The document summarizes preclinical vaccine studies of the Plasmodium vivax circumsporozoite (CS) protein in mice. The studies showed that immunizing mice with synthetic peptides from the N-terminal, central repeat, and C-terminal regions of the CS protein using multiple antigen peptides or long synthetic peptides induced antibody and T cell responses. The antibodies recognized the native CS protein on sporozoites. Further studies found that a mixture of the three peptide regions was safe, well-tolerated, and immunogenic in mice when formulated with adjuvant. The vaccine elicited antibodies that remained detectable for months and recognized the native CS protein.

1. PRECLINICAL VACCINE STUDIES OF THE CS Plasmodium vivaxPROTEININ MICE

2. Malaria Indices Authors: World Health OrganizationPublication date: 2009 Source: http://www.rollbackmalaria.org/endemiccountries/endemiccountry Malaria Indices for species and geography regions Latin America 1999 Malaria Indices for species and geography regions Colombia 2004 Log scale IVA/IFA Source: Siviligila, Held National Institute, 2004 Source: Transmissible Illness Programme (HTC) from OPS

4. P. vivax vaccine candidates currently in development 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

5. The circumsporozoite (CS) protein CS is a thrombospondin type I repeat (TSR) domain family (Dharmendar Rathore et al. 2002) CS is a multifunctional protein essential for many steps of the sporozoite’s life (Robert Ménard 200) It binds specifically to salivary glands of Anopheles stephensi (Sidjanski SP., et al 1997) CS binds to the surface of hepatocytes cells in mice(Cerami C., et al 1994) CS protein is involved in sporozoite infection of hepatocytes (Potocnjak P. et al 1980) Source: N Engl J Med 358;17 www.nejm.org april 24, 2008

6. P. vivaxcircumsporozoite protein CSP events Mabs against CS show protection in mice and neutralize infectivity in vitro (Hollingdale, et al 1982) VK247 Variant (Rosenberg et al.1989) Induction of T cell responses by several epitopes CS (Herrera S, et al 1992) Recombinant P. vivax CSP vaccine with VK210 and VK247 (Anjali Yadava 2007) Human Malaria parasite identified in the mosquito Cloning of the first Plasmodium antigen CSP P. vivax CSP synthetic peptides N, C and R Pre-clinic studies (Herrera S, et al 2004) 1899 1948 1982 1984 - 1987 1992 1998 - 2002 2004 2005 2007 Identifying multiple B, T-helper and CD8+ T cell epitopes (Arevalo Herrera, et al 1998-2002) Synthetic CS protein vaccine Phase I clinical (Herrera S, et al 2005) Discovery of pre-eritrocite stage Recombinant P. vivaxCSP (rPVCS-1) vaccine candidate proposed Vaccine trials in human volunteers did not show significant immune responses(Herrington DA, et al 1991, Fries LF et al 1992) 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

7. P. vivaxcircumsporozoite protein CSP B-cell epitope Universal T-cell epitope 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

8. Developing a Malaria vaccine Source: Socrates Herrera et al., Procesopara el desarrollo de unavacuna contra la fase hepatica de Plasmodium vivax. Colombia Medica. Vol. 36 N° 1, 2005 Objectives Asses the immunogenicity in BALB/c Mice of Multiple Antigen Peptides and Long Synthetic Peptides of P. vivaxCS protein

9. Preclinical studies in mice Morphology Physiology Safety Formulation Immunogenicity protection toxicity studies Toevaluate Potency A.BY C57L C57BL/6 C3H.SW 129/J H-2b H-2d H-2k Genetic restriction: MHC haplotypes influence the ability to respond to an antigen Inbred Syngeneic BALB/c C57BL/6 C3H.SW 129/J Outbred CBA C3H/HeJ C57BR/cd C58/J B10.BR The complexity of protective immunity against liver-stage malaria Doolan DL, Hoffman SL. J Immunol. 2000 Aug 1;165(3):1453-62.

10. MultipleAntigenPeptides (MAPs) Synthetic peptides solid-phasefluorenylmethoxycarbonyl (F- Moc) chemistry 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.

11. Miceimmunization Six-weeks-old BALB/c (3 mice/group) Subcutaneous (S.C.) immunization 50 µg/ml MAP/Complete Freund adjuvant (CFC)+PBS O & 20 day Serum collected 30 day Humoral immuneresponse ELISA Anti-sporozoiteantibodiesdeterminedby IFAT Lymphocyte proliferation assay T cell proliferation test 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.

12. Antibody Responses 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.

13. Antibody Responses P11(3x)-ptt30(1x) 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.

14. T cellProliferation In vitro Proliferative T-cell Response 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.

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 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.

16. Immunogens Synthetic peptides Lab condition: Bench quality (BC) and Good laboratory practices (GLP) solid-phasefluorenylmethoxycarbonyl (F- Moc) chemistry 28 peptides x 20 aa overlapped in 10 aa Small peptides Characterization of protein 6 peptides 9-10 residues Purification by HPLC and MS N= N-terminal (20-96aa) R= 3 repeat peptide p11 (96-104 aa) + ptt30 C= C-terminal (301-372aa) Long peptides more than 70 aa Measure of vaccine effect Pre-clinic and clinic assay phase 1 Microbiology assay pyrogenicity analysis Evaluation of sterility Socrates Herrera et al. Colombia Médica. Vol. 36 N° 1, 2005

17. Preclinical studies N,C & R peptides Toxicity LCG Bioscience (Turin) 5 female mice CD-1 BR 2 female guinea-pigs Dunkin Hartley Albino Intraperitoneal Immunization (IP) Peptide 100 µg in 1 ml SS N R C Systematic and local changes Behaviour alterations Mortality at 30 min and 2,4,6 h after immunization Evaluation each 2 h until 6th day Clinical sings Socrates Herrera et al. Colombia Médica. Vol. 36 N° 1, 2005

18. Pre-Clinic Studies N,C & R peptides Potency Instituto de Inmunología del Valle (MVDC-260901) BALB/c n=60 Female 3-4 weeks age S.C. tail base (TB or BC) N C R 20 mice A = 0,1 B = 0,3 C = 3 D = 10 E = 30 20 mice A = 0,1 B = 0,3 C = 3 D = 10 E = 30 20 mice A = 0,1 B = 0,3 C = 3 D = 10 E = 30 Dose= µg ELISA 0, 15 and 30 day post-immunization (PI) Positive title ≥ 1:100 Socrates Herrera et al. Colombia Médica. Vol. 36 N° 1, 2005

19. Results Repeat region Bench Quality (BQ) Good Laboratory Practices (GLP) Socrates Herrera et al. Colombia Médica. Vol. 36 N° 1, 2005

20. Immunogenicity in experimental animals Immunogenicity in mice Instituto de Inmunología del Valle BALB/c 12 mice 3-4 weeks 50 µg peptide/50:50 Freund adjuvant and saline solution (SS) 3 Inoculations N C R Peptide BC Peptide IP Mix C,N & R BC Mix C, N & R IP Mix 1= C & N 2&3= C,N & R ELISA 0, 14, 35 & 42 days positive title ≥ 1:100 Socrates Herrera et al. Colombia Médica. Vol. 36 N° 1, 2005

21. Results Socrates Herrera et al. Colombia Médica. Vol. 36 N° 1, 2005

22. The vaccine was well tolerated and showed good safety and immunogenicity inpreclinical studies facilitating progression to further stages of clinical research

23. Preclinical Studies in miceP. vivax CS synthetic vaccine Vaccineformulation Mix of N, R, C proteinpeptides 50-100 µg each 150-300 µg/dose Emulsification 24h beforeimmunization A Peptidemix Emulsification in Montanide ISA 720 B Peptidemix Emulsification in Montanide ISA 51 C SS + Montanide ISA 720 D SS + Montanide ISA 51 Source: Arévalo-Herrera, et al. Preclinical Vaccine Study of Plasmodium vivax, American Journal of Tropical Medicine & Hygiene 2010 for peer review

24. Immunogenicitystudy in BALB/c mice n= 24 BALB/c Female 3-5 weeks age 50 µg each individual peptide D= 150 µg V= 0.3 ml SS+ respective adjuvant V= 0.3 ml C n=6 B n=6 D n=6 A n=6 TB Immunization N+ C N + C+ R N + C+ R Blood collected (150 µl) ELISA using N, R or C- proteins P values < 0.05 Source: Arévalo-Herrera, et al. Preclinical Vaccine Study of Plasmodium vivax, American Journal of Tropical Medicine & Hygiene 2010 for peer review

25. Wilcoxonsigned-rank test. P values < 0.05 Source: Arévalo-Herrera, et al. Preclinical Vaccine Study of Plasmodium vivax, American Journal of Tropical Medicine & Hygiene 2010 for peer review

26. Recognition of the CS P. vivaxprotein Experimental infection of Anophelesalbimanus usinghumanblood P. vivaxinfected P. vivax sporozoites antigen acquired by salivary gland dissection An. albimanus IFAT Serum mice control group Serummice test group Fluorescence microscope Mice serum were reactive with native CS protein by IFAT Source: Arévalo-Herrera, et al. Preclinical Vaccine Study of Plasmodium vivax, American Journal of Tropical Medicine & Hygiene 2010 for peer review

27. Conclusions Anti-peptidesantibodiesremained detectable forseveralmonths and recognizednativeproteinonsporozoites Unlikemonkeys, micedevelopedbetterantibody responses against C peptide There are no significantdifferencesbetweenMontanide ISA 720 and Montanide ISA 51 formulations Source: Arévalo-Herrera, et al. Preclinical Vaccine Study of Plasmodium vivax, American Journal of Tropical Medicine & Hygiene 2010 for peer review

28. The long peptides (N, C, R) have great potential as antigenic sub-units that could be included in a multivalent vaccine against Malaria

32. Immunogenicity in experimental animals Immunogenicity in primate Fundacion Centro de Primates Short peptides Long peptides Aoutus lemurinus griseimembra Male o female W=800g n=24 Aoutus lemurinus griseimembra Male o female W=800g n=18 Immunization SC vi 0, 40 & 120 days N=6 2 doses 100 µg peptide /Freund adjuvant SC vi PV 1, PV 2, PV5 & PV 6 inductor of (CD8+) Group A n=6 100 µg (N+R+C)/ Montanide ISA-720 adjuvant Group B n=6 100 µg (N+R+C)/Freund reference adjuvant Group C n=6 H2O Montanide IS-720/ placebo Before and 10 days after immunization measure γ-IFN lymphocytes stimulated By ELISA ELISA after each immunization IFAT 30 & 130 days after last immunization Colombia Médica. Vol. 36 N° 1, 2005

33. Results γ-INF Production in Aotus monkey immunized with PV1, PV2, PV5 & PV6 Colombia Médica. Vol. 36 N° 1, 2005

34. Clinic Studies Phase I n=23 Young adults naive 16 women 7 men Vaccine long peptide R n=7 N n=7 C n=7 Control n=2 Montanide ISA-720/100 µg peptide Vf= 500 µl 3:7 Montanide ISA-720/ SS Vf= 500 µl double-blind 3 Immunization Intramuscularly vi Colombia Médica. Vol. 36 N° 1, 2005

35. Clinic Studies Phase I Immunogenicity in human Safety evaluation 7 evaluation to determinate Immune humoral responds Against immunogen by ELISA Against protein native IFAT Observation for 1 h post-immunization and physical examination 8, 24 h and 7th days Clinical laboratory test Colombia Médica. Vol. 36 N° 1, 2005

36. Results Specific antibodies title against N, R & C peptides after each immunization Colombia Médica. Vol. 36 N° 1, 2005

38. The malarial parasite's life cycle. Images from Purves et al., Life: The Science of Biology, 4th Edition,