Immune thrombocytopenic purpura (ITP) has recently been described as an AIDS-related condition in homosexual men. The goal of our project is to define the pathogenesis of AIDS-related immune platelet destruction in relation to viral infection in this group of patients. We have previously identified a target platelet antigen recognized by serum antibody from homosexual men with ITP. This antigen appears to be carried by Herpes simplex virus. We propose to investigate the pathogenesis of antibody formation against this target antigen in three steps. In the first step, we will isolate the target protein in large quantities by means of a gel electroelution technique. The purified protein will be analyzed for peptide sequence, and the antigenic portion will be determined. In the second step, a mouse monoclonal antibody directed against this target protein will be produced. This antibody will be used to localize the site of the antigen on the platelet membrane, and to investigate its presence in Herpes simplex virus and in virally-infected and uninfected cells. In addition, the role of the antibody in macrophage-mediated platelet destruction will be evaluated by in vitro techniques. We will also attempt to construct an animal model of AIDS-related ITP by examining the relationship between immune thrombocytopenia and herpesvirus infection in macaque monkeys that have been infected with simian AIDS virus. In the third step, the cDNA for the target platelet antigen will be cloned in order to characterize the expression and function of the antigen. A green monkey kidney (Vero) cell cDNA library in gamma gtll will be screened using monoclonal anti-GP25 antibody. In addition, the amino acid sequence of GP25 peptide fragments will be used to construct oligonucleotide probes. These probes will be used to screen the Vero cell library for GP25 cDNA clones. Confirmation of cDNA clones will be done by hybrid-selected in vitro translation. Sequencing of the cDNA for GP25 will be performed by restriction mapping and insertion into the bacteriophage M13 sequencing vector. In addition, the chromosome on which the gene coding for GP25 resides will be identified by hybridizing a labeled cDNA probe for GP25 with individual chromosomes separated by a cell sorter. Expression of the target protein in herpes virus-infected cell systems will also be investigated at the molecular level. By these techniques, we hope to gain an understanding of autoimmune mechanisms in AIDS and AIDS- related conditions, so that a rational approach to the treatment of AIDS-related ITP can be achieved. We also hope to define the role of virus-induced antigen expression and antibody formation in autoimmune platelet destruction.
| Stricker, R B (1991) Hemostatic abnormalities in HIV disease. Hematol Oncol Clin North Am 5:249-65 |
| McHugh, T M; Stites, D P; Busch, M P et al. (1988) Relation of circulating levels of human immunodeficiency virus (HIV) antigen, antibody to p24, and HIV-containing immune complexes in HIV-infected patients. J Infect Dis 158:1088-91 |
