Data_Sheet_2_On the Evolution and Function of Plasmodium vivax Reticulocyte Binding Surface Antigen (pvrbsa).pdf

The RBSA protein is encoded by a gene described in Plasmodium species having tropism for reticulocytes. Since this protein is antigenic in natural infections and can bind to target cells, it has been proposed as a potential candidate for an anti-Plasmodium vivax vaccine. However, genetic diversity (a challenge which must be overcome for ensuring fully effective vaccine design) has not been described at this locus. Likewise, the minimum regions mediating specific parasite-host interaction have not been determined. This is why the rbsa gene’s evolutionary history is being here described, as well as the P. vivax rbsa (pvrbsa) genetic diversity and the specific regions mediating parasite adhesion to reticulocytes. Unlike what has previously been reported, rbsa was also present in several parasite species belonging to the monkey-malaria clade; paralogs were also found in Plasmodium parasites invading reticulocytes. The pvrbsa locus had less diversity than other merozoite surface proteins where natural selection and recombination were the main evolutionary forces involved in causing the observed polymorphism. The N-terminal end (PvRBSA-A) was conserved and under functional constraint; consequently, it was expressed as recombinant protein for binding assays. This protein fragment bound to reticulocytes whilst the C-terminus, included in recombinant PvRBSA-B (which was not under functional constraint), did not. Interestingly, two PvRBSA-A-derived peptides were able to inhibit protein binding to reticulocytes. Specific conserved and functionally important peptides within PvRBSA-A could thus be considered when designing a fully-effective vaccine against P. vivax.

RBSA蛋白由存在于嗜网织红细胞（reticulocytes）的疟原虫属（Plasmodium）物种中的基因编码。鉴于该蛋白在自然感染中具有抗原性，且可结合靶细胞，因此被提议作为抗间日疟原虫（Plasmodium vivax）疫苗的潜在候选靶点。然而，该基因座（locus）的遗传多样性——这是保障疫苗设计完全有效的关键挑战之一——此前尚未被系统阐明。同样，介导寄生虫与宿主特异性互作的最小功能区域也尚未被确定。为此，本研究对rbsa基因的演化历史、间日疟原虫rbsa（pv rbsa）的遗传多样性，以及介导寄生虫黏附网织红细胞的特定功能区域进行了系统分析与报道。与既往报道不同，rbsa在多个隶属于猴疟原虫演化支（monkey-malaria clade）的寄生虫物种中均有存在；同时在侵袭网织红细胞的疟原虫属物种中也发现了其旁系同源基因（paralogs）。pv rbsa基因座的遗传多样性低于其他裂殖子表面蛋白（merozoite surface proteins），自然选择（natural selection）与重组（recombination）是导致该位点观测到多态性（polymorphism）的主要演化驱动力。PvRBSA的N端区域（PvRBSA-A）具有序列保守性，且处于功能约束之下，因此我们将其制备为重组蛋白（recombinant protein）用于结合实验（binding assays）。该蛋白片段可结合网织红细胞，而包含于重组蛋白PvRBSA-B（该区域未受功能约束）中的C端区域则无此结合活性。值得注意的是，两种源自PvRBSA-A的肽段能够抑制该蛋白与网织红细胞的结合。因此，在设计针对间日疟原虫的高效疫苗时，可将PvRBSA-A内的特异性保守且具有关键功能的肽段纳入考量。