T-cell ELISpot responses elicited over time. Shown are IFN-γ (left panels), IL-2 (middle panels), and IL-4 (right panels) ELISpots from individual animals immunized with SHIV immunogens delivered by DNA/MVA or DNA/NYVAC to HIV-1_89.6P Env and SIV_mac239 Gag, Pol, and Nef peptides. Background responses (mean numbers of SFC plus twice the standard deviations of triplicate assays with medium alone) were subtracted. Responses are presented as the number of SFC per 10⁶ PBMC. Arrows indicate immunization time points at weeks 0, 4 (DNA), 20, and 24 (poxvirus).

Vaccine efficacy. The viral RNA load (top), absolute number of circulating CD4⁺ T cells (middle), and percentage of CD4⁺ Tcm cells (bottom) after the challenge of each individual animal are presented. The left column represents results from animals immunized with SHIV immunogens delivered by DNA/MVA, the middle column shows results from animals immunized with SHIV immunogens delivered by DNA/NYVAC, and the right column presents results from control animals.

T-cell ELISpot responses after challenge. IFN-γ production by PBMC of individual control animals (black diamonds) or animals immunized with SHIV immunogens delivered by DNA/MVA (open squares) or DNA/NYVAC (black dots) to HIV-1_89.6P Env and SIV_mac239 Gag, Pol, and Nef peptides at 22 weeks after challenge (pc) (left panels) and at 41 weeks after challenge (right panels). Box-whisker plots indicate the interquartile ranges and the medians (horizontal lines) of the groups. Background responses (mean numbers of SFC plus twice the standard deviations of triplicate assays with medium alone) were subtracted. Responses are presented as the number of SFC per 10⁶ PBMC.

Neutralizing antibody titers. Relative inhibition of virus infection of indicator cells with the challenge virus SHIV_89.6p is shown. The infection of indicator cells with virus incubated with individual sera from before immunization or before challenge (controls) was set at 0% inhibition.

Schematic representation of the Gag-Pol-Nef polyprotein and Env protein in relation to the SIV_mac239 virus genome and the peptide pools used for the analysis of immune responses. Different genes are indicated by different colors (gag in red, pol in blue, nef in green, and env in yellow). A detailed description of the construction can be found in Materials and Methods. LTR, long terminal repeat; GAG, group specific antigen; POL, polymerase; VIF, virion infectivity factor; VPR, viral protein regulator; VPU, viral protein unknown; TAT, transactivator of viral transcription; REV, regulator of viral protein expression; NEF, negative factor; PROT, protease; RT, reverse transcriptase; INT, integrase; ENV, envelope.

Vaccine-induced antigen-specific CD4⁺ and CD8⁺ T-cell responses 2 weeks prior to challenge. (A) Representative flow cytometry profiles of vaccine-induced CD4⁺ (left) and CD8⁺ (right) T-cell responses directed against Env in monkeys R99005 and R00033 immunized with SHIV immunogens delivered by DNA/MVA or DNA/NYVAC, respectively. CD4 and CD8 T-cell responses were defined using polychromatic flow cytometry. Blood mononuclear cells were stimulated with the relevant peptide pools and stained with CD4, CD8, IFN-γ, and IL-2 antibodies. (B) Cytokine production by CD4⁺ (left) and CD8⁺ (right) T cells of all individual animals immunized with SHIV immunogens delivered by DNA/MVA (open squares) or DNA/NYVAC (black dots) to HIV-1_89.6p Env (gray boxes) and SIV_mac239 Gag peptides (clear boxes) as measured by ICS assays 2 weeks before challenge. Box-whisker plots indicate the interquartile ranges and the medians (horizontal lines) of the groups. Responses are presented as the numbers of cytokine-producing CD3⁺ T cells per 10⁶ lymphocytes. Pie charts represent the average response for all animals of each group of the CD4⁺ and CD8⁺ HIV-1_89.6p Env- and SIV_mac239 Gag-specific T-cell responses grouped by function (expressing either one cytokine, IFN-γ [light gray] or IL-2 [dark gray], or the two simultaneously [black]) relative to the total antigen-specific response. (C) Representative flow cytometry profiles of CFSE-labeled PBMC of monkeys R99005 and R00033 immunized with SHIV immunogens delivered by DNA/MVA or DNA/NYVAC, respectively, unstimulated (Neg) or stimulated with Env peptides, cultured for 6 days, and stained with CD3, CD4, and CD8 antibodies. (D) Percentage of antigen-specific proliferating CD4⁺ and CD8⁺ T cells of all individual animals (dots) immunized with SHIV immunogens delivered by DNA/MVA (open squares) or DNA/NYVAC (black dots) to HIV-1_89.6p Env (gray boxes) and SIV_mac239 Gag peptides (clear boxes) 2 weeks before challenge. Responses are presented as percentages of proliferating (CFSE low) CD3⁺/CD4⁺ and CD3⁺/CD8⁺ T cells. Background responses (medium alone) were subtracted.

Survival. The percentages of animals that remained disease free after SHIV_89.6P challenge are shown. The animals were immunized with SHIV immunogens delivered by DNA/MVA (solid line) or DNA/NYVAC (dashed line) or were control animals (declining line).

Antigen-specific CD4⁺ and CD8⁺ T-cell responses at the time of euthanasia. (A) Cytokine production by CD4⁺ (left) and CD8⁺ (right) T cells of control animals (black diamonds) or individual animals (dots) immunized with SHIV immunogens delivered by DNA/MVA (open squares) or DNA/NYVAC (black dots) to HIV-1_89.6p Env (gray boxes) and SIV_mac239 Gag peptides (clear boxes) as measured by ICS assays at the time of euthanasia. Box-whisker plots indicate the interquartile ranges and the medians (horizontal lines) of the groups. Responses are presented as the number of cytokine-producing CD3⁺ T cells per 10⁶ lymphocytes. Pie charts represent the average response for all animals of each group of the CD4⁺ and CD8⁺ HIV-1_89.6p Env- and SIV_mac239 Gag-specific T-cell responses grouped by function (expressing either one cytokine, IFN-γ [light gray] or IL-2 [dark gray], or the two simultaneously [black]) relative to the total antigen-specific response. (B) Percentage of antigen-specific proliferating CD4⁺ (left) and CD8⁺ (right) T cells of control animals (black diamonds) or individual animals (dots) immunized with SHIV immunogens delivered by DNA/MVA (open squares) or DNA/NYVAC (black dots) to HIV-1_89.6p Env (gray boxes) and SIV_mac239 Gag peptides (clear boxes). Responses are presented as the percentage of proliferating (CFSE low) CD3⁺/CD4⁺ and CD3⁺/CD8⁺ T cells. Background responses (medium alone) were subtracted. Statistically significant differences between immunization groups are given by showing the P values (Mann-Whitney test).