This project represents the initial stages of a proposed research program to develop a model Simian Immunodeficiency Virus (SIV) vaccine system that can be used to evaluate vaccine formats and immunization protocols for candidate vaccines directed against the Human Immunodeficiency Virus. The objective of the Phase I aspect of this proposal are two fold: to construct a pre-vaccine vector containing SIV envelope sequences and to establish that these sequences are expressed in cultured monkey cells. A biologically active clone of macaque isolate SIV MAC251 will be isolated from HUT 78 cells using a lambda EMBL 3 vector and subcloned into plasmid pBR327. SIV MAC sequences will be evaluated for biologic activity by transfecting COS-1 monkey kidney cells with calcium phosphate precipitates of plasmid DNA and monitoring for progeny virus production on the basis of immunocytochemical staining for the viral p24 protein and reverse transcriptase activity. SIV envelope coding sequences excised for the pBR327 vector will be cloned into a previously constructed pre-vaccine vector under transcriptional control of the adenovirus in monkey cells. Monkey kidney cell lines derived from African green and rhesus monkeys will be transfected with the plasmid DNA and transient expression of SIV envelope-specific RNA and protein will be analyzed. Having established that SIV envelope sequences are expressed in monkey cells, the pre-vaccine plasmid will be used in the next phase of the project to construct the recombinant virus.
