A study of genetic diversity in the gene encoding the circumsporozoite protein (CSP) of Plasmodium falciparum from different transmission areas—XVI. Asembo Bay Cohort Project

Abstract

We have investigated the genetic diversity of the gene encoding the CS protein. A total of 75 complete and 96 partial sequences are studied. We find high levels of genetic polymorphisms as evidenced by 50 and 24 alleles at the Th2R and Th3R epitopes, respectively. Overall, we find that African isolates are more polymorphic as compared with parasites from other geographic regions. We conclude that the uneven geographic polymorphism may have an adverse impact on the effectiveness of vaccines based on this antigen alone. We find extensive polymorphism in the repeat allotypes, or RATs. In order to explore how the protein structure may impose restrictions in the number of repeats, we have simulated the stability of the structure of the tandem repeat region. Our analysis suggests that the protein structure may play an important role in the observed polymorphism in the number of CS repeats in Plasmodium falciparum. We explored the linkage and recombination events among the polymorphic sites. We found that putative recombination events overlap with linked sites. We discuss how this pattern is explained by the action of positive natural selection, where the recombination events detected are convergent mutations. We conclude that it is inappropriate to use linkage-recombination patterns on genes under positive selection for assessing the structure of parasite populations.

Introduction

The studies conducted on the genetic diversity of malaria parasites could be categorized into two major groups: (a) those directed to understand the population structure, specifically, the extent and role of recombination in natural parasite populations [1], [2]; and (b) studies assessing the diversity of specific genes encoding antigens [3], [4]. In addition to yielding information on evolutionary genetics, these studies answer key questions related to the development and predictive effectiveness of intervention strategies for malaria control programs.

Studies of population structure provide an overall picture of the origin, dispersal and stability of multi-locus genotypes. This information is essential in predicting effectiveness of intervention strategies, such as usefulness of drugs and the emergence multi-drug resistant strains [5], long-term impact of physical interventions such as bed nets, and the origin of genotypes that may elude the immune response elicited by vaccines.

Studies of the diversity of specific genes, on the other hand, provide information about how alleles are generated and maintained in the population. Specifically it addresses the relevance of factors like intragenic recombination and natural selection. It can also assess geographic differentiation to explain the spatial distribution of alleles at a given loci. This approach answers fundamental questions related with the association of specific alleles with drug resistance [6], and how the genetic variation may affect vaccine development [7] and deployment.

As part of our program to investigate the genetic diversity of human malaria parasites, with a particular focus on genes encoding vaccine antigens, we have conducted a detailed study of the diversity of the Circumsporozoite protein (CS). The CS is the predominant protein found on the surface of the CS; it has approximately 420 residues and a molecular weight of 58 kDa. The CSP can be subdivided into two non-repetitive regions (5′ and 3′ ends) and a variable central region consisting of multiple repeats of four-residues-long motifs [8]. There is substantial point mutation polymorphism in the 3′ region of the protein where T-cell epitopes have been identified [9], [10]; this polymorphism has been explained as consequence of positive natural selection by the host immune system [4], [11].

Polymorphism in the CS protein is also observed in the number of tandem repeats in the central region. The extensive diversity in the tandem repeat region is even more evident when the nucleotide sequences of the tandem repeats, or so called repeat allotypes (RATs sensu [12]), are taken into account. It has been proposed that these repeats may be generated by sexual intragenic recombination [8]; however, slipped-strand mismatch repair during mitosis has been suggested [12]. Molecular epidemiologists have used these polymorphisms in malarial antigens as genetic markers even when the available data suggest that the distribution of alleles is maintained by positive natural selection [13].

We report a comprehensive study on the genetic diversity of the gene encoding the CS protein using complete and partial sequences from field isolates from Kenya, India, Cameroon, and Venezuela. We find that African isolates are more polymorphic as compared with parasites from other geographic regions. We conclude that the uneven geographic polymorphism may have an adverse impact on the effectiveness of CS-based vaccines. We explore the linkage and recombination events among the polymorphic sites. We find that putative recombination events overlap with linked sites. We discuss how this pattern is explained by the action of positive natural selection, where the recombination events detected are convergent mutations. In order to explore how the protein structure may impose restrictions in the number of repeats polymorphisms, we have simulated the stability of the structure of the tandem repeat region. Our analysis suggests that the protein structure may play an important role in the observed polymorphism in the number of CS repeats in Plasmodium falciparum.