Plasmodium vivax is the most widespread cause of human malaria. Recent reports of drug resistant vivax malaria and the challenge of eradicating the dormant liver forms increase the importance of vaccine development against this relapsing disease. P. vivax reticulocyte binding protein 1a (PvRBP1a) is a potential vaccine candidate, which is involved in red cell tropism, a crucial step in the merozoite invasion of host reticulocytes. As part of the initial evaluation of the PvRBP1a vaccine candidate, we investigated its genetic diversity and antigenicity using geographically diverse clinical isolates. We analysed pvrbp1a genetic polymorphisms using 202 vivax clinical isolates from six countries. Pvrbp1a was separated into six regions based on specific domain features, sequence conserved/polymorphic regions, and the reticulocyte binding like (RBL) domains. In the fragmented gene sequence analysis, PvRBP1a region II (RII) and RIII (head and tail structure homolog, 152-625 aa.) showed extensive polymorphism caused by random point mutations. The haplotype network of these polymorphic regions was classified into three clusters that converged to independent populations. Antigenicity screening was performed using recombinant proteins PvRBP1a-N (157-560 aa.) and PvRBP1a-C (606-962 aa.), which contained head and tail structure region and sequence conserved region, respectively. Sensitivity against PvRBP1a-N (46.7%) was higher than PvRBP1a-C (17.8%). PvRBP1a-N was reported as a reticulocyte binding domain and this study identified a linear epitope with moderate antigenicity, thus an attractive domain for merozoite invasion-blocking vaccine development. However, our study highlights that a global PvRBP1a-based vaccine design needs to overcome several difficulties due to three distinct genotypes and low antigenicity levels.
Plasmodium vivax is the most widespread and difficult to treat cause of human malaria. The development of vaccines against the blood stages of P. vivax remains a key objective for the control and elimination of vivax malaria. Erythrocyte binding-like (EBL) protein family members such as Duffy binding protein (PvDBP) are of critical importance to erythrocyte invasion and have been the major target for vivax malaria vaccine development. In this study, we focus on another member of EBL protein family, P. vivax erythrocyte binding protein (PvEBP). PvEBP was first identified in Cambodian (C127) field isolates and has subsequently been showed its preferences for binding reticulocytes which is directly inhibited by antibodies. We analysed PvEBP sequence from 316 vivax clinical isolates from eight countries including China (n = 4), Ethiopia (n = 24), Malaysia (n = 53), Myanmar (n = 10), Papua New Guinea (n = 16), Republic of Korea (n = 10), Thailand (n = 174), and Vietnam (n = 25). PvEBP gene exhibited four different phenotypic clusters based on the insertion/deletion (indels) variation. PvEBP-RII (179-479 aa.) showed highest polymorphism similar to other EBL family proteins in various Plasmodium species. Whereas even though PvEBP-RIII-V (480-690 aa.) was the most conserved domain, that showed strong neutral selection pressure for gene purifying with significant population expansion. Antigenicity of both of PvEBP-RII (16.1%) and PvEBP-RIII-V (21.5%) domains were comparatively lower than other P. vivax antigen which expected antigens associated with merozoite invasion. Total IgG recognition level of PvEBP-RII was stronger than PvEBP-RIII-V domain, whereas total IgG inducing level was stronger in PvEBP-RIII-V domain. These results suggest that PvEBP-RII is mainly recognized by natural IgG for innate protection, whereas PvEBP-RIII-V stimulates IgG production activity by B-cell for acquired immunity. Overall, the low antigenicity of both regions in patients with vivax malaria likely reflects genetic polymorphism for strong positive selection in PvEBP-RII and purifying selection in PvEBP-RIII-V domain. These observations pose challenging questions to the selection of EBP and point out the importance of immune pressure and polymorphism required for inclusion of PvEBP as a vaccine candidate.
Traditionally, patient travel history has been used to distinguish imported from autochthonous malaria cases, but the dormant liver stages of Plasmodium vivax confound this approach. Molecular tools offer an alternative method to identify, and map imported cases. Using machine learning approaches incorporating hierarchical fixation index and decision tree analyses applied to 799 P. vivax genomes from 21 countries, we identified 33-SNP, 50-SNP and 55-SNP barcodes (GEO33, GEO50 and GEO55), with high capacity to predict the infection's country of origin. The Matthews correlation coefficient (MCC) for an existing, commonly applied 38-SNP barcode (BR38) exceeded 0.80 in 62% countries. The GEO panels outperformed BR38, with median MCCs > 0.80 in 90% countries at GEO33, and 95% at GEO50 and GEO55. An online, open-access, likelihood-based classifier framework was established to support data analysis (vivaxGEN-geo). The SNP selection and classifier methods can be readily amended for other use cases to support malaria control programs.