Team:Arizona State/Data

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<h3>Original Technical Design</h3>
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<h3>Technical Design</h3>
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E. coli Nissle 1917 will be engineered to secrete granulocyte colony-macrophage stimulating factor (GM-CSF) to attract dendritic cells (DCs). GM-CSF is a well-studied immunostimulant used in multiple clinical vaccines. The modular nature of this secretion system allows the bacteria to also secrete adjuvants and other immunostimulants to provoke a variety of immune responses. The bacteria will target dendritic cells via cell surface expression of the anti-DEC-205 single-chain variable fragment (scFv), which targets the DC endocytic protein DEC-205 and facilitates bacterial uptake by the DC.  Once enside the dendritic cell endosome, the acidic conditions will activate the expression of the listeriolysin O (LLO) protein in the bacteria, which will shear the endosome and allow the cancer cell markers expressed in the bacteria to enter to DC cytosol. Expression of the LLO protein is inhibited in neutral and basic conditions by an antisense RNA to the RBS of the composite LLO sequence, functionally creating a genetic pH switch for LLO expression. Cytosolic presence of cancer cell antigens allows them to be expressed on DC major histocompatibility complex (MHC) Class I molecules, which are critical to activate a Cytotoxic T Lymphocyte (CTL) response.  
E. coli Nissle 1917 will be engineered to secrete granulocyte colony-macrophage stimulating factor (GM-CSF) to attract dendritic cells (DCs). GM-CSF is a well-studied immunostimulant used in multiple clinical vaccines. The modular nature of this secretion system allows the bacteria to also secrete adjuvants and other immunostimulants to provoke a variety of immune responses. The bacteria will target dendritic cells via cell surface expression of the anti-DEC-205 single-chain variable fragment (scFv), which targets the DC endocytic protein DEC-205 and facilitates bacterial uptake by the DC.  Once enside the dendritic cell endosome, the acidic conditions will activate the expression of the listeriolysin O (LLO) protein in the bacteria, which will shear the endosome and allow the cancer cell markers expressed in the bacteria to enter to DC cytosol. Expression of the LLO protein is inhibited in neutral and basic conditions by an antisense RNA to the RBS of the composite LLO sequence, functionally creating a genetic pH switch for LLO expression. Cytosolic presence of cancer cell antigens allows them to be expressed on DC major histocompatibility complex (MHC) Class I molecules, which are critical to activate a Cytotoxic T Lymphocyte (CTL) response.  
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The aDEC-205 scFv and the pH switch for LLO expression were later delegated as future tasks to improve vaccine safety and efficiency.  
The aDEC-205 scFv and the pH switch for LLO expression were later delegated as future tasks to improve vaccine safety and efficiency.  
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<h3><center>Vaccine Chassis</center></h3>
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<center><img src="https://static.igem.org/mediawiki/2013/a/a2/E._Coli_Nissile_Probiotic_Vaccine.png" width="800"></center>
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<center><p><i>Figure 1: Assembled biobricks expressed in E.coli Nissile.</i></p></center>
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<center><p><b>Blue</b>: BBa_K1190002 Coding sequence for Lysteriolysin O pore-forming complex.</p></center>
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<center><p><b>Red</b>: BBa_K1190000 and BBa_K1190004 Modular pathogenic antigen of choice. For our project, we used Melan-A (MART-1) and FluM1 target antigens</p></center>
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<center><p><b>Green</b>: BBa_K1190003 Granulocyte Macrophage Colony-Stimulating Factor with Yeb bacterial secretion tag</p></center>
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<h3><center>Overview of Dendritic Cell Response Process</center></h3>
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<center><img src="https://static.igem.org/mediawiki/2013/0/0d/Overview_of_Dendritic_Cell_Respose_Process.png" width="800"></center>
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<p><center><i>Figure 2: Uptake of genetically engineered probiotic vaccine leads to mounted cytotoxic T-cell response to pathogenic antigen of choice</i></center></p>
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<h3><center>1. Pinocytosis of E. coli vaccine by a Dendritic Cell</center></h3>
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<center><img src="https://static.igem.org/mediawiki/2013/3/34/1._Phagocytosis_of_E._Coli_Nissile_Cell_by_a_Dendritic_Cell.png" width="800"></center>
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<p><center><i>Figure 3: Immunological sampling at the Peyer's Patches leads to uptake of probiotic vaccine.</i><center></p>
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<h3><center>2. Lysosome and E. Coli Nissile Undergoing Lysis within a Dendritic Cell</center></h3>
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<center><img src="https://static.igem.org/mediawiki/2013/a/ae/2._Lysosome_and_E._Coli_Nissile_Undergoing_Lysis_within_a_Dendritic_Cell.png" width="800"></center>
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<p><center><i>Figure 4: Lysosome degrades cellular membrane of E.coli Nissile, causing free diffusion of Lysteriolysin O and expressed pathogenic antigen. Lysteriolysin O integrates into lysosome membrane, causing lysosome degradation and free diffusion of pathogenic antigen in dendritic cell cytoplasm.</i></center><p>
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<h3><center>3. MHC Class 1 Complex Assembled within Endoplasmic Reticulum</center></h3>
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<center><img src="https://static.igem.org/mediawiki/2013/d/d9/3._MHC_Class_1_Complex_Assembled_within_ER.png" width="800"></center>
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<p><center><i>Figure 5: Pathogenic antigens shuttled to Endoplasmic reticulum. Development of MHC class 1 complex.</i></center><p>
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<h3><center>4. MHC Class 1 Complex Elicits Cytotoxic T Cell Response</center></h3>
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<center><img src="https://static.igem.org/mediawiki/2013/5/57/4._MHC_Class_1_Complex_Elicits_Cytotoxic_T_Cell_Response.png" width="800"></center>
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<p><center><i>Figure 6: MHC class 1 complex displays pathogenic antigen epitope on dendritic cell, eliciting cytotoxic T cell response to pathogenic antigen.
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Latest revision as of 01:56, 28 September 2013

Technical Design

E. coli Nissle 1917 will be engineered to secrete granulocyte colony-macrophage stimulating factor (GM-CSF) to attract dendritic cells (DCs). GM-CSF is a well-studied immunostimulant used in multiple clinical vaccines. The modular nature of this secretion system allows the bacteria to also secrete adjuvants and other immunostimulants to provoke a variety of immune responses. The bacteria will target dendritic cells via cell surface expression of the anti-DEC-205 single-chain variable fragment (scFv), which targets the DC endocytic protein DEC-205 and facilitates bacterial uptake by the DC. Once enside the dendritic cell endosome, the acidic conditions will activate the expression of the listeriolysin O (LLO) protein in the bacteria, which will shear the endosome and allow the cancer cell markers expressed in the bacteria to enter to DC cytosol. Expression of the LLO protein is inhibited in neutral and basic conditions by an antisense RNA to the RBS of the composite LLO sequence, functionally creating a genetic pH switch for LLO expression. Cytosolic presence of cancer cell antigens allows them to be expressed on DC major histocompatibility complex (MHC) Class I molecules, which are critical to activate a Cytotoxic T Lymphocyte (CTL) response.  


The aDEC-205 scFv and the pH switch for LLO expression were later delegated as future tasks to improve vaccine safety and efficiency.

Vaccine Chassis

Figure 1: Assembled biobricks expressed in E.coli Nissile.

Blue: BBa_K1190002 Coding sequence for Lysteriolysin O pore-forming complex.

Red: BBa_K1190000 and BBa_K1190004 Modular pathogenic antigen of choice. For our project, we used Melan-A (MART-1) and FluM1 target antigens

Green: BBa_K1190003 Granulocyte Macrophage Colony-Stimulating Factor with Yeb bacterial secretion tag


Overview of Dendritic Cell Response Process


Figure 2: Uptake of genetically engineered probiotic vaccine leads to mounted cytotoxic T-cell response to pathogenic antigen of choice

1. Pinocytosis of E. coli vaccine by a Dendritic Cell

Figure 3: Immunological sampling at the Peyer's Patches leads to uptake of probiotic vaccine.


2. Lysosome and E. Coli Nissile Undergoing Lysis within a Dendritic Cell

Figure 4: Lysosome degrades cellular membrane of E.coli Nissile, causing free diffusion of Lysteriolysin O and expressed pathogenic antigen. Lysteriolysin O integrates into lysosome membrane, causing lysosome degradation and free diffusion of pathogenic antigen in dendritic cell cytoplasm.


3. MHC Class 1 Complex Assembled within Endoplasmic Reticulum

Figure 5: Pathogenic antigens shuttled to Endoplasmic reticulum. Development of MHC class 1 complex.


4. MHC Class 1 Complex Elicits Cytotoxic T Cell Response

Figure 6: MHC class 1 complex displays pathogenic antigen epitope on dendritic cell, eliciting cytotoxic T cell response to pathogenic antigen.


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