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Transcript
OPTIMALVAC
Initiative on Optimising Malaria Vaccine
Laboratory Assays Evaluation
About the project
• EC FP7 Coordination and Support Action
• EC Budget €1 mil.
• Complementary contributions from the PATH Malaria
Vaccine Initiative and the Centers for Disease Control
and Prevention (€0.5 mil ).
• 13 Partners; Coordinator : EVI; Global Coordinator:
WHO
• Start Date: 01 April 2009, three years
Objectives
The goal is to identify and harmonise key
immunoassays to facilitate comparison of
results and improve decision-making in
malaria vaccine development.
3
Immunoassays in Malaria Vaccine
Development
Assess immunogenic
potential of candidate
vaccines
4
Keyword: Correlates of protection
• Correlates of immunity/protection to
a virus or other
infectious pathogen are measurable signs that a
person (or other potential host) is immune, in
the sense of being protected against becoming
infected and/or developing disease.
• Without knowing the correlates of immunity,
scientists cannot know exactly what sort of
immune response a vaccine would need to
stimulate
Y
Vaccine Candidate A
Naturally aquired Immunity or
immune potection demonstrated in
clinical trials or challenge studies
Vaccine Candiate B
Down selection of vaccine candidates
• Malaria vaccines: correlates of protection
not fully characterised
• 2/3 of malaria vaccines use IgG assays,
1/3 T cell based assays as readout
• Immunoassays used to determine intake of
vaccines
• Robust assays requried to compare
different approaches
Malaria Vaccine Assay
Harmonisation
Identify Key Assays
and Labs
Establishment of
Reference Centre
1st Comparison with
Existing SOPs/
Reagents
Harmonisation of
SOPs/Reagents
Agreed Community
Harmonised SOPs &
Reagent Repository
Iterative Comparisons
with Harmonised
SOPs
Assay Validation
8
Key immunoassays identified for harmonisation in
OPTIMALVAC
Cell mediated
immunity
• ICS
• ELISPOT
Humoral Assays
• Blood Stage IFA
Functional Assays
• Antibodydependent cellular
inhibition assay
9
T cell mediated immunity
Work package leader: Dr Patrice Dubois, Immunovacc Consulting
• Assess number of cytokine producing T cells in peripheral blood mononuclear
cells after stimulation
• Cell-mediated immunity (CMI) does not involve antibodies but rather the
activation of macrophages and NK-cells, the production of antigen-specific
cytotoxic T-lymphocytes , and the release of various cytokines in response to an
antigen .
Intracellular Cytokine staining (ICS)
Enyzme linked Immunospot (ELISpot)
Labelled CD4 AB
Y
T cell
Labelled anti-cytokine AB
10
ICS – Intracellular cytokine staining
Labelled CD4 AB
Y
• Whole blood is cultured and
stimulated
• Brefeldin A added shortly before
measurement to trap cytokines
in the cell
• T cell subtypes are marked with
fluorescent labelled antibodies
• Cells are fixed and
permeabilised
• Staining with fluorescentlabelled AB against cytokine
• Analysis by Fluorescence
activated Cell Sorting (FACS)
T cell
Labelled anti-cytokine AB
ELISpot - Enzyme Linked ImmunoSpot
• PBMCs cultured and
stimulated in 96well plates
with antibody coated
membrane
• Secreted IFN binds to
antibody on membrane
• Detection with secondary
antibody coupled to enzyme
• Add substrate and develop
• Analysis in ELISpot plate
reader
T cell mediated immunity
Identify Key Assays
and Labs
• Barcelona Centre for International Health Research (CRESIB), Spain
• Biomedical Primate Research Centre, BPRC, The Netherlands (Only ICS)
• Infectious Disease Research Institute (IDRI), USA (Only ELISpot)
• Institut Pasteur, France
• Kenya Medical Research Institute (KEMRI), Kenya
• National Institutes of Health (NIH), USA
• Radboud University Medical Center, The Netherlands (Only ICS)
• Seattle Biomedical Research Institute, USA (Only ELISpot)
• University of Oxford, UK
• Walter Reed Army Institute for Research (WRAIR), USA
Five african laboratories will be added at a later stage
13
T cell mediated immunity
Identify Key Assays
and Labs
•
•
•
•
1st Comparison with
Existing SOPs/
Reagents
Harmonisation of
SOPs/Reagents
Iterative Comparisons
Harmonised ICS and ELISpot SOP from the HIV
with Harmonised
community will be adapted
SOPs
Stimulation (activation of T cells in ELISpot and ICS):
– Tetanus Toxoid (Donation from Serum Institute India
Ltd.)
– CEF peptides (a pool of MHC class 1 binding peptides
derived from the CMV, EBV and Flu viruses (JPT
Peptide Technologies GmbH).
Positive control:
– Human PBMCs selected for reactivity to TT and CEF
peptides (Dr Gepi Pantaleo, CHUV in Lausanne)
14
Three separate rounds of testing planned starting from Q1
Blood stage Immunofluorescence
Assay (IFA)
Work package leader: Dr David Cavanagh, University of Edinburgh
• Assess capacity of purified murine and human antibodies to
recognise malaria parasites in infected red blood cells.
• As quantification of the IFA, anti-parasite IgG endpoint titers
are determined
Labelled Secondary AB
Y
Primary AB
Parasite
Infected RBC
15
Blood stage Immunofluorescence
Assay (IFA)
Identify Key Assays
and Labs
• Participating laboratories
– Biomedical Primate Research Centre, BPRC, Netherlands
– Radboud University Nijmegen, RUNMC, Netherlands
– University of Edinburgh, UK
16
Blood stage Immunofluorescence
Assay (IFA)
Identify Key Assays
and Labs
•
•
•
1st Comparison with
Existing SOPs/
Reagents
Harmonisation of
SOPs/Reagents
Identification of standard reagents:
– Positive controls
• 4G2 (rat anti-AMA1 mAb) – BPRC,
• polyclonal anti-AMA-1 rabbit IgG (lyophilized) - BPRC
• mAb 12.8 (mouse anti-MSP1-19) – UEDIN
• pooled anti-MSP-1-19 rabbit sera
– Negative control: naïve rabbit IgG - UEDIN
– Single, synchronised batch of mature P.falciparum schizont rich IFA slides
(Wellcome isolate)
Test reagents diluted, coded and shipped by National Institute for Biological standards
and Control (NIBSC)
17
First round of testing currently finalised and data analysed by NIBSC
Antibody Dependent Cell Inhibition
(ADCI) Assay
Work package leader: Dr Patrice Dubois, Immunovacc Consulting
• ADCI is based on the capacity of purified human or murine
antibodies to inhibit malaria parasite growth in cooperation
with effector cells (monocytes)
• Mechanism shown to correlate with protection after passive
transfer of antibodies1
• Low IgG concentrations required for activity
1
Bouharoun-Tayoun et al., JEM, 1990
18
ADCI (Antibody Dependent Cell
Inhibition) Assay - Protocol
• Serum IgG preparation using
ion exchange chromatography
• Monocyte isolation from a
healthy blood donor
• Preparation of P. falciparum
parasites including
synchronisation and schizont
enrichment
• Parasite culture, for 96h, in the
presence of antibodies and
monocytes
• Inhibition effect assessed by
microscopic observation and
parasite counting
Y
Y
Antibody Dependent Cell Inhibition
(ADCI) Assay
Identify Key Assays
and Labs
•
•
•
•
Biomedical Primate Research Centre, BPRC, Netherlands
Centers for Disease Prevention and Control, CDC, USA
Institut Pasteur, IP, France
International Centre for Genetic Engineering and Biotechnology,
ICGEB, India
• Radboud University Nijmegen, RUNMC, Netherlands
• University of Edinburgh, UK
20
Antibody Dependent Cell Inhibition
(ADCI) Assay
Identify Key Assays
and Labs
1st Comparison with
Existing SOPs/
Reagents
Harmonisation of
SOPs/Reagents
Identification of standard reagents:
• Positive standard:
– Pools of human sera collected in malaria endemic regions with
shown ADCI reactivity – ethical clearance from NIBSC ethical
review board
– Monoclonal RAM-1 Ab (Human IgG1 specific for P.falciparum
MSP-3) with shown ADCI activity – quality control under way
Existing protocols will be compared using standardised positive controls
and from this a consensus SOP determined.
21
Reference Reagent Repository
Identify Key Assays
and Labs
1st Comparison with
Existing SOPs/
Reagents
Harmonisation of
SOPs/Reagents
Agreed Community
Harmonised SOPs &
Reagent Repository
Iterative Comparisons
with Harmonised
SOPs
www.malariaresearch.eu
22
Outlook
• Harmonised SOP and control reagents available in reference
reagent repository by the end of the project
• Sharing of harmonisation activities in- and outside of the
malaria vaccine community
→ Contacts established:
Sylvia Janetzki, Cancer Vaccine Consortium (T cell harmonisation/Cancer)
Tom H.M. Ottenhoff, Leiden University Medical Center (T cell
harmonisation/TB)
Thomas N.Denny, Barton F.Haynes, Duke University Human
Vaccine Institute (T cell harmonisation/HIV)
23
Questions?
Project website: www.optimalvac.eu
Coordinator Dr Odile Leroy
Project Manager Dr Agnes Kisser
European Vaccine Initiative
UniversitätsKlinikum Heidelberg
Im Neuenheimer Feld 326 - 3. OG
69120 Heidelberg
Germany
www.euvaccine.eu
Global Coordinator Dr Vasee Moorthy
WHO Initiative for Vaccine Research
Geneva
24