Immunization with HIV Gag targeted to dendritic cells followed by recombinant New York vaccinia virus induces robust T-cell immunity in nonhuman primates

Abstract

Protein vaccines, if rendered immunogenic, would facilitate vaccine development against HIV and other pathogens. We compared in nonhuman primates (NHPs) immune responses to HIV Gag p24 within 3G9 antibody to DEC205 ("DEC-HIV Gag p24"), an uptake receptor on dendritic cells, to nontargeted protein, with or without poly ICLC, a synthetic double stranded RNA, as adjuvant. Priming s.c. with 60 μg of both HIV Gag p24 vaccines elicited potent CD4(+) T cells secreting IL-2, IFN-γ, and TNF-α, which also proliferated. The responses increased with each of three immunizations and recognized multiple Gag peptides. DEC-HIV Gag p24 showed better cross-priming for CD8(+) T cells, whereas the avidity of anti-Gag antibodies was ∼10-fold higher with nontargeted Gag 24 protein. For both protein vaccines, poly ICLC was essential for T- and B-cell immunity. To determine whether adaptive responses could be further enhanced, animals were boosted with New York vaccinia virus (NYVAC)-HIV Gag/Pol/Nef. Gag-specific CD4(+) and CD8(+) T-cell responses increased markedly after priming with both protein vaccines and poly ICLC. These data reveal qualitative differences in antibody and T-cell responses to DEC-HIV Gag p24 and Gag p24 protein and show that prime boost with protein and adjuvant followed by NYVAC elicits potent cellular immunity.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Immunization with DEC HIV Gag p24 or nontargeted HIV Gag p24 protein and poly ICLC elicits IFN-γ–producing T cells. (A) NHPs (four per group) were immunized s.c. three times at wks 0, 8, and 27 with DEC Gag p24 or HIV Gag p24 and poly ICLC. Additional groups were DEC Gag p24 only (n = 2), HIV Gag p24 protein only (n = 3), or DEC “empty” and poly ICLC (n = 2). IFN-γ elispots (SFC)/106 PBMCs were quantified after stimulation by HIV Gag p24 pooled peptides. (B) As in A but isolated, CD8+ T cells were analyzed after the second and third immunizations (means of four NHPs analyzed in triplicate ± SE from a single experiment. **P < 0.05 from animals immunized with HIV Gag p24 protein and poly ICLC.

Fig. 2.

Breadth of HIV Gag p24 responses. As in Fig. 1A, but IFN-γ responses to individual peptides spanning the entire HIV Gag p24 protein at wk 14 by ELISPOT with each animal as a separate plot. The number of HIV Gag peptides with >55 SFC/106 cells (dotted line) is noted for each animal.

Fig. 3.

Features of HIV Gag-specific CD4+ and CD8+ T-cell cytokine responses. (A) Frequency of Gag-specific cytokine (IFN-γ, IL-2, or TNF-α) producing CD4+ and CD8+ T cells in PBMCs by multiparameter flow cytometry at indicated times. (B) As in A, but polyfunctionality of the cytokine responses in frozen PBMCs in a batch analysis, was assessed by SPICE analysis and depicted as pie charts to show relative proportions of seven populations producing different combinations of IFN-γ, IL-2, or TNF-α. Mean of each population's percentages from four NHPs per group at wks 10 and 29. # denotes a difference from animals immunized with HIV Gag p24 protein and poly ICLC (P < 0.05) at wk 29. (C) CFSE-labeled PBMCs were cultured 5 d ± the entire pool of HIV Gag p24 peptides. The entire pool of HIV Gag peptides was added for 12 h and the frequency of IFN-γ–producing cells assessed by flow cytometry. Dots are individual animals and bars the interquartile range.

Fig. 4.

Antibody binding titers and avidity following protein vaccines. (A) HIV Gag-specific antibodies in serum at various times postimmunization. (B) Surface plasmon resonance binding avidity map to Gag p24 in RU versus dissociation rate (kDa, s-1). Ranking of relative avidity is based on response magnitude (binding response in RU) and dissociation rate (kDa, s-1). Binding RU and kDa of the control antibody HIVIG at 10 mg/mL is the red circle. Higher avidity antibody responses show higher binding RU and slower kDa values. Ranking of groups based on their relative avidity is outlined in clusters.

Fig. 5.

Magnitude of HIV Gag-specific CD4+ and CD8+ T-cell IFN-γ responses after prime-boost immunization. As in Fig. 3, HIV Gag-specific IFN-γ–producing CD4+ (A) and CD8+ (B) T cells at 31 wk after the third immunization with the protein vaccines and poly ICLC, which was the time of the NYVAC HIV Gag/Pol/Nef boost (wk 0) and then 2, 6, and 10 wk later. Results are from frozen PBMCs analyzed at the same time. The box plots show the mean of each group. Dots are from individual animals and bars, the interquartile range. (C) As in A and B but responses are shown before and 2 wk after a second boost of NYVAC, which was given 16 wk after the first dose.

Fig. 6.

Breadth of HIV Gag-specific CD8+ T-cell IFN-γ responses before and after boosting with replication defective NYVAC. The breadth analysis is shown by pooling responses by each NHP in each vaccine group before and after NYVAC boost.