Richard Forde. M.Sc. Immunology and Global Health Molecular Analysis of MSP1 and MSP2 Genotypes of Plasmodium falciparum Obtained from Adults Participating in the P. Falciparum Malaria Protein 10 (FMP10) Vaccine Trial in Kombewa Division, Western Kenya. Richard Forde. M.Sc. Immunology and Global Health

Overview of Presentation: Background – Life Cycle / Genetic Diversity / Multiplicity of Infection/ Capillary Electrophoresis Objectives Results: Plasmodium falciparum genotype diversity Temporal Distribution of P. falciparum Genotypes Conclusions Questions

Background: Malaria Burden 300 million clinical cases reported every year - African Region (85%) Annual death toll of 0.7 – 2.7 million Majority = Children. Each 30 seconds, a child dies from malaria Plasmodium Falciparum Most common - 2.2 billion people worldwide Most pathogenic - kills over 1 million children annually in sub - Saharan Africa alone

P. falciparum: Life Cycle Bite of an infected Anopheles female mosquito. Sporozoites invade hepatocytes = the pre- erythrocytic stage (Liver) Asexual multiplication Merozoites rupture from the infected hepatocytes Infect the red blood cells = erythrocyitic stage Associated with the clinical manifestation of the disease due to rosetting and cytoadherance.

P. Falciparum : Genetic Diversity The P. falciparum genome sequence is comprised of 5,268 predicted protein-encoding genes - 60% of which have not been assigned function Extensive polymorphism - particularly those expressed on the parasite’s surface This study - polymorphism that exists within merozoite surface proteins (MSP) 1 and 2 Markers to determine the genotype of the parasite and to describe the dynamics of parasite infection.

MSP 1+2 – Genetic Diversity MSP1 allelic variants fall under three major types – K1, MAD20 and RO33 MSP2 allelic variants fall under two dimorphic allelic families, designated FC27 and IC3D7 Analysing the size polymorphism in the block 2 and block 3 regions of the MSP1 and MSP2 genes, respectively, allows for the unique identification of the main allelic variants K1, MAD20 and RO33 for MSP1 and FC27 and IC3D7 for MSP2, and the number of distinguishable alleles for these genes within a parasitic infection can be detected

Multiplicity of Infection (MOI) The extensive genetic diversity displayed by P. falciparum results in complex infections consisting of multiple parasite clones Endemic areas - infected individuals can harbor several different parasite clones with distinct genotypes Distinguishing between parasitic clones based on their genotype allows for the calculation of Multiplicity of Infection (MOI). The number of distinguishable alleles for MSP 1 and MSP 2 will be determined and largest of those numbers will be the MOI of that sample

Traditionally… Discrimination of P. falciparum genotypes was performed using agarose-gel electrophoresis to make size comparisons of the amplified products of these genes. ease of use and low cost However, provides inadequate discrimination of alleles. subjective results may lead to a number of genotyping misclassifications.

Capillary Electrophoresis Capillary Electrophoresis (CE) = high-resolution genotyping Allows for the recognition of changes between allele types down to 1bp in size. Studies show: CE detected more MSP 1 and 2 alleles with more precise sizing compared to gel electrophoresis (Liljander et al 2009) Direct comparison of gel and capillary electrophoresis for two anti malarial drug trials showed CE to detect more alleles and provide higher discriminatory power than gel elctrophoresis (Gupta et al 2010)

Objectives: To genotype P. falciparum obtained from volunteers participating in the FMP10 vaccine trial through Capillary Electrophoresis analysis of the MSP1 and MSP2 allelic variants. To analyse the temporal changes in MSP1 and MSP2 allelic frequency and MOI during the course of the FMP10 vaccine trial.

Study Cohort: FMP-10 Vaccine Trial Volunteers

1. P. falciparum genotype diversity A total of 285 MSP1 and MSP2 alleles were detected. 76 (26.7%) K1 36 (12.6%) MAD 20 33 (11.5%) R033 50 (17.5%) FC27 90 (31.7%) IC3D7.

2. Temporal Distribution of P. falciparum genotypes and MOI

Conclusions: Our findings corroborate previous studies that show that CE is a highly sensitive technique that allows for the precise sizing and discrimination of MSP1 and MSP2 alleles The observed temporal changes of P. falciparum infection dynamics were susceptible to the effects of season and the clearance of parasitic infections through anti-malarial treatment.

Future Research: Further assessment of this data comparing vaccinated and non-vaccinated populations is suggested, which would allow an exploration of the workings of the FMP10 vaccine at the molecular level. It is recommended that further genotyping of volunteers associated with clinical malaria be undertaken.

Acknowledgements: Dr. Noel Murphy Combat Diseases of Poverty Consortium Dr. John Waitumbi and all KEMRI staff in Kisumu

Questions????