Sporozoite vaccination of both humans and rodents elicits potent anti-malarial immunity, but the dose of sporozoites and the number of immunizations required varies with vaccination approach. Here we examine the immunological basis for superior protection afforded from single-dose vaccination with virulent sporozoites administered under prophylatic chloroquine-cover, referred to as infection-treatment-vaccination (ITV), compared to the well-studied approach of administering radiation-attenuated Plasmodium sporozoites (RAS). Earlier rodent studies utilizing ITV and RAS vaccination suggested a major role of CD8 T cells in reducing liver parasite burden after sporozoite challenge in a BALB/c mouse model. Consistent with this, we find that in C57Bl/6 mice ITV elicits substantially higher parasite-specific CD8 T cell responses than RAS vaccination and enhances immunity against P. yoelii infection. However, we show ITV-induced CD8 T cells are not necessary for protection following liver-stage sporozoite or blood-stage parasite challenge. Mechanistically, we found protection afforded from single-dose ITV is associated with low grade, transient parasitemia shortly following cessation of chloroquine treatment and generation of potent antibody responses to blood-stage parasites. Collectively, our data show the mechanistic basis for enhanced protective immunity against P. yoelli elicited by ITV in highly susceptible C57Bl/6 mice is independent of CD8 T cells. These studies may be relevant in understanding the potent immunity observed with ITV in humans.

Despite decades of research, malaria remains a global health crisis. Current subunit vaccine approaches do not provide efficient long-term, sterilizing immunity against Plasmodium infections in humans. Conversely, whole parasite vaccinations with their larger array of target antigens have conferred long lasting sterilizing protection to humans. Similar studies in rodent models of malaria reveal that CD8+ T cells play a critical role in liver-stage immunity after whole parasite vaccination. However, it is unknown whether all CD8+ T cell specificities elicited by whole parasite vaccination contribute to protection, an issue of great relevance for enhanced subunit vaccination. Here we show that robust CD8+ T cell responses of similar phenotype are mounted following prime-boost immunization against Plasmodium berghei GAP5041-48, S20318-325, TRAP130-138 or CSP252-260 protein-derived epitopes in mice, but only CSP252-260- and TRAP130-138-specific CD8+ T cells provide sterilizing immunity and reduce liver parasite burden following sporozoite challenge. Further, CD8+ T cells specific to sporozoite surface-expressed CSP and TRAP proteins, but not the intracellular GAP50 and S20 proteins, are efficiently recognized by sporozoite-infected hepatocytes in vitro. These results suggest that 1) protection-relevant antigenic targets, regardless of their immunogenic potential, must be efficiently presented by infected hepatocytes for CD8+ T cells to eliminate liver-stage Plasmodium infection and 2) proteins expressed on the surface of sporozoites may be good target antigens for protective CD8+ T cells.

Human infection with Plasmodium parasites remains a serious global health crisis, leading to more than 600,000 deaths annually. Currently, no licensed vaccine is available to alleviate this malaria disease burden and vaccination with the most advanced anti-malarial vaccine candidate, RTS,S, provides limited protection that wanes over time. To date, the only vaccination strategy capable of inducing complete, longlasting protection in human subjects is administration of attenuated whole sporozoites. Several approaches for vaccination with attenuated whole sporozoites have been clinically tested in humans and include vaccination with radiation or genetically attenuated sporozoites or with virulent sporozoites concurrent with administration of anti-malarial drug cover. Rodent studies with these three attenuated whole sporozoite vaccination approaches provide insights into the immune-correlates of vaccine-induced protection. The majority of these studies have identified a critical role for liver-stage parasite directed CD8 T cells in providing protection with possible contributions from Plasmodium-specific CD4 T cells or antibodies. Together, rodent and human vaccination studies with attenuated whole sporozoite vaccination may lead to an understanding of the correlates of protective immunity against malarial disease, and the development of new, highly efficacious vaccines.

CD8 T cells are critical mediators of protection against Plasmodium liver-stage infection. Most studies of the CD8 T cell response to whole parasite Plasmodium vaccines address a single T cell epitope in BALB/c mice, and thus provide limited information. Here, we describe a surrogate activation marker approach that uses the coordinate downregulation of the CD8α chain and upregulation of the integrin CD11a to track the total CD8 T cell response to Plasmodium vaccination via flow cytometry. With this approach, quantitative (magnitude, kinetics) and qualitative (distribution, phenotype, and function) features of the total CD8 T cell response to vaccination with attenuated Plasmodium or other pathogens can be studied.

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Despite decades of research, malaria remains a global health crisis. Currently, subunit vaccine approaches do not provide long-term, sterilizing immunity against Plasmodium infection. Conversely, immunization using whole parasites can confer protection in rodents through eliciting protective CD8 T cell responses, likely against a broad spectrum of Plasmodium antigens. However, it is unknown whether all of these CD8 T cell specificities contribute to protection. Recently, four novel H-2b-restricted Plasmodium-specific CD8 T cell epitopes were described from the following antigens: GAP50, PbT, S20, and TRAP. Here, we show that priming with peptide-loaded mature dendritic cells followed by boosting with recombinant L. monocytogenes expressing these epitopes results in memory antigen-specific CD8 T cell responses constituting 5-40% of the CD8 T cell compartment. To date, only TRAP-specific CD8 T cells generated through this vaccination approach can provide sterilizing immunity following a sporozoite challenge. This result occurs despite similar numbers, function and phenotype for each of the memory CD8 T cell populations. These data suggest that timing of antigen presentation during liver-stage infection coupled with a requirement for direct MHC presentation predicts the protective capacity of a CD8 T cell specificity. These data may aid in identifying novel target antigens to elicit sterilizing protection against Plasmodium infection.

Despite decades of research, malaria remains a global health crisis. Currently, the most advanced anti-malarial vaccine in human clinical trials (RTS,S) does not provide long-term, sterilizing immunity against Plasmodium infection. Plasmodium-specific CD8 T cells can provide sterilizing immunity to Plasmodium infection in rodent models. However, the numerical requirements for memory CD8 T cell protection against a defined epitope, P. berghei CS252-260 (a CSP epitope) exceeds 1% of peripheral blood lymphocytes - a 103-104 fold higher number than required for protection against some bacterial or viral infections. Recently, three novel H-2b-restricted Plasmodium-specific CD8 T cell epitopes were described from the following antigens: GAP50, S20, and TRAP. Priming with peptide-loaded mature dendritic cells followed by boosting with recombinant L. monocytogenes expressing these epitopes results in antigen-specific CD8 T cell responses constituting 0.3-6% of the PBL in CB6.F1 mice. The capacity of these single CD8 T cell responses to provide protective immunity in a stringent model of liver stage infection with P. berghei will be described. Moreover, we are determining whether combining multiple epitope-specific responses can reduce the total numerical requirement for CD8 T cell-mediated protection. These studies may aid in the preclinical design of a sterilizing CD8 T cell based anti-Plasmodium subunit human vaccine containing multiple CD8 T cell epitopes.