The Progression of Epitope-specific Antibody Responses to<i>Plasmodium falciparum C</i>ircumsporozoite Protein Following Consecutive Malaria Infections in Naïve Adults: A Repetitive Controlled Human Malaria Infection Study

Abstract Background With an estimated 249 million cases and 608,000 deaths in 2022 malaria continues to cause substantial morbidity and mortality worldwide, mostly in children in Sub-Saharan Africa due to Plasmodium falciparum. Circumsporozoite protein (CSP) coats the surface of the P. falciparum sporozoite and is the target of the two vaccines approved by the World Health Organization (WHO). CSP has three major regions: the N-terminus, the central repeat region, and the C-terminus, which contains highly variable T cell epitopes; only a fragment of the central repeat region and the C-terminus are represented by the two approved vaccines. The junction between the N-terminal Region 1 and the NANP repeat region (“junctional region”) is not represented in the approved vaccines and was recently found to be a target of protective antibody responses. Methods We used a repetitive controlled human malaria infection (rCHMI) model in which eight malaria-naïve participants were infected with P. falciparum up to three times over two years to identify immune responses associated with acquisition of natural immunity. We evaluated samples from the day of each CHMI and 18 days after each CHMI. We measured antibody responses using a peptide microarray inclusive of hundreds of P. falciparum proteins and focused our current study on the response to CSP. The amino acid sequences of CSP from the NF54 strain and 4 additional CSP variants were represented as 16-amino acid peptides with 13-amino acid overlap. Signal intensities at each amino acid position were estimated using a sliding window approach that averaged signals from adjacent overlapping peptides. Participants who comprised a “delayed parasitemia group” (n = 5) had onset of PCR-confirmed parasitemia 13 or more days after the last CHMI, a sign of developing humoral immunity. A “non-delayed parasitemia group” (n = 3) was comprised of those had onset of parasitemia within 12 or fewer days after the last CHMI. Along each amino acid position, the median signal intensities for all CSP variants are presented to describe the participants’ antibody responses at each time point. Wilcoxon signed rank test was used to compare medians between timepoints; p-values &amp;lt; 0.05 without adjustment for multiple comparisons were considered statistically significant. Results After three rCHMIs, participants developed antibody responses to amino acids corresponding to the junctional region and the central repeat region (P&amp;lt;0.05). When participants are grouped by time to parasitemia, those who showed delayed parasitemia development had strong immune responses to the junctional region while the non-delayed group did not. Antibody responses to the C-terminal region were more variable among the 5 CSP variant sequences compared to other regions. Conclusion Our results showed higher junctional region antibody response in participants with delayed time to parasitemia. We also demonstrate variable antibody response to the C-terminal region, a region with known greater genetic variability that has been previously associated with allele-specific vaccine escape and concern for decreased efficacy of current WHO-approved vaccines. Taken together, our results support future vaccine development that focuses on the conserved junctional region over the polymorphic C-terminal region.