This research plan continues and refines the long-term goal of developing immunocontraceptives based on the testis-specific lactate dehydrogenase isozyme (LDH-C4; LDH-X). Previous studies have identified several epitopes of mouse LDH-C4 and located them within the crystallographic structure of the molecule. We propose to extrapolate these findings to study the immunocontraceptive properties of the human isozyme based on the cDNA sequence which we have recently determined. Genetic engineering will be employed to produce large quantities of the human protein for biochemical and immunological studies. The 5' and 3' ends of the Ldh-c cDNA will be engineered such that the coding region is flanked by restriction enzyme sites. This fragment will be inserted into: 1) PKK223-3 for transformation of E. coli, and 2) pAc373 for recombination with Baculovirus and subsequent infection of Sf9 insect cells. The human LDH-C4 will be purified and characterized. Structural information on mouse and human LDH-C4 will be used to design synthetic peptides which mimic the topographic determinants of the molecule. Particular emphasis will be placed on stabilizing amphipathic peptides into predetermined supersecondary structures that represent their configuration in the native molecule. These peptides will be studied for their chemical and immunological properties. As an alternative to peptide antigens, we will also develop a viral vaccine by cloning the human gene into vaccinia virus. This will be accomplished by recombination between the thymidine kinase sequences of wild-type vaccinia and pGS20+Ldh-c. Recombinants (tk-) grown in human 143B cells (tk-) will be selected by pGS20+Ldh-c. Recombinants (tk-) grown in human 143B cells (tk-) will be selected by resistance to 5' bromodeoxyuridine. Each of the peptides and viral vectors produced will be tested for its ability to evoke antibodies in an experimental animal model. Putative T-cell epitopes will be used to stimulate secondary T-cell proliferation in vitro. The effect of the antibodies on sperm will be assessed using human sperm and hamster ova in an in vitro fertilization assay. Candidate contraceptive antigens will be tested for pre- and post-fertilization effects in vivo.
