IABS Non Animal testing Rabies focus Dr Cat Stirling

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1 IABS Non Animal testing Rabies focus Dr Cat Stirling 1

2 IMI2: OVERVIEW AND OBJECTIVES IMI: 2008, IMI2: 2014 as Public-Private Partnership (PPP) between European Union and European Federation of Pharmaceutical Industries and Associations (EFPIA) World s largest PPP in health research: total budget : 3.28 billion 50% in cash from EC, 50% in kind from EFPIA and other organisations Brings together companies, universities, public laboratories, small and medium-sized enterprises (SMEs), patient groups and regulators in collaborative projects Aims to speed up development of next generation of drugs, vaccines and treatments 2

3 OVERVIEW 21 participants: 15 public partners, 6 EFPIA companies Total budget: 7.85M EU funding in cash 8.13M from EFPIA partners in kind Seven work packages WP 1: Physicochemical methods WP 2: Immunochemical methods WP 3: Cell-based assays WP 4: Multi-parametric assays and bioinformatics WP 5: (Pre)validation WP 6: Promotion of consistency testing to regulatory acceptance WP 7: Consortium management 3

4 CONSORTIUM PARTNERS 4

5 OBJECTIVES AND AMBITION Proof of concept of consistency approach for batch release testing of established vaccines using sets of in vitro and analytical methods Develop, optimise & evaluate non-animal methods that cover key-parameters for demonstrating batch consistency, safety and efficacy (Pre-)validate methods and define with regulators guidance for regulatory approval and routine use 5

6 CONSISTENCY TESTING: INTRO * Quality, safety and efficacy of a vaccine batch should be ensured * Consistency testing might be a strategy to move away from animal use Consistency testing aims to combine manufacturing controls and non-animal assays - instead of animal tests - to ensure that each vaccine batch produced is consistent with a (clinical/historical) batch already proven to be safe and efficacious in registration studies or clinical use 6

7 CONSISTENCY APPROACH IN BATCH RELEASE TESTING OF ESTABLISHED* VACCINES VACCINE BATCH RELEASE TESTING Current approach Consistency approach Uniqueness of each batch Emphasis of Q.C. on final product Use of international reference preparation (in some cases) Each batch is one of a series Emphasis on every step of production process (seed lot, in-process, final product) Read out is: non deviation from consistency Benchmarking to clinical/ historical batch * An established vaccine is a vaccine which is being produced by classical procedures: inactivation, detoxification or attenuation 7

8 POTENTIAL ADVANTAGES Increase in depth knowledge on the product Improved product quality and consistency Simplification and easier standardisation of methods Global streamlining of batch release methods Reduction animal use Time and cost saving (ensuring product availability) 8

9 MODEL VACCINES Selected human and veterinary model vaccines Type Vaccine Final batch testing Human Inactivated viral Tick-borne encephalitis virus (TBEV) Challenge (mice) Toxoid, purified protein Diphtheria (D), tetanus (T), acellular pertussis(ap); (DTaP) Challenge, serology (mice, guinea pigs) Veterinary Inactivated viral Infectious Bronchitis Virus (IBV) Serology (chickens) Inactivated viral Newcastle disease virus (NDV) Challenge, serology (chickens, target species) Inactivated viral Porcine circovirus (PCV) Serology (pigs) Inactivated viral Feline leukaemia virus (FeLV) Serology (mice) Inactivated viral Veterinary rabies Challenge, serology (cats, dogs, mice) Inactivated bacterial Bovine leptospira Challenge, serology (cattle, guinea pigs) Inactivated bacterial Canine leptospira Challenge, serology (dogs, hamsters) Inactivated bacterial Clostridium chauvoei Challenge (guinea pigs) Toxoid Clostridium tetani Serology (target species, guinea pigs, rabbits) Toxoid Clostridium perfringens C Challenge (mice) 9

10 WORK PACKAGE STRUCTURE WP1 Physicochemical methods New biochemical and physicochemical methods to assess the: Proteome of whole cell and toxoid vaccines Conformational integrity of toxoid vaccine (with/without adjuvants, ) WP2 Immunochemical methods New or better methods for Ag quantity/quality throughout production Assays suitable for different manufacturers? In vitro methods detect Ag changes relevant to biological function? 10

11 WORK PACKAGE STRUCTURE WP3 Cell-based assays New or better cell-based assays for in vitro monitoring of the vaccine: Protective immune response and/or Safety WP4 Multi-parametric assays and bioinformatics Transcriptomics/proteomics to identify bacterial biomarkers suitable for characterization of Clostridium tetani seed strains Alternative for the histamine sensitisation test for pertussis vaccine safety based on kinome analysis Platform technology and identification of vaccine quality biomarkers with in vitro antigen presenting cells (vaccines/adjuvants) 11

12 WORK PACKAGE STRUCTURE WP5 (Pre)validation Criteria for method development and validation (Pre)validation of the selected methods developed in WP1-4 Guidance document for the design of multi-centre validation studies Biological Standardisation Programme (BSP) of EDQM WP6 Regulatory acceptance Roadmap for regulatory acceptance of consistency approach as guidance Facilitate discussion between stakeholders on questions and issues linked to consistency approach implementation 12

13 EXPECTED RESULTS AND IMPACT R Replacement, Reduction or Refinement of animal use (3R) Development, optimization and evaluation of analytical methods for vaccine QC Proof of concept of consistency approach for veterinary and human vaccines Secure European leadership in vaccine quality control (QC) testing Develop roadmap for regulatory acceptance for Europe and beyond Global dissemination of knowledge 13

14 PROGRESS YEAR 2 Physicochemical methods: - Mass spectrometry a(lc-ms) ssays for Leptospira and DTaP vaccines were successfully set up confirming suitability of MS for further analysis. - Circular dichroism and fluorescence spectroscopy standout as promising candidate tests to assess structural conformation and stability of the TTs. - LC-MS of the toxoid bulks showed that a purity profile can be generated. - To facilitate these studies, tailor-made desorption protocols were developed for human DTaP vaccines. - Enzymatic assays simulating antigen degradation by immune cells has started. 14

15 PROGRESS YEAR 2 Immunochemical methods - Characterisation and selection of monoclonal antibodies. - Veterinary tetanus, the results suggest that an ELISA method can be used for antigen detection in a variety of products. - DTaP vaccines showed potential of at least one antibody to be suitable for use in development and validation of immunoassays. - TBEV, a suitable immunoassay format (depleting ELISA) has been identified for use with adjuvanted vaccines and optimization of the assay conditions is progressing well. - Clostridium chauvoei vaccine, an ELISA format has been developed and appears to be sufficiently sensitive for testing vaccine products. Outreach to USDA on regaents 15

16 PROGRESS YEAR 2 Cell based assays for consistency testing - The conditions for the monocyte activation test (MAT) were adapted to analyze TBEV vaccine. A final protocol was designed to analyze different vaccine lots to validate these optimized MAT conditions. - An inflammasome activation assay is being developed for evaluating in vitro adjuvant activity of DTaP vaccines. Apart from that, cell based assays to study IBV and leptospira using antigen-presenting cells (APC) are under development. - For Innate immune fingerprinting of FeLV, activating signal of one vaccine component was quantified and now multiple batches of this component are tested. - Protocols for detection of antigen-specific B cells and quantification of immunoglobulin production in PBMC in response to bulk antigen (DTaP) were developed. 16

17 PROGRESS YEAR 2 Cell based assays for consistency testing - To assess toxoid processing and subsequent peptide presentation by APC, an in vitro co-culture assay of toxoid-primed human APC and tetanus- or diphtheria toxoid-specific CD4+ T cell hybridoma s is being set up. - In vitro safety test: a detection limit for tetanus toxin as low as 0.03 nm was reached and modifications to assay conditions will be tested to improve sensitivity of the assay and bring it close to what is detectable with in vivo assays. - For veterinary C. perfringens C it was found that both A10 and THP-1 cells are susceptible to the C. perfringens C non-inactivated antigen (containing β-toxin as main antigen) in a concentration-dependent manner and with a sensitivity in the required range. 17

18 PROGRESS YEAR 2 Work on Pre-validation of selected methods - With the workshop on the design of multi-centre validation studies organised by WP5 in 2017, the consortium started an open discussion with all stakeholders - vaccine manufacturers of major human and animal health companies, competent authorities, OMCLs, EDQM, etc. - signalling a common commitment by all parties to the 3Rs principles. The summary of the discussion and recommendations was published in Biologicals ( 18

19 PROGRESS YEAR

20 PROGRESS YEAR 2 Work on Regulatory acceptance of the consistency approach - The focus on outreaching to international regulatory agencies. - Working on a document on in vivo testing of altered batches to avoid that the project partners perform unnecessary in vivo tests when introducing altered batches. - Further, work is co-ordinated with actions done at EDQM, particularly groups 15, 15V and OCABR (Official Control Authority Batch Release at EDQM). 20

21 MODEL VACCINES Selected human and veterinary model vaccines Type Vaccine Final batch testing Human Inactivated viral Tick-borne encephalitis virus (TBEV) Challenge (mice) Toxoid, purified protein Diphtheria (D), tetanus (T), acellular pertussis(ap); (DTaP) Challenge, serology (mice, guinea pigs) Veterinary Inactivated viral Infectious Bronchitis Virus (IBV) Serology (chickens) Inactivated viral Newcastle disease virus (NDV) Challenge, serology (chickens, target species) Inactivated viral Porcine circovirus (PCV) Serology (pigs) Inactivated viral Feline leukaemia virus (FeLV) Serology (mice) Inactivated viral Veterinary rabies Challenge, serology (cats, dogs, mice) Inactivated bacterial Bovine leptospira Challenge, serology (cattle, guinea pigs) Inactivated bacterial Canine leptospira Challenge, serology (dogs, hamsters) Inactivated bacterial Clostridium chauvoei Challenge (guinea pigs) Toxoid Clostridium tetani Serology (target species, guinea pigs, rabbits) Toxoid Clostridium perfringens C Challenge (mice) 21

22 RABIES IN VAC2VAC Rabies is part of WP2 and WP6 Accepted by all that Glycoprotien ELISAs are the best in vitro test approach WP2 BI/Merial shared their mabs for Zoetis and MSD to test WP6 Work with regulators to understand what data packages are required to support variations to switch from challenge/serology to GP ELISA D C V M N W O R K S H O P, H Y D E R A B A D 22

23 PROGRESS TO DATE BI/Merial - Have validated ELISA - GP ELISA only is now approved for legacy Merial vaccine in EU Zoetis tested the BI mabs - they do work on one Zoetis strain - Zoetis vaccines are currently tested by commercial GP ELISA and serology for release (over 5 years of concurrent testing data) - Zoetis is working on data packages to remove the serology test MSD tested by antibodies did not work well they have their own in house ELISA - MSD vaccines are currently tested by serology - MSD is working towards the switch to ELISA D C V M N W O R K S H O P, H Y D E R A B A D 23

24 VALIDATION APPROACH FOR GP ELISA Zoetis example Validation of ELISA as per VICH GL1/2 Commercial kit (INGENESA) - antibodies confirmed as neutralising by manufacturer - Specific characterisation may be required Product specific validation - Based in consistency approach - Dose titration using GP/serology - Ph.Eur. DOI challenge data with known GP content for one vaccine is available D C V M N W O R K S H O P, H Y D E R A B A D 24

25 SN titre (log10 IU/ml) GLYCOPROTEIN TEST DISCRIMINATING POWER Scatterplot of SN titre (log10 IU/ml) vs Input Glyco 2.5 Exp sub formulated lots Commercial lots ISPAH lots Pfizer lots with ISPAH ag Pfizer lots with Pfizer ag IU/ml Input Glyco (IU/ml) To guarantee no subformulated batches will be released, a minimum input of 6 IU/ml in order having at least 4 IU/ml at release is proposed

26 SEROLOGICAL RESPONSES OF DOG INOCULATED WITH SUB FORMULATED BATCHES Dogs have been injected once with batches listed below (2 dogs / batch) and serological response measured Aim is to verify if sub formulated batches are still potent (serological response > 0.5 IU/ml) Input Glyco (IU/ml) Input antigen CCID50/ml Mouse potency test (log10 IU/ml) Dog response Bleeding day 14 (IU/ml) Specification NLT (100%) VMRD (3%) VMRD (36%) 0.94 Dog # 2223: 7.07 Dog # 2322: Dog # 9575: 8.17 Dog # 9626: 7.07

27 Clinical data with known GP content DOI for dogs - 3 years from a single dose Ph.Eur. Challenge test GP content of batch used was 4.38 IU/ml Potency of this batch was 1.68 IU/ml Current release potency level is > 2IU/ml Current vaccines are released with a min 10IU/ml theoretical content based on testing adjuvented bulk Rabies Ag Batch with GP content less than half that of commercial vaccines is full potency in dogs giving protection for up to 3 years Based on testing using proposed test method

28 NEXT STEPS All companies agree that a single common GP ELISA for Rabies for EU is not currently viable Work through WP6 and internal processes to complete variations to move to GP ELISA only Work with OMCLs to transfer release ELISAs 28

29 PH.EUR. CHANGE Manufacturers have GP ELISAs that work on adjuvented vaccines Request change to Vet Rabies monograph (0451) Specifically include GP ELISA for release Maintain other tests Similar to Human Rabies text (0216) The following is listed for bulk vaccine and under final product tests Glycoprotein content. Determine the glycoprotein content by a suitable immunochemical method (2.7.1), for example, single-radial immunodiffusion, enzyme-linked immunosorbent assay or an antibody-binding test. The content is within the limits approved for the particular product D C V M N W O R K S H O P, H Y D E R A B A D 29

30 Acknowledgement The VAC2VAC project has received funding from the Innovative Medicines Initiative 2 Joint Undertaking under grant agreement Nº This Joint Undertaking receives support from the European Union s Horizon 2020 research and innovation programme and EFPIA. Disclaimer This communication reflects the author's view, neither IMI nor the European Union, EFPIA, or any Associated Partners are responsible for any use that may be made of the information contained therein Useful links

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