UNISEC Universal Influenza Vaccines Secured
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1 UNISEC Universal Influenza Vaccines Secured Partners in the UNISEC Consortium UNISEC has been made possible by contributions from the European Commission DG-Research and the European member states Project Coordinator: Erik Frijlink Project Manager: Ed Schmidt Rijks Universiteit Groningen (RUG) The Netherlands University Medical Center Groningen (UMCG) The Netherlands PEPTCELL (SEEK) United Kingdom BiondVax Pharmaceuricals Israel Retroscreen United Kingdom Medicines and Healthcare products Regulatory Agency (MHRA) (NIBSC) United Kingdom Statens Serum Institut (SSI) Denmark National Center for Epidemiology (NCE) Hungary Norwegian Institute of Public Health (NIPH) Norway Robert Koch Institute (RKI) Germany Goeteborgs Universiteit (UGOT) Sweden Isconova AB Sweden
2 The UNISEC pipeline of of comparative product development Network of Clinical Study Sites
3 Multiparametric Analysis of T cell Responses to Vaccination EDUFLUVAC Workshop, Brussels 12th 13th June 2017 Steve Norley, RKI
4 UNISEC Workpackage 5 Original Tasks Task 1: Dissemination of standardised protocols Task 2: Co ordination of sample analyses Task 3: Multicentre collaborative studies to standardise selected assays
5 T Cell Based Vaccines Problems and Solutions Correlates of CMI induced immunity unknown Measure as many responses as possible Limited number of cell aliquots available Simultaneous measurement Interlaboratory standardisation of cell based assays very difficult Carry out core assay in one central laboratory 'Core Assay' Selected: IntracellularCytokineStaining (ICS)
6 NIPH (Oslo): COP related immuno assays: B cell asays: Hemagglutination inhibition assay Micro neutralization assay Ig ELISA IgG subclass analysis (multiplex) T cell assays: Elispot assay (IFN g/granzb) Multiparameter flowcytometry
7 NIPH (Oslo): Elispot assays: INF g Best documented Correlate of Protection (COP) assay for symptomatic influenza disease Dual IFN g/granz B enzymatic Elispot assay (CTL) Validated assay and PC assisted data analysis CTL reader: ISO certified for clinical trials (GCLP)
8 T cell receptor Peptide MHC molecule
9
10 Core Assay: Multi Parametric Intracellular Cytokine Staining Designed to maximise the information derived from limited cell numbers Developed, optimised and validated for large scale AIDS vaccines trials Adapted and evaluated for the UNISEC candidate vaccines
11 Clinical Trial #1 Biondvax M 001 peptide based vaccine. Trial site: Budapest (NCE) 222 volunteers recruited (four drop outs) Blood samples taken pre and 42d post vaccination PBMC isolated and frozen using the UNISEC protocol (RKI). Shipped to RKI. 436 PBMC samples (blinded). Six stimulants (peptide pools) plus pos and neg controls >3488 FACS samples Ten markers: Viability, CD3, CD4, CD8, CD107a, IFNg, IL2, TNFa, IL4, IL17 Twelve 'results' per sample >
12 Preparation of Stimulants
13 Thaw and wash PBMC. Rest overnight Count PBMC 1 PBMC 2 PBMC 3 PBMC 4 Neg Con Peptide Pool 1 Peptide Pool 2 PBMC 5 PBMC 6 PBMC 7 Peptide Pool 3 Peptide Pool 4 Peptide Pool 5 Peptide Pool 6 PBMC 8 PBMC 11 PBMC 10 PBMC 9 PBMC 12 PTIC anti CD28 anti CD49d anti CD107a BV605 Stimulants SEB Incubate 24hrs Anti CD3 Alexa Fluor 700 Anti CD4 FITC Anti CD8a PerCP efluor 710 Anti IL 2 PE Anti IL 4 APC Anti IL 17A efluor 506 Anti IFN gamma PE Cyanine7 Anti TNF alpha efluor 450 Wash 3x, viability stain, wash 2x, fix, permeabilize, wash 2x, FC block Wash 3x
14 Unstimulated Controls
15 Stimulated with SEB
16 Stimulated with Peptides
17 Dual Plots (Stimulated)
18 Assay Validation PBMC frozen in aliquots from two different donors Used in three independent assays on different days in triplicate Preset permissible mean intra assay variation: 20% Preset permissible mean inter assay variation: 30%
19 Assay Validation BC#8. SEB: 1:1000. Run BC#8. SEB: 1:1000. Run IFNg+ IL2+ TNFa+ CD107a+ IL4+ IL17+ IFNg+ IL2+ TNFa+ CD107a+ IL4+ IL17+ IFNg+ IL2+ TNFa+ CD107a+ IL4+ IL17+ IFNg+ IL2+ TNFa+ CD107a+ IL4+ IL17+ %Specific Responders %Specific Responders 0 CD4 CD8 CD4 CD BC#8. SEB: 1:1000. Run IFNg+ IL2+ TNFa+ CD107a+ IL4+ IL17+ IFNg+ IL2+ TNFa+ CD107a+ IL4+ IL17+ %Specific Responders CD4 CD
20 Assay Validation Mean Variation Intra assay 6.2% Inter assay 5.9% BC#8. SEB: 1: BC#9. SEB: 1: Run 1 Run 2 Run Run 1 Run 2 Run IFNg+ IL2+ TNFa+ CD107a+ IL4+ IL17+ IFNg+ IL2+ TNFa+ CD107a+ IL4+ IL17+ IFNg+ IL2+ TNFa+ %Specific Responders 5 CD107a+ IL4+ IL17+ IFNg+ IL2+ TNFa+ CD107a+ IL4+ IL17+ %Specific Responders CD4 CD8 CD4 CD BC#8. SEB: 1: BC#9. SEB: 1: IFNg+ IL2+ TNFa+ CD107a+ IL4+ IL17+ IFNg+ IL2+ TNFa+ CD107a+ IL4+ IL17+ IFNg+ IL2+ TNFa+ %Specific Responders CD4 CD8 0 CD107a+ IL4+ IL17+ IFNg+ IL2+ TNFa+ CD107a+ IL4+ IL17+ %Specific Responders CD4 CD
21 Preparation of SOPs SOP 01. Thawing and recovery of PBMC SOP 02. Stimulation of PBMC SOP2a. Stimulants and concentrations SOP 03. FACS Staining SOP 04. Compensation controls SOP 05. FACS Analysis SOP 06. Analysis of FACS data in FlowJo SOP 08. Counting cells using the Coulter Counter SOP 09. Stimulation of PBMC for IFNg ELISA SOP 11. Receipt of PBMC samples SOP 12. Working in the biosafety flow hood SOP 13. Communications Plan SOP 15. Data Management SOP 17. Cold Storage SOP 18. Criteria for Retesting SOP 19. Data Archiving
22 Preparation of SOPs
23 Audit
24
25 Stimulated with Peptides
26 Database Input Vial Barcode Sample Name FACS Tube No. Date of CD4/TNFa CD4/CD107a CD8/TNFa CD8/CD107a Visit Number Rand. Code Stimulant Total Counts Lymphocytes Viable CD3 Pos CD4 Pos CD4/IFNg Pos CD4/IL2 Pos CD4/IL4 Pos CD4/IL17 Pos CD8 Pos CD8/IFNg Pos CD8/IL2 Pos CD8/IL4 Pos CD8/IL17 Pos Sampling Pos Pos Pos Pos C110V2 C110V2a Con C110V2 C110V2b Pep C110V2 C110V2c Pep C110V2 C110V2d Pep C110V2 C110V2e Pep C110V2 C110V2f Pep C110V2 C110V2g Pep C110V2 C110V2h SEB C110V4 C110V4a Con C110V4 C110V4b Pep C110V4 C110V4c Pep C110V4 C110V4d Pep C110V4 C110V4e Pep C110V4 C110V4f Pep C110V4 C110V4g Pep C110V4 C110V4h SEB C173V2 C173V2a Con C173V2 C173V2b Pep C173V2 C173V2c Pep C173V2 C173V2d Pep C173V2 C173V2e Pep C173V2 C173V2f Pep C173V2 C173V2g Pep C173V2 C173V2h SEB C173V4 C173V4a Con C173V4 C173V4b Pep C173V4 C173V4c Pep C173V4 C173V4d Pep C173V4 C173V4e Pep C173V4 C173V4f Pep C173V4 C173V4g Pep C173V4 C173V4h SEB C182V4 C182V4a Con C182V4 C182V4b Pep C182V4 C182V4c Pep C182V4 C182V4d Pep C182V4 C182V4e Pep C182V4 C182V4f Pep C182V4 C182V4g Pep C182V4 C182V4h SEB
27 Statistical Analysis of Data Generated Measurement of multiple responses to multiple stimulants raises the real risk of 'p hacking'. The 'MIMOSA' package was specifically developed to avoid this problem MIMOSA uses a Bayesian approach to the statistical analysis of data from the ICS assay to provide a measurement of confidence that the response is 'real'
28 MIMOSA Input NSUB CYTNUM ANTIGEN CYTOKINE TCELLSUBSET UID NSUB CYTNUM ANTIGEN CYTOKINE TCELLSUBSET UID NOSTIM CD107a CD4 C1010V STIM Pool4 CD107a CD4 C1010V NOSTIM IFNg CD4 C1010V STIM Pool4 IFNg CD4 C1010V NOSTIM IL17 CD4 C1010V STIM Pool4 IL17 CD4 C1010V NOSTIM IL2 CD4 C1010V STIM Pool4 IL2 CD4 C1010V NOSTIM IL4 CD4 C1010V STIM Pool4 IL4 CD4 C1010V NOSTIM TNFa CD4 C1010V STIM Pool4 TNFa CD4 C1010V STIM Pool1 CD107a CD4 C1010V STIM Pool5 CD107a CD4 C1010V STIM Pool1 IFNg CD4 C1010V STIM Pool5 IFNg CD4 C1010V STIM Pool1 IL17 CD4 C1010V STIM Pool5 IL17 CD4 C1010V STIM Pool1 IL2 CD4 C1010V STIM Pool5 IL2 CD4 C1010V STIM Pool1 IL4 CD4 C1010V STIM Pool5 IL4 CD4 C1010V STIM Pool1 TNFa CD4 C1010V STIM Pool5 TNFa CD4 C1010V STIM Pool2 CD107a CD4 C1010V STIM Pool6 CD107a CD4 C1010V STIM Pool2 IFNg CD4 C1010V STIM Pool6 IFNg CD4 C1010V STIM Pool2 IL17 CD4 C1010V STIM Pool6 IL17 CD4 C1010V STIM Pool2 IL2 CD4 C1010V STIM Pool6 IL2 CD4 C1010V STIM Pool2 IL4 CD4 C1010V STIM Pool6 IL4 CD4 C1010V STIM Pool2 TNFa CD4 C1010V STIM Pool6 TNFa CD4 C1010V STIM Pool3 CD107a CD4 C1010V STIM SEB CD107a CD4 C1010V STIM Pool3 IFNg CD4 C1010V STIM SEB IFNg CD4 C1010V STIM Pool3 IL17 CD4 C1010V STIM SEB IL17 CD4 C1010V STIM Pool3 IL2 CD4 C1010V STIM SEB IL2 CD4 C1010V STIM Pool3 IL4 CD4 C1010V STIM SEB IL4 CD4 C1010V STIM Pool3 TNFa CD4 C1010V STIM SEB TNFa CD4 C1010V
29 Analysis of the 'Quality' of the T cell Response
30 COMPASS: Visualisation of Multifunctionality BC#01 BC#02 BC#03 BC#04 BC#05 BC#06 BC#07 BC#08 BC#09 BC#10 BC#11 BC#12 BC#13 BC#14 BC#15 BC#16 BC#17 BC#18 BC#19 BC#20 BC#21 BC#22 BC#23 BC#24 Expt 009. CD8/CEF Probability Plot IL17 IFNg IL2 TNFa CD107a
31 Thanks to everyone in UNISEC that helped and advised.
32 Special thanks to Nicky, my technician (and wife), for still being my technician (and wife)
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