Jennings, R.M.; (2004) Murine and human antibody responses to Plasmodium falciparum merozoite surface protein-1. Doctoral thesis , University of London.

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Abstract

A major surface protein complex on the Plasmodium falciparum merozoite, Merozoite Surface Protein-1 (MSP-1) undergoes a proteolytic cleavage at the time of erythrocyte invasion by the parasite. Two murine monoclonal antibodies, mAb 12.8 and mAb 12.10, are specific for a 19 kDa subunit of MSP-1 (MSP-119) and can prevent both this proteolytic cleavage and erythrocyte invasion. Such invasion-inhibitory mAbs may be generated by natural P. falciparum infection in man and may contribute to protection against malaria. To isolate naturally occurring human anti-MSP-119 monoclonal antibodies, two phage display libraries were constructed, consisting of single chain Fv antibody fragments (scFvs) derived from malaria-exposed donors in The Gambia, West Africa. The libraries were panned with recombinant MSP-119 and a number of anti-MSP-119 scFvs were identified. Their ability to bind recombinant MSP-119 and native parasite antigen was demonstrated and their sequences were found to be diverse. The anti-MSP-119 scFv were shown to partially block the binding of mAbs 12.8 and 12.10 to MSP-119 but none of the scFvs isolated could be shown to inhibit erythrocyte invasion in vitro. In order to further characterise the invasion-inhibitory murine hybridoma mAbs 12.8 and 12.10, the DNA sequences encoding the variable, antigen-binding, regions of these mAbs were obtained and confirmed by mass spectrometry of the antibodies themselves. The mAbs 12.8 and 12.10 were reconfigured into scFv format to serve as a future model in which to test ways of restoring invasion-inhibitory activity to scFvs. The scFv 12.10 was shown to bind its target antigen but could not be shown to inhibit erythrocyte invasion in vitro. Preliminary experiments were performed to examine ways in which any invasion-inhibitory properties of the parent mAbs might be restored to the human and murine scFvs. Chimeric murine/human versions of mAbs 12.8 and 12.10, engineered from the mAb sequences obtained, were shown to bind recombinant and native antigen, and were also shown to inhibit erythrocyte invasion in vitro. In the future, the chimeric mAbs will be suitable reagents to test the ability of invasion-inhibitory mAbs to modify P. falciparum infection in a primate model, and the phage display libraries should provide a resource from which naturally occurring human invasion-inhibitory mAbs can be isolated.

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