For the triple regimens, analyses of the challenge virus loads were carried not only in splenocytes, where respective 2.7- and 5.5-fold decreases were detected for the DCM and DMC regimens (Fig. 4D), but also in the pooled superficial cervical and

MLNs and thymus. In all of these nonsplenic sites, a considerable clearance of the EcoHIV/NDK virus was detected (Fig. 4E). At 42 days postvaccination, in vivo killing of AMQ peptide-pulsed R788 in vivo and re-infused splenocytes showed close to 100% killing efficiency by T cells elicited by both triple regimens (Fig. 4F). Finally, to assess the longevity of the triple vaccine-induced responses, the third subgroup of animals receiving either the DCM or DMC regimens was rested for 115 days prior to the late surrogate virus challenge. Collected pooled PBMCs maintained polyfunctionality upon the AMQ peptide restimulation and the IFN-γ+-cell frequencies remained at respective 5.6 and 2.2% of total CD8+ cells for the DCM and DMC regimens (Fig. 5A). After challenge and measured in spleen, these frequencies rose to means of 9.6 and 6.7%, respectively (Fig. 5B). In the same animals, the DCM- and DMC-induced memory T cells decreased the EcoHIV/NDK DNA copy numbers 3.5-

and 5.2-fold, respectively. Using ANOVA analysis, the means of the challenge virus loads among individual treatments were significantly different, but in pairwise comparisons, this significance was lost after the Bonferroni adjustment (Fig. 5C). Thus, a sequential combination of three different vaccine modalities into a single regimen induced robust, durable, and polyfunctional CD8+ learn more T-cell responses. It remains that the real benefit of triple regimens may only become apparent in more challenging situations such as protection of humans against HIV-1. Finally, we assessed the AMQ-specific,

IFN-γ-producing CD8+ T cells for expression of proliferation-promoting IL-2, L-selectin CD62L, memory marker Atazanavir IL-7 receptor α-chain CD127 and CD27, which is required for generation and maintenance of T-cell immunity and is lost from terminally differentiated effector cells. Central memory (TCM), but not effector memory (TEM), T cells possess ability to express high levels of CD62L, have a high proliferative potential, and are primarily found in lymphoid tissue. Thus in spleen for both DCM and DMC regimens, the AMQ-specific postchallenge responses increased from the peak to the memory phase for IL-2 production and CD62L and CD127 expression (Fig. 6A). Memory PBMCs prior to the challenges differed from immune splenocytes the most in lower levels of IL-2 and higher expression of CD62L and CD27 (Fig. 6B). In addition to memory markers, vaccine-elicited CD8+ T cells were also analyzed for expression of the α4β7 adhesion molecule linked to migration of lymphoytes to the GALT. Vaccine-induced T-cell population expressing low level of α4β7 was found among immune splenocytes (Fig. 6C), but not PBMCs (Fig. 5D).