FIG. 3.

A prime/boost lentiviral vector-based vaccination strategy induces robust cellular immunity. The longitudinal follow-up of the SIVmac239 GAG specific T-cell responses was performed at various time points postprime (light gray), postboost (medium gray), and postchallenge (dark gray) by IFN-γ ELISPOT assay after restimulation of whole PBMC with pools of overlapping peptides encompassing SIVmac239 GAG p55. The individual GAG-specific cumulative responses of all six vaccinated animals injected with TRIP-SIVmac239 GAG (low dose, animals 20022 and 20089; medium dose, animals 20293 and 20056; and high dose, animals 20195 and 20158) (A), two control animals immunized with an irrelevant antigen (TRIP-GFP) at a high p24 dose (animals 21544 and 20456) (B), and unvaccinated animals (animals 15661, 14184, 15885, and 14468) (C) are shown. Arrows indicate when the first injection (three doses), the second injection (identical dose), and the SIVmac251 challenge were performed. Aldrithiol AT-2 inactivated SIVmac239 was also used to restimulate GAG-specific CD4⁺ and CD8⁺ T cells 2 weeks postboost in a whole PBMC IFN-γ ELISPOT assay. Background after coculture with the control microvesicles was subtracted (D). When the frequency of specific T cells was high and spots overlapped, the number of IFN-γ SFC/million was underestimated to 1,400 before the background was subtracted. Indeed, the saturation curve of the ELISPOT reader was calculated by using serial dilutions of PBMC, and the maximum number of spots enumerated per well appeared to be 280 spots, corresponding to 1,400 spots/million PBMC when 200,000 cells were cultured. The symbol “+” was added at the top of the histogram when saturation was reached for at least one pool of peptides. At 2 weeks postchallenge, it was not possible to quantify the number of spots in the control medium without peptide wells and thus to calculate the cumulative response for animal 20022 (noted as “++”). nd, Not determined.