Uthaipibull, Chairat; (2001) Plasmodium falciparum merozoite surface protein 1: Antigenicity, immunogenicity and structure. Doctoral thesis (Ph.D.), University College London.

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Abstract

The antigenicity, immunogenicity and structure of Plasmodium falciparum C- terminal merozoite surface protein 1 (MSP-119), a leading malaria vaccine candidate, were investigated. Specific antibodies to MSP-119 can be either inhibitory antibodies that inhibit erythrocyte invasion and proteolytic processing of MSP-1, or blocking antibodies that block both the inhibition of erythrocyte invasion and inhibition of proteolytic processing mediated by the inhibitory antibodies. In order to identify the residues contributing to the epitopes of different antibodies on MSP-119, a series of single amino acid substitutions has been made by site-directed mutagenesis. The reactivity of the MSP-119 mutant proteins with a panel of monoclonal antibodies (mAbs) was assessed by western blotting and BIAcore analysis. It was found that, of 27 MSP-119 mutant proteins made, 7 of the substitutions each abolished the binding of one or more mAbs. By combining the substitutions that were found to prevent the binding of blocking mAbs, individual proteins with multiple amino acid substitutions were made. It was found that these proteins continued to bind inhibitory, but not the targeted blocking mAbs; suggesting that it is possible to produce an additive effect within a single protein by combining individual point mutations. In order to investigate the effects of amino acid substitutions on the proteins' immunogenicity and their ability to induce protective antibodies, selected mutant proteins were used to immunise laboratory animals and raise polyclonal antibodies. Total immunoglobulin G (IgG) was then purified from the serum of animals immunised with the proteins and tested for inhibitory activities in the MSP-1 secondary processing-inhibition and the erythrocyte invasion-inhibition assays. We found that both mouse and rabbit IgGs induced by the mutant protein 12+28 (Cysl2 Ile and Cys28 Trp substitutions) significantly inhibited the secondary processing of MSP-1 and erythrocyte invasion by the parasite. Rabbit IgG induced by the mutant protein 27+31+43 (Glu27,Tyr, Leu31,Arg and Glu43 Leu substitutions) significantly inhibited erythrocyte invasion by the parasite, but not MSP-1 secondary processing. These results suggest that the mechanism of inhibition of erythrocyte invasion by inhibitory antibodies is not mediated only by the inhibition of the secondary processing of MSP-1, and other mechanisms may be involved. Using a computational biology approach, the molecular structures and surfaces of the 12+28 and 27+31+43 mutant proteins were studied and compared to those of the wild type protein. We showed that although the overall structures of the mutant proteins and the wild type protein were not different, the molecular surfaces of the mutant proteins at the substituted sites were changed. The changes to the molecular surface of the mutant proteins at these specific sites may contribute to their ability to induce protective antibodies.

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