Engineering of recombatinant antimalria antibodies for application in paratransgenesis

Anani, B. 2011. Engineering of recombatinant antimalria antibodies for application in paratransgenesis. PhD thesis University of Westminster School of Life Sciences

TitleEngineering of recombatinant antimalria antibodies for application in paratransgenesis
TypePhD thesis
AuthorsAnani, B.
Abstract

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.

Year2011
FileBernard_ANANI.pdf
Publication dates
Completed2011

Related outputs

Module based antibody engineering: a novel synthetic REDantibody
Markiv, A., Anani, B., Durvasula, R.V. and Kang, A.S. 2011. Module based antibody engineering: a novel synthetic REDantibody. Journal of Immunological Methods. 364 (1-2), pp. 40-49.

Permalink - https://westminsterresearch.westminster.ac.uk/item/8zwx8/engineering-of-recombatinant-antimalria-antibodies-for-application-in-paratransgenesis


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