Poly(I:C) is an effective adjuvant for antibody and multi-functional CD4+ T cell responses to Plasmodium falciparum circumsporozoite protein (CSP) and αDEC-CSP in non human primates

Abstract

Development of a fully effective vaccine against the pre-erythrocytic stage of malaria infection will likely require induction of both humoral and cellular immune responses. Protein based vaccines can elicit such broad-based immunity depending on the adjuvant and how the protein is formulated. Here to assess these variables, non human primates (NHP) were immunized three times with Plasmodium falciparum (Pf) circumsporozoite protein (CSP) or CSP cloned into MG38, a monoclonal antibody that targets DEC-205 (αDEC-CSP), an endocytic receptor on dendritic cells (DCs). Both vaccines were administered with or without poly(I:C) as adjuvant. Following three immunizations, the magnitude and quality of cytokine secreting CD4+ T cells were comparable between CSP+poly(I:C) and αDEC-CSP+poly(I:C) groups with both regimens eliciting multi-functional cytokine responses. However, NHP immunized with CSP+poly(I:C) had significantly higher serum titers of CSP-specific IgG antibodies and indirect immunofluorescent antibody (IFA) titers against Pf sporozoites. Furthermore, sera from both CSP or αDEC-CSP+poly(I:C) immunized animals limited sporozoite invasion of a hepatocyte cell line (HC04) in vitro. To determine whether CSP-specific responses could be enhanced, all NHP primed with CSP or αDEC-CSP+poly(I:C) were boosted with a single dose of 150,000 irradiated Pf sporozoites (PfSPZ) intravenously. Remarkably, boosting had no effect on the CSP-specific immunity. Finally, immunization with CSP+poly-ICLC reduced malaria parasite burden in the liver in an experimental mouse model. Taken together, these data showing that poly(I:C) is an effective adjuvant for inducing potent antibody and Th1 immunity with CSP based vaccines offers a potential alternative to the existing protein based pre-erythrocytic vaccines.

Copyright © 2010 Elsevier Ltd. All rights reserved.

Figures

Figure 1. Frequency of IFN-γ producing cells after immunization with CSP or αDEC-CSP + poly(I:C)

NHP were immunized three times with CSP or αDEC-CSP with or without poly(I:C) at weeks 0, 5 and 13. As controls, animals were immunized with poly(I:C) alone. PBMCs were analyzed for production of IFN-γ by ELISPOT assay at weeks 2, 7, 15 (two weeks after each immunization) and 26 post primary immunization. At week-27 all animals were boosted with 150,000 irradiated PfSPZ i.v.. As controls for the boost 5 animals were immunized with irradiated PfSPZ alone. At week-2 post irradiated PfSPZ boost (week-29) PBMCs were analyzed for production of IFN-γ by ELISPOT assay. Shown are the IFN-γ SFC/106 PBMCs for individual animals in each group (4–5 animals/group). The # denotes p<0.05 using non-parametric Wilcoxon Rank test comparing CSP with CSP + poly(I:C) and αDEC-CSP with αDEC-CSP + poly(I:C) immunized groups respectively.

Figure 2. Magnitude and quality of CSP-specific CD4+ T cell responses following immunization with CSP or αDEC-CSP + poly(I:C)

PBMCs were analyzed by multi-parameter flow cytometry at week-2 following the 3rd immunization (week-15). The frequency of total cytokine producing CD95+CCR7 hi and lo CD4+ T cells (IFN-γ, IL-2 or TNF-α) was analyzed (A). Data are shown for individual animals in each group. Using SPICE analysis the cytokine producing CD4+ T cell responses were divided into 7 distinct subpopulations producing any combination of IFN-γ, IL-2, and TNF-α. The total frequency of combination cytokine producing CD4+ T cells were analyzed for groups immunized with CSP + poly(I:C) (B) and αDEC-CSP + poly(I:C) (C). Pie chart representation of distinct combinations of cytokine producing CD4+ T cells is shown for individual animals from CSP + poly(I:C) immunized group (D) and αDEC-CSP + poly(I:C) group (E). The pie charts highlighted in boxes show the mean frequency for each group. The # denotes p<0.05 using non-parametric Wilcoxon Rank test comparing CSP with CSP + poly(I:C) and αDEC-CSP with αDEC-CSP + poly(I:C) immunized groups respectively.

Figure 3. Immunization with CSP + poly(I:C) elicits high titer of CSP-specific IgG antibodies

CSP-specific IgG antibodies were assessed in the plasma or serum of animals immunized with CSP or αDEC-CSP with or without poly(I:C) at indicated time points post-immunization. Data is shown for 5 individual animals at 4 weeks following the 3rd immunization (week-17) with CSP + poly(I:C) (A) and αDEC-CSP + poly(I:C) (B). A comparison of CSP-specific antibodies was assessed pre-immunization and 4 weeks following the 2nd and 3rd immunization with CSP or αDEC-CSP with or without poly (I:C) (C). At week-27 post primary immunization NHP were boosted with 150,000 irradiated PfSPZ i.v.. CSP-specific IgG antibodies were assessed one week prior to irradiated PfSPZ boost and at weeks 2 and 11 post-boost in the indicated groups (D). The kinetics of CSP-specific IgG antibodies were determined in the plasma of animals immunized with CSP or αDEC-CSP with or without poly(I:C) and boosted with irradiated PfSPZ or irradiated PfSPZ only at indicated time points (E). Data shown in C, D and E is the mean ± S.D. for 4–5 animals/group. The asterisks denote p<0.05 using the unpaired student’s t-test with Welch’s correction.

Figure 4. Immunization with CSP + poly-ICLC reduces malaria parasite burden in mice upon challenge

C57BL/6 mice were immunized with 3 doses of CSP with or without poly-ICLC or poly-ICLC alone. At week-3 following the 3rd immunization CSP-specific IgG antibodies were assessed in the serum of all immunized animals as well as naïve controls (A). Data shown represents the mean ± S.D. for 4 – 8 animals/group. Two months following last immunization all animals were challenged with 10,000 chimeric Pb-Pf intravenously. At 40 hrs post-challenge the total frequency of CSP-specific cytokine producing CD4+ T cell responses (IFN-γ, IL-2 or TNF-α) were measured in the spleen using multi-parameter flow cytometry as described in Fig. 2(B). The total frequency and pie chart representation of combination cytokine producing CD4+ T cells are shown for individual animals immunized with CSP + poly-ICLC (C and D). The pie chart highlighted in the box shows the mean frequency for the group (C). P. berghei 18S rRNA copies were measured in the liver extracts of immunized animals at 40 hrs post-challenge (E). The asterisks denote p<0.05 using the unpaired student’s t-test with Welch’s correction. The # denotes p<0.05 using the non-parametric Wilcoxon Rank test.