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

Abstract

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.

Vaccine-induced human monoclonal antibodies to PfRH5 show broadly neutralizing activity againstP. falciparumclinical isolates

“…( C ) heat maps and ( D ) Volcano plots depicting the difference between the Bliss additivity predicted percent GIA and the actual percent GIA from mAb combinations. Bliss analysis was calculated as previously described 35 and the multiple unpaired t-test was used to assess the statistical difference. Volcano plots show -log10 of the q-values.…”

“…While vaccine-induced mAbs to PfRH5 are associated with varying levels of neutralizing activity against P. falciparum laboratory lines from different geographical locations 25 , these had not been tested on P. falciparum clinical isolates, the ultimate target of any given malaria vaccine. Importantly, we and others have recently reported an emergence of rare PfRH5 variants from naturally circulating P. falciparum clinical isolates across Africa [34][35][36][37][38] .…”

“…Another limitation of this study worth highlighting is related to our sequencing strategy. Indeed, here we opted for deep targeted sequencing, which is a powerful tool to accurately identify rare variants occurring at very low frequencies within the parasite populations, however, due to its resulting short reads, we were unable to accurately assemble full-length PfRH5 gene haplotypes within a given polygenomic infection to compare their genetic relatedness to earlier reported parasite haplotypes [34][35][36][37][38] .…”

“…Given parasite genotype can impact susceptibility to vaccine-induced antibodies, we sought to investigate the breadth of PfRH5 genetic diversity in our study population. As in our previous study, we implemented a deep amplicon sequencing of the PfRH5 gene using a sensitive discovery threshold of 2% prevalence 35 . This analysis resulted in the discovery of 12 SNPs, the majority of which were present at low prevalence, and detected in single isolates.…”

Reticulocyte Binding Protein Homologue 5 is a target of balancing selection in the Plasmodium falciparum population of Papua New Guinea

“…Of the 11 nonsynonymous polymorphisms identified globally, eight are considered 'common' (>1% frequency) (Naung et al, 2022) but previous investigations did not identify any evidence of immune selection (Ouattara et al, 2018). Nevertheless, RH5 gene sequences from African isolates were recently shown to be more diverse than previously described (Mangou et al, 2022). However, analysis of RH5 gene sequences in parasite populations in the Asia-Pacific suggest that diversity may be driven by balancing selection (Naung et al, 2022).…”