Background: Novel AIDS vaccine approaches are needed that optimize safety and efficacy. We created SIV recombinants that are limited to a single cycle of infection and express 8 of the 9 viral genes. These strains are being evaluated for their ability to serve as non-replicating vaccine strains.

Methods: Single-cycle SIV (scSIV) was produced using a 2-plasmid transfection strategy that was specifically designed to minimize the chances of generating replication-competent virus through recombination or nucleotide reversion. One (1) plasmid contains a full-length SIV genome with nucleotide substitutions in the gag/pol-frameshift site and a deletion in integrase to doubly inactivate Pol expression. To provide Pol in trans, we created a Gag-Pol expression construct with a single nucleotide insertion to place gag and pol in the same reading frame. We also mutated the frameshift site in this construct so that any recombinants would still be replication incompetent. Co-transfection of these 2 plasmids into 293 T-cells resulted in the release of Gag-Pol-complemented virus that was capable of one round of infection but was unable to spread. Four (4) Mamu-A*01⁺ rhesus macaques were inoculated intravenously on wks 0 and 8 with concentrated mixtures of T-cell (SIVmac239) and macrophage (SIVmac316) tropic scSIV (8.6 µg p27) and the development of SIV-specific immune responses was monitored.

Results: Four (4) days after the first inoculation, plasma viral RNA loads between 10⁴ and 10⁵ copies per ml were detected in each animal. Consistent with a single round of infection, viral loads steadily declined to undetectable levels at subsequent time points. Viral RNA in plasma almost certainly represents cellular production of non-infectious virus by scSIV-infected cells. Virus-specific T-cell responses were detected by Mamu-A*01-Gag_181-189 tetramer staining (0.07% to 0.2% of CD8⁺ T-cells at week 2) and IFN-γ ELISpot assays with Gag peptides (50–250 SFC/10⁶ PBMC at wk 3). Following the second inoculation, plasma viral RNA loads peaked between 10² and 10⁴ copies per ml on day 2. In addition to boosting Gag-specific responses, T-cell responses to other SIV antigens were also observed. SIV-specific antibody responses were observed in all 4 animals.

Conclusions: The induction of immune responses to multiple SIV antigens in macaques inoculated with scSIV suggests that single-cycle HIV may represent a promising approach to a vaccine for AIDS.