Novel empirical and bioinformatic approaches to characterising Plasmodium falciparum antigens and their application to a merozoite-stage vaccine candidate

A highly efficacious vaccine against the malaria parasite Plasmodium falciparum is needed. Repeat sequences are common in P. falciparum proteins and some are known immune targets. Short read sequence data are available for thousands of parasite isolates, but aligning and assembling repetitive sequences remains a challenge. A combined mapping and de novo assembly approach was developed to resolve highly complex and polymorphic allelic repeat sequences in the merozoite protein MSP-1. This approach gave an unbiased call of allele frequencies and full repeat sequence for a majority of clinical isolates tested. These data were used to design polyvalent hybrid sequences that would containing motifs from multiple alleles. Potential construct designs representing a greater spectrum of sequence diversity than that of previously designed polyvalent hybrid antigens were generated. Assays of mechanisms of antibody mediated inhibition of parasite growth are needed to identify which antigen sequences are functional targets of immunity. Such assays are hard to standardise and would be benefitted by availability of human monoclonal antibody reagents. As an approach towards obtaining these, a technique was developed using a tetramerised P. falciparum MSP-1 recombinant antigen to isolate cognate B-cells from the blood of exposed Ghanaian adult donors. Despite lower than expected viability of cryopreserved samples, 82 memory antigen specific B-cells were successfully isolated by single cell flow sorting of lymphocytes from 16 donors. Complimentary DNA encoding both the heavy and light chain immunoglobulin variable regions was sequenced and analysed for two of these cells, revealing some distinct features. This is the first time a tetrameric antigen has been used to isolate human B-cells recognising a P. falciparum antigen, demonstrating their potential for use in the study of malarial immunity. The modest numbers of specific B-cells sorted from cryopreserved samples encourage the application of this approach to freshly obtained samples.