Engineering a single-chain antibody against Trypanosoma cruzi metacyclic trypomastigotes to block cell invasion

Abstract

Trypanosoma cruzi is a flagellate protozoan pathogen that causes Chagas disease. Currently there is no preventive treatment and the efficiency of the two drugs available is limited to the acute phase. Therefore, there is an unmet need for innovative tools to block transmission in endemic areas. In this study, we engineered a novel recombinant molecule able to adhere to the T. cruzi surface, termed scFv-10D8, that consists of a single-chain variable fragment (scFv) derived from mAb-10D8 that targets gp35/50. The synthetic gene encoding scFv-10D8 was cloned and fused to a 6×His tag and expressed in a prokaryotic expression system. Total periplasmic or 6xHis tag affinity-purified fractions of scFv-10D8 retained the capacity to bind to gp35/50, as shown by Western blot analyses. Pre-incubation of metacyclic trypomastigotes with scFv-10D8 showed a remarkable reduction in cell invasion capacity. Our results suggest that scFv-10D8 can be used in a paratransgenic approach to target parasites in insect vectors, avoiding dissemination of infective forms. Such advances in the development of this functional molecule will surely prompt the improvement of alternative strategies to control Chagas disease by targeting mammalian host stages.

Fig 1. Amplification and assembly of a scFv.

a Amplification of the light and heavy variable regions of mAb-10D8. Light variable portion amplification products are about 350 bp and heavy variable portion amplification products are about 400 bp. PCR products were obtained using cDNA as template. b Bidimensional representation of the scFv-10D8 (accession # MN106365) by IMGT/Collier de Perles highlighting the hypervariable regions (CDRs) of each variable chain. The amino acid residues were numbered according to the standard IMGT. Residues at positions 23, 41, 89, 104 and 118 are critical for the antibody structure and function. The coloured circles correspond to the CDRs of each chain, and the hatched circle indicates gaps that have been introduced for better alignment with the sequences of deposited variable regions.

Fig 2. 3D structure of scFv-108.

Panel a shows the potential site of interaction between scFv-10D8 and its ligand, β-D-galactofuranose(1–4)N-acetylglucosamine. Model obtained in silico with the most stable interaction between the two molecules using Hex 8.0 software. β-D-galactofuranose(1–4)N-acetylglucosamine is represented by the sticks model, while scFv-10D8 is represented by the surface model for better visualisation of the antigen-binding site. Panel b represents scFv-10D8 in a VH-linker-VL format, highlighting the β-sheets, turns and flexible regions. In yellow are the cysteine residues and the disulfide bonds that are critical for the antibody structure.

Fig 3. Expression, solubility and reactivity of scFv-10D8.

a Western blotting of extracts obtained from uninduced (0 h) or induced (4 h) cultures of Escherichia coli Rosetta strain without (control) or with pET22b-scFv-10D8. Samples from uninduced and induced cultures were harvested, normalized by absorbance at 600 nm (cells from 1.5 mL culture at 0.5 OD are resuspended in 100 μL of sample buffer) and subjected to SDS-PAGE followed by Western blot using Anti-His antibody. b Bacterial cells were fractionated in periplasm, soluble cytoplasmic proteins and insoluble proteins and then tested for scFv-10D8 presence by Western blot using anti-6×His antibody. Insoluble and soluble fractions were prepared keeping the ratio during lysis process of 20 mL culture, and the total extracts corresponds to the sample from previous Western blot (4h after induction). Panel c shows immunoblots of epimastigote cell extracts (2×10⁶ parasites/lane) probed with scFvs or mAbs as primary antibodies and their controls. The scheme below the blots shows the sequence of antibody incubation used to detect their reactivity. Curly bracket corresponds to the antibodies used during Western blot detection step. “C” corresponds to soluble fraction of bacteria without periplasm, and “P” corresponds to a fraction containing soluble proteins located in the periplasm. Anti-6×His and an unrelated scFv (scFv Loxo) were used as negative controls and mAb-10D8 as a positive control.

Fig 4. Effect of pre-incubation of metacyclic trypomastigotes (MTs) with scFv-10D8 on LLC-MK2 cell invasion.

a MTs of T. cruzi G strain were pre-incubated with different dilutions of periplasmic fractions containing sFv-10D8 or an unrelated scFv for 2 h. b MTs of T. cruzi G strain were pre-incubated with different concentrations (250 and 2500 ng) for both purified protein fractions (scFv-10D8) and unrelated scFv (scFv Loxo) for 2 h and a negative control (NC, without antibody presence). FR.A and FR.B correspond to two independent fractions enriched for scFv-10D8. Statistically significant differences are represented by letters above the graph bar. The parasites were washed and incubated with LLC-MK2 cells at MOI 100:1. The percentage of infected cells was determined by the mean of blind counts of 300 cells from three independent experiments. The main statistically significant differences are indicated by horizontal bars above the columns (p ≤ 0.05).