The identification of antigens targeted by protective immune responses and development of vaccines against complex pathogens based on these antigens has been problematic. In particular, vaccines inducing strong cellular immune responses against pathogens for which cell mediated immunity is implicated in protection, such as the Plasmodium spp. parasite, remain elusive. Plasmodium spp. has a multi-stage life cycle, expressing ~5,300 putative proteins in a stage-specific manner. Infection with attenuated sporozoites whose development is arrested in the liver, and continuous natural exposure to the Plasmodium parasite, both induce protective immunity, thereby demonstrating the feasibility of successful vaccination against malaria. However, the antigens targeted by these protective immune responses have not been identified. Immunomics offers a potential solution for the identification of novel antigenic targets from genome sequence information based on reactivity with biological samples, such as sera or PBMC.
The hypothesis tested in this thesis was that proteins identified using immunomics platforms might be excellent targets for next-generation malaria vaccines. Thus, the overall objective of this thesis was to evaluate novel antigens identified in genome-wide screening approaches for immunogenicity and protective capacity against Plasmodium parasite challenge, in a rodent model system. The specific aims were (1) to develop and optimise methods for in vivo and in vitro validation of Plasmodium antigens; (2) to evaluate antigens identified via serological screening; and (3) to evaluate antigens identified via cellular screening. Target antigens were selected from (i) an antibody-based screening platform using protein microarrays to profile antigen-specific responses in protected and un-protected populations; and (ii) from a novel T cell epitope-based PBMC screening platform identifying antigenic targets of naturally acquired IFN-γ responses. Specific questions addressed by individual chapters included whether antigens identified in serological screening approaches are targets of cellular immunity and whether antigens targeted by naturally acquired cellular responses can induce sterile protection.
In total, I have evaluated eight novel previously uncharacterized proteins identified using two different immunomics approaches and established all of these proteins as targets of cell-mediated immunity and protection in a rodent malaria model. Robust sterile protection was induced by two of the antigens identified as targets of naturally acquired cellular immune responses. The combination of two other antigens synergistically enhanced protective capacity of the individual antigens. Furthermore, two partially protective antigens induced cross-species reactive immunity between P. yoelii and P. falciparum species. Importantly, protective capacity for all our antigens was significantly correlated to the induction of IFN-γ/IL-2/TNF multifunctional T cell populations.
Additionally, to facilitate validation of novel antigens in challenge models and in vitro assays, I tested and optimised novel high-throughput cloning and protein expression systems, and methodologies for evaluation of immunogenicity and liver-stage parasite burden.
Taken together, the herein described findings provide new tools for the in vivo evaluation of novel vaccine candidates and identify eight novel antigens as targets of cellular immunity and protective immune responses in a rodent malaria model. Thus, this thesis consolidates the use of immunomics for antigen identification and provides a base for further evaluation of multifunctional T cell responses and novel antigenic targets for the development of next-generation malaria vaccines.