WO1989002277A2

WO1989002277A2 - Prophylaxis and therapy of acquired immunodeficiency syndrome

Info

Publication number: WO1989002277A2
Application number: PCT/US1988/002970
Authority: WO
Inventors: Ralph B. Arlinghaus
Original assignee: Board Of Regents, The University Of Texas System
Priority date: 1987-08-28
Filing date: 1988-08-26
Publication date: 1989-03-23
Also published as: AU2914889A; WO1989002277A3

Abstract

The present invention involves a process for inducing resistance of an individual to infection by human immunodeficiency virus. The process involves vaccinating said individual with a synthetic peptide of mixture of peptides. The synthetic peptide(s) comprises an amino acid sequence derived at least in part from human immunodeficiency virus envelope protein conserved region. Upon antigenic presentation to an animal, this peptide induces directed cell-mediated immunity (i.e., T-cell cytotoxicity) to a substantially greater extent than production of antibody directed against native human immunodeficiency virus is elicited. The vaccine of the present invention comprises a synthetic peptide having an amino acid sequence derived at least in part from T-cell epitopes of human immunodeficiency virus envelope protein conserved region and preferably consists exclusively of T-cell epitopes.

Description

PROPHYLAXIS AND THERAPY OF

ACQUIRED IMMUNODEFICIENCY SYNDROME

The present invention concerns a method to prevent or treat acquired immunodeficiency syndrome (AIDS) and involves a new and novel approach for making a vaccine. The vaccine comprises synthetic peptides which exhibit certain immunologicar characteristics of one or more proteins encoded by the viral causative agent of this disease.

AIDS was first recognized in the United States in 1981; the number of cases has been increasing at a dramatic pace since then. Since 1978 more than 2.4 million AIDS infections have been reported in the United States, alone (Rees, Nature, 326:343, 1987). Once significant immunosuppressive symptoms appear in an infected individual, the expected outcome of the infection is death. There is currently no known treatment that can indefinitely delay or prevent the fatal consequences of the disease. Although the disease first manifested itself in homosexual or bisexual males and intravenous drug abusers, it has now spread to others by means such as intimate sexual contact with or receipt of blood products from a carrier of the virus. The causative agent, associated with AIDS has been identified as a group of closely related retroviruses commonly known as Human T Cell Lymphotrophic Virus-type III (HTLV-III), Lymphoadenopathy Viruses (LAV), AIDS-Related Viruses (ARV), or more recently named Human Immunodeficiency Virus (HIV). These viruses will be collectively referred to herein for convenience as HIV.

Like other retroviruses, HIV has RNA as its genetic material. When the virus enters the host cell, a viral enzyme known as reverse transcriptase copies the viral RNA into a double stranded DNA. The viral DNA migrates to the nucleus of the cell where it serves as a template for additional copies of viral RNA which can then be assembled into new viral particles. The viral RNA can also serve as messenger RNA for certain viral proteins [either the viral core proteins (known as p18, p24 and p13)] or the reverse transcriptase, or be "spliced" into specific viral messenger RNAs necessary to produce several other viral proteins including two glycosylated structural proteins known as gp41 and gp120 which are inserted in the outer membrane of the virus (Wain-Hobson et al., Cell 40:9, 1985). A recent study has shown that purified gp120 induces antibody in the goat, horse and rhesus monkey that neutralizes HIV in lab tests (Robey et al., Proc. Natl. Acad. Sci., USA 83:7023, 1986).

Vaccines have been used for many years to prevent infections caused by agents such as viruses. The general approach has been to inject healthy individuals with, for example, a killed or modified virus preparation in order to prime the individual's immune systems to mount an assault on the infecting virus. Recent advances in recombinant DNA technology have allowed safer methods of vaccination that involve use of exposed viral components produced by microbial systems. After sufficient purification, the viral component, for example a protein subunit, is administered as a vaccine in a suitable vehicle and/or an adjuvant. The latter stimulates the host's system in a way that improves the immune response to the viral subunit.

Another potential method of making a vaccine is by using chemically synthesized peptide fragments of a viral protein subunit. This method has several advantages over the other methods of producing vaccines, including purity of the product, reproducibility and specificity of the immune response.

Surface antigens of an infecting virus may elicit T cell and B cell responses. From the work of Milich and coworkers (Milich et al., J. Exp. Med. 164:532, 1986; Milich and McLachlan, Science, 234:1398, 1986) it is clear that some regions of a protein's peptide chain may possess either T cell or B cell epitopes. These epitopes are frequently distinct from each other and may comprise different peptide sequences. Other examples include the work of Maizel et al., (Eur. J. Immunol. 10: 509, 1980) for hen egg-white lysozyme, and Senyk et al., (J. Exp. Med., 133:1294, 1971) for glucagon. Thus, short stretches of a protein sequence (e.g. 15 amino acids) may elicit a T cell response but not a B cell response. A more complete review of. these and other observations pertinent to this point is included in the work of Livingstone and Fathman (Ann. Rev. Immunol., 5: 477, 1987).

A short peptide region within the surface protein of infectious Hepatitis B virus has been shown to elicit only a T cell response in mice (Milich et al., 1986). Specifically, a synthetic peptide, whose sequence is derived from amino acids numbered 120-132 located within the pre-S(2) domain of the Hepatitis B surface antigen gene, elicited a very strong T cell priming response to the peptide but stimulated only a very weak antibody response. In other words, mice mounted a poor antibody response to that peptide, but the T cells of immunized mice were efficiently primed (i.e. activated) to recognize that peptide as measured in T cell proliferation assays (Milich et al., 1986). The low level of the antibody produced by mice immunized with this peptide did not bind to the native viral surface antigen. The sequence of this T cell active peptide is:

Amino terminal-MQWNSTTFHQTLQ-carboxy terminal.

The single letter code for amino acids used throughout this application is: A, alanine; C cysteine; D, aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H, histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N, asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T, threonine; V, valine; W, tryptophan; and Y, tyrosine.

In contrast to the above-described results, a second peptide sequence (amino acids 132-145) elicited a very weak T-cell response in mice (Milich et al., 1986). This second peptide did, however, efficiently bind antibody raised against it under conditions where a T cell epitope is provided.

The sequence of the second or B cell active peptide is:

Amino terminal-DPRVRGLYFPAGG-carboxy terminal.

Mice were also immunized with a longer peptide made up of both of the above-mentioned T- and B-active peptide sequences. In this case high titers of antibody were produced against the B site peptide but not the T site peptide. The combination of both T- and B-sites within one peptide should stimulate both T and B cell responses, as measured by producing a specific antibody to the B cell epitope of the peptide chain. Synthetic peptide antigens may be constructed to produce two types of immune responses: T-cell only and T cell combined with a B cell response.

Cellular immune responses provide a major mechanism for reducing the growth of virus-infected cells (Doherty et al., Adv. Cancer Res., 42:1, 1985). A report by Earl et al., (Science, 234:728, 1986) demonstrated T-lymphocyte priming and protection against the Friend virus (a retrovirus)-induced mouse leukemia by a viral surface protein vaccine. Direct evidence for the role of a subset of T-lymphocytes (OKT8/LEU2 positive) in suppressing HIV growth in vitro was recently obtained by Walker et al., (Science, 234:1563, 1986). This study further demonstrated that, after depletion of CD8+ T-lymphocytes from the blood of HIV-infected individuals, large quantities of HIV were isolated from peripheral blood mononuclear cells of four of seven asymptomatic, seropositive homosexual men who were initially virus-negative or had very low levels of virus. Thus, the CD8+ subset of T-lymphocytes may play a role in virus infected individuals to prevent HIV replication and disease progression.

The present invention involves a process for inducing resistance of an individual to infection by human immunodeficiency virus. The process involves vaccinating said individual with a synthetic peptide or mixtures of synthetic peptides. The synthetic peptide(s) comprises an amino acid sequence derived at least in part from human immunodeficiency virus envelope protein conserved region. Upon antigenic presentation, such a peptide induces directed cell-mediated immunity (i.e. T-cell cytotoxicity) to a substantially greater extent than production of antibody directed against native human immunodeficiency virus is elicited. The vaccine of the present invention comprises a synthetic peptide (s) having an amino acid sequence derived at least in part from T-cell epitopes of human immunodeficiency virus envelope protein conserved region and preferably consists exclusively of T cell epitopes.

The invention further predicts that the chemical nature and properties of the HIV surface proteins are similar to or may resemble proteins products of the immunoglobulin gene family in one or more biological characteristics. This group of genes includes the various immunoglobulins, the T cell receptor protein involved in antigen recognition, the major histocompatibility genes, the T4 antigen and others. This similarity will likely render HIV resistant to vaccines that induce an antibody response.

Just as HIV infects certain lymphoid cells, viruses like Human T Cell Leukemia virus type 1 (HTLV-1) and Feline Leukemia virus (FeLV) also infect and alter lymphocytes. HTLV-1 is associated with a T cell malignancy known as Adult T cell Leukemia (Yoshida and Seiki, Ann. Rev. Immunol 5:541, 1987). It is likely then that the surface proteins of both of these viruses also share one or more biological properties with the protein products of the immunoglobulin gene family and therefore will be resistant to vaccines that depend on antibody-induced inactivation of the infectious virus.

In greater detail, the process of the present invention for inducing resistance to human immunodeficiency virus comprises several steps. Amino acid sequences of human immunodeficiency virus envelope protein conserved region able to form helical structures and further characterized by the presence of amphipathically interrelated amino acids are first identified. Peptides or peptide derivatives comprising at least a substantial part of the identified sequences are then synthetically prepared. Said peptides or peptide derivatives are then administered to a test animal in a manner stimulating an immune response. The T cell response and humoral antibody response in said test animal are monitored to screen for peptides or peptide derivatives which stimulate T-cell immunity without inducing substantial production of humoral antibody directed against native human immunodeficiency virus. An individual is then inoculated with an immunogenic composition comprising said screened peptide or peptide derivative to induce resistance to human immunodeficiency virus infection.

The peptides or peptide derivatives of the present invention useful in prophylaxis of AIDS preferably comprise an amino acid sequence of human immunodeficiency virus envelope glycoproteins' conserved regions. The human immunodeficiency virus envelope glycoproteins includes human immunodeficiency virus glycoprotein gp 120 and human immunodeficiency virus glycoprotein gp 41.

In situations where the treatment of individuals already infected with HIV is desired, a T cell mediated immunity toward HIV-infected cells is also warranted. Such HIV-infected cells may express, on their surface, T cell epitopes of HIV envelope proteins and/or HIV core proteins. Thus, for such treatment, an immunizing peptide or peptide derivative may have an amino acid sequence substantially comprising one or more T cell epitopes of a HIV envelope protein or HIV core protein. The synthetic peptides of the present invention may be prepared by techniques involving solid-phase chemical synthesis, liquid-phase chemical synthesis or biological synthesis involving recombinant DNA, all well-known to those skilled in the relevant arts.

The HIV agent is unique in that it infects cells involved in the immune response and can kill these cells. The host cell often involved is the T4 lymphocyte, a white blood cell that plays a central role in regulating the immune system. The virus binds to cell surface T4 protein which is implicated in the mediation of efficient T cell-target cell interactions. T4+ lymphocytes interact with target cells expressing major histocompatibility (MHC) class II gene products. Both T4 and MHC genes are members of the immunoglobulin gene family (Maddon et al., Cell, 47:333, 1986). The observation that T4 interacts with the exterior HIV envelope protein, gp120, prompted a structural comparison of the viral protein to immunoglobulin proteins. Interestingly, two regions of gp120 were found to share sequence homology with human immunoglobulin heavy chain constant regions (Maddon et al., Cell, 47:333, 1986). Extrapolating from these observations, the present invention may hinge upon the fact that gp120 has certain properties unique to human immunoglobulins. Furthermore, this similarity in structure may allow the virus to escape inactivation by antibody interaction. Furthermore, viral-antibody interaction may, in certain situations, increase the infectivity of the virus. Recent work suggests that AIDS patients can and do have antibodies that neutralize the virus, as determined by in vitro lab tests. Yet these same patients die of the disease. The present invention predicts that antibodies binding to the virus may not interfere with and in some cases may even increase the virus' inherent ability to infect the patient's lymphoid cells. Recently retrovirus infectivity was shown to be increased by binding of anti-retrovirus antibodies (Legrain et al., J. Virol., 60:1141, 1986). Therefore, an AIDS vaccine that primes the individual's immune system to make antibodies to viral surface proteins may enhance the infectivity of an already deadly virus. What is needed then is to stimulate only the individual's T cell immunity (for example, cytotoxic T cells or CD8+ T cells) without involving an antibody response to viral proteins.

Synthetic peptide immunogens can certainly achieve this result.

The vaccine of the present invention is preferably a totally synthetic vaccine made using a synthetic peptide(s) linked to a fatty acid compound, or polymerized through natural or extra cysteine residues. Important facets or considerations may be listed as follows for a vaccine of the present invention.

The vaccine of the present invention comprises short synthetic peptides. These short synthetic peptides (10-30 amino acids in length) have sequences from one or more conserved regions of either of the two HIV envelope. These peptides should elicit a T cell response but not a substantial antibody response. Therefore, when suitably prepared, the peptide vaccine of the present invention will stimulate T cell immunity (i.e., cytotoxic T cells) without producing a substantial humoral antibody response. The peptide-vaccine of the present invention should prime T cells in a way that, when the infecting virus appears at a later date, memory T cells will be activated to result in a cell-mediated immune response that will destroy the virus. The activation of only T cells without an antibody response is important because it is believed that antibodies to most regions of the viral envelope protein may stimulate the infectivity of the virus. This latter point will render most viral surface envelope antigen preparations (e.g., intact gp120 and gp41 that contain both B- and. T-cell epitopes) ineffective as vaccines (see article by D. Barnes in Science, 236:255, 1987). This article reported that about 20 chimpanzees had been given various prototype vaccines (containing B- and T-cell epitopes) and some were challenged by injecting virus, but the results indicated that none of the vaccines prevented infection by infectious HIV). In contrast, this invention predicts that a suitable T cell response will produce cytotoxic T cells or other types of T-cell responses that will neutralize the virus in a newly infected individual.

It should be emphasized that an effective peptide may in some cases induce a low to moderate level antibody response and still be useful as an effective vaccine. In this case, the induced anti-peptide antibodies must be incapable of recognizing or detecting the mature protein from which the vaccinating peptide was derived. Thus, the anti-peptide antibody induced by the T cell active peptide must not be substantially capable of binding to the intact, infectious virus. It is well known that antipeptide antibodies to certain regions of a given protein may not recognize the native protein (for example, see the work of Ho et al., J. Virol., 61:2024, 1987).

The use of synthetic peptides that are T cell-active but that are not immunogenic for native virus (antipeptide antibodies that are unable to detect the virus particle) may have some advantages in that inherent immunological memory should be superior for peptide vaccines of the present invention.

The first step in preparation of the vaccine of the present invention is to prepare a number of peptides 10-30 amino acids in length and having an amino acid sequence derived from the two envelope proteins or their genes. Conserved protein sequence regions of each envelope protein will be selected for investigation. For example, a large portion of gp41 is conserved among the seven strains of HIV-sequenced to date (Modrow et al., J. Virol., 61:570, 1987).

Computer programs have been developed that are useful in predicting T cell recognition sites and antibody binding sites within antigens (the latter known as B cell sites). Several computer programs can be used such as the

De. Lisi and Berzofsky program for T cell sites (Proc.

Natl. Acad. Sci. USA, 82:7048, 1985), and for B cells- the Hopp and Woods program (J. Mol. Biol., 157:105, 1982) and the Sette et al., program (Mol. Immunol., 23:807, 1986).

Short synthetic peptides are made from predicted T cell regions.

Using the computer program of Sette et al., (1986) to analyze the linear sequence of the HIV envelope proteins, several proposed T cell epitopes were selected from a first conserved segment of gpl20 (Modrow et al., J. Virol., 61:570-578). Their sequences are as follows, with the amino terminus at the left and carboxy terminus on the right, in standard fashion:

(1) CSAVEQLWVTVY;

( 2 ) TTLFCASDAKAY;

( 3 ) EWLGNVTENFNM; (4) QMHEDIISLWDQS; and

(5) QSLKPCVKLTPLC.

These peptides are predicted T cell epitopes within a 100 amino acid stretch of conserved sequences near the amino terminus of the gp120 protein. A recent report indicated that this region is active in stimulating T cell immunity (Ahearne et al., III International Conference on AIDS, held in Washington, D.C., June 1-5, 1987, abstract # M.10.3, page 8).

Antigenic sites recognized by T cells have been reported to correlate with helical structures (either alpha helices or another type helix called a 3₁₀ helical structure). Such antigenic sites are also thought to be protein segments displaying a polar/apolar character, forming a stable amphipathic structure with separated hydrophobic and hydrophilic surfaces and/or protein segments displaying a marked change in hydrophilicity between the first-half and the second-half of a block of amino acids (differential amphipathic structures).

In practice, using computer programs, the helical structures are identified by a consistent stretch of blocks of amino acids (each block being 6-7 residues in length) with angles (termed delta values) of 100' ± 20' (alpha helix) or 120' ± 15' (3₁₀ helical structure). Differential amphipathic structures are identified by peaks of differential hydrophilicity (See Table 1). For the purpose of selecting regions that are predicted to be poor antibody eliciting and/or binding sites, these structures should have negative mean hydrophilicity values. All of these values are listed below in Table 1 as the computer analysis of a conserved gp120 sequence (residues 35-137).

Five peptides were selected from within residues 35 through 137 of the gp120 surface protein of HIV.

Peptide number (1) which spans blocks 1-5 (6 amino acids per block) has delta values (termed ANGLE) consistent with a helical structure as predicted by both the Hopp/Woods computer program (block length of 6 amino acids) and the Kyte/Doolittle computer program (block length of 7 amino acids).

Peptide number (2) which spans blocks 23-28 has a peak of differential hydrophilicity (a marked change in mean hydrophilicity between the first-half and second-half of a block of amino acids) that is predicted by both programs.

Peptide number (3) which spans blocks 56-63 has delta values consistent with a helical structure (Kyte/Doolittle) and a peak of hydrophilicity (both programs).

Peptide number (4) which spans blocks 76-83 has a peak of differential hydrophilicity (both programs).

Peptide number (5) which spans blocks 87-94 has delta values consistent with. helical structures (both programs).

All five of these peptides exhibit negative mean hydrophilicity values indicating that they are poor antibody binding sites.

Five other conserved regions of the two HIV envelope proteins can be similarly analyzed and putative T cell-active peptides selected. These regions include residues 204-279 (C2 or conserved region 2), 415-458 (C3), 470-510 (C4), 511-616 (C5) and 654-745 (C6) (Modrow et al., J.

Virology, 61:570,1987).

As an alternate approach to identify T cell active peptides, it may be necessary to thoroughly cover the protein sequence in question. In this case, overlapping 15-amino acid peptides (15 mers) can be made (the second peptide overlaps with the C-terminal 5 amino acids of the first peptide, the third overlaps the second, etc.) across the complete conserved amino acid sequence of both gp120 and gp41.

All of these peptides may be made, for example, by the solid phase Merrifield-type synthesis but may also be made by liquid phase synthesis or recombinant DNA-related methods known to those skilled in the relevant arts. A further description of the basic solid phase synthesis method, for example, can be found in the literature (i.e., M. Bodansky et al., Peptide Synthesis, John Wiley and Sons, Second Edition, 1976, as well as in other reference works known to those skilled in this type of chemistry. Appropriate protective groups usable in such synthesis and their abbreviations will be found in the above reference, as well as in J.F.W. McOmie, Protective Groups in Organic Chemistry, Plenum Press, New York, 1973).

In one type of synthesis, the N-terminal end of each peptide is linked to a dipalmityl-lysyl-glycyl-glycyl sequence to serve as a carrier as described by T.P. Hopp (Mol. Immunol., 21:13, 1984).

An example of this type of structure is shown below: = alpha amino group of lysine

= epsilon amino group of lysine

Alternately, peptides can be made without the use of the dipalmitate carrier and otherwise tested. In this case, peptides containing two natural cysteines as part of their natural sequence may be selected and synthesized. Peptides lacking such cysteines can be modified by the addition of extra cysteines to the N- and C-terminal ends, respectively. The presence of two cysteines per peptide allow polymerization of the subunit peptide by air oxidation to form cysteine-linked polymers and/or cyclic peptides. Such polymers should enhance immune recognition of the peptide without the need of a carrier.

An example of this type of structure is shown below:

Each peptide preparation will first be tested in mice, for example, to screen for appropriate T cell active peptides. T cell active peptides will be assayed by injecting the peptide into mice, and then testing T cells recovered from the murine lymph nodes one to three weeks after inoculation with the peptide. The measurement of activation or priming of T cells will be done by T cell proliferation tests and/or interleukin-2 production (Milich et al., J. Exp. Med., 164:532, 1986). Two types of T cell active peptides should be found. The more prevalent group of peptides will prime T cells that respond in test tube assays to only the peptide and not the corresponding native HIV surface protein. The second group of peptides will prime T cells to respond to both the peptide and the native HIV protein. It is this latter group of peptides that will induce protective immunity in the vaccinated host. Several strains of mice will be used which vary in their histocompatibility genes. Peptides that have a broad response in the various MHC genotypes will be selected for further study in primates, finally humans.

T cell active peptides will then be screened to identify those peptides that lack B cell stimulatory activity. This will be accomplished by injecting each peptide into small animals (various strains of mice) to identify those peptides that fail to generate an antibody response. These animals should not produce anti-peptide antibodies binding to native viral proteins. These same selected peptides will be tested in baboons and monitored to confirm the lack of anti-peptide antibody production in baboon sera. At this stage, mixtures of peptides will be employed because it is quite possible that one peptide sequence will not provide the broad spectrum coverage needed for an effective vaccine. Candidate peptide mixtures will then be incorporated into a vaccine. Candidate peptide mixtures will then be tested in a suitable animal that allows replication of the AIDS virus (Chimpanzees) to test for priming of T cells. Peptides that are more active will be used to vaccinate chimpanzees in a virus challenge experiment. A successful protection experiment will prevent viremia without eliciting a significant humoral antibody response but will prime T cells for in vitro responses to the envelope antigens. The virus will be neutralized by cell mediated immunity. The present invention involves the prediction that antibody responses to most if not all surface antigen epitopes will increase or at least not impede the infectivity of the AIDS virus.

As described above, it may not be necessary to select a peptide that completely lacks the capability to raise anti-peptide antibodies. In this situation, the anti- peptide antibody must not be capable of recognizing the native envelope proteins as measured, for example, either by immunoblotting procedures or by other immunoabsorbent (ELISA) tests. What is important in this particular response is that anti-peptide antibodies against a certain peptide sequence must not induce antibodies that bind to the infectious virus. Thus, in this case, T cell active peptides that raise low or moderate levels of anti-peptide antibodies will be screened to identify those that fail to detect either intact virus preparations or viral surface proteins by immunoabsorbent tests (ELISA) and/or immunoblot procedures.

An important issue in considering the effectiveness of this invention is whether the cell mediated immune system can function in a previously vaccinated individual when at a later time the vaccinee is exposed to HIV which is infecting and altering the function of T4 helper cells. The research findings of Buller et al. (Nature, 328: 77, 1987) provide evidence that is consistent with the hypothesis that a T cell active peptide can invoke a cell mediated response in the absence of T4 helper cells.

Their work demonstrates that cytotoxic T cell responses can be induced in mice in the absence of T helper cells; the end result was that mice being studied recovered from a viral disease without T helper cells.

Therapy for HIV-infected people is also an object of the present invention. Although the synthetic vaccine of the present invention will focus on peptides sequences predicted from one of the viral surface proteins in order to prevent virus infection of the exposed individual, this approach might also be used to treat individuals who are already infected with HIV. In this particular situation, it is important to consider that the target for cell-mediated immunity includes not only the virus but more importantly the virus-infected cell. Infected cells will have not only viral envelope proteins on their surfaces but possibly glycosylated core proteins (gag gene products) or their higher molecular weight precursors as well (Naso et al., J. Virol., 45:1200, 1983). Therefore, T cell active peptides from the gag gene of HIV can also be selected and tested for their affects on virus infected cells.

Computer analysis of the gag gene of HIV has revealed several T cell epitopes from within the core or gag gene of HIV (Coates et al., Nature, 326:549. 1987).

56 62

EGCRQIL 74 85 ELRSLYNTVAT 170 180 VIPMFSALSEG 199 206

AMQMLKET 298 305 YVDREYKT

333 342

KTILKALGPA 346 355 EMMTACQGV 367 375 AEAMSQVTN

Such synthetic peptides (either from the surface proteins or the core proteins) should be able to induce a cell-mediated response sufficient to destroy virus-infected cells bearing the expected epitopes, or as suggested by the work Walker et al., (Science, 234:1563-1566, 1986) inhibit the growth of the virus.

The T helper cell independent cytotoxic T cell response, described by Buller et al., bodes well for the use of T cell active peptides in the therapy of AIDS. Such a peptide or mixture of peptides would be expected to mount an effective cell mediated immune response at a time when T4 cells are being infected and killed by the HIV. Since T8 cells are resistant to HIV infection, the proposed peptide(s) (either polymerized or coupled to fatty acids as described in a previous section) should activate and prime T8 cytotoxic cells allowing a specific virus-killing response in the AIDS patient even though the virus may be infecting and altering the immune helper function of T4 cells.

Studies of Walker et al., (Nature, 328: 345, 1987) have demonstrated the presence HIV-specific cytotoxic T cells in persons infected with HIV. These cytotoxic T cells were able to kill HIV-antigen containing B lymphocytes derived from the same patient in laboratory tests. Their study showed that monoclonal antibody specific for cytotoxic T cells was able to inhibit the cell killing activity. These results support the vaccine approach described in this patent, and may have important implications for the use of T-cell active peptides in the treatment of AIDS patients.

* * * * *

Changes may be made in the construction, operation and arrangement of the various parts, elements, steps and procedures described herein without departing from the concept and scope of the invention as defined in the following claims.

Claims

CLAIMS :

1. A process for inducing resistance of an individual to infection by human immunodeficiency virus, the process involving the steps of vaccinating said individual with a synthetic peptide or mixture of peptides comprising a sequence of from about 10 to about 30 amino acids derived at least in part from human immunodeficiency virus envelope protein conserved regions and which, upon antigenic presentation to an animal, induces directed cell-mediated immunity (including a T-cell cytotoxicity response to AIDS virus) to a substantially greater extent than it elicits production of antibody directed against native human immunodeficiency virus.

2. A process for inducing resistance of an individual to infection by human immunodeficiency virus, the process involving the steps of treating said individual with a vaccine consisting essentially of a synthetic peptide, or mixture of peptides, having an amino acid sequence derived from T-cell epitopes of human immunodeficiency virus envelope protein conserved region.

3. A process for inducing resistance to human immunodeficiency virus, the process involving the steps of: identifying amino acid sequences of about 10 to about 30 amino acids from human immunodeficiency virus envelope protein conserved region able to form a helical structure and being further characterized by the presence of amphipathically interrelated amino acids; preparing peptides or peptide derivatives comprising at least substantial parts of the identified sequences;

administering said peptide, or mixture of peptides, or peptide derivatives to a test animal in a manner stimulating an immune response;

monitoring T-cell activity and humoral antibody response in said test animal to screen for a peptide which stimulates T-cell activity without inducing substantial production of antibody directed against native human immunodeficiency virus to identify peptides exclusively having T cell epitopes; and

treating an individual with an immunogenic composition comprising exclusively with T cell epitopes peptides to induce resistance to human immunodeficiency virus infection.

4. A process for inducing resistance to human immunodeficiency virus, the process involving the steps of:

preparing peptides substantially comprising the structure:

CSAVEQLWVTVY,

TTLFCASDAKAY,

EWLGNVTENFNM,

QMHEDIISLWDQS, or

QSLKPCVKLTPLC; screening for a peptide, or mixture of peptides, which, upon administration to an animal, stimulates T- cell activity without induction of substantial production of antibody directed against native human immunodeficiency virus; and

administering said screened peptide to a human to induce resistance to human immunodeficiency virus infection.

5. The process of claim 4 wherein the peptide is comprised in a human immunodeficiency virus envelope glycoprotein.

6. The process of claim 4 wherein the peptide is comprised in human immunodeficiency virus glycoprotein gp 120.

7. The process of claim 4 wherein the peptide is comprised in human immunodeficiency virus glycoprotein gp 41.

8. A process for suppressing infection by human immunodeficiency virus, the process involving the steps of:

preparing a peptide, or mixture of peptides, substantially comprising the sequence: EGCRQIL; ELRSLYNTVAT; VIPMFSALSEG; AMQMLKET; YVDREYKT;

KTILKALGPA; EMMTACQGV; or AEAMSQVTN;

screening for a peptide (or peptides) which, upon administration to an animal, stimulates T-cell activity without inducing substantial production of antibody directed against native human immunodeficiency virus; and

administering said screened peptide(s) to a human to induce resistance to human immunodeficiency virus infection.

9. The process of claim 8 wherein the peptide(s) is comprised in a human immunodeficiency virus core protein and T-cell cytotoxicity is directed toward cells infected with human immunodeficiency virus.

10. A vaccine for the prevention of infection by human immunodeficiency virus, the vaccine comprising a synthetic peptide(s) having an amino acid sequence derived from that of conserved regions of human immunodeficiency virus envelope protein, said vaccine inducing a T-cell mediated response against human immunodeficiency virus but not a substantial production of humoral antibody against human immunodeficiency virus.

11. The vaccine of claim 10 wherein the synthetic peptide(s) comprises a sequence of between about 6 and about 30 amino acids.

12. The vaccine of claim 10 wherein the synthetic peptide is prepared by solid-phase chemical synthesis, liquid- phase chemical synthesis or biological synthesis involving recombinant DNA.

13. A method for therapy of a human immunodeficiency virus-infected patient with AIDS, the method comprising treatment with a vaccine comprising a synthetic peptide(s) having an amino acid sequence derived from that of a human immunodeficiency virus protein, said synthetic peptide eliciting a T-cell response but not a substantial production of humoral antibody against native human immunodeficiency virus protein.

14. The method of claim 13 wherein the human immunodeficiency virus protein is a core protein.

15. The method of claim 13 wherein the human immunodeficiency virus protein is an envelope protein.