P. Sebo Institute of Microbiology Academy of Sciences of the Czech Republic PRAGUE

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P. SeboInstitute of Microbiology Academy of
Sciences of the Czech RepublicPRAGUE
"A Tool for Detecting and Inducing T Cell
Immunity against TB, Malaria and HIV"
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Sebolab
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Bacterial protein toxins are smart, pretty and
useful
You will not find a cellular process that is not
a target of a toxin
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Adenylate cyclase toxin - cytolysin
AC domain
RTX hemolysin moiety
Principal CD11b/CD18 binding segment
T18
T25
1
400
500
700
1000
1706
N
C
Secretion signal
Hydrophobic segments
I
II
III
CBS
42 calcium binding repeats X-(L/I/F)-X-G-G-X-G-(D/
N)-D
Palmitoylation on K860 and K983
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• Rational
• CyaA targets the aMß2 integrin CD11b/CD18
• specifically present on professional antigen
  presenting cells
• - dendritic cells
• - macrophages
• Make CyaA to a novel tool for antigen delivery to
  dendritic cells
• for vaccination against infections
• Immunotherapy of certain tumors
• diagnostics of infections and cancer

Exploit for antigen presentation
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dACT as a novel antigen delivery tool
Ag
Ag
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Immunization with CyaA-LCMV affords protection
against a lethal challenge by LCMV
Saron, M. F. et al. (1997) Proc. Natl. Acad. Sci.
USA 94, 3314-3319
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Insertion point CTL
induction SSLAHG107 VR-V3-LCMV-OVA-VH 108HTAVDL
LKEYIG335 VR-V3-LCMV-OVA-VH 336QQRGEG
SEATGG232 VR-V3-LCMV-OVA-VH 233LDRERI

Mice are protected against an LCMV challenge
Fayolle et al. (2001) J. Virol. 75, 7330-8
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dACT allows induction of antigen-specific T cell
responses
CD8 Antigens CD4 Antigens OVA

MalE LCMV
MAGE Apa, Cfp

ESAT-6, CFP10 gp120

TB-10.4 E7
Ag85A CSP
Melanomy ESAT-6, CFP10 TB-10.4

Sebo et al., 1995, Infect. Immun.
HIV, LCMV in
vitro Fayolle et al., 1996, J. Immunol.
HIV Saron et al.,
1997, Proc. Natl. Acad. Sci. U.S.A.
LCMV in vivo Osicka et al., 2000, Infect. Immun.
permisive sites
Fayolle et al., 2001, J.
Virol.
polyvalent CTL response Loucka et al., 2002,
Infect. Immun.
CD4 T cell response Schlecht et al., 2004, J.
Immunol.
Mechanisms Mackova et al., 2006, Cancer Immunol.
Immunother. Tumor immunotherapy Tartz
et al., 2006, Infect. Immun.,
protection against malaria Wilkinson
et al. 2005 Infect Immun.
Improvement of LTBI detection Anderson et al.
2006 Am. J. Crit. Care Resp. Med.
Improvement of LTBI detection Majlessi et al.,
2006, Infect. Immun., IFN? and
immunity against MTB Hervas-Stubs et al., 2006,
Infect Immun IFN? and immunity
against MTB
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Prime/Boost Immunotherapy of HPV16-induced
tumors by combimations of CyaA-E7 and MVA-E7
vaccines (higher challenge dose)
Mackova et al. (2006) Cancer Immun.
Immunother.55, 39-46
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(No Transcript)
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New constructs for T cell immunity against HIV
PLoS ONE. 2007 2(10) e984. Design and Pre-Clinical Evaluation of a Universal HIV-1 Vaccine Sven Létourneau,1a Eung-Jun Im,1 Tumelo Mashishi,1 Choechoe Brereton,1b Anne Bridgeman,
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Differential diagnosis using antigens whose genes
haven been deleted from genome of Mycobacterium
bovis BCG Pasteur compared to M. bovis
oriC
Mycobacterium bovis
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Dose response to native and CyaA toxoid antigens
in HUMAN whole blood
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CyaA toxoid presentation improves recognition of
CFP-10
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CyaA presentation of antigens increases the
sensitivity of recognition in low responding
subjects
250
300
200
250
200
150
IFN-g SFC / 106 PBMC
IFN-g SFC / 106 PBMC
150
100
100
50
50
0
0
high
medium
low
High responders n20
medium responders n17
low responders n27
responders
responders
responders
n22
n6
n34
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Conclusions MTB story
dCyaA fusions improve the efficiency of antigen
recognition, especially in low responders
Both CD8 and CD4 recall responses can be
enhanced ! MIGHT WORK ALSO IN CD4-depleted HIV
infected !
The mechanism of enhancement is classical
Whole blood based detection of CyaA toxoid
responses potentially simplifies in vitro
immunodiagnosis of latent tuberclosis both in man
and cattle
Wilkinson et al. (2005) Infect. Immun. 73,
2991 Vordemeir et al. (2004) Infect. Immun. 72,
6255
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Scientific background 2
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Results of a study in Harrow
N 75 BCG vaccinated 59/75 (79) Heaf Grade
N NEGATIVE 0 15 (20) 1 47 (63) 2 9
(12) POSITIVE 2 2 (3) 3 0 4 0 UKN 2
16 (22)
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Current status of CyaA-antigen delivery technology

• 1997 - Protective immunity against a virus (LCMV)
• 1999 - Immunotherapy of transplanted tumors in
  mice
• 2004 - Enhanced detection of latent tuberculosis
• 2005 - Protective immunity against Plasmodium
  (mouse malaria model)
• 2005 - immunotherapy of experimental tumors (such
  as HPV16 induced)
• (US Patent No. 5,503,829, No. 5,679,784, No.
  5,935,580, EU Patent application No. 03291486.3,
  US Prov 03495, 6094 (2003) )
• preclinical data in vivo
• toxicological studies
• Phase III clinical trial in preparation for
• melanoma (EU FP consortium THERAVAC)
• HPV16-induced cervical carcinoma (BT Phar,a S.A.
  Toulouse France)

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Induction of a protective immune response against
Plasmodium berghei liver stage malaria by
vaccination with recombinant adenylate cyclase
toxoid of Bordetella pertussis in combination
with CTLA-4 blockade
Susanne Tartz Bernhard Nocht Insitute for
Tropical Medicine, Hamburg
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Vaccination against malaria

• liver stage
• few days, asymptomatic replication of the
• parasites within hepatocytes
• blocking antibodies against sporozoites
• CTL response against infected hepatocytes

• blood stage
• replication of the parasites within the
  erythrocytes
• causes pathology of the infection
• blocking antibodies against merozoites and
• gametocytes

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Bockade of CTLA-4 during prime or boost

• blockade of CTLA-4 during secondary immunisation
  leads to increased numbers of CSP-specifc
  IFN?-producing T cells
• the same effect is seen in liver and blood

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Induction of protective immunity against malaria
Mice immunized with infected protection
PBS 10/10 0
?-CTLA-4 5/5 0
ACT-CSP 9/9 0
ACT-CSP ?-CTLA-4 4/10 60

• prime/boost immunisation with ACT-CSP does not
  induce protective immunity
• blockade of CTLA-4 during boost immunisation
  leads to significantly enhanced protection
  against P. berghei challenge

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Acknowledgments for MTB story
Institut Pasteur Claude Leclerc Laleh
Majlessi Sandra Hervas
Institute of Microbiology Prague Zdenka
Abrhamova-Jarvis Jiri Mašín Marcela Šimšová



Imperial College Robert Wilkinson Katalin
Wilkinson
VLA Surrey Martin Vordemeier

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Acknowledgments for malaria story
Institute of Microbiology Prague Jana Kamanova


Bernard Nocht Institute Thomas Jacobs Susanne
Tartz
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Institut Pasteur Claude Leclerc Daniel
Ladant Geraldine Schlecht Gilles
Dadaglio Catherine Fayolle Pierre
Guermonprez M.-F. Saron M. El-Azami
El-Idrissi Cecile Bauche
Institute of Microbiology Prague Jana
Kamanova Marek Basler Jiri Masin Jana
Vodolanova Radim Osicka Adriana Osickova Irena
Linhartova Marcela Simsova



Charles University Prague Radek Fiser Ivo
Konopasek
University of Wurzburg Roland Benz Oliver
Knapp Elke Meyer

Institute of Physiology Prague Radek Fiser Ivo
Konopasek
Institute of Hematology and blood
Transfusion Jana Mackova Sarka Nemeckova