Limited variation in vaccine candidate Plasmodium falciparum Merozoite Surface Protein-6 over multiple transmission seasons

Abstract

Background: Plasmodium falciparum Merozoite Surface Protein-6 (PfMSP6) is a component of the complex proteinacious coat that surrounds P. falciparum merozoites. This location, and the presence of anti-PfMSP6 antibodies in P. falciparum-exposed individuals, makes PfMSP6 a potential blood stage vaccine target. However, genetic diversity has proven to be a major hurdle for vaccines targeting other blood stage P. falciparum antigens, and few endemic field studies assessing PfMSP6 gene diversity have been conducted. This study follows PfMSP6 diversity in the Peruvian Amazon from 2003 to 2006 and is the first longitudinal assessment of PfMSP6 sequence dynamics.

Methods: Parasite DNA was extracted from 506 distinct P. falciparum infections spanning the transmission seasons from 2003 to 2006 as part of the Malaria Immunology and Genetics in the Amazon (MIGIA) cohort study near Iquitos, Peru. PfMSP6 was amplified from each sample using a nested PCR protocol, genotyped for allele class by agarose gel electrophoresis, and sequenced to detect diversity. Allele frequencies were analysed using JMP v.8.0.1.0 and correlated with clinical and epidemiological data collected as part of the MIGIA project.

Results: Both PfMSP6 allele classes, K1-like and 3D7-like, were detected at the study site, confirming that both are globally distributed. Allele frequencies varied significantly between transmission seasons, with 3D7-class alleles dominating and K1-class alleles nearly disappearing in 2005 and 2006. There was a significant association between allele class and village location (p-value = 0.0008), but no statistically significant association between allele class and age, sex, or symptom status. No intra-allele class sequence diversity was detected.

Conclusions: Both PfMSP6 allele classes are globally distributed, and this study shows that allele frequencies can fluctuate significantly between communities separated by only a few kilometres, and over time in the same community. By contrast, PfMSP6 was highly stable at the sequence level, with no SNPs detected in the 506 samples analysed. This limited diversity supports further investigation of PfMSP6 as a blood stage vaccine candidate, with the clear caveat that any such vaccine must either contain both alleles or generate cross-protective responses that react against both allele classes. Detailed immunoepidemiology studies are needed to establish the viability of these approaches before PfMSP6 advances further down the vaccine development pipeline.

Figure 1

Genotyping PfMSP6 using nested PCR and agarose gel electrophoresis. This study utilizes a nested PCR protocol to amplify the region of PfMSP6 where most inter- and intra-allele genetic diversity has been shown to occur. The two major PfMSP6 allele types, K1-class and 3D7-class alleles, result in nested PCR products of significantly different sizes, and allele genotyping was scored using agarose gel electrophoresis, as shown.

Figure 2

PfMSP6 allele distribution changes significantly over consecutive transmission seasons. Distribution of the K1- and 3D7-like allele frequencies across the 2003-2006 transmission seasons showed a significant decline in K1-allele frequency between 2003/2004 and 2005/2006. Each bar represents the percentage of each allele type detected in n samples for the given year, and * denotes significant differences between paired years with p = 0.0001 in χ² analysis.

Figure 3

Distribution of P. falciparum samples analysed. Zungarococha is a small community in the Peruvian Amazon near Iquitos consisting of four separate villages of varying size: Zungarococha village (population = 805), Puerto Almendra (population = 272), Ninarumi (population = 590), and Llanchama (population = 203). Since 2003, the MIGIA cohort study has monitored P. falciparum transmission throughout the community using both active and passive sample detection (for details, see Methods section). Samples were collected from all four villages; the number genotyped from each village reflects variation in both the size of the villages and the burden of P. falciparum infection.