Quantifying immunological memory and optimising immunoprofiling platforms in Plasmodium falciparum malaria

Plasmodium falciparum (Pf) malaria remains one of the world’s most detrimental infectious diseases and the search for a highly effective vaccine remains a global priority. Human malarial infection is known to require repeated infections and sustained exposure to develop immunity, and although sterile immunity is not typically achieved, individuals develop naturally acquired immunity (NAI) that offers protection against disease. Both the humoral and cellular immune responses play vital roles in this process, although the underlying mechanisms – particularly those governing the development and maintenance of immunological memory – are not fully understood. Given the complexity of both innate and adaptive responses and their association with protection, there is a need for deeper investigation using sensitive and innovative tools. This thesis aims to identify key knowledge gaps in our understanding of adaptive immunity to malaria, and to optimise methodologies that support current and future research. The central focus is to altogether better characterise the generation and persistence of immune memory in malaria. First, and following a review of immunological memory in malaria, the process of memory B cell (MBC) and antigen-specific antibody development following malaria exposure was explored. Using a longitudinal cohort from a low transmission setting of The Gambia, responses to a broad panel of Pf antigens representing diverse parasite life stages were analysed. Antibody responses remained detectable and durable over a four-year period, with variations in titre and avidity. Antigens associated with cumulative exposure elicited higher responses than those linked to recent infection. Notably, MBCs were consistently detectable even when corresponding antibody levels were low or of low affinity, underscoring the durability of the cellular basis of humoral memory. Building on this, the thesis presents the first evidence of gametocyte antigen-specific MBCs. In a cross-sectional cohort from the same setting, memory responses to sexual stage antigens were detected at frequencies comparable to those of asexual antigens. Interestingly, correlations between MBC and antibody responses were weaker for sexual stage antigens, suggesting distinctive immunological dynamics. These 4 antigens also showed potential as markers of recent infection, with promising implications for seroepidemiological applications. Second, the thesis focusses on the development of tools to investigate immune responses to Pf malaria. A customised multiplex platform was optimised to assess antibody responses to linear B cell epitopes of a Pf antigen in symptomatic and asymptomatic individuals from Ghana and Burkina Faso. The platform revealed substantial differences in responses to epitope peptides versus full-length proteins, and highlighted the importance of assay configuration – including peptide length and spacer inclusion – in enhancing sensitivity. Critical in protection against disease is the T cell coordination and effector response. To examine T cell responses, a flow cytometry assay capable of detecting the activation of CD4+ and CD8+ T cell subsets following Pf-specific stimulation was optimised. The utility of this assay was demonstrated, along with a discussion of its advantages and disadvantages relative to conventional cytokine release assays. In summary, the thesis presents robust evidence for the generation and persistence of immunological memory to Pf malaria and refines platforms for its investigation. It reports the most comprehensive panel of Pf antigens assessed to date in a single study and provides the first demonstration of MBC responses to sexual stage antigens. These findings support a more nuanced understanding of NAI in malaria and contribute valuable tools to advance research toward improved disease control and elimination strategies.