THE YIN AND YANG OF A BACTERIAL TOXIN
Fooling host Immunity and
delivering T cell vaccines
P. Šebo
PRAGUE Institute of Microbiology Academy of Sciences of the CR
Bordetella pertussis The agent of whooping cough (pertussis)
Pertussis used to be a major cause of infant morbidity and death in the pre-vaccine era (slide dr. Fabianova NIPH)
Died of pertussis according to age groups in a 15 million population of Czechoslovakia 1949-1957 Total Age (months)
Unspec Total
Total
We thought the problem was solved, but in CR we expect this year 3000 cases… Pertussis incidence in CR in 1995 – 2010 per 100,000 wP vaccine introduced
wP production deteriorated
aP vaccine Introduced in CR
Thanks to “democracy” and introduction of acellular vaccines the whooping cough is back to the most developed countries….
the 2010 and 2014 California outbreaks (we all wake up when something happens in the US…)
10 infants < 2 month old died in 2010
We`ve got a problem again…. Confirmed clinical pertussis in 1990-2013 in CR (10 million people) We had 2,518 cases in 2014 (like in 1961…) 3000
No. cases
2500 2000 1500 1000 500 0 1990
1994
1998
2002
year
2006
2010
2014
So, does pertussis resemble this?
Ministerstvo zdravotnictví Palackého nám. 4 128 01 Praha 2 http://www.mzcr.cz/Verejne/obsah/doporuceni-pro-ockovani-tehotnych-proti-pertusi-v-ceske-republice_3249_5.html
6. 4. 2015 Doporučení pro očkování těhotných proti pertusi v České republice Doporučení pro těhotné: Těhotné je doporučeno očkovat jednou dávkou kombinované vakcíny proti pertusi, difterii a tetanu (Tdap) během (každého) těhotenství, ideálně mezi 28. a 36. týdnem těhotenství. Tdap - vakcína se sníženým množstvím difterického toxoidu, s tetanickým toxoidem a acelulární pertusovou složkou. Registrované očkovací látky: Adacel – výrobce Sanofi Pasteur, vakcína proti difterii, tetanu a pertusi (acelulární), (adsorbovaná se sníženým obsahem antigenů) Boostrix – výrobce GSK, vakcína proti difterii, tetanu a pertusi (acelulární komponenta) se sníženým obsahem antigenů Hlavním cílem u očkování v těhotenství je chránit nejmenší děti prostřednictvím posílení mateřských protilátek. Hladina mateřských protilátek je považována za nejdůležitější faktor ochrany kojenců před onemocněním. Většina žen byla v dětství proti pertusi očkována nebo pertusí onemocněla. Očkování ani prožité onemocnění však neposkytuje celoživotní ochranu. Očkování v posledním trimestru těhotenství proti pertusi dočasně zvýší ochranné mateřské protilátky, které přechází od matky přes placentu jejímu nenarozenému dítěti. Přenesené mateřské protilátky pasivně chrání dítě v prvních dvou měsících života, než může být očkováno proti pertusi. Nebylo prokázáno zvýšené riziko vedlejších reakcí po Tdap vakcinaci u těhotných žen ve třetím trimestru a ani u jejich dětí.
B. pertussis is armed with numerous parallel and redundant virulence systems (cytotoxins, immunomodulators and adhesins)
Slide: Courtesy of C. Locht, Institut Pasteur de Lille
B. pertussis makes two of the ‘smartest’ toxins that subvert cell signaling fools cells by cAMP – the second messenger…! TII
Cya
Sec
TIV
AC
TI
Pertussis toxin
Cholera toxin
ATP cAMP
B. pertussis AC
B. anthracis EF
TTSS P. aeruginosa Exo Y
Gia
AC ADPR
Gsa
ADPR
AC ATP cAMP
ATP cAMP
AC ATP cAMP
Slide: Courtesy of S. Lory, Harvard Medical School
Adenylate cyclase toxin - cytolysin AC domain
T25
RTX hemolysin moiety Principal CD11b/CD18 binding segment
T18
1
400
500
700
1000
1706
N
C I
II CBS
III
Hydrophobic segments
42 calcium binding repeats X-(L/I/F)-X-G-G-X-G-(D/N)-D Palmitoylation on K860 and K983
T25
T18
Guo Q. et al. (20005) EMBO J. 24, 3190–3201
C-CaM
Secretion signal
A conserved block of C-proximal residues is required also for activities of other RTX toxins
*T*+XWF
- a conserved motif
ACT is an RTX protein secreted by a type I system… >100 mM Ca2+ Outer membrane
Inner membrane
<100 nM Ca2+ Need to unfold and refold on the way to target…
Láďa
Calcium-dependent folding of CyaA starts from the C-terminus and proceeds towards the N-terminus of the RTX domain
Calcium-driven formation of an intramolecular ratchet directs movement of large RTX proteins through type I secretion system conduits
THE YIN
Fooling host Immunity
ACT/cAMP signaling breakes the hell loose… and supresses TLR signaling of the bug on the mucosa mucosa…
LPS ACT
signal transduction events: NF-κB, MAPK – p38, ERK, JNK expression and upregulation of TLR: TLR1-6, 9, TLR4, TLR2
mucin: MUC2, MUC5AC
AEC other cells
cytokine and chemokines: IL-1α, IL-1β, IL-6, IL-8, IL-10, TNFα, IFNβ, TGF-β, GM-CSF, MCP-1, MIP-1α, RANTES,..
ciliary beating defensins and other antimicrobial peptides: hβdefensin2 , βdefensin1, cathelicidin
other soluble factors: NO, PGE2
cAMP
expression of costimulatory x inhibitory molecules: CD80, CD86, CD40, CD54, B7-H2, B7-H3 x FasL, PD-L1, PD-L2
the
Yin: ACT as a SWIFT SABOTEUR
low ACT (CyaA) concentrations make a difference on respiratory mucosa… Capsule Periplasm
Pertactin
PT
DNT TCT
CyaA
┴
fimbriae
FHA
IL-6
invasion
Macrophage Mucus layer Respiratory epithelium
Ciliated cells
Cilliated cells
Nonciliated cells (Goblet cells, Absorptive cells) Submucosa
Type I
CD11b+
IL-8 IL-6
B.pertussis secreting CyaA
intraepithelial DC Immature dendritic cell
?
Macrophage
CyaA Cations Neutrophil ATP cAMP Intoxication
↓ IL-12, TNFα, CCL3 ↑ IL-10, IL-6, IL-1beta, IL-17 (Semi-mature state of DC ?)
↓ T regulatory response ?
CD11b/CD18
Channel formation ?
Phagocytic functions Phagocytosis Superoxid production
Apoptosis
T cell
IL-1β Dendritic cell Neutrophil Macrophage
B cell
Cell lysis
Osičková et al., (1999) J. Biol. Chem. 274, 37644)
Vojtová et al., 2006, Curr. Op.. Microbiol. 9, 69-75
The three cytotoxic activities of ACT adenylate cyclase toxin & pore-forming
Intoxication
Repeats
Repeats
Ca2+
CR3 = Mac-1 aMb2-integrin receptor
AC
AC
hemolysin/Cytolysin
CD11b/ CD18
Na Na++
Cytoplasmic membrane
AC CaM
ATP cAMP
Translocation precursor
Sebo et al. (1991) Gene 104:19 Sakamoto et al. (1992) J. Biol. Chem. 267, 13598 Benz et al. (1994) J. Biol. Chem. 269, 27231 Hackett et al. (1995) J. Biol. Chem. 270, 20250 Gray et al. (1998) J. Biol. Chem. 273, 18260 Osickova et al. (1999) J. Biol. Chem. 274, 37644 Basler et al. (2007) J. Biol. Chem. 282, 12419 Fiser R. et al. (2007) J. Biol. Chem. 282, 2808 Osickova et al. (2010) Mol. Microbiol. 75:15450-1562
K+
Channel precursor
K+
Oligomeric Cation-selective Channel
AC
The toolbox: A panel of mutations characterized that block ACT activity at each individual step of toxin action
Repeats
Repeats
b2-integrin receptor
E509K+E516K Intoxication
C+
AC
(CD11b/ CD18)
E570Q+K860R E570Q E581P
AC
CaM
cAMP ATP
Translocation precursor
E570P E570K+E581P
Channel precursor
Oligomeric Cation-selective (hemolytic) Channel
E570K+E581P E509K+E516K drop of channel cation selectivity
Basler et al., (2007) J. Biol. Chem. 282, 12419
Marek
AC
AC- Hly- toxoid under evaluation as pertussis vaccine candidate
b2-integrin receptor
Ca2+
(CD11b/ CD18) AC
Intoxication
Repeats
Cytoplasmic membrane
AC CaM
ATP cAMP
Translocation precursor
Osickova et al. (2010) Mol. Microbiol. 75:15450-1562 Osickova et al., (1999) J. Biol. Chem. 274, 37644 Basler et al., (2007) J. Biol. Chem. 282, 12419 Fiser R. et al.(2007) J. Biol. Chem. 282, 2808
Bordetella adenylate cyclase toxin hijacks its β2 integrin receptor into lipid rafts to accomplish membrane translocation in two steps
Bumba et al. (2010). PLoS Pathog 6(5): e1000901.
CyaA-induced morphological rearrangements Mouse macrophage-like cell line J774 A.1:
Buffer, 5 min
CyaA,
CyaA-AC-, 10 ng/ml, 5 min
Kamanova et al. (2008) J. Immunol. 181, 5587-97
10 ng/ml, 5 min
db-cAMP, 2mM, 10 min
A
cAMP
B
phagocytosis
Phagocytosis (% of control)
pmol cAMP/6x105 J744A.1 cells
ACT at low doses ablates complement-mediated phagocytosis (through RhoA inactivation) 1000
10 ng/ml 100 ng/ml
100 10 1 0 0
150
5
20 Time (minutes)
*
*
* *
50
ng/ml
60
FcR-mediated phagocytosis CR3-mediated phagocytosis
100
Buffer control
30
*
*
* *
*
* 0
CyaA ( ) CyaA-AC- (ng/ml) db-cAMP (mM)
+ -
-
-
1
5
-
-
10 -
50 -
100 -
100 -
1
Kamanova et al. (2008) J. Immunol. 181, 5587-97
cAMP signaling wipes out receptor signaling through Syk, blocking bactericidal functions of phagocytes
iC3bZymosan iC3b-Zymosan Syk
IP: anti-PY, IB: anti-Syk
Syk
Syk
Lysate, IB: anti-Syk
Syk
Syk activation (%)
Syk activation (%)
IP: anti-PY, IB: anti-Syk Lyzate, IB: anti-Syk
iC3bZymosan
iC3b-Zymosan
Toxin added after Syk signaling has Toxin added with iC3b zymosan been pre-activated with iC3b zymosan
CyaA-produced cAMP signaling through PAK activates SHP-1 tyrosine phosphatase that blocks iNOS gene transcription Hydrophobic domain
Cell-invasive Adenylate cyclase
RTX-hemolysin repeats
AC
RTX ?
?
Palmitoylated Lys860 and Lys983
Translocation RTX
AC ATP cAMP
cAMP Reg
RTX
AC
Plasma membrane
a
Ca2+
Potassium efflux K+
b RTX
Cation selective pores
Calcium influx
PKA
SH2 SH2 Cat
SHP-1 active Catconformation
AP1 Černý et al. (2015) J. Immunol 194:4901-13
RTX
RTX
c-Fos
X
iNOS
†
Upper band Lower band
†
a
a
a
a
a 2
‡ iNOS expression
3 2,5 2 1,5 a 1
**
0,5
0
P-c-Fos
SHP-1 activity (A.U.)
actin LPS CyaA NSC87877
-
+ -
+ 10 -
+ 10 +
0,7 0,6
**
**
+ +
LPS + CyaA LPS
0,5 0,4 0,3
0,5
30 min
60 min
+ -
+ 10 -
+ 10 +
+ + - LPS + LPS
150
+ LPS + CyaA
100 50 0
SHP-2 10 min
-
200
SHP-1
0 min
**
0
siRNA:
0
**
1
0,2 0,1
†
1,5
iNOS actin LPS CyaA NSC87877
NO production [%]
Relative phosphorylation
CyaA-produced cAMP signaling through PKA activates SHP-1 tyrosine phosphatase that blocks iNOS gene transcription
**
**
**
THE YANG OF A BACTERIAL TOXIN"
delivering T cell vaccines
Drugs from bugs
Ag
epitope insert
AC
dACT as a novel antigen delivery tool
AC
dACT
AC
Ag
Repeats
epitope insert
Repeats
b2integrin receptor CD11b/ CD18
Ca+
dACT
Channel formation in plasma membrane
Translocation into cytosol
Lysosome
Endosome integrin
Antigenic peptides
Proteasome
Endogenous antigen
(CD11b CD18) Antigenic peptides
Transporter associated with antigen procesing Endoplasmatic reticulum MHC I peptide MHC I
MHC II peptide
MHC II
Golgi
Proteasome processing TAP transport to ER MHC I binding Epitope presentation on MHC class I molecules
Receptor mediated endocytosis endosomal processing CD8+ T cell
MHC II binding Epitope presentation on MHC class II molecules
CD4+ helper T cell
www.genticel.com
Vaccines that are built with the CyaA vector are chimeric recombinant proteins consisting of the CyaA protein and the antigen of choice. 1: Sebo P, Fayolle C, d'Andria O, Ladant D, Leclerc C, Ullmann A. (1995) Infect Immun. 63(10):3851-7. 2: Fayolle C, Sebo P, Ladant D, Ullmann A, Leclerc C. (1996) J Immunol. 156(12):4697-706. 3. Saron MF, Fayolle C, Sebo P, Ladant D, Ullmann A, Leclerc C. Proc Natl Acad Sci U S A. 1997;94(7):3314-9.
www.genticel.com September 2012 - Genticel S.A. completed Phase I clinical trial for HPV16/18-induced cervical carcinoma
Using a cGMP batch of the adenylate cyclase (CyaA-AC-) toxoid for delivery of HPV E7 antigen as immunotherapeutic vaccine safe, immunogenic, inducing CD8+ CTLs and HPV 16/18 virus load reduction demonstrated
Entered phase II trial = will be of interest to see pertussis incidence in CyaA-E7 toxoid treated woman… IPO on April 4 at Euronext Paris and Brussels - 34 millions Euro
Knowledge is useful… We still have 20 years of work on ACT ahead of us…
Occasionally it is more fun…
Acknowledgments Institut Pasteur: teams:
Claude Leclerc Laleh, Catherine, Gilles
Daniel Ladant Nicole Guiso
Institute of Microbiology
Lída Tučková Marek Kovář and their teams University Wurzburg
Roland Benz and his team
Imperial College
Bernhard Nocht Institut:
Robert Wilkinson Katalin Wilkinson
Thomas Jacobs Susanne Tartz
VLA Surrey
MH Hannover
Martin Vordemeier
Ingo Just Harald Genth
