Next generation sequencing to detect variation in the Plasmodium falciparum circumsporozoite protein

Abstract

The malaria vaccine RTS,S/AS01, based on immunogenic regions of the Plasmodium falciparum circumsporozoite protein (CSP), has partial efficacy against clinical malaria in African children. Understanding how sequence diversity in CSP T- and B-cell epitopes relates to naturally acquired and vaccine-induced immunity may be useful in efforts to improve the efficacy of CSP-based vaccines. However, limitations in sequencing technology have precluded thorough evaluation of diversity in the immunogenic regions of this protein. In this study, 454, a next generation sequencing technology, was evaluated as a method for assessing diversity in these regions. Portions of the circumsporozoite gene (cs) were sequenced both by 454 and Sanger sequencing from samples collected in a study in Bandiagara, Mali. 454 detected more single nucleotide polymorphisms and haplotypes in the T-cell epitopes than Sanger sequencing, and it was better able to resolve genetic diversity in samples with multiple infections; however, it failed to generate sequence for the B-cell epitopes.

Figure 1.

Determination of a minor allele frequency (MAF) threshold for calling SNPs from the 25 ng/μL concentration of standardized mixed infections. In 454 output (Left), the largest increase in number of SNPs occurred below the MAF of 0.025 (broken line). The solid line marks the MAF chosen for inclusion in this study. In Sanger sequencing output (Right), the largest increase in number of SNPs occurred below the MAF of 0.2 (solid line).

Figure 2.

Primers for amplification of 454 PCR products.

Figure 3.

Mean difference between observed allele frequency in sequence output for 454 and Sanger sequencing and expected allele frequency in five dilutions of standardized mixed infections. The average difference between observed and expected allele frequencies in 454 output was less than 0.1 for each concentration (100, 50, 25, 12.5, and 6.25 ng/μL) and greater than 0.1 for each concentration in Sanger sequencing output. The largest differences occurred at the highest concentration for both technologies. Statistical significance was calculated using a Student t test and is denoted by an asterisk.

Figure 4.

The number of SNPs (Left), haplotypes (Center), and mixed infections (Right) detected in P. falciparum circumsporozoite genes by 454 and Sanger sequencing in 45 dried blood spot field samples from Mali, West Africa.

Figure 5.

Concordance between Sanger sequencing and 454 in determination of majority alleles in the Th2R and Th3R regions of the circumsporozoite gene. Concordant SNPs are those SNPs that are determined to be majority alleles in an infection by both technologies. Discordant SNPs are those SNPS on which the two technologies disagree on the majority allele.