Malaria is a major health problem and an effective vaccine is essential for the eradication of the disease. Despite extensive efforts, a malaria vaccine remains elusive due to the parasite's complex life cycle, diverse morphology, and immune system evasion mechanisms. Antibodies against C terminal domain of merozoite surface protein 1 (MSP1-19), a highly conserved protein and the main vaccine candidate for blood-stage malaria, can inhibit erythrocyte invasion by the parasite and alleviate the disease symptoms. However, MSP1-19 is poorly immunogenic and classic protein-in-adjuvant MSP1-19-based vaccine formulations failed to induce strong immune responses due to low immunogenicity and generation of ineffective antibodies. The aim of this study was to use hepatitis B virus core (HBc) particles to increase the immunogenicity of MSP1-19. HBc forms particles with protruding spikes and induces a strong and specific immune response against foreign epitopes inserted at the tips of the spikes. In addition, positioning of MSP1-19 on the particle can influence the accessibility of certain antibody binding sites, possibly altering elicited antibody fine specificity and vaccine efficiency. MSP1-19 domain was inserted into the middle of the HBc sequence so that it is displayed at the tips of the HBc particle. Two HBc-MSP1-19 constructs, having different insert flanking linkers, displayed soluble particle formation after bacterial expression and lysis optimization. The particles were purified and the suitability of these two constructs as malaria vaccine candidates was assessed. Firstly, binding of the conformational anti-MSP1-19 antibodies indicated that MSP1-19 domain in the chimeric proteins has the correct disulphide bond pattern which is crucial for the protective properties of an MSP1-19-based vaccine. Furthermore, electron microscopy imaging and determination of initial 3D structures confirmed that both HBc MSP1-19 constructs form particles resembling the wild-type HBc particles, meaning the insertion of MSP1-19 did not heavily distort the overall HBc particle structure. In addition, it was shown that MSP1-19 domains are displayed at the tips of the particle spikes. Particle formation and foreign epitope display are important for the epitope's immunogenicity improvement. The immunogenicity of the chimeric particles was then assessed in mice. Both constructs elicited similar high antibody titres without the use of additional adjuvants, but no difference was observed between the particulate constructs and a non-particulate control (an MSP1-19-based protein). Interestingly, although both HBc-MSP1-19 and non-particulate MSP1-19-elicited antibodies recognized native malarial parasite, only the particulate construct antibodies demonstrated a moderate parasite growth inhibition while the antibodies from the control group did not show parasite inhibition above the background levels. In conclusion, it was shown that MSP1-19 can be expressed in bacteria as a soluble correctly folded protein fused to HBc. More importantly, the fusion protein is capable of forming immunogenic particles which generate antibodies that recognize native MSP1 and inhibit parasite growth more effectively than the protein without the HBc. Therefore, this work lays grounds and supports further chimeric HBc-MSP1-19 research and development.

Natural exposure to Plasmodium falciparum’s asexual blood-stage results in protection against severe disease, but no vaccine using the widely-studied blood-stage antigens apical membrane antigen 1 (AMA1) or merozoite surface protein 1 (MSP1) has proven convincingly protective in clinical trials. Challenges include antigenic polymorphism, the apparent requirement for exceptionally high antibody concentrations for protection, and clinical-grade production of conformationally-accurate recombinant protein antigens followed by formulation with a human-compatible adjuvant. This thesis describes the generation of viral-vectored vaccines targeting ten less-studied blood-stage antigens, focusing upon antigens implicated in erythrocyte invasion. These vaccines were immunogenic in mice and rabbits. The rabbit antibodies raised were functionally active in the in vitro assay of parasite growth inhibitory activity (GIA). GIA with antibodies against one antigen, RH5, exceeded that achieved with antibodies against the ‘gold standard’ AMA1 or MSP1 antigens. This antigen’s amino acid sequence is relatively conserved between parasite strains. Importantly, and unlike anti-AMA1 and MSP1 antibodies, the GIA effects transcend genetically diverse strains. It was hypothesised that blockade of the interaction of RH5 with its receptor basigin was likely to be a mechanism of action of anti-RH5 antibodies. Vaccine-induced polyclonal anti-RH5 serum was found to be capable of blocking this interaction, as well as merozoite attachment to erythrocytes. A panel of RH5-specific monoclonal antibodies were raised: those which block the RH5-receptor interaction were capable of neutralising parasites. Minimal linear epitopes recognised by these antibodies were mapped, and are likely to be within or close to RH5’s receptor binding site. These data support prompt clinical testing of RH5-based vaccines, and shed light upon the mechanism of action of anti-RH5 antibodies. However substantial challenges remain in establishing whether this antigen, selected on the basis of the in vitro assay of GIA, will be capable of achieving in vivo protection against P. falciparum. Further work presented in this thesis addresses the use of quantitative PCR data to assess blood-stage vaccine efficacy in experimental human challenge with P. falciparum, and the use of surface plasmon resonance to establish more detailed characterisation of vaccine-induced antibody responses. Finally, the results of P. falciparum challenge of RH5-vaccinated Aotus nancymaae non-human primates are presented.

Immunity to asexual blood-stage of Plasmodium falciparum malaria is believed to be associated with protective antibodies of certain immunoglobulin classes and subclasses. This thesis addressed the importance of antibodies in relation to malaria infection and their effective interactions with Fc gamma receptor IIa (FcyRIIa) polymorphisms. Our data indicate that the frequency of FcyRIIa-R/R131 genotype was statistically significantly higher in Sudanese patients with severe malaria, while the FcyRIIa-H/H131 genotype showed a significant association with mild malaria. The levels of IgG1 and IgG3 subclass antibodies were statistically higher in the mild malaria patients. The Fulani ethnic group in West Africa has been shown to be relatively resistant to malaria. We investigated the possible impact of FcyRIIa polymorphisms in the Fulani and non-Fulani in Mali and Sudan, and analysed their malaria-reactive IgG subclass profiles. The FcyRIIa-H/H131 genotype and H131-allele were found to be prevalent in the Fulani while R131-allele was prevalent in non-Fulani. The Fulani had higher serum levels of IgG1-3, with higher proportion of IgG2 than the non-Fulani. Most clinico-epidemiology studies have been in areas with holo- and hyper-malaria endemicity. We have analysed antibody responses to a panel of six blood-stage antigens in relation to clinical malaria outcome in mesoendemic Sudan. Our results revealed a linear association with anti-AMA-1 IgG1 antibodies and reduced risk of severe malaria while a non-linear relationship with IgG3 antibodies was observed for MSP-2, MSP-3 and GLURP. In the combined final model, the highest levels of IgG1 subclass antibodies to AMA-1, GLURP-R0, and the highest levels of IgG3 subclass antibodies reactive to 3D7 MSP-2 were independently associated with a reduced risk of clinical malaria. Taken together, these data suggest a possible association between FcyRIIa-R/H131 and anti-malarial antibody responses in the clinical outcome of malaria.