Synthetic vaccine against aids virus.

Description: TECHNICAL FIELD [Detailed description of the invention]

The present invention relates to the general development of vaccines against the human immunodeficiency virus (HIV). More specifically, the present invention stimulates helper T lymphocytes that specifically recognize HIV membrane proteins, thereby enhancing antibody production, and cells that suppress the onset of AIDS virus and infections caused by AIDS virus. It relates to providing toxic T cells. Background of the invention The development of vaccines against the human immunodeficiency virus (HIV) is a decisive way to prevent the further spread of the widespread immunodeficiency syndrome (AIDS). For safety reasons, not all viral vaccines can be applied for the case of HIV. However, any subunit vaccine should contain immunodominant helper T cell sites that can elicit helper T cell immunity in response to exposure to the progenitor antigen. Helper T cells are required for both B cell proliferation and activation of B cells by differentiating them against antibody-producing cells, as well as induction of cytotoxic T cells. It should be noted that helper T cells recognize unique sites within the protein molecule rather than the artificial protein antigens. Of the sites, a few usually elicit a mass of responses and are therefore referred to as "immune-dominant." Thus, in order to elicit an effective cytotoxic or immune response, the synthetic antigen should preferably contain an immunodominant region within the molecule. In addition, synthetic antigenic peptides should overcome the problems of MHC restriction often encountered in synthetic fragment vaccines. Abstract of the invention Therefore, an object of the present invention is a synthetic peptide antigen consisting of immunodominant sites that are recognized by all or most MHC-type T cells and at least in part elicit a high degree of helper T cell responsiveness to HIV infection. Is to provide. A further object of the present invention is to provide a synthetic or recombinant fragment vaccine against HIV infection that is substantially unconstrained due to restrictions caused by different major histocompatibility forms. Another object of the present invention is to provide reagents for determining the specificity and / or level of T cell immunity to AIDS virus. Other objects and advantages of the present invention will become apparent from the following detailed description of the present invention. A brief description of the drawing The above and other objects, features and many notable advantages of the present invention will be better understood by reading the detailed description below when considered with reference to the accompanying drawings. Figure 1 shows the amino acid residues of HIV membrane proteins. The amino acid linkages of 44 overlapping synthetic peptides are represented. In HP-6 (R), arginine is replaced by carboxy-terminated lysine. Peptides HP-17 and 33 have an external carboxy-terminal cysteine residue that is absent in the HIV chain. Figure 2 shows T₂(HP-5) Shows the amino acid residues of overlapping peptides in the cluster. Abbreviated amino acid codes are used: A = Ala, C = Cys, D = Asp, E = Glu, F = Phe, G = Gly, H = His, I = Ile, K = Lys, L = Leu, M = Met, N = Asn, P = Pro, Q = Gln, R = Arg, S = Ser, T = Thr, V = Val, W = Trp, Y = Tyr. FIG. 3 shows the amino acid residues of the overlapping peptides in the immunodominant region HP-52-57. Figure 4 shows B10.BR (H-2)^k) The dose response curve for HP-52 to 57 in mice is shown. B10.BR mice were immunized with gp160, and lymphocyte nodule proliferation analysis was performed as described in the text. Responses to peptides HP-52-57 were assessed by dose response testing. The stimulus index indicates an experimental count ratio that exceeds the antigen-free background count. Figure 5 shows B10.D2 (H-2)^d) The dose response curve for HP-52 to 57 in mice is shown. B10.D2 mice were immunized with gp160, and lymphocyte nodule proliferation analysis was performed as described in the text. Responses to peptides HP-52-57 were assessed by dose response testing. The stimulus index indicates an experimental count ratio that exceeds the antigen-free background count. Figure 6 shows B10.S (9R) (H-2)^t4) The dose response curve for HP-52 to 57 in mice is shown. B10.S (9R) mice were immunized with gp160, and lymphocyte nodule proliferation analysis was performed as described in the text. Responses to peptides HP-52-57 were assessed by dose response testing. The stimulus index indicates an experimental count ratio that exceeds the antigen-free background count. Figure 7 shows B10.A (5R) (H-2)ⁱ⁵) The dose response curve for HP-52 to 57 in mice is shown. B10.A (5R) mice were immunized with gp160, and lymphocyte nodule proliferation analysis was performed as described in the text. Responses to peptides HP-52-57 were assessed by dose response testing. The stimulus index indicates an experimental count ratio that exceeds the antigen-free background count. FIG. 8 represents a bar graph showing a large number of peptides using a response by geometric means lying within the indicated range. The lower limit of the range is shown on the X-axis (eg, "1.5" indicates the range 1.5-2.0). Figures 9A and 9B show three peptides HP-5, HP-26 and HP- in an emulsion of Resus monkeys 1-4 (= RhM1 to RhM4) 1) Group 1: Complete Freund Aid (CFA). 39 nanomoles of each of 53; 2) Group 2: CFA is used to show the results of treatment on days 0 and 39. These monkeys were immunized on day 47 with 0.2 nanomoles of gp160 in Independent Financial Adviser (IFA). Anti-gp41 antibody was measured on days 0,27,52,55,59,62,68 and 80 days. Results are expressed as'net cpms': 125-I-protein A bound to antibody on the gp41 band of Western Blot is drawn from the background (pre-bleeding serum: 642 ± 187 cpm). Brief description of the invention The above and various other purposes and advantages of the present invention are achieved by peptide antigens that stimulate the proliferation of T cells capable of recognizing cells exhibiting HIV membrane proteins without limitation across a number of different MHC types. Will be done. Unless otherwise stated, all technical and scientific expressions used herein have the same meaning as commonly understood by one of ordinary skill in the art in the art to which the present invention belongs. Any method or material that is the same as or equivalent to the methods and materials described herein can be used in the examples or tests of the present invention, but preferred methods and materials are described below. All references in the text below have been added to the text for reference. Unless otherwise stated, the techniques used herein are standard methodologies known to those of skill in the art in the art. The expression "many MHCs or types" used in the text means at least three or more MHCs or types. The expression "substantially pure" as used in the text means as pure as possible obtained by standard purification techniques. The expression "synthetic" peptide used in the text means that it has been chemically synthesized by some suitable means such as genetic recombination technology. Materials and methods mouse B10.BR / SgSn (H-2)^k) And B10.D2 / nSn (H-2)^d) Mice were obtained from Jackson Laboratories (Bar Harbor, ME). B10.S (9R) / Sg (H-2)^t4) And B10.A (5R) /SgSn (H-2)ⁱ⁵) Mice were bred in NIH breeding colonies from DJ Stimpfling [Great Falls, MT] and from breeding pairs obtained from the Jackson laboratory. Mice from B10.S (9R) and B10.A (5R) strains are H-2^sAnd H-2^{b b}Both haplotyped IA and IE molecules are expressed in the recombinant strain, whereas IE is non-recombinant H-2.^{b b}And H-2^sI chose it because it is not expressed in mice. antigen Recombinant gp160 was prepared from cells infected with recombinant bacrovirus that expresses a gene for gp160 in HTLVIIIB isolates of HIV as described in Rusche et al., 1987, PNAS, United States, 84: 6924. did. It was specifically purified by standard lentil lectin chromatography and gel filtration techniques. Synthetic peptides were made by multiple peptide synthesis or the "Tea Bag method" as described in Houghten et al., 1986, Bio Techniques, 4: 522. Peptides were degraded from their resins by the "low-high" hydrogen fluoride method. Degrading peptides were desalted by Sep-pak [Waters, Milford, MA] and then their purity and concentration were determined by analytical HPLC. If the peptides were not pure enough, they were repurified using standard production HPLC. Lymph node T cell purification analysis Mice were subcutaneously immunized with 10-30 ug of recombinant gp160 1: 1 against complete Freund's aid (CFA) relative to the tail. (This dose was determined based on the purity of the gp160 preparation.) After 8-10 days, the periaortic and inguinal lymph nodules were found in Berkower et al., 1984, J. Immunol., 132: 1370. Was removed and prepared in a single cell suspension in a complete T cell medium according to the method of. 4x10 per hole⁵Lymphatic nodule cells of cells were added to each hole in a flat-bottomed 96-well microtitration plate [Costar, Cambridge, MA]. Forty-four peptides were added to the holes with a solvent to give a final volume of 0.2 ml. On the 40th day of culture, [³H] Thymidine luCi [6.7 Ci / mmol; New England Nuclear, Boston, MA] was added to each hole. After 18 hours, as a measure of proliferation, for DNA [³The H] thymidine compounding ratio was determined using a liquid scintillation count. The stimulus index represents the ratio of experimental counts to background counts without antigen. Example H-2^k, H-2^d, H-2ⁱ⁵, And H-2^t4Haplotype mice were subcutaneously immunized to the tail with recombinant gp160 in CFA at a dose of 20-30 ug per mouse. After 8-10 days, the mice were sacrificed, peri-aortic and inguinal lymph nodules were isolated, and then proliferation of lymph nodule cells in the presence or absence of antigen is described herein. It was decided like this. Table I summarizes the results of T cell proliferation analysis of the four haplotype mice tested. Strong proliferative response, pre-identified membrane T₂H-2 for peptides HP-4 to 8 composed surrounding the site (HP-5) (Fig. 2)^kSeen in mice. The best response was seen with the peptides HP-5 (residues 112-124, HEDIISLWDQSLK) and HP-6 (residues 112-124, HEDIISLWDQSLR), which differ only in the substitution of arginine for lysine. .. A tapering response was seen for the peptides HP-4 (residues 110-124, QMHEDIISLWDQSLK), HP-7 (residues 114-128, DIISLWDQSLKPCVK), and HP-8 (residues 119-134, WDQSLKPCVKLTPLCV). A positive proliferative response was observed for peptide HP-26 (residue 428-443, KQIINMWQEVGKAMYA), which is identical to helper T cell outer membrane, membrane T. This response was seen at a dose of 1uM. At a dose of 2uM, the growth index was greater than 2, but not statistically sufficient. Peptide HP-29 (residues 437-451, VGKAMYAPPISGQIR), which has six amino acids overlapping with peptide HP-26, also elicited a positive response. In the four haplotype mice tested, a positive proliferative response was observed with one or more of the peptides in the group overlapping peptides HP-52-57 (Fig. 3) at the COOH group terminal of the gp41 molecule. It was. H-2^kThe administration response curve to the peptide in mice (FIG. 4) shows the preferred response to peptides HP-52 (residues 828-842, AVAEGT DRVIEVVQG) and HP-53 (residues 834-848, DRVIEVVQGAYRAIR). H-2^dThe T cell proliferation response to 44 peptides in haplotype mice is shown in column 2 of Table I. Membrane T₂A sufficient proliferative response to the peptide HP-3 (residues 109-123, EQMHEDIISLWDQSL) in the region was observed. Peptide HP-3 is HP-5 (membrane T)₂), But it is longer by three amino acids in the amino-terminal direction of the molecule and shorter by one amino acid in the carboxy-terminal direction of the molecule. A positive response was seen for HP-26 and for overlapping HP-29. In the initial screening, a positive response was observed only for HP-55 (residues 841-855, QGAYRAIR HIPRRIR) in groups HP-52 to 57. A more detailed study using dose response tests showed a positive and favorable response to HP-55 and HP-56 (residues 846-860, AIRHIPRRIRQGLER) (Fig. 5). H-2^t4Haplotype mouse (A^SEβ^SEα^k) (Table I) is the membrane T₂No response to peptides HP-3-8 in the region. Mice of this strain have membrane T₁They responded to the cytopeptide HP-26, as well as the overlapping peptides HP-28 (residues 432-446, NMWQEVGKAMYAPPI) and HP-29. In early screening, a positive response was seen for peptides HP-55 and HP-56 at a dose of 1uM. Dosage response testing confirmed the view with a favorable response to HP-55 and HP-56 (Fig. 6).<img file="JP2676166B2_D0001.tif" /><img file="JP2676166B2_D0002.tif" />Stimulation index (SI) using p-value (Example cpm / Control cpm) For each peptide in four strains of mouse (student's t-test examples compared to controls) is shown. Peptides were tested at a concentration of 2 μM unless otherwise noted. Stimulation indices with a p-value greater than 2, which is statistically sufficient, are shown in thick type.^aPeptide concentration 1.3 μM^{b b}Peptide concentration 1 μM^cPeptide concentration 0.3 μM^dgp160 concentration 0.1 μM^egp160 concentration 0.02 μM^fPPD concentration 25 μg / ml<img file="JP2676166B2_D0003.tif" /> The 10 peptides with the strongest response within each strain were identified. The peptides are said to strongly stimulate the strain for the purposes of this table (25% maximal stimulating peptides). For each of the 41 peptides tested, we are in the absence of a strain (0) or 25% maximal stimulating peptide for all 1, 2, 3, or 4 strains. I decided whether or not. Numerous peptides in each category have been shown to strongly stimulate a large number of given strains. H-2ⁱ⁵(A^{b b}Eβ^{b b}Eα^k) Haplotype mouse is H-2^dMembrane T rather than HP-5, as haplotype mice do₂Responds to peptide HP-3 in the region. In early screening, all peptides in groups HP-52-57 elicit a positive response. However, further studies using dose response tests (Fig. 7) showed maximal responses in peptides HP-53 and HP-55. T₁Or T₂Peptides from sites and regions other than HP-52-57 groups were found to be positive for some of the strains tested. Positive responses were for the peptides HP-30 (residues 483-497, RDNWRSELYKYKVVK), HP-35 (residues 560-581, NNLLRAIEAQQHLLQLTVWGIK), HP- for 3 of the 4 strains tested. 47 (residues 787-801, RIVELLGRRGWEALK), HP-50 (residues 801-815, KYWWNLLQYWSQELK), and HP-51 (residues 806-820, LLQYWSQELKNSAVS) were found. Peptides HP-29 and HP-30 elicit a positive response for all strains. Some of the immunized peptides mentioned above overlap. Peptides HP-30 and HP-33 overlap at 5 residues. H-2ⁱ⁵Haplotype mice did not respond to HP-30, but to peptide HP-33, which is as long as nine residues in the carboxy-terminal direction of the molecule. Similarly, peptide HP-26 (membrane T)₁The response to the site) is H-2ⁱ⁵Not seen in mice, but the response was seen for the overlapping peptide HP-29, which is as long as eight residues in the carboxy-terminal direction of the molecule. The most prominent response to overlapping peptides for all strains is the response seen for peptides in groups HP-52-57. The above results reflecting the T cell proliferation response to overlapping peptides of the gp160 membrane protein in four MHC haplotype mice immunized with the gp160 molecule show that the membrane T₁And T₂Confirm that the site represents an immunized area. H-2^k, H-2^d, And H-2^t4Haplotype mice have membrane T₁Respond to the site. H-2ⁱ⁵Mice respond to the overlapping peptide HP-29. Membrane T₂Site, peptide HP-5, H-2^kRecognized by haplotype mice, while H-2^dAnd H-2ⁱ⁵Mice recognize the overlapping peptide HP-3. Therefore, five peptides (HP-30,35,47,50, and 51) were recognized and identified as T cell membranes by three of the four strains tested, and two peptides. (HP-29 and 33) were recognized by all strains tested. Peptides 47, 50, and 51 form a group of overlapping outer membranes. In addition, a group of peptides (HP-52-57) were identified as helper T cell outer membranes in the COOH group region of the gp41 molecule, which each mouse strain responds to. This feature was found in the gp41 molecule, which is a relatively conserved region compared to the many highly variable regions of the gp120 molecule, and is particularly sufficient in the production of vaccines. The four haplotype mice tested respond to different peptides in this group. H-2^dHaplotype mice responded favorably to the peptides HP-55 and HP-56. This result suggests that the T cell outer membrane of the linkage may be the amino acid linkage AIRHIPRRI (representing overlapping amino acids of HP-55 and HP-56) (Fig. 4). Conversely, two endemic sites can be present in two overlapping peptides. H-2^t4Haplotype mice showed similar responses; again, the peptides HP-55 and HP-56 were favorably recognized. Therefore, H-2^t4Recognition sites for haplotype T cells may map the same nine amino acid linkages. In contrast, H-2^kHaplotype mice first show a proliferative response to the peptides HP-52 and HP-53, and then map this site to nine amino acids (DRVIEVVQG) shaped by the two peptides. H-2ⁱ⁵Haplotype mice respond best to the peptides HP-53 and HP-55. The duplication of the peptides suggests that the T cell outer membrane is the eight amino acid linkages QGAYRAIR, but again, the two peptides can, on the contrary, represent two unique sites. The discovery of four overlapping adventitial groups (peptides 3-8, 26-29, 47-51, and 52-56) that make up the site of immunization against the AIDS virus is the development of vaccines against the AIDS virus. Has important implications for. Although a single peptide is not immunized in all mouse strains tested, the extension of the peptide chain by some amino acids is from more haplotyped mice, as in the case of HP-5, 26, and 30. To elicit the response of. Therefore, the extended peptides are more suitable for intertribal marriage populations such as humans. The discovery of the group of peptides HP-52-57 located in the relatively observed region of the AIDS membrane is particularly satisfactory for human inoculum vaccines. Although this group of peptides resides on the intracellular portion of gp41, unlike antibodies, T cells do not require the protoprotein to be donated or present on the target cell, so T cell recognition is not compromised. Would; they can recognize processed fragments bound to MHC molecules. Obviously, an important feature of the invention is the discovery of the outer membrane group recognized by most MHC haplotypes, and the peptide outer membrane extended by a few residues is of all haplotypes tested. It is an observation that each produces a single peptide recognized by each. This early finding provides a means to overcome the difficult problems of MHC restriction and Ir genes in peptide or fragment vaccines aimed at eliciting T cell immunity, especially with respect to HIV infection. To assess whether the peptides were also immunized in primates and whether they could provide helper T cells to help the second antibody in vitro in response to the proto-HIV membrane protein. Lisa's monkeys were immunized with three representative species of the group, in other words peptides HP-5, HP-26, and HP-53. As shown in FIGS. 9A and 9B, two monkeys in Group 1 (RhM1, RhM2; these were immunized with a mixture of the above three peptides prior to immunization with the membrane protein gp160. T) produced highly enhanced antibodies in response to membrane proteins (gp41 portion) compared to two group 2 monkeys (RhM3, RhM4; these did not receive the peptide prior to total gp160). To do. Thus, the peptide can be used to immunize primates against the first helper T cells to induce highly enhanced antibodies that respond to the HIV virus membrane. In addition, Group 1 monkeys showed a good peripheral blood mononuclear cell proliferation response to both HP-26 and HP-53. This result indicates that the peptides can induce T cell help as well as proliferation in vitro, and they can be used to immunize primates as well as mice. It would be appropriate to state here that live viral vaccines for AIDS and all-killed or subunit viral vaccines pose a potential safety risk. In contrast, synthetic peptides such as those described in the text are inherently safe. In addition, molecules that correspond to all viral proteins, but made by recombinant DNA technology, contain, in addition to the protective outer membrane, a structure that potentially elicits suppression of immune responsiveness and a structure that elicits antibodies, rather than being protective. Rather, it enhances viral uptake and can therefore be toxic. From this, vaccines containing only selected peptides that elicit suitable immune types and have no other toxic effects should, of course, be more desirable for difficult viruses such as HIV. The present invention provides such a vaccine. Our study by Case et al., 1987, Proc. Natl. Adad. Sci. 84: 4249-4253, defines two synthetic peptides that stimulate helper T cell immunity to the AIDS virus. However, the peptide is not found in almost all individual viruses of histocompatibility type. Additional peptides will be needed to cover the entire population. The present invention in the early stages provides such additional peptides, including those recognized by T cells from all MHC types of tested mice and several types of monkeys tested. For diagnostic or prognostic use, antibodies are the only commonly measured immune response. However, there is an interval in the range of weeks to months after infection, as infected individuals will not produce antibodies and will therefore be tested as negative. During this period, the individual infects others or feeds infected blood. Helper T cells are believed to express faster in the immune response than antibodies, and therefore tests to detect helper T cells detect infection faster than commonly used antibody-based tests. Can be done. Only a limited amount of purified protein can be obtained from the virus, and recombinant molecules are usually contaminated with other substances from vectors that can give false positive results. In addition to this, many of the substances have proven to be mitotic, and may therefore give false positive results. In contrast, synthetic peptides such as those described in the text are highly purified and not mitotic. Furthermore, by using the different peptides described in the text, different sets of T cells that can vary with disease progression can be detected, and this is not characterized by the use of whole protein antibodies. Let's go. Therefore, the synthetic peptides of the invention are quite effective in determining disease progression. This is readily accomplished by performing standard immunoassays, such as peripheral blood proliferative blast conversion analysis known to those skilled in the art. In summary, the invention provides a set of synthetic peptides that have been shown by helper T cells to be multiple recognition sites within AIDS viral membrane proteins in four different histocompatibility types. The peptide is a residue of the AIDS virus membrane linkage (BH10 isolate) described in Nature 313: 277, 1985 by Ratner et al., Along with the specific amino acid residues described herein. Corresponds to 860 (and overlapping peptides therein), 109-124, 432-446, 437-451, 483-497, 492-506, 560-581, 787-801, and 806-820NI. The present invention overlaps or overlaps the same site on all recombinant or protopeptides selected to contain the site and, for example, other denatured isolates of the AIDS virus. All synthetic peptides, including variants of; their use for immunization in any carrier, aid, drug pathway, or in combination with other substances that elicit T cell immunity; and T cell immunity against the AIDS virus. Includes diagnostic or prognostic reagents for assessing the nature and extent of sex. Of course, the peptide can be used alone or as a combination with more than one peptide of the present invention. Methods for inducing immune responsiveness provide an effective amount of the antigen of the invention to induce the growth of helper T cell immunity to HIV infection to a host who can tolerate infection with HIV membrane protein. It consists of things. Tests to determine exposure to HIV membrane proteins are performed by culturing peripheral blood mononuclear cells from a blood sample with the peptides of the invention and then proliferating, blast conversion or phosphokine production using conventional immunological techniques such as. It consists of determining by the above technique (a positive reaction indicates that the solid has been exposed to the HIV membrane protein). The diagnostic or prognostic kit for AIDS of the invention consists of a container containing the antigenic peptide of the invention, either alone or in admixture, and a commanding material for testing. The examples and embodiments described herein are for illustration purposes only, and various improvements and changes within that scope will be suggested to those skilled in the art and the ideas of the present application. And it is understood that it is included within the scope of the claims and the scope of the attached claims.