Participating in MSP-142 vaccine trials in Kombewa division, Western Kenya

Abstract

Phase Ib and lIb paediatric clinical trials in children between the ages of 12 and 48 months were conducted in Kombewa division, western Kenya between 2003 and 2006, where the safety and efficacy of Falciparum Merozoite Protein-1 (FMP 1), a merozoite surface protein- 142(MSP-142) malaria vaccine were tested. MSP-142 comprises MSP-119, which elicits protective humoral immune responses according to preclinical studies, and MSP-133 that contains T cell epitopes believed to be essential for supporting the induction of immunity. Limited but significant polymorphisms occur within the conserved C-terminal block of the msp-l gene, where four key single nucleotide polymorphisms (SNPs) (E/Q, T/K, SIN and RIG) are present and allow for the definition of the dominant MSP-119 haplotypes in mixed clone infections. FMP1 was developed from the 307 Plasmodium strain which has the ETSR MSP-119 haplotype and it is not known whether this vaccine would generate an effective immune response against parasite with various MSP-l19 haplotypes in natural infections and whether this would be significant with regard to overall vaccine efficacy. The work described here constitutes the molecular analysis of MSP-119 haplotypes of infecting parasites in vaccinated and non-vaccinated children in a phase Ib and a phase Ilb trial. • The phase 1b trial had three FMP 1 vaccine dose cohorts (10 ug, 25 ug and 50 ug) and a rabies vaccine control arm, while the Phase lIb had the full vaccine dose (50 ug) arm and a rabies vaccine control arm. In the phase I trial genotyping of DNA 'samples was done at enrolment and one month post-vaccination by PCR-sequencing and by Real Time quantitative PCR (RT-qPCR) so as to validate the latter assay. For sequencing analysis, a 400 bp amplicon covering all of block 17 of the msp-l gene which encodes MSP-119 was amplified from each sample and then sequenced before determining the MSP-l19 haplotype. Haplotypes resolved by RT-qPCR involved first determining the dominant allele at each MSP-119 SNP using the threshold cycle (Ct) values of each allele before linking together these alleles. Analysis of MSP-119 haplotypes for the Phase lIb study was conducted at enrolment, one month post-vaccination and for children with clinical malaria using RT-qPCR. Haplotype prevalence was then compared between the vaccine and control groups. Eight different MSP-119 haplotypes were identified from the sequence data (EKNG, EKSG, EKSR, ETNG, ETSR, QKNG, QKSG and QKSR) and by RT-qPCR (EKNG, EKSR, ETNG, ETSR, QKNG, QKSR, QTNG and QTSR). Haplotype prevalence at enrolment determined through direct sequencing of PCR products revealed a predominance of EKNG (49%) and QKNG (40%) while ETSR, the vaccine haplotype, represented only 3% of these infections with other lesser prevalent haplotypes (EKSR, EKSG, ETNG and QKSG) together constituting the remaining 8%. The RT-qPCR results for predicted MSP-119 haplotypes were highly comparable to sequencing: 73% agreement at Day 0 and 91% agreement at Day 90. Six different MSP-119 haplotypes (EKNG, EKSR, ETNG, ETSR, QKNG and QKSR etc) were predicted from the RT-qPCR allele data in the phase lIb trial. At enrolment, EKNG and QKNG were still the major MSP-119 haplotypes as predicted from RT-qPCR (EKNG 65%, QKNG 16%). Other haplotypes were at lower levels with EKSR at 13%, the vaccine haplotype (ETSR) at 4% and QKSR and ETNG together making up the remaining 2%. While the predicted haplotype frequencies following vaccination did not differ between the vaccine and control groups among asymptomatic children at one month post-vaccination, significantly fewer T alleles were observed in the vaccine group (49%) at one month post-vaccination (95% CI 38% - 60%) compared to the control group (64%, 95% CI 53% - 73%, P = 0.04). Comparison of the complete haplotypes in 381 children (176 vaccinees and 205 controls) who had one or more malaria episodes post-vaccination indicated no significant difference in haplotype distribution between the vaccine and control groups when considered altogether or as separate episodes. However, among children who fell sick during the first four months following vaccination there was a significant lower prevalence of the E allele among vaccines: 54% (95%CI 42% - 65%) compared to 69% (95%CI 58% - 79%, P = 0.04) in the controls. In addition, a trend towards significance (p = 0.08) was noted in the time to first clinical episode with the vaccine haplotype (ETSR) among vaccinees suggesting a vaccine-induced delay. The results from the phase Ib showed that the RT-qPCR assay developed was reliable for haplotyping. Furthermore, the observations from the phase lIb trial implied that FMP 1 induced a subtle homologous effect on parasites with the ETSR haplotype. This was the expectation based on previous investigations that examined the specificity of antibodies generated by FMP 1. The results also support previous observations that suggest that effective immune responses induced by FMP 1 seem to be diminished in malaria exposed populations. Given the apparent low level homologous impact and the absence of any detectable effect on heterologous haplotype strains, consideration must be given toward boosting the efficacy ofFMPI through the testing and possible combination with antigens from other strains or other vaccine candidate antigen altogether. Future studies should address these concerns and also unequivocally substantiate the vaccine effects noted in this study.