Temporal changes in Plasmodium falciparum reticulocyte binding protein homolog 2b (PfRh2b) in Senegal and The Gambia.

BACKGROUND: The Plasmodium falciparum reticulocyte binding protein homolog 2b (PfRh2b) is an important P. falciparum merozoite ligand that mediates invasion of erythrocytes by interacting with a chymotrypsin-sensitive "receptor Z". A large deletion polymorphism is found in the c-terminal ectodomain of this protein in many countries around the world, resulting in a truncated, but expressed protein. The varying frequencies by region suggest that there could be region specific immune selection at this locus. Therefore, this study was designed to determine temporal changes in the PfRh2b deletion polymorphism in infected individuals from Thiès (Senegal) and Western Gambia (The Gambia). It was also sought to determine the selective pressures acting at this locus and whether prevalence of the deletion in isolates genotyped by a 24-SNP molecular barcode is linked to background genotype or whether there might be independent selection acting at this locus. METHODS: Infected blood samples were sourced from archives of previous studies conducted between 2007 and 2013 at SLAP clinic in Thiès and from 1984 to 2013 in Western Gambia by MRC Unit at LSHTM, The Gambia. A total of 1380 samples were screened for the dimorphic alleles of the PfRh2b using semi-nested Polymerase Chain Reaction PCR. Samples from Thiès were previously barcoded. RESULTS: In Thiès, a consistent trend of decreasing prevalence of the PfRh2b deletion over time was observed: from 66.54% in 2007 and to 38.1% in 2013. In contrast, in Western Gambia, the frequency of the deletion fluctuated over time; it increased between 1984 and 2005 from (58.04%) to (69.33%) and decreased to 47.47% in 2007. Between 2007 and 2012, the prevalence of this deletion increased significantly from 47.47 to 83.02% and finally declined significantly to 57.94% in 2013. Association between the presence of this deletion and age was found in Thiès, however, not in Western Gambia. For the majority of isolates, the PfRh2b alleles could be tracked with specific 24-SNP barcoded genotype, indicating a lack of independent selection at this locus. CONCLUSION: PfRh2b deletion was found in the two countries with varying prevalence during the study period. However, these temporal and spatial variations could be an obstacle to the implementation of this protein as a potential vaccine candidate.

Functional Analysis Reveals Geographical Variation in Inhibitory Immune Responses Against a Polymorphic Malaria Antigen.

Background: Plasmodium falciparum reticulocyte-binding protein homologue 2b (PfRh2b) is an invasion ligand that is a potential blood-stage vaccine candidate antigen; however, a naturally occurring deletion within an immunogenic domain is present at high frequencies in Africa and has been associated with alternative invasion pathway usage. Standardized tools that provide antigenic specificity in in vitro assays are needed to functionally assess the neutralizing potential of humoral responses against malaria vaccine candidate antigens. Methods: Transgenic parasite lines were generated to express the PfRh2b deletion. Total immunoglobulin G (IgG) from individuals residing in malaria-endemic regions in Tanzania, Senegal, and Mali were used in growth inhibition assays with transgenic parasite lines. Results: While the PfRh2b deletion transgenic line showed no change in invasion pathway utilization compared to the wild-type in the absence of specific antibodies, it outgrew wild-type controls in competitive growth experiments. Inhibition differences with total IgG were observed in the different endemic sites, ranging from allele-specific inhibition to allele-independent inhibitory immune responses. Conclusions: The PfRh2b deletion may allow the parasite to escape neutralizing antibody responses in some regions. This difference in geographical inhibition was revealed using transgenic methodologies, which provide valuable tools for functionally assessing neutralizing antibodies against vaccine-candidate antigens in regions with varying malaria endemicity.