Field of the Invention

The present invention relates to the field of treatments and diagnostics for viral infection. More particularly, this invention relates to the field of treatments and diagnostics for infection by the human immunodeficiency virus-l. Reference to Related Applications This application is a continuation-in-part application of U.S. patent application serial number 183,840 filed April 20, 1988 in the names of Mark I. Greene, William V. Williams and David Weiner, entitled "Methods of Modulating Retro-Virus Host Cell Interactions", which application is specifically incorporated as if fully set forth herein. Background of the Invention

Acquired Immune Deficiency Syndrome (AIDS) is one of the most feared diseases in the world today. Infection with the human immunodeficiency virus (HIV-1) , believed to be the cause of AIDS is almost always fatal. Symptoms of the disease can take years to develop, thus facilitating the spread of this fatal disease by persons unknowingly harboring the virus. Treatments for AIDS are limited and have been unsuccessful in controlling the disease.

HIV-1 has been shown to preferentially infect cells expressing the CD4, a 55,000 dalton cell surface glycoprotein. This tropism is believed to result from interactions between the virus envelope gpl20 and a high affinity binding site on the CD4 glycoprotein which permits viral adsorption. gpl20 is part of the envelope glycoprotein gpl60. This larger glycoprotein consists of two main glycoprotein portions - gpl20 and gp41. gpl20 is believed to be the outermost part of the complex made up of these two glycoproteins. gp41, the inner portion of the complex, is embedded in the viral membrane. Following the initial attachment of virus to the cell surface CD4 molecule, gp41 pierces the membranes of the target cell and initiates fusion. This interaction precedes viral entry, uncoating, and replication. U.S. Patent 4,520,113 issued May 28, 1985 to Gallo et al. discloses methods of detecting HTLV-III (now named HIV-1) in sera of AIDS and pre-AIDS patients. These methods detect the presence of antibodies in the patient's serum which bind to antigenic sites on HIV-1 or fractions of HIV-1, thus signalling the presence of the virus itself in the patient. A fraction known as p41, a 41,000 dalton viral envelope protein was found to be particularly useful in the diagnostic methods of the invention because many persons having AIDS or pre-AIDS illnesses were found to have antibodies against this viral protein. U.S. patent 4,725,669 issued February 16, 1988 to

Essex and Lee discloses novel polypeptides along with assays which use the polypeptides to detect infection of cells by human T-cell lymphotrophic virus-Ill (i.e. HIV-1) . The polypeptides may be purified forms of glycoproteins found in the cell surface membrane of cells infected with human T-cell lymphotrophic virus-Ill. The polypeptides contain antigenic determinants immunologically cross-reactive with glycoproteins having a molecular weight of 120,000 daltons and 160,000 daltons which occur on the surface of cells infected with HTLV-III (HIV-1) .

. Treatment of individuals infected with HIV-1 has bee complicated by the binding capacity of the virus to mammalia cells and the extreme toxicity of infection with the virus. Th potential for inadvertently infecting healthy individuals wit only partially inactivated whole HIV-1 or components of th virus as part of a vaccine is very high. One commerciall available compound which is useful as an anti-infective agen is a form of CD4. The compound is believed to work because i binds the gpl20 as tightly as the natural CD4. In this way, i given in high enough concentrations, the free (administered) CD will bind all of the viral gpl20 and prevent its binding CD4 o host cells. Efforts to control the virus through drugs has no succeeded. Alternate means of treating individuals infecte with HIV-1, as well as alternate means of preventing o inhibiting infection of cells with HIV-1 are needed which ar not toxic to the individual infected with HIV-1 and are safe fo individuals not infected with the virus.

A major problem with retroviral infections is th manner in which the retroviruses are able to subvert the hos organism's immune response to the detriment of the host. Thi is illustrated most vividly by the effect of HIV infection o human helper T cells. HIV-1 infects cells by first binding t host cell CD4 molecules utilizing the viral envelop glycoprotein gpl20, and subsequently fusing with the cel membrane. In an infected cell, gpl20 is expressed on th surface of the cell, creating a potential target for cytolyti antibody responses. Superficially, an antibody response t gpl20 would seem to be advantageous to the host. However, during HIV infection, large amounts of gpl20 are also shed fro infected cells, with subsequent binding to CD4 molecules o uninfected cells. These uninfected cells also become target for cytolytic antibody binding and subsequent lysis. Among CD4 bearing cells in the host are helper T cells, the very cell necessary for the development of antibody responses. B

shedding gpl20 from infected cells, HIV is able to cripple an essential arm of the immune response that would lead to the elimination of HIV.

Accordingly, it is an object of the invention to provide agents and methods to inhibit infection of cells by HIV- 1. A further object is to provide polypeptides capable of effecting such inhibition. Another object is to interfere with the binding of HIV-1 to host cells. A further object is to provide methods for detecting the presence of HIV-1 in biological specimens or of detecting the presence of antibodies for HIV-1 in such specimens. Yet another object is to provide methods of treatment of cells to avoid HIV-1 infection.

It would obviously be beneficial to the host to develop antibody responses that are able to recognize and destroy infected cells, while leaving uninfected cells intact. Thus it is also an object of the invention to provide such antibodies. It is a further objedt of the inention to provide methods for the recognition of sites on retroviruses that are immunogenic and lead to the development of beneficial antibodies of this type. These and other objects will become apparent from a review of the instant specification. Brief Description of the Drawings

Figures 1 through 9 show binding of anti-H156 sera to murine L cells. Figure 10 shows competitive immunoprecipitation of virus envelope glycoproteins.

Figure 11 shows idiotypic specific binding to H156. Figures 12 and 13 show gel electrophoresis of second receptor glycoproteins.

Summary of the Invention

The present invention discloses novel receptors present on cells which bind gp41 which have not heretofore been described. These receptors appear to regulate the entry of HIV-

1 into cells subsequent to the binding of the virus to the ce at the CD4 receptor by gpl20.

The present invention further discloses nov antibodies which are specific for the antigenic site of gp which binds the novel receptors. It is now believed that the antibodies are at least partly responsible for the inhibiti of syncytia formation which occurs when human cells are infect with HIV-1. It is believed that gp41 has at least two antigen determinants and that at least one of them binds to polypeptid on the surface of a host cell. Antibodies specific for t antigenic determinant of gp41 which binds to the novel recepto have the ability to -inhibit formation of syncytia, whereas oth antibodies to gp41 previously known do not inhibit formation syncytia. Antibodies to gp41 are known to occur in perso infected with HIV-1, however these antibodies are now believ to be specific for an antigenic determinant or determinants n associated with binding of HIV-1 to the .host cell.

The discovery of a second binding site which appea to regulate the entry of HIV-1 into cells and novel antibodi specific for gp41 at the antigenic determinant which binds the second receptor provides a substantial advance in treatme of cells by inhibiting infection. The discovery of a seco binding site and novel antibodies to gp41 also provide metho and agents for inhibiting infection of cells. This seco binding site is believed to be comprised of one or mo glycoproteins having molecular weights of 25,000 to 35,0 daltons, 45,000 daltons to 60,000 daltons, 80,000 to 100,0 daltons and 180,000 to 220,000 daltons. The second binding si binds gp41 an envelope glycoprotein of HIV-1. The present invention provides novel agents f interfering with the effects of HIV-1 upon host cells havi surface polypeptides capable of binding HIV-1. The inventi provides substantially pure polypeptides having an antigen determinant or determinants immunologically cross-reactive wi

determinants of a glycoprotein having a molecular weight of approximately 41,000 daltons, and determinants of a glycoprotein having a molecular weight of approximately 160,000 daltons; each of the glycoproteins being obtained from cells infected with human immunodeficiency virus - 1. Polypeptides suitable for use in the invention include anti-idiotype antibodies having the appropriate antigenic determinants. The glycoprotein known as gp41 which has a molecular weight of 41,000 daltons and is obtained from cells infected with HIV-1 contains a polypeptide region which has an appropriate antigenic determinant and is suitable for use in the invention.

By "polypeptides containing immunologically cross-reactive antigenic determinants" is meant polypeptides having in common antigenic determinants with which a given antibody will react.

The invention also provides novel polypeptides comprising substantially pure polypeptides having an antigenic determinant or determinants specific for a glycoprotein having a molecular weight of approximately 41,000 daltons which is obtained from cells infected with human immunodeficiency virus - 1, the polypeptides further having an antigenic determinant or determinants immunologically cross-reactive with at least one glycoprotein having a molecular weight of 25,000 to 35,000 daltons, 45,000 daltons to 60,000 daltons, 80,000 to 100,000 daltons or 180,000 to 220,000 daltons. The glycoproteins are preferably obtained from HSB, ST, HeLa and human cells. The polypeptides are also useful in methods of interfering with the effects of HIV-1 upon host cells having surface polypeptides capable of binding HIV-1. Antibodies and glycoproteins are examples of polypeptides which may provide a suitable antigenic determinant or determinants for use in the invention.

The invention further provides methods of interferin with the effects of human immunodeficiency virus - 1 upon hos cells having cell surface polypeptides capable of binding huma

immunodeficiency virus - 1. These comprise polypeptide havin an antigenic determinant or determinants immunologically cross reactive with determinants of a glycoprotein having a molecula weight of approximately 41,000 daltons, and determinants of glycoprotein having a molecular weight of approximately 160,00 daltons, each of which glycoproteins are obtained from cell infected with human immunodeficiency virus - 1 is contacted wit the cells under conditions selected to permit the polypeptid to bind to the host cell surface polypeptides thereby inhibitin the virus from binding to the cell surface polypeptide to effec the interference.

In accordance with other embodiments of the invention methods of interfering with the effect of human immunodeficienc virus-1 upon host cells having cell surface polypeptides capabl of binding human immunodeficiency virus - 1 are provided. Thes comprise contacting human immunodeficiency virus - 1 with polypeptide having an antigenic determinant or determinant specific for a glycoprotein having a molecular weight o approximately 41,000 daltons, which is obtained from cell infected with human immunodeficiency virus - 1; the polypeptid further having an antigenic determinant or determinant immunologically cross-reactive with at least one glycoprotei having a molecular weight of 25,000 to 35,000 daltons, 45,00 daltons to 60,000 daltons, 80,000 to 100,000 daltons or 180,00 to 220,000 daltons. These glycoproteins are obtained from HSB ST, HeLa and human cells. The contacting is effected unde conditions selected to permit the polypeptide to bind to th virus thereby inhibiting binding of the virus to the host cell and effecting the interference. Methods for detecting the presence of neutralizin antibodies to HIV-1 in biological specimens suspected o containing HIV-1 are also provided. The biological specimen i contacted with a polypeptide having an antigenic determinant o determinants immunologically cross-reactive with determinant

of a glycoprotein having a molecular weight of approximately 41,000 daltons, and determinants of a glycoprotein having a molecular weight of approximately 160,000 daltons, each of th glycoproteins being obtained from cells infected with huma immunodeficiency virus - 1, under conditions selected to permi binding of the polypeptide to neutralizing antibodies in th biological specimen. The polypeptide is then detected. I preferred embodiments of the invention the polypeptide i detectably labeled with a label known in the art. Using thes methods the course of treatment of cells with neutralizin antibodies or polypeptides can be followed.

The invention additionally provides further method of detecting the presence of human immunodeficiency virus - in biological specimens suspected of containing the virus. These methods comprise contacting the biological specimen wit a polypeptide having an antigenic determinant or determinant specific for a glycoprotein having a molecular weight o approximately 41,000 daltons, which is obtained from cell infected with human immunodeficiency virus - 1; the polypeptid further having an antigenic determinant or determinant immunologically cross-reactive with at least one glycoprotei having a molecular weight of 25,000 to 35,000 daltons, 45,00 daltons to 60,000 daltons, 80,000 to 100,000 daltons or 180,00 to 220,000 daltons, these glycoproteins being obtained from HSB ST, HeLa and human cells, under conditions selected to permi binding of the polypeptide to the virus in the biologica specimen. The peptide is then detected. In preferre embodiments of the invention, the peptide is detectably labele with a label known in the art. The invention further provides methods of determinin the presence of neutralizing antibodies to huma immunodeficiency virus-1 in serum of humans which antibodie inhibit formation of syncytia. Human serum is contacted with mixture of cells capable of forming syncytia in the presence o

HIV-1 and cells infected with HIV-l with human serum und conditions selected to allow binding of neutralizing antibodi to said cells. The formation of syncytia are then detected. I preferred embodiments of the invention, the neutralizin antibodies have an antigenic determinant or determinan specific for a glycoprotein having a molecular weight o approximately 41,000 daltons which is obtained from cell infected with human immunodeficiency virus - 1; the polypeptid further having an antigenic determinant or determinant immunologically cross-reactive with at least one glycoprotei having a molecular weight of 25,000 to 35,000 daltons, 45,00 daltons to 60,000 daltons, 80,000 to 100,000 daltons or 180,00 to 220,000 daltons, these glycoproteins being obtained from HSB ST, HeLa and human cells. The invention also provides methods of treating cell having cell surface polypeptides capable of binding huma immunodeficiency virus - 1 to inhibit infection by HIV-1. Agent which block the gp41 binding site on the cells are provided an these agents are administered to the cells under condition selected to allow binding of the agents to the cells thereb blocking the gp41 binding site and inhibiting infection of th cells. In preferred embodiments of the invention, agents whic block the gp41 binding site on the cells are those polypeptide which have an antigenic determinant or determinant immunologically cross-reactive with determinants of glycoprotein having a molecular weight of approximately 41,00 daltons, and determinants of a glycoprotein having a molecula weight of approximately 160,000 daltons; each of th glycoproteins being obtained from cells infected with HIV-l. The invention additionally provides methods o inhibiting HIV-l infection of cells having cell surfac polypeptides capable of binding gp41 on HIV-l. Agents whic bind to gp41 are provided and these agents are administered t HIV-l under conditions selected to allow binding of the agen

to gp41, thereby blocking gp41 and making it unavailable for binding to cells and thus inhibiting infection of the cells. In preferred embodiments of the invention, the agents which bind to gp41 are polypeptides having an antigenic determinant or determinants specific for a glycoprotein having a molecular weight of approximately 41,000 daltons, this glycoprotein being obtained from cells infected with human immunodeficiency virus - 1; the polypeptide further having an antigenic determinant or determinants immunologically cross-reactive with at least one glycoprotein having a molecular weight of 25,000 to 35,000 daltons, 45,000 daltons to 60,000 daltons, 80,000 to 100,000 daltons or 180,000 to 220,000 daltons, these glycoproteins being obtained from HSB, ST, HeLa and human cells.

The invention further provides peptides having an amino acid sequence of about 10 to about 50 amino acids that corresponds to at least a portion of a protective epitope of HIV. These peptides correspond to regions of the HIV envelope glycoprotein gpl60 that "protect" or inhibit infection and syncytia formation of human lymphocytes or other susceptible cell, when peptides corresponding to at least a portion of the region are contacted with cells having receptors for HIV-l. Without wishing to be bound by any theory or mode of action, it is believed that the peptides correspond to regions of gpl60 (or gpl20 and gp41) that are involved with binding of HIV to receptors on the surface of human cells. The peptides of the invention may bind to these receptors, thus making the receptors unavailable for binding HIV. The peptides of the invention may also be used to elicit antibodies to prevent virus from binding to receptors on the surface of human cells. The peptides of the invention are preferably selected from the group consisting of gly-glu-ile-lys-asn-cys-ser-phe-asn-ile-ser-thr-ser- ile-arg-gly-lys-val-gln-lys-glu-tyr-ala;

asn-gly-asn-ala-glu-glu-val-val-ile-arg-ser-ala-asn phe-thr-asp-asn-ala-lys-thr-ile-ile-val; cys-asn-ile-ser-arg-ala-lys-trp-asn-asn-thr-leu-lys gln-ile-asp-ser-lys-leu-arg-glu-gln-phe; gly-ser-asp-thr-ile-thr-leu-pro-cys-arg-ile-lys-gln ile-ile-asn-met-trp-gln-glu-val-gly-lys; val-gln-gln-gln-asn asn-leu-leu-arg-ala-thr-glu-ala-gln-gln-his-leu-leu-gln-leu- thr val-trp- gly-ile-lys-gln-leu-gln; and peptides containing these sequences.

The invention also provides antibodies specific fo a peptide of the invention described above.

The invention provides methods of developing o synthesizing biologically active peptides. The binding pattern of antibodies from a healthy individual infected with retrovirus and antibodies from a symptomatic individual infecte with the retrovirus are compared to determine at least on binding region unique to antibodies from healthy, infecte individual. A peptide corresponding to at least a portion of unique binding region is then synthesized. The comparing step preferably comprises the steps o providing at least one test peptide derived from the amino aci sequence of a component of said retrovirus; contactin antibodies from a healthy, infected individual and symptomatic, infected individual with at least one test peptid to determine the presence of antibodies bindable with the tes peptide; comparing the results of the second step for th healthy, infected individual and the symptomatic, infecte individual to determine at least one binding region unique t antibodies from the healthy, infected individual. Mouse cells do not have the CD 4 receptor on thei surface and cannot be infected with the AIDS virus under norma conditions. Mouse cells in which DNA encoding for CD 4 has bee inserted into the expressed genetic material, however, can b

shown to express CD 4 on their surfaces and are capable of binding the AIDS virus. They are not, however, infected by the virus. Human cells which do not express CD 4 are also generally not infectable by the virus, but if they are made to have CD 4 on their surfaces they can be infected, indicating that CD 4 alone is insufficient to produce infectability, but that there is some other element expressed on human cell surfaces required for actual infectability.

Mouse antibodies which are similar in structure to the functional part of the GP 41 act as probes to cell surface proteins which function as a GP 41 receptor. Cells that have CD 4, but do not bind the GP 41 look-alike are not infectable with the AIDS virus. Once cells are infected with AIDS virus, they begin to manifest the glycoproteins encoded for by the AIDS virus on their own surfaces. Because these cells have the proteins which lead to fusion of the AIDS virus membrane with the human cell membrane, they tend to fuse together themselves, through the same molecular mechanisms involved in infection, forming what are called syncytia. Both the mouse antibodies, and the human antibodies from which they are derived are capable of blocking the formation of syncytia between infected cells, indicating that either binding of the GP 41 active site, or binding of the human receptor site is capable of preventing fusion. This is believed to mean that either is capable of preventing cellular infection, since fusion is an essential step to such infection.

Serum from patients with high levels of antibodies to GP 120 does not prevent the fusion of such cellular membranes and is further evidence that antibodies to GP 120 are not protective against infection.

It is known that persons infected with HIV-l produce antibodies to gp41; however, not all of these antibodies have a neutralizing or inhibiting effect upon the ability of HIV-l to enter susceptible cells. Those antibodies which have a

neutralizing effect are within the scope of the invention. It ' is believed that gp41 has at least two antigenic determinants and that at least one of them binds to polypeptides on the surface of a host cell. Non-neutralizing antibodies are believed to be specific for an antigenic determinant or determinants not principally associated with binding of HIV-l to the host cell, whereas neutralizing antibodies are believed to be directed towards an antigenic determinant or determinants involved with binding of HIV-l to the host cell. It is these latter antibodies which are especially useful the practice of this invention.

These neutralizing antibodies, which are a source of some of the polypeptides useful in the invention, can be obtained for example by testing the serum of persons with AIDS, ARC or who may have been infected with the virus through contacts with persons infected with the virus. Serum from these persons is tested in the Fusion Inhibition or Syncytia Assay set forth herein or by other methods which determine fusion inhibition. Antibodies which inhibit syncytia formation are selected and used in the invention as sources of polypeptides having an antigenic determinant or determinants specific for a glycoprotein having a molecular weight of approximately 41,000 daltons which are obtained from cells infected with human immunodeficiency virus - 1, and which further have an antigenic determinant or determinants immunologically cross-reactive with at least one glycoprotein having a molecular weight of 25,000 to 35,000 daltons, 45,000 daltons to 60,000 daltons, 80,000 to 100,000 daltons or 180,000 to 220,000 daltons, said glycoproteins being obtained from HSB, ST, HeLa and human cells. In accordance with this invention, these antibodies may also serve as a source of antigens for use in making anti- idiotype antibodies. Anti-idiotype antibodies are in effect antibodies to an antibody. The active site of an anti-idiotype antibody contains a functional equivalent of the antigen region

for which the antibody is specific. These antibodies can be a - source of polypeptides having an antigenic determinant or determinants immunologically cross-reactive with determinants of a glycoprotein having a molecular weight of approximately 41,000 daltons, and determinants of a glycoprotein having a molecular weight of approximately 160,000 daltons, both glycoproteins being obtained from cells infected with human immunodeficiency virus - 1.

Those of ordinary skill in the art will recognize that various modifications can be made in the compounds (polypeptides) of the present invention without departing from the scope hereof. It is contemplated that molecular modelling techniques will permit compounds of different primary and secondary structures to be substituted for the polypeptides of this invention, provided equivalent tertiary structures can be determined. All such modifications may be within certain embodiments of the invention.

Other polypeptides which may be suitable for use in the invention include the unglycosylated moieties of glycoproteins. Other useful polypeptides or proteins, which have the necessary immunogenic determinants, include synthetic polypeptides. Polypeptide fragments of antibodies and anti- idiotype antibodies may also be suitable for use in the invention as may polypeptides produced from recombinant DNA techniques. For example, genes encoding a polypeptide which binds to gp41 or the second receptor can likely be cloned into an expression vector or plasmid which could then be made to produce the polypeptide. Cell lines containing the expression vector which encodes genes for the polypeptide then would provide a source of the polypeptide.

Some of the polypeptides useful in the invention can be purified by electrophoresis of cell lysates or extracts containing the polypeptides with subsequent removal of the polypeptides from the electrophoresis gel to give substantially

pure polypeptides. Gel electrophoresis and removal of the '■ polypeptides from gels are readily accomplished using methods known in the art. Other forms of purification may be employed either in addition to or in lieu of the foregoing without deviating from the spirit of the invention. Polypeptides which are immunologically cross-reactive with a glycoprotein having a molecular weight of approximately 41,000 daltons (gp41) which is obtained from cells infected with human immunodeficiency virus - 1 are useful as diagnostic agents to measure levels of anti-gp41 antibodies in biological systems and patients' sera. gp41 is believed to be a viral envelope protein. Antibodies to gp41 have been found in the sera of many AIDS and ARC patients; the presence of antibodies to gp41 is believed to be a reliable indicator of the presence of the virus in cells.

Polypeptides which are immunologically cross-reactive with a glycoprotein having a molecular weight of approximately 41,000 daltons (gp41) which is obtained from cells infected with human immunodeficiency virus - 1 are also useful as agents to detect the presence of polypeptide receptors on host cells which are specific for a polypeptide having an antigenic determinant or determinants specific for a glycoprotein having a molecular weight of approximately 41,000 daltons which is obtained from cells infected with human immunodeficiency virus - 1, and which polypeptides further have an antigenic determinant or determinants immunologically cross-reactive with at least one glycoprotein having a molecular weight of 25,000 to 35,000 daltons, 45,000 daltons to 60,000 daltons, 80,000 to 100,000 daltons or 180,000 to 220,000 daltons. It is believed that the presence on the surface of cells of polypeptides having the above characteristics determines the capability of cells to become infected with HIV-l. Cells which have CD4 receptors to bind gpl20 but which do not have polypeptide receptors as described above do not become infected with HIV-l. Thus the

presence of the above polypeptides is a marker for cells that : can become infected with HIV-l. Polypeptides which are immunologically cross-reactive with a glycoprotein having a molecular weight of approximately 41,000 daltons (gp41) which is obtained from cells infected with human immunodeficiency virus - 1 can be used in methods designed to determine the infectability of cells with HIV-l. These polypeptides are contacted with test cells under conditions selected to permit binding of the polypeptides to the test cells. The polypeptides which have bound to the test cells are then detected. The polypeptides may be detectably labeled using any of the methods known in the art, such as enzymes, for later detection with chro ogenic substrates, radiolabels, enzyme-linked immunosorbent assays and the like. Methods of detecting infectability of cells may also combine the use of antibodies to CD4 or other molecules which are capable of binding CD4 to determine the exact infectability status of the cell.

Conventional assay procedures for detecting labeled antigens, antibodies and the like are suitable for use in the methods of the invention which detect the presence of HIV-l in biological specimens. In preferred embodiments of the invention, for example, the polypeptides may be labeled a radiolabel such as ¹²⁵ I or ³⁵ S for use in radioimmunoassay, with fluorescein for fluorescent immunoassay, with an enzyme for enzyme immunoassay or with biotin for biotin-avidin linked assays. Immobilization assays wherein the polypeptide is bound to an insoluble phase and detection of the virus or antibodies is carried out by measuring their binding to the insoluble phase are also suitable for use in the invention. These methods are exemplary only and other methods may be useful in the invention.

Biological specimens such as blood, serum, lymphocytes, urine, tissues, saliva, feces, and the like may be tested using the methods of the invention. The particular method employed to prepare a specimen for use in the methods of the

invention will vary according to the type of specimen and preparation may be easily accomplished using methods known in the art. Screening of blood-derived products, such as vaccines, can also be done by the methods of the invention. Polypeptides which are immunologically cross-reactive with a glycoprotein having a molecular weight of approximately 41,000 daltons (gp41) which is obtained from cells infected with human immunodeficiency virus - 1 may also be used as antigenic substances for the production of antibodies protective against infection of cells by HIV-l. Thus presenting such polypeptides to the immune system of patients is believed to be capable of producing protective antibodies against such infection.

Polypeptides having an antigenic determinant or determinants specific for a glycoprotein having a molecular weight of approximately 41,000 daltons which is obtained from cells infected with human immunodeficiency virus - 1 and which further have an antigenic determinant or determinants immunologically cross-reactive with at least one glycoprotein having a molecular weight of 25,000 to 35,000 daltons, 45,000 daltons to 60,000 daltons, 80,000 to 100,000 daltons or 180,000 to 220,000 daltons which are obtained from HSB, ST, HeLa and human cells are useful agents for interfering with the infection of cells by HIV-l. These polypeptides are contacted with HIV- 1 under conditions selected to allow binding of the polypeptides to the virus and thereby interfere with binding of the virus to host cells. It is believed that these polypeptides would bind to polypeptides which are immunologically cross-reactive with a glycoprotein having a molecular weight of approximately 41,000 daltons (gp41) which is obtained from cells infected with human immunodeficiency virus - 1, notably gp41 on the surface of HIV- 1. In this way a substantial number of sites by which the virus binds to host cells would already be occupied by the polypeptides of the invention and thus be unavailable for binding to the host. This would result in the virus being

unable or severely handicapped in binding to host cells and consequently reduce the rate of infection of cells by the virus.

A wide range of retroviral agents infect mammalian hosts, including HIV-l & 2, HTLV-1-4, SIV, FeLV, Bovine leukemia virus, and many others. These viruses share a common structural feature in the organization of their membrane glycoproteins. These envelope glycoproteins are synthesized as a single unit, and then cleaved into an external glycoprotein (e.g. gpl20) and an integral membrane protein (e.g. gp41) which acts as an anchor for the external glycoprotein. The interaction of these envelope glycoproteins with cellular elements determines the tissue and species tropism of these retroviruses. The i munodominant nature of the external glycoprotein, along with its ability to be shed, is likely to play an important role in the pathogenesis of retroviral infections. The development of substances that bind to the integral membrane protein, without interacting with the external glycoprotein, therefore has utility in targeting virally infected cells and eliminating them without adverse effects on "innocent bystander" cells. Accordingly, this outlines a general method whereby antibody responses from infected, healthy individuals are utilized to develop substances that bind to the integral membrane protein on infected cells, without binding to uninfected cells, even if they bear the external glycoprotein in their surface.

Thus, antibodies raised to the peptide F560 derived from the sequence of gp41 the integral membrane prtoein of HIV- 1, and preferentially recognized by a healthy infected individual's antibodies, binds to gp41 bearing cells and targets them for complement mediated lysis. In contrast, antibodies to the peptide F160, derived from the sequence of gpl20 the external glycoprotein of HIV-l, bind to gpl20 bearing cells and target them for complement mediated lysis regardless of the nature of the association of gpl20 with the cell surface (i.e.

either expressed endogenously, or adsorbed to the surface of the cells) . This strategy is expected to have general utility in developing substances that are capable of binding specifically to retrovirally infected cells without interacting with uninfected cells that bear viral components or receptors.

For other retroviruses, the antibody responses from infected individuals who are healthy or otherwise do not exhibit symptoms of disease associated with infection can be compared with the immune response of infected symptomatic individuals who exhibit smyptoms of disease associated with infection to determine epitopes that are unique to the invected healthy individual and not shared by the infected symptomatic individual. Peptides corresponding to at least a portion of the unique protective epitope are synthesized. The peptides can then be used as vaccines, for production of antibodies or in diagnostic assays.

Comparison of the immune responses can be done as described herein or by other appropriate methods. Test peptides having a length of from about 10 to about 50 amino acids corresponding to portions of retroviral envelope glycoproteins are synthesized or purified from natural sources. Test peptides are selected by arbitrarily dividing the amino acid sequence of the envelope glycoprotein into portions and synthesizing corresponding peptides. Alternatively, test peptides corresponding to exposed portions of the molecule or other regions of interest can be synthesized. Where the amino acid sequence of the glycoprotein has not been determined, peptides can be generated by limited digestion of the molecule that has been isolated from natural sources. The amino acid sequence of peptides that have "protective" properties can be determined subsequently by conventional techniques for amino acid sequencing.

The test peptides are used to screen serum from healthy, infected individuals and symptomatic, infected

individuals in binding assays to determine the presence of - antibodies in the sera that bind to the peptides. The results of binding assays constitute the binding profile of the serum. The binding profiled of the sera from the healthy infected individual and the symptomatic infected individual are compared. It may be preferable in some circumstances to use the pooled serum from a number of symptomatic infected individuals in the comparison with the healthy infected individual so that individual variation of immune response is averaged an a representative immune response is used for the comaprison. Reactivities in both sera to the same peptide are disregarded. Unique reactivities found in serum from the healthy infected individual thus correspond to regions of the retroviral envelope important in the development of protective immunity. Peptides corresponding to the unique reactivities may then be further tested to determine their usefulness in inhibiting viral replication and for producing antibodies that are capable of binding specifically to retrovirally infected cells without interacting with uninfected cells that bear viral components or receptors.

The protective peptides of the invention have been derived from epitopes of gpl60 the 160,000 dalton envelope glycoprotein of HIV-l. Regions of gpl60 have been found to contain amino acid sequences which "protect" susceptible cells from infection with the virus or inhibit syncytia formation with infected cells, when peptides corresponding to at least a portion of the region are contacted with susceptible cells. These peptides are set forth in Table 1.

TABLE 1 Approximate

Linear Position

On gpl60 Amino Acid Sequence gly-glu-ile-lys-asn-cys-ser-phe-asn- 155-175 ile-ser-thr-ser-ile-arg-gly-lys-val-

(F160) gln-lys-glu-tyr-ala

asn-gly-asn-ala-glu-glu-val-val-ile- 265-284 arg-ser-ala-asn-phe-thr-asp-asn-ala- lys-thr-ile-ile-val cys-asn-ile-ser-arg-ala-lys-trp-asn- 333-351 asn-thr-leu-lys-gln-ile-asp-ser-lys- leu-arg-glu-gln-phe gly-ser-asp-thr-ile-thr-leu-pro-cys- 415-430 arg-ile-lys-gln-ile-ile-asn-met-trp- gln-glu-val-gly-lys val-gln-gln-gln-asn-asn-leu-leu-arg-

552-575 ala-thr-glu-ala-gln-gln-his-leu-leu-

(F560) gln-leu-thr-val-trp-gly-ile-lys-gln- leu-gln

Preferred peptides have an amino acid sequence of about 10 to about 50 amino acids that correspond to at least a portion of a protective epitope of HIV and inhibit syncytia formation of human lymphocyte cells. Other portions of the gpl60 molecule that also provide "protection" are also within the scope of the invention. It will be appreciated that modifications of these peptides that retain the protective function are also within the scope of the invention. Such modifications include peptides having an amino acid sequence extending beyond the region of the synthesized peptides in either direction; peptides containing amino acid sequences corresponding to at a least portion of two or more protective regions; peptides having one or more amino acids substituted with other amino acids or other compounds but which still retain the protective function; peptides having a cytotoxic or other molecule attached; or any combination of these. Additionally, the peptides may form part of a larger molecule, such as an antibody or fragment of an antibody. Further it is contemplated that molecular modelling techniques will permit compounds of different primary and secondary structures to be substituted for the polypeptides of this invention, provided equivalent tertiary structures can be determined. All such modifications may be within certain embodiments of the invention.

The peptides of the invention are selected by comparing binding patterns of antibodies from a healthy, infected individual and antibodies from a symptomatic, infected individual to determine at least one binding region unique to antibodies from the healthy, infected individual. These unique regions define protective regions or epitopes. Once the protective regions or epitopes have been determined, peptides corresponding to at least a portion of at least one of these regions is prepared. The peptides may be prepared by any convenient methods such as synthesis with the appropriate amino acids and a peptide synthesizer, or by recombinant DNA techniques, where a DNA sequence coding for the amino acid sequence is synthesized or prepared from cellular sources and inserted into an appropriate host cell for production of the peptide. The test peptides may also be prepared by chemical synthesis, recombinant DNA techniques or by purification from natural sources.

For some embodiments of the invention it may be preferable to conjugate the "protective" peptides to a carrier protein such as keyhole limpet hemocyanin. Peptides can be conjugated to carrier proteins by conventional techniques for conjugating proteins. A preferred method for conjugating the peptides and carrier protein is the method described herein. For this method a cysteine residue is added to the amino terminal end of the peptide before conjugation with the carrier protein. This can be conventiently accomplished by chemical symthesis when the peptide is being made or at a later time.

The peptides of the invention are useful as diagnostic reagents and vaccines. The presence of the protective epitopes in antibodies of persons infected with HIV is a measure of the likelihood of that person developing symptoms of viral infection and progressing to Acquired Immunodeficiency Disease (AIDS) at a later date; the presence of protective antibodies indicating

a decreased likelihood of that individual developing symptoms of AIDS. The peptide diagnostic reagents can be used i conventional immunoassays for detecting antigens or antibodies and the presence of protective antibodies in the test sample ma be determined by any suitable method, including radiolabel suc as ¹²⁵ I or ₃ ⁵ S for use in radioimmunoassay, with fluorescein fo fluorescent immunoassay, with an enzyme for enzyme immunoassa or with biotin for biotin-avidin linked assays. These methods are exemplary only and other methods may be useful in the invention.

For example, the peptides of the invention can be bound to a solid phase such as a multi-well plate. Test samples suspected of containing protective antibodies for HIV are contacted with the peptides under conditions that allow binding of protective antibodies in the test sample to the peptides. Bound protective antibodies are then contacted with an antibody such as anti-human IgG labeled with ¹²⁵ I under conditions that allow binding of the labeled antibody to bound protective antibodies. The label is then detected by autoradiographical means. The presence of radiolabel indicates the presence of protective antibodies in the test sample.

The antibodies of the invention can be made by conventional methods for the production of polyclonal or monoclonal antibodies. POlyclonal antibodies can be produced by methods such as the method described herein for producing rabbit antibodies. For monoclonal antibodies, an animal such as a mouse is first injected with the antigen, its spleen cells are removed and fused with myeloma cells to form hybridoma cells, the latter are cloned in a serum-containing medium and the monoclonal antibodies are separated from the medium. Experimental Fusion Inhibition Assay

Sup - Tl cells are favored as target cells for their rapid degree of cell fusion when co-cultured with HIV - 1

producing cell lines. Cell culture is performed according the method of Dalgleish et al. Nature 312; 763, (1984). S -Tl cells are plated in 96 well plates (10 ⁵ cells/well in RP 1640 + 10% FCS) and incubated with or without dilutions patient sera mouse sera, or control monoclonal antibodies f 30 minutes at 37C. HTLV-III B (HIV-l) infected H9 cells a then added 5xl0*/well and the number of multinucleated gia cells per 16 X field counted with a Zeiss inverted field pha contrast microscope after 18 hours. Syncytia are easi identified and inhibition of syncytia by patient sera or ant idiotypic antisera can be compared with anti CD4 monoclon antibody induced syncytia inhibition.

For screening of patient sera, samples were collect and directly added to syncytia assays and described above various dilutions. In later assays the HTLV-IIIb (HIV- infected H9 cells were replaced with a noninfectious CHO-HIV envelope expressing constructed cell line with fusi capabilities similar to infectious virus, according to t method of Sodrosfsky et al.. Nature 322: 470, (1986) . At hi concentrations, inhibition of syncytia was observed. contrast, no inhibition of syncytia formation at a concentration was observed when anti-idiotypic antise generated against pooled AIDS immunoglobulin were used.

Antibodies from one patient, H156, were found significantly inhibit syncytia formation. These antibodies we used in subsequent experiments and were used to generate ant idiotype antibodies. References herein to H156 refer to t antibodies from this patient which were obtained through t described screening process. Syncytia Inhibiting Activity

1/8 1/16 1/32 1/64 1/128 1/256

Protein A 0 0 0 0 0 IS purified IgM 0 0 2M IM 2M 2M

fraction

H156 sera 0 0 0 0 IM 2M

Normal 4L 4L 4L 4L 4L 4L human sera Degree of syncytia formation Size of syncytia forme

4=Full S=8mall

0=None M=Medium

LsLarge

HELS Patient sera was extensively dialyzed against phosphat buffer and subjected to gel filtration on high-performanc liquid chromatography columns TOYOSDA TSK G 4000 - 3000 set i series. Samples were analyzed in lOmM sodium phosphate buffe (PH 7.2). Fractions were run at 0.5 ml/min for 120 min tota run. The column was calibrated utilizing molecular weigh standards (Sigma) . 1ml fractions were analyzed by syncyti inhibition assays; immunoglobulin fractions were visualized b SDS-PAGE followed by Commassie blue staining. Purified IgG an IgM fractions were stored at -70° until use. Active fraction were observed in two approximate molecular weight ranges o 170kd and greater than 570 kd. When combined with data from th syncytia inhibiting assay above, this size fractionatio suggested syncytia inhibiting activity segregated in the IgG an IgM molecular weight ranges. SDS-polyacryla ide ge electrophoresis of tested fractions revealed the presence o characteristic immunoglobulin bands IgM at molecular weight greater than 590 Kd and IgG in 170Kd fractions supporting thi interpretation. Most inactivity was found in the IgG fraction.

Affinity Chromatography To further demonstrate that antisyncytia activity wa mediated by IgG, immunoglobulin fractions were purified b affinity chromatography on protein A agarose beads (Sigma) .

The protein A-purified antibody mediated significant antisyncytia activity, whereas non protein A binding materials had little activity.

Generation of Anti-idiotypic Antisera 8 to 10 week old female Balb/c mice were inoculated subcutaneously with 100 ugs of protein A-purified H156 IgG or pooled AIDs patient IgG emulsified in complete Freunds adjuvant, following the primary immunization mice were boosted bimonthly with 100 ug antibody emulsified in incomplete Freunds adjuvant. One week following the fourth and subsequent boost, mice were bled through their tail veins and serum saved for analysis, collected serum was extensively absorbed on HIV Ab negative human antibody columns before being sterile filtered and reconcentrated back to their original volumes. Absorbed samples were stored as small samples at -70C until screened.

Purification of Sera

Serum antibodies were purified from hybridoma ascites flui by sequential ammonium sulfate precipitation and protein A- sepharose (Sigma) chromatography. Sera was gradually made 50% ammonium sulfate by the addition of an equal volume of saturate ammonium sulfate at 4°C with stirring. The solution was stirre for an additional 60 minutes to allow im unoglobulins t precipitate completely. The precipitate was collected b centrifugation at 15000x g for 15 min, and resuspended i phosphate-buffered saline (PBS; 188 mM NaCl, lOmM P0 ₄ , pH 7.2). The resulting immunoglobulin solution was dialyzed for 24 h against PBS with at least three changes. The ammonium sulfat cut was then clarified by centrifugation and passed over protein A-sepharose column. The column was washed with norma saline until the OD ₂₈₀ of the filtrate was less than 0.1. Th bound immunoglobulin was then eluted with 3.5 M MgCl ₂ . Relevan fractions were pooled and dialyzed extensively against norma

saline and then PBS, and filtered through a 0.45 urn filter. T antibody solution was concentrated using an Amicon concentrat under nitrogen pressure, and the protein concentration w determined using a Protein Assay Kit (BioRad Labs, Richmon CA) .

Flow Cytometry

Cells were removed from tissue culture and washed twice FACS medium (Hanks' balanced salt solution (Gibco) supplement with 2% fetal calf serum, 0.2% sodium azide, and lOmM Hepes) 1 x 10 ⁵ to 1 x 10 ⁶ cells were incubated on 0.1 ml of FACS medi with antibody or control supernatant in a volume of 0.1 ml f 1 hr at 4°C. Cells were diluted in 2.5 ml of FACS mediu pelleted by centrifugation at 1000 x g and washed twice with 2. ml of FACS medium per wash. Following the final wash, the ce pellet was gently resuspended and cells incubated with 0.1 of FITC-conjugated rabbit anti-mouse IgG (reactive with antibo heavy and light chains, Miles Laboratories) diluted 1:20-1: in FACS medium for 1 hr at 4°C. Cells were diluted and wash as after the first incubation. The cell pellet was final resuspended and the cells fixed in 0.5-1.0 ml 2 parafor aldehyde-PBS. Samples were run on an Becton Dickins FACS IV. 20,000 cells per sample were routinely analyze Specific fluorescence was quantitated by subtracting the medi fluorescence channel of cells stained with FITC-conjugat rabbit anti-mouse immunoglobulin alone (negative control) fr the median fluorescence channel of cells stained with specifi antibody followed by FITC-conjugated rabbit anti-mous immunoglobulin (positive staining) .

CD4+ cell lines, Molt 4 and Sup Tl both demonstrated stron specific reactivities with anti-H156. In addition, human cel lines including HSB (American Type Culture Collection numbe

CCL120.1), a CD4-T cell line, was also reactive. Thi

demonstrates that the determinant recognized by the anti- idiotypic sera is not CD4. To determine if the reactivity pattern of the antiidiotype antisera was similar to the reported species tropism of HIV, the binding of the anti-H156 sera to urine cells was examined. Negligible reactivity was observed (Figures 1-9) . Absorption with murine L cells prior to staining both human and other murine cell lines removed all reactivity to murine cells without affecting the reactivity to human cells. When the experiment was performed with the pooled AIDS immunoglobulin generated anti-idiotypic antisera, no staining of human or murine cell lines was seen. The surface reactivity pattern of the anti-H156 antisera appears to be due to components of the human cell lines examined, and distinct from the HIV-l receptor CD4. This structure correlates with the species tropism reported for HIV-l in inducing productive syncytia formation.

Immunoprecipitation Protocol

Cell lines are precultured in methionine and cysteine free RPMI (Gibco) + 10% dialyzed FCS and labeled for 16 hrs. with media supplemented with ³⁵ S-cysteine and ³⁵ S-methionine (lOOuCi/ml) and lysates prepared and precleared as described in Sodrosfsky et al., Nature 322: 470, (1986). Portions (200ul) of cleared lysates are added to 20ul of Protein A-Agarose beads preincubated with serum and rotated for 3 hours at 4°C. Beads are washed sequentially in lysing buffer (LB) ; LB containing 0.5 M NaCl; and LB with 0.1% sodium dodecyl sulfate (SDS) . The adsorbed material is eluted by heating at 100°C for 3 minutes in 50 ul of sample buffer [0.01 M tris, pH 8.0, containing 2% SDS, 5% 2-mercaptoethanol (by volume) , bromophenol blue 25 ug/ l, and 10% glycerol (by volume)], and analyzed on 7.5% SDS-PAGE. The gels are then fixed, dried and autoradiographed at -70° on XAR (Kodak) film.

For competitive immunoprecipitation of virus envelope glycoproteins the basic procedure was modified. H156 protein A purified IgG was coupled to CnBr sepharose 4B as per manufacturers instructions (Sigma) . 40ul of H156 beads (approximately 32ugs of H156 based on 80% coupling efficiency) were preincubated at 37°C for 30 minutes on a circular rotator with 50ul of the following reagents (see Figure 10) : nothing (lane 1) , H156 serum (lane 2) , normal mouse sera (NMS) (lane 3) , anti-pAIg mouse sera (lane 4) , or anti-H156 mouse sera, for 30 minutes on a circulator rotator. The serum was then chilled on ice for 15 minutes and equal counts of 35S-met labeled precleared cell lysate was added to each Eppendorf test tube and precipitated as described in Sodrosfsky et al. , Nature 322:

470, (1986) , which is incorporated herein by reference.

The anti-idiotypic antibodies blocked the ability of H156 antibody to precipitate gpl60 but only minimally blocked gpl20 immunoprecipitation. In contrast the pooled AIDS patient anti- idiotypic antisera did not exhibit complete blocking of either glycoprotein (Figure 10) . This result supports competitive immunoblotting data in that the predominant anti-idiotypic response is directed against antibodies specific for gp41. The studies also corroborate previously published observations such as those of McCune et al. Cell J52: 55, (1988) that the epitopes of the free gpl20 and covalently linked gpl20-gp41 (gp 160) are not identical.

While H156 sera blocks all reactivity with HIV-l envelope glycoproteins gpl60 and gpl20, NMS exhibits no blocking ability, anti-pAIg exhibits the ability to partially block both gpl60 and gpl20 reactivities. Anti-Hl56 mouse sera partially blocks gpl20 reactivity but specifically and repeatedly blocks all reactivity of H156 with gpl60 envelope glycoprotein precursor protein.

Twelve out of twelve mice immunized with H156 produced thi identical dominant immune response supporting our observatio of a dominant idiotype in H156 sera directed at gp41. Thi result supports competitive im unoblotting data in that th predominant anti-idiotypic response is directed agains antibodies specific for gp41.

Immunoblotting

Cell lines productively infected with HIV-l are lysed i lysing buffer (0.02M tris and 0.12 M NaCl, pH 8.0, with 0.2m phenylethylsulfonyl fluoride, 0.2 mM EGTA, 0.2 mM NaF, 5ug/m of aprotinin, 0.2% sodium deoxycholate, and 0.5% by volum Nonidet P-40) . Lysates are boiled for 5 minutes in 3% SDS, an approximately 15 ug of protein per lane is separated on 10% SDS PAGE, electrotransferred to nitrocellulose and reacted wit serum on control antisera. For competitive Western analysis th electrotransferred filters were reacted with pooledAIDS patien immunoglobulin on control sera at a concentration of lmg/ after a 30 minute pre-incubation 50ngs of I ¹²⁵ labeled H15 purified IgG or IgM was added and allowed to incubate a furth 1 hr. with agitation at 25°C. After extensive washing the bl was exposed to XAR (Kodak) autoradiography film at -70°C for 2 hrs. Relevant bands were subjected to densitometry tracing fo quantitation of specific reactivity with virus envelop glycoprotein gp41 or gpl20.

Most reactivity to gpl20 was inhibited by pretreatment o the nitrocellulose with pooled AIDS immunoglobulin. I contrast, significant reactivity for envelope glycoprotein gp4 remained after binding pooled AIDS immunoglobulin to it. Thi reflects H156 reactivity with unique epitopes expressed on gp4 These reactivities are not present in significant amounts in t pooled AIDS sera.

A 95% reduction in the reactivity to gpl20 was produced b pretreatment of the nitrocellulose with pooled AIDS patien immunoglobulin (pAIg) . In contrast, 90% of the reactivity fo envelope glycoprotein gp41 remained after blocking with pAIg. This reflects H156 reactivity with unique epitopes expressed o gp4l. Radioimmunoassay

Radioimmunoassay (RIA) analysis of the anti-idiotypi antibody following the procedure of Burstin et al. , Virolog 117: 146, (1982) . RIA analysis demonstrated specific response against the immunizing im unoglobulins with minimal binding t normal human immunoglobulin (Figure 11) . Mouse anti-H156 wa compared with mouse anti-pAIg (pooled AIDS patien immunoglobulin) and normal mouse serum (NMS) for idiotyp specific binding to H156. Both the anti-H156 and anti-pAI showed specific binding to pAIg consistent with the presence o public idiotypes in both pAIGg and H156 relevant to HIV- exposure. However, anti-H156 demonstrated much greate idiotypic specific binding activity to H156 than anti-pAIg, while NMS showed negligible binding to either immunoglobulin.

These data demonstrate the anti-idiotypic nature of anti-H156 as well as the presence of private idiotypes present in the H156 sera not represented in the pAIg sera.

Characterization of Host Cell Receptor (second receptor) Cell lines (human HSB (American Type culture Collection number CCL120.1), ST and HeLa (American Type Culture Collection number CCL2) and murine L cells and NIH 3T3 cells) are precultured in methionine and cysteine free RPMI (Gibco) + 10% dialyzed FCS and labeled for 16 hrs. with media supplemented with ³⁵ S-cysteine and ³⁵ S-methionine (lOOuCi/ml) and lysates prepared and precleared as described in Sodrosfsky et al. , Nature 322: 470, (1986). Portions (200ul) of cleared lysates

are added to 20ul of Protein A-Agarose beads preincubated with serum and rotated for 3 hours at 4°C. Beads are washed sequentially in lysing buffer (LB); LB containing 0.5 M NaCl; and LB with 0.1% sodium dodecyl sulfate (SDS). The adsorbed material is eluted by heating at 100°C for 3 minutes in 50 ul of sample buffer [0.01 M tris, pH 8.0, containing 2% SDS, 5% 2- mercaptoethanol (by volume) , bromophenol blue 25 ug/ml, and 10% glycerol (by volume)], and analyzed on 7.5% SDS-PAGE. The gels are then fixed, dried and autoradiographed at -70° on XAR (Kodak) film.

As shown in Figures 12 and 13, anti-H156 specifically immunoprecipitates, from human cells and not from murine cells, several polypeptides including a major band at 25-35Kd, as well as minor bands at 45-60 Kd, 80-l00Kd and 180-220Kd.

Protective Peptides

PEPTIDES: Peptides were synthesized by the Protei Chemistry Laboratory of the University of Pennsylvania usin conventional techniques. Amino terminal cysteine residues wer added to the sequence of some peptides during synthesis fo coupling to proteins. Peptides were purified, and conjugate to keyhole limpet hemocyanin (KLH) by the following method. T 15 mg of keyhole limpet hemocyanin (KLH, Sigma) in 1 ml of 5 mM sodium bicarbonate was added 5 mg of sulfo-MBS (Pierce) . After thirty minutes at room temperature, the KLH was separate from excess sulfo-MBS by gel filtration on Sephadex G50. Fift mg of the peptide in 1 ml of sodium bicarbonate was added to th KLH and allowed to react for a further 3 hours. Th macromolecular conjugate was separated fromunconjugated peptid by gel filtration on Sephadex G50. The peptide-KLH conjugat was suspended in phosphate-buffered saline (20 mM sodiu phosphate, 154 mM NaCl, pH 7.2) at a concentration of 1 mg/ml.

PATIENTS: H156 represents serum from an HIV-l infecte individual who was asymptotic, and which demonstrated a uniquel

high degree of syncytia inhibitory activity by several isolates of HIV-l, Weiner et al..1989. Non-CD4 molecules on human cells important in HIV-l cell entry. Vaccines 89. Cold Spring Harbor Laboratories, CSH, N.Y. 115-120. EPITOPE DETERMINATION: H156 was utilized to obtai purified IgG and directly radioiodinated as in Williams et al. , Proc. Natl. Acad. Sci. USA 85:6488 (1988). Radioimmunoassay plates were coated with various peptides derived from the HIV- 1 gpl60 sequence, including the peptides shown in Table 1, and binding of purified radioiodinated H156 IgG to these peptides was carried out as described in Williams supra.

Several peptides were chosen for further study on the basis of the ability of H156 to bind these peptides at a much higher level than IgG from control AIDS patients IgG prepared in the same manner. These are detailed in Table 2. Two of these peptides were designated F160 (bearing the sequence of residues 150-170 of gpl60) , and F560 (bearing the sequence of residues 550-570 of gpl60) (see Table 1) .

IMMUNIZATION: NZW rabbits were injected subcutaneously with 50-100 μg of peptide-KLH conjugate or unconjugated peptide emulsified with 50-100 μl of Freund's complete adjuvant. After two weeks, the rabbits received a similar injection using incomplete adjuvant. Further booster injections were performed subcutaneously at two-week intervals using 50 μg of the conjugate without adjuvant. Serum was obtained from immune animals following the third boost, and utilized without heat inactivation.

Serum was obtained one week after the fifth injection and assayed for anti-peptide antibodies using a solid-phase radioimmunoassay. For this assay, 2.5 μug of peptide in 50 μl of water was dried onto each well of a 96-well polyvinyl chloride microtiter place. A solution of bovine serum albumin (20 g/ml) in phosphate-buffered saline containing 0.1% sodium azide was added to fill each well. These plates were stored at

4*C until use. Preimmune and immune sera from each mouse wer diluted into albumin-containing buffer and added to the draine wells. The plates were incubated overnight at room temperature and the wells were then washed with phosphate-buffered saline [1251]-goat anti-mouse light chains (40,000 cpm/well. Souther Biotechnology Associates, iodinated using a modification of th chloramine T method as described in Hunter and Greenwood, (1962 Nature 194: 495-496 in 100 μl of phosphate-buffered salin containing bovine serum albumin was added to each well an incubated for two hours at 37*C. The plate was then washe several times with water and the radioactivity in each wel determined with an automated gamma counter.

CYTOFLOORIMETRY: The ability of immune rabbit sera t stain gpl20 and gpl60 bearing cells was determined b cytofluori etry as described in Williams et al.. Proc. Natl Acad. Sci. USA 85:6488 (1988). Rabbit sera were used a dilutions of 1:10 to 1:100. Positive staining was determine by % positive of >10 or Δ mean channel florescence of >8.

CELLS LYSIS: Cells were lysed by antibody and compleme treatment as described in Williams et al.. (1987) "The cellul basis for the la restriction in murine experimental autoimmu thyroiditis", Cell. Immunol. 110:35-45. Rabbit antisera w utilized at dilutions of 1:2 - 1:128. Those considered positi lysed cells at dilutions of at least 1:16. Lysis of cells w determined by direct visualization and cell counting. Lysis w considered present if >90% of the cells originally present the sample were deleted. Staining and Lysis of Cells bv Immune Rabbit Sera

To construct cells that bore gpl20 without gp41, cultu supernatants from CHO/gpl60 cells, which shed gpl20, we incubated with H9 cells, which bear large amounts of CD4. T cells were washed, and utilized in the cell lysis a cytofluorimetry assays described herein.

CELLS: Chinese hamster ovary (CHO) cells, and CHO cell that are infected with and express HIV-l gpl60 (CHO/gpl60) hav been described in Weiner et al.. 1989. Non-CD4 molecules o human cells important in HIV-l cell entry. Vaccines 89. Col Spring Harbor Laboratories, CSH, N.Y., 115-120. H9 is a huma T cell line that expresses large amounts of CD4 molecules. Al cells were grown in RPMI 1640 with adde penicillin/streptomycin, L-glutamine, and 10% fetal calf seru (culture media) . PREPARATION OF gpl20 CONTAINING SUPERNATANT: CHO/gpl6 cells were selected for secretion of large amounts of gpl20 These cells were grown in culture media to a high cell density supernatants harvested, centrifuged, and filtered through a 0.4 urn filter prior to use. By comparing results utilizing these cells with result from CHO/gpl60 and H9 cells infected with HTLV-lllb, Weiner g al. , 1989 Non-CD4 molecules on human cells important in HIV- cell entry. Vaccines 89. Cold Spring Harbor Laboratories, CSH N.Y. 115-120, binding and lysis via gpl20 and gp41 wer distinguished. The results are shown in Table 2.

TABLE 2

STAINING AND LYSIS OF CELL LINES BY IMMUNE RABBIT SERA

Complement Flow Cytometry

Cell Line Antibody To: Mediated Lysis Staining

CHO F560-KLH CHO F160

H9 F560-KLH H9 F160

H9+GP120* F560-KLH H9+GP120 F160

CHO/gpl60 F560-KLH + + CHO/gpl60 F160 + +

H9/lll ^b F560-KLH + + H9/lllb F160 + +

^m H9 cell preincubated with culture supernatant from CHO/gpl6 cells containing gpl20 9 cells infected with the HTLV-lllb isolate of HIV-l.

As shown in Table 2, antibodies to peptide F560 coupled t KLH do not lyse H9 cells that have been preincubated wit culture supernatant from CHO/gpl60 cells containing gpl2 whereas antibodies to peptide F160 did bind to these cells This result indicates that these anti-F560-KLH antibodies ar specific for a region of gpl60 that becomes gp41 and that th peptide F560 defines an epitope of gp41. Anti-F160 antibodie are specific for a region of gpl60 that becomes gpl20 and th peptide thus defines an epitope of gpl20. Significance

An important structural component of HIV virions is th membrane spanning molecule gp41. During viral replication, th HIV envelope protein is synthesized as a large unit, terme gpl60, which is subsequently cleaved into gpl20 and gp41. Gpl2 forms the outer membrane glycoprotein of HIV, while gp41 remain anchored in the membrane acting as an anchor to which gpl2 attaches. While gpl20 is capable of being shed into the media gp41 remains anchored in the membrane of the virion or of th infected cell. It is important to note that the sequence of th F560 defined epitope is completely covered in all HIV-l isolate examined to date. It is expected that analogous regions wil be present in other retroviruses.

Serum with potent syncytia inhibitory activity was obtaine from a healthy HIV-l infected individual. It was hypothesize that this individual's antibody response would recognize region of the HIV envelope important in the development of protectiv immunity. By comparing this individual's antibody profile fo binding to gpl60 derived peptides to the profile of antibodie from symptomatic HIV-l infected individuals, Applicants wer able to detect unique reactivities to several peptide regions This included amino acids 150-170 of gpl60 (contained on gpl20)

and amino acids 550-570 of gpl60 (contained on gp41) . Applicant then immunized rabbits with these peptides (either uncoupled o coupled to KLH) , and tested their antisera or binding t gpl20/gp41, and for their ability to lyse cells bearing gpl2 and/or gp41. Antibodies specific for the peptides bound t gpl20/gp41 and were able to lyse cells bearing gpl20 and/o gp41. The peptide corresponding to amino acids 550-570 of gpl6 (F560) which is contained on gp41 is especially useful becaus it defines an epitope on gp41, the integral membrane protein o HIV-l. gp41 is not shed by the virus or infected cells bu remains anchored in the membrane. Antibodies specific for th peptide F560 bind to gp41 on infected cells, initiatin complement mediated lysis of the infectd cell. Antibodie specific for the peptide F560 do not bind to uninfected cell that express CD4 receptors that have gpl20 bound thereon thu sparing the "innocent bystander" cells from lysis.