Limited global diversity of the Plasmodium vivax merozoite surface protein 4 gene

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Abstract

Merozoite surface proteins (MSPs) of the malaria parasites are major candidates for vaccine development targeting asexual blood stages. However, the diverse antigenic repertoire of these antigens that induce strain-specific protective immunity in human is a major challenge for vaccine design and often determines the efficacy of a vaccine. Here we further assessed the genetic diversity of Plasmodium vivax MSP4 (PvMSP4) protein using 195 parasite samples collected mostly from Thailand, Indonesia and Brazil. Overall, PvMSP4 is highly conserved with only eight amino acid substitutions. The majority of the haplotype diversity was restricted to the two short tetrapeptide repeat arrays in exon 1 and 2, respectively. Selection and neutrality tests indicated that exon 1 and the entire coding region of PvMSP4 were under purifying selection. Despite the limited nucleotide polymorphism of PvMSP4, significant genetic differentiation among the three major parasite populations was detected. Moreover, microgeographical heterogeneity was also evident in the parasite populations from different endemic areas of Thailand.

Introduction

Plasmodium vivax malaria is considered as the most neglected, highly prevalent, and potentially dangerous disease (Baird, 2007, Price et al., 2007). Currently, it is estimated that 2.6 billion people live at the risk of vivax malaria and there are 130–145 million vivax malaria cases per year (Hay et al., 2004, Guerra et al., 2006). Although rarely fatal, a P. vivax infection is by no means clinically benign, and the episode of debilitating intensity can be repeated several times due to relapses (Price et al., 2007). Recently, several prospective studies have reported that clinical and severe vivax malaria, manifested by severe anaemia, respiratory distress and impaired consciousness was common in hyperendemic areas of Asia and South America (Barcus et al., 2007, Rodriguez-Morales et al., 2008, Genton et al., 2008, Tjitra et al., 2008). Furthermore, the first-line therapies comprised of chloroquine and primaquine remain unchanged for the past 50 years, and the appearance of resistant parasites to these drugs are of great concern for vivax malaria control. As malaria eradication is once again on the agenda of international malaria control communities (Roberts and Enserink, 2007), these efforts must also include elimination of vivax malaria.

Effective control of malaria requires comprehensive approaches integrating multiple strategies such as chemotherapy, vaccination, and vector control. For vaccine development, antigens located on the surface or apical organelles of merozoites are prime candidates, given their potentials of eliciting immunity that blocks merozoite invasion of host erythrocytes (Richie and Saul, 2002). The effect of vertebrate host immunity on shaping the parasite population structure is well known and considered mostly responsible for the diverse antigenic repertoire of the parasite. There is strong evidence showing the induction of strain-specific protective immunity in human (e.g., Hodder et al., 2001, Cortes et al., 2005). Therefore, the significant levels of polymorphism present in these antigens create a major challenge for the development of a successful vaccine. Since the antigenic repertoire of a specific antigen is shaped by multiple factors such as the selection strength of host immune responses, functional constraints, and frequency of genetic recombination, detailed studies of antigenic diversity of a vaccine candidate in different malaria-endemic areas must be undertaken (Cui et al., 2003).

At least ten distinct P. vivax merozoite surface proteins (PvMSPs) have been identified experimentally or by in silico analysis of the P. vivax genome (Carlton et al., 2008). Among them, PvMSP4 shows significant homology to PfMSP4 in terms of sequence identity and gene structure (Black et al., 2002). In P. falciparum and P. vivax, MSP4 and MSP5 have similar gene organizations and are located in tandem on chromosome 2 and 4, respectively, suggesting that these two genes might have originated from an ancient gene duplication event. In rodent malaria parasites, a single gene MSP4/5 is found with similarity to both MSP4 and 5. The PvMSP4 gene has a two-exon structure, with a 300 bp exon 1 and 387 bp exon 2 separated by a 156 bp intron for the Thai-NYU strain. Like PfMSP4, PvMSP4 is a GPI-anchored surface protein with an epidermal growth factor (EGF)-like domain encoded in the second exon (Black et al., 2002).

PfMSP4 is being considered as a component of a subunit vaccine (Dooland and Steward, 2007). Immunization of mice with Plasmodium yoelii MSP4/5 is able to confer significant protection against lethal challenges with blood stage P. yoelii (Goschnick et al., 2004). Recombinant PfMSP4 protein is immunogenic in laboratory animals and recognized by antibodies from humans living in malaria endemic areas (Wang et al., 1999, Wang et al., 2001). Limited population studies indicate that PfMSP4 has low sequence diversity (Wang et al., 2002, Benet et al., 2004, Polson et al., 2005), and analysis of PvMSP4 sequences from 30 Colombian samples revealed a similar result of limited polymorphisms (Martinez et al., 2005). Yet, it is not clear whether this reflects a recent expansion of similar strains in this region or global conservation of the PvMSP4 locus. To determine whether this observation can be extended to other vivax endemic regions, we have sequenced 195 samples from several vivax endemic countries. Our analysis supports the finding that PvMSP4 has limited genetic diversity.

Section snippets

Source of P. vivax samples

We have procured 195 P. vivax finger-prick samples on filter papers from Thailand (n = 130), Indonesia (n = 34), Brazil (n = 24), India (n = 1), Papua New Guinea (n = 1), Solomon islands (n = 1), China (n = 2) and Vietnam (n = 2). This study was approved by the Institutional Review Board at Chulalongkorn University. Parasite genomic DNA was extracted by using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). The DNA purification procedure was essentially as described in the manufacturer's instruction manual

Genetic diversity of the PvMSP4

To assess the global genetic diversity of P. vivax parasites at the PvMSP4 locus, we determined the PvMSP4 sequence from 195 parasite clinical samples from various vivax malaria-endemic areas. Together with two PvMSP4 sequences from the Sal-1 and Thai-NYU strain, 53 haplotypes were identified (Table 1). Geographically, the 130 Thai, 34 Indonesian, and 24 Brazilian samples contained 35, 15, and 12 PvMSP4 haplotypes, respectively. Hyplotype 1, 3, and 5 were the most abundant and they accounted

Discussion

In this study, we have characterized the genetic diversity of PvMSP4 gene using 195 field samples representing diverse endemic areas of P. vivax parasite. Our results confirmed the previous finding about the conservation of this gene. Most of the observed haplotype diversity of PvMSP4 is due to polymorphism in repeat copy number of two repeat arrays. The overall nucleotide diversity of PvMSP4 is much lower than what has been determined for other P. vivax antigens such as MSP1 (Putaporntip et

Acknowledgements

We are grateful to all patients who donated their blood samples for this analysis. This work was supported by research grants from The National Research Council of Thailand and The Thai Government Research Budget to S.J. and C.P., a grant from The Thailand Research Fund (RMU5080002) to C.P. and a grant (D43-TW006571) from Fogarty International Center, National Institutes of Health to L.C.

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