The present invention relates to a synthetic peptide antigen the sequence of which corresponds to a region of an immunologically important protein of HIV-2. This peptide is useful as a diagnostic reagent for detecting the presence of antibodies to HIV-2. The peptide may also be useful as an immunogen in compositions to elicit the production of antibodies against HIV-2 in animals including man.
The acquired immunodeficiency syndrome (AIDS) is a major worldwide health problem. The etiologic agent of AIDS has been identified as HIV (for human immunodeficiency virus) which is the name given to a group of highly related viruses, formerly called HTLV-III, LAV and ARC (now collectively HIV-1).
HIV isolates from individuals with AIDS and ARC (AIDS-related complex) from North America, Western Europe and Central Africa generally have the same biological properties and antigenically cross-reacting proteins. Genetic studies, however, have characterized differences in nucleotide sequence of the genomes of North American and African HIV isolates (Benn et al., Science (1985) 230:949-951). In addition, small differences in nucleotide sequences in different HIV isolates from the USA have also been documented.
Several other viruses that are genetically and structurally related to HIV have been recently isolated. These viruses which genetically and immunologically resemble HIV have been isolated from captive rhesus macaques (Macaca mulatta) ill with an AIDS-like disease and healthy wild-caught African green monkeys (Cercocithecus sp.) (Kanki et al., Science (1985) 230:951-954). The viruses which were previously designated STLV-III.sub.AGM (Africa green monkey isolate) and STLV-III.sub.MAC (macaque isolate) are now known as SIV (for simian immunodeficiency virus).
A new human virus, designated HTLV-IV, has been isolated from apparently healthy individuals in West Africa (Kanki et al., Science (1986) 232:238-243. This virus, which produces retroviral type particles in infected cells, has growth characteristics and major viral proteins that are similar to those of HTLV-III/LAV and STLV-III. Serologic data indicated that HTLV-IV is more related to STLV-III.sub.AGM than to the prototype HTLV-III/LAV isolates from patients in Europe and the United States.
Recently, cases of AIDS were identified in West African patients. Although the individuals had classic symptoms of AIDS, no detectable titers of antibodies to known HIV antigens could be detected in patient sera. However, a retrovirus originally termed LAV-2 which is structurally and biolgically related to HIV was isolated from the West African patients (Clavel et al., Science (1986) 233: 343-346. The West African virus, which has now been isolated from a number of individuals with AIDS, ARC and no symptoms, is now known as HIV-2 to distinguish it from HIV (now HIV-1) isolates previously identified as the etiologic agent of AIDS in Europe, North America and Central Africa (Clavel et al., N. Eng. of Med. (1987) 316:1180-1185; Guyader et al. Nature (1987) 326:662-669).
Like HTLV-IV, HIV-2 is more related to SIV than to HIV-1. However, HIV-2 and HTLV-IV are not the same virus since HIV-2 kills human helper T cells infected in vitro whereas HTLV-IV does not.
The complete nucleotide sequence of HIV-2 which has recently been reported (Guyader et al., supra) indicates a genetic sequence homology with HIV-1 of only 42%. Significant differences occur in most viral protein but are most pronounced in the glycoproteins encoded by the HIV-1 and HIV-2 env genes. In fact, the HIV-2 envelope glycoproteins appear to be more closely related to those of SIV than to HIV-1.
The issue of serologic non cross-reactivity between HIV-1 and HIV-2 is of major importance in developing diagnostic tests for detection of and vaccines against HIV-2 infection. Studies have shown that the patients with HIV-2 infections were not identified by serologic tests which detect HIV-1. Any cross reactions which have been seen for the two viruses generally are mediated by antibodies which react with common epitopes on the major core proteins, p25 and p26, encoded by the gag gene of the two viruses. Antibodies to the viral envelope glycoproteins gp120 and gp41 and their precursor gp160 of HIV-1 do not cross-react with the envelope glycoproteins of HIV-2 (Clavel et al., Science (1986) 233:343-346; Clavel et al., N. Engl. J. Med. (1987) 316:1180-1185). Currently available tests for detection of HIV-1, which are mainly based on detection of antibodies to the HIV-1 glycoproteins, e.g., gp160/gp120/gp41, and portions thereof, cannot be used to detect antibodies to HIV-2 in samples for diagnostic and screening purposes. Thus specific HIV-2 antigens should be included with HIV-1 antigens in reagents for effective diagnostic and therapeutic use.
Methods being developed for detecting HIV-2 infection, in general, will measure exposure to the virus by detecting and quantifying antibodies to HIV-2 antigens in blood, sera, and blood-derived products. Such assays can be used to aid diagnosis of AIDS and ARC (AIDS-Related Complex) and to screen blood and blood products for previous exposure to HIV-2.
The current attempts to diagnose HIV-2 infections and screen blood for exposure to HIV-2 include enzyme-linked immunosorbent assay (ELISA) techniques to detect the presence of antibodies to immunogenic components of HIV-2 in a test sample. Other methods may utilize Western blotting techniques to detect HIV-2 specific antibodies in test samples. In general, almost any known immunoassay, such as radio-immunoassays, can be adapted, by use of specific reagents, for the detection of HIV-2 and antibodies thereto.
The source of antigens for these assays may include inter alia HIV-2 proteins obtained from HIV-2 infected T cell lines and antigens produced by recombinant DNA techniques. The use of antigens obtained from these sources, however, has significant drawbacks.
The production of HIV-2 per se in continuous cell lines must be performed in high risk (P3 containment) laboratories due to the danger to investigators who may become adversely exposed to the virus. In addition, since there have been false negative and false positive results reported with ELISA tests using whole virus HIV-1 antigens obtained from cell lines; it is likely that similarly unreliable results will be obtained with cell-derived HIV-2 antigens. Western blot analyses, for HIV-2 detection using electroblotted whole virus antigens, may provide greater specificity but are more laborious and time-consuming then ELISA tests. Furthermore, since HIV-2 producing cells are of human origin, viral antigen preparation obtained from these cell lines, unless exhaustively purified, may be contaminated with normal cellular antigens, such as HLA antigens, which could produce false positive reactions in an ELISA test.
Exhaustive purification of viral antigens from cell lines can also conceivably destroy immunogenicity of immunologically important proteins or otherwise inactivate antigens, thereby producing reagents that result in false negative reactions. In addition, false negative reactions using live-virus-derived antigens may occur because of steric hinderance whereby antibodies cannot react with their specific antigens because the reaction is blocked by the presence of other antigens and antibodies in the reaction mixture.
ELISA tests to detect HIV-2 infection may also employ immunologically important viral proteins produced by cloning portions of the HIV-2 genome in bacteria. The complete nucleotide sequence of HIV-2 has now been reported (Guyander et al., Nature (1987) 326: 662-669) with the genes coding for various HIV-2 proteins identified by comparison to homologous HIV-1 genes. The viral envelope glycoproteins and core proteins respectively encoded by the env and gag genes of HIV-2, are apparently the antigens recognized by antibodies in the sera of patients with HIV-2 infection.
Immunologically important HIV-2 antigens such as gp160 and its cleavage products gp120 and gp41, which are present in the viral envelope, may be prepared by cloning portions of the HIV-2 genome in various expression systems such as bacteria, yeast or vaccinia. Such recombinant antigens may be used in diagnosis and as potential vaccine compositions as has been done for HIV-1 proteins (See, e.g., Cabradilla et al., Biotechnology (1986)4: 128-133; Chang et al., Biotechnology (1985) 3: 905-909; Putney et al., Science (1986) 234: 1392-1395; Kieny et al. Biotechnology (1986) 4: 790-795). HIV-2 antigens produced by recombinant DNA methods, however, will still have to be exhaustively purified to avoid false positive reactions in the ELISA due to any antibody reactivity to antigens of the expression system which may contaminate the HIV-2 antigen preparation. Also, denaturation of HIV-2 antigens during purification may destroy important antigen activity.
While HIV-2 antigens produced by recombinant techniques may be an improvement over antigens obtained from virus-infected cell cultures, the recombinant proteins still may not provide reagents that give as accurate a diagnosis as possible. Because of the nature of the disease and the need for accurate results, other reagents must be developed to approach 100% accuracy in diagnosis of HIV-2.
Protein antigens contain a number of epitopes or antigenic determinants which are the regions of the proteins which comprise the binding sites for specific antibodies. In general, protein antigens contain between 5 to 10 epitopes, each of which contains a sequence of 6 to 8 amino acids. Epitopes can be either continuous, in which the 6 to 8 amino acids are present in linear sequence, or discontinuous, in which the amino acids that form the epitope are brought together by the three dimensional folding of the protein. Even though an epitope constitutes only a relatively few amino acids, its reactivity with an antibody is influenced by the amino acids in the protein which surround the epitope.
Studies aimed at mapping antigenic sites or epitopes of proteins have been aided by the use of synthetic peptides corresponding to various regions of the proteins of interest (See e.g., Lerner et al., in The Biology of Immunological Disease: A Hospital Practice Book, (1983) Dixon and Fisher, eds., pp. 331-338; Lerner, Adv. Immunol. (1984) 36: 1). In addition to their usefulness in epitope mapping studies, synthetic peptides, if encompassing major antigenic determinants of a protein, have potential as immunogenic compositions, including vaccines, and diagnostic reagents. Synthetic peptide antigens have several advantages in regard to specific antibody production and reactivity. The exact sequence of the synthesized peptide can be selected from the amino acid sequence as actually determined by amino acid sequencing of a protein or predicted from the DNA sequence coding for the protein. The use of specific synthetic peptides eliminate the need for using the full-length protein in the production of or assay for specific antibodies. Furthermore, the solid phase peptide synthetic techniques of Merrifield and coworkers allow for essentially unlimited quantities of the synthesized peptide of interest to be chemically produced. (See, e g., Erickson and Merrifield in The Proteins, 3rd Edit. (1976), Vol. 2, Academic Press, New York, Chapter 3). The availability of automated peptide synthesizers has further advanced such techniques.
Synthetic peptide antigens corresponding to regions of immunologically important proteins of HIV-2 would find immediate use in diagnostic methods and as potential vaccines for HIV-2.