Plasmodium CSP - Based vaccines Past - prsesent - future - Conférence de la 6e édition du Cours international « Atelier Paludisme » - SNOUNOU Georges - Singapour - snounou@mnhn.fr

1. Plasmodium CSP-Based Vaccines
Past - Present - Future
Georges Snounou
Parasitologie Comparée et Modèles
Comparé Modè Muséum National d’Histoire Naturelle
Musé
Expérimentaux
Expé Paris - France

2. OVERVIEW
Malaria & Anti-Malarial Vaccination
CSP-Based Pre-Erythrocytic Vaccines
Present Status
Perspectives
Quest for Novel Protective Antigens

3. A Simple Life Cycle with Complexity
Protozoans of the Genus Plasmodium
Transmitted by Mosquitoes of the Genus Anopheles
Established after 100 years of research

4. Discovery of the Pathogen
Alphonse Charles Louis Laveran

5. Transmission By Mosquitoes
Manson Ross
London India
The Roman Dispute
Grassi
Rome

6. Spot the Error

7. A Single Observation
Schaudinn 1903 Arb. Kaiserl. Gesundh.
Schaudinn

8. The Liver Fate of the Sporozoite
Shortt Garnham

9. Life Cycle Landmark Dates
1898
Plasmodium
Sexual Stages
1880 1948 1980
Plasmodium Plasmodium Plasmodium
Asexual Stages Liver Stages Liver Hypnozoites
1903 1924
1898
Anopheles
Sporozoites

10. The Control of Malaria
Drug & Insecticide Resistance
Former Times DDT
Chloroquine CSP Gene Plasmodium Genomes
Drainage
1955 1970 1983 2001…
2001…
Larvicide
Nets
Quinine WHO
Scramble for a Vaccine
Eradication Campaign
1970s
mAb - Cloning - In vitro Culture

11. Many Vaccine Candidates…
Erythrocytic Asexual Targets
AMA1 DBP EBA175 EBP2 GLURP GPI MSP1 MSP2 MSP3 PfEMP1 RESA SERP
Erythrocytic Sexual Targets
PfS16 PfS28 PvS25
Pre-erythrocytic Targets
CSP LSA1 LSA3 TRAP
…Little Protection
Unknown Mechanisms of Naturally Acquired Immunity
Requires Repeated Lengthy Exposure - Wanes Rapidly
Not Sterilising
Species and Strain-Specificity

12. VACCINES TARGETING THE P. falciparum CSP

13. Gold Standard Vaccine : The Attenuated Sporozoite
1941 & 1966 1973 - 1990
Avian parasites Human parasites
1910 UV-inactivated sporozoite Radio-inactivated sporozoite
Avian parasites
Heat-inactivated sporozoite
1967
Murine parasites
Radio-inactivated sporozoite
Sterile Protection Requires Many Doses
Quest for a Subunit Vaccine

14. Incrimination of a Sporozoite Surface Protein
Normal Serum Immune Serum
Circumsporozoite Protein

15. Pfcsp - Repeats and Variations
NANP NVDP
1200 - 1400 bp
30 - 50 Repeat Units
Oocyst Development - Gliding Motility - Salivary Gland Invasion - Interaction with Hepatocyte

16. Immunological Mechanisms of Action
Immunisation
Hepatocyte Arrested
Invasion Development
Blood
Sporozoite Liver
Infection
Challenge
Invasion Liver Stage
Inhibition Killing
Blood
Sporozoite Liver
Infection
Antibody
T Cells

17. Pfcsp-based Vaccines - Trials and Tribulations
Immunogens
DNA
Peptides
Proteins (E. coli, Baculovirus)
Recombinant Virus (Pox, Adenovirus)
Recombinant Bacteria (Salmonella)
Recombinant Parasite (Leishmania, Toxoplasma)
Trials
Thirty in Mice
Four in Monkeys
Twenty-seven in Humans
Adequate Levels of Sterile Protection Remain Elusive

18. The RTS,S Vaccine
Naïve Volunteers - Homologous Challenge
Adjuvant-Dependent Efficacy
48% Sterile Protection
NEJM 1997 336:86

19. The RTS,S Vaccine
1998
First
1999
Boost
Strain-Transcending Protection
Short-lived Sterile Protection
Lancet 2001 357:1927

20. The RTS,S Vaccine
≥ One episode Malaria infection
Lancet 2004 364:1411

21. Contrasting Interpretations
Alonso et al.
29.9 % against first clinical episode
57.7 % against severe malaria
45.0 % against infection
Snounou et al.
The vaccine is ineffective

22. Contrasting Interpretations
Placebo is inadequate
Detection by microscopy
Statistical measures obscure
No primary data available

23. Contrasting Interpretations
≥ One episode
35.3 % against first clinical episode
48.6 % against severe malaria
Lancet 2005 366:2012

24. More RTS,S Trials
About 7 Phase IIb are underway in Africa
About 12 Phase III are planned in Africa
Estimated/Projected costs = > 600 Million USD

25. Suitability of Pfcsp as an Anti-Infection Vaccine
Role in Naturally Acquired Protection
Epidemiological Surveys
Role in Experimentally-Induced Sterile Protection
Experimental Approach

26. Role of PfCSP in Naturally Acquired Immunity
Prevalence of αCSP Antibodies
Prevalence of CSP-specific T-Cells
Presence of Th Epitope Polymorphisms
Lack of Formal Evidence
Exploit Th-Epitope Polymorphisms
Formal Tests in Animal Models

27. Diversity of Pfcsp Th Epitopes
Th2R Th3R
Th2R*01 P S D K H I E Q Y L K K I K N S I
Th3R*01 N K P K D E L D Y E N D
Th2R*02 Q K T Q L
Th2R*03 K E N Q L Th3R*02 Q A
Th2R*04 Q K R Q L Th3R*03 A
Th2R*05 Q L Th3R*04 D Q
Th2R*06 Q K T Q L Th3R*05 Q N
Th2R*07 Q K Q Q L Th3R*06 R A
Th2R*08 Q K Q L Th3R*07 A D
Th2R*09 T N Q L Th3R*08 Q
Th2R*10 Q K Q R L Th3R*09 G S
Th2R*11 Q K Q Y L Th3R*10 N
Th2R*12 T E Q L Th3R*11 D Q C S
Th2R*13 Q Y L Th3R*12 D
Th2R*14 K E T Q Th3R*13 G E
Th2R*15 E Q L .
. .
. .
. .
.

28. Study Sites in Thailand
Mae La Kanchanaburi
Ratchaburi
EIR 0.1 - 0.5 EIR 1 - 5
1996 N = 35 2001 N = 183
Children and adults
1999 N = 45
2001 N = 20
Children and adults

29. Conclusion
CSP-induced Immunity is Short-Lived
CSP-induced Immunity is Not Protective
CSP-Induced Natural Immunity is Not Selective
Brazil Papua New Guinea
*01/*01 *04/*01 *05/*04
23/23 19/23 22/22
Hypothesis
Biological Selection by the Mosquito
Kumkhaek et al. 2005 J. Immunol. 175:3935

30. Role in Experimentally Induced Sterile Immunity
P. berghei CSP versus P. falciparum CSP

32. Hypothesis
IF
CSP plays a role in the acquisition of sterile immunity
THEN
Immunization with P. berghei/Pbcsp irradiated sporozoites
WILL NOT
Confer protection against a challenge with P. berghei/Pfcsp
Provided anti-CSP cross-reactive immune responses are minimal

33. αCSP Antibody Responses after Immunisation
N-Terminus Repeats C-Terminus
P. berghei
P. falciparum
0 100 200 300 400 0 100 200 300 400 0 100 200 300 400
Fluorescence (AU x 10-3)
The Majority of Induced Antibodies are Directed against the CSP

35. Sterile Protection Induced by Immunisation
Challenge Parasite
P. berghei/Pbcsp P. berghei/Pfcsp
Immunising Parasite
None
P. berghei/Pbcsp
0 20 40 60 80 100 0 20 40 60 80 100
Sterile protection (%)

36. Conclusion
Immune Responses Induced against CSP
Do not Confer Sterile Immunity
Other Antigens from Irradiated Sporozoites must have a
Determining Role in Sterile Protection
Grüner et al. 2007 PLoS ONE e-1371

37. Quest for Novel Protective Pre-Erythrocytic Antigens
A strategy for Identification

38. Discriminating Immunological Reagents
Suboptimal Immunisation
Irradiated Sporozoite
Challenge
Protected Infected
Antibodies Antibodies
T Cells T Cells
Animal Models of Malaria
Ethical Considerations
Collection of Reagents

39. Liver Stage Transcriptomes
High Efficiency In vitro Infection
CD81-HepG2 Cells
Irradiated Sporozoite Hepatic Trophozoite Arrested Growth
mRNA Purification
cDNA Construction
Normal Sporozoite Hepatic Trophozoite Hepatic Schizont
Gene Identification by Microarray Analyses
DNA Chip for the P. yoelii Genome

40. Immunological Screening
Bioinformatic Selection
Confirmation of in vivo Expression
RT-PCR
RT-
Cloning in Appropriate Expression Vectors
Quantify B Cell and T Cell Responses
Correlates of Protection

41. Proof-of-Principle Validation of Protective Role
Immunisation / Challenge in Mice
Identification of Homologues in P. falciparum
Reagents from Humans
Volunteers Immunised with Sporozoite Naturally Protected Endemic Residents
under Chloroquine Cover following Radical Cure

43. Pfcsp Th Repertoire Diversity - Age and Exposure
RepTh Non-Immunes > RepTh Immunes
?
Parasites
Age
Relatively Low Endemicity
Mosquito Bites Lead to Clinical Symptoms
Prompt & Effective Treatment
Blood Parasites Reflect Sporozoite Inoculum

44. An Unexpected Biased Distribution
0.8
Th2R 0.15 Th3R

45. A Dominant Pfcsp Haplotype
Th2R*5 Th3R*1 *5/*1
n % % %
Ratchaburi 144 78 81 76
Kanchanaburi 43 84 91 84
Mae La 100 82 84 82
Frequencies did not Vary with Age or Time of Collection

46. Fixation of the Pfcsp Haplotype
msp1 msp2 glurp
K1 MAD20 RO33 FC27 IC
C B B
A B E D
D E A
Children G C C
D H B
C B C
1996 D B DK B
F B F
F B C
Adults D DK F
C AB C
E G B
D D B
C B DG CD
D B B
A C D
C BD A AB EG CD
Children A B B
BC B E A
E F A B EF AB
1999
D
D
C AB D
J
F
C
*5/*1
D C F
C B E
C E A
Adults AC I C
C B A
D C C
D E C
D D C
EG I C
C F A
Children D EK C
D EK C
E CK C
2001 C H A
BD B D DF
D C AB
Adults C C C
C DE B G BC
E B C