Post on 03-Jun-2015
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
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
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.
P11(3x)-ptt30(1x)
T cell Proliferation
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.
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
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.
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
Socrates Herrera et al. Colombia Médica. Vol. 36 N° 1, 2005
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
Socrates Herrera et al. Colombia Médica. Vol. 36 N° 1, 2005
Results
Bench Quality (BQ)Good Laboratory Practices (GLP)
Socrates Herrera et al. Colombia Médica. Vol. 36 N° 1, 2005
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
Socrates Herrera et al. Colombia Médica. Vol. 36 N° 1, 2005
Results
Socrates Herrera et al. Colombia Médica. Vol. 36 N° 1, 2005
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
Source: Arévalo-Herrera, et al. Preclinical Vaccine Study of Plasmodium vivax, American Journal of Tropical Medicine & Hygiene 2010 for peer review
TB Immunization
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
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
Source: Arévalo-Herrera, et al. Preclinical Vaccine Study of Plasmodium vivax, American Journal of Tropical Medicine & Hygiene 2010 for peer review
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
Source: Arévalo-Herrera, et al. Preclinical Vaccine Study of Plasmodium vivax, American Journal of Tropical Medicine & Hygiene 2010 for peer review
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
Colombia Médica. Vol. 36 N° 1, 2005
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
Colombia Médica. Vol. 36 N° 1, 2005
Specific antibodies title against N, R & C peptides after each immunization
Results
Colombia Médica. Vol. 36 N° 1, 2005
The malarial parasite's life cycle. Images from Purves et al., Life: The Science of Biology, 4th Edition,