Presentacion 1 cs protein y ratones

PRECLINICAL VACCINE STUDIES OF

THE CS Plasmodium vivax PROTEININ MICE

Malaria Indices

Malaria Indices for species and geography regions Latin America

1999

Log

scal

e IV

A/IF

A

Malaria Indices for species and geography regions Colombia

2004

Source: Siviligila, Held National Institute, 2004

Source: Transmissible Illness Programme (HTC) from OPS

Source: http://www.rollbackmalaria.org/endemiccountries/endemiccountry

Authors: World Health OrganizationPublication date: 2009

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

• Sterile protective immunityradiation-attenuated sporozoites

• Anti-disease immunity

Vaccine against Malaria

• Transmission-blocking vaccine

The RTS.S a P. falciparum vaccine candidate based on the circumsporozoite (CS)protein is ready to enter Phase III clinical trials. (Arevalo-Herrera et al. For peer review )

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

The circumsporozoite (CS) protein

Source: N Engl J Med 358;17 www.nejm.org april 24, 2008

CS is a multifunctional protein essential formany 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)

CS is a thrombospondin type I repeat (TSR) domain family (Dharmendar Rathore et al. 2002)

1899 1948 1982 1984 - 1987 1992 1998 - 2002 2004 2005 2007

Human Malaria parasite identified in the

mosquito

Discovery of pre-eritrocite

stage

Cloning of the first Plasmodium

antigen CSP

Recombinant P. vivax CSP (rPVCS-1) vaccine candidate proposed

Identifying multiple B, T-helper and CD8+ T cell epitopes

(Arevalo Herrera, et al 1998-2002)

Recombinant P. vivax CSP vaccine with VK210 and VK247

(Anjali Yadava 2007)

P. vivax CSP synthetic peptides N, C and R

Pre-clinic studies(Herrera S, et al 2004)

Synthetic CS protein vaccine Phase I clinical

(Herrera S, et al 2005)

P. vivax circumsporozoite protein CSP events

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

Induction of T cell responses by several

epitopes CS (Herrera S, et al 1992)

Mabs against CS show protection in mice

and neutralize infectivity in vitro (Hollingdale, et al 1982)

Vaccine trials in human volunteers did not show significant immuneresponses (Herrington DA, et al 1991, Fries LF et al 1992)

VK247 Variant (Rosenberg et al.1989)

P. vivax circumsporozoite protein CSP

B-cell epitopeUniversal 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

Developing a Malaria vaccine

ObjectivesAsses the immunogenicity in BALB/c Mice of Multiple Antigen Peptides and Long Synthetic Peptides of P. vivax CS protein

Source: Socrates Herrera et al., Proceso para el desarrollo de una vacuna contra la fase hepatica de Plasmodium vivax. Colombia Medica. Vol. 36 N° 1, 2005

Preclinical studies in mice

To evaluate

Safety

Formulation

Immunogenicityprotection

toxicity studies MorphologyPhysiology

Genetic restriction:MHC haplotypes influence the ability to respond to an antigen

H-2b

H-2d

H-2k

A.BYC57LC57BL/6C3H.SW129/J

BALB/cC57BL/6C3H.SW129/JCBAC3H/HeJC57BR/cdC58/JB10.BR

Inbred Syngeneic

Outbred

The complexity of protective immunity against liver-stage malariaDoolan DL, Hoffman SL. J Immunol. 2000 Aug 1;165(3):1453-62.

Potency

Multiple Antigen Peptides (MAPs)Synthetic peptides

solid-phase fluorenylmethoxycarbonyl (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.

Mice immunizationSix-weeks-old BALB/c

(3 mice/group)

Subcutaneous (S.C.) immunization50 µg/ml MAP/Complete Freund adjuvant (CFC)+PBS

O & 20 day

Serum collected 30 day

Humoral immune responseELISA

Anti-sporozoite antibodies determined by IFAT

Lymphocyte proliferation assayT 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.

Antibody Responses


Antibody Responses


P11(3x)-ptt30(1x)

T cell Proliferation


In vitro Proliferative T-cell Response

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


ImmunogensSynthetic peptides

Lab condition: Bench quality (BC) and Good laboratory practices (GLP) solid-phase fluorenylmethoxycarbonyl (F- Moc) chemistry

28 peptides x 20 aa overlapped in 10 aa

6 peptides 9-10 residues

N= N-terminal (20-96aa)R= 3 repeat peptide p11 (96-104 aa) + ptt30C= C-terminal (301-372aa)

Small peptidesCharacterization of protein

Long peptides more than 70 aaMeasure of vaccine effect

Pre-clinic and clinic assay phase 1

Purification by HPLC and MS

Microbiology assay pyrogenicity analysis

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

Evaluation of sterility

Preclinical studies N,C & R peptides

ToxicityLCG Bioscience (Turin)

5 female mice CD-1 BR2 female guinea-pigs Dunkin Hartley Albino Intraperitoneal Immunization (IP) Peptide 100 µg in 1 ml SS

Systematic and local changesBehaviour alterations Mortality at 30 min and 2,4,6 h after immunizationEvaluation each 2 h until 6th day

Clinical sings

N

R

C


Pre-Clinic Studies N,C & R peptidesPotency

Instituto de Inmunología del Valle (MVDC-260901)

BALB/c n=60Female 3-4 weeks age S.C. tail base (TB or BC)

20 miceA = 0,1 B = 0,3C = 3D = 10E = 30

Dose = µg

ELISA 0, 15 and 30 day post-immunization (PI)Positive title ≥ 1:100

20 miceA = 0,1 B = 0,3C = 3D = 10E = 30

20 miceA = 0,1 B = 0,3C = 3D = 10E = 30

N C R


Results

Bench Quality (BQ)Good Laboratory Practices (GLP)


Repeat region

Immunogenicity in experimental animalsImmunogenicity 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 BCPeptide IPMix C,N & R BCMix C, N & R IP

Mix 1= C & N2&3= C,N & R

ELISA 0, 14, 35 & 42 days positive title ≥ 1:100


Results


The vaccine was well tolerated and showed good safety and immunogenicity in

preclinical studies facilitating progression to further stages of clinical research

Preclinical Studies in miceP. vivax CS synthetic vaccine

Mix of N, R, C protein peptides 50-100 µg each 150-300 µg/dose

Emulsification 24h before immunization

APeptide mix

Emulsification inMontanide ISA 720

BPeptide mix

Emulsification inMontanide ISA 51

CSS + Montanide

ISA 720

Vaccine formulation

DSS + Montanide

ISA 51

Source: Arévalo-Herrera, et al. Preclinical Vaccine Study of Plasmodium vivax, American Journal of Tropical Medicine & Hygiene 2010 for peer review

n= 24 BALB/c Female 3-5 weeks age

A n=6 C n=6B n=6 D n=6

50 µg each individual peptideD= 150 µg V= 0.3 ml

N+ C N + C+ R N + C+ R

Blood collected (150 µl) ELISA using N, R or C- proteins

Immunogenicity study in BALB/c mice

SS+ respective adjuvantV= 0.3 ml


TB Immunization

P values < 0.05


Wilcoxon signed-rank test. P values < 0.05

Recognition of the CS P. vivax protein

P. vivax sporozoites antigen acquired bysalivary gland dissection An. albimanus

Experimental infection of Anopheles albimanususing human blood P. vivax infected

IFATSerum mice control group Serum mice test group

Fluorescence microscope


Mice serum were reactive with native CS protein by IFAT

Conclusions

• Anti-peptides antibodies remained detectable for several months and recognized native protein on sporozoites

• Unlike monkeys, mice developed better antibody responses against C peptide

• There are no significant differences between Montanide ISA 720 and Montanide ISA 51 formulations


The long peptides (N, C, R) have great potential as

antigenic sub-units that could be included in a multivalent vaccine

against Malaria

Immunogenicity in experimental animalsImmunogenicity in primate

Fundacion Centro de Primates

Aoutus lemurinus griseimembraMale o female W=800g

n=24

Before and 10 days after immunization

measure γ-IFN lymphocytes stimulated

By ELISA

Short peptides Long peptides

Immunization SC vi 0, 40 & 120 days

Group A n=6100 µg (N+R+C)/ Montanide ISA-720 adjuvant

Group B n=6100 µg (N+R+C)/Freund reference adjuvant

Group C n=6H2O Montanide IS-720/ placebo

ELISA after each immunization IFAT 30 & 130 days after last immunization

N=62 doses

100 µg peptide /Freund adjuvant SC vi

PV 1, PV 2, PV5 & PV 6 inductor of (CD8+)

Aoutus lemurinus griseimembraMale o female W=800g

n=18

Colombia Médica. Vol. 36 N° 1, 2005

Results

γ-INF Production in Aotus monkey immunized with PV1, PV2, PV5 & PV6


Clinic Studies Phase I

n=23Young adults naive16 women 7 men

Vaccine long peptide

Rn=7

Nn=7

Cn=7

Controln=2

Montanide ISA-720/100 µg peptideVf= 500 µl

3:7 Montanide ISA-720/ SSVf= 500 µl

double-blind3 Immunization

Intramuscularly viColombia Médica. Vol. 36 N° 1, 2005

Clinic Studies Phase I

Safety evaluation Immunogenicity in human

7 evaluation to determinate Immune humoral responds

Against immunogen by ELISAAgainst protein native IFAT

Observation for 1 h post-immunization andphysical examination 8, 24 h and 7th days

Clinical laboratory test


Specific antibodies title against N, R & C peptides after each immunization

Results


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

Presentacion 1 cs protein y ratones

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