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BackgroundElicitation of broad immune responses is understood to be required for an efficacious preventative HIV vaccine. This Phase 1 randomized controlled trial evaluated whether administration of vaccine antigens separated at multiple injection sites vs combined, fractional delivery at multiple sites affected T-cell breadth compared to standard, single site vaccination.MethodsWe randomized 90 participants to receive recombinant adenovirus 5 (rAd5) vector with HIV inserts gag, pol and env via three different strategies. The Standard group received vaccine at a single anatomic site (n = 30) compared to two polytopic (multisite) vaccination groups: Separated (n = 30), where antigens were separately administered to four anatomical sites, and Fractioned (n = 30), where fractions of each vaccine component were combined and administered at four sites. All groups received the same total dose of vaccine.FindingsCD8 T-cell response rates and magnitudes were significantly higher in the Fractioned group than Standard for several antigen pools tested. CD4 T-cell response magnitudes to Pol were higher in the Separated than Standard group. T-cell epitope mapping demonstrated greatest breadth in the Fractioned group (median 8.0 vs 2.5 for Standard, Wilcoxon p = 0.03; not significant after multiplicity adjustment for co-primary endpoints). IgG binding antibody response rates to Env were higher in the Standard and Fractioned groups vs Separated group.InterpretationThis study shows that the number of anatomic sites for which a vaccine is delivered and distribution of its antigenic components influences immune responses in humans.FundingNational Institute of Allergy and Infectious Diseases, NIH.

T cell responses have been implicated in reduced risk of HIV acquisition in uninfected persons and control of viral replication in HIV-infected individuals. HIV Gag-specific T cells have been predominantly associated with post-infection control, whereas Env antigens are the target for protective antibodies; therefore, inclusion of both antigens is common in HIV vaccine design. However, inclusion of multiple antigens may provoke antigenic competition, reducing the potential effectiveness of the vaccine. HVTN 084 was a randomized, multicenter, double-blind phase 1 trial to investigate whether adding Env to a Gag/Pol vaccine decreases the magnitude or breadth of Gag/Pol-specific T cell responses. Fifty volunteers each received one intramuscular injection of 1 × 1010 particle units (PU) of rAd5 Gag/Pol and EnvA/B/C (3:1:1:1 mixture) or 5 × 109 PU of rAd5 Gag/Pol. CD4+ T cell responses to Gag/Pol measured 4 weeks after vaccination by cytokine expression were significantly higher in the group vaccinated without Env, whereas CD8+ T cell responses did not differ significantly between the two groups. Mapping of individual epitopes revealed greater breadth of the Gag/Pol-specific T cell response in the absence of Env compared to Env coimmunization. Addition of an Env component to a Gag/Pol vaccine led to reduced Gag/Pol CD4+ T cell response rate and magnitude as well as reduced epitope breadth, confirming the presence of antigenic competition. Therefore, T cell-based vaccine strategies should aim at choosing a minimalist set of antigens to reduce interference of individual vaccine components with the induction of the maximally achievable immune response.

The interferon gamma, enzyme-linked immunospot (IFN-γ ELISpot) assay is widely used to identify viral antigen-specific T cells is frequently employed to quantify T cell responses in HIV vaccine studies. It can be used to define T cell epitope specificities using panels of peptide antigens, but with sample and cost constraints there is a critical need to improve the efficiency of epitope mapping for large and variable pathogens. We evaluated two epitope mapping strategies, based on group testing, for their ability to identify vaccine-induced T-cells from participants in the Step HIV-1 vaccine efficacy trial, and compared the findings to an approach of assaying each peptide individually. The group testing strategies reduced the number of assays required by >7-fold without significantly altering the accuracy of T-cell breadth estimates. Assays of small pools containing 7-30 peptides were highly sensitive and effective at detecting single positive peptides as well as summating responses to multiple peptides. Also, assays with a single 15-mer peptide, containing an identified epitope, did not always elicit a response providing validation that 15-mer peptides are not optimal antigens for detecting CD8+ T cells. Our findings further validate pooling-based epitope mapping strategies, which are critical for characterizing vaccine-induced T-cell responses and more broadly for informing iterative vaccine design. We also show ways to improve their application with computational peptide:MHC binding predictors that can accurately identify the optimal epitope within a 15-mer peptide and within a pool of 15-mer peptides.