The Plasmodium falciparum Rh5 invasion protein complex reveals an excess of rare variant mutations

The invasion of the red blood cells by Plasmodium falciparum merozoites involves the interplay of several proteins that are also targets for vaccine development. The proteins PfRh5-PfRipr-PfCyRPA-Pfp113 assemble into a complex at the apical end of the merozoite and are together essential for erythrocyte invasion. They have also been shown to induce neutralizing antibodies and appear to be less polymorphic than other invasion-associated proteins, making them high priority blood-stage vaccine candidates. Using available whole genome sequencing data (WGS) and new capillary sequencing data (CS), this study describes the genetic polymorphism in the Rh5 complex in P. falciparum isolates obtained from Kilifi, Kenya.

162 samples collected in 2013 and 2014 were genotyped by capillary sequencing (CS) and re-analysed WGS from 68 culture-adapted P. falciparum samples obtained from a drug trial conducted from 2005 to 2007. The frequency of polymorphisms in the merozoite invasion proteins, PfRh5, PfRipr, PfCyRPA and PfP113 were examined and where possible polymorphisms co-occurring in the same isolates.

From a total 70 variants, including 2 indels, 19 SNPs [27.1%] were identified by both CS and WGS, while an additional 15 [21.4%] and 36 [51.4%] SNPs were identified only by either CS or WGS, respectively. All the SNPs identified by CS were non-synonymous, whereas WGS identified 8 synonymous and 47 non-synonymous SNPs. CS identified indels in repeat regions in the p113 gene in codons 275 and 859 that were not identified in the WGS data. The minor allele frequencies of the SNPs ranged between 0.7 and 34.9% for WGS and 1.1-29.6% for CS. Collectively, 12 high frequency SNPs (> 5%) were identified: four in Rh5 codon 147, 148, 203 and 429, two in p113 at codons 7 and 267 and six in Ripr codons 190, 259, 524, 985, 1003 and 1039.

This study reveals that the majority of the polymorphisms are rare variants and confirms a low level of genetic polymorphisms in all proteins within the Rh5 complex.