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Background: Measuring malaria transmission intensity using the traditional entomological inoculation rate is difficult. Antibody responses to mosquito salivary proteins like SG6 have been used as biomarkers of exposure to Anopheles mosquito bites. Here, we investigate four mosquito salivary proteins as potential biomarkers of human exposure to mosquitoes infected with P. falciparum: mosGILT, SAMSP1, AgSAP, and AgTRIO., Methods: We tested population-level human immune responses in longitudinal and cross-sectional plasma from individuals with known P. falciparum infection from low and moderate transmission areas in Senegal using a multiplexed magnetic bead-based assay., Results: AgSAP and AgTRIO were the best indicators of recent exposure to infected mosquitoes. Antibody responses to AgSAP, in a moderate endemic area, and to AgTRIO in both low and moderate endemic areas, were significantly higher than responses in a healthy non-endemic control cohort (p-values = 0.0245, 0.0064, and <0.0001 respectively). No antibody responses significantly differed between the low and moderate transmission area, or between equivalent groups during and outside the malaria transmission seasons. For AgSAP and AgTRIO, reactivity peaked 2-4 weeks after clinical P. falciparum infection and declined 3 months after infection., Discussion: Reactivity to AgSAP and AgTRIO peaked after infection, with no differences between transmission seasons within region or between low and moderate transmission regions. This suggests that reactivity reflects exposure to infectious mosquitoes or recent bites rather than general mosquito exposure. Kinetics suggest reactivity is relatively short-lived. AgSAP and AgTRIO are promising candidates to incorporate into multiplexed assays for serosurveillance of population-level changes in P. falciparum-infected mosquito exposure., (© The Author(s) 2024. Published by Oxford University Press on behalf of Infectious Diseases Society of America.)

The Plasmodium falciparum cysteine-rich protective antigen (PfCyRPA) is a promising target as a next-generation blood-stage malaria vaccine and together with PCRCR complex members, the reticulocyte binding-like homologous protein 5 (PfRh5) and the Rh5-interacting protein (PfRipr), are currently being evaluated in clinical trials. PfCyRPA is essential for merozoite invasion and appears to be highly conserved within the P. falciparum parasite populations. Here, we used a targeted deep amplicon next-generation sequencing approach to assess the breadth of PfCyRPA genetic diversity in 95 P. falciparum clinical isolates from Kédougou, an area with a high seasonal malaria transmission in Senegal. Our data show the dominant prevalence of PfCyRPA wild type reference allele, while we also identify a total of 15 single nucleotide polymorphisms (SNPs). Of these, only five have previously been reported, while the majority of the SNPs were present as singletons within our sampled population. Moreover, the variant read frequency of the identified SNPs varied from 2.6 to 100%, while the majority of the SNPs were present at frequencies greater than 25% in polygenomic samples. We also applied a structure-based modelling approach to thread these SNPs onto PfCyRPA crystal structures and showed that these polymorphisms have different predicted functional impacts on the interactions with binding partner PfRH5 or neutralizing antibodies. Our prediction revealed that the majority of these SNPs have minor effects on PfCyRPA antibodies, while others alter its structure, stability, or interaction with PfRH5. Altogether, our present findings reveal conserved PfCyRPA epitopes which will inform downstream investigations on next-generation structure-guided malaria vaccine design.