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Abstract

Vaccine development relies on identifying target proteins that can elicit protective immune responses. However, highly immunogenic regions within these proteins can divert B cell responses from generating protective antibodies specific for less immunogenic regions. This is exemplified by the major surface protein circumsporozoite protein (CSP) of Plasmodium falciparum (Pf), the primary causative agent of malaria. The only available malaria vaccine, RTS,S/AS01, induces strong antibody responses against repeating NANP amino acid motifs in the central repeat domain of PfCSP. Despite their correlation with protection, these antibodies are not long-lived, and the vaccine's efficacy is moderate. Recent studies identified highly potent antibodies that target alternating NANP and NANP-like-motifs in the N-terminal junction of PfCSP, which is not part of RTS,S/AS01, with high affinity. However, these antibodies are overall rare and seem to develop frequently from NANP-specific precursors through affinity maturation. I hypothesized that a large number of repeating NANP-motifs in PfCSP prevents the development of anti-PfCSP-junction responses, while fewer repeating motifs would allow the induction of higher-quality antibody responses. To determine the impact of differing numbers of NANP-motifs on the anti-PfCSP-junction response, I immunized C57BL/6J mice with Helicobacter pylori-ferritin-based immunogens presenting the PfCSP-junction either in combination with a NANP5- or a NANP18.5-repeat. The humoral data showed that the NANP5-immunogen effectively directed the IgG response towards epitopes in the PfCSP-junction without compromising the strength of the anti-PfCSP-repeat response. Furthermore, immunization with the NANP5-nanoparticle induced a higher proportion of cross-reactive antibodies binding both the PfCSP-repeat and -junction. Additionally, reducing the number of repetitive amino acid motifs led to antibodies with overall higher avidity for PfCSP and an increased parasite inhibitory capacity. These differences were also reflected in the immunoglobulin repertoire of the B cells. I found that heavy chain variable (VH) and light chain variable (VL) gene segment combinations associated with antibody cross-reactivity and high affinity were enriched in germinal center B cells and plasma cells of mice immunized with the NANP5-immunogen. Finally, I showed that reducing the number of repetitive amino acid motifs led to higher frequencies of PfCSP-reactive memory B cells after the booster immunization. By highlighting the impact of epitope repetition on B cell affinity maturation, antibody quality, and the development of robust B cell memory, these findings hold promising implications for optimizing malaria immunogens and for guiding the rational design of other vaccine candidates, unlocking the ideal immunization strategy against various pathogens.