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Abstract

SUMMARY Plasmodium falciparum malaria, a mosquito-borne, unicellular parasitic disease, remains a major health concern, particularly in the sub-Saharan Africa. Despite many attempts to develop effective malaria vaccines in recent decades, only one vaccine (RTS,S/AS01) targeting the pre- erythrocytic stage of the Plasmodium falciparum life cycle, has passed the Phase III clinical trial. However, the protective efficacy of this vaccine is low, and the protection is not long-lasting. Thus, a deeper understanding of the immune responses elicited by currently available vaccines and the identification of protective and non-protective B-cell epitopes are essential for the development of next-generation vaccine candidates that will elicit a robust and long-lasting humoral immune response against protective epitopes while avoiding the induction of non-protective antibodies. The vast majority of pre-erythrocytic malaria vaccines elicit antibodies that primarily target the Plasmodium falciparum circumsporozoite protein (PfCSP), a major protein on the surface of sporozoites with a basic structure consisting of the N-terminus (N-CSP), the junction (N-Junc), the central repeat motifs, and the C-terminus (C-CSP), which comprises a linker (C-linker) and a structured α-TSR subdomain that hosts almost all PfCSP T cell epitopes. While antibodies targeting the central repeat with or without cross-reactivity to the N-Junc or C-CSP have attracted much interest due to their promising protective capacity, little is known about the molecular dynamics, fine epitope specificity and the overall protective efficacy of antibodies specific for the N-Junc or the C-CSP domain.

To address these questions, I analyzed a large collection of human monoclonal antibodies (mAbs) against the N-Junc and C-CSP domains using a high-throughput single-cell immunoglobulin (Ig) gene repertoire analysis, following a three-dose immunization with Plasmodium falciparum radiation-attenuated sporozoites (Pf RAS). The naïve human B cell repertoire contains numerous anti-N-Junc and ant-C-CSP antibodies that undergo efficient affinity maturation and IgG class-switching, providing an explanation for the high immunogenicity of the N-Junc and C-CSP domains. Specificities for the N-Junc and C-CSP epitopes were found in both germline and mutated mAbs, and were frequently encoded by IGHV3-23/IGLV1-47 and IGHV3-21/IGLV3-1 or IGLV3-21 gene combinations, respectively. With the exception of one mAb that recognized the C-linker subdomain, all C-CSP specific mAbs targeted the α-TSR subdomain, demonstrating the immunodominance of the α-TSR subdomain. However, while the N-Junc- specific mAbs showed a lower Plasmodium falciparum parasite inhibitory capacity than the cross- reactive mAbs, none of the C-CSP-specific mAbs showed parasite inhibition, regardless of their affinity, gene usage, and epitope specificity. Direct comparison of these antibodies in mice showed that protection from blood stage parasitemia was limited only to C-CSP reactive mAb that cross-reacted with the central repeat domain and N-Junc. Taken together, these findings highlight the molecular features associated with N-Junc and C-CSP specific antibodies at B cell repertoire level and provide supportive evidence that antibodies elicited specifically against the C-CSP domain, whether through natural malaria exposure or vaccine immunization, will not contribute to protection.

Hence, to develop a broadly effective PfCSP-based subunit vaccine against Plasmodium falciparum malaria, careful consideration must be given to whether integrating the C-CSP domain into PfCSP-based immunogens is the optimal means of delivering T-cell epitopes. Instead, vaccine design should focus on the N-Junc and the conserved central repeat domains to elicit robust and cross-neutralizing antibodies.