Comparative population structure of Plasmodium falciparum circumsporozoite protein NANP repeat lengths in Lilongwe, Malawi

Abstract

Humoral immunity to Plasmodium falciparum circumsporozoite protein is partly mediated by a polymorphic NANP tetra-amino acid repeat. Antibody response to these repeats is the best correlate of protective immunity to the RTS,S malaria vaccine, but few descriptions of the natural variation of these repeats exist. Using capillary electrophoresis to determine the distribution of NANP repeat size polymorphisms among 98 isolates from Lilongwe, Malawi, we characterised the diversity of P. falciparum infection by several ecological indices. Infection by multiple distinct variants was common, and 20 distinct repeat sizes were identified. Diversity of P. falciparum appeared greater in children (18 variants) than adults (12 variants). There was evidence of genetic distance between different geographic regions by Nei's Standard Genetic Distance, suggesting parasite populations vary locally. We show that P. falciparum is very diverse with respect to NANP repeat length even on a local level and that diversity appears higher in children.

Figure 1. Distribution of variants based upon the number of NANP repeats in all participants, children, and adults.

The x-axis indicates the number of NANP repeats and the y-axis indicates the number of study participants with that variant detected in their blood.

Figure 2. Variant species accumulation and rarefaction curves with 95% confidence intervals.

Variant accumulation and rarefaction curves for all participants (a), adults (b), and children (c) are shown. Variant accumulation curves of the raw data are shown in dark blue. Computed smoothed rarefaction curves determined by simulation are shown as thick red lines, and 95% confidence intervals for the rarefaction curves are demonstrated by thin red lines. The computed rarefaction curve represents the expected average rate of variant accumulation that would be produced by repeated sampling of the same population.

Figure 3. Analysis of genetic similarity between different parts of the city of Lilongwe.

Panel A shows the geographic regions within and around Lilongwe used for the spatial analysis. Arrows show the pairwise comparison between each region and Nei's standard genetic distance between parasites identified in each population. The East Urban Region was the most diverse and had the highest number of participants (48 individuals, 61 variants), followed by the West Urban (25 individuals, 32 variants), West Peri-urban (10 individuals, 11 variants), and East Peri-urban (9 individuals, 11 variants). The map was prepared using ArcGIS (ESRI, Redlands, CA) and Microsoft Powerpoint (Microsoft, Seattle, WA). Panel B shows a principle coordinate analysis of genetic relatedness between regions. East and west urban regions cluster more closely to each other than either periurban region. Coordinate 1 explains 78.9% of the variation and coordinate 2 explains 18.8% of the variation.

Figure 4. Frequency distribution of specific NANP variants within each region.

The east urban region demonstrated the greatest diversity of parasites, but it also had the largest number of samples. As seen in the figure, the distribution of individual variants differed by geographic region.