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.

Introduction

Malaria is an infectious disease of tropical regions infecting approximately 500 million people and causing 1–2 million deaths each year [1]. Adults living in malaria endemic region acquire a form of adaptive immunity over time which provides protection against clinical disease, but is dependent upon continuous exposure to the parasite for its maintenance [2]. Among the five species of Plasmodium known to infect humans, P. falciparum (Pf) is the leading cause of morbidity and mortality. While public health measures such as insecticide treated bed nets and anti-malarial therapy have significant effects on morbidity and mortality, vaccines offer the most compelling intervention for effective and durable prevention of this infection.

At present, the most clinically advanced pre-erythrocytic malaria vaccine candidate uses circumsporozoite protein (CSP), which is expressed abundantly on the surface of the sporozoite stage of the parasite [3], [4]. CSP-specific antibodies [5], CD8+ and CD4+ T cells [6], [7], [8] have been shown to elicit protective immunity in mouse models of malaria. In humans it seems clear that antibodies against CSP may be necessary but not entirely sufficient for the protection seen. Indeed, CSP-specific Th1 responses have also been suggested to correlate with protection in humans following vaccination or natural infection [9], [10]. Based on these findings there has been substantial effort to develop vaccines using CSP as an antigen. Currently, a phase III efficacy trial is underway using the RTS,S vaccine. RTS,S is a complex formulation comprised of two polypeptide chains of Pf CSP (amino acid 207–395) linked to hepatitis B surface antigen (HBsAg) to form a particle. This is then mixed with the TLR4 ligand, MPL and QS-21 in an oil-in-water (AS02A) or liposome (AS01B) formulation. Immunization of malaria naïve individuals with RTS,S and AS02A or AS01B induces CSP-specific CD4+ T cells and humoral immune responses with ∼30–50% efficacy [11]. Importantly, Th1 and CSP-specific humoral immunity are increased with AS01B compared to AS02A [11], [12], [13], [14] suggesting that the vaccine formulation may have a critical role in optimizing immunity. Finally, protective immunity induced following immunization with RTS,S appears to wane over time and is not boosted upon natural infection [15]. Thus, developing alternative CSP based vaccines with improved adjuvants, formulations or both may improve the durability of humoral and T cell immunity and enhance protection.

In terms of formulations, recombinant proteins can be administered as soluble antigens or as a particle such as the RTS,S vaccine. Alternatively, more efficient processing and presentation of proteins with increase in immunogenicity can be achieved by targeting the protein directly to dendritic cells (DCs) through monoclonal antibodies against cell surface receptors [16]. In this regard, DEC-205, an endocytic receptor expressed at high levels on lymphoid tissue DCs has been extensively characterized for targeting protein antigens in mice [16], [17], [18]. Indeed, DEC-205 mediated delivery of protein antigens improves the induction of both Th1 and CD8+ T cell responses in mouse models [16], [19]. T cell immunity with αDEC-205 requires poly(I:C) as an adjuvant [17]. Poly(I:C), a synthetic double stranded RNA, is a potent inducer of IL-12 and type I IFNs through activation of innate immunity via endosomally expressed TLR3 and the cytoplasmic receptor MDA-5 [20]. Moreover, poly(I:C) through induction of type I IFNs enhances DC maturation and B cell activation leading to induction of potent CD4+ T cell and humoral immune responses, respectively, in mice with protein antigens [21], [22], [23]. Finally, type I IFN is critical for cross presentation of protein antigens to generate CD8+ T cell responses in mice [24], [25]. Collectively, these data strongly support poly(I:C) as an adjuvant for improving humoral and cellular immunity with protein based vaccines. While the ability of poly(I:C) to induce broad-based immunity in mice has been established with protein and αDEC vaccines, there is only initial data on the potency of poly(I:C) as an adjuvant in NHP [26]. As innate immune mechanisms are far more similar between humans and NHP than mice, evaluation of NHP may provide a more predictive model for what would be observed in humans.

The primary aim of this study was to compare the adaptive immune responses generated in NHP following immunization with CSP or αDEC-CSP (CSP cloned into the carboxyl terminus of the heavy chain of mAb against DEC-205) with or without poly(I:C) as adjuvant. In addition, as prime-boost immunization with heterologous vaccine formulations has been shown to enhance immunity in a variety of experimental settings compared to either vaccine modality alone, all animals immunized with CSP or αDEC-CSP with or without poly(I:C) were boosted with irradiated PfSPZ. As irradiated PfSPZ are the gold standard for eliciting complete protection in humans, this study provides the first assessment of how they would influence an existing CSP-specific response in NHP. Finally, to determine whether this vaccine approach could have a biologic effect in vivo, C57BL/6 mice were immunized with CSP with or without poly-ICLC [26] and challenged with chimeric P. berghei sporozoites expressing Pf CSP (Pb-Pf) [27]. Overall, this study shows that poly(I:C) is an effective adjuvant for inducing potent multi-functional CD4+ T cells and antibody responses in mice and NHP with CSP. Thus, poly(I:C) offers a promising adjuvant for application in humans as part of a pre-erythrocytic Pf vaccine regimen with protein based vaccines.