Immunization with whole pre-erythrocytic (sporozoite and liver stage) Plasmodium falciparum (Pf) vaccines confers sterilizing immunity in human clinical trials. Replication-deficient vaccines, such as radiation- attenuated sporozoites infect the liver as sporozoites but do not develop into liver stage schizonts. Replication- competent vaccines, however, infect the liver and replicate as tissue schizonts. Multiple lines of evidence in animal models of malaria have shown that protection is dependent on antigen-specific CD8+ T cells that recognize liver stage-infected hepatocytes, leading to their elimination. Replication-competent parasite vaccination confers superior durable sterilizing immunity against infection, and this appears to be, in animal models, associated with broader and better CD8+ T cell responses. However, it remains largely unknown how the distinct molecular cell biological features of whole attenuated parasite vaccines drive differences in the priming of protective CD8+ T cells and of equal importance, which liver stage antigens are directly presented by wildtype liver stage-infected hepatocytes that are the targets of vaccine-elicited protective CD8+ T cells. We will address these critical knowledge gaps.
In Aim 1, we will identify the distinct time points during which the demise of liver stage-infected hepatocytes results in optimal cross-presentation of liver stage antigens by antigen presenting cells to CD8+ T cells. For this, we will use the Plasmodium yoelii (Py) rodent malaria model to inform vaccine design of Pf, with which mechanistic host studies cannot be done. We will also determine at what time points of wildtype liver stage development infected hepatocytes are most vulnerable to effector CD8+ T cell- mediated elimination. In concert with this, we will determine dynamic liver stage transcriptomes and proteomes throughout development and down-select the subset of liver stage proteins most prone to intrahepatocytic processing and MHC class I-restricted peptide presentation.
In Aim 2, we will directly determine the MHC class I peptidome of Py and Pf presented on infected hepatocytes, specifically at timepoints of highest vulnerability and test their reactivity with whole parasite-vaccine-elicited CD8+ T cells. We will then test reactive epitopes as well as nonreactive epitopes (covert epitopes) as vectored subunit vaccines in mice. As in Aim 1, mechanistic testing cannot be done in Pf and thus we will conduct studies of Py to guide our Pf work.
In Aim 3, we will genetically engineer the ultimate Pf replication-competent parasite strain that is built with gene deletions and dominant negative transgenes of parasite origin and will also over-express protective CD8+ T cell epitopes that target liver stages at the point of their greatest vulnerability. Thus, in a multi-pronged approach our project will develop the next generation of pre-erythrocytic vaccines including both vectored subunit vaccine candidates and whole genetically attenuated parasite vaccine candidates, designed to generate optimal and durable protective CD8+ T cell responses against Pf infection.
Live-attenuated Plasmodium parasite vaccines confer sterilizing immune protection against liver infection via CD8+ T cells, yet their molecular biological and immunological features that elicit this response and the target MHC class I epitopes of liver stage parasites remain largely unknown. We will elucidate protective features of replication-competent parasite vaccines and directly identify MHCI-restricted liver stage antigens from the surface of infected hepatocytes. This will allow us to build next generation vectored subunit vaccines as well genetically engineer optimally safe and potent live-attenuated vaccines.