1. 1 The Reduction of Animal Use in the Critical Path to Vaccines VRBPAC Meeting February 17, 2005 Sadhana Dhruvakumar Sr. Scientific Research Specialist People for the Ethical Treatment of Animals (PETA) SadhanaD@peta.org (757) 622-PETA ext. 8110

2. 2 Animals in the Critical Path “We must modernize the critical development path that leads from scientific discovery to the patient” - Critical Path report, 3/04 Assessing Safety Animal safety testing is “laborious, time-consuming, requires large quantities of product, and may fail to predict the specific safety problem that ultimately halts development.” “The traditional tools used to assess product safety -- animal toxicology and outcomes from human studies -- have changed little over many decades and have largely not benefited from recent gains in scientific knowledge.” Demonstrating Medical Utility (efficacy) “Currently available animal models… have limited predictive value in many disease states” Many animal methods of vaccine potency testing have low reproducibility, have an ill-defined relevance to humans, and utilize non-physiological routes of infection (e.g., intracerebral inoculation) and so the results are often not an adequate measure of human protection Industrialization (manufacturing) With respect to drugs, FDA is emphasizing advanced engineering principles and control technologies, GMP, and in-process characterization procedures and standards over the traditional requirement for multiple conformance batches to ensure manufacturing standards. This mentality should be carried over into vaccine manufacturing: an emphasis on production consistency can lead to reduction in routine batch testing on animals (more below).

3. 3 Used in: Vaccine development (research, validation of efficacy) Production (sometimes in animals, primary cell cultures, eggs) Batch control testing (safety and potency testing) Routine batch control testing is responsible for 80% of animal use in vaccine industry and regulation Batch control testing of vaccines accounts for ~10% of all animal use in biomedical research, using 10 million animals every year Biologicals testing has the highest proportion and number of experiments causing severe pain and distress to animals out of various types of experiments (basic research, toxicity testing, etc.) Animal Use in Human and Veterinary Vaccines

4. 4 Replace: Antigen quantification (e.g., with ELISA or newer technologies for detection of binding interactions such as fluorescence polarization or surface plasmon resonance) Deletion of certain tests that are no longer needed due to increased production consistency Refine: Use of humane non-lethal endpoints Vaccination + estimation of immune response instead of virulent challenge (usually antibody estimation using ELISA or ToBI) Reduce: Vaccination + estimation of immune response Move testing upstream in production process (e.g., final bulk rather than final lot) Single dilution rather than multi-dilution vaccination-challenge or vaccination-serology 3Rs impact on vaccine batch control testing

5. 5 US Department of Agriculture (USDA) Center for Veterinary Biologics (CVB) Replace: USDA has developed an in vitro potency test (antigen quantification) for leptospira vaccine USDA is developing in vitro potency test (antigen quantification) for clostridial vaccine – this involves identifying protective immunogens and establishing references “We can’t expect industry to go out and develop the alternatives. It’s a static market so the demand is there for alternatives but not the financial incentive. CVB must be the leader.” Held their first alternatives meeting in April 2004: “Technology and Approaches to Reduce, Refine & Replace Animal Testing” with very active participation from over 150 attendees from industry and the CVB Has presented to U.S. Interagency Coordinating Committee on Validation of Alternative Methods (ICCVAM) advisory committee on their development of an antigen quantification test (sandwich ELISA) for leptospira vaccine Embarking on comprehensive effort to change legislation and regulations to encourage use of non-animal methods including: “Umbrella” policy holding animal tests to the same standards as new in vitro tests Changing language to include novel non-animal technologies and encourage movement away from animal testing

6. 6 Since 1993, Biologicals is one of 11 “key areas” set up with a permanent Biologicals Working Group and a Pyrogenicity Task Force; ECVAM also works with Advisory Group on Alternatives to Animal Testing in Immunobiologicals (AGAATI) on these issues Workshops and other communication efforts: Has organized 9 workshops on alternatives in biologicals production and quality control (Including “Three Rs Approaches in the Quality Control of Inactivated Rabies Vaccines”) Has organized 5 “VACTRAINING” sessions to provide hands-on lab training in alternatives Financially contributes to other relevant conferences, expert reports, manuals, training Has published many workshop reports and scientific papers and reports Replace: Validated commercially available ELISA kits for rabies potency testing (1999) Validated human-blood-based pyrogenicity test (just completed) Prevalidation of Vero cell test for specific toxicity testing of diphtheria toxoid Prevalidation of ELISA, ToBI and rocket immunoelectrophoresis assay for batch potency testing of tetanus antisera and immunoglobin – validation of ToBI recommended (2000) Refine: Sponsored development of humane endpoints for rabies, pertussis, and erysipelas challenge tests (1999) Validated ELISA and ToBI test for batch potency testing of human tetanus vaccine (2000) Validated ELISA test for swine erysipelas vaccine Reduce: Validated ELISA and ToBI test for batch potency testing of human tetanus vaccine (2000) Validated ELISA test for swine erysipelas vaccine European Center for the Validation of Alternative Methods (ECVAM)

7. 7 European Directorate for Quality Medicine(EDQM)/ European Pharmacopeia (Ph. Eur.) Replace: Accepts antigen quantification test for rabies (1998) Deleted Abnormal Toxicity Test (in favor of production consistency approach) Deleted guinea pig test for diphtheria (residual toxin and irreversibility of diphtheria toxoid) Deleted in vivo test for polio (for some manufacturers*) Deleted residual pertussis toxin test for acellular pertussis (for some manufacturers*) Refine: Accepts vaccination-serology tests for tetanus, diphtheria, and cholera vaccines (in lieu of vaccination-challenge) Recommends use of humane endpoints in vaccination-challenge procedures Reduce: Accepts single dilution assays for diphtheria, tetanus, and acellular pertussis vaccines (in lieu of multiple dilution assays) Accepts vaccination-serology tests for tetanus, diphtheria, and cholera vaccines (in lieu of vaccination-challenge) *A licensing authority can waive tests in monographs if it is assured of production consistency Has organized various international conferences: “Replacement, reduction and refinement of the use of animals in the quality control of vaccines” in November 2002 “Serological Potency Tests for Diphteria and other Vaccines” in October 2004

8. 8 World Health Organization (WHO) Replace: Accepts antigen quantification for batch release of Hep B vaccines (1999) Studying deletion of Abnormal Toxicity Test Refine: Replaced live poliomyelitis vaccine neurovirulence monkey test with a transgenic mouse test and MAPREC (mutant analysis by polymerase chain reaction and restriction enzyme cleavage) (2000) Accepts ToBI test for tetanus toxoid and Vero cell test for diphtheria toxoid (1995) Reduce: Accepts single dilution assays for tetanus and diphtheria toxoids Accepts TOBI test for tetanus toxoid and Vero cell test for diphtheria toxoid (1995)

9. 9 Status of alternatives in common vaccines: bacterial vaccines TypeExamplesAnimal testAlternatives (accepted by) ToxoidsTetanus Safety: absence of toxin, irreversibility of toxoid, specific toxicity Potency: multidilution vaccination challenge on guinea pigs or mice Deletion of specific toxicity test (EU) Combined absence and irreversibility of toxin tests (EU) In vitro endopeptidase test for toxin detection has been developed but not validated Single dilution test (EU) Antibody estimation by ELISA or ToBI (EU, WHO) Diphtheria Safety: absence of toxin (5 guinea pigs for bulk lot), irreversibility of toxoid, specific toxicity Potency: multidilution vaccination challenge on guinea pigs with ~ 20 control animals Deletion of specific toxicity test (EU) Combined absence and irreversibility of toxin tests (EU) In vitro Vero cell test for toxin detection (WHO) Single dilution test (EU, WHO) Antibody estimation by Vero cell test (WHO) – ELISA and ToBI have also been developed Acellular pertussis vaccines (ACPVs) Safety: absence of toxin (5 mice), irreversibility of toxoid (5 mice) Potency: multidilution vaccination + serology on 6 groups of mice In vitro CHO clustering test can be done on bulk but not final lot Single dilution test (EU) BacterinsWhole cell pertussis vaccines Safety: mouse weight gain test with 10 mice for testing specific toxicity Potency: Kendrick test - multidilution vaccination and intracerebral challenge in 136 mice – large numbers of animals, severe distress, poor precision and reliability Modified to use 5 guinea pigs (EU) In vitro alternatives include LAL pyrogen test (WHO) Humane non-lethal endpoints (EU) Aerosol challenge instead of intracerebral Antibody estimation by whole cell ELISA (validated in 2000) Cholera Potency: multidilution vaccination + serology on 6 mice, guinea pigs, or rabbits This serology test is accepted by the EU Haemophil us type B conjugate Potency: multidilution vaccination + serology on 16 mice This serology test is accepted by the EU Moving testing upstream: if final bulk testing is satisfactory, can omit potency testing of final lot

10. 10 Status of alternatives in common vaccines: viral vaccines ExamplesAnimal testAlternatives (accepted by) Rabies Safety: extraneous agent testing Potency: NIH test (multi-dilution vaccination + intra-cerebral challenge test in up to 170 mice per batch) Cell culture test (WHO) + EU for vet vaccines Humane endpoints (EU) Single dilution (EU) Vaccination + antibody estimation using 5 mice (EU) Antigen quantification (WHO) Hep A (inactivated) and Hep B (recombinant) Vaccination + serological test in mice or guinea pigs Antigen quantification (EU, WHO) Inactivated Poliovirus (IPV) Multi-dilution vaccination + serology in at least 60 rats Molecular analyses, e.g., MAPREC for poliovirus type 3 (WHO) Neurovirulence in transgenic mice for poliovirus type 3 (WHO) Oral Poliomyelitis (OPV) Neurovirulence testing in over 80 monkeys by intra-spinal injection Molecular analyses, e.g., MAPREC for poliovirus type 3 (WHO) Neurovirulence in transgenic mice for poliovirus type 3 (WHO) Already not tested in animals: Influenza (tested in eggs), Meningococcal and Pneumococcal (only need pyrogen test which can be done in vitro), Oral typhoid, Varicella, Measles, Mumps, Rubella Currently no alternatives available: BCG (2 safety tests on 6 guinea pigs each) Not covered here: Yellow Fever, Smallpox, Japanese Encephalitis, Anthrax

11. 11 Critical Path opportunities for promoting progress and change Devote more FDA research to better defining pathogens, vaccines, human- based tissue engineered models of infection, and adjuvants– with a goal of developing of modern non-animal technologies such as antigen quantification and enabling rational vaccine design Validate and accept newer non-animal technologies (fast-tracking technologies already validated by other regulatory bodies) Promote switching licensed products over to modern non-animal technologies for ongoing batch testing (e.g., fee waivers, incentives, research assistance) Better familiarize FDA reviewers/researchers with new technologies Publish guidances and organize workshops on animal testing alternatives In 50-100 years, we will be far beyond using animals as surrogates for humans in vaccine testing – how do we get there from here?