New HIV infections worldwide occur every 25 seconds and a vaccine is the only long-term solution to this epidemic. A new attenuated strain of Listeria monocytogenes is a vector with potential for use as an oral vaccine against the disease. In Project 1 of this application several recombinants of attenuated Listeria that individually express various foreign genes will be generated by standard cloning methods: the flourochrome DsRed, for use in mouse experiments, SIV gag and nef, 89.6 tat, and the envelope genes of SHIV 89.6P and SHIV 1157ip for use in trials in juvenile and adult rhesus monkeys, and new recombinants of the attenuated Listeria that express a consensus-type clade B env sequence, JRFL, and a clade B'C env sequence from Beijing for potential use in future human studies. This core project is designed to (1) generate amounts of these recombinant bacteria commensurate with the needs of the various projects using them and (2) to assure the authenticity of the organisms by characterizing the preparations with respect to their genetic correctness, their lack of reversion to wild-type, their safety and immunogenicity in mice, and their expression of the desired heterologous proteins. We will also explore the ability of a synthetic medium to be used for the growth of the organisms so as to eliminate from future preparations, if possible, the complex, poorly characterized molecules that currently are used in their growth medium.
