Immunobricks bricks Immuno Immunobricks Immunobricks ���������� to�prepare a�hi-tech synthetic vaccine against Helicobacter pylori Helicobacter pylori • About 50 % of world population infected • Main cause of peptic ulcers and gastric cancer Alternative = • 7 million get sick every year • 8 th leading cause of death by 2010 • Antibiotic treatment is effective, BUT: – costs – antibiotic resistance – reinfection
Vaccines Save more lives than any other medical treatment • Requirements – Safety – Efficacy – Cost, stability, method of application... • Current vaccine problems – Whole microbes Safety ? – Subunit vaccines Efficacy ? • Need for adjuvants “Little dirty secret” Immune system • Adaptive immune response – Antibody production – Strong cellular response • Innate immune response – Particularly Toll-like receptors (TLRs) – PAMP recognition (MPLA, flagellin, dsRNA)
Master of disguise Hexaacylated LPS H. pylori E. coli Tetraacylated LPS TLR4 Flagellin Flagellin TLR5 TLR5 does Cell activation: not bind H. •chemokines pylori •cytokines flagellin •interferon... NF κ B P50/52 The idea Track 1 • Reengineer flagellin of H.pylori to activate TLR5 Track 1 Track 2 • Activate TLRs by dimerization, colocalization with antigen Track 2
Modular composition of vaccine in track 1 Track 1 Urease B Antigens Multiepitope Flagellin TLR agonists Flagellin Cell adhesion RGD tripeptide Peptide tags His 6 tag Chimeric flagellin Track 1 Chimeric flagellin H. pylori FlaA E. coli FliC Antigenicity, functionality TLR5 activation No TLR5 activation
Chimeric flagellin Track 1 Innate response Adaptive response against H. pylori activation of innate H. pylori variable immunity (TLR5) flagellin domain protein antigens H. pylori antigen (activation of (e.g. UreB) adaptive immunity) Multiepitope Track 1
Implementation Track 1 protein vaccine engineered bacteria DNA vaccine CMV ss HF CMV-HF-UreB.. Antigen Implementation1 Protein vaccine Track 1a Circular dichroism Western Isolated chimeric flagellin is correctly folded. Isolated recombinant chimeric flagellin activates cells through TLR5. Cells internalize fluorescently labelled vaccine
Implementation 2 DNA vaccine Track 1b chimeric flagellin- H. pylori antigen cellular and DNA humoral vaccine immune response TLR5 activation NF κ B P50/52 antigen presentation, Host activation tissue cytokines, ... host macrophage cells Implementation 2 DNA vaccine Track 1b Cell, cotransfected with TLR5 and DNA vaccine Chimeric flagellin needs to be secreted Transactivation of TLR5 by secreted in order to activate cells with TLR5. chimeric flagellin.
Implementation 3 Modified bacteria Track 1c E. coli express chimeric flagellin E. coli express chimeric flagellin at their (Western blot). surface (confocal microscopy, anti-His Ab) The idea Track 1 • Reengineer flagellin of Track 1 H. pylori to activate TLR5 Track 2 • Activate TLRs by dimerization, colocalization with antigen Track 2
TLR signaling Track 2 TLR signaling activates antigen processing Track 2 Adjuvant (PAMP -TLR ligand) protein antigen protein antigen MHCII phagosome maturation, antigen degradation antigen processing and presentation
Constitutively active antigen-TLR fusion Track 2 Antigen and TLR signal are colocalized No additional adjuvant is required MHCII phagosome maturation, antigen processing and presentation Modular composition of vaccine in track 2 Track 2 Urease B Antigens Multiepitope Dimerization domain CD4e CD4sh TM domain TLR3 TM TIR3 Active domain TIR4 His 6 tag Peptide tags HA tag
The function of TLR-fusion vaccine B CELL Track 2 BASIC STRUCTURE OF ANTIGEN –TLR VACCINE ANTIGEN DIMERIZATION CELL MEMBRANE DOMAIN TRANSMEMBRANE T CELL DOMAIN ACTIVATION DOMAIN ACTIVATION OF T CELL RECEPTOR COSTIMULATORS NUCLEUS ENDOSOME APC TLR4 activation Track 2 Localized at cell membrane and in cellular vesicles.
Selecting localization Track 2 Localization Signaling network of TLR-fusion vaccine Track 2 Combined activation of several TLRs mimics stimulation by complete microbe (+synergy between MyD88- and TRIF-dependent signaling) CellDesigner 4.0
Final test - in vivo efficiency of the vaccine Immune serum against designed epitope (MULTI) recognizes live H. pylori 3 weeks! Relative flourescence intensity neg. control Secondary Ab to H. pylori CF-sera to H. pylori Relative flourescence intensity Electroporated DNA vaccine is expressed in mouse leg Strong immunoreactivity in the sera of immunized mice (CF-MULTI). +Serum titer after only 10 days is encouraging ! BioBricks • 132 new parts in the registry • More than 35 composite bricks • More than 20 bricks expressed in eucaryotic and bacterial cells and characterized with various experiments • All basic bricks were sequenced
Achievements • Synthetic biology and BioBrick principles were used to produce synthetic vaccine • Reengineered H. pylori flagellin that became visible to the immune system with additional designed antigens attached – Tested three vaccine implementations • Reengineered TLR signaling network to colocalize activation of innate signaling and antigen processing in a single molecule • Demonstrated intense in vivo antibody response Beyond iGEM • Complete animal studies of DNA and bacterial vaccines • Clinical studies - eradication of H. pylori as the ultimate goal • Expand immunobrick library for more efficient synthetic vaccines against other infectious diseases – Track 1: bacteria that have TLR5- unresponsive flagellin ( Bartonella , Campylobacter , Brucella ...) – Track 2: potentially universally applicable principle
Dream Team Students: •Eva Č eh •Vid Ko č ar •Katja Kolar •Ana Lasi č •Jan Lonzari ć •Jerneja Mori •Anže Smole Mentors: •Roman Jerala •Simon Horvat •Mojca Ben č ina •Monika Cigli č •Karolina Ivi č ak •Nina Pirher •Alojz Ihan TollImmune, Roche, MEDILINE, Anthron, Lotri č , Mercator, Fementas, Micro+Polo
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