Genetic diversity and recombination at the C-terminal fragment of the merozoite surface protein-1 of Plasmodium vivax (PvMSP-1) in Sri Lanka

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Abstract

Extensive polymorphism in the genes encoding for surface antigens of Plasmodium falciparum and Plasmodium vivax has been a serious impediment for malaria vaccine development. One such antigen is the merozoite surface protein-1 (MSP-1). The MSP-1 precursor after proteolytic cleavage generates a C-terminal fragment of 42 kDa (MSP-142), which subsequently produces 33 kDa (MSP-133) and 19 kDa (MSP-119) fragments. Since MSP-142 is currently being considered as a candidate for vaccine development against blood stage malaria it is important to catalogue the existing diversity in this antigen in natural P. vivax infections. Here we investigated the level of genetic diversity in the PvMSP-142 gene fragment in 95 single clone P. vivax infections in Sri Lanka. We observed that the PvMSP-119 fragment was highly conserved among these samples, whereas the PvMSP-133 fragment exhibited extensive diversity with 39 polymorphic amino acid positions (corresponding to 27 haplotypes, 19 of which were unique to Sri Lanka). Of these 27 PvMSP-142 haplotypes, 24 belonged to hypervariable region (HVR) T1-T7 types, while 3 haplotypes were generated by interallelic recombination between T1/T3 (HVRT8-T9) and T2/T3 (HVRT10). In addition, we analysed 107 PvMSP-142 sequences (corresponding to 62 haplotypes, H28 to H89) deposited in the NCBI GenBank database from other regions of the world. Seventy-four of these correspond to 9 of the 10 HVR types (HVR-T7 was unique to Sri Lanka). Two novel HVR types, T11 and T12, with a double recombination between HVR-T1/T3 and HVRT6/T2, were derived from South America and Thailand, respectively. T cell epitope polymorphism arising due to non-synonymous substitutions in PvMSP-133 may result in differential binding of the polymorphic peptides to class II MHC alleles, inducing different host immune responses. In conclusion, under low transmission and unstable malaria conditions prevalent in Sri Lanka, extensive allelic polymorphism was evident at PvMSP-133 due to recombination, mutation, and balancing selection. In contrast, PvMSP-119 is highly conserved, greatly enhancing its suitability as a malaria vaccine candidate.

Research highlights

▶ Recombination, mutation and balancing selection direct polymorphism at PvMSP-133 in Sri Lanka. ▶ 19 of the 27 PvMSP-142 a.a. haplotypes, and HVR type-7 out of 10 such global types, unique to Sri Lanka. ▶ Natural selection determines diversity of predicted B and T cell epitopes at PvMSP-142. ▶ Highly conserved PvMSP-119 with protective antibody response is a veritable vaccine candidate.

Introduction

Malaria is endemic in 109 countries, with an estimated 247 million cases among 3.3 billion people at risk in 2006 (WMR, 2008). Although most of the malaria related deaths are due to Plasmodium falciparum, there have been several reports in the last few years which have documented severe complications and deaths due to Plasmodium vivax infections (Price et al., 2007). P. vivax is responsible for up to 400 million infections each year, representing the most widespread Plasmodium species. Although both species are endemic in Sri Lanka, the majority of reported malaria cases (65–80%) are due to P. vivax (Konradsen et al., 2000). Sri Lanka has two seasonal peaks, one at the beginning of the year and a larger one around June (Briët et al., 2003). The incidence of malaria in Sri Lanka has decreased significantly over the past few years, with only 196 cases reported for both species in 2007 (Annual Report of the Anti-malaria Campaign, 2007).

The malaria control program has traditionally relied on vector control (e.g. insecticides and bed nets) and case management through chemotherapy. Unfortunately, because of increasing resistance to both insecticides and anti-malarials, these two measures alone may not be sufficient to durably reduce the global burden of malaria. Thus effective, long-lasting malaria control may depend on developing cheap, broadly protective vaccines to both species (Gardiner et al., 2005). A blood stage vaccine generally aims to prevent or significantly reduce blood stage parasitemia either by reducing merozoite invasion of red cells or by targeted destruction of parasitized red cells. While progress in the development of such vaccines has been hampered by a number of factors, antigenic diversity has played a major role (Ferreira et al., 2004). This diversity is generated and maintained by several factors, including genetic recombination during the sexual phase of the parasite reproduction in the mosquito and positive natural selection by the host immune system (Chen et al., 2000, Escalante et al., 2004). This variation may partly explain why the acquisition of natural immunity to malaria is slow since the immune system is exposed to a constantly changing parasite population.

The merozoite is the parasite stage that invades circulating erythrocytes and reticulocytes during the parasite life cycle (Cowman and Crabb, 2006). Vaccine development efforts have focused on merozoite surface proteins (MSPs) because they are accessible to antibodies and complement, and they play critical roles in erythrocyte invasion (Holder, 2009). P. vivax merozoite surface protein-1 (PvMSP-1), like P. falciparum MSP-1 (PfMSP-1), after proteolytic processing generates a C-terminal fragment of 42 kDa (MSP-142), which subsequently produces 33 kDa (MSP-133) and 19 kDa (MSP-119) fragments. The MSP-119 remains on the merozoite surface during and just after erythrocyte invasion (Holder, 2009). Both MSP-142 and MSP-119 fragments are under consideration for vaccine development (Galinski and Barnwell, 2008, Holder, 2009). In an immuno-epidemiological study carried out in Sri Lanka, individuals responded more to PvMSP-142 than to PvMSP-119 (Wickramarachchi et al., 2007). The baculovirus produced PcMSP-119 antigen, closely related to PvMSP-119, was highly protective in vaccination trials carried out in the natural simian host-parasite system involving Plasmodium cynomolgi and the toque monkey Macaca sinica (Perera et al., 1998, Amaratunga, 2004).

Although sequence variation in PvMSP-1 genes has been studied extensively, few studies have focused on the diversity in the regions coding for PvMSP-142 (PvMSP-133 and PvMSP-119) (Tanabe et al., 1987, Putaporntip et al., 2002, Putaporntip et al., 2006, Escalante et al., 1998, Pacheco et al., 2007, Thakur et al., 2008, Sawai et al., 2010). This lack of interest in vaccine candidate diversity is surprising, given that host immune responses have specifically maintained it as an effective parasite survival strategy (Tanabe et al., 2007), and that subunit vaccines based on polymorphic polypeptides are destined to have short-lived efficacy at best. Like P. falciparum MSP-142, P. vivax MSP-142, exhibits extensive genetic polymorphism in natural infections (Escalante et al., 1998, Conway et al., 2000, Putaporntip et al., 2002, Pacheco et al., 2007, Thakur et al., 2008). In previous studies it has been shown that in P. falciparum, MSP-119 fragment was under positive selection and the MSP-133 was neutral or under purifying selection, while the opposite pattern was observed in P. vivax (Pacheco et al., 2007, Thakur et al., 2008).

Protection from infectious disease by the host immune response requires specific molecular recognition of unique epitopes of a given pathogen. The complex interplay of B and T cell epitopes of a parasite antigen, with relevant host MHC molecules are central to the specific stimulation of humoral and cell mediated host immune response(s). Therefore, polymorphism in predicted B and T cell epitopes of a parasite antigen in different parasite strains will enable parasites to escape host immune responses (Tanabe et al., 2007).

In this study we aimed to evaluate how the genetic diversity in the PvMSP-142 locus is generated and maintained in natural P. vivax infections in Sri Lanka, where low transmission and unstable malaria prevails. Tests of diversity and of neutrality, statistical analysis of recombination and linkage disequilibrium, phylogenetic analysis, fixation index values, and polymorphism of predicted B and T cell epitopes were examined. Furthermore, we compared our data with worldwide PvMSP-142 sequences obtained from the NCBI GenBank database to understand the global picture of PvMSP-142 diversity.

Section snippets

P. vivax isolates

This study was approved by the ethics review committee of the University of Colombo, Sri Lanka (EC/04/103). Following informed voluntary consent from patients tested positive for P. vivax infection via Giemsa stained thick and thin blood smears, 5 ml of venous blood was collected from each patient (age >15) prior to anti-malarial therapy. The samples were collected from December 1998 to March 2000 from three different regions; (i) General Hospital, Anuradhapura (8°22′N, 80°20′E; N = 42); (ii)

Results

Of 167 P. vivax infected blood samples initially tested, only 95 (Colombo, N = 37; Anuradhapura, N = 22 and Kataragama, N = 36) were successfully amplified for the 1016 bp PvMSP-142 fragment. We performed all genetic analyses for the PvMSP-142, PvMSP-133 and PvMSP-119 fragments separately in the two endemic study populations, and in the entire local population and compared the latter with previously published global isolates.

Acknowledgements

Financial assistance by the National Science Foundation, Sri Lanka (grant no NSF/RG/2005/HS/06) and the National Research Council of Sri Lanka (grant no NRC-05-34) is acknowledged. The assistance rendered by Drs. Shiroma M. Handunnetti and Thilan Wickramarachchi, and Messers L. Perera and S. Bandara of the Malaria Research Unit, Department of Parasitology, Faculty of Medicine in sample collection (through PVUR's grant No. F/3008-1 from IFS, Sweden), and by Ms Anoma de Silva of the Department of

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