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

To increase the immunogenicity of H. pylori antigens, we constructed fusion proteins with flagellin. Fusion flagellins with H. pylori antigens were tested as recombinant proteins expressed in bacteria (see Results - Modified bacteria as mucosal vaccines) as a delivery system, as well as a DNA vaccine.

We tested several constructs listed below:

Construct name	vector	Biobrick number
CMV-ssCF	BBa_J52017	BBa_K133021
CMV-ssCF-UreB	BBa_J52017	BBa_K133024
CMV-ssCF-multi-CF	BBa_J52017	BBa_K133023
CMV-CF-multi-CF	BBa_J52017	BBa_K133020
CMV-ssUreB	BBa_J52017	BBa_K133129

Stimulation of TLR5 occurs only when chimeric flagellin is secreted.

HEK293 cells were co-transfected with TLR5, luciferase reporter plasmids and CMV-ss-CF-multi-CF or CMV-CF-multi-CF. Cells expresing only TLR5 were stimulated with flagellin from S. typhimurium (positive control) or with MQ (negative control). 6 h after stimulation, cells were lysed and luciferase activity was measured.

We found out that constructs without signal sequnce did not activate the TLR5 signaling pathway. This proves the hypothesis, that chimeric flagellin with fused H. pylori antigens, had to be transported outside the cell to activate the TLR5 signaling pathway. To confirm these results we made further tests on cell lines (see TLR5 activation in mixed cell lines).



Activation of TLR5 requires flagellin, but is not affected by antigen adding.

HEK293 cells were co-transfected with TLR5, CMV-CF-multi-CF, CMV-ss-CF-UreB or CMV-ss-UreB and luciferase reporter plasmids. Cells expresing only TLR5 were stimulated with FliC from S. typhimurium (positive control) or with MQ (negative control). 6 h after stimulation, cells were lysed and luciferase activity was measured.

There has been activation of TLR5 with chimeric flagellin fused with antigen (multiepitope or urease B). On the other hand there was no activation with urease B. This proves, that chimeric flagellin is needed to activate TLR5 signaling and to stimulate immune response against H. pylori antigene.
Taken together, for TLR5 activation secretion of chimeric flagellin is needed, whereas antigens do not affect flagellin's agonistic function.

TLR5 TRANSACTIVATION IN MIXED CELL CULTURES

The main goal of this experiment was to find out if cells transfected with DNA coding for chimeric flagellin, activate the TLR5 signaling pathway in co-culture cells that expressed TLR5 on their surface.



Schematic illustration of expeimental design of cellular transactivation by chimeric flagellin DNA vaccine.



Chimeric flagellin DNA vaccine produced by transfected cells activates neighboring cells that express TLR5.
The purpose of this experiment was to demonstrate that introduction of DNA vaccine with chimeric flagellin into epithelial cells can trigger activation of near/underlying dendritic cells which have flagellin receptor TLR5 on their surface in vivo. The experiment was performed with a mixed culture of HEK293 cells transfected with CMV-ss-CF-multi-CF DNA and HEK293 cells transfected with the TLR5 plasmid and reporter luciferase plasmids which detect its activation (green bar) (see also scheme above).

Pattern of cytokine production triggered by chimeric flagellin construct

Production of cytokines was determined by the muliplexing analysis on flow cytometer. DNA construct CMV-ss-CF-multi-CF produced the following concentrations of cytokines: 9 ng/ml IL-8, 4 ng/ml IL-6, 16 ng/ml IFN-alph and 30 ng/ml IL-1beta.



Multiplexing flow cytometric determination of cytokines IL-8, IL-6, IFN-alpha and IL-1beta in HEK293 cells transfected with TLR5 and CMV-ss-CF-multi-CF.