Noi principii in producerea si realizarea protocoalelor de vaccinare, videoconferinta Martie 2014 Leclerc C.

1. Nouvelles perspectives en
vaccinologie
AAEIP, Université Paris Sud, 31 Mars 2014
Claude Leclerc

2. DEVELOPPEMENT of HUMAN VACCINES
Live attenuated
vaccines
Genetically
engieneered
Purified protein
or polysaccharide
Killed vaccines
Smallpox, 1798
Rabies, 1885
BCG, 1927
Yellow fever, 1935
Polio (oral)
Measles
Mumps
Rubella
Adenovirus
Typhoid (Ty21a)
Varicella
Rotavirus
Diphteria, 1923
Tetanus, 1927
Pneumococcus
Meningococcus
Haemophilus influenzae PRP
Hepatitis B (plasma derived)
Tick-birne encephalitis
H. influenzae PRP conjugate
Typhoid (Vi)
Acellular pertussis
Typhoid 1896
Cholera, 1896
Plague, 1897
Pertussis, 1926 (killed bacteria)
Influenza, 1936
Rickettsia, 1938
Polio (injected)
Rabies (new)
Japanese Encephalitis
Hepatitis A
Hepatitis B (recombinant)
Human Papilloma virus
Rotavirus
18th Century
19th Century
Early 20th Century
After World War II (cellular culture)

3. Vaccines have been made
for 36 of >400 human
pathogens
Immunological Bioinformatics, The MIT press.
+HPV & Rotavirus

4. The different types of vaccines
Attenuated
Vaccines
Killed
Vaccines
Acellular sub-
unit vaccines
Pertussis
Diphteria
Hepatitis B
Tetanus
Cholera
Pertussis
Hepatitis A
Polio
Polio
Yellow fever
BCG

5. New and improved technologies and resulting
vaccines
R Rappuoli, CW. Mandl, S Black & E De Gregorio
Nature Reviews Immunology Published online 4 November 2011

6. New and improved technologies and resulting
vaccines
R Rappuoli, CW. Mandl, S Black & E De Gregorio
Nature Reviews Immunology Published online 4 November 2011

7. New and improved technologies and resulting
vaccines
R Rappuoli, CW. Mandl, S Black & E De Gregorio
Nature Reviews Immunology Published online 4 November 2011

8. New and improved technologies and resulting
vaccines
R Rappuoli, CW. Mandl, S Black & E De Gregorio
Nature Reviews Immunology Published online 4 November 2011

9. New and improved technologies and resulting
vaccines
R Rappuoli, CW. Mandl, S Black & E De Gregorio
Nature Reviews Immunology Published online 4 November 2011

10. R Rappuoli, CW. Mandl, S Black & E De Gregorio Nature
Reviews Immunology Published online 4 November
2011

11. R Rappuoli, CW. Mandl, S Black & E De Gregorio Nature
Reviews Immunology Published online 4 November
2011

12. Dengue epidemiology

13. Nature Reviews Microbiology 2010
Dengue vaccines under development

14. Dengue vaccines under development
Sanofi Pasteur dengue vaccine enters phase III
clinical study in October 2010

15. The yellow fever 17D virus as a platform for new
live attenuated vaccines

17. Worldwide map of phase II/III dengue clinical trials, and major results
obtained so far in humans
Guy et al, Vaccine, 2011, 7229-7241

18. Lancet, Published Online, September 11, 2012

19. Serotype-specific and overall efficacy of CYD tetravalent dengue vaccine against
virologically confirmed dengue disease

21. Reverse cumulative distribution of serotype-specific PRNT 50 antibody titres curves for DENV
serotypes 1–4 by baseline FV-serostatus, pre-vaccination and after two and three doses of CYD-TDV
(Full Analysis Set).
Vaccine, Volume 31, 2013, 5814 - 5821

22. sReuters, March 25, 2014

23. R Rappuoli, CW. Mandl, S Black & E De Gregorio Nature
Reviews Immunology Published online 4 November
2011

24. Schematic representation of the CSP and the RTS,S
vaccine
P D. Crompton, SK. Pierce, L H. Miller J Clin Invest. 2010

26. Malaria cuts risk in half in late-stage trial
H Waters Nature Medicine Nov 2011

27. N Eng J Med 2012

28. Malaria cuts risk in half in late-stage trial
H Waters Nature Medicine Nov 2011
31%

31. R Rappuoli, CW. Mandl, S Black & E De Gregorio Nature
Reviews Immunology Published online 4 November
2011

32. How to discover protective
antigens?

33. Identification of new target antigens: impact of genomics
Whole genome sequences of most bacterial pathogens and
parasites completed
E. coli K-12
B. burgdorferi
B. subtilis
M. tuberculosis
R. prowazekii
H. influenzae
C. pneumoniae
C. trachomatis
N. gonorrhoeae
S. aureus
H. pylori
P. horikoshü
E. faecalis
N. meningitidis
S. epidermitis
M. genitalium
S. pneumoniae
L. pneumophila
P. falciparum
S. pyogenes
M. pneumoniae
T. pallidum
L. major
P. aeruginosa
T. cruzi
M. leprae
P. aerophilum
V. cholerae

34. Genomic-based vaccine development
Whole genomic sequence
Computer prediction
Expression of recombinant proteins
DNA preparation
In silico vaccine candidates
Immunogenicity testing in animal models
Vaccine development

35. 600 potential vaccine candidates
identified
350 proteins successfully expressed
in E.coli
344 proteins purified and used
to immunize mice
355 sera tested
91 novel surface-exposed
proteins identified
28 novel proteins
have bactericidal
activity
Meningoccocal B Vaccine: A Genomic Approach
5 vaccine candidates Rappuoli et al, 2002
Clinical trials

37. 2000
2013

38. R Rappuoli, CW. Mandl, S Black & E De Gregorio Nature
Reviews Immunology Published online 4 November
2011

39. Front Immunol 2014
Structural vaccinology

40. A new computational method to design epitope-focused
vaccines, illustrated with a neutralization epitope from RSV
Nature 507, 201–206 (13 March 2014)

41. Nature 507, 201–206 (13 March 2014)
Induction of neutralizating antibodies against RSV

42. R Rappuoli, CW. Mandl, S Black & E De Gregorio Nature
Reviews Immunology Published online 4 November
2011

43. Overview of the problems and methodologies of systems vaccinology
Seminars in Immunology, 2013, 209 - 218

46. R Rappuoli, CW. Mandl, S Black & E De Gregorio Nature
Reviews Immunology Published online 4 November
2011

47. Alum adjuvants are non-cystalline gels based on aluminum oxyhydroxide (referred
to as Aluminum hydroxide gel), aluminum hydroxyphosphate (referred to as
aluminum phosphate gel) or various proprietary salts such as aluminum hydroxy-
sulfate)
Alum is used in several licensed vaccines including:
• diphtheria-pertusis-tetanus
• diphtheria-tetanus (DT)
• DT combined with Hepatitis B (HBV)
• Haemophilus influenza B
• inactivated polio virus
• Hepatitis A (HAV)
• Streptoccucus pneumonia
• Menngococccal
• Human papilloma virus (HPV)
Vaccines containing Alum Adjuvant

48. Dendritic cells initiate antigen-specific immune
responses
• most efficient of all APCs
• high MHC class I, II & costimulators
• efficient cross presentation
• stimulate naïve T cells (CD4, CD8)
All immunization strategies must target DCs
Initiate Ag-specific immune responses

49. Multiple inducers of DC maturation
Immature DC
Mature DC
various T cell
responses
Microbial products / TLR ligands
Viral products
Inflammatory cytokines
Signaling receptors

50. Antigen-presenting cells serve as the bridge between
innate and antigen-specific responses
2003, 2, 727-735

51. Rappuoli, CW. Mandl, S Black & E De Gregorio Nature Reviews Immunology Nov 2011
Vaccine adjuvants

52. Innate immune responses

53. Innate Lymphoid Cells (ILC)

54. T cell differentiation pathways
Coomes S M et al. Open Biol. 2013;3:120157
©2013 by The Royal Society

55. Therapeutic vaccines
for chronic infections or cancers

56. Cancer, a worldwide burden
 1st cause of mortality in France
 In Europ, in 2012:
- 1.75 million deaths from cancer
- 3.45 million new cases of cancer
 In the world, in 2012:
- 8.2 millions deaths
- 14 million new cases diagnosed

57. Cancer, a cell disease
uncontrolled proliferation
Tumor
Surgery
ChimiotherapyRadiotherapy
Anti-angiogenic
drugs

58. Immune responses can control the growth of tumor
cells
The immunosurveillance theory
“It is by no means inconceivable that small
accumulations of tumour cells may develop
and because of their possession of new
antigenic potentialities provoke an effective
immunological reaction, with regression of
the tumor and no clinical hint of its existence”
British Med Journal, April 1957Burnet

59. Tumor specific/associated antigens
Overexpressed self
antigens
Differentiation antigens
Mutated self antigens
Non self
oncoviral antigens
Altered self antigens:
Abnormal post-
translational/
transcriptional
modification:
underglycosylation

60. The concept of therapeutic anti-cancer vaccines
 Induction of specific immune responses
against tumor specific/associated antigens to
kill tumor cells or prevent their growth
without affecting normal cells

61. Tumor vaccines
- Whole tumor cells: + BCG or DETOX, e.g. Melacine vaccine (cell lysates), CancerVax
( irradiated melanoma cell lines), M-Vax (hapten-treated autologous cells) and gene-
modified, irradiated tumor cells (GM-CSF)
- Tumor antigens: MAGE-1, MAGE-3, MART-1/Melan-A, tyrosinase, gp100, MUC-1,
CEA, etc.
- Peptide vaccines: mutated ras, mutated p53, Her-2/neu, MART -1, gp100, MUC-1
- Heat shock proteins
- DNA vaccines
- Dendritic cell vaccines

62. Response rate = 3. 8%
Current human cancer vaccines show very low objective clinical response rate
Rosenberg, Yang & Restifo
Nature Med 10:909 (2004)

63. Response rate = 3. 8%
Current human cancer vaccines show very low objective clinical response rate
Rosenberg, Yang & Restifo
Nature Med (2004)
Benefit of passive immunotherapy
(antibodies)
in cancer patients
Lack of efficacy of most
current therapeutic cancer vaccines

64. Problems
Tumor derived antigens are weakly
immunogenic
Need for better adjuvants
or immunisation strategies

65. Dendritic cells initiate antigen-specific
immune responses
• most efficient of all antigen-presenting cells
• stimulate naïve T cells (CD4, CD8)
All immunization strategies must target DCs

66. An Approach to Initiating Immunity to Cancer:
Dendritic Cells Loaded with Tumor Antigens ex vivo
DC precursors
expanded
immature DCs
add
disease-
related
antigens
maturing DCs
presenting antigen(s)
Tumor-
specific
T cells
responding
to
dendritic
cells

67. 2010: FDA panel passes first cancer vaccine

68. Original Article
Sipuleucel-T Immunotherapy for Castration-
Resistant Prostate Cancer
Philip W. Kantoff, M.D., Celestia S. Higano, M.D., Neal D. Shore, M.D., E. Roy
Berger, M.D., Eric J. Small, M.D., David F. Penson, M.D., Charles H. Redfern, M.D.,
Anna C. Ferrari, M.D., Robert Dreicer, M.D., Robert B. Sims, M.D., Yi Xu, Ph.D., Mark
W. Frohlich, M.D., Paul F. Schellhammer, M.D., for the IMPACT Study Investigators
N Engl J Med
Volume 363(5):411-422
July 29, 2010

70. Source: www.provenge.com/
Provenge clinical trials : prostate cancer

71. Source: www.provenge.com/
Provenge clinical trials : prostate cancer

72. Cancer vaccine pipeline

75. Problems
Tumor derived antigens are weakly
immunogenic
Need for better adjuvants
or immunisation strategies

76. CD8+
T cell
CD4+
T cell
Dendritic cell
Induction of optimized T cell responses
by in vivo dendritic cells targeting
Antigen
targeting
Maturation signals
2
1
Adjuvant

77. CyaA: a new proteinic vector targeted to dendritic cells
Bordetella pertussis
Dermonecrotic Toxin
BrkA
FHA
TCF
FIM
TCT
Pertussis Toxin
cAMP
Pertactin
Adenylate cyclase
Toxin
Dendritic
Cell
CD11b/CD18
AC
domain
RTX
domain
1 400 1706
Internalization
Endosomes
Cytosol
CyaA binds to CD11b
allowing efficient targeting
to dendritic cells
Guermonprez et al, J. Exp. Med, 2001
Adenylate cyclase (CyaA)

78. Recombinant CyaA
+
Activation of CD8+
Cytotoxic T lymphocytes
Dendritic Cell
ϕ
CD11b/CD18
Antigen
Th
CD4+
MHC-II
endosomes
lysosomes
ϕ
ϕ
MHC class II presentation
Activation of CD4+
Helper T lymphocytes
MHC class I presentation
Endoplasmic
Reticulum
CTL
CD8+
MHC-I/β2
ϕ
Translocation
Endocytosis
Antigens grafted in CyaA are delivered to both MHC class I & MHC
class II presentation pathways

79. Immunisation in mice and non-human primates by
recombinant CyaA carrying a variety of antigens (such as
from M. tuberculosis or HIV) stimulates strong CTL and Th1
responses, even in the absence of adjuvant.
Préville et al, Cancer Res, 2005, Mascarell et al, J. Virol 2005, Majlessi et al, Inf Immun,
2005, Hervas-Stubb et al, Inf Immun, 2006, Mascarell et al, Vaccine 2006, Berraondo et al,
Cancer Res, 2007, Fayolle et al, Vaccine 2010.
CyaA: a new proteinic vector targeted to
dendritic cells

80. 80
HPV infection life cycle
Few months to few years Up to 20 years
Goodman A., Wilbur D. C.

81. Human papilloma virus

82. - The HPV E6 and E7 oncoproteins are expressed
throughout the replicative cycle of the virus and are
necessary for the onset and maintenance of malignant
transformation.
- HPV E6 and E7 antigens are potential targets for specific
immunotherapy.
Rational

83. MHGDTPTLHEYMLDLQPETTDLYCYEQLN
CyaAE5
GQAEPDRAHYNIVTFCCKCDSTLRLCVQSTHVDIRTLEDLLMGTLGIVCPICSQKP
CyaA-E7∆30-42
Catalytic domain
224 235
319 320
1 400 1706
LQ
LQ
Recombinant CyaA carrying E7 from HPV16
GMP batch produced in recombinant E. coli

84. Therapeutic vaccination with recombinant
HPV16-E7 CyaAs eradicates established tumors
No treatment
CyaA-OVA
0
500
1000
1500
2000
0
10
5/5
10/10
0/10
20
30
40
50
60
70
80
90
0
500
1000
1500
2000
CyaA-HPV16E7∆30-42
- Graft of TC-1 cells at day 0
- At day 10, one injection of:
- 50 µg of CyaA-E7 or
of control CyaA-OVA
Preville et al, Cancer Res. 2005; Berraondo, K. et al. Cancer Res. 2007

85. A therapeutic vaccine candidate against HPV chronic infection
and/or cervical cancer
Clinical trials started in 2010
0 102030405060708090
0
500
1000
1500
2000
0 102030405060708090
0
500
1000
1500
2000
0 102030405060708090100
0
20
40
60
80
100
0 5 10 15 20 25 30 35
0.0
0.2
10
20
daysdays days
CyaAE5 HPV16E7∆30-42CyaAE5 CysOVA
Controls
One injection at
Day 10
CyaAE5 HPV16E7∆30-42
Préville et al, Cancer Res, 2005

86. http://www.genticel.com
ProCervix - Phase 2 Clinical Trial

87. Merci de votre attention !
claude.leclerc@pasteur.fr