The long-term objectives of the research plan are to identify antigenic determinants that may be used in the development of a contraceptive vaccine. Immunologic infertility has been described clinically in some cases of failed conception. While no specific epitope(s) has been isolated as the causative agent most studies implicate circulating antibodies to spermatozoa. Sera containing such antibodies will be useful probes of a human testes cDNA library packaged in a lambdagtll expression vector. Preliminary results have been obtained with sera that have a biological effect on spermatozoa. These data support the proposal that recombinant DNA techniques, cloning, and expression will permit identification of antisperm epitopes on naturally occurring antigens responsible for immunologic infertility. Techniques of molecular biology and protein chemistry will be used to (1) screen a human testis cDNA library and subtraction library with sera from patients with known or suspected immunologic infertility, (2) subclone and sequence the cDNA inserts and analyze for homologies with known amino acid and nucleotide sequences in computerized data bases, and to determine transcript size and specificity by Northern analysis of human testis and somatic RNAs, (3) clone novel testis antigens into an appropriate plasmid such as pGEX for expression, e.g. as a gluthathione transferase fusion protein, which will be used for immuno-affinity purification of antigen- reactive antibodies from human """"""""infertility"""""""" sera. Purified antibodies will be tested for ability to mimic anti-fertility effects of original sera via in vitro assays. A rigorous system of triage will be instituted, where at each step of the selection process, false positives are discovered and eliminated, (4) use production-scale fermentation methods and produce large quantities of fusion protein to affinity purify and cleave putative """"""""infertility"""""""" antigen from fusion partner, and (5) test contraceptive effectiveness by active immunization of female baboons. These studies should contribute to the development of a contraceptive vaccine based upon antigenic determinants that presumably provoke antibodies found in sera of patients described as immunologically infertile. In addition this work may be useful in establishing the cause of such infertility and determining the nature of sperm surface epitopes by which such antibodies perturb sperm function.
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