Figure 3

T-cell responses to S are greater for hAd5 S-Fusion + N-ETSD as compared to hAd5 S-WT and CD4 + T cells were more responsive to N. Intracellular cytokine staining (ICS) of interferon-γ (IFN-γ), IFN-γ/tumor necrosis factor α (TNF-α), and IFN-γ/TNF-α/interleukin-2 (IL-2) in response to S peptide pools 1 and 2 as well as the N peptide pool is shown for (a–c) CD4 + and (d–f) CD8β + T lymphocytes. CD4 + T cells were more responsive to N peptides and CD8 + T cells to S peptide pool 1 that contains the S RBD as compared to S peptide pool 2 or the N pool. Media and SIV nef are negative controls. ELISpot detection of (g) IFN-γ secretion by T-lymphocytes also reveals responses to S peptide pool 1 and the N pool. (h) Interleukin-4 (IL-4) secretion was very low. SIV nef is a negative control and Con A a positive control. (i) The IFN-γ/IL-4 ratios of > 1 (dashed line) reflects the Th1 bias of all T-cell responses. hAd5 Null n = 4, hAd5 S-WT n = 3, and hAd5 S-Fusion + N-ETSD n = 8. Statistical analyses performed using one-way ANOVA with Dunnett’s post-hoc comparison of the S-Fusion + N-ETSD group to the S-WT or Null groups, where *p < 0.05 and **p ≤ 0.01. Data graphed as the mean with SEM.