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

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		<a href="https://2008.igem.org/Team:Slovenia/Background/The_problem" target="_self">The problem</a>
		<a href="https://2008.igem.org/Team:Slovenia/Background/Modern_vaccines" target="_self">Modern vaccines</a>
<a href="https://2008.igem.org/Team:Slovenia/Background/Immune_response" target="_self">Immune response</a>
<a href="https://2008.igem.org/Team:Slovenia/Background/Flagellin" target="_self">Flagellin</a>
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		<a href="https://2008.igem.org/Team:Slovenia/Project/Motivation" target="_self">Motivation</a>
		<a href="https://2008.igem.org/Team:Slovenia/Project/Engineered_flagellin_vaccine" target="_self">Engineered flagellin vaccine</a>
		<a href="https://2008.igem.org/Team:Slovenia/Project/Antigen-TLR_fusion_vaccine" target="_self">Antigen-TLR fusion vaccine</a>
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		<a href="https://2008.igem.org/Team:Slovenia/Results/Engineered_flagellin_vaccine" target="_self">Engineered flagellin vaccine</a>
		<a href="https://2008.igem.org/Team:Slovenia/Results/Antigen-TLR_fusion_vaccine" target="_self">Antigen-TLR fusion vaccine</a>
		<a href="https://2008.igem.org/Team:Slovenia/Results/Real-life_results" target="_self">"Real-life" results</a>
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		<a href="https://2008.igem.org/Team:Slovenia/Notebook/Methods" target="_self">Methods</a>
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		<a href="https://2008.igem.org/Team:Slovenia/Notebook/References" target="_self">References</a>
                <a href="https://2008.igem.org/Team:Slovenia/Notebook/Notebook" target="_self">Notebook</a>
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		<a href="https://2008.igem.org/Team:Slovenia/Acknowledgements" target="_self">Acknowledgements</a>
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<font size="6" color="#C73E4A"><i>Engineered bacterial vaccine</i></font> 
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Various immunization ways have been tested for delivery of <i>H. pylori</i> antigens. Recent studies have shown that oral vaccination is the optimal mucosal route for induction of antigen-specific IgA response. However, antibody production has no role in protective immunity in mice. To increase the immunogenicity of <i>H. pylori</i> antigens, we made fusion proteins with flagellin. Studies have shown that genetically fusing an antigen to flagellin significantly increases the immunogenicity and protective potency of the antigen.  
<br /><br />
 
For our delivery system we selected <i>Escherichia coli</i>, strain JW1908-1 with mutations in genes coding for flagellin, that prevent expression of <i<E. coli</i> own flagellin. We cloned several constructs, which were transformed into <i>E. coli</i>, strain JW1908-1 (+T7 polymerase and bacterial ghost plasmid (BG)).
<br /><br />
 
<strong>PROTEIN EXPRESSION<br /><br /></strong>

To test wheather proteins are expressed in bacteria we analyzed bacterial lysates with Western blotting and surface expression of proteins was checked with confocal microscopy.
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<center><img src="https://static.igem.org/mediawiki/2008/5/5d/Westrn.gif" width="372" height="254" border="0" /></center>
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<strong>The majority of chimeric flagellin constructs in bacteria was expressed as soluble proteins. </strong>
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Western blot analysis of expressed proteins was done as indicated in Methods. Sample 1: T7-CF-UB33-CF expressed in soluble fraction of cell lysate (SN1) or in insoluble fraction (IB1); sample 3: T7-CF-AK3 expressed in soluble fraction (SN3); sample 8: TetR-CF-UB33-CF expressed in soluble fraction (SN8) and insoluble fraction (IB8); sample 9: T7-CF-pET expressed in soluble fraction (SN9) and in insoluble fraction (IB9). Expected protein sizes: T7-CF-UB33-CF and TetR-CF-UB33-CF 57.9 kDa, T7-CF-AK3 and T7-CF-pET 54.8 kDa. 
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With Western blot analysis we detected high expression of our proteins of expected sizes.
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<img src="https://static.igem.org/mediawiki/2008/3/36/ControlKoncna2.gif" width="590" height="296" border="0" /><br /><br />
<img src="https://static.igem.org/mediawiki/2008/e/e1/T7-CF-AK3%2BKoncna.gif" width="591" height="296" border="0" /><br /><br />
<img src="https://static.igem.org/mediawiki/2008/f/f5/T7-CF-UB33-CF%2BKoncna.gif" width="590" height="295" border="0" /><br /><br />
<img src="https://static.igem.org/mediawiki/2008/c/c2/TetR-CF-UB33-CF%2BKoncna.gif" width="589" height="295" border="0" /><br /><br />

<strong>Confocal microscopy showed expression of chimeric flagellins on the surface of bacteria.</strong>
<br /><br />
Confocal microscopy images of surface-stained bacteria expressing chimeric flagellin. Flagellin knockout <i>E. coli</i> strain JW1908-1 was transformed with constructs coding for: T7-CF-AK3, T7-CF-UB33-CF and TetR-CF-UB33-CF and grown in liquid culture. Bacteria were incubated with mouse anti-His antibodies and after that another 1h with goat anti-mouse IgG-FITC. Stained bacteria were visualized using a Leica TCS SP6 confocal microscope.
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<strong>Cell activation with live bacteria and bacterial lysate</strong>
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To symulate in vivo responses of the immune system to bacteria, we stimulated TLR5 transfected HEK293 cells with bacteria expressing recombinant proteins on their surface. We tested live bacteria and bacterial lysate symultaneously.
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<img src="https://static.igem.org/mediawiki/2008/5/57/Bakterije.gif" width="451" height="326" border="0" /><br /><br />
<strong>Activation of innate immunity with live or lysed bacteria expressing our engineered flagellin.</strong>
<br /><br />
Flagellin knockout <i>E. coli</i> strain JW1908-1 was transformed with FliC from <i>E. coli</i> in pET19b vector and with chimeric flagellin with urease B as an antigen from <i>H. pylori</i>. Bacteria were grown in liquid culture and harvested 5 h after IPTG induction. One half of bacteria samples was incubated at 4°C and the other one was incubated at 70°C (cell lysate). 2000x diluted bacteria were tested on HEK293 cells transfected with TLR5 and luciferase reporter plasmids to prove TLR5 signaling activation with dual luciferase assay as indicated in Methods.
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<strong>Bacterial ghost system</strong>
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We also used the bacterial ghost (BG) system as a novel vaccine delivery vehicle. BG are nonliving Gram-negative empty bacterial envelopes. They enhance immune responses against envelope bound antigens, including mucosal immunity and T-cell activation. Further advantages of bacterial ghost vaccines are safety, simplicity of the production method and the fact that they can be stored and processed without the need for refrigeration. The major advantage of the approach would be easy distribution throughout remote and third world countries.
<br /><br />
 

<i>E. coli</i>, strain JW1908-1, was transformed with a BG plasmid, coding for the lysis gene E under control of a temperature induced promoter in order to lyse and empty the bacteria of their cytoplasmic contents. Controlled expression was induced at 42° C.
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<img src="https://static.igem.org/mediawiki/2008/1/1a/BGkoncnacopy.gif" width="650" height="394" border="0" /><br /><br />
 

Schematic presentation of bacterial ghost plasmid function<br /><br />

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