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PRECLINICAL VACCINE STUDIES OF THE  CS Plasmodium vivaxPROTEININ MICE <br />
Malaria Indices<br />Authors: World Health OrganizationPublication date: 2009<br />Source: http://www.rollbackmalaria.org/...
<ul><li>Sterileprotectiveimmunity</li></ul>radiation-attenuatedsporozoites<br /><ul><li>Anti-diseaseimmunity</li></ul>Sour...
P. vivax vaccine candidates currently in development <br />Source:  Myriam Arévalo-Herrera, Chetan Chitnis and Sócrates He...
The circumsporozoite (CS) protein<br />CS is a thrombospondin type I repeat (TSR) domain family (Dharmendar Rathore et al....
P. vivaxcircumsporozoite protein CSP events<br />Mabs  against CS show protection in mice <br />and neutralize infectivity...
P. vivaxcircumsporozoite protein CSP<br />B-cell epitope<br />Universal T-cell epitope<br />Source: Socrates Herrera et al...
Developing a Malaria vaccine<br />Source: Socrates Herrera et al., Procesopara el desarrollo de unavacuna contra la fase h...
Preclinical studies in mice<br />Morphology<br />Physiology<br />Safety<br />Formulation<br />Immunogenicity<br />protecti...
MultipleAntigenPeptides (MAPs)<br />Synthetic peptides <br />solid-phasefluorenylmethoxycarbonyl  (F- Moc) chemistry<br />...
Miceimmunization<br />Six-weeks-old BALB/c  <br />(3 mice/group)<br />Subcutaneous (S.C.) immunization<br />50 µg/ml MAP/C...
Antibody Responses<br />Source: Immunogenic of Multiple Antigen Peptides Containing Plasmodium vivax CS Epitopes in BALB/c...
Antibody Responses<br />         P11(3x)-ptt30(1x)<br />Source: Immunogenic of Multiple Antigen Peptides Containing Plasmo...
T cellProliferation<br />In vitro Proliferative T-cell Response <br />Source: Immunogenic of Multiple Antigen Peptides Con...
MAPs containing B and T cell epitopes induced high titters of anti-peptides antibodies, which recognized the native CS pro...
Immunogens<br />Synthetic peptides <br />Lab condition: Bench quality (BC) and Good laboratory practices (GLP)<br />solid-...
Preclinical studies N,C & R peptides<br />Toxicity<br />LCG Bioscience (Turin)<br />5 female mice CD-1 BR<br />2 female gu...
Pre-Clinic Studies  N,C & R peptides<br />Potency<br />Instituto de Inmunología del Valle (MVDC-260901)<br />BALB/c n=60<b...
Results<br />Repeat region<br />Bench Quality (BQ)<br />Good Laboratory Practices (GLP)<br />Socrates Herrera et al. Colom...
Immunogenicity in experimental animals<br />Immunogenicity in mice<br />Instituto de Inmunología del Valle <br />BALB/c 12...
Results<br />Socrates Herrera et al. Colombia Médica. Vol. 36 N° 1, 2005<br />
The vaccine was well tolerated and showed good safety and immunogenicity inpreclinical studies facilitating progression to...
Preclinical Studies in miceP. vivax CS synthetic vaccine<br />Vaccineformulation<br />Mix of N, R, C proteinpeptides<br />...
Immunogenicitystudy in BALB/c mice<br />n= 24 BALB/c <br />Female 3-5 weeks age<br />50 µg each individual peptide<br />D=...
Wilcoxonsigned-rank test. P values < 0.05<br />Source: Arévalo-Herrera, et al. Preclinical Vaccine Study of Plasmodium viv...
Recognition of the CS P. vivaxprotein<br />Experimental infection of Anophelesalbimanus<br />usinghumanblood P. vivaxinfec...
Conclusions<br />Anti-peptidesantibodiesremained detectable forseveralmonths and recognizednativeproteinonsporozoites<br /...
The long peptides <br />(N, C, R) have great potential as antigenic sub-units that could be included in a multivalent vacc...
Immunogenicity in experimental animals<br />Immunogenicity in primate<br />Fundacion Centro de Primates<br />Short peptide...
Results<br />γ-INF Production in Aotus monkey immunized with PV1, PV2, PV5 & PV6  <br />Colombia Médica. Vol. 36 N° 1, 200...
Clinic Studies Phase I<br />n=23<br />Young adults naive<br />16 women 7 men<br />Vaccine long peptide <br />R<br />n=7<br...
Clinic Studies Phase I<br />Immunogenicity in human <br />Safety evaluation<br /> 7 evaluation to determinate Immune humor...
Results<br />Specific antibodies title  against N, R & C peptides after each immunization<br />Colombia Médica. Vol. 36 N°...
The malarial parasite's life cycle. Images from Purves et al., Life: The Science of Biology, 4th Edition,<br />
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  1. 1. PRECLINICAL VACCINE STUDIES OF THE CS Plasmodium vivaxPROTEININ MICE <br />
  2. 2. Malaria Indices<br />Authors: World Health OrganizationPublication date: 2009<br />Source: http://www.rollbackmalaria.org/endemiccountries/endemiccountry<br />Malaria Indices for species <br />and geography regions Latin America 1999<br />Malaria Indices for species <br />and geography regions Colombia <br />2004<br />Log scale IVA/IFA<br />Source: Siviligila, Held National Institute, 2004<br />Source: Transmissible Illness Programme (HTC) from OPS<br />
  3. 3. <ul><li>Sterileprotectiveimmunity</li></ul>radiation-attenuatedsporozoites<br /><ul><li>Anti-diseaseimmunity</li></ul>Source: The malarial parasite's life cycle. Images from Purves et al., Life: The Science of Biology, 4th Edition,<br />Vaccine against Malaria<br /><ul><li>Transmission-blocking vaccine </li></ul>The RTS.S a P. falciparum vaccine candidate based on the circumsporozoite (CS)<br />protein is ready to enter Phase III clinical trials. (Arevalo-Herrera et al. For peer review )<br />
  4. 4. P. vivax vaccine candidates currently in development <br />Source: Myriam Arévalo-Herrera, Chetan Chitnis and Sócrates Herrera. Current status of Plasmodium vivax vaccine. Human Vaccines 6:1, 1-9; January 2010; Landes Bioscience<br />
  5. 5. The circumsporozoite (CS) protein<br />CS is a thrombospondin type I repeat (TSR) domain family (Dharmendar Rathore et al. 2002)<br />CS is a multifunctional protein essential for<br />many steps of the sporozoite’s life (Robert Ménard 200) <br />It binds specifically to salivary glands of Anopheles stephensi (Sidjanski SP., et al 1997) <br />CS binds to the surface of hepatocytes cells in mice(Cerami C., et al 1994)<br />CS protein is involved in sporozoite infection of hepatocytes (Potocnjak P. et al 1980)<br />Source: N Engl J Med 358;17 www.nejm.org april 24, 2008<br />
  6. 6. P. vivaxcircumsporozoite protein CSP events<br />Mabs against CS show protection in mice <br />and neutralize infectivity in vitro <br />(Hollingdale, et al 1982)<br />VK247 Variant (Rosenberg et al.1989)<br />Induction of T cell responses by several epitopes CS <br />(Herrera S, et al 1992)<br /> Recombinant P. vivax CSP vaccine with <br />VK210 and VK247<br />(Anjali Yadava 2007) <br />Human Malaria parasite identified in the mosquito<br />Cloning of the<br /> first Plasmodium antigen CSP <br />P. vivax CSP synthetic peptides <br />N, C and R <br />Pre-clinic studies<br />(Herrera S, et al 2004)<br /> 1899 1948 1982 1984 - 1987 1992 1998 - 2002 2004 2005 2007 <br /> Identifying multiple <br />B, T-helper and CD8+ T cell epitopes <br />(Arevalo Herrera, et al 1998-2002)<br /> Synthetic CS protein vaccine <br />Phase I clinical<br />(Herrera S, et al 2005)<br />Discovery of pre-eritrocite stage<br />Recombinant P. vivaxCSP (rPVCS-1) vaccine candidate proposed <br />Vaccine trials in human volunteers did not show significant immune<br />responses(Herrington DA, et al 1991, Fries LF et al 1992)<br />Information source: Myriam Arévalo-Herrera,1,2 Chetan Chitnis3 and Sócrates Herrera. Current status of Plasmodium vivax vaccine Human Vaccines 6:1, 1-9; January 2010; © 2010 Landes Bioscience<br />
  7. 7. P. vivaxcircumsporozoite protein CSP<br />B-cell epitope<br />Universal T-cell epitope<br />Source: Socrates Herrera et al., Use of long synthetic peptides to study the antigenicity and immunogenicity of the Plasmodium vivax circumsporozoite protein. International journal of Parasitology 34 (2004) 1535-1546<br />
  8. 8. Developing a Malaria vaccine<br />Source: Socrates Herrera et al., Procesopara el desarrollo de unavacuna contra la fase hepatica de Plasmodium vivax. Colombia Medica. Vol. 36 N° 1, 2005<br />Objectives<br />Asses the immunogenicity in BALB/c Mice of Multiple Antigen Peptides and Long Synthetic Peptides of P. vivaxCS protein <br />
  9. 9. Preclinical studies in mice<br />Morphology<br />Physiology<br />Safety<br />Formulation<br />Immunogenicity<br />protection<br />toxicity studies <br />Toevaluate<br />Potency<br />A.BY<br />C57L<br />C57BL/6<br />C3H.SW<br />129/J<br />H-2b<br />H-2d<br />H-2k<br />Genetic restriction:<br />MHC haplotypes influence the ability to respond to an antigen<br />Inbred Syngeneic<br />BALB/c<br />C57BL/6<br />C3H.SW<br />129/J<br />Outbred<br />CBA<br />C3H/HeJ<br />C57BR/cd<br />C58/J<br />B10.BR<br />The complexity of protective immunity against liver-stage malaria<br />Doolan DL, Hoffman SL. J Immunol. 2000 Aug 1;165(3):1453-62.<br />
  10. 10. MultipleAntigenPeptides (MAPs)<br />Synthetic peptides <br />solid-phasefluorenylmethoxycarbonyl (F- Moc) chemistry<br />Source: Immunogenic of Multiple Antigen Peptides Containing Plasmodium vivax CS Epitopes in BALB/c Mice. Herrera MA, et al 1994 Mem Inst Oswaldo Cruz. 1994;89 Suppl 2:71-6.<br />
  11. 11. Miceimmunization<br />Six-weeks-old BALB/c <br />(3 mice/group)<br />Subcutaneous (S.C.) immunization<br />50 µg/ml MAP/Complete Freund adjuvant (CFC)+PBS<br />O & 20 day<br />Serum collected 30 day<br />Humoral immuneresponse<br />ELISA<br />Anti-sporozoiteantibodiesdeterminedby IFAT<br />Lymphocyte proliferation assay<br />T cell proliferation test<br />Source: Immunogenic of Multiple Antigen Peptides Containing Plasmodium vivax CS Epitopes in BALB/c Micw. Herrera MA, et al 1994 Mem Inst Oswaldo Cruz. 1994;89 Suppl 2:71-6.<br />
  12. 12. Antibody Responses<br />Source: Immunogenic of Multiple Antigen Peptides Containing Plasmodium vivax CS Epitopes in BALB/c Micw. Herrera MA, et al 1994 Mem Inst Oswaldo Cruz. 1994;89 Suppl 2:71-6.<br />
  13. 13. Antibody Responses<br /> P11(3x)-ptt30(1x)<br />Source: Immunogenic of Multiple Antigen Peptides Containing Plasmodium vivax CS Epitopes in BALB/c Micw. Herrera MA, et al 1994 Mem Inst Oswaldo Cruz. 1994;89 Suppl 2:71-6.<br />
  14. 14. T cellProliferation<br />In vitro Proliferative T-cell Response <br />Source: Immunogenic of Multiple Antigen Peptides Containing Plasmodium vivax CS Epitopes in BALB/c Micw. Herrera MA, et al 1994 Mem Inst Oswaldo Cruz. 1994;89 Suppl 2:71-6.<br />
  15. 15. MAPs containing B and T cell epitopes induced high titters of anti-peptides antibodies, which recognized the native CS protein on sporozoites as determined by IFAT <br />Source: Immunogenic of Multiple Antigen Peptides Containing Plasmodium vivax CS Epitopes in BALB/c Micw. Herrera MA, et al 1994 Mem Inst Oswaldo Cruz. 1994;89 Suppl 2:71-6.<br />
  16. 16. Immunogens<br />Synthetic peptides <br />Lab condition: Bench quality (BC) and Good laboratory practices (GLP)<br />solid-phasefluorenylmethoxycarbonyl (F- Moc) chemistry<br />28 peptides x 20 aa <br />overlapped in 10 aa<br />Small peptides<br />Characterization of protein<br />6 peptides <br />9-10 residues<br />Purification by HPLC and MS<br />N= N-terminal (20-96aa)<br />R= 3 repeat peptide p11 (96-104 aa) + ptt30<br />C= C-terminal (301-372aa)<br />Long peptides more than 70 aa<br />Measure of vaccine effect <br />Pre-clinic and clinic assay phase 1 <br />Microbiology assay <br />pyrogenicity analysis <br />Evaluation of sterility <br />Socrates Herrera et al. Colombia Médica. Vol. 36 N° 1, 2005<br />
  17. 17. Preclinical studies N,C & R peptides<br />Toxicity<br />LCG Bioscience (Turin)<br />5 female mice CD-1 BR<br />2 female guinea-pigs Dunkin Hartley Albino<br />Intraperitoneal Immunization (IP)<br /> Peptide 100 µg in 1 ml SS<br />N<br />R<br />C<br />Systematic and local changes<br />Behaviour alterations <br />Mortality at 30 min and 2,4,6 h after immunization<br />Evaluation each 2 h until 6th day <br />Clinical sings <br />Socrates Herrera et al. Colombia Médica. Vol. 36 N° 1, 2005<br />
  18. 18. Pre-Clinic Studies N,C & R peptides<br />Potency<br />Instituto de Inmunología del Valle (MVDC-260901)<br />BALB/c n=60<br />Female 3-4 weeks age S.C. tail base (TB or BC) <br /> N C R<br />20 mice<br />A = 0,1 <br />B = 0,3<br />C = 3<br />D = 10<br />E = 30<br />20 mice<br />A = 0,1 <br />B = 0,3<br />C = 3<br />D = 10<br />E = 30<br />20 mice<br />A = 0,1 <br />B = 0,3<br />C = 3<br />D = 10<br />E = 30<br />Dose= µg<br />ELISA 0, 15 and 30 day post-immunization (PI)<br />Positive title ≥ 1:100<br />Socrates Herrera et al. Colombia Médica. Vol. 36 N° 1, 2005<br />
  19. 19. Results<br />Repeat region<br />Bench Quality (BQ)<br />Good Laboratory Practices (GLP)<br />Socrates Herrera et al. Colombia Médica. Vol. 36 N° 1, 2005<br />
  20. 20. Immunogenicity in experimental animals<br />Immunogenicity in mice<br />Instituto de Inmunología del Valle <br />BALB/c 12 mice 3-4 weeks<br /> 50 µg peptide/50:50 Freund adjuvant and saline solution (SS)<br />3 Inoculations<br />N C R<br />Peptide BC<br />Peptide IP<br />Mix C,N & R BC<br />Mix C, N & R IP<br />Mix <br />1= C & N<br />2&3= C,N & R<br />ELISA 0, 14, 35 & 42 days positive title ≥ 1:100<br />Socrates Herrera et al. Colombia Médica. Vol. 36 N° 1, 2005<br />
  21. 21. Results<br />Socrates Herrera et al. Colombia Médica. Vol. 36 N° 1, 2005<br />
  22. 22. The vaccine was well tolerated and showed good safety and immunogenicity inpreclinical studies facilitating progression to further stages of clinical research<br />
  23. 23. Preclinical Studies in miceP. vivax CS synthetic vaccine<br />Vaccineformulation<br />Mix of N, R, C proteinpeptides<br />50-100 µg each<br />150-300 µg/dose<br />Emulsification 24h beforeimmunization<br />A<br />Peptidemix<br />Emulsification in<br />Montanide ISA 720 <br />B<br />Peptidemix<br />Emulsification in<br />Montanide ISA 51<br />C<br />SS + Montanide<br />ISA 720 <br />D<br />SS + Montanide<br />ISA 51<br />Source: Arévalo-Herrera, et al. Preclinical Vaccine Study of Plasmodium vivax, American Journal of Tropical Medicine & Hygiene 2010 for peer review<br />
  24. 24. Immunogenicitystudy in BALB/c mice<br />n= 24 BALB/c <br />Female 3-5 weeks age<br />50 µg each individual peptide<br />D= 150 µg V= 0.3 ml<br />SS+ respective adjuvant<br />V= 0.3 ml<br />C n=6<br />B n=6<br />D n=6<br /> A n=6 <br />TB Immunization <br /> N+ C<br />N + C+ R<br />N + C+ R<br />Blood collected (150 µl) <br />ELISA using N, R or C- proteins<br />P values < 0.05<br />Source: Arévalo-Herrera, et al. Preclinical Vaccine Study of Plasmodium vivax, American Journal of Tropical Medicine & Hygiene 2010 for peer review<br />
  25. 25. Wilcoxonsigned-rank test. P values < 0.05<br />Source: Arévalo-Herrera, et al. Preclinical Vaccine Study of Plasmodium vivax, American Journal of Tropical Medicine & Hygiene 2010 for peer review<br />
  26. 26. Recognition of the CS P. vivaxprotein<br />Experimental infection of Anophelesalbimanus<br />usinghumanblood P. vivaxinfected<br />P. vivax sporozoites antigen acquired by<br />salivary gland dissection An. albimanus<br />IFAT<br />Serum mice control group <br />Serummice test group<br />Fluorescence microscope<br />Mice serum were reactive with native CS protein by IFAT<br />Source: Arévalo-Herrera, et al. Preclinical Vaccine Study of Plasmodium vivax, American Journal of Tropical Medicine & Hygiene 2010 for peer review<br />
  27. 27. Conclusions<br />Anti-peptidesantibodiesremained detectable forseveralmonths and recognizednativeproteinonsporozoites<br />Unlikemonkeys, micedevelopedbetterantibody responses against C peptide<br />There are no significantdifferencesbetweenMontanide ISA 720 and Montanide ISA 51 formulations<br />Source: Arévalo-Herrera, et al. Preclinical Vaccine Study of Plasmodium vivax, American Journal of Tropical Medicine & Hygiene 2010 for peer review<br />
  28. 28. The long peptides <br />(N, C, R) have great potential as antigenic sub-units that could be included in a multivalent vaccine against Malaria<br />
  29. 29.
  30. 30.
  31. 31.
  32. 32. Immunogenicity in experimental animals<br />Immunogenicity in primate<br />Fundacion Centro de Primates<br />Short peptides<br />Long peptides<br />Aoutus lemurinus griseimembra<br />Male o female W=800g<br />n=24<br />Aoutus lemurinus griseimembra<br />Male o female W=800g<br />n=18<br />Immunization <br />SC vi 0, 40 & 120 days<br />N=6<br />2 doses<br />100 µg peptide /Freund adjuvant <br />SC vi <br />PV 1, PV 2, PV5 & PV 6 inductor of (CD8+)<br />Group A n=6<br />100 µg (N+R+C)/ Montanide ISA-720 adjuvant<br />Group B n=6<br />100 µg (N+R+C)/Freund reference adjuvant <br />Group C n=6<br />H2O Montanide IS-720/ placebo <br />Before and <br />10 days after immunization<br />measure γ-IFN<br />lymphocytes stimulated <br />By ELISA<br />ELISA after each immunization <br />IFAT 30 & 130 days after last immunization<br />Colombia Médica. Vol. 36 N° 1, 2005<br />
  33. 33. Results<br />γ-INF Production in Aotus monkey immunized with PV1, PV2, PV5 & PV6 <br />Colombia Médica. Vol. 36 N° 1, 2005<br />
  34. 34. Clinic Studies Phase I<br />n=23<br />Young adults naive<br />16 women 7 men<br />Vaccine long peptide <br />R<br />n=7<br />N<br />n=7<br />C<br />n=7<br />Control<br />n=2<br />Montanide ISA-720/100 µg peptide<br />Vf= 500 µl<br />3:7 Montanide ISA-720/ SS<br />Vf= 500 µl<br />double-blind<br />3 Immunization <br />Intramuscularly vi<br />Colombia Médica. Vol. 36 N° 1, 2005<br />
  35. 35. Clinic Studies Phase I<br />Immunogenicity in human <br />Safety evaluation<br /> 7 evaluation to determinate Immune humoral responds<br />Against immunogen by ELISA<br />Against protein native IFAT<br />Observation for 1 h post-immunization and<br />physical examination 8, 24 h and 7th days<br />Clinical laboratory test<br />Colombia Médica. Vol. 36 N° 1, 2005<br />
  36. 36. Results<br />Specific antibodies title against N, R & C peptides after each immunization<br />Colombia Médica. Vol. 36 N° 1, 2005<br />
  37. 37.
  38. 38. The malarial parasite's life cycle. Images from Purves et al., Life: The Science of Biology, 4th Edition,<br />
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