We have developed a technique for the antigen-specific stimulation of SIV-specific CTL using autologous B-LCL infected with recombinant vaccinia viruses expressing SIV antigens. Using this technique we have been able to detect relatively vigorous SIV specific CTL activity in macaques vaccinated with either SIV nef or SIV 3. This CTL activity is directed against gag, pol and env and has been detectable in almost all vaccinated animals that have been studied at time points ranging from 12 months to several years after infection. Progress over the last year has been made in several areas including 1. Characterization of SIV-specific CTL activity. CTL assays using effector cells from multiple vaccinated animals have confirmed that lysis of target cells expressing SIV antigens occurs only in the autologous setting and not using MHC-mismatched target cells. 2. SIV-specific CTL activity is mediated by CD8+ cells. 3. SIV-specific CTL can be induced by vaccination with SIV vpr/vpx. Dr. Desrosiers' laboratory has developed a panel of live attenuated retroviruses with a variety of different deletions. A subset of these viruses has been used to infect animals and we recently had the opportunity to analyze CTL activity in animals infected with SIV vpr/vpx and SIV 4 ( nef, vpr, vpx, NRE). SIV-specific CTL activity was found in 2/2 SIV vpr/vpx infected animals. In contract, no CTL activity was detected in the 4 vaccinated animals. 4. Optimization of limiting dilution precursor frequency assays for the detection of SIV-specific CTL. Over the past we have defined conditions using freshly irradiated human cells and restimulation at 7 days that permit quantification of SIV-specific CTL activity using limiting dilution assays. This precursor frequency assay will be used to correlate CTL activity with protection from challenge.