In situ gastrointestinal protection against anthrax edema toxin by single-chain antibody fragment producing lactobacilli

Abstract

Background: Anthrax is caused by the bacterium Bacillus anthracis and is regarded as one of the most prominent bioterrorism threats. Anthrax toxicity is induced by the tripartite toxin complex, composed of the receptor-binding anthrax protective antigen and the two enzymatic subunits, lethal factor and edema factor. Recombinant lactobacilli have previously been used to deliver antibody fragments directed against surface epitopes of a variety of pathogens, including Streptococcus mutans, Porphyromonas gingivalis, and rotavirus. Here, we addressed whether or not anthrax toxins could be targeted and neutralised in the gastrointestinal tract by lactobacilli producing recombinant antibody fragments as a model system for toxin neutralisation in the gastrointestinal lumen.

Results: The neutralising anti-PA scFv, 1H, was expressed in L. paracasei as a secreted protein, a cell wall-anchored protein or both secreted and wall-anchored protein. Cell wall display on lactobacilli and PA binding of the anchored constructs was confirmed by flow cytometry analysis. Binding of secreted or attached scFv produced by lactobacilli to PA were verified by ELISA. Both construct were able to protect macrophages in an in vitro cytotoxicity assay. Finally, lactobacilli producing the cell wall attached scFv were able to neutralise the activity of anthrax edema toxin in the GI tract of mice, in vivo.

Conclusion: We have developed lactobacilli expressing a neutralising scFv fragment against the PA antigen of the anthrax toxin, which can provide protection against anthrax toxins both in vitro and in vivo. Utilising engineered lactobacilli therapeutically for neutralising toxins in the gastrointestinal tract can potential be expanded to provide protection against a range of additional gastrointestinal pathogens. The ability of lactobacilli to colonise the gastrointestinal tract may allow the system to be used both prophylactically and therapeutically.

Figure 1

Plasmid constructs for expression of the 1H scFv in lactobacilli based on the expression cassette from the apf gene from Lactobacillus crispatus M247. Variations in the anchoring domain and placement of the translational stop codon gives respectively secreted, cell wall anchored or attached production of the scFv. The APF promoter (P), APF signal peptide (blue), APF anchoring domain (light blue), translational stop codon (arrowhead), 1H scFv (white), prtP anchor (yellow), E-tag (orange) and transcriptional terminator (lollipop) are indicated.

Figure 2

(A) Detection of the scFv expressed by recombinant L. paracasei by immunoblotting. Cell extract (c) of cell wall anchored strain (KKA307), secreted strain (KKA308) and attached strain (KKA317). Culture supernatant (s) from cell wall anchored strain (KKA307), secreted strain (KKA308) and attached strain (KKA317). The expected size of L. paracasei produced scFvs was 57.1, 29.2 and 42.2 KDa for the anchored, secreted and attached constructs respectively. (B) Binding and quantification of anti-PA scFv secreted into the growth media of the recombinant lactobacilli as measured by ELISA, with 1H scFv purified from E. coli as a reference (average of 4 experiments).

Figure 3

Flow cytometry analysis of lactobacilli surface displayed scFvs. (A) Visualisation of the production and display of the 1H scFv on the surface of recombinant L. paracasei presenting the scFv anchored (KKA307) or attached (KKA317) (wt L. paracasei was as a negative control). ScFvs were visualised by detection of the E-tag fused to the scFv using a mouse anti-E-tag antibody in conjunction with a FITC conjugated rabbit anti-mouse immunoglobulin antibody. (B) Binding of recombinant L. paracasei through surface displayed 1H scFv to PA coated fluorescent beads. The display of scFv on surface visualised through binding of mouse anti-E-tag antibody and FITC conjugated rabbit anti-mouse immunoglobulin antibody. 1,1 quarter: unlabeled L. paracasei, 2,1 quarter: scFv displaying L. paracasei, 1,2 quarter: PA conjugated fluorescent beads and 2,2 quarter: PA conjugated beads bound by L. paracasei.

Figure 4

In vitro neutralisation of anthrax LT by purified Lactobacillus produced scFv. (A) In vitro neutralisation by L. paracasei produced scFvs of anthrax LT in a macrophage toxicity assay (cell viability) visualised by staining with MTT. Comparison of neutralising capabilities of the secreted 1H scFv produced by KKA308 and the attached 1H scFv produced by KKA317 with the corresponding secreted and attached SAI/II as negative controls. (B) Quantification of in vitro neutralisation by colorimetric measurement of the break down of MTT into dark blue MTT-formazan.

Figure 5

Re-attachment of 1H scFv to the cell wall of L. paracasei detected by immunoblotting. Pellet fraction of attached strain (KKA317) and (KKA101) grown in conditioned media containing 1H scFv (lane 1 and 3 respectively). Pellet fraction of strain KKA101 grown in conditioned media containing no 1H scFv, lane 2.