Transcript Harmonisations of assays – experiences and lessons

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.
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Immunoassays in Malaria Vaccine
Development
Assess immunogenic
potential of candidate
vaccines
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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
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Key immunoassays identified for harmonisation in
OPTIMALVAC
Cell mediated
immunity
• ICS
• ELISPOT
Humoral Assays
• Blood Stage IFA
Functional Assays
• Antibodydependent cellular
inhibition assay
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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
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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
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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)
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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
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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
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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)
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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
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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
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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.
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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
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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)
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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
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