Immunobricks bricks Immuno Immunobricks Immunobricks - - PDF document

Immunobricks Immunobricks

toprepare ahi-tech synthetic vaccine against Helicobacter pylori

Immuno Immunobricks bricks

Helicobacter pylori

• About 50 % of world population

infected

• Main cause of peptic ulcers and

gastric cancer

• 7 million get sick every year
• 8th leading cause of death by

2010

• Antibiotic treatment is effective,

BUT: – costs – antibiotic resistance – reinfection

Alternative =

• Requirements

– Safety – Efficacy – Cost, stability, method of application...

• Current vaccine problems

– Whole microbes Safety ? – Subunit vaccines Efficacy ?

• Need for adjuvants

“Little dirty secret”

Vaccines

Save more lives than any other medical treatment

Immune system

• Adaptive immune

response

– Antibody production – Strong cellular response

• Innate immune response

– Particularly Toll-like receptors (TLRs) – PAMP recognition (MPLA, flagellin, dsRNA)

NFκB

• E. coli

Hexaacylated LPS TLR4

Cell activation:

• chemokines
• cytokines
• interferon...

Master of disguise

Flagellin

• H. pylori

Tetraacylated LPS

TLR5 does not bind H. pylori flagellin

Flagellin TLR5

The idea

Track 1

• Reengineer flagellin of

H.pylori to activate TLR5

Track 2

• Activate TLRs by

dimerization, colocalization with antigen

Track 2 Track 1

Modular composition of vaccine in track 1

Urease B Multiepitope Flagellin TLR agonists Cell adhesion Peptide tags Antigens Flagellin RGD tripeptide His6 tag

Track 1

Chimeric flagellin

Track 1

No TLR5 activation

• H. pylori FlaA
• E. coli FliC

Chimeric flagellin Antigenicity, functionality TLR5 activation

Chimeric flagellin

Track 1

• H. pylori variable

flagellin domain

• H. pylori antigen

(e.g. UreB) protein antigens (activation of adaptive immunity) activation of innate immunity (TLR5)

Adaptive response against H. pylori Innate response

Multiepitope

Track 1

Implementation

protein vaccine engineered bacteria DNA vaccine

CMV-HF-UreB..

Antigen HF ss CMV

Track 1

Implementation1 Protein vaccine

Track 1a

Cells internalize fluorescently labelled vaccine Isolated recombinant chimeric flagellin activates cells through TLR5. Isolated chimeric flagellin is correctly folded. Western Circular dichroism

cellular and humoral immune response antigen presentation, activation cytokines, ...

host macrophage

activation

NFκB

TLR5

chimeric flagellin-

• H. pylori antigen

Track 1b

Implementation 2 DNA vaccine

Host tissue cells

DNA vaccine

Chimeric flagellin needs to be secreted in order to activate cells with TLR5. Transactivation of TLR5 by secreted chimeric flagellin.

Track 1b

Implementation 2 DNA vaccine

Track 1c

Implementation 3 Modified bacteria

• E. coli express chimeric flagellin at their

surface (confocal microscopy, anti-His Ab)

• E. coli express chimeric flagellin

(Western blot).

The idea

Track 1

• Reengineer flagellin of
• H. pylori to activate TLR5

Track 2

• Activate TLRs by

dimerization, colocalization with antigen

Track 2 Track 1

TLR signaling

Track 2

TLR signaling activates antigen processing

Track 2 protein antigen Adjuvant (PAMP -TLR ligand) MHCII protein antigen

antigen degradation phagosome maturation, antigen processing and presentation

Constitutively active antigen-TLR fusion

Track 2 MHCII

phagosome maturation, antigen processing and presentation

Antigen and TLR signal are colocalized No additional adjuvant is required

Modular composition of vaccine in track 2

Track 2

Urease B Multiepitope Antigens Peptide tags His6 tag HA tag TM domain CD4sh TLR3 TM Dimerization domain CD4e Active domain TIR3 TIR4

BASIC STRUCTURE OF ANTIGEN –TLR VACCINE

ANTIGEN DIMERIZATION DOMAIN TRANSMEMBRANE DOMAIN ACTIVATION DOMAIN B CELL T CELL T CELL RECEPTOR CELL MEMBRANE ENDOSOME NUCLEUS

The function of TLR-fusion vaccine

APC

ACTIVATION OF COSTIMULATORS

Track 2

TLR4 activation

Localized at cell membrane and in cellular vesicles.

Track 2

Selecting localization

Track 2

Localization

Signaling network of TLR-fusion vaccine

CellDesigner 4.0 Combined activation of several TLRs mimics stimulation by complete microbe (+synergy between MyD88- and TRIF-dependent signaling)

Track 2

Final test - in vivo efficiency of the vaccine

Strong immunoreactivity in the sera

• f immunized mice (CF-MULTI).

Immune serum against designed epitope (MULTI) recognizes live H. pylori

Relative flourescence intensity Relative flourescence intensity

Electroporated DNA vaccine is expressed in mouse leg +Serum titer after only 10 days is encouraging !

3 weeks!

• neg. control

Secondary Ab to H. pylori CF-sera to H. pylori

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

Students:

• Eva Čeh
• Vid Kočar
• Katja Kolar
• Ana Lasič
• Jan Lonzarić
• Jerneja Mori
• Anže Smole

Mentors:

• Roman Jerala
• Simon Horvat
• Mojca Benčina
• Monika Ciglič
• Karolina Ivičak
• Nina Pirher
• Alojz Ihan

TollImmune, Roche, MEDILINE, Anthron, Lotrič, Mercator, Fementas, Micro+Polo

Dream Team