HIV Gag protein conjugated to a Toll-like receptor 7/8 agonist improves the magnitude and quality of Th1 and CD8+ T cell responses in nonhuman primates

Abstract

Induction and maintenance of antibody and T cell responses will be critical for developing a successful vaccine against HIV. A rational approach for generating such responses is to design vaccines or adjuvants that have the capacity to activate specific antigen-presenting cells. In this regard, dendritic cells (DCs) are the most potent antigen-presenting cells for generating primary T cell responses. Here, we report that Toll-like receptor (TLR) agonists and ligands that activate DCs in vitro influence the magnitude and quality of the cellular immune response in nonhuman primates (NHPs) when administered with HIV Gag protein. NHPs immunized with HIV Gag protein and a TLR7/8 agonist or a TLR9 ligand [CpG oligodeoxynucleotides (CpG ODN)] had significantly increased Gag-specific T helper 1 and antibody responses, compared with animals immunized with HIV Gag protein alone. Importantly, conjugating the HIV Gag protein to the TLR7/8 agonist (Gag-TLR7/8 conjugate) dramatically enhanced the magnitude and altered the quality of the T helper 1 response, compared with animals immunized with HIV Gag protein and the TLR7/8 agonist or CpG ODN. Furthermore, immunization with the Gag-TLR7/8 conjugate vaccine elicited Gag-specific CD8+ T responses. Collectively, our results show that conjugating HIV Gag protein to a TLR7/8 agonist is an effective way to elicit broad-based adaptive immunity in NHPs. This type of vaccine formulation should have utility in preventive or therapeutic vaccines in which humoral and cellular immunity is required.

Fig. 1.

IFN-γ- and IL-2-producing cells are increased after immunization with HIV Gag protein and a TLR7/8 agonist or CpG ODN. NHPs were immunized four times at 4-week intervals with HIV Gag protein, with or without CpG ODN, TLR7/8, or TLR8 agonist. An additional group was immunized in a similar manner with HIV Gag protein conjugated to the TLR7/8 agonist (Gag-TLR7/8 conjugate). PBMCs were harvested at various times postimmunization, and the frequency of IFN-γ- (A) and IL-2- (B) producing cells was determined by ELISPOT assay. Data show the means (±SD) of five animals for all time points except week 20 (Gag plus CpG ODN = 1 animal only). P < 0.05, compared with Gag group (*) or compared with all other groups (**). ↑ indicates time of immunization; ND, not determined.

Fig. 2.

Functional characterization of IL-2-, IFN-γ- or TNF-α-producing memory T cells by multiparameter flow cytometry. PBMCs were analyzed after the fourth immunization by nine-color flow cytometry. The gating strategy is shown on a representative animal immunized with Gag-TLR7/8 conjugate. The pie charts show the quality of the cytokine response, comprised of seven functionally distinct populations producing IL-2, IFN-γ, and TNF-α, individually or in any combination. The percentages shown are based on the production of the respective cytokines within the CD45RA-CD95⁺ CD4⁺ or CD8⁺ population. FSC, forward scatter; EMA, ethidium monoazide bromide; SSC, side scatter.

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.

Fig. 4.

Immunization with HIV Gag protein and the TLR7/8 agonist, CpG ODN or Gag-TLR7/8 conjugate elicits high-titer antibody responses. HIV Gag-specific antibodies were assessed in serum obtained from NHPs after the fourth immunization. Data are shown from four to five individual animals per group. *, P < 0.05.