Preferential Targeting of Conserved Gag Regions after Vaccination with a Heterologous DNA Prime-Modified Vaccinia Virus Ankara Boost HIV-1 Vaccine Regimen

Abstract

Prime-boost vaccination strategies against HIV-1 often include multiple variants for a given immunogen for better coverage of the extensive viral diversity. To study the immunologic effects of this approach, we characterized breadth, phenotype, function, and specificity of Gag-specific T cells induced by a DNA-prime modified vaccinia virus Ankara (MVA)-boost vaccination strategy, which uses mismatched Gag immunogens in the TamoVac 01 phase IIa trial. Healthy Tanzanian volunteers received three injections of the DNA-SMI vaccine encoding a subtype B and AB-recombinant Gag_p37 and two vaccinations with MVA-CMDR encoding subtype A Gag_p55 Gag-specific T-cell responses were studied in 42 vaccinees using fresh peripheral blood mononuclear cells. After the first MVA-CMDR boost, vaccine-induced gamma interferon-positive (IFN-γ⁺) Gag-specific T-cell responses were dominated by CD4⁺ T cells (P < 0.001 compared to CD8⁺ T cells) that coexpressed interleukin-2 (IL-2) (66.4%) and/or tumor necrosis factor alpha (TNF-α) (63.7%). A median of 3 antigenic regions were targeted with a higher-magnitude median response to Gag_p24 regions, more conserved between prime and boost, compared to those of regions within Gag_p15 (not primed) and Gag_p17 (less conserved; P < 0.0001 for both). Four regions within Gag_p24 each were targeted by 45% to 74% of vaccinees upon restimulation with DNA-SMI-Gag matched peptides. The response rate to individual antigenic regions correlated with the sequence homology between the MVA- and DNA Gag-encoded immunogens (P = 0.04, r² = 0.47). In summary, after the first MVA-CMDR boost, the sequence-mismatched DNA-prime MVA-boost vaccine strategy induced a Gag-specific T-cell response that was dominated by polyfunctional CD4⁺ T cells and that targeted multiple antigenic regions within the conserved Gag_p24 protein.IMPORTANCE Genetic diversity is a major challenge for the design of vaccines against variable viruses. While including multiple variants for a given immunogen in prime-boost vaccination strategies is one approach that aims to improve coverage for global virus variants, the immunologic consequences of this strategy have been poorly defined so far. It is unclear whether inclusion of multiple variants in prime-boost vaccination strategies improves recognition of variant viruses by T cells and by which mechanisms this would be achieved, either by improved cross-recognition of multiple variants for a given antigenic region or through preferential targeting of antigenic regions more conserved between prime and boost. Engineering vaccines to induce adaptive immune responses that preferentially target conserved antigenic regions of viral vulnerability might facilitate better immune control after preventive and therapeutic vaccination for HIV and for other variable viruses.

Keywords: Gag; T cells; human immunodeficiency virus; vaccines.

FIG 1

Diversity of HIV-1 Gag protein sequences originating from the Mbeya region. (A) The distribution of subtypes and unique recombinant forms of Gag polyprotein sequences from 91 HIV-infected subjects from the Mbeya region is shown in the pie chart. (B) Shannon entropy plot generated from these Gag sequences is shown.

FIG 2

Immunogen sequences included in the DNA-Gag prime and modified vaccinia virus Ankara (MVA)-Gag boost and their coverage by peptide pools. The seven DNA peptide pools covered the Gag_p37 region consisting of p17 (blue) and p24 (gray), whereas the nine MVA-Gag peptide pools covered the Gag_p55 precursor protein, including the p15 region (red) in addition to p17 and p24. The p15 region was only covered by MVA-Gag peptide pools 8 and 9.

FIG 3

Phenotype, function, and breadth of vaccine-induced Gag-specific T-cell responses. (A) Representative dot plots for the analyses of Gag-specific CD4 and CD8 T-cell functions are shown. (B) Frequencies of IFN-γ⁺, CD4⁺, and CD8⁺ T cells after stimulation of freshly isolated PBMC with whole MVA-CMDR-Gag_p55 (left) or DNA-SMI-Gag_p37 (right) peptide pools in 41 vaccinees are shown. (C) Coexpression of additional functions (TNF-α, Mip-1β, IL-2, and the degranulation marker CD107) for IFN-γ⁺ CD4⁺ (left) and IFN-γ⁺ CD8⁺ (right) T cells. The four color-coded arcs indicate the proportion of cells coexpressing the four additional functions. The color-coded pies symbolize the 16 possible functional combinations. Intracellular cytokine staining was performed using fresh PBMC stimulated overnight with the indicated antigens as well as the control antigens Staphylococcus enterotoxin B and CMV_pp65. (D) The number of different antigenic regions (linear peptide pools, x axis) recognized by TaMoVac vaccinees (n = 42) is shown and was determined using the IFN-γ ELISPOT assay. The frequency of subjects with a given Gag response breadth is indicated on the y axis. Nine and 7 linear peptide pools matching MVA-CMDR-Gag_p55 (gray bars) or DNA-SMI-Gag_p37 (black bars), respectively, subdivided Gag into distinct antigenic regions. Statistical comparison for panel B was performed using Wilcoxon matched-pairs signed-rank test.

FIG 3

Phenotype, function, and breadth of vaccine-induced Gag-specific T-cell responses. (A) Representative dot plots for the analyses of Gag-specific CD4 and CD8 T-cell functions are shown. (B) Frequencies of IFN-γ⁺, CD4⁺, and CD8⁺ T cells after stimulation of freshly isolated PBMC with whole MVA-CMDR-Gag_p55 (left) or DNA-SMI-Gag_p37 (right) peptide pools in 41 vaccinees are shown. (C) Coexpression of additional functions (TNF-α, Mip-1β, IL-2, and the degranulation marker CD107) for IFN-γ⁺ CD4⁺ (left) and IFN-γ⁺ CD8⁺ (right) T cells. The four color-coded arcs indicate the proportion of cells coexpressing the four additional functions. The color-coded pies symbolize the 16 possible functional combinations. Intracellular cytokine staining was performed using fresh PBMC stimulated overnight with the indicated antigens as well as the control antigens Staphylococcus enterotoxin B and CMV_pp65. (D) The number of different antigenic regions (linear peptide pools, x axis) recognized by TaMoVac vaccinees (n = 42) is shown and was determined using the IFN-γ ELISPOT assay. The frequency of subjects with a given Gag response breadth is indicated on the y axis. Nine and 7 linear peptide pools matching MVA-CMDR-Gag_p55 (gray bars) or DNA-SMI-Gag_p37 (black bars), respectively, subdivided Gag into distinct antigenic regions. Statistical comparison for panel B was performed using Wilcoxon matched-pairs signed-rank test.

FIG 4

Recognition of Gag antigenic regions by vaccine-induced T cells. (A) A comparison of the magnitude of Gag-specific T-cell responses (y axis) targeting antigenic regions within p17, p24, and p15 normalized per 15mer peptide is shown. (B) The frequency of responders is shown for different Gag regions. Vaccine-induced T-cell responses were characterized using IFN-γ ELISPOT assay in 42 participants after stimulation of fresh PBMC with 9 and 7 peptide pools matching MVA-CMDR-Gag_p55 (gray bars) and DNA-Gagp₃₇ (black bars), respectively. (C) The frequency of responders for individual MVA-CMDR-Gag_p55 matched peptides is shown and is based on 23 subjects with at least 1 detectable response against an individual peptide during fine mapping using cryopreserved instead of fresh PBMC. (D) The key data for the most frequently recognized peptides are shown. The cutoff for a positive response was 2-fold above the level of the unstimulated control. Corresponding Gag regions p15, p17, and p24 are indicated in panels B and C.

FIG 5

Linear regression analyses between DNA-SMI-Gag_p37 and MVA-CMDR-Gag_p55 sequence mismatches and induced T-cell responses targeting specific antigenic regions. (A) A linear regression analysis was performed to study the association of immunogen-located amino acid mismatches and the respective T-cell response rate to the DNA-SMI-Gag_p37 pools 1 to 6. T-cell responses were detected using the IFN-γ ELISPOT assay, and freshly isolated PBMC pool 7 was excluded because it contains only 3 instead of 11 peptides. A comparison of mismatches between MVA-CMDR-Gag_p55 and the DNA-SMI-Gag_p37B sequence and the respective T-cell response rate (B) or the magnitude of responses (C) for single-peptide-specific T-cell responses was detected using the IFN-γ ELISPOT assay and cryopreserved PBMC.