Team:Slovenia/Results/Engineered flagellin vaccine/Protein vaccine

A) ISOLATION AND CHARACTERIZATION OF CHIMERIC PROTEINS

Flagellin is known to act as the main virulence factor of many pathogenic bacteria and induces proinflammatory signaling. FlaA, a major flagellin that forms the bulk of the H. pylori filament, is essential for motility, colonization and infection of the stomach mucus, and has very low immune stimulatory activity on gastric and intestinal epithelial cells. In contrast to other bacteria, this seems to be an intrinsic property of H. pylori. Flagellin consists of a middle hypervariable domain and conserved N- and C-terminal domains, which are both required for pro-inflammatory activity.

Amino acid sequence of H. pylori FlaA was compared to those of other well known pathogenic bacteria. We found some differences in N -and C- terminal regions, which might prevent TLR5 binding or even actively inhibit TLR5-dependent innate immune response. Besides, flagellins in general have not only potent proinflammatory activity, but also play a role in triggering adaptive immune responses by stimulating chemokine secretion, migration and maturation of DCs, and by modulating T-cell activation.

In this respect, we decided to construct a chimeric protein vaccine for the use in preventing and fighting chronic inflammatory and infectious diseases. Critical amino acid sequences of N- and C-terminal domain were determined based on the structure of Salmonella typhimurium flagellin. For successful TLR5 activation we decided to use N- and C-terminal conserved regions of E. coli flagellin FliC, and keep the hypervariable domain of H. pylori flagellin for optimal adaptive immune system activation. Chimeric flagellin was additionally fused to a H. pylori antigen, urease B or with a computationally constructed multiepitope, as outlined in detail in the Project section. At the end we added RGD tripeptide for better binding to integrins on M cells of the Peyer's patches and His-tag for Ni-NTA purification. Finally, alpha helix structure of chimeric flagellin was determined by CD spectroscopy.

Chimeric flagellins were produced in E. coli, purified and demonstrated by circular dichroism that they are correctly folded. A: Western blot analysis of specific recombinant proteins, CF-multi and CF-UreB. These proteins were expressed in BL21(DE3)pLysS cells and purifed using Ni-NTA chromatography. Elution fractions were subjected to 10% SDS-PAGE and analyzed by immunoblotting as described in Methods. Expressed proteins were determined by using anti-His monoclonal antibody (dilution 1:2000) and goat anti-mouse IgG-HRP secondary antibody (dilution 1:3000). Lane 1: CF-multi (73 kDa), Lane 2: CF-UreB (116.5 kDa). B: Circular dichroism spectrum of isolated chimeric flagellin (CF) at concentration 0.5 mg/mL in MQ water. The CD spectrum showing alpha helix secondary structure was recorded using an optical cell with 1 mm pathway. ACTIVATION OF TLR5 Purified proteins were tested for their biological activity as TLR5 receptor activators using a dual luciferase reporter assay. Among all known PAMPs that stimulate TLR signaling pathways, TLR5 is activated only by flagellin. By using deletion and site-directed mutagenesis and conformational analysis, it was predicted that only evolutionary conserved N- and C- terminal domains of flagellin play an important role in TLR5 activation. Engineered chimeric flagellin stimulates activation of TLR5. HEK293 cells were transfected with TLR5 plasmid and luciferase reporter plasmids. 24 h post transfection cells were stimulated with chimeric flagellin (CF) or chimeric flagellin with multiepitope (CF-multi) for 6 h, lysed and assayed for luciferase activity as described in Methods. Our results demonstrate that chimeric proteins efficiently activate TLR5 signaling pathway. ENDOCYTOSIS OF CHIMERIC VACCINE We analysed flagellin uptake into cells by labeling isolated protein CF-multi and S. typhimurium flagellin FliC with Alexa Fluor 555 hydrazide as described in Methods. In HEK293 cells chimeric flagellin was internalized similarly as native FliC flagellin from S. typhimurium. Our results show that internalisation of chimeric flagellin into HEK293 was comparable with S. typhimurium flagellin FliC. As shown above from negative control Alexa dye without protein also got internalised into cells via endocytosis, but to much lesser extent than our chimeric protein. To ascertain, whether chimeric protein was localized in early endosomes, fluorescently labeled transferrin, which labels endocytic recycling, was co-administered. Labeled chimeric and S. typhimurium flagellin was endocytosed in both - TLR5-transfected and TLR5-untransfected cells, what suggests that it is internalised in a TLR5-independent manner. Taken together, above results suggest that N- and C- terminal amino acid domains of flagellin are likely to be important for strong TLR5 activation, while the variable domain with the antigen inside or at the C-terminal ensures antigen processing. Therefore, our experiments provide strong evidence that combining both domains in a single recombinant chimeric protein vaccine should lead to a more effective immune system activation.

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