Fig. 3.

Gag-TLR7/8 conjugate immunization enhances the magnitude and alters the quality of Th1 and CD8⁺ T cell responses. Nine-color flow cytometry was performed on PBMCs from NHPs immunized with the TLR7/8, TLR8 agonist, CpG ODN, or Gag-TLR7/8 conjugate after the fourth immunization. Cytokine analysis for IL-2, IFN-γ, or TNF-α was performed on CD45RA-CD95⁺ CD4⁺ or CD8⁺ T cells, as described in Fig. 2. (A) The frequencies of the total memory CD4⁺ T cell cytokine response from four individual animals in all vaccine groups, except CpG ODN (n = 3 animals). (B) Quality of CD4 memory response. The total memory CD4⁺ T cell cytokine response is further divided into seven distinct subpopulations producing any combination of IFN-γ, IL-2, or TNF-α; these data are shown as the means of their respective percentages from the four animals per group (except CpG ODN; n = 3 animals). (C) The frequencies of the total memory CD8⁺ T cell cytokine response from four individual animals (except CpG ODN; n = 3 animals). (D) The quality of the memory CD8⁺ T cell cytokine response is shown as the means from the four animals postimmunization with Gag-TLR7/8 conjugate Vaccine groups (A and C), •, Gag; ▴, Gag plus CpG; ▾, Gag plus TLR7/8; ▪, Gag plus TLR8; ○, Gag-TLR7/8 conjugate.