Unique Th1/Th2 phenotypes induced during priming and memory phases by use of interleukin-12 (IL-12) or IL-28B vaccine adjuvants in rhesus macaques

Abstract

Adjuvant compounds are usually included in vaccinations in order to bolster total vaccine-specific responses or to tailor an immune response toward a desired endpoint, such as the production of gamma interferon or an increase in antibody titers. While most adjuvants are studied in regard to their impact on vaccine-specific responses during and just after the vaccination period, a detailed analysis of how adjuvants skew the Th1/Th2 axis at more distant time points is not often undertaken. In the current study, we present data that suggests that adjuvants differ in their relative abilities to bolster and skew immune responses in the short term compared with more distant time points. To that end, we have employed interleukin-12 (IL-12) and IL-28B as adjuvants for DNA vaccination of rhesus macaques. While both adjuvants were able to bolster Th1-biased responses, our analysis shows that this skewing was achieved through different mechanisms. Moreover, analysis 3 months after the final immunization revealed the activity of the IL-12 adjuvant to be short lived, while the IL-28B adjuvant continued to exert its influence on the immune system. Taken together, these data suggest that the scientific and medical communities would benefit from a more detailed analysis of adjuvant function, including the determination of long-term influences of administered adjuvants.

FIG. 1.

Group design, immunization schedule, and gating strategy. (A) Rhesus macaques were divided into 3 groups that received various immunizations consisting of HIV antigens with or without IL-12 or IL-28B/IFN-λ3 adjuvant. (B) Animals were immunized three times 1 month apart, with an initial immune analysis 2 weeks after the final vaccination. Immune analysis also occurred 3 months after the final vaccination. (C) A representative gating strategy is shown for flow cytometry-based identification of IFN-γ- and IL-4-positive lymphocytes (in this case CD4⁺ T cells). FSC, forward scatter; SSC, side scatter.

FIG. 2.

Analysis of the effects of the IL-12 and IL-28B/IFN-λ3 adjuvants on antigen-specific IFN-γ and IL-4 production in T cells. (A) Two weeks after the final vaccination, peripheral CD4⁺ and CD8⁺ T cells were assayed for the production of IL-4 and IFN-γ. (B) Total IL-4 and IFN-γ responses are graphed to allow intragroup and intergroup comparison of responses. *, P < 0.05 for intragroup comparison of IL-4 and IFN-γ responses. IFNg, IFN-γ.

FIG. 3.

Analysis of the effects of IL-12 and IL-28B/IFN-λ3 adjuvants on long-lived responses from T cells. (A) Three months after the final vaccination, peripheral CD4⁺ and CD8⁺ T cells were again assayed for the production of IL-4 and IFN-γ. (B) Total IFN-γ production from all T cells (top left) and IL-4 production from all responding T cells (top right) are shown. Relative contributions of IFN-γ and IL-4 to the T cell immune response are shown by group (bottom). *, P < 0.05 for intragroup comparison of IL-4 and IFN-γ responses. IFNg, IFN-γ.

FIG. 4.

Cumulative overview of the ability of the IL-12 and IL-28B/IFN-λ3 adjuvants to affect the Th1/Th2 axis in the short term and long term. (A) Pie charts show relative contributions of Th1 and Th2 cytokines to the total T cell response, as well as broken down into CD8⁺ and CD4⁺ T cell responses, 2 weeks after the final immunization. (B) Pie charts show relative contributions of Th1 and Th2 cytokines to the total T cell response, as well as broken down into CD8⁺ and CD4⁺ T cell responses, 3 months after the final immunization. (C) Before-and-after plots show the Th1-skewing capabilities of the adjuvants 2 weeks after the final immunization in comparison with the Th1 levels 3 months after the final immunization in CD8⁺ T cells, CD4⁺ T cells, and total T cells. Tables display the hierarchies of Th1-biased responses driven by the adjuvants.