Global characterization of diversity and selective pressures in five Plasmodium falciparum loci identified by whole-genome sieve analysis as putative antigens

Previously conducted, whole-genome sieve analyses (SA WG ) using whole genome sequencing data from Plasmodium falciparum isolates collected in two placebo-controlled vaccine trials identified twelve single-copy loci as putative protective antigens. Here, five of those loci were investigated to characterize their diversity and mode of evolution . The five selected loci were PF3D7_0711200, PF3D7_0808100, PF3D7_1324300, PF3D7_1335900 and PF3D7_1361800. Each locus was reference-based reconstructed in 2053 clinical isolates sampled from 24 geographic locations throughout Africa, Asia, and Oceania, using publicly available data from MalariaGEN Pf7. Nucleotide diversity ( π ) was estimated for each location, partitioned by synonymous ( π S ) and nonsynonymous ( π N ) sites, and examined on a sliding window. Encoded protein variants were used to determine protein heterozygosity and to infer networks on which to visualize the geographic distribution of common variants. Tajima’s D and McDonald and Kreitman (MK) tests were conducted to identify putative signals of positive selection. Higher malaria transmission was associated with increased π . PF3D7_1335900 (Pf trap ) was the most diverse locus by several fold, indicative of a markedly different mode of evolution. A π N / π S > > 1 in Pf trap revealed strong positive selection. Heterogenous π across the length of all loci, with peaks in diversity common to all continental regions, indicated common selective pressures. Protein variant networks showed geographic structure in all three chromosome 13 loci, suggestive of recent region-specific polymorphisms originating independently in each region. Protein networks and haplotype diversity results showed extensive global diversity of variants and that the Pf3D7 protein variant is rare or altogether absent in most geographic regions. This may result in vaccine evasion when vaccine efficacy is allele-specific and only the Pf3D7 allele is used in vaccine formulations. Our results show that all three loci in chromosome 13 have negligible synonymous diversity, possibly due to recent selective sweep(s) or chromosome region-specific phenomena, but that positive (diversifying) selection acting on Pf trap is sufficiently strong to have enabled new nonsynonymous polymorphisms to rise to detectable frequencies, consistent with a mode of evolution characteristic of antigens. These results suggest that despite their speculative role in naturally acquired malaria immunity, pre-erythrocytic antigens can be subjected to selective pressure imposed by the vertebrate immune system.