant fusion proteins biologically expressed or lysates or extracts of HIV cultured, are particularly advantageous for use in the purification protocols. Generally the antibodies have affinity association constants on the order of 10 to 10 m such antibodies can be used to purify the recombinant proteins from the culture medium of the recombinant expression system if the expressed protein is secreted or from the constituents of the biological expression system broken unless it is secreted. Generally, the monoclonal antibodies which are capable of reacting with gpllO or other antigenic determinants are attached or immobilized on a substrate or support. The solution containing the antigenic determinants HIV is then contacted with the immobilized antibody of in. the correct conditions for the formation of immune complexes between the antibodies and polypeptides containing the antigenic determinants gpllO. Unbound material is separated from the bound immune complexes, in which the complexes or antigenic fragments are then comstically parés.dugpllO medium.

Typically, the monoclonal antibodies are purified from a crude ℮ ascites fluid or supernatants, cell culture prior to attachment to a support. Such processes are well known to those familiar technology, and may include a fractionation of neutral salts with high concentration. Other methods, such as chromatography with DEAE, gel filtration chromatography, preparative gel electrophoresis, or affinity chromatography with protein, can also be used for purifying the monoclonal antibody before its use as a carbonized.

The support on which the monoclonal antibodies are immobilized must have the following general characteristics: (has) weak interactions with proteins in general to minimize non-specific binding, (d) good flow - which allow flow through materials of high molecular weight,

(e) possession of chemical groups that can be activated or modified to allow the chemical binding of the monoclonal antibody, (D.) physical and chemical stability under the conditions used for linking the monoclonal antibody (th) and weatherability and constituents buffers required for 1¹ adsorbing et .1 ' éluti.on of the antigen. Some media commonly used are agarose, polystyrenes derivatives, polysaccharides, polyacrylamide granules, the activated cellulose, glass and the like. Various chemical methods exist for the binding of the antibodies on the supports forming substrates. See generally Cuatrecasas, e., In Advances in Enzymology 3 6:29 (1972). The antibodies of the present invention may be fixed directly on the support or, alternatively, via a linker or spacer.

The general conditions required for the immobilization of monoclonal antibodies on the chromatographic media are well known in the technique. See e.g. Tijssen, P-. 1985 ., A practice NDA to partition an enzyme Immunoassay kits, where the publication is incorporated herein as a reference. The coupling current processes depend slightly characteristics and the type of the antibody coupling. The monoclonal antibodies have characteristics that are usually constant of a mixture to the other, thereby to such conditions or characteristics of being^V. optimized. The attachment typically occurs via covalent bonds.

A suspension of extracts or lysates of HIV virus, the supernatant of a cultivated biological expression system, or a suspension cells ruptured or broken is then added to the separation matrix. The mixture is incubated under conditions and for a time sufficient for the occurrence of immune complex formation, usually for at least 30 min, more usually for a period of 2 to 24 hours. The immune complexes containing polypeptides with portions antigenic gpllO are then separated from the mixture. Typically, the mixture is removed, such as by elution, and immune complexes bound are washed exhaustively with a buffer

* d-adsorbing. The immune complexes can then be eluted from the separation matrix using an eluent compatible with the particular carrier that is used, which are well known by eluants to those familiar technology. Also, the polypeptides containing the gpllO or other antigenic moieties can be selectively removed. For example, the peptides which contain an epitope recognized by the antibody, can be used to compete with the binding site of the antibody, which is a technique of élûtion AC that can be performed under conditions of mild élûtion. The polypeptide selectively adsorbsgpllO containing the antigen can be eluted from an adsorbent of antibody affinity by altering the pH and/or the ionic strength of the buffer. Chaotropic agents also may find use in the removal of the bound antigen. The selection of the chaotropic agent, its concentration and other elution conditions depend on the characteristics of the interaction antigen antibody, but once they are determined, these should not be subject to change usually required in the polyclonal affinity separation systems.

The material éluépeut requiring adjustment at physiological pH if buffers of low pH or ionic strength or higher are used to separate antigens gpllO linked the separation matrix. A dialysis or gel filtration chromatography may also be required to remove excess salts used in the eluent to enable reconstitution of gpllO or polypeptides containing antigenic moieties of gpllO on nascent conformations.

The methods of the invention provide, for example, the gpllO substantially, or purified polypeptides containing antigenic fragments thereof, is produced naturally by cell cultures infected by recombinant expression systems of bacteria, yeasts, or. mammalian cells or cultured insect. The gpllO and fragments or other purified proteins will be typically of a purity of greater than 50%, more usually at least 75% and frequently greater than 95 to 99%. GHG molecules can then find further use in a wide variety of applications.

The HTV gpllO proteins, polypeptides containing antigenic fragments thereof, or other proteins substantially purified according to the methods of the present invention, may find use in a wide variety of applications, including the formulations subunit vaccine aids, wherein the immunogen comprises an effective amount of antigen determinants of, for example, gpllO or region inactivating gpllO. Others of the formulation constituents may include antigenic proteins fractions HIV or glycoproteins that stimulate the production of antibodies (preferably neutralizing antibodies) in a host immunized, which antibodies are capable to protect against subsequent infection by HIV.

Uses in diagnostics monoclonal antibodies

The monoclonal antibodies of the present invention are also useful diagnostically. They can either be labeled or unlabeled for this purpose. Typically, diagnostic tests enable to detect the formation of a complex through the binding of the monoclonal antibody on an HIV antigen. If they are unlabeled, the antibodies find use e.g. in the agglutination assays. In addition, unlabeled antibodies can be used in combination with other labeled antibodies (second antibodies) that are reactive with the monoclonal antibody, such as antibodies specific for immunoglobulin. Alternatively, the monoclonal antibodies can be labeled directly. A wide variety of labels can be used, such as radionuclides, fluorescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, ligands (particularly haptens), so on various types of immuno-assays are available, and, for example, those described in the patents Americans no. 3,817 827; 3,850 752; 3,901 654;

are given herein for reference.

In the usual manner, the monoclonal antibodies and peptides of the present invention find use in enzyme immunoassays where, for example, the antibodies of the invention, or 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 suspension cell culture infectéespar viruses, is combined with the antibodies, binding occurs between the molecules and the antibodies having the desired epitope.

Such proteins or viral 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 bound to the antigen is determined. Other conventional techniques well known to familiar technology can also be used

Kits may also be designed for use with the antibodies in the detection of infection HIV or for the presence of hiv. Thus, the monoclonal antibody compositions of the present invention may be proposed, usually in lyophilized form, either alone or in conjunction with additional antibodies specific for other epitopes of HIV.

The antibodies, which may be conjugated or non-conjugated to a label, are included in the kits with buffers, such as tris-, phosphates, carbonates and c..., stabilizers, biocides, inert proteins, e.g. bovine serum albumin, or the like. In general, these materials will be present in an amount of less than about 5% 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 to include an inert extender or excipient to dilute the active ingredients, where the excipient may be present in an amount of 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 antigens - p25 or gpllO, or whole virus, in various biological samples can find use in the diagnosis of infection by HIV current. The biological samples may include, but are not limited to, blood serum, saliva, semen, the tissue samples obtained by biopsy (brain, skin, nodulation lymph, spleen, and so forth.), culture supernatants of cells, bacterial expression systems and eukaryotic broken or shattered, and the like. The presence of the virus is tested by incubating the monoclonal antibody with the biological sample under conditions contributing to the formation of an immune complex, which is checked in the formation of the complex. In one embodiment, the complex formation is detected by using a second antibody capable of binding the flight monoclonal antibody that is typically conjugated to a label and formulated in a manner analogous to the antibody formulations described above. In another embodiment, the monoclonal antibody is attached to a solid phase support which is then contacted with a biological sample. After an incubation phase, the labeled monoclonal antibody is added to detect bound antigen.

The present invention concerns novel peptides which, inter alia, immunologically mimic protein epitopes encoded by the HIV retrovirus, particularly epitopes encoded in the regions the gag or clade the viral genome encoding gpllO p25 or respectively.

To meet variations in strain to strain, among different isolates, tolerances for the conservative substitutions, and selecting among the alternative if the non-conservative substitutions are required, can be realized. These peptides can be used as immunogens, for inhibiting or eliminating the production of hiv in. in vitro 00 or I in vivo, for detection of 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 compounds, labeled or unlabeled, bound to a solid surface, or the like.

According to one embodiment, peptides of interest may drift region gpllO virus. The region which pre feel of particular interest is that which is in the open reading frame clade extending from about the base pair 6688 LP up to about 6750 7246 LP LP and from about up to about 7317.

The peptides of interest, comprising the blocking peptides, comprise at least five, sometimes six-, sometimes eight, sometimes twelve, sometimes 21, usually somewhat less than 50, more usually somewhat less that about 35 and preferably less than about 25 amino acids in an encoded sequence by a HIV retrovirus. In a manner desirable, the peptide will be as small as chargeable while still maintaining substantially all of the antiviral activity of the peptide of immunoreactivity or larger. In some cases, it may be desirable to join two or more oligopeptides overlapping to form a structure of unique peptide at using them as individual peptides - simultaneously, which together provide an equivalent sensitivity or separately parent peptide.

The peptide can be modified by introducing substitutions conservative or non-conservative in the 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. Oans many cases to achieve chemical stability, methionine can be substituted by norleucine (nor-).

It should be understood that the peptide used in the present invention need not be identical to a polypeptide sequence of HIV particular, provided that the compound is capable of providing a competition immunoassay with proteins of at least one of the strains of the HIV retrovirus. Therefore, the peptide of the invention can be subject to various changes, such as insertions, deletions and substitutions, either conservative or non-conservative, if such changes may provide certain advantages in use. By conservative substitutions, is meant substitutions in groups such than Gly, Ala-; of val, lle, Leu-; PSA, the Glu; of Asn, Gln; RCS, of Thr; at Lys, Lys; to Phe, Tyr to; and nor, puts. Usually, the sequence does not differ by more than 20% with respect to the sequence of at least one strain of a HIV retrovirus except where additional amino acids can be added on one or the other terminal so as to provide a "arm" by which the peptide of the invention can appropriately be immobilized. The arms can have a length that is usually at least 1 amino acid and can be of 50 or more amino acid, most often from 1 to 10 amino acids.

The peptide wherein the amino acid sequence

is modified by substitution, addition or removal of amino acid residues, must retain substantially all of the antiviral activity or of immunoreactivity of unmodified peptides, which can be conveniently measured by various techniques described herein test. The form d-isomer of one or more amino acids may be used, if desired, to alter the biological properties, such as the activity, the breakage rate andc..

•further, a, two or more amino acids are added on the end portions of an oligopeptide or peptide to facilitate the binding of the peptides to each other, for the coupling. to a support or a peptide larger, for reasons that will be discussed further, for modifying the physical or chemical properties of the. peptide or 1' an oligopeptide, or the like.

Amino acids such as tyrosine, cysteine, lysine, glutamic or aspartic acid, or the like, may be introduced to the c or n-terminus, the peptide

or of the oligopeptide to provide useful functionality in the link. Cysteine is particularly preferred to facilitate covalent coupling to other peptides or to form polymers by oxidation.

Further, the peptide or oligopeptide sequences can differ from the natural sequence by the sequence being modified by acylating the NH ^ - terminus, for example acetylation or thioglycolic acid amidation by, amidation of the carboxy-terminus, e.g., with ammonia or methylamine, to provide stability, increased hydrophobicity for linking to a support or other molecule, or for polymerization.

Thus, for example, in the peptides in I-VIII and IX-Xv frames 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 cysteine residues with amino acids spacer. Glycine constitutes a spacer means or a spacer particularly preferred. Preferred peptides for use in an oxidative polymerization are those in which Y or Y ' represents at least two cysteine residues die. If two cysteine residues are present at the same end of the peptide, a preferred embodiment exists when cysteine residues are separated by means of a three-residue spacer amino acid, preferably glycine. The presence of cysteine residues may allow the formation of dimers of the peptide and/or increase the hydrophobicity of the resultant peptide which facilitates immobilization of the peptide immobilized test systems or systems with solid phase.

Use of particular interest is that of the thiol group of cysteine or thioglycolic acids used for the acylation of amino end groups or the like for bonding two peptides or oligopeptides or combinations thereof by a disulfide bond or a greater binding to form polymers that contain a number of epitopes. Such polymers provide the advantage of increased immunological reaction. If different peptides are used to make the polymer, they have the additional ability to induce antibodies that immunoreact with several antigenic determinants of different isolates of HIV.

To achieve the formation of antigenic polymers (synthetic multimers), can be used compounds having groups of bis-haloacétyles, nitroarylhalogénures of, or the like, when the reagents are specific for thio groups. Thus, the connection between the two mercapto groups of various peptides or oligopeptides may be a single bond or a linking group of at least 2, usually at least 4, and not more than about 16, usually not more than about 14 carbon atoms.

The peptide according to the invention can be used by being bonded to a support-soluble macromolecular (e.g. not less than 5 kDal). Conveniently, the support may be a poly (amino acid), either natural or synthetic, against which antibodies have little chance of being encountered in human serum. Examples of such carriers are the poly-L lysine, 1' keyhole limpet Hemocyanin hemocyanin, thyroglobulin, albumins, such as bovine serum albumin, tetanus toxoid. so on the choice of the support is substantially dependent on the ultimate use of the antigen, and depends on utilities and the availabilities.

With such conjugates, there will be at least one molecule of hands at a peptide of the invention by macromolecule, and not more than about 1 by 0, 5kdal, usually not more than about 1 by 2kdal of the macromolecule. One or more different peptides can be bound to the same macromolecule.

How to accomplish the connection is conventional, and uses reagents such as p-maléïmidobenzoique, p-méthyldithiobenzoïque, maleic acid anhydride, succinic acid anhydride, glutaraldehyde like binding can occur on the amino terminus, carboxy terminus or at a site intermediate the ends of the molecule.

The peptide according to the invention can be derivatized by binding, may be bonded while being coupled to a support, or the like.

Various trial protocols known to those familiar technique may be used to detect the presence of either the antibodies on the retroviral proteins is the retroviral proteins themselves. There is a particular interest to use the peptide as the labeled reagent, if the label provides a detectable signal, or to bind the peptide, either directly or indirectly onto a surface, whether the antibody on the peptide in the sample becomes bound to the peptide to the surface. The presence of a human antibody bound to the peptide may then be detected using an antibody specific for human immunoglobulin xenogenic, normally both IgM and human igg, or a labeled protein specific for immune complexes, for example factor RF the protein has Staphylococcus aureus.

As an illustration of a testing technique, is the use of a sample container, for example the well plates having microwells, wherein the OspA polypeptide of the invention or the conjugates thereof are ad. sorbed on the bottom of the container and/or the walls either covalently or non-covalently.

The sample, which is normally of the human blood or serum diluted in a buffered medium appropriately, is put into the container and sufficient time is left for the formation of the complex between the polypeptide and all parent antibodies in the sample. The supernatant is removed and the container scrubbed to remove non-specifically bound proteins. A labeled specific binding protein that specifically binds to the complex, such that the anti-xenogeneic for human immunoglobulin, is used for detection.

The peptide can be prepared in a variety of ways. The peptide, because of its dimensions in relatively short, can be synthesized in solution or on a solid support according to conventional techniques. Various automatic music synthesis are commercially available today and can be used with known protocols. See e.g. Stewart platform and Young, segregation peptide that constraint, 2è DE._T- Pierce's Disease of Chemical CO, 1984;

and trna and others, Dermatitis. Mal. Share. (1983) 105: 6442.

Alternatively, hybrid DNA technology may be used if a synthetic gene can be prepared using single strands which encodes for the polypeptide or substantially complementary strands thereof, if the single strands overlap and can be put together in a medium annealing so as to permit hybridization. The strands or filaments hybridized can then be ligated to form the complete gene, and, by selection of appropriate terminations, the gene can be inserted into expression vectors, which are readily available today.

See e.g. Maniatis and other, trial for Cloning,

Ultra-Isolation Manual, HSC, at Cold Spring Harbor Laboratory in, 1982. Or the region of the viral genome encoding the peptide can be cloned by conventional techniques and the recombinant AON are expressed (see Maniatis, above).

Coding sequences AON are the LAV isolates_DDL , and ARV1-to-2 DnU HIV which can be used to express the peptides, are as follows:

THE CCC LAVBRUTGT AAA AAA TAA AAC AGM ACA HAS THE AGT AAA-AGM

SGC TCA TCA AGC MGA GGAA CCAS GGG AGM TCA DTCS

TWG AAC ATA-GGAA AAA-TAA TGA CAA ASSAY AGM ATA-GGAA

CAT-TCA TGT

ARV1 AND 2 THE CCC AAA AAA TGT AAC AGM AGM THE AGT AAA-TAA AAC

THE TAT TCA ATA-GGAA CCAS GGG AGM LGA DTCS CAT-AAC

AAC GGAA AGM FOR ATA AND ATA AND ATA AGM AAA-TCA GAT ABSORBENCY GGAA

CAT-TGT

Fragments of a sequence may be used for the expression of peptide fragments, changes may be made conservative base, or if the codons encode the same amino acids, or changes of non-linearly the coding sequence can be made, if the amino acid resulting can be a conservative change or non-cpnservatif in the amino acid sequence, as has been discussed previously.

The coding sequence can be extended either at

5 'or 3' terminal or to. two terminals to elongate the peptide, while retaining its epitopic sites.

The extension may provide an arm for connection, for example to a label, such as an enzyme, for joining two or all peptides together in the same chain, so as to provide an antigenic activity, restriction sites suitable for cloning, or the like.

The DNA sequence by itself, its fragments, or larger sequences, usually of at least 15 base, preferably at least 18 base, can be used as nucleic acid probes for the detection of retroviral RNA or proviral DNA, or to identify homologous regions for cloning or formation of sequence. Many techniques are described, such for example the technique Grunstein-to-Hogriess, blotting technique, the technique northern blots, DOTs blot, their improvements, as well as other methodologies, such as those described in U.S. Patent № 4,358 535 which is used herein as reference.

The peptides of the invention, including blocking peptides, and analogs thereof find use in themselves or in combination in vaccines. Similarly, anti-idiotype antibodies it is to say reactive with idiotype antibody of the present invention and therefore containing epitopes mimicking HIV neutralizing regions, can also be used in vaccines. The peptides or anti-idiotype antibodies can be formulated in a suitable way, generally at concentrations in the range of 1 to 20 mg/kg of jjg host. Physiologically acceptable media may be used as carriers, such as sterile water, saline, phosphate buffered saline or the like. Adjuvants can be used such as aluminum hydroxide gel, the surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, proteins (e.g. cholera toxin or diphtheria) and emulsions of oil.

The peptides may also be incorporated into liposomes, or conjugated to polysaccharides, polypeptides or polymers for use in a vaccine formulation. Administration may be by injection, e.g. intramuscular, intraperitoneal, subcutaneous, intravenous administration of like immunogenically effective can be made of one or several times, usually in a range of one to four weeks. A "immunogenically effective amount" corresponds to an amount of vaccine appropriate to achieve an immune response in a host, demonstrating increased infection in the host.

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

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

SUCH AS I

The example I described the production of hybrid cell lines that produce monoclonal antibodies specific for the envelope glycoproteins of HIV. This method involves the use of purified extracts of lectin LAVgp ^ j is attached to agarose of lentil lectin as the immunogen. The monoclonal antibodies produced subsequently by the hybrid cell lines are characterized by their ability to precipitate radioimmunitairement and "blotting" gpllO from the LAV and as purified recombinant fusion protein biologically expressed. The monoclonal antibodies that bind to epitopes on gpllO are equally reactive in ELISA with whole virus broken, 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 characterization of the antibody are as follows.

Or LAV virus purified from infected CEM cells (A.T.T.C.CRL8904), was ruptured or broken in 50 mm tris, pH of 7.4, 0.15 m in the NaCl, 1% aprotinin, Nonidet P 40 (NP 40) ^ (octylphenoxypolyethoxyethanol) 2%. The extract was clarified by centrifugation twice and adjusted to 0.5% NP 40 with the addition of three volumes of buffer 100 dislocation NP 40. Sepharose of lentil lectin (Pharmacia is, Piscataway, NJ) was prewashed dislocation in the buffer 100 NP 40 and then equilibrated in the adsorption buffer (tris-50 mm, . at pH 7.4, the NaCl 0.15 M., aprotinin 1%, ' NP 40 0.5%).

The extract was clarified virus adsorbed with lentil lectin sepharose 42 hours at 4 °c during. The non-adsorbed material was removed by washing with excess of the adsorbing pad. Eluting the adsorbed material was performed with alpha methyl-mannoside 0.2 m in the adsorption buffer. The eluent was dialyzed against PBS and remove the sugar and the material was réadsorbé on sepharose of lentil lectin.

The lectin Sepharose complex lentilleglycoprotéine was used to immunize Balb/c by three intraperitoneal injection without adjuvant and data separately over a period of 2 - 3 weeks. SSO spleens were removed mice immunized, which demonstrated the presence of antibody circulating on the glycoproteins of HIV by immunoblot, RIP and/or ELISA.

Protocols used for the producing cell lines were typically those Kohler and Milstein (In nature 256:495 (1985)) with changes Goldstein and S.C. and other. (Disgusting. Immune. (1982) 38:273). B cells were fused to mice immunized spleen cells with myeloma NS 1 using 40% (weight/volume of) polyethylene glycol. After melting, the cell mixture was resuspended in a hat in medium (medium■R.P.M.I. 164, 0 - 15% supplemented with fetal calf serum, of

hypoxanthine 1 x 10^{4 _} The m, aminopterin~4 x 10⁷ M and

5 _

1, 6 x 10 m-thymidine) for choosing the growth of hybrid cells, and then ' the mixture was delivered in

microculture trays to 96 wells at a concentration

grams

of 1 to 3 x 10 cells/ml and incubated at 37 °c in a moist atmosphere containing 6% of co2. The cultures were fed by replacing one half of the supernatant with the hat in fresh medium. The wells were observed using an inverted microscope for the indications of cell proliferation and when cells were of sufficient density supernatants were tested for anti-LAV antibodies.

The wells containing antibody-producing hybrid cells facing the LAV were identified by ELISA test by measuring the binding to either purified whole virus disrupted the fusion proteins or biologically expressed. Of ELISA assays using virus broken were performed on plates the LAV Electronic Industries Association EIA (phage systems that, in Seattle, Washington State).

The plates were incubated with cell culture fluids during 45 minutes and 37 °c then washed three times with 0.5% tween 20 in phosphate buffered saline (PBS-to-Tvveen).

Of igg anti-mouse goat-peroxidase (diluting 1:2.000 in PBS-to-Tvveen; Zymed Laboratories Ltd, Inc. or, Francisco south, California) was added (100 pl per well), plates were incubated for and 45 minutes at 37 °c and washed as above. A substrate (citric acid 0,025 m to, dibasic sodium phosphate 0.05 meters, at pH 5 containing 14 mg of O-phenylenediamine and 10 pL hydrogen peroxide 30% by 50 ml) was added and the plates were incubated for 30 minutes at room temperature in the dark. The reaction barrel stopped with sulfuric acid 3n, and colorimetric reactions were quantified with an automatic microplate reader. Wells that are scientifically positive results were subcloned by limited dilution, retesting for specificity, then extended.

The monoclonal antibodies secreted by the resulting hybrid cell lines were then characterized with respect to specificity and reactivity by immunoblot, immunoprecipitation and ELISA test using the LAV virus broken, the LAV recombinant fusion proteins and peptides to the LAV synthesis. All antibodies were determined to be of the IgG isotype ^. Cell lines HIVtgpllO-to-1, hiv gpllO and 2 and hiv gpllO and 3 were deposited on (the American 11432) prior to the deposition of this application and are referenced in the n°s A.T.C.C.

H8 9175, Sulf 917g and HB 9177 respectively.

The recombinant fusion proteins tested for

r. éactivité previously called ENV2, ENV3, ENV4 and ENV5. The protein is expressed from ENV2pENV2 (A.T.C.C. no. 53071), which is a region of lav databases 6598 7178 BP-LP(numbered to tourists for a-Hobson and other Glial cell 4 4:9 (1985)), Is expressed from ENV3pENV3 (A.T.C.C. № 53072) that includes the region to the LAV 7178 BP

lP 7698; ENV4 is expressed from pENV4 (A.T.C.C. no. 53073)" and comprises BPs 7698 BP 8572; and ENV5 is expressed from pENV5 (A.T.C.C. № 53074) that includes the region the LAV 5889 7698 BP-LP.

The peptide i (29) and VIII (110 - 2 - 2) were assembled on a benzhydrylamine resin (polystyrene/divinylbenzene) (average of the Biosystems, Inc. or, Foster on the City, California). The peptide is V (177) assembled on a resin P-butyloxy carbonyl (Boc or) - (PAMs) éthylbenzylcystéine-to-phénylacétamidométhyl polystyrene/divinylbenzene. Couplings anhydride

•symmetrical build in a synthesizer 430a (Amazon Biosystems). Cysteine was added as a first residue dansdans.les two peptides.

Couplings of dicyclohexylcarbodiimide in the presence of hydroxylbenzotriazole were used to asparagine and glutamine. Protection. side chain or branched based benzyl protection and alpha-amino Boc-were used. Other guards side chain used in the usual manner were made by tryptophan (formil) through Boc, Boc or methionine sulfoxide, an arginine (tosyl or) through Boc, Boc or cysteine (methylbenzyl)_F. (tosyl or) through Boc of histidine, lysin (chlorobenzyloxycarbonyl) Boc and tyrosin (bromobenzyloxycarbonyl) through Boc.

When the peptides were radiolabeled, in the barrel by acetylation of the amino terminal-acetic acid - H and an excess of dicyclohexylecarbodiimide.

The deprotection and cleavage of the peptide from

of the resin were performed by the RF protocol "low-high" TAMs (trna and other, above). Extracting the resin was made with acetic acid 5% and the extract was subjected to gel filtration chromatography in 5% acetic acid.

The synthetic peptides tested for the HTV reactivity with the monoclonal antibodies had the peptides 29, 36 and 39. The peptide 29 is encoded by the region of genome LAVgpu 6688 from LP to 6750 LP; 36 the peptide is encoded by the region from about 7246 LP until 7317 LP;

39 and the peptide is encoded by the region from about 7516 LP 7593 to LP. The peptides 36 and 39 are described in detail in U.S. Patent no. 4,629 783 which is used herein as reference.

Blocking peptides, protoporphyrin-IX Xv frames were assembled essentially as described above on a methyl-a benzhydrylamine resin (polystyrene/divinylbenzene) (average of the Biosystems, Inc. or, Foster on the City, California). The symmetrical anhydride couplings were made on a synthesis apparatus 430a (Amazon Biosystems). The couplings of dicyclohexylcarbodiimide in the presence of d'hydroxylbenzotriazole were used to asparagine. For protection, protection boc-amine and benzyl-based side chain was used, while Boc or (bromobenzyloxycarbonyl) was used specifically for branched chain of tyrosine. The acetylation, if present, was performed using acetic anhydride or glacial acetic acid and dicyclohexyl carbodiimide. The deprotection and cleavage of the peptide from the resin were accomplished by the "high" a standard RF protocol (Stewart platform and other, see above).

Extracting the resin was carried out with 50% acetic acid, and the extract was then subjected to gel filtration chromatography in 20% acetic acid. As desired, a high-performance liquid chromatography was performed on a column Vydac cl8 (Rainin implement CO, Emeryville, AC) using a 0.1% trifluoacetic acid, gradient of acetonitrile.

Blotting

By immunoblot characterization was done on supernatants clone or ascites fluid using the LAV virus purified and recombinant fusion proteins as antigens. The antigens were first separated by gel electrophoresis (7 - 15%) of polyacrylamide gradient in th and transferred to a nitrocellulose membrane (Mem) during 4 hours by electrophoresis to 25 V in 25 mm sodium phosphate (pH of 7). After transfer, the Ncm can was blocked to prevent non-specific interactions by incubation

In PBS-to-Tvveen or•Blotto (5% non-fat dry milk in PBS) for one hour at room temperature. The Ncm can was incubated with cell culture supernatant or ascites fluid diluted in PBS-to-Tvveen for one hour at room temperature and was rinsed with three changes in PBS-to-Tvveen.

In the second phase, the Ncm can was incubated with a horseradish peroxidase goat anti-mouse IgG, diluted in PBS-to-Tvveen for one hour at room temperature. Such an incubation was followed by washing with PBS-to-Tvveen. and then dipping into a color developing solution of horseradish peroxidase (bio-research Laboratories, in Richmond, California) during 20 min. The reaction was stopped by immersion in deionized water. The reactivity of the monoclonal antibody was compared ee.à a positive control serum which react with purified virus disrupted or an expressed fusion protein. The results showed that all antibodies bind th gpllO and its precursor molecule, gpl50, using virus preparations is broken or separated. 110 - 1 And 110 - 2 antibodies also recognized the fusion protein ENV3, while the antibodies 110 - 3, 110 - 4, 110 - 5 and 110 - 6 were a ENV2 immunocomplex.

Extracts of virus for radioimmune precipitation were prepared from CEM cells infected with the isûlatLAVgpy HIV adapted to lytic growth by passage. continuous. When effects were apparent early cytopathes, the cells were transferred to a medium containing methionine labeling of £^ e J

(iOM/ml to 0.05) or glucosamine ^ [pyramid (0,025 ICM/ml.), then incubated for 24 hours until most

Cells have been lysed•, which releases the virus in the culture supernatant. The virus was transformed into pills or tablets (one hour to 100,000 named xG) from the cell-free supernatant, and extracts of detergent were prepared in a buffer the P-RIPA effected (phosphate buffered saline containing 1% Triton x * 100, 1% deoxycholate, 0.1% of SOS and 1% aprotinin). Similar extracts were prepared from the supernatants of uninfected CEM cells.

The tests were made with 100 immunoprecipitation PL extract virus incubated with 100 yl derivatives of culture supernatant from hybrid cell lines for one hour on ice. Four microliters Ig rabbit anti-mouse (. Zymed Laboratories Ltd, OS. Francisco, California) were added to each sample and incubated

30 mins. Of 1' immunoprécipitine (100 jjI; the Bethesda a research laboratory, the Bethesda, Maryland and) resuspended in a buffer containing 1% P RIPA effected ovalbumin was added to each sample and incubated for 30 minutes.

Bound complexes were washed and separated by SDS-polyacrylamide gel electrophoresis (15% acrylamide derivatives; DATD freezing). Following die electrophoresis, the gels were fixed, soaked in the product "maturity of" (nu NewEngland Radionuclide, terrier, am), dried and exposed to a film Camera FXR-a 5. Positive sera

Reference•immunoprécipitait all HIV viral proteins was carried. in reaction with supernatants of infected CEM cells infected by viruses and falsely as negative and positive controls.

The results showed that all six monoclonal antibodies specifically immunoprecipitated gpllO and gpl50.

Establish that the gpllO epitopes were recognized by the monoclonal antibodies of the present invention, culture supernatants of from hybrid cell lines or ascites fluids were further characterized by reactivity in ELISA with biologically expressed fusion proteins and synthetic peptides.

Processes were the same as those described above except that the th fusion proteins or synthetic peptides replaced the purified virus as the antigen adsorbed on the surface of the microtiter well.

When peptides were used as antigen, the protocol on plate was the following. The lyophilized peptide was dissolved in 6m guanidine HCI. Just before the has brought on plate in 96 well plates, the solution was diluted in a guanidine bicarbonate buffer 0.05 m in the carbonate/(at pH 9.6) to a final concentration of peptide up to 100/ml of the same table. A volume of 50 UL or peptide diluted was placed in each well of microtiter plates and the ES were then incubated overnight at 4 °c.

The solutions. excess peptide was "shaken", the plates closed or blocked Blotto, and the process described above was done for the remainder of the ELISA test. 0th similarly, a recombinant protein was diluted to a final concentration of about 2 pg/ml in sodium carbonate/bicarbonate buffer (pH of 9.6) 0.05 hr before 1 ℮· same process is followed.

The results are given in Table II. The monoclonal antibodies produced by the cell lines HlV to-gpllO-a 1 and hiv gpllO-to-2 reacted with ENV3, ENV5, the peptide 36 and the. viruses disrupted. The antibodies from lines ceilules hiv gpllO-to-3, in hiv gpiiO above 4, in hiv gpllO above 5 and in hiv gpllO-to-6 ENV2 and reacted with the peptide 29 as well as the virus disrupted or broken.

TABLE II

ELISA test showing the reactivity of monoclonal antibodies with recombinant proteins and

The results in Table II show that the 110 - 1 and 110 - 2 monoclonal antibodies recognized an epitope encoded by a DNA sequence in the region pENV3, more particularly by the region of the genome of HIV defined by a sequence of amino acids in the peptide 36.

In other words, the monoclonal antibodies gpllO-a 1 and IIO72 bind to a peptide region of gpllO encoded in 7246 BP 7317, as evidenced by the formation of immune complexes with the peptide 36 and ENV3. This region of the genome of HIV has previously been identified as conserved, it is to say little change in the sequence and Aon in the encoded region by peptide 36 among different viral isolates from various geographical locations. See Starcich and others, Glial cell (1986) 46:637. By contrast, the monoclonal antibodies gpll0 and 3, -4, -5 -6 and peptide-binding HIV defined by the region encoded by the peptide 39 of 6688 bp to about 6750 LP. The region in gpllO defined by peptide 29 has been identified as containing more nucleotide substitutions among different virus isolates. The monoclonal antibodies that selectively bind to the polypeptides gpllO that contain conserved epitopes, such as 110 - 1 and 110 - 2 antibodies may possess enhanced utility in a variety of circumstances, such as affinity chromatography and the like also, in an ELISA, the peptide 110 - 2 - 2 reacted with sera of the individual from whom the LAV-a 2 was isolated.

An indirect immunofluorescent assay testing

Indirect immunofluorescents tests using monoclonal antibodies directed to the antigen gpllO HIV were performed on living and fixed cells by acetone. The frames or frame attached by acetone, prepared from cells th an OEM infected by the LAV incubated with culture supernatant diluted or ascites fluid 37 °c for up to one hour, while living cells were incubated with culture supernatant or ascites fluid for one hour at 4 °c before the cells are placed on frames or the like and fixed by acetone.

Both methods employed IgG labeled anti-mouse 1' fluorescein isothiocyanate for detecting cells bearing the antigen gpllO reagent. The monoclonal antibody in HIV gpllO-to-1 gave positive results using either. living cells or cells infected with the LAV fixed by acetone.

SUCH AS II

Neutralizing the infectivity of HIV by

monoclonau antibodiesthe X-gpllO

This example describes and characterizes the neutralization of infectivity of HIV using monoclonal antibodies which bind to gpllOgpllO and peptides. The results indicate that the monoclonal antibodies GpllO and 3,

-4, -5 and -6 possess neutralizing activity, and that gpliO and 3 and -4 have, especially high levels of neutralizing activity.

A substantial neutralization assay was developed to quantify the effect of antibody on the infectivity of monqclonauxHTV. Cell line CD4 + highly susceptible or sensitive HIV infection related, EMC was selected as a target for the comparisons of the infectivity. Ascites fluid prepared as described in the example-I, or its fraction IgG purified using ammonium sulfate precipitation, was deactivated with the heat during 30 minutes at 56 °c th, and diluted as required in a RPMI containing 10% fetal calf serum. A suspension of THE LAV_lnd strain HTV was harvested from cultures of about 4 days of EMC in a logarithmic growth phase, filtered through 0.2 or 0.45 micron in filters, formed into aliquots and frozen to -70^I C. an aliquot was thawed, titrated to determine the TCIDgg, and subsequent tests were performed to. freshly thawed aliquots, diluted to 1:500 in a culture medium at a concentration of up th approximately ten times the amount required to infect 50% of CEM cells in the culture (10 TCIDgg). Virus suspension was mixed with an equal volume (250 dd 1) of a monoclonal antibody preparation from 1:5 to 1:9 dilution five, 765,625. The mixture was incubated for viruses/antibody 45 minutes at 37 °c and then duplicated 1:5 th 1:9, 765,625. The mixture was incubated for viruses/antibody 45 minutes to 37^I C and then of

duplicate samples of the UL were used to inoculate 200

5

pits containing 1 ml of approximately 2 x 10 CEM cells per well. The cultures were incubated th 37 °c in an atmosphere of 5% of wetted in 14 days COGs. The cells were harvested, stored tablets, and lysed with 1% Triton-X 100 in PBS for about 10 min. The

■amount of virus (or viral antigen) present in the lysed cells was quantified using an enzyme-immunoassay ' "sandwiches" HIV antigen capture enzyme-sensitive. Titration of the neutralizing activity, if any, was determined as the reciprocal dede.la dilution of monoclonal antibody, which inhibits the production of antigen over more than 50% virus control cultures incubated without antibody, or a monoclonal antibody of the same isotype in that had been previously been shown as missing neutralizing activity.

. Web capture testing antigenic HTV described above was using two monoclonal antibodies directed against antigens p25 as capture reagents. These hybridoma cell lines were produced by the methods described above with minor modifications, including by use of a ' recombinant fusion of the gag purified■as the immunogen and characterizing the monoclonal antibodies resulting in specificity and reactivity using the fusion proteins recambinantes previously called gag 1, gag 2 and gag-3, and the peptide synthesis 141.

La - gag 1 protein is expressed from - of pGAG-a 1 (A.T.C.C. no.-to-53379), gag 2 is expressed from pGAG-to-2 (A.T.C.C. no. 53111) and gag-3 is expressed from ' of pGAG and 3 (A.T.C.C. № 53112).

The synthetic peptide is encoded by the 141 - r.égion of LAVgRu genome corresponding to amino acid residues 198 - 242.

Monoclonal antibodies produced by hybridoma cell lines p25-a 2 25 3 - ρ and have been found to react with recombinant fusion proteins gag 1, gag 2 and gag-3 and the monoclonal ρ 25 - 3 is also ' reactive with the synthetic peptide 141.

To perform the test antigen capture, the capture reagents were initially adsorbed on a solid support. A fluid ascites derived from hybridoma cell lines 25 - 2 and ρ ρ 25 - 3 was diluted 1:5.000 in 25 mm tris buffer, pH of 8.5 and 200 jjI were placed in wells of micro-well plates. The wells were closed, sealed and incubated for about 16 hours at 4 °c. The solution was removed by suction wells prior to the addition of a blocking solution of 0.3% Blotto in BSS. The blocking. - was made during, 15 minutes at room temperature.

The blocking solution was drawn and the sample was. added. Two hundred microliters of the lysed cell suspension and 5 jul of detection conjugate, prepared as described below, were added to each well. The plates or strips were incubated for 2 hours well to 37^I C., after which the suspension was drawn and the wells washed four times with buffer (0.05% of Tween 20 in PBS). The product detection conjugate was prepared as follows. The monoclonal antibodies ρ ρ 25 - 6 and 7 - 25 were conjugate with the horseradish peroxidase (HRP-) in a molar ratio 3:1 (Aβ: the HRP) for three hours by using a method by periodate oxidation (Nakane and others, J Histochem.

Cytochem. 22:1084 (1974). The conjugates were diluted 1:1500 in 2.5% (weight/volume of) non-fat dry milk, 0.05% and 0.01% thimerosal of the anti one-foam has in the citrate salt. 20 mm sodium. The remaining the ELISA was performed as described in the example-I agonist.

The results of testing the neutralizing activity with the antibodies gpll0 and 3, ' -4, =5 and -6 are as follows.

The highest titles which retained the neutralizing activity were determined to be: gpll0 and 3=15,625;

gpllO and 4=9,765,625; gpllO and 5=125; and gpllO and 6=625.

Because of the genetic variability predicted the region defined by the peptide 29, the ability of the monoclonal antibody gpllO and 4 to recognize other HIV isolates was examined. The tests were performed immunofluorescence as described above. The antibody detected a gpllO thereof 4.

antigen in cultures of at least 3 to 16 HTV isolates tested.

The antibody gpllO and 4 was capable of neutralizing viruses isolated on fifteen weeks from a chimpanzee inoculated with LAVg_R y as a control animal in a test vaccine aids.. The monoclonal antibody was capable of neutralizing the isolates. same if 1' animals had. serum neutralizing antibodies iji HTV in vitro, and had developed a measurable immune response generated by the cells, HIV infection related. This indicates a fault. of. antigenic movement vivo for the epitope recognized by the antibody gpl.10 thereof 4.

SUCH AS III

a cocktail of monoclonal anti-gpllQ

and anti-p25

This example describes the neutralization of infectivity HIV using monoclonal antibodies which bind to the peptides in and gpl10gpllO in combination with monoclonal antibodies that bind to p25 p25 and peptides, which only show little or pas.d ' neutralizing activity. The results indicate that the monoclonal antibody gpllO-a 2 in combination with either 25 p25-a 6 or 7 - ρ has especially high levels of neutralizing activity.

Ρ hybridoma cell lines 25 - 6 and 7 - 25 ρ

were produced by the methods described above with modifications that include the use of the virus disrupted or inactivated or disrupted or protein fusionnrecombinante of the gag purified expressed in e ^ _ Escherichia coli, as an immunogen, and characterizing the monoclonal antibodies resulting in 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. The synthetic peptides are encoded by the region of genome LAVg_LAN) corresponding to amino acid residues as follows: a peptide 15, amino acid residues 329 to 350; the peptide 88, amino acid residues 315 to 350;

the peptide 150, amino acid residues 318 to 363; 147 the peptide, amino acid residues 278 to 319; 148 and peptide, amino acid residues 319 to 290. The monoclonal antibodies produced by the hybridoma cell lines 25 - 6 and ρ. ρ 25 - 7 react with recombinant gag 3, 25 - ρ 6 reacts with the synthetic peptides 147 and 148, and ρ 25 - 7 reacts with the synthetic peptides 15, 88, and 15ô.

SSO disablement were made as described above, except when cocktails were used;

monoclonal antibodies of individuals were first diluted 1:5 in culture medium and then mixed depending•ratios equal to give a final dilution of 1:10.

The remainder of the assay was carried out as described above. '

The monoclonal antibodies gpllO-a 2, 6 - ρ ρ 25 and 25 - 50% of 7 show less neutralizing activity when used alone. When used in a combination which comprises the monoclonal antibodies gpliO and 2 - 6 or 25 with ρ gpllO and 2 with 25 - 7 according ρ 1:10 dilution, there occurs a total neutralization. A cocktail comprising the antibodies, monoclonal ρ 25 - 6 in combination with ρ 25 - 7 - gave a rate of 60 - 90% of neutralizing activity.

EXAMPLE VI .

The péptideImmunopotentialité 29 and its homologs The ability of the peptide 2s and homologous peptides, including the peptide 177, to stimulate an immune response against HIV was first examined in two mouse strains.

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

The 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 type) for conjugation according to the procedure emphasized in U.S. Patent № 4,629 783, column 10, lines 28 to 51. As the second. immunogenic, thyroglobulin was derived with glutaraldehyde as follows.

Thyroglobulin swine, 27 mg, was dissolved in '1 ml sodium bicarbonate 0.1 meters, which 8.3 jul'd' 25% glutaraldehyde solution were added dropwise, and the mixture was stirred overnight at room temperature;

1 ml of bicarbonate buffer/sodium carbonate, pH of 9.3, was added to the solution and then dialyzed during 8 - hours on 2 liters of the same buffer à,4 °C, with a complete change of the dialysate after 4 hours. The peptide 29 was then added to follow approximately derived from thyroglobulin, a molar excess of 100, and the mixture was stirred overnight, at room temperature. The glutaraldehyde which had not responded was blocked with 200 μ 1 solution 0.2 m to lysine, which mixture was stirred several hours - (or overnight) at room temperature. The conjugate was then dialyzed thyroglobulin peptide a extensively on PBS to 4 °c.

Two mouse strains (black c57 and Corporate /. C.) inoculated with peptides prepared by each method of conjugation. All animals had an age of about 2 - 4 weeks at the time of inoculation. Inoculation routes include the neck of the lug, scarring of the tail, subcutaneous, intranasal, intraperitoneal or. The inoculum consisted of 25 jjg of conjugated peptide suspended in 0.5 ml of Freunds complete adjuvant, with repeated and reinforced inoculations to weeks 2, 3 and 5, with the same immunogen suspended in incomplete Freund's adjuvant. Mouse serum samples were collected before immunization individual, 4 days after immunization reinforced to 3 weeks, and

4 days after the inoculation to 5 weeks reinforced. The serum samples were analyzed for antibodies against the homologous peptides or whole virus by "Mutation" in the ELISA test. The sera with activity of antibodies for LAV are further sorted for neutralizing activity, followed analysis sera in immunoblots for the antigen the LAV broken and tests with radiolabeled gpllO radioimmunoprecipitation.

The results show that the immunizations of mouse immunized with the peptide 29 developed antibodies reactive with the peptide 29 and the LAV virus-a 1 ruptured or broken in the ELISA. The conjugation of the peptide 29 through glutaraldehyde revealed generally higher titer of antibodies for the peptide 29 in Balb/C., although conjugation through the maleimide was successfully updated to provide anti-peptide antibodies 29 in few Balb/c-.

Mice immunized with the peptide 177 developed antibodies for I_I the peptide, and a conjugation of the peptide 177 through glutaraldehyde was better for the discovery an immunological response. C57 mice were more susceptible to immune stimulation with the peptide 177 that the Balb/C.. Mice immunized with the peptide 110 - 2 - 2 the LAV-to-2 developed antibodies against the virus and 110 - 2 - 2 as shown by lâv and 2 the ELISA. A peptide 110 - 2 - 2 conjugate through glutaraldehyde was immunogenic in both Balb/c and c57, although the titles for the peptide lld and 2 and 2 are generally, higher in the Balb/C as c57, while the.

securities at LAV virus-to-2 were typically higher to mouse c57 than in the Balb/c-.

Typically, the serum samples from mice of which (the I) have antibodies against the whole virus; (ll) are capable of neutralizing the HIV, such as in the assays described in example II; and (lll) react with the peptide 29 in ELISA, indicate cumulatively utilization efficiency of the peptide 29 in a vaccine formulation.

_■EXAMPLE V. ".

■ Parting¹ immunoaffinity D.¹ a monoclonal antibody using gpllO

The monoclonal antibodies against antigen gp110. hIV can be used in processes rather for parting ' immunoaffinity purify substantially. the recombinant proteins of bacterial expression. If the expressed protein is secreted by the bacteria, the protein. can be isolated from the culture supernatant; if, the protein is not secreted.., rupture of the bacterial cells may be necessary.

The plasmid ENP-a 5 (A.T.C.C. no. 53074) encodes a major fraction of the carboxyl terminus of gpllO and a fraction of the terminal amine. of gp41 LAV inserted into the vector.•expression a Trp . TH.Escherichia coli C600 transformed, by this vecte.ur " expresses but does not produce the protein gpl.10.

Of _E. Escherichia coli C. 600 containing the plasmid ENP and 5 are subjected to the growth in medium containing tryptophan (20 pg/ml) and ampicillin (100/ml at jjg) overnight at 37 °c with aeration. The crops of the night are then inoculated as 1:100 in a minimal medium containing fresh ampicillin (100 μ / g at m1) but not of tryptophan. These cultures grown with aerating for 2 - 3 hours (until the early log phase) to 37 °c. The inductor, acid 3 and 8 indole-acrylic (delta

CO•of Chemical, St. Louis, Mo alloy) is added to a final concentration of 20 pg/ml freshly obtained from inventory of 20 mg/ml in 95% ethanol. Induced cultures are then subjected to growth at 37 °c with 4 to aerating for 5 hours and then made into tablets and, optionally, frozen. The yields of proteins - from ENP and 5 are typically lower than 1 mg/liter.

The bacterial cells in grain or pills are lysed using the P-RIPA effected the buffer (PBS 1% Triton-X 100, 1% deoxycholate, 0.1% sodium dodecyl sulfate, and 1% aprotinin) that will induce the lysing cells E :. Escherichia coli . The suspension may be subjected to sonic waves for shearing of DNA and RNA, followed performs centrifugation to remove the particles. A phase dilution or concentration may be necessary to standardize the protein concentration.

The monoclonal antibody in HIV gpllO-a 1 is precipitated initially from ascites fluids or culture supernatants of cells at room temperature or in cold solutions (the NH₄ )₂ S0₄ or Na₂ S0₄ 7.3 pH buffered until saturation final 33 or 18% respectively. The precipitated proteins were removed by centrifugation and redissolved in PBS and precipitated a second time with 33% of (the NH₄ )₂ S0₄ or 12 - 15% Na₂ S0_4< This phase can be repeated if necessary. The tablet is re-dissolved in PBS and the excess salts are removed by gel filtration through a die or by dialyseexhaustive desalting on a PBS.

The monoclonal antibody purified 110 - 1 may then be coupled to sepharose activated by cyanogen bromide. The necessary amount of gel is inflated in

3 _

hCl 10 m on a glass filter (1 g of dried material by freezing gives a final volume of approximately 3.5 ml gel) and 15 minutes with washing during the same solution, and the antibody is added immediately after. In general, the coupling reaction takes place more effectively in a range of pH 8 - 10, but a lower pH can be used when needed for the stability of the antibody. The antibody is to be dissolved in PBS or a borate buffer or bicarbonate/carbonate high ionic strength with 150 mm NaCl. The suspension of antibodies and activated Sepharose is stirred gently for 2 - 4 hours at ambient temperature, or overnight at 4 °c, and then washed on a glass filter sintered with coupling buffer. All remaining active groups are blocked by treatment with 1' ethanolamine 1 m to 2 hours at pH 8 during, the the probro end-Sepharose antibody is then washed alternately with solutions tamponsà high pH and low (borate buffer, 0, 1 Μ, at pH 8.5, acetate buffer and NaCl 1 Μ, 0, 1 Μ, at pH 4, 1 the m NaCI, respectively) four or five times. This washing removes traces of materials adsorbed non-covalently immunoaffinity separation matrix finished is stored below 8 °c in the presence of a bacteriostatic agent suitable, such as 0.01% thimerosal.

Adding the suspension of expressed protein to the immunoaffinity separation matrix causes selective elimination of antigen gpllO. The mixture is allowed to interact during 24 hours 2, preferably 12 - 18 hours, oscillation or with slow stirring. A column system can also be used wherein the matrix is poured onto an immunoaffinity column, balanced and suspension of expressed protein added slowly to the column. After the suspension of protein was added, the flow can be stopped to allow the maximum immune complex formation.

Unbound material is washed or removed by separation by extensive washing with a buffer for¹ adsorbing. A sintered glass filter with vacuum can be used or use is made of a flow through the column. The bound material is then eluted using buffers of low pH or high (acetate buffer, at pH 4 or borate buffer, at pH 8.6) or a chaotropic agent.

EXAMPLE VI.

Immunoaffinity purification of recombinant gpllO of a mammalian expression system The monoclonal antibodies of the present invention find use in the immunoaffinity purification of gpllO of bacterially expressed recombinant mammalian cells.

The mammalian cells are infected with recombinant vaccines (Mackett and others, the j. Virol. (1984) 49:857 that contain sequences encoding at^- least the portion of gpllO which is antigen and updates the neutralizing antibody.

The recombinant vaccines are constructed in the manner described in U.S. Patent № b42 984 which is incorporated herein as a reference. Briefly speaking, sequences encoding glycoprotein .'d ' envelope hIV are inserted in a plasmid vector (pgs20) downstream of a transcription control element vaccine. This chimeric gene is flanked by sequences encoding the gene (TC) viral thymidine kinase.

Plasmid vectors containing a promoter of chimeric vaccine virus envelope gene ligated to the LAV are used to transform the strain E. Escherichia coli MC1000.

The insertion of the LAV env chimeric sequences in the genome. viral vaccines was produced recombinantly vivo , which is achieved by the fact that the chimeric genes in plasmids ρν - ℮ην 5 are flanked or arranged next to sequences encoding the vaccine virus TK gene. This plasmid is then introduced into cells previously infected with a virus vaccine of wild type and allowed to recombination between the TK sequences on the plasmid and the: e homologous sequences in the viral genome of vaccine, thereby ensuring the chimeric gene is inserted. Cells of African green monkey kidney (strain BSCs and 40, derived lineage of BSC cells and 1, as ATCC № CCL26) are used as a host cell in the expression system.

The confluent BSC cells and 40 are infected as a multiple of infection of 10 by recombinant vaccine virus. Is allowed infection for 12 hours at which time, the cells are harvested, washed once with PBS, and collected by centrifugation. The grains cellular suspended in a lysis buffer (1% NPs 40, 2.5% sodium deoxycholate, NaCI 0.1 meters, the m TrisHCl 0.01 meters, at pH 7.4, 1 mm EDTA to), and the lysate is then clarified pax-centrifuging. A separation of immunoaffixité ' of. the ' expressed recombinant protein is carried out - as described, above for the bacterial expression system ., in■using -1 ' monoclonal antibody gpllO-a 1. The proteins produced by the expression system more closely approximate gpllO at HTV product naturally due to the treatment and the glycosylation provided by the mammalian cells.

EXAMPLE VII.

Inhibiting hiv infection with

Effectiveness of blocking peptides in inhibiting infection of tissue cells by the strain LAVg ^ HIV was interest therein using a modification of the protocol for the evaluation of peptide t, as published by PERT and other, see above. Inhibiting testing HIV include preferred combining equal volumes of blocking peptide and CEM cells (2, 5 x 10⁵ ) in a medium (R.P.M.I., 10% FCS and 2 mg/ml of polybrene), and incubated for 45 minutes at 37 °c virus is then added to different doses (10, 50, 500 a TCID 50) the mixture is incubated for 14 days at 37 °c the supernatant and then tested for the production of virus antigen (e.g. p25 core).

Preincubation of cells with the peptides prior EMC■1 ' addition of virus in the cultures enhances the inhibition effect in the tests. Inhibition was found to be dose dependent viruses, and exhibits high activity at medium and low doses, but less effect for very high virus doses.

About 60% to 90% inhibition of antigen produced in the experiments with low doses virus, were obtained with peptide T additional experiments with peptides of X-Xv frames, typically to carboxy-terminal amidated and NHg-terminus acetylated, gave similar results, while the peptide Xl was particularly effective over a broad range of dosage. The neutralizing antibodies can be added either during the preincubation period or as the virus is added to produce a synergistic inhibition or additive production of viral antigen.

It is understood from the above that the antibody lonauxmonoc. and peptides, comprising the blocking peptides, of the present invention, constitute improved methods for neutralizing and/or inhibiting HIV infections. This allows therapeutic and prophylactic compositions to be more easily developed and which can be effective against infections for most, if not all, strains of HIV. Further, the novel, inventive materials find use in diagnostic testing and other processes well known.

The vaccine following an array including references from a number of microorganisms that form part of the present invention and which have been deposited with the American Type Culture Collection, 12301 Parklawn driving distance, of Rockville, in Maryland 20852, Revenue.

It should be noted that the hybridomas of HB 9175, Sulf 9176 and HB 9177 were tested and found viable 26 August 1986.

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

Of course, the present invention is in no limited to the given examples which must be considered as clarifying the invention but do not restrict the.

I. e. 1' invention comprises all the technical equivalents of the means described as well as their combinations if they are carried out according to its spirit.