OPTIMALVAC Initiative on Optimising Malaria Vaccine Laboratory Assays Evaluation

1. OPTIMALVAC Initiative on Optimising Malaria Vaccine Laboratory Assays Evaluation

2. 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

3. Objectives The goal is to identify and harmonise key immunoassays to facilitate comparison of results and improve decision-making in malaria vaccine development.

4. Immunoassays in Malaria Vaccine Development Assess immunogenic potential of candidate vaccines

5. 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

6. 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

7. 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

8. Malaria Vaccine Assay Harmonisation Harmonisation of SOPs/Reagents 1st Comparison with Existing SOPs/ Reagents Identify Key Assays and Labs 1st Comparison with Existing SOPs/ Reagents Agreed Community Harmonised SOPs & Reagent Repository Iterative Comparisons with Harmonised SOPs Agreed Community Harmonised SOPs & Reagent Repository Assay Validation Establishment of Reference Centre Assay Validation

9. Key immunoassays identified for harmonisation in OPTIMALVAC Cell mediated immunity • ICS • ELISPOT Humoral Assays • Blood Stage IFA Functional Assays • Antibody-dependent cellular inhibition assay

10. T cell mediated immunity Work package leader: Dr Patrice Dubois, Immunovacc Consulting Enyzme linked Immunospot (ELISpot) Intracellular Cytokine staining (ICS) Labelled CD4 AB Y T cell Y Labelled anti-cytokine AB • 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 .

11. ICS – Intracellular cytokine staining • 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 fluorescent-labelled AB against cytokine • Analysis by Fluorescence activated Cell Sorting (FACS) Labelled CD4 AB Y T cell Y Labelled anti-cytokine AB

12. ELISpot - Enzyme Linked Immuno-Spot • 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

13. T cell mediated immunity •  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 Identify Key Assays and Labs

14. T cell mediated immunity • Harmonised ICS and ELISpot SOP from the HIV community will be adapted • 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 GepiPantaleo, CHUV in Lausanne) • Three separate rounds of testing planned starting from Q1 2011 Harmonisation of SOPs/Reagents Harmonisation of SOPs/Reagents 1st Comparison with Existing SOPs/ Reagents 1st Comparison with Existing SOPs/ Reagents Identify Key Assays and Labs Iterative Comparisons with Harmonised SOPs

15. 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 Y Labelled Secondary AB Y Primary AB Parasite Infected RBC

16. Blood stage Immunofluorescence Assay (IFA) • Participating laboratories • Biomedical Primate Research Centre, BPRC, Netherlands • Radboud University Nijmegen, RUNMC, Netherlands • University of Edinburgh, UK Identify Key Assays and Labs

17. Blood stage Immunofluorescence Assay (IFA) • 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) • First round of testing currently finalised and data analysed by NIBSC Harmonisation of SOPs/Reagents 1st Comparison with Existing SOPs/ Reagents Identify Key Assays and Labs

18. 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

19. ADCI (Antibody Dependent Cell Inhibition) Assay - Protocol Y • 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

20. Antibody Dependent Cell Inhibition (ADCI) Assay • 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 Identify Key Assays and Labs

21. Antibody Dependent Cell Inhibition (ADCI) Assay 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. Harmonisation of SOPs/Reagents 1st Comparison with Existing SOPs/ Reagents Identify Key Assays and Labs

22. Reference Reagent Repository Harmonisation of SOPs/Reagents 1st Comparison with Existing SOPs/ Reagents Identify Key Assays and Labs Agreed Community Harmonised SOPs & Reagent Repository Iterative Comparisons with Harmonised SOPs www.malariaresearch.eu

23. 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)

24. 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