Allelic diversity and naturally acquired allele-specific antibody responses to Plasmodium falciparum apical membrane antigen 1 in Kenya

Abstract

Although Plasmodium falciparum apical membrane antigen 1 (AMA1) is a leading malaria vaccine candidate, extensive allelic diversity may compromise its vaccine potential. We have previously shown that naturally acquired antibodies to AMA1 were associated with protection from clinical malaria in this Kenyan population. To assess the impact of allelic diversity on naturally acquired immunity, we first sequenced the ectodomain-encoding region of P. falciparum ama1 from subjects with asymptomatic, mild, and severe malaria and measured allele frequency distributions. We then measured antibodies to three allelic AMA1 proteins (AMA1_3D7, AMA1_FVO, and AMA1_HB3) and used competition enzyme-linked immunosorbent assays (ELISAs) to analyze allele-specific antibodies. Seventy-eight unique haplotypes were identified from 129 alleles sampled. No clustering of allelic haplotypes with disease severity or year of sampling was observed. Differences in nucleotide frequencies in clinical (severe plus mild malaria) versus asymptomatic infections were observed at 16 polymorphic positions. Allele frequency distributions were indicative of balancing selection, with the strongest signature being identified in domain III (Tajima's D = 2.51; P < 0.05). Antibody reactivities to each of the three allelic AMA1 proteins were highly correlated (P < 0.001 for all pairwise comparisons). Although antibodies to conserved epitopes were abundant, 48% of selected children with anti-AMA1 IgG (n = 106) had detectable reactivity to allele-specific epitopes as determined by a competition ELISA. Antibodies to both conserved and allele-specific epitopes in AMA1 may contribute to clinical protection.

FIG. 1.

Amino acid differences in the surface-exposed ectodomain of AMA1 between the three alleles AMA1_HB3 (EMBL accession number U33277), AMA1_3D7 (accession number U33274), and AMA1_FVO (accession number AJ277646). AMA1_HB3 differs from AMA1_3D7 at 23 amino acid residues and differs from AMA1_FVO at 18 residues, while AMA1_3D7 differs from AMA1_FVO at 23 residues. Codon positions are numbered according to the numbering reported previously by Hodder et al. (23).

FIG. 2.

(a) Cluster dendrogram used to visualize 129 AMA1 alleles. There was no evidence of clustering of particular alleles in children presenting with severe malaria (squares), mild malaria (circles), or asymptomatic malaria (triangles). (b) Linkage disequilibrium (LD) calculated by using the R² index. Black squares indicate significant values, while gray squares indicate nonsignificant values. The LD decreased rapidly with increasing nucleotide distance, indicating a high meiotic recombination rate leading to the generation of many new alleles, as shown in a.

FIG. 3.

Sliding-window plot of Tajima's D using a window size of 100 nucleotides and including all 129 ama1 alleles. Positive values generally indicate that balancing selection is acting to maintain alleles at intermediate frequencies.

FIG. 4.

Amino acid frequencies at each polymorphic site are shown for the entire sequenced region. Blue indicates a common allele, red indicates the second most common allele, and other colors indicate additional rarer alleles at some sites. Asterisks indicate the positions at which the polymorphisms were observed more commonly for children with clinical malaria than for those with asymptomatic infections, or vice versa, at a P value of <0.10 level. After correction for multiple testing by the Bonferroni method (6), this remained significant only for amino acid residues at position 230 (P < 0.001).