Polymorphism in the gene encoding the apical membrane antigen-1 (AMA-1) of Plasmodium falciparum. X. Asembo Bay Cohort Project

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Abstract

We have investigated the genetic diversity of the gene encoding the apical membrane antigen-1 (AMA-1) in natural populations of Plasmodium falciparum from western Kenya and compared it with parasite populations from other geographic regions. A total of 28 complete sequences from Kenya, Thailand, India, and Venezuela field isolates were obtained. The genetic polymorphism is not evenly distributed across the gene, which is in agreement with the pattern reported in earlier studies. The alleles from Kenya exhibit 20 and 30% more polymorphism than that found in Southeast Asia and Venezuelan alleles, respectively. Based on the gene genealogies derived from sequencing data, no evidence for allele families was found. We have found evidence supporting limited gene flow between the parasite populations, specifically, between the Southeast Asian and Venezuelan isolates; however, no alleles could be linked to a specific geographic region. This study reveals that positive natural selection is an important factor in the maintenance of genetic diversity for AMA-1. We did not find conclusive evidence indicating intragenic recombination is important in the generation of the AMA-1 allelic diversity. The study provides information on the genetic diversity of the AMA-1 gene that would be useful in vaccine development and testing, as well as in assessing factors that are involved in the generation and maintenance of the genetic diversity in P. falciparum.

Introduction

Malaria is arguably one of the most important parasitic diseases in the world. Today more than 40% of the world's population lives in areas at risk for malaria. Each year there are between 500–600 million cases and 2–3 million deaths attributed to malaria most of which are of young children in sub-Saharan Africa [1]. While antimalarial drugs, insecticides, and bednets are being used against this disease, there is a realization that an effective vaccine is required for the control of malaria.

Different complementary approaches to vaccine development are being undertaken in several laboratories. However, irrespective of the approach, a common concern is the nature and extent of genetic diversity in natural populations of malaria parasites [2]. The obvious question is whether the vaccine-elicited immune response will be equally effective against all the genetic variants of the parasite, and how quickly resistance to the vaccine will develop.

In malaria, the issue of diversity is compounded by the multistage nature of the parasite. We have undertaken a comprehensive study to determine the genetic diversity of Plasmodium falciparum parasites from an area of high malaria transmission in western Kenya [3]. Our interest is also to compare it with the genetic diversity found in natural populations of P. falciparum from other geographic regions. For this purpose, we have included parasite isolates from Venezuela, Thailand, and India.

In this study, we describe the diversity of the gene encoding the apical membrane antigen-1 (AMA-1 gene). The AMA-1, also known as PF83, is a protein of 622 residues and a molecular weight of 83 kDa with three major domains defined by eigth disulfide bonds [4], [5]. This antigen first appears in the apical complex and then migrates to the merozoite's surface. Data from animal models suggests that this protein elicits protective immune responses [4], [6], [7]. We have earlier shown that synthetic peptides complementary to the putative T-cell determinants of AMA-1 are recognized by PBMCs from persons naturally exposed to malaria parasites in western Kenya [8].

In an earlier study of genetic diversity, which only focused on parasites from western Kenya, we reported that the AMA-1 was a conserved antigen as compared with other blood-stage antigens [9], [10]. Conversely, Marshall et al. [11] reported the presence of several alleles, suggesting that this antigen gene had high levels of genetic diversity. Unfortunately, the terms ‘low’ or ‘high’ do not have any genetic meaning. For this reason, we have striven to incorporate appropriate analytical tools in studies of genetic diversity [10].

Based on enzyme restriction sites, four allele families have been proposed for AMA-1 using a hypervariable region (HVR) [12]. The HVR was originally reported by Thomas et al. [13] between residues 160 and 210. However, others have defined the HVR as a larger portion in the domain I [5]; thus defining it between residues 138 and 307 [11], or residues 148–337 [12]. Oliveira et al. [9] subdivided the AMA-1 into 12 segments or blocks based on their level of polymorphism. The block 4 sensu Oliveira et al. [7] is a segment of 46 residues that overlaps with the HVR [9], [13].

This study is a comprehensive analysis of the diversity of the gene encoding the AMA-1 protein using primary isolates of P. falciparum. We have taken an approach to obtain a limited set of sequences from different geographic locations, which increases the probability of sampling the most divergent alleles in order to infer the history and processes involved in the evolution of the observed polymorphism [14]. A total of 28 complete sequences of P. falciparum were obtained from Kenya, Thailand, India, and Venezuela field isolates. The homologous gene in Plasmodium reichenowi, which is the closest related species to P. falciparum, was characterized for comparative analysis [15], [16]. Our P. reichenowi sequence differs in two nucleotide substitutions from the one available in GeneBank under the accession number AJ25087 [17].

Section snippets

Materials and methods

The parasite DNA used in this study was obtained from field isolates from Kenya, India, Thailand, and Venezuela. The gene encoding the AMA-1 protein was amplified by polymerase chain reaction (PCR) using specific primers [9]. The amplification conditions were as follows, first, 1 min at 94°C, followed by 30 cycles with 0.5 min of denaturation at 94°C, annealing at 40°C for 0.5 min, and elongation at 72°C for 1.5 min. After 30 cycles, a final elongation step at 72°C for 3.0 min was carried out.

Results

Table 1 describes genetic variation observed by analyzing complete sequences of the gene encoding the AMA-1. In addition, we have included published sequences in the analysis [11], [12], [13], [18]. π is a measure of genetic polymorphism that estimates the average number of substitutions between any two sequences. The overall genetic diversity in 44 sequences (π=0.01402) is lower than we reported earlier using a smaller sample size (π=0.01635) [10], indicating that the earlier estimation was

Discussion

The AMA-1 protein is considered an important antigen to be included in a multivalent vaccine. Studies based on synthetic peptides have indicated that this vaccine candidate elicits a specific immune response in persons naturally exposed to malaria parasites in western Kenya [8]. A long-term goal of our vaccine-related field studies is to identify naturally immunogenic determinants of key vaccine antigens to assess how the parasite genetic diversity relates to natural immunity. A step in

Acknowledgements

This research is supported by grants from the ‘Consejo Venezolano de Investigaciones Cientı́ficas’, (G97000634) and The National Institutes of Health (R01 GM60740-01) to A.A.E. This work was supported in part by the US Agency for International Development grant HRN-60010-A-00-4010-00 to A.A.L. We thank Oralee Branch, Shannon L. Takala, and Lauren M. Singer for helpful comments on the manuscript.

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