Two mosquito salivary antigens demonstrate promise as biomarkers of recent exposure to P. falciparum infected mosquito bites.

Background: Measuring malaria transmission intensity using the traditional entomological inoculation rate is difficult. Antibody responses to mosquito salivary proteins such as SG6 have previously 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 samples 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 both AgSAP and AgTRIO peaked after infection and did not differ seasonally nor between areas of low and moderate transmission, suggesting reactivity is likely reflective of exposure to infectious mosquitos or recent biting 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.

Structure-guided insights into potential function of novel genetic variants in the malaria vaccine candidate PfRh5.

The recent stall in the global reduction of malaria deaths has made the development of a highly effective vaccine essential. A major challenge to developing an efficacious vaccine is the extensive diversity of Plasmodium falciparum antigens. While genetic diversity plays a major role in immune evasion and is a barrier to the development of both natural and vaccine-induced protective immunity, it has been under-prioritized in the evaluation of malaria vaccine candidates. This study uses genomic approaches to evaluate genetic diversity in next generation malaria vaccine candidate PfRh5. We used targeted deep amplicon sequencing to identify non-synonymous Single Nucleotide Polymorphisms (SNPs) in PfRh5 (Reticulocyte-Binding Protein Homologue 5) in 189 P. falciparum positive samples from Southern Senegal and identified 74 novel SNPs. We evaluated the population prevalence of these SNPs as well as the frequency in individual samples and found that only a single SNP, C203Y, was present at every site. Many SNPs were unique to the individual sampled, with over 90% of SNPs being found in just one infected individual. In addition to population prevalence, we assessed individual level SNP frequencies which revealed that some SNPs were dominant (frequency of greater than 25% in a polygenomic sample) whereas most were rare, present at 2% or less of total reads mapped to the reference at the given position. Structural modeling uncovered 3 novel SNPs occurring under epitopes bound by inhibitory monoclonal antibodies, potentially impacting immune evasion, while other SNPs were predicted to impact PfRh5 structure or interactions with the receptor or binding partners. Our data demonstrate that PfRh5 exhibits greater genetic diversity than previously described, with the caveat that most of the uncovered SNPs are at a low overall frequency in the individual and prevalence in the population. The structural studies reveal that novel SNPs could have functional implications on PfRh5 receptor binding, complex formation, or immune evasion, supporting continued efforts to validate PfRh5 as an effective malaria vaccine target and development of a PfRh5 vaccine.

Leveraging genome editing to functionally evaluate Plasmodium diversity.

The ambitious goal of malaria elimination requires an in-depth understanding of the parasite's biology to counter the growing threat of antimalarial resistance and immune evasion. Timely assessment of the functional impact of antigenic diversity in the early stages of vaccine development will be critical for achieving the goal of malaria control, elimination, and ultimately eradication. Recent advances in targeted genome editing enabled the functional validation of resistance-associated markers in Plasmodium falciparum, the deadliest malaria-causing pathogen and strain-specific immune neutralization. This review explores recent advances made in leveraging genome editing to aid the functional evaluation of Plasmodium diversity and highlights how these techniques can assist in prioritizing both therapeutic and vaccine candidates.