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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.
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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.
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