Genetic diversity and neutral selection in Plasmodium vivax erythrocyte binding protein correlates with patient antigenicity

Abstract

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.

Fig 1. Pvebp primary structure and phenotypic clustering.

(A) Disc shape shows pvebp exon and solid line represents intron part. Region separation was followed by PvDBP region divided strategy on cluster 1. The line in the box represents indels variation sites (GK or DERS). Cluster 3, 4, and PcyDBP2 (M strain) sequences have large insertions (oblique line box) in RIII-V domain. (B) Amino acid sequence alignment with PvEBP cluster shows in yellow background for Gly-Lys (GK), and gray background shows large indels variation.

Fig 2. Median-joining networks of PvEBP-ecto haplotype.

The geographical haplotype network shows the relationships among 67 haplotypes present in 316 isolates sequence. Distances between nodes are generated by NetWork 5.0 software. The orange cutting line connected to cluster 2 and cluster 3, the blue cutting line connected to cluster 4. The cluster 3 and cluster 4 contained large insertion sequence in RIII-V domain and cluster 4 connected to PcyM DBP2.

Fig 3. Pvebp domains nucleotide diversity (π) based on the geographical areas.

PvEBP region divided for region I (RI, 1–534 bp), RII (535–1,437 bp), RIII-V (1,438–2,070 bp), and RVI (2,071–2,307 bp) based on the PvDBP homologue region.

Fig 4. Humoral immune response of PvEBP-RII and PvEBP-RIII-V.

(A) Purity confirmation by SDS-PAGE of recombinant PvEBP-RII (30.1 kDa) and PvEBP-RIII-V (25.8 kDa) expression. (B) Total IgG prevalence of each domain with the vivax patient (red dot) and healthy individual (blue dot) sera. The bar indicates the mean fluorescence intensity (MFI) ± 95% CI. The p values were calculated by Student’s t-test. Significant differences are shown as triple asterisks p <0.001. (C) IgG prevalence visualized for comparison between RII and RIII-V with each patient sera by normalized reactivity index. Significant differences are shown as single asterisks p <0.05 and triple asterisks p <0.001. (D) Correlation between RII and RIII-V total IgG reactive indices using Pearson correlation test (r). Blue dot and dash line represent patient sera reactive index from ROK and its regression line. Black and yellow dot and dash line represent reactivity indices and its regression lines from Myanmar and Thailand patient sera, respectively. Red line indicates total regression.

Fig 5. The correlation of parasitaemia and age with PvEBP domains.

(A and B) PvEBP-RII and (C and D) PvEBP-RIII-V total IgG indices obtained from mean fluorescence intensity (MFI) were evaluated correlation with patient age (years) and parasitaemia (%) using Pearson correlation test (r), respectively. The horizontal dash line indicates MFI+2S.D. value as positive reactivity and vertical dash line in parasitaemia (>0.2) considered high parasitaemia.