Polymorphism patterns in Duffy-binding protein among Thai Plasmodium vivax isolates

Molecular mechanisms of invasion by Plasmodium vivax merozoites are mediated by the Duffy-binding-like (DBL) family of homologous erythrocyte binding protein (EBPs) located within the micronemes of merozoites that recognize specific receptors on red blood cell [1]. PvDBP is a 140-kDa protein belonging to a family of erythrocyte binding proteins characterized by a functionally conserved cysteine-rich region. The similarity among DBL families of EBPs is the greatest within two adjacent cysteine-rich domains, designated as the amino cysteine-rich domain (N-cys) and carboxyl cysteine-rich domain (C-cys) [2]. The amino cysteine-rich domain has been identified as the principle adhesion region binding to erythrocyte receptors, while the function of the carboxyl cysteine-rich domain remains unclear. This amino-cysteine-rich domain is present within the 330 amino acid region II, which has been shown to contain the binding motifs necessary for adherence to the Duffy antigen receptor for chemokines (DARC), required for erythrocyte invasion [3]. Critical binding motifs in PvDBPII have been mapped to a 170 amino acid spanning region between cysteines 4 and 7 (amino acids 291–460) [4]. Cysteine residues are conserved within the identified binding motif, whereas other amino acids are highly polymorphic, having a high ratio of nonsynonymous to synonymous mutations [5].

The polymorphic nature of the PvDBP, particularly its region II (DBPII) is a major impediment to the development of a broadly protective vaccine against vivax malaria. A study in Papua New Guinea showed 43 and 18 unique nonsynonymous mutations in 10 Madang [6] and 24 Wosera vivax isolates [7], respectively, whereas 19 unique nonsynonymous mutations were described in 10 Colombian vivax isolates [8]. Often the mutations were found in corresponding locations in the protein sequence of isolates from different geographical areas. Amino acids substitutions at D384G, K386(N/Q), N417K, L424I, W437R, and I503K were found in both Papua New Guinea isolates and Colombia isolates [5‐8]. Such results demonstrate that many similar alleles are widely distributed among P. vivax from different geographical areas. Polymorphic patterns in the PvDBP cluster within the critical erythrocyte-binding segment imply the existence of a selection pressure on PvDBP [9]. Under the influence of immune pressure, the distribution of existing antigenic polymorphisms affects the population structure in which newly selected mutants spread and may provide insight to the evolution and selection of parasite populations over time [10]. Polymorphisms of PvDBPII require further exploration among vivax isolates from different geographic areas, particularly south-east Asia where a large proportion of vivax infections take place. Therefore, the gene polymorphisms within the region II domain of the P. vivax DBP among Thai P. vivax isolates were investigated.

The process invasion into erythrocyte is essential for survival of P. vivax parasite and requires DBP to bind the Duffy blood group antigen that act as erythrocyte receptor, Duffy antigen receptor for chemokines (DARC). Plasmodium vivax utilizes genetic diversity of individual functional proteins to evade the host immune system and resist many anti-malarial drugs [21]. In this study, polymorphisms and natural selection was assessed and suggested positive natural selection in PvDBPII of Thai isolates. Although, non significant could be observed trending positive selection. As this is only a glimpse at the overall population, a wide range study including more subjects from different areas of Thailand would give a more detailed picture.

Analysis of 300 amino acids of PvDBPII protein relative to the Sal I reference strain, resulted in 25 nonsynonymous mutations being found. In addition, 142 nonsynonymous mutations were reported in 76 Papua New Guinea isolates [22]. The nonsynonymous mutations in PvDBPII might have an effect on parasite binding to erythrocyte receptors. Nonsynonymous mutations often times result in amino acid substitutions that alter charge and therefore host immune/antibody recognition [6]. Thus, excessive stable polymorphism generation within the PvDBPII ligand domain from high rate of nonsynonymous polymorphisms may promote the parasite escape of host immune response [9].

The high frequency (>50%) of L424I (86%), D384G (76%), W437R (63%), R390H (56%), and I503K (56%) residues were found in Thai isolates relative to Sal I PvDBP sequence. The finding is in contrast to previous studies showing R308S (67%), D384G (66%), and S447K (59%) in Papua New Guinea, D384G (59%) in Colombia and D384G (85%) in Brazil isolates [23]. Of the following variants N417K, W437R and I503K, all having been shown to be involved in evasion to antibody neutralization, variant N417K was found in low frequency (36%) while W437R and I503K were high (63% and 56% respectively) in Thai isolates. These results were different from what has been currently reported in that the low frequencies of variants N417K (27.5%) and W437R (27.5%) and the high frequency of variant I503K (55%) found in Brazil and low frequencies of all three variants (N417K, W437R, I503K) found in either Colombia (47%, 18%, 12%, respectively) and/or Papua New Guinea (23%, 26%, 29%, respectively) (Table 4) [23]. N417K, W437R and I503K have recently been shown to not be directly involved in erythrocyte binding, but possibly in inhibition of antibody binding to erythrocyte receptor [24]. Moreover, the variant amino acid T404R was identified in PvDBPII domains of three Thai isolates (Table 2) which is similar to the recent data in Papua New Guinea [22]. Other Thai variants, namely R308S, K371E, D384G, E385K, N386(N/Q), R390H, T404R, N417K, L424I, W437R, S447S and I503K, were also found in isolates of other regions, demonstrating commonality with a progenitor clone (Table 4). The other 18 polymorphic residues of PvDBPII ligand domain were recognized only among Thai isolates, indicative of divergence from a progenitor clone.

The study showed that 25 PvDBPII haplotypes clustered into five main groups (dominant haplotypes) (Table 3) on the basis of nonsynonymous mutations is similar, but not entirely the same, with data from Papua New Guinea isolates whose 27 different PvDBPII haplotypes clustering into three dominant PvDBPII haplotypes. Potentially dominant PvDBPII haplotypes observed in these populations could be less immunogenic, thereby may help promoting parasite success and escaping the host immune response [22].

Phylogenetic analysis of PvDBPII suggested that Thai isolates fell into 6 different alleles groups. Among these, group 1 formed a group with a subset of Papua New Guinea isolates. Groups 4 and 6 were unique among Thai P. vivax isolates. However, the rest of the allele groups that Thai isolates fell into were more related to the isolates from Korea, India, and Colombia than from Papua New Guinea (Figure 3). The observations derived from the PvDBPII phylogeny suggested that single Indian isolate found in group 1 appeared to share a common ancestor close to a subset of Thai and Papua New Guinea isolates, demonstrating common ancestoral origins. Thai and Papua New Guinea isolate subsets in group 7 also may have shared common ancestor heritage. Recently, it was reported that travellers' malaria among foreigners at the Hospital for Tropical Diseases, Bangkok, Thailand including India and Papua New Guinea patients were though to have acquired their infections in their countries. Malaria importations from country to country can occur by either immigration or travel, and changing malaria attack rates in the countries of exposure are likely to influence the incidence of imported disease [25]. Additionally, these results demonstrated that there is some difference in the nucleotide or amino acid variation in PvDBPII among isolates from Thai, Korea, India, Papua New Guinea and Colombia, but most importantly, unique variants from Thailand do exist and should be considered in future vaccine development of PvDBPII. It should be noted that the present highly diverse phylogenetic tree was constructed relying on polymorphisms within a single gene, demonstrating the high degree of topological variation that can be found using this technique, but also highlighting the problems that can be associated with gene sampling in phylogenetic studies [26]. Use of a combination of several genes with different rates of evolutions has been suggested as an efficient way to overcome this difficulty to prove the existence of a unique tree relating these sequences [27]. Finally, it must also be pointed out that in many situations a single-gene phylogeny may be interesting in itself. Awareness of the problems of orthology (genes in different species that derive from a common ancestor) assignment and tree reconstruction artifacts should be considered.

The results indicated that PvDBPII gene among Thai isolates is genetically diverse. Nonsynonymous polymorphism in PvDBPII was the predominant result of which variant L424I showed the highest frequency (86%). Five dominant PvDBPII haplotypes could be clustered among Thai isolates. The high frequency of polymorphisms and the presence of distinct alleles within PvDBPII gene among P. vivax from different geographical areas could provide complication in malaria vaccine development, but should be considered important parts of the development process.