Refolding and characterization of a diabody against Pfs25, a vaccine candidate of Plasmodium falciparum

Anal Biochem. 2022 Oct 15:655:114830. doi: 10.1016/j.ab.2022.114830. Epub 2022 Aug 6.

Abstract

Pfs25, a vaccine candidate, expressed on the surface of the malarial parasite, plays an important role in the development of Plasmodium falciparum. 1269, a monoclonal antibody targeting the epidermal growth factor-like domain 1 and epidermal growth factor-like domain 3 of Pfs25, blocks the transmission of parasites in mosquitoes. In this study, we refolded 1269-Db, a dimeric antibody fragment referred as diabody, designed from 1269, with a yield of 3 mg/litre of bacterial culture. Structural integrity of the protein was validated with thermal stability, disulphide bond analysis and glutaraldehyde crosslinking experiments. To evaluate the functionality of 1269-Db, recombinant monomeric MBP-Pfs25 was produced from bacteria. Qualitative binding assays demonstrated that 1269-Db recognized the epitopes on Pfs25 in its native, but not the denatured state. An apparent KD of 2.6 nM was determined for 1269-Db with monomeric MBP-Pfs25, using isothermal titration calorimetry. 1269-Db recognized the periphery of zygotes/ookinetes, demonstrating recognition of Pfs25, expressed on the surface of the parasite. As the established refolding method resulted in a functional diabody, the optimized method pipeline for 1269-Db can potentially facilitate engineering of antibody fragments with desired properties.

Keywords: 1269; 4B7; Diabody; Inclusion bodies; Malaria; Monoclonal antibody; Pfs25; Plasmodium; Refolding; Transmission blocking.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies, Protozoan
  • Antigens, Protozoan
  • EGF Family of Proteins
  • Malaria Vaccines*
  • Malaria, Falciparum* / prevention & control
  • Plasmodium falciparum
  • Protozoan Proteins / chemistry

Substances

  • Antibodies, Protozoan
  • Antigens, Protozoan
  • EGF Family of Proteins
  • Malaria Vaccines
  • Protozoan Proteins