The use of transformed cell substrates for prophylactic vaccine manufacturing is widely debated. Extensive characterization is required to address the suitability of neoplastic cell substrates for vaccine manufacture. The HeLa-based cell substrate used in the manufacture of a prophylactic rAAV-HIV vaccine, AAV2-gagPR delta RT (tgAAC09) was tested in vivo for its tumor-forming potential, the oncogenic potential of its high molecular weight DNA and the potential presence of occult oncogenic adventitious agents. This data from these in vivo studies, in conjunction with prion gene and protein characterization, cell and viral clearance studies and quantity of residual host-cell DNA levels in the purified tgAAC09 vaccine, were used to establish what we believe to be an acceptable safety profile for the vaccine manufacturing process. The tumor-producing dose in 50% of the animals was consistent with that in a published report from FDA staff for HeLa cells. High molecular weight cellular DNA was not oncogenic and no occult oncogenic agents were detected by testing in nude mice and newborn rodent models, respectively. Endogenous prion protein was also normal and genomic sequence analysis detected no mutations associated with increased risk of prion disease. In addition, the purification process used to produce this vaccine candidate removed all detectable cells (clearance of greater than 22 log10), viral clearance study showed 6-17 log10 clearance of three model viruses and host-cell DNA in the bulk product was less than 100pg host-cell DNA per dose of 3 x 10(11) DNase resistant particles (DRP) of the vaccine. Taken together, the data from the in vivo and in vitro tests that were performed to characterize the HeLa based producer cell line (T3B12-5B) and HeLa S3 cells support the use of these cells as substrates for the manufacture of a purified rAAV-HIV vaccine candidate. The data also supports the ability of the process, employing the HeLa cell substrate, used to manufacture the rAAV-HIV vaccine to produce a product as free of adventitious agents as current testing procedures can document. Safety of the rAAV-HIV vaccine is currently being assessed in a Phase I clinical trial.