Praziquantel facilitates IFN-γ-producing CD8+ T cells (Tc1) and IL-17-producing CD8+ T cells (Tc17) responses to DNA vaccination in mice

Abstract

Background: CD8(+) cytotoxic T lymphocytes (CTLs) are crucial for eliminating hepatitis B virus (HBV) infected cells. DNA vaccination, a novel therapeutic strategy for chronic virus infection, has been shown to induce CTL responses. However, accumulated data have shown that CTLs could not be effectively induced by HBV DNA vaccination.

Methodology/principal findings: Here, we report that praziquantel (PZQ), an anti-schistoma drug, could act as an adjuvant to overcome the lack of potent CTL responses by HBV DNA vaccination in mice. PZQ in combination with HBV DNA vaccination augmented the induction of CD8(+) T cell-dependent and HBV-specific delayed hypersensitivity responses (DTH) in C57BL/6 mice. Furthermore, the induced CD8(+) T cells consisted of both Tc1 and Tc17 subtypes. By using IFN-γ knockout (KO) mice and IL-17 KO mice, both cytokines were found to be involved in the DTH. The relevance of these findings to HBV immunization was established in HBsAg transgenic mice, in which PZQ also augmented the induction of HBV-specific Tc1 and Tc17 cells and resulted in reduction of HBsAg positive hepatocytes. Adoptive transfer experiments further showed that PZQ-primed CD8(+) T cells from wild type mice, but not the counterpart from IFN-γ KO or IL-17 KO mice, resulted in elimination of HBsAg positive hepatocytes.

Conclusions/significance: Our results suggest that PZQ is an effective adjuvant to facilitate Tc1 and Tc17 responses to HBV DNA vaccination, inducing broad CD8(+) T cell-based immunotherapy that breaks tolerance to HBsAg.

Figure 1. Induction of DTH was mediated by CD8⁺ T cells in pcD-S2 and PZQ immunized group.

A: Twelve days after the second immunization with indicated the combinations, all groups were challenged with the rHBsAg in the right foot pad as test and with a saline solution in the left foot pad as control. The thickness of footpads was measured at 24, 48 and 72 h. B, Mice were depleted of CD4⁺ (CD4del) or CD8⁺ (CD8del) T cells by in vivo injection of specific mAbs. The efficiency of depletion was determined by flow cytometry of splenocytes. The numbers indicate the percentages of CD4⁺ or CD8⁺ T cells. C: Ten days after the second immunization of pcD-S2 plus PZQ, mice were injected i.p. with anti-CD4 mAb, anti-CD8 mAb, or rat IgG and challenged 2 days later with rHBsAg. DTH was measured at 24 h. D: The immunization schedule is shown. The data shown summarizes one of three experiments, all of which demonstrated similar results (* p<0.05; ** p<0.01).

Figure 2. Tc1 and Tc17 cells induction in response to HBsAg DNA vaccination with PZQ.

A–B, Splenic T cells were isolated on day 14 after the third immunization and were stimulated with the HBsAg-derived peptide S208-215 in the presence of brefeldin A (5 µg/ml) for 6 h in culture and immunostained for surface CD8, and intracellular IFN-γ and IL-17. C, Summary of the percentage of IFN-γ- and IL-17-expressing CD8⁺ T cells. D, Intracellular staining for IFN-γ and IL-17 in CD8⁺ T cells. C57BL/6 mice were immunized with pcD-S2 and PZQ and CD8⁺ T cells isolated on day 7 after third immunization were stimulated in vitro. Data shown are representative of 3 independent experiments (* p<0.05; ** p<0.01).

Figure 3. Impairment of DTH in IFN-γ KO mice and IL-17 KO mice immunized with pcD-S2 and PZQ.

Mice were challenged with rHBsAg 12 days after the second immunization. DTH was measured at 24, 48 and 72 h. A and B, DTH of IFN-γ and IL-17 KO mice, respectively. Data shown are representative of three independent experiments (* p<0.05; ns, p>0.05).

Figure 4. PZQ enhancement of the induction of Tc1 and Tc17 cells in HBsAg-transgenic mice.

A, Splenic T cells were isolated on day 14 after the third immunization and stimulated with S208-215 in the presence of brefeldin A (5 µg/ml) for 6 h in culture. CD8⁺ T cells were intracellularly immunostained for IFN-γ and IL-17. B, 12 days after the second immunization, DTH was measured at 24, 48 and 72 h later. Results were representative of three independent experiments (* p<0.05).

Figure 5. Histopathology of the liver and serology assays.

A: livers from each group were obtained on day 14 after the third immunization and fixed, sectioned, and stained with H&E. Bar, 50 µm. CD8-specific immunostaining of the liver from HBsAg transgenic mice on day 14 after final immunization. Bar, 20 µm. Specific immunostaining of HBsAg on day 14 after final immunization. Bar, 50 µm. B: Percentage of HBsAg-positive liver cells in indicated groups on day 14 after final immunization. C: HBsAg antigen in the serum on day 14 after final immunization. D: ALT activity in the serum on weeks 0, 1, 2, 3, and 4 after the third immunization. Results were representative of three independent experiments. There were four mice in each group (* p<0.05; ns, p>0.05).

Figure 6. Adoptive transfer of PZQ-induced CD8⁺ T cells.

A: In vivo cytotoxic lysis was performed in the wild type, IFN-γ KO and IL-17 KO mice on day 7 after the third immunization with pcD-S2 and PZQ. B: The percentage of specific lysis was summarized. C: HBsAg-specific immunostaining of HBsAg of livers from the HBsAg transgenic mice was analyzed on day 14 after adoptive transfer. Bar, 20 µm. D: Percentage of HBsAg-positive liver cells. E, ALT level in the serum was detected on weeks 0, 1 and 2 after adoptive transfer of CD8⁺ T cells. The data shown are representative of three independent experiments (* p<0.05; ** p<0.01; ns, p>0.05).