rAd5 and HSV-1 amplicon vectors encoding HIV-1 gp120 under the transcriptional control of different promoters were used in this experiment. Promoter elements including the standard CMV major immediate early promoter-enhancer (cmv), a hybrid CMV promoter and HTLV-1 LTR enhancer (cmvr) or the HSV-1 4/5 immediate early promoter ((hsv); see text for details). These vectors were inoculated either alone or in combination (HSV & rAd5 group) into the left hind quadriceps of BALB/c mice (4/group; doses are indicated in Table 1A). Animals were sacrificed 12 days later and splenocytes were harvested. Env-specific responses were assayed by intracellular cytokine staining for CD8⁺T-cell (A) and CD4⁺T-cell (B) production of IFNγ, TNFα and IL2. The data represent the total percentage of T cells that produced any of the measured cytokines in response to stimulation with the Env peptide pool. Data shown are for individual mice; horizontal bars represent the mean cytokine production for each group. Statistical analysis was performed by a non-parametric one-way ANOVA. All values from immunized animals showed statistically significant induction of Env-specific T cell responses, versus mice immunized with amplicon particles encoding an irrelevant protein (HSV:LacZ; p < 0.05).

rAd5 and HSV-1 amplicon vectors encoding HIV-1 gp120 from the indicated promoter elements (cmv, cmvr, hsv; see Fig. 1 legend), were inoculated either alone or in combination (HSV & rAd5 group) into the left hind quadriceps of BALB/c mice (4/group) at the doses denoted in Table 1A. Animals were sacrificed 12 days later and splenocytes were harvested. Env-specific responses were assayed by intracellular cytokine staining for CD8⁺T-cell and CD4⁺T-cell production of IFNγ, TNFα and IL2. Analysis was performed by gating on CD8⁺ and CD4⁺ T-cells for the production of IFNγ. IFNγ-positive and IFNγ-negative cells were then separately gated, to assess antigen-specific production of IL2 and TNFα. The frequency of cells producing a single cytokine, or two or more cytokines was calculated using FlowJo software. The CD8⁺ (A) and CD4⁺ T-cell (B) data are presented here as pie charts; each pie chart represents the mean cytokine profile for splenocytes from each group of mice. Each slice (different color) of a pie denotes a distinct subset of cytokine producing cells for that group of immunized mice, after subtraction of non-specific background staining (determined from mice immunized with HSVLacZ). (C) Quantitative data for the CD4⁺ T-cell response. The percentage of CD4⁺ T-cells that produced each of the seven possible combinations of IFNγ, TNFα and IL2 in response to stimulation with the Env peptide pool is shown. Data represent mean values for each group of four mice; horizontal bars represent the standard error of the mean. Statistical analysis was performed by two-way ANOVA with Bonferroni’s post-test. * Different from all groups (p < 0.01); ** different from all groups (p < 0.001). Note that panel C includes background staining data (determined from mice immunized with HSVLacZ); no background subtraction was applied to the data in this panel.

BALB/c mice (4/group) were primed with rAd5 (1×10⁷ genome units) and HSV-1 amplicon (1×10⁶ genome units), encoding HIV-1 gp120, by inoculation into the left hind quadriceps at day 0. Mice were boosted 72 days post prime, with HSVgp120 (1×10⁶ g.u.) or rAd5gp120 (1×10⁷ g.u.) as summarized in Table 1B. Animals were sacrificed 12 days post boost and splenocytes were harvested. Env-specific responses were assayed by intracellular cytokine staining for: CD8⁺T-cell production of IFNγ, TNFα and IL2 in splenocytes from HSV-1 amplicon (A) and rAd5 (B) primed mice; CD4⁺T-cell production of IFNγ, TNFα and IL2 in splenocytes from HSV-1 amplicon (C) and rAd5 (D) primed mice. The data represent the total percentage of T cells that produced any of the measured cytokines in response to stimulation with the Env peptide pool. Data shown are for individual mice; horizontal bars represent the mean cytokine production for each group. Statistical analysis was performed by a non-parametric one-way ANOVA followed by a Tukey-Kramer post test, using GraphPad Prism. Statistically significant enhancement in T-cell responses, versus mice that were not boosted (None) is denoted by “+” signs as follows: +, p<0.01; ++p<0.001. Statistically significant enhancement in T cell responses, versus mice that were boosted with rAd5, is denoted by “Δ” signs as follows: Δ: p<0.01; ΔΔ: p<0.001.

BALB/c mice (4/group) were primed with rAd5 (1×10⁷ genome units) and HSV-1 amplicon (1×10⁶ genome units), encoding HIV-1 gp120, by inoculation into the left hind quadriceps at day 0. Mice were boosted 72 days post prime, with HSVgp120 (1×10⁶ g.u.) or rAd5gp120 (1×10⁷ g.u.) as summarized in Table 1B. Animals were sacrificed 12 days post boost and splenocytes were harvested. Env-specific responses were assayed by intracellular cytokine staining for CD8⁺T-cell and CD4⁺T-cell production of IFNγ, TNFα and IL2. Analysis was performed by gating on CD8⁺ and CD4⁺ T-cells for the production of IFNγ. IFNγ-positive and IFNγ-negative cells were then separately gated, to assess antigen-specific production of IL2 and TNFα. The frequency of cells producing a single cytokine, or two or more cytokines was calculated using FlowJo software. (A) The CD8⁺ and CD4⁺ T-cell data are presented here as pie charts; each pie chart represents the mean cytokine profile for splenocytes from each group of mice. Each slice (different color) of the pies denotes a distinct subset of cytokine producing cells for that group of immunized mice, after subtraction of non-specific background staining (determined from mice immunized with HSVLacZ). (B) Quantitative data for the CD4+ T-cell response. The inset Figure legend denotes the various prime-boost combinations used (Ad prime only = rAd5gp120 prime only; Ad-Ad = rAd5gp120 prime and boost; Ad-HSV = rAd5gp120 prime plus HSVgp120 boost; HSV prime only = HSV(hsv)gp120 prime only; HSV-Ad = HSVgp120 prime plus rAd5gp120 boost; HSV-HSV = HSVgp120 prime and boost; lacZ = HSVlacZ only). The percentage of CD4⁺ T-cells that produced each of the seven possible combinations of IFNγ, TNFα and IL2 in response to stimulation with the Env peptide pool is shown. Data represent mean values for each group of four mice; horizontal bars represent the standard error of the mean. Statistical analysis was performed by two-way ANOVA with Bonferroni’s post-test. * Different from all groups (p < 0.01); ** different from all groups (p < 0.001). Note that panel B includes background staining data (determined from mice immunized with HSVLacZ); no background subtraction was applied to the data in this panel.