SIV antigen-specific effects on immune responses induced by vaccination with DNA electroporation and plasmid IL-12

Abstract

Molecular adjuvants are important for augmenting or modulating immune responses induced by DNA vaccination. Promising results have been obtained using IL-12 expression plasmids in a variety of disease models including the SIV model of HIV infection. We used a mouse model to evaluate plasmid IL-12 (pIL-12) in a DNA prime, recombinant adenovirus serotype 5 (rAd5) boost regimen specifically to evaluate the effect of IL-12 expression on cellular and humoral immunity induced against both SIVmac239 Gag and Env antigens. Priming with electroporated (EP) DNA+pIL-12 resulted in a 2-4-fold enhanced frequency of Gag-specific CD4 T cells which was maintained through the end of the study irrespective of the pIL-12 dose, while memory Env-specific CD4+T cells were maintained only at the low dose of pIL-12. There was little positive effect of pIL-12 on the humoral response to Env, and in fact, high dose pIL-12 dramatically reduced SIV Env-specific IgG. Additionally, both doses of pIL-12 diminished the frequency of CD8 T-cells after DNA prime, although a rAd5 boost recovered CD8 responses regardless of the pIL-12 dose. In this prime-boost regimen, we have shown that a high dose pIL-12 can systemically reduce Env-specific humoral responses and CD4T cell frequency, but not Gag-specific CD4+ T cells. These data indicate that it is important to independently characterize individual SIV or HIV antigen immunogenicity in multi-antigenic vaccines as a function of adjuvant dose.

Keywords: Adenovirus; Antibody; DNA; IL-12; T cell.

Fig. 1.

Frequency of CD4+ (A and B) and CD8+ (C and D) SIV-specific cytokine secreting T cells following DNA immunization. Mice were immunized twice, 4 weeks apart with SIV Gag and Env DNA (30μg) by IM or EP +/− pIL-12 (0.3μg). Splenocytes were harvested two weeks after the second immunization, incubated ex vivo for 5h with Gag pooled peptides, the dominant Gag peptides: DD13 (CD4) and AL11 (CD8) (A and C) or Env pooled peptides (B and D), anti-CD28 and brefeldin A before being analyzed by flow cytometry for production of IFNγ, IL-2 and TNFα. The total frequency of cytokine secreting T cells (%) producing IFNγ, IL-2 and TNFα are shown and bars represent the median, n = 4 and data are representative of 2 experiments. *p < 0.05, significantly different from animals immunized by IM.

Fig. 2.

Quality of SIV Gag-specific (A) and SIV Env-specific (B) CD4+ Th1 cells secreting IFNγ, IL-2 and TNFα in any combination after DNA immunization. Mice were immunized twice, 4 weeks apart with SIV Gag and Env DNA (30μg) by IM or EP +/− pIL-12 (0.3μg). Splenocytes were harvested two weeks after the second immunization, incubated ex vivo for 5h with Gag pooled peptides or the dominant peptide DD13 (A) or Env pool 1 and Env pool 2 peptides (B), anti-CD28 and brefeldin A before being analyzed by flow cytometry for production of IFNγ, IL-2 and TNFα. The frequency of cytokine secreting T cells (%) producing IFNγ, IL-2 and TNFα in any combination are shown with bars representing the median, n = 4 and data are representative of 2 experiments. *p < 0.05, significantly different from animals immunized by IM.

Fig. 3.

Effect of pIL-12 on the frequency of SIV Gag and Env-specific cytokine secreting T cells following EP immunization. Mice were immunized by EP twice, four weeks apart with Gag and Env plasmids and low dose (0.3μg) or high dose (25μg) pIL-12 before a boost with rAd5 vectors expressing Gag and Env four weeks after the second DNA immunization. Splenocytes were harvested at week 6 and week 10, incubated ex vivo for 5h with Gag pooled peptides or the dominant peptides DD13/AL11 (A/C) or Env pool peptides (B/D), anti-CD28 and brefeldin A before being analyzed by flow cytometry for combined production of IFNγ, IL-2 and TNFα. The frequency of multifunctional cytokine secreting Gag-specific (E) or Env-specific (F) CD4+ T cells (%) at week 10 producing IFNγ, IL-2 and TNFα in any combination are shown, bars represent the median, n =4 and data are representative of 2 experiments. *p < 0.05, significantly different from animals immunized by EP or ^#p < 0.05, significantly different from animals immunized by EP+pIL-12 low.

Fig. 4.

Frequency of memory Gag and Env-specific CD4+ (A) or CD8+ (C) T cells secreting IFNγ, IL-2 and TNFα or the quality of the memory CD4+ Gag-specific T cell response (B). Mice were immunized twice, 4 weeks apart with SIV Gag and Env DNA (30μg) by IM or EP +/− pIL-12 (0.3μg) before a boost with rAd5 vectors expressing Gag and Env. Splenocytes were harvested at week 14, incubated ex vivo for 5h with Gag pooled peptides, the dominant DD13/AL11 peptides or Env pooled peptides, anti-CD28 and brefeldin A before being analyzed by flow cytometry for total production of IFNγ, IL-2 and TNFα (A/C) or IFNγ, IL-2 and TNFα in any combination (B), bars represent the median, n = 4 and data are representative of 2 experiments. *p < 0.05, significantly different from animals immunized by IM.

Fig. 5.

Effect of high dose pIL-12 on the frequency (A) and quality (B/C) of memory CD4 and CD8 (D) SIV-specific cytokine secreting T cells. Mice were immunized twice, 4 weeks apart with SIV Gag and Env DNA (30μg) by EP with low dose (0.3μg) or high dose (25μg) pIL-12 before a boost with rAd5 vectors expressing Gag and Env. Splenocytes were harvested at week 14, incubated ex vivo for 5h with Gag pooled peptides, the dominant DD13/AL11 peptides or Env pooled peptides, anti-CD28 and brefeldin A before being analyzed by flow cytometry for total production of IFNγ, IL-2 and TNFα (A/D) or IFNγ, IL-2 and TNFα in any combination (B/C) with bars representing the median, n = 4 and data are representative of 2 experiments, *p < 0.05, significantly different from animals immunized by EP.

Fig. 6.

SIVmac239 (A) and cross reactive SIVmac251 (B) and SIVsmE660 (C) Env-specific IgG endpoint titers following DNA vaccination (week 6) and rAd5 boost (week 10) in mice immunized by IM or EP with SIV Env DNA +/− pIL-12. Serum was collected after vaccination and endpoint titers calculated by ELISA. Bars represent median endpoint titer, n = 4 and data are representative of 2 experiments. *p < 0.05, significantly different from animals immunized by EP+pIL-12 high, ^#p < 0.05, significantly different from week 6 to week 10.