GENETIC ENGINEERING AND EXPRESSION ANALYSIS

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The objective of this proposal is to develop the use of recombinant vaccinia virus as a live viral vector for the delivery of a vaccine which yields total and long-term protection against multiple strains of HIV-1. Studies of poxvirus biology in this laboratory have made specific contributions to our understanding of virus growth and replication which have had direct application in the preparation of vaccinia recombinants. A program is in place which is deriving attenuated forms of the virus without affecting its efficacy as a vaccine, and a drug- and mutagen-free selection scheme has been developed for the rapid selection of recombinants. The modulated expression of multiple antigens in a single virus has been achieved, and novel concepts in the application of homologous recombination are being pursued. For the development of an effective vaccine against AIDS, recombinant vaccinia virus which express numerous antigens from HIV-1 and SIV will be constructed and used in studies of the immune response to these pathogens. These recombinants will include those expressing the gene products of env, gag, pol, and sor, in monovalent, divalent, and trivalent conformations. A multivalent recombinant virus represents the most likely candidate vaccine as it presents the largest number of antigens from HIV-1 and will potentially elicit the broadest immune response. The monovalent and divalent recombinant vaccinia will aid in delineating the contribution of individual antigens to the immune response, and possible synergy between antigens. Future generations of recombinants will be constructed based on the assimilation of results from studies of humoral and cell-mediated immunity using the first generation of recombinants, with studies of the immunogenicity of specific epitopes and mutated genes. For example, the genes encoding the individual polypeptide products of env, gag, and pol will be isolated and manipulated for expression, and immunodominant, non-neutralizing domains in the envelope glycoprotein will be deleted utilizing site-directed mutagenesis. The issue of the strain diversity observed in the envelope glycoprotein will also be addressed. All studies combined will ultimately lead to the preparation of candidate vaccines for use in human Phase I clinical trials.