Genetic diversity and recombination at the C-terminal fragment of the merozoite surface protein-1 of Plasmodium vivax (PvMSP-1) in Sri Lanka
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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2020, Infection, Genetics and EvolutionCitation Excerpt :The PvMSP1-42 fragment was amplified using the Pv1SF 5´-AGAAGAAAACGTAGCAGCAA-3′ and Pv1SR 5´-AAGCCCAGTTCAGAACTCA-3′ primers (Zhou et al., 2017) or nested PCR primers P1: 5´-GATGACGACGGGGAGGAAGACC-3′, P2: 5´-AAGCTCCATGCACAGGAGG-3′, followed by N1: 5´-GACCAAGTAACAACGGGAG-3′ and N2: 5´-GGACAAGCTTAGGAAGCTGG-3′ primers (Dias et al., 2011). Cycling parameters and primary and secondary reactions were performed as described previously (Dias et al., 2011). Ultraviolet visualization of the amplified product following 1% agarose gel electrophoresis revealed an approximately 1400 bp band using the Pv1SF and Pv1SR primers, and approximately 1200 bp using the N1 and N2 primers.
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2017, Infection, Genetics and EvolutionCitation Excerpt :MSP-142 and MSP-119 fragments have received attention as immunogens, given that antibodies directed against MSP-142 and MSP-119 were shown to interrupt merozoite invasion in vitro (Patino et al., 1997; Nwuba et al., 2002). Sequence variation in the central repeat region of MSP-142 of P. falciparum (see Mehrizi et al., 2008; Zamani et al., 2009; Pan et al., 2010; Viputtigul et al., 2013) and P. vivax (see Dias et al., 2011; Kang et al., 2012) has been relatively well studied, but nothing is known about the structure, function or genetic variation in MSP-1 in P. knowlesi. In the present study, we explored, for the first time, sequence variation in P. knowlesi MSP-142 (designated Pk-MSP-142) from Peninsular Malaysia and Sabah Borneo, Malaysia.