Mice were vaccinated and boosted, as described above, peripheral blood Gag-specific CD8 T cells were enumerated by Flow cytometric analyses. Whole blood (100 ml) was collected in heparinized PBS on days 26, 69, and 84 following initial vaccination. Leukocytes were collected using lymphocyte separation medium (Mediatech. Inc. Manassas, VA). Cells were collected and washed twice in 1%BSA, 2mM EDTA in PBS and counted. One to two million lymphocytes were stained with or without Gag Tetramer (APC) (created by Emory University) and a cocktail of CD4-Alexa488 and CD8-PE antibodies (BD Pharmingen, San Jose, California). Cells were washed with 1%BSA, 2mM EDTA in PBS. Cells were fixed with 1% paraformaldehyde and subsequently analyzed for flow cytometry on LSR II cytometer. Analysis of 3,000 CD8 T cells was collected per sample.