Genetic polymorphisms in malaria vaccine candidate Plasmodium falciparum reticulocyte-binding protein homologue-5 among populations in Lagos, Nigeria

Abstract

Background: Vaccines are the most reliable alternative to elicit sterile immunity against malaria but their development has been hindered by polymorphisms and strain-specificity in previously studied antigens. New vaccine candidates are therefore urgently needed. Highly conserved Plasmodium falciparum reticulocyte-binding protein homologue-5 (PfRH5) has been identified as a potential candidate for anti-disease vaccine development. PfRH5 is essential for erythrocyte invasion by merozoites and crucial for parasite survival. However, there is paucity of data on the extent of genetic variations on PfRH5 in field isolates of Plasmodium falciparum. This study described genetic polymorphisms at the high affinity binding polypeptides (HABPs) 36718, 36727, 36728 of PfRH5 in Nigerian isolates of P. falciparum. This study tested the hypothesis that only specific conserved B and T cell epitopes on PfRH5 HABPs are crucial for vaccine development. Methods:One hundred and ninety-five microscopically confirmed P. falciparum samples collected in a prospective cross-sectional study of three different populations in Lagos, Nigeria. Genetic diversity and haplotype construct of Pfrh5 gene were determined using bi-directional sequencing approach. Tajima's D and the ratio of nonsynonymous vs synonymous mutations were utilized to estimate the extent of balancing and directional selection in the pfrh5 gene.Results: Sequence analysis revealed three haplotypes of PfRH5 with negative Tajima's D and dN/dS value of − 1.717 and 0.011 ± 0.020, respectively. A single nucleotide polymorphism, SNP (G → A) at position 608 was observed, which resulted in a change of the amino acid cysteine at position 203 to tyrosine. Haplotype and nucleotide diversities were 0.318 ± 0.016 and 0.0046 ± 0.0001 while inter-population genetic differentiation ranged from 0.007 to 0.037. Five polypeptide variants were identified, the most frequent being KTKYH with a frequency of 51.3%. One B-cell epitope, 151 major histocompatibility complex (MHC) class II T-cell epitopes, four intrinsically unstructured regions (IURs) and six MHC class I T-cell epitopes were observed in the study. Phylogenetic analysis of the sequences showed clustering and evidence of evolutionary relationship with 3D7, PAS-2 and FCB-2 RH5 sequences. © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article' s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article'

Assessing the functional impact of PfRh5 genetic diversity on ex vivo erythrocyte invasion inhibition

“…Six samples contained no SNPs. These SNPs have been previously described in both published 9 , 11 , 13 , 19 , 20 and unpublished data generated by the Pf3k project ( www.malariagen.net/pf3k ), representing 3,248 samples from 40 separate locations in 20 countries 9 . ( B ) Genotype–phenotype associations were performed for samples with the C203Y SNP and those without (C203Y vs C203 respectively), at each concentration of anti-BSG (MEM-M6/6) antibody.…”

“…At this discovery threshold of 1% three samples (320322, 320379, and 320558) are shown to contain more than one SNP, with two of these samples (320322 and 320558) containing three SNPs. Most of these SNPs have been previously described in both published 9 , 11 , 13 , 19 , 20 and unpublished data generated by the Pf3k project ( www.malariagen.net/pf3k ), representing 3248 samples from 40 separate locations in 20 countries 9 . Additionally, novel SNPs (T216A and N354S) were identified.…”

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

“…The majority of the polymorphisms in this complex or merozoite invasion antigens were rare, which is in contrast to previous findings from surface exposed and abundant merozoite antigens such as apical membrane antigen 1 (AMA1) [ 45 ], merozoite surface protein 1 (MSP1) [ 45 ], MSP3 [ 46 ] and erythrocyte binding antigen-175 (EBA175) [ 47 ], which are under balancing selection and exhibit allele-specific immunity in vaccine trials. In a recent study of samples from Nigeria, only 5 non-synonymous SNPs were identified in Rh5: K62R, T81Q, P197S, C203Y and H240R [ 48 ], of which only the C203Y mutation was identified in our study, while codon 197 was described in the global MalariaGEN dataset, codons 62, 81 and 240 are potentially rare variant sites. Of note, the high frequency sites of codons 147 and 148 in this study were not identified in the Nigerian study.…”

Hotspots in Plasmodium and RBC Receptor-Ligand Interactions: Key Pieces for Inhibiting Malarial Parasite Invasion

“…Such a characteristic, added to limited genetic polymorphism concerning multiple P. falciparum laboratory strains and geographic isolates regarding other candidates has led to a significant amount of research on this protein in the field of antimalarial vaccines [ 80 , 81 , 82 ]. Although few studies have involved using clinical isolates, previous Pfrh5 genetic diversity studies using isolates from Nigeria and Mali have coincided by confirming that Pf rh5 was highly conserved in a setting where other vaccine antigens had extensive polymorphism [ 83 , 84 ]…”

“…Such a characteristic, added to limited genetic polymorphism concerning multiple P. falciparum laboratory strains and geographic isolates regarding other candidates has led to a significant amount of research on this protein in the field of antimalarial vaccines [80][81][82]. Although few studies have involved using clinical isolates, previous Pfrh5 genetic diversity studies using isolates from Nigeria and Mali have coincided by confirming that Pf rh5 was highly conserved in a setting where other vaccine antigens had extensive polymorphism [83,84] Crystallographic studies have shown that Pf Rh5 (residues 140-526 lacking 248-296) (Figure 4A) has a flat rigid structure constituted by two domains, each mainly formed by three helices. The N-terminal domain starts with a short β-sheet, followed by a short α-helix and two large α-helices connected by a truncated loop, while the C-terminal domain is formed by three large α-helices covering the domain's whole length.…”