p19-Targeting ILP Protein Blockers of IL-23/Th-17 Pro-Inflammatory Axis Displayed on Engineered Bacteria of Food Origin

Abstract

IL-23-mediated Th-17 cell activation and stimulation of IL-17-driven pro-inflammatory axis has been associated with autoimmunity disorders such as Inflammatory Bowel Disease (IBD) or Crohn’s Disease (CD). Recently we developed a unique class of IL-23-specific protein blockers, called ILP binding proteins that inhibit binding of IL-23 to its cognate cell-surface receptor (IL-23R) and exhibit immunosuppressive effect on human primary blood leukocytes ex vivo. In this study, we aimed to generate a recombinant Lactococcus lactis strain which could serve as in vivo producer/secretor of IL-23 protein blockers into the gut. To achieve this goal, we introduced ILP030, ILP317 and ILP323 cDNA sequences into expression plasmid vector containing USP45 secretion signal, FLAG sequence consensus and LysM-containing cA surface anchor (AcmA) ensuring cell-surface peptidoglycan anchoring. We demonstrate that all ILP variants are expressed in L. lactis cells, efficiently transported and secreted from the cell and displayed on the bacterial surface. The binding function of AcmA-immobilized ILP proteins is documented by interaction with a recombinant p19 protein, alpha subunit of human IL-23, which was assembled in the form of a fusion with Thioredoxin A. ILP317 variant exhibits the best binding to the human IL-23 cytokine, as demonstrated for particular L.lactis-ILP recombinant variants by Enzyme-Linked ImmunoSorbent Assay (ELISA). We conclude that novel recombinant ILP-secreting L. lactis strains were developed that might be useful for further in vivo studies of IL-23-mediated inflammation on animal model of experimentally-induced colitis.

Keywords: IL-23; albumin-binding domain; binding protein; cytokine; lactococcus; surface display.

Figure 1

Binding of p19-TRX fusion protein to immobilized ILP317 variant in ELISA. Left: Recombinant p19 protein, alpha subunit of human IL-23, was produced as a fusion protein with Thioredoxin A. Protein was expressed in E. coli BL21(λDE3), purified from inclusion bodies and refolded from 8 M urea extracts. Final product of calculated molecular weight 40 kDa is shown as a stained band after SDS polyacrylamide gel electrophoresis. Right: 96-well Polysorp ELISA plate was coated with the ILP317 protein variant in the form of a fusion with TolA-Avitag protein. p19-TRX was used as an analyte, detected by anti-IL-23 (p19) polyclonal antibody and anti-mouse IgG-HRP conjugate. The result represents three individual measurements and the error bars indicate standard deviations.

Figure 2

Analysis of protein expression by Western blot (A) and Coomassie Brilliant Blue-stained SDS PAGE gel (B) of lysates of L. lactis cells expressing ILP binding proteins and ADN23. All proteins are in fusion with Usp45 secretion signal, FLAG tag and LysM-containing cA surface anchor.

Figure 3

Flow cytometry of L. lactis cells displaying ILP proteins, or ADN23, detected with Anti-FLAG tag antibodies demonstrating mean fluorescence intensity (MFI; A) or a shift in fluorescence intensity (B). Vertical bars denote standard error. Significant differences (p < 0.05) are marked with an asterisk.

Figure 4

Flow cytometry of L. lactis cells displaying ILP proteins, or ADN23, without FLAG tag (A,B) or with FLAG tag (C,D) detected with recombinant p19-TRX protein. Mean fluorescence intensity (MFI; A,C) or a shift in fluorescence intensity (B,D) are depicted. Vertical bars denote standard error. Significant differences (p < 0.05) are marked with an asterisk.

Figure 5

ELISA-determined removal of IL-23 from the solution by L. lactis displaying ILP binding proteins or ADN23. Concentration of remaining IL-23 is shown. Vertical bars denote standard error. Significant difference (p < 0.05) in comparison to control (8148) is marked with an asterisk.

Figure 6

Modeling of p19/ILP interactions. (A) Comparison of homology model of mouse p19 protein (green) to crystal structure of human IL-23/IL-23R complex (PDB ID 5MZV [39]), with p19 (blue), p40 (red) and IL-23R (magenta). Representative binding modes from docking of ILP variants ILP030 (B), ILP317 (C) and ILP323 (D) to the homology model of the mouse p19 protein (green) are shown. Yellow and orange colors indicate common binding areas predicted on the mouse p19. The residues involved in the interaction with the orange binder overlap with p19/p40 interaction interface (Figure 6A blue/red) while the yellow binding mode corresponds to p19/IL-23R interface (Figure 6A blue/magenta).