Global Population Structure of the Genes Encoding the Malaria Vaccine Candidate, Plasmodium vivax Apical Membrane Antigen 1 (PvAMA1)

Abstract

Background: The Plasmodium vivax Apical Membrane Antigen 1 (PvAMA1) is a promising malaria vaccine candidate, however it remains unclear which regions are naturally targeted by host immunity and whether its high genetic diversity will preclude coverage by a monovalent vaccine. To assess its feasibility as a vaccine candidate, we investigated the global population structure of PvAMA1.

Methodology and principal findings: New sequences from Papua New Guinea (PNG, n = 102) were analysed together with published sequences from Thailand (n = 158), India (n = 8), Sri Lanka (n = 23), Venezuela (n = 74) and a collection of isolates from disparate geographic locations (n = 8). A total of 92 single nucleotide polymorphisms (SNPs) were identified including 22 synonymous SNPs and 70 non-synonymous (NS) SNPs. Polymorphisms and signatures of balancing (positive Tajima's D and low FST values) selection were predominantly clustered in domain I, suggesting it is a dominant target of protective immune responses. To estimate global antigenic diversity, haplotypes comprised of (i) non-singleton (n = 40) and (ii) common (≥10% minor allele frequency, n = 23) polymorphic amino acid sites were then analysed revealing a total of 219 and 210 distinct haplotypes, respectively. Although highly diverse, the 210 haplotypes comprised of only common polymorphisms were grouped into eleven clusters, however substantial geographic differentiation was observed, and this may have implications for the efficacy of PvAMA1 vaccines in different malaria-endemic areas. The PNG haplotypes form a distinct group of clusters not found in any other geographic region. Vaccine haplotypes were rare and geographically restricted, suggesting potentially poor efficacy of candidate PvAMA1 vaccines.

Conclusions: It may be possible to cover the existing global PvAMA1 diversity by selection of diverse alleles based on these analyses however it will be important to first define the relationships between the genetic and antigenic diversity of this molecule.

Figure 1. Population genetics of the genes encoding the PvAMA1 ectodomain.

A) Polymorphism. Schematic of the Pvama1 region analysed indicating the locations of all nonsynonymous (NS, red lines), synonymous (SP, blue lines) and NS and SP singleton (black and grey lines, respectively) SNPs. Black arrows indicate the positions of PCR primers. B) Diversity. Sliding window analysis showing nucleotide diversity (Π; Pi) values in Pvama1 for the 372 sequences analysed. A window size of 100 bp and a step size of 3 bp were used. C) Natural selection. Sliding window calculation of Tajima's D statistic was performed for the two PNG populations (blue = Madang, n = 61; red = East Sepik, n = 41). A window size of 100 and a step size of 3 were used. Horizontal dashed lines indicates the significance threshold of p = 0.05; A single asterisk indicates significance values for which p<0.05; and double asterisk indicates p<0.01. D) Amino acid allele frequencies. The frequencies of 40 non-singleton NS amino acid polymorphisms are indicated by the proportion of each bar shaded. Polymorphisms with minor allele frequencies (MAF)≥10% as indicated by an asterisk, were used for further analyses. E) Interpopulation differentiation. Pairwise F _ST values were calculated for the two PNG populations at each of the 40 non-singleton NS amino acid polymorphisms. Those with a MAF≥10% are indicated by an asterisk.

Figure 2. Worldwide distribution of PvAMA1 haplotypes.

The frequencies of 210 haplotypes based on the analysis of 23 common amino acid polymorphisms are depicted as pie charts and mapped to their geographic origin. Coloured segments indicate shared haplotypes and grey indicates haplotypes unique to one population. Only two haplotypes were identical to reference strains (Belem/Palo Alto and Chesson I), therefore haplotypes from the remaining reference strains are shown in grey. Sample size (n) and origin are indicated.

Figure 3. Location of 22 polymorphic PvAMA1 residues predicted to be under balancing selection.

A) Ribbon diagram of the PvAMA1 model showing each of the PvAMA1 domains (DI in cyan, DII in magenta, and DIII in orange) and the hydrophobic ligand binding cleft (dark blue). Each of the 22 residues under selection and the 12 hydrophobic cleft residues are shown by CPK-models of their atoms (spheres) and are coloured according to location. B) Solvent-accessible surface representation of the ‘active face’ of the PvAMA1 model. The hydrophobic cleft and polymorphic residues are shown, with colouring as described for panel A. C) Solvent-accessible surface representation of the ‘silent face’ of the PvAMA1 model. The hydrophobic cleft and polymorphic residues are shown, with colouring as described for panel A.

Figure 4. Proximity of the PvAMA1 residues under selection to the hydrophobic ligand-binding cleft.

Solvent-accessible surface representation of the PvAMA1 model showing a top-view of the hydrophobic ligand-binding cleft. Binding cleft residues are highlighted in dark blue, DI polymorphic residues in cyan and a DII residue in magenta. Residues labeled with bold, underlined type are located in close proximity to the cleft and are predicted to be of functional importance.

Figure 5. Clustering patterns of PvAMA1 haplotypes.

Structure analysis of PvAMA1 haplotypes composed of 23 common amino acid polymorphisms. Haplotypes are shown according to A) the membership coefficient (Q) and B) geographic distribution of eleven clusters (K = 11).

Figure 6. Network analysis of PvAMA1 haplotypes.

Haplotypes composed of 23 common amino acid polymorphisms were analysed using the Median Joining algorithm implemented in Phylogenetic Network version 4.6.1.1 software. Nodes represent the haplotypes and lines indicate connections between them. The size of each node indicates haplotype frequency. Colours indicated by the key depict the cluster membership as defined by Structure analyses.