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Plasmodium CSP - Based vaccines Past - prsesent - future
- 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
- 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