DataSheet_1_Reticulocyte Binding Protein Homologue 5 is a target of balancing selection in the Plasmodium falciparum population of Papua New Guinea.docx

Plasmodium falciparum Reticulocyte Binding Protein Homologue (RH5), a leading malaria vaccine candidate, is essential for erythrocyte invasion by the parasite, interacting with the human host receptor, basigin. RH5 has a small number of polymorphisms relative to other blood-stage antigens, and in vitro studies have shown that vaccine-induced antibodies raised against RH5 are strain-transcending, however most studies investigating RH5 diversity have been done in Africa. Understanding the genetic diversity and evolution of malaria antigens in other regions is important for their validation as vaccine candidates. In this study the rh5 gene was sequenced in 677 samples from a longitudinal cohort of Papua New Guinean (PNG) children aged 1-3 years. Of 677 samples successfully sequenced, 566 were identified as independent infections (i.e. one of each pair of identical sequences within hosts were removed). A total of 14 non-synonymous polymorphisms were identified, eight that are ‘common’ in the population (minor allele frequency > 1%), with 44 haplotypes ranging in frequency from 1% to 21%. Modeling of common SNPs to the cryo-EM structure of the RH5/CyRPA/RIPR complex mapped them to the Basigin binding site and near the contact point of CyRPA. Tajima’s D analyses of the corresponding nucleotide sequences produced positive values indicating potential hotspots of balancing selection. We attempted to confirm whether these signals were due to immune selection by measuring the rate of polymorphism between independent infections within the same host, and the association with clinical symptoms, however, no such associations were identified. Together these results suggest that while there is evidence of balancing selection driving RH5 diversity in the PNG P. falciparum population, immune escape was not observed within the cohort of young children. Limited immunity and therefore low selective pressure may explain this result, alternatively other evolutionary forces may contribute to balancing selection at the RH5-BSG binding interface in PNG.

恶性疟原虫裂网红细胞结合蛋白同源物（RH5），作为领先的疟疾疫苗候选物，对于寄生虫侵入红细胞至关重要，并与人类宿主受体basigin相互作用。与其它血阶段抗原相比，RH5具有较少的多态性。体外研究表明，针对RH5诱导的疫苗抗体具有跨株性，然而，大多数研究RH5多样性的工作都是在非洲进行的。了解其他地区疟疾抗原的遗传多样性和进化对于验证其作为疫苗候选物的有效性具有重要意义。在本研究中，对来自巴布亚新几内亚（PNG）1-3岁儿童纵向队列的677个样本中的rh5基因进行了测序。在成功测序的677个样本中，566个被确认为独立感染（即，在宿主体内每对相同序列中的一条被去除）。共鉴定出14个非同义多态性，其中8个在人群中较为常见（次要等位基因频率>1%），44个单倍型频率从1%到21%不等。将常见单核苷酸多态性建模到RH5/CyRPA/RIPR复合物的冷冻电镜结构中，将其映射到Basigin结合位点及其与CyRPA接触点附近。对应核苷酸序列的Tajima's D分析产生了正值，表明潜在的平衡选择热点。我们试图通过测量同一宿主内独立感染的多态性率以及与临床症状的相关性来确认这些信号是否由免疫选择引起，然而，并未发现此类关联。这些结果共同表明，尽管有证据表明平衡选择在PNG恶性疟原虫种群中驱动RH5的多样性，但在年轻儿童的队列中并未观察到免疫逃逸。有限的免疫力和因此低选择性压力可能解释了这一结果，或者其他进化力量可能有助于PNG中RH5-BSG结合界面的平衡选择。