IL-10 blockade facilitates DNA vaccine-induced T cell responses and enhances clearance of persistent virus infection

Abstract

Therapeutic vaccination is a potentially powerful strategy to establish immune control and eradicate persistent viral infections. Large and multifunctional antiviral T cell responses are associated with control of viral persistence; however, for reasons that were mostly unclear, current therapeutic vaccination approaches to restore T cell immunity and control viral infection have been ineffective. Herein, we confirmed that neutralization of the immunosuppressive factor interleukin (IL)-10 stimulated T cell responses and improved control of established persistent lymphocytic choriomeningitis virus (LCMV) infection. Importantly, blockade of IL-10 also allowed an otherwise ineffective therapeutic DNA vaccine to further stimulate antiviral immunity, thereby increasing T cell responses and enhancing clearance of persistent LCMV replication. We therefore propose that a reason that current therapeutic vaccination strategies fail to resurrect/sustain T cell responses is because they do not alleviate the immunosuppressive environment. Consequently, blocking key suppressive factors could render ineffective vaccines more efficient at improving T cell immunity, and thereby allow immune-mediated control of persistent viral infection.

Figure 1.

IL-10R blockade enables effective stimulation of antiviral T cell responses by therapeutic vaccination. (A) Schematic representation of anti–IL-10R antibody treatment and DNA vaccination. LCMV-Cl 13–infected mice were treated with an isotype control antibody, treated with isotype control antibody and DNA vaccine (encoding the entire LCMV-GP), treated with an anti–IL-10R blocking antibody, or cotreated with DNA vaccine plus anti–IL-10R antibody. Anti–IL-10R treatment was initiated on day 25 after infection and continued every 3 d for 2 wk. DNA vaccination was administered on day 29 and 34 after infection. T cell responses were then analyzed on day 39 after infection. (B) Cytokine production was quantified by ex vivo peptide stimulation and intracellular staining. Flow plots are gated on CD8 T cells and illustrate the frequency of IFNγ- and TNFα-producing, LCMV-GP_33-41–specific CD8 T cells. Data are representative of four to five mice per group. (C) The graphs indicate the number of IFNγ-producing LCMV-GP_33-41-, GP_276-286-, and NP_396-404-specific CD8 T cells after each treatment regimen. Circles represent individual mice. *, P < 0.05 compared with untreated and DNA vaccination alone. **, P < 0.05 compared with all other treatment groups. (D) The bar graph represents the mean fold increase in the number of TNFα-producing, LCMV-GP_33-41–specific CD8 T cells in each treatment group compared with isotype treatment (which is set to 1). Values are the mean ± the SD of three experiments using four to six mice. *, P < 0.05 compared with untreated and DNA vaccination alone; **, P < 0.05 compared with all other treatment groups.

Figure 2.

CD4 T cell responses are enhanced by IL-10R blockade and vaccination. (A) LCMV-Cl 13–infected mice were treated and analyzed as described in Fig. 1. The frequency of cytokine-producing, LCMV-GP_61-80–specific CD4 T cells was quantified by ex vivo peptide stimulation and intracellular staining. Flow plots are gated on CD4 T cells and illustrate the frequency of IFNγ- and IL-2–producing, LCMV-GP_61-80–specific CD4 T cells after each treatment (39 d after infection). Data are representative of 4–5 mice per group. (B) The graph illustrates the number of IFNγ-producing, LCMV-GP_61-80–specific CD4 T cells (quantified as described in Fig. 1 C). *, P < 0.05 compared with untreated and DNA vaccination alone. **, P < 0.05 compared with all other treatment groups. (C) The bar graph illustrates the mean fold increase in the number of IL-2–producing, LCMV-GP_61-80–specific CD4 T cells after each treatment regimen compared with isotype treatment. Individual bars represent the mean value ± the SD of 3 experiments containing for 4–6 mice per group. *, P < 0.05 compared with untreated and DNA vaccination alone. **, P < 0.05 compared with all other treatment groups.

Figure 3.

IL-10R blockade combined with vaccination restores T cell function. Before infection, mice were seeded with LCMV-specific, Thy1.1⁺, TCR tg CD8⁺ (P14; A), and CD4⁺ (SMARTA; B) T cells and infected with LCMV-Cl 13. Mice were treated with isotype control antibody, anti–IL-10R blocking antibody, and/or DNA vaccine, as described in Fig. 1 A. Bar graphs indicate the fold increase in the number of P14 and SMARTA cells on day 39 after infection. Individual bars represent the mean ± the SD of five mice per group. *, significant (P < 0.05) increase in the mean number versus isotype and DNA vaccine alone. **, significant (P < 0.05) increase versus all other conditions.

Figure 4.

Accelerated control of persistent viral infection by alleviating the immunosuppressive environment and vaccinating to stimulate T cell responses. (A) LCMV-Cl 13–infected mice were infected and treated as described in Fig. 1 A. Serum viral titers were quantified on day 25 (gray circles, before treatment) and day 40 (white circles, after the completion of all therapeutic treatments) after infection. Each circle represents the virus titers in a single mouse on day 25 and 40 after infection. The dashed line indicates the level of detection of the plaque assay (200 PFU/ml). *, indicates a significant (P < 0.05) decrease in virus titers on day 40 after infection compared with untreated or to DNA vaccination alone. **, indicates a significant (P < 0.05) decrease in virus titers on day 40 after infection compared with isotype antibody treatment, DNA vaccine alone, or IL-10R antibody treatment alone. The numbers above each group indicate the mean fold decrease in virus titers between day 25 and 40 after infection. The graph contains data from three separate experiments. (B) The graph illustrates the virus titer in the liver of individual mice 3 d after the withdrawal of therapy. Each circle represents the liver virus titers in an individual mouse on day 40 after infection. The dashed line indicates the level of detection of the plaque assay (200 PFU/ml). *, indicates a significant (P < 0.05) decrease in virus titers on day 40 after infection compared with untreated mice or with DNA vaccination alone. **, indicates a significant (P < 0.05) decrease in virus titers on day 40 after infection compared with isotype antibody treatment, DNA vaccine alone, or IL-10R antibody treatment alone. (C) The graph illustrates a longitudinal analysis of the mean fold decrease in serum virus titers after isotype antibody treatment (white circles), IL-10R antibody blockade (gray circles), or IL-10R antibody blockade plus DNA vaccination (black circles). The mean fold decrease in each group was determined by dividing the pretreatment virus titers in each mouse (on day 25 after infection) by the virus titer in the same mouse on the indicated day after infection. Each circle represents the mean fold decrease ± the SD in viral titers of four to five mice per group. The gray box indicates the duration of antibody treatment. (D) Longitudinal analysis of serum viral titers were performed after LCMV-Cl 13 infection after isotype antibody treatment (white circles), IL-10R antibody blockade (gray circles), or IL-10R antibody blockade plus DNA vaccination (black circles). To better standardize virus titers, all groups were initially normalized to the virus titer of the isotype-treated group on day 25 after infection (i.e., before treatment). Each circle represents the mean virus titer of 4–5 mice on the indicated day after infection. The gray box indicates the duration of antibody treatment. The dashed line indicates the level of detection of the plaque assay (200 PFU/ml).