Engineering of recombatinant antimalria antibodies for application in paratransgenesis
Malaria, transmitted by Anopheles mosquitoes is responsible for millions of deaths worldwide, especially in the developing countries. The emergence of drug resistant parasites and insecticide resistant vectors has stimulated efforts to develop novel genetic strategies to modify the insect vector and reduce its competence to transmit the parasite. One proposed approach is the genetic manipulation of insect’s midgut symbionts to express anti-parasite molecules. Recombinant antibodies that target specific antigens expressed on the parasites’ surface could be used as anti-parasite molecules, especially if they could not only bind but agglutinate the target. The murine antibody 4B7 binds to Pfs25 epitope expressed on the zygote and ookinette stages of the parasite. Pf-NPNA-1 is a human antibody that specifically binds to the NPNA (Asn-Pro-Asn-Ala) repeats of the circumsporozoite protein expressed on the sporozoite stage of the malaria parasite. This study aimed to characterise these antibodies for their application in symbiont control. For this purpose, the antibodies have been codon optimised for bacterial expression and formatted as single chain variable fragments (scFv). Synthetic genes encoding the scFv 4B7 and Pf-NPNA-1 were constructed, with varying linker length, in the VH-VL and VL-VH orientation. The scFvs were cloned into different expression plasmids to evaluate a suitable expression system. The orientation of the variable domains on secretion of the scFv 4B7 was investigated. No secretion was observed for the scFv 4B7 in the VH-VL orientation. For the reverse orientation, scFv 4B7 (VL-VH) was poorly secreted with no antigen binding. Secretion was observed for a variant of scFv 4B7 but this did not show significant antigen binding. Pf-NPNA-1 scFv constructs, in the VH-VL orientation, were efficiently secreted and showed detectable binding to antigen. Multimeric assembly of the scFv constructs was evaluated by varying the linker length. 4B7 and Pf-NPNA-1 scFv constructs exhibited monomeric, dimeric and multimeric assembly. Fusion of the human kappa constant domain to the scFvs resulted in formation of monomeric and higher ordered forms. Transfer of the scFv gene fragments into a broad-host vector facilitated evalution of recombinant antibody expression in the acetic acid bacterium, Asaia SF2.1. In summary, the results from this study demonstrate the potential utility of the antibodies, 4B7 and Pf-NPNA-1, as anti-parasite molecules for blockade of malaria transmission via mosquito midgut symbionts.
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