Background: The recent phase IIB STEP trial tested the efficacy of a replication-defective Ad5 vaccine expressing HIV-1 Gag, Pol, and Nef. We randomized 3000 HIV-1 high-risk HIV-1-uninfected volunteers (1:1) within baseline Ad5 strata to receive 3 injections of vaccine or placebo. The interim analysis found no evidence that vaccination prevented infection or lowered viral set-point, and a greater incidence of infections occurred in male Ad5 seropositive vaccine than placebo recipients. We describe laboratory investigations to explore underlying reasons for lack of efficacy, and apparent enhanced infection rates in the Ad5 seropositive vaccinees.

Methods: Using STEP participant pripheral blood mononuclear cells (PBMC) cryopreserved within 8 hours of venipuncture for immunoassays, we characterized the frequency, magnitude, and breadth of vaccine-induced T cell responses. General immune activation and effector-memory phenotypes were assessed by multiparameter flow cytometry.

Results: The trivalent vaccine was highly immunogenic in the STEP trial, consistent with observations in previous phase I studies. The vaccine-induced, anti-HIV CD8⁺ and CD4⁺ cells were polyfunctional. Potency and breadth of the vaccine-induced responses were comparable between cases and non-cases within Ad5 ≤200 and Ad5 >200 strata; 4 weeks after the third dose, the circulating levels of activated (Ki67^hiBcl2^lo) CD4⁺CCR5⁺ cells were higher in subjects with baseline Ad5 titers >200 than in subjects with Ad5 titers ≤200; however, within each stratum, level of activation did not differ between vaccine and placebo recipients. Characterization of adenovirus vector-specific T cell responses, anti-HIV T cell epitopes, virus sequencing, and host genetic analyses are also ongoing to determine whether these parameters will help to explain the clinical outcome.

Conclusions: Laboratory investigations are underway to gain insight into immunological factors that may explain in part the clinical outcomes of the STEP trial. From data thus far, no obvious immune profile distinguishes the cases from non-cases or appears to explain the greater infection rates in vaccinees with prior Ad5 immunity. Results from future laboratory investigations, in progress and planned, of responses to the trans-gene and vector itself, as well as analysis of immune activation in blood and mucosally, will have important implications on the directions of other T cell-based vaccine development and the HIV vaccine field in general.