Scholarly article on topic 'Alternative methods and strategies to reduce, refine, and replace animal use for human vaccine post-licensing safety testing: state of the science and future directions'

Alternative methods and strategies to reduce, refine, and replace animal use for human vaccine post-licensing safety testing: state of the science and future directions Academic research paper on "Biological sciences"

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Abstract of research paper on Biological sciences, author of scientific article — Richard Isbrucker, Robin Levis, Warren Casey, Richard McFarland, Michael Schmitt, et al.

Abstract NICEATM and ICCVAM convened an international workshop to review the state of the science of human and veterinary vaccine potency and safety testing methods, and to identify opportunities to advance new and improved methods that can further reduce, refine, and replace animal use (the 3Rs). Six topics were addressed in detail by speakers and workshop participants and are reported in a series of reports. This workshop report, the fifth in the series, addresses methods and strategies for human vaccine post-licensing safety testing that can reduce, refine, and replace animal use. It also provides recommendations for priority research and other activities necessary to advance the development and/or implementation of 3Rs methods for human vaccine post-licensing safety testing. Workshop participants agreed that future 3Rs activities should give highest priority to vaccine safety tests that (1) use the most animals per test and for which many vaccine lots are tested annually, (2) produce high variability and/or require frequent repeat tests, (3) are associated with severe animal pain and distress, and/or (4) involve nonhuman primates. Based on these criteria, safety tests for diphtheria, pertussis, oral polio, and tetanus vaccines were identified as the highest priorities. Recommended priority research, development, and validation activities included (1) expanding use of the transgenic mouse model for oral polio vaccine, and (2) developing alternatives to the monkey neurovirulence test for preclinical safety and lot release neurovirulence testing of mumps vaccines. Implementation of the workshop recommendations is expected to advance alternative in vitro methods for human vaccine post-licensing safety testing that will benefit animal welfare while ensuring continued production of safe human vaccines and protection of human and animal health.

Academic research paper on topic "Alternative methods and strategies to reduce, refine, and replace animal use for human vaccine post-licensing safety testing: state of the science and future directions"

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NICEATM-ICCVAM# International Workshop on Alternative Methods to Reduce, Refine, and Replace the Use of Animals in Vaccine Potency and Safety Testing: State of the Science and Future Directions Bethesda, Maryland, USA, 14-16 September 2010

Alternative methods and strategies to reduce, refine, and replace animal use for human vaccine post-licensing safety testing: state of the science and future directions

Richard Isbruckera, Robin Levisb, Warren Caseyc*, Richard McFarlandb, Michael Schmittb, Juan Arciniegab, Johan Descampsd, Theresa Finnb, Coenraad Hendriksene, Yoshinobu Horiuchif, James Kellerb, Hajime Kojimag, Dorothea Sesardich, Paul Stickingsh, Nelson W Johnsoni, David Alleni

"Health Canada, Center for Vaccine Evaluation, Ottawa, Ontario, Canada, bUnited States Food and Drug Administration- Center for Biologies

Evaluation and Research, Rockville, Maryland, USA,, eNational Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA, dGlaxoSmithKline Biologicals, Rixensart, Belgium, "Netherlands Vaccine Institute, ALL Bilthoven, Netherlands, J.Pharmaceuticals andMedical Devices Agency Japan, Tokyo, Japan, Japanese Center for the Validation of Alternative Methods, Tokyo, Japan, hNational Institute for Biological Standards and Control, Health Protection Agency, Hertfordshire, United Kingdom, iIntegrated Laboratory

Systems, Inc., Research Triangle Park, North Carolina, USA.

Abstract

NICEATM and ICCVAM convened an international workshop to review the state of the science of human and veterinary vaccine potency and safety testing methods, and to identify opportunities to advance new and improved methods that can further reduce, refine, and replace animal use (the 3Rs). Six topics were addressed in detail by speakers and workshop participants and are reported in a series of reports. This workshop report, the fifth in the series, addresses methods and strategies for human vaccine post-licensing safety testing that can reduce, refine, and replace animal use. It also provides recommendations for priority research and other activities necessary to advance the development and/or implementation of 3Rs methods for human vaccine post-licensing safety testing. Workshop participants agreed that future 3Rs activities should give highest priority to vaccine safety tests that (1) use the most animals per test and for which many vaccine lots are tested annually, (2) produce high variability and/or require frequent repeat tests, (3) are associated with severe animal pain and distress, and/or (4) involve nonhuman primates. Based on these criteria, safety tests for diphtheria, pertussis, oral polio, and tetanus vaccines were identified as the

# The National Toxicology Program Interagency Center for the Evaluation of Alternative Toxicological Methods and the Interagency Coordinating Committee on the Validation of Alternative Methods

* Corresponding author e-mail address: warren.casey@nih.gov

This article may be the work product of an employee or group of employees of the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH), however, the statements, opinions or conclusions contained therein do not necessarily represent the statements, opinions or conclusions of NIEHS, NIH, the United States government, or other organizations.

1877-282X © 2011 Published by Elsevier Ltd. Selection and/or peer-review under responsibility of the National Toxicology Program Interagency Center

for the Evaluation of Alternative Toxicological Methods (NICEATM).

doi:10.1016/j.provac.2011.10.004

highest priorities. Recommended priority research, development, and validation activities included (1) expanding use of the transgenic mouse model for oral polio vaccine, and (2) developing alternatives to the monkey neurovirulence test for preclinical safety and lot release neurovirulence testing of mumps vaccines. Implementation of the workshop recommendations is expected to advance alternative in vitro methods for human vaccine post-licensing safety testing that will benefit animal welfare while ensuring continued production of safe human vaccines and protection of human and animal health.

© 2011 Published by Elsevier Ltd. Selectlon and/or peer-review under responsibility bf the National Toxicology Program Interagency C enter for the Evaluation of Alternative Toxic ological Methods(NIC EATM).

Keywords: human vaccines; vaccine safety testing; vaccine potency testing; 3Rs alternatives; ICCVAM

1. Introduction

Vaccines contribute to improved animal and human health and welfare by preventing and controlling infectious diseases agents that can cause disease and death. However, the test methods necessary to ensure vaccine effectiveness and safety can involve subjecting large numbers of animals to significant pain and distress. In the United States, the Interagency Coordinating Committee on the Validation of Alternative Methods (ICCVAM) and the National Toxicology Urogram Interagency Center for the Evaluation of Alternative Toxicological Methods (NICEAeM) promote the scientific validation and regulatory acceptance of test methods that accurately assess the safety of chemicals and products while reducing, refining (less or no pain and distress), and replacing animal use (3Rs). Accordingly, NICEATM and ICCVAM recently identified vaccine potency and safety testing as one of their four highest priorities [1].

ICCVAM is an interagency committee of United States Federal agencies that is charged by law with evaluating new, revised, and alternative testing methods with regulatory applicability. ICCVAM members represent 15 U.S. Federal regulatory and research agencies that require, use, generate, or disseminate safety testing data. Member agencies include the Department of Agriculture (USD A), which regulates veterinary vaccines, and the Food and Drug Administration (FDA), which regulates human vaccines. ICCVAM is a permanent interagency committee of the National Institute of Environmental Health Sciences (NIEHS) under NICEATM. NICEATM administers ICCVAM, provides scientific and operational support for ICCVAM-related activities, and conducts validation studies on promising new safety testing methods. NICEATM and ICCVAM serve a critical public health role in translating research advances from the bench into standardized safety test methods that can be used in regulatory practice to prevent disease and injury.

To promote and advance the development and use of scientifically valid alternative methods for human and veterinary vaccine testing, NICEATM and ICCVAM organized the International Workshop on Alternative Methods to Reduce, Refine, and Replace the Use of Animals in Vaccine Potency and Safety Testing: State of the Science and Future Directions. The workshop was held at the National Institutes of Health in Bethesda, Maryland, on September 14-16, 2010. It was organized in conjunction with the European Centre for the Validation of Alternative Methods (ECVAM), the Japanese Center for the Validation of Alternative Methods (JaCVAM), and Health Canada.

The workshop addressed the state of the science and developed recommendations for future progress in three major areas: (1) in vitro replacement methods for potency testing; (2) reduction and refinement methods for potency testing; and (3) reduction, refinement, and replacement methods for vaccine safety testing [2]. Workshop reports were prepared for each of the three topics for human vaccines and for each of the three topics for veterinary vaccines. Each report, including this one, incorporates the contributions of invited experts and the general public during the various plenary presentations and dedicated breakout group sessions [3, 4, 5, 6, 8]. This report addresses methods and strategies for the reduction, refinement, and replacement of procedures requiring animal use for post-licensing safety testing of human vaccines.

2. Goals and organization of the workshop

The goals of the international workshop were to (1) identify and promote the implementation of currently available and accepted alternative methods that can reduce, refine, and replace the use of animals in human and

veterinary vaccine potency and safety testing; (2) review the state of the science of alternative methods and identify knowledge and data gaps that need to be addressed; and (3) identify and prioritize research, development, and validation efforts needed to address these gaps in order to advance alternative methods that will also ensure continued protection of human and animal health.

The workshop was organized with four plenary sessions and three breakout group sessions. In the breakout sessions, workshop participants:

• Identified criteria to prioritize vaccine safety and potency tests for future alternative test method development and identified high priorities using these criteria

• Reviewed the current state of the science of alternative methods and discussed ways to promote the implementation of available methods

• Identified knowledge and data gaps that need to be addressed

• Identified and prioritized research, development, and validation efforts needed to address these gaps in order to advance alternative methods while ensuring continued protection of human and animal health

The workshop opened with a plenary session in which expert scientists and regulatory authorities from the United States, Europe, Japan, and Canada outlined the importance of vaccines to human and animal health [9, 10] and described national and international regulatory testing requirements for human and veterinary vaccines [2, 11, 12, 13, 14, 15, 16]. Authorities emphasized that, following the approval of a vaccine, testing is required to ensure that each subsequent production lot is pure, safe, and potent enough to generate a protective immune response in people or animals [11, 12].

The second plenary session addressed methods that have been accepted and methods that are in development that do not require the use of animals for assessing the potency of vaccines [17, 18, 19, 20]. This was followed by breakout sessions to discuss the state of the science and recommendations for future progress for in vitro potency tests for human and veterinary vaccines. Workshop recommendations to advance the use and development of alternative methods that can replace animals for the potency testing of human [3] and veterinary vaccines [4] are available elsewhere in these proceedings.

The third plenary session addressed (1) potency test methods that refine procedures to avoid or lessen pain and distress by incorporating earlier humane endpoints or by using antibody quantification tests instead of challenge tests and (2) methods and approaches that reduce the number of animals required for each test [21, 22, 23, 24, 24, 25, 26, 27]. Breakout groups then discussed the state of the science and developed recommendations for future progress. Workshop recommendations to advance the use and development of alternative methods that can reduce and refine animal use for potency testing of human [5] and veterinary [6] vaccines are available elsewhere in these proceedings.

The final plenary session addressed methods and approaches for reducing, refining, and replacing animal use to assess the safety of production lots of veterinary and human vaccines [11, 28, 29, 30]. Breakout groups then discussed the state of the science and developed recommendations for advancing alternative methods for vaccine safety testing. Workshop recommendations to advance the use and development of alternative methods for safety testing of veterinary vaccines are available in these proceedings [7], while recommendations for safety testing of human vaccines are provided in this paper.

3. Requirements for human vaccine post-licensing safety testing

For both human and veterinary vaccines, strict regulatory guidelines ensure that vaccines released for sale are pure, safe, potent, and effective [31]. Vaccine safety is ensured by extensive in-process and end-product testing, consistent manufacturing processes, and post-marketing surveillance. The post-licensing safety tests performed to support release of vaccine lots for clinical use include general safety tests to evaluate systemic toxicity associated with the vaccine or any of its components, and more specific tests such as:

• Assessment for the presence of residual toxin and reversion of inactivated toxins to their active form

• Neurovirulence tests for live attenuated vaccines (e.g., oral polio vaccine [OPV])

• Pyrogen tests

• Tests for extraneous viral contamination of virus seed lots

Due to the large number of animals used annually for safety testing of human vaccines, many regulatory agencies actively encourage the development and implementation of novel approaches that reduce, refine, and replace the use

of animals in vaccine safety testing. Tables 1 and 2 provide examples of alternative methods that have been successfully implemented for some of the required tests.

Table 1: Examples of human vaccine safety tests that replace the use of animals

Available In Vitro Alternative Method Traditional Safety

Vaccine Product

Alternative Method

References

Traditional Safety Test Reference

All human vaccines'1

Bacterins and Toxoids

Diphtheria toxoid vaccine

(Corynebacterium diphtheriae)

Viral Vaccines

Oral polio vaccine (live virus)

GMP/consistency of manufacture (replacement)

Vero cell testa b c for residual toxin and reversion to toxicity (replacement)

Mutation analysis by PCR and restriction enzyme cleavage (MAPREC) test (type 3)f (replacement)

Schwanig et.al. 1997 [36]

Ph. Eur. Monograph 0443 [32]

General safety test in mice and guinea pigse

Guinea pig test

Ph. Eur. Monograph 215 [37]; WHO TRS 904, Annex 1, 2002 [38]

Nonhuman primate neurovirulence test

21 CFR 610.11 [35]; Japanese Minimum Requirements for Biological Products, 2006 [33]

Japanese Minimum Requirements for Biological Products, 2006 [33]; WHO TRS 800, Annex 2, 1990 [34]

Japanese Minimum Requirements for Biological Products, 2006 [33]; WHO TRS 800, Annex 1, revised 1989 [39]

"Published in the European Pharmacopoeia (Ph. Eur.).

bValidated in Japan.

cWHO Technical Report Series.

d Licensed in the United States unless an exemption has been granted.

e General safety tests in mice/guinea pigs not required in Europe for routine lot release testing. fValidated by the WHO for type 3 oral polio vaccine .

4. Prioritizing vaccine safety tests for future research, development, and validation activities

Although progress has been made in the introduction of alternative methods for post-licensing vaccine safety testing, many vaccines still require the use of animals to document that they are safe prior to release for sale. Many alternative safety tests have been developed or are currently in development but have not yet been validated for routine use. Workshop participants recommended that these should be given a high priority for further development and validation studies. The following vaccine safety tests were identified as priority for alternative test method activities:

• Safety tests that require a lethal endpoint

• Safety tests that require the largest number of animals and/or cause severe animal pain and distress

• Safety tests that are highly variable and/or require frequent repeats

• Safety tests that are associated with vaccines that have the largest number of lots produced annually

• Safety tests that involve nonhuman primates

Based on these criteria, the highest-priority vaccine safety testing methods recommended for further development and validation activities were:

• The Vero cell assay for diphtheria toxoid to replace the guinea pig test for specific toxicity/reversal to toxicity

• Histamine sensitization (HIST or HSA) for acellular pertussis (aP) vaccine: proposed humane endpoint (decrease in dermal temperature); in vitro replacement assays (enzyme-coupled HPLC, carbohydrate-binding assays, and cAMP assay) in development [29, 40, 41]

• Transgenic mouse test for OPV, particularly for seed lots where the monkey neurovirulence test (MNVT) is still used

• Development of a functional in vitro assay for tetanus toxoid to replace the guinea pig test

• Massively parallel sequencing (MPS) and other emerging technologies for OPV testing. (Participants noted that this would require substantial investment in the development of an adequate database with which to create a profile).

Table 2: Examples of human vaccine safety tests that reduce and/or refine animal use

Vaccine Product

Alternative Method to Reduce/Refine

Alternative Method References

Traditional Safety Test

Traditional Safety Test Reference

Bacterins and Toxoids

Whole-cell pertussis vaccine (Bordetella pertussis)

Acellular pertussis vaccine for whooping cough

(Bordetella pertussis)

Viral Vaccines

Oral polio vaccine (Poliovirus)

Reduction in animal numbera

Nonlethal endpoint HIST (refinement)11

Transgenic mouse modela (refinement)

Hendriksen 2008 [43]

Ph. Eur.

Monographs1356 [45] and 1595 [46]; Arciniega et al. 1998 [47]; Japanese Minimum Requirements for Biological Products,

2006 [33]; Ochiai et al.

2007 [48]

Ph. Eur. Monograph 218 [50]; WHO TRS 889, 1999 [51]; WHO TRS 904, 2002 [38]

Mouse weight gain test

Histamine sensitization test in mice

Nonhuman primate neurovirulence test

Ph. Eur. Monograph 0161 [44]; WHO TRS 941, 2007 [42]

Japanese Minimum Requirements for Biological Products, 2006 [33]; WHO TRS 878, 1998 [49]

Japanese Minimum Requirements for Biological Products, 2006 [33]; WHO TRS 800, 1989 [39]

aPublished in the European Pharmacopoeia (Ph. Eur.).

bIncluded in the European Pharmacopoeia as an option to lethal endpoint and subject to validation per product.

5. Human vaccine post-licensing safety testing: reduction, refinement, and replacement alternative methods

5.1. State of the science

5.1.1. General safety test

The general safety test (GST) is also known as the abnormal toxicity test (ATT) by the European Pharmacopoeia (Ph. Eur.) and the test for innocuity by the WHO. It is typically conducted using groups of approximately five mice

and two guinea pigs administered the equivalent of one or more human doses of vaccine. A vaccine passes the test if none of the treated animals shows clinical signs of illness or adverse effects. The Ph. Eur. and the U.S. Code of Federal Regulations recently changed the requirement for GST/ATT by allowing the test to be omitted. Specifically, the Ph. Eur. states that

the test can be waived by the competent authority in the interests of animal welfare when a sufficient number of consecutive production batches (generally 10) are found to comply with the test, thus demonstrating consistency of the manufacturing process, provided the vaccine is produced according to the principles of good manufacturing practice and quality assurance [36]. Similarly, the U.S. Code of Federal Regulations states that

a manufacturer may request an exemption from the general safety test by submitting information as part of a biologics license application submission or supplement to an approved biologics license application

establishing that because of the mode of administration, the method of preparation, or the special nature of the product a test of general safety is unnecessary to assure the safety, purity, and potency of the product or cannot be performed [35].

5.1.2. Specific toxicity tests

An important part of the safety evaluation of vaccines that contain an inactivated toxin or inactivated virus (e.g., rabies) is a test to confirm that the inactivation has been effective. Examples of vaccines comprising or containing inactivated toxins include diphtheria, tetanus, aP, and whole-cell pertussis (wP) vaccines. Inactivation has historically been confirmed using tests in mice or guinea pigs with potential lethal outcomes. However, during the past decade, alternative methods have been introduced that reduce, refine, and replace the use of animals in these more specialized safety tests (Tables 1 and 2).

Diphtheria is an upper respiratory tract illness caused by Ceryneb/cterium diphtheri/e, a facultative anaerobic Gram-positive bacterium. A highly sensitive in vitre Vero cell assay for determining the presence of residual diphtheria toxin and reversal to toxicity is currently approved for use in Europe and by the WHO to replace the guinea pig test. The in vitre assay is more sensitive than the animal model and is suitable for use during vaccine production to detect toxicity in pre-adsorbed toxoid bulks. A notable limitation of this assay is that it cannot be used for testing final vaccine lots of diphtheria vaccine adsorbed to aluminum adjuvant [32, 52, 53]. However, the assay is available and suitable for use as an in-process safety test. Workshop participants recommended further activities to bring about broader international acceptance and use of this assay where appropriate.

Pertussis, also known as whooping cough, is a highly contagious disease caused by the bacterium Berdetell/ pertussis. For wP vaccines, the mouse weight gain (MWG) test for residual toxicity is recommended by the WHO [42]. Recently it has been proposed to reduce the number of mice required from 10 per group to 5 per group [43, 44]. It should be noted that the Ph. Eur. now also includes a semiquantitative Chinese hamster ovary (CHO) cell assay that can be used to assess the presence of active pertussis toxin (PT) as an in-process control. However, due to technical limitations, it cannot be used for ensuring detoxification or on the final product containing an aluminum adjuvant.

A number of other alternatives have also been investigated as replacements for the MWG test, including the HIST/HSA, the CHO cell assay mentioned above, and the leukocyte promotion (LP) test. For example, Straaten-van de Kappelle et al., [54] concluded that HSA, CHO, or LP test might identify wP residual toxicity more effectively than the MWG test.

In the late 1970s, concerns about the side effects associated with wP vaccines prompted the development of aP vaccines. The aP vaccines, which contain up to five purified pertussis antigens, are less reactogenic than wP vaccines, but have been shown to confer adequate protection to vaccinees. One of these antigens, PT, is included in its chemically detoxified form in all U.S.-licensed vaccines that contain an aP component [Arciniega 2011]. The HIST, a lethal challenge assay, is the required test that measures the residual PT toxicity and the possible reversion of toxoid to toxin in aP vaccines [29]. However, dermal measurement of body temperature reduction has been proposed as a nonlethal, humane endpoint to allow early termination of the study, thereby reducing animal pain and distress [48].

5.1.2.1. Neurevirulence

A potential hazard associated with live attenuated strains is the possibility that they could regain virulence during vaccine manufacturing. Thus, vaccines derived from neurotropic viruses (e.g., poliovirus, mumps virus, Japanese encephalitis virus, yellow fever virus) are evaluated to ensure that they are free of residual neurotoxicity. Differences are seen among regional regulatory testing requirements for these vaccines with respect to performing the test on master seed lots, working seed lots, or final manufacturing lots; and whether histological sections must be reviewed. Historically, the test for neurovirulence has been performed using monkeys in the MNVT. However, progress has been made in the development of alternative methods for OPV with the introduction of an in vive transgenic mouse neurovirulence model.

Although animals are still used, the transgenic mouse model replaces the use of nonhuman primates for OPV neurovirulence testing. The mouse strain used expresses the human poliovirus receptor [55, 56], and protocols have been developed for use with poliovirus types 1, 2, and 3 [38, 51]. However, there are practical limitations due to the cost, availability, and increased training requirements associated with this model [57].

An entirely in vitrc approach to neurovirulence testing of OPV is the mutant analysis by PCR and restriction enzyme cleavage (MAPREC) test, which uses molecular biology tools to identify single-base mutations associated with reversion to neurovirulence of poliovirus type 3 [58]. The MAPREC test has undergone extensive validation and is currently being used in conjunction with in vivc tests for poliovirus type 3 [38]. More recent efforts have been directed at its applicability to poliovirus type 1 and type 2. It has been approved as a screening test prior to in vivc testing, such that any vaccine that fails this test would not be further tested in animals. Although the MAPREC test quantifies individual mutations (one per serotype) at a few genomic loci, it does not detect other mutations that may contribute to neurovirulence. To overcome this shortcoming, the FDA Center for Biologics Evaluation and Research is investigating the usefulness of new technologies such as MPS for identifying and quantifying the mutational profile in OPV lots [30, 59] in order to monitor their genetic stability.

5.1.2.2. Bncterinl endotoxins

Part of the required safety testing for vaccines is an evaluation for the presence of pyrogenic (fever-inducing) substances in each lot that is released. The traditional pyrogenicity test described by the Ph. Eur. [60] and the United States Pharmacopeia (USP) requires the use of three rabbits to detect increases in body temperature during 3 hours after injection of the vaccine. The most common pyrogenic substances are lipopolysaccharide (LPS) cell wall components of Gram-negative bacteria. In vitrc alternatives to the rabbit test are accepted for Gram-negative endotoxins including the Limulus amoebocyte lysate test (LAL test, also known as the bacterial endotoxin test) [61]. The Ph. Eur. and the FDA accept the LAL test for vaccines to hepatitis A, Haemophilus influenzae type B, influenza, rabies, typhoid, and yellow fever [62]; however, the LAL test cannot detect non-LPS pyrogens such as any derived from Gram-positive bacteria. One additional limitation for vaccine pyrogen testing with the LAL test is the fact that aluminum hydroxide and certain proteins can interfere with the LAL test [63, 64]. Therefore, vaccines that contain such components often cannot be tested in the LAL test.

Other in vitrc pyrogenicity assays have been developed that employ human monocytes or monocytoid cell lines and measure the release of fever-inducing cytokines such as IL-1P, IL-6, and TNFa [65, 66]. These alternatives can be accepted in certain circumstances by regulatory authorities in the United States and Europe on a case-by-case basis, subject to product-specific validation [66, 67]. For some vaccines, the limitations of the currently available alternatives necessitate that the animal test for pyrogens still be performed for some vaccines (e.g., polysaccharide vaccines, and tick-borne encephalitis vaccines [62]).

5.2. Knowledge gnpr nndpriority rerenrch, development, nnd vnlidnticn activities

Advancing the prioritized vaccine safety tests from concept to regulatory acceptance and implementation will require substantial investment in basic research, time, and money. Participants identified a number of knowledge and data gaps that currently exist and the key research that is necessary to address these gaps:

• Basic understanding of the in vivo and in vitro mode of action(s) of specific toxins

• Relevance of the in vitro test to product safety (correlation with the current in vivo safety test)

• Identification of interactions between toxin and other components in the final vaccine

Participants noted that there is a need for assays (for diphtheria and PT, specifically) that provide a measurement of function such as toxin cell binding, cell entry, or enzymatic activity that can then be related to vaccine safety. However, varying national and international regulatory requirements can complicate the adoption of any new alternative assays. Therefore, a roadmap for development of alternatives was considered important to guide manufacturers through the process for approval. An important part would be guidance on how to meet the requirements for a safety testing waiver in each region.

In discussing ways to address these gaps, participants considered the advantages provided by measurements that demonstrate consistency in manufacturing. They suggested ways to better apply these principles to vaccine safety testing to facilitate the reduction of animal use. The key issue of consistency emerged from the category of vaccines that are manufactured using new technology, as in the case of recombinant DNA (rDNA) vaccines. These vaccine products are manufactured in a consistent way, and the focus of quality control is on in-process monitoring rather than on final batch testing [68]. Increased in-process monitoring of existing vaccines can be accomplished using newer, more sensitive technologies, but it could also create new challenges in understanding the relevance of small changes that are identified.

Participants identified a number of product-specific research activities that should be considered high priorities to reduce, refine, and replace the use of animals in safety testing for these vaccines, including the following:

• Refinement of the aP lethal-endpoint HIST/HSA by the use of a dermal temperature endpoint

• Selection and validation of combined in vitre assays as replacement alternatives to HIST/HSA (e.g., enzyme-linked HPLC, carbohydrate-binding assays, cAMP assay)

• Use of the Vero cell assay to monitor diphtheria toxin inactivation

• Development of a fully functional in vitre assay for tetanus toxin

• Research to expand the use of the transgenic mouse model for OPV

• Expanded validation efforts for the sequence-based approach to OPV neurovirulence safety

• Development of alternatives to the MNVT for preclinical safety and lot release neurovirulence testing of mumps (and possibly measles) vaccines

6. Achieving broader acceptance and use of 3Rs methods for human vaccine post-licensing safety testing

Workshop participants discussed how to facilitate the broader acceptance of alternative vaccine safety tests. In addition to the more general efforts aimed at improving interaction, communication, and harmonization of international regulatory requirements, they saw a need for the targeted research efforts:

• Improve collaborative work to facilitate validation of the Vero cell assay for diphtheria toxin inactivation/reversion

• Organize international collaborative studies once a test is considered validated, in an effort to improve worldwide acceptance

• Ensure that collaborations are maintained and fostered throughout the process

• Discuss the barriers preventing OPV manufacturers from accepting the FDA- and WHO-approved test that uses transgenic mice as an alternative to the use of monkeys in the neurovirulence testing of OPV

• Increase international coordination in developing new safety tests to avoid the need to perform multiple tests to fulfill the requirements for different countries

• Encourage broader access to information that describes vaccine safety test methods that have been successfully implemented to reduce, refine, and replace the use of animals. For example, publication of testing methods, open online access to the USP and Ph. Eur. monographs, understanding that the USP does not have vaccine specific monographs

• Task focused work groups, such as the ICCVAM Interagency Biologics Working Group, with specific challenges as they arise

7. Other issues to be addressed to facilitate the 3Rs in human vaccine post-licensing safety testing

The workshop highlighted the following additional actions needed to either advance specific alternative vaccine safety testing methods or impact the general application of the 3Rs to post-licensing vaccine safety testing:

• Harmonize and mutually recognize alternative test methods using the 3Rs principles

• Clearly delineate the validation requirements for any new test method for acceptability to regulatory bodies around the world

• Acknowledge, within the scientific community, that a 3Rs method may not agree with the current method when that test method is of questionable relevance (e.g., MNVT for OPV). Results of the "new" and the "old" method may not need to agree completely for an acceptable validation; however, there must be assurances that safety will be equivalent or better than that provided by the existing method

• Delineate the role of international organizations (e.g., WHO, ICH) in facilitating the 3Rs and developing a framework to facilitate advances at the global level (recognizing that the ICH does not have vaccine specific guidance)

• Accept that some validation studies using large numbers of animals may be required in some cases to provide the necessary information for new method acceptance

• Address whether the GST/ATT/innocuity test for human vaccines can be replaced worldwide with principles of manufacturing consistency

• Address whether any replication by National Control Laboratories of in vivc safety testing performed by manufacturers can be eliminated (note: FDA and Health Canada regulations do not require replication of testing performed by manufacturers). Participants noted that proven consistency of production would be required before implementation on a case-by-case basis

• Establish interaction and information exchange between human and veterinary stakeholders to provide a means of sharing best practices and facilitate adoption of 3Rs methods

Participants also discussed the need for incentives for manufacturers and/or the academic research community that could speed up the development, validation, and implementation of 3Rs methods in post-licensing vaccine safety testing. Suggestions for regulatory authorities to consider included (1) offering fast-track regulatory review and (2) providing a fee waiver for biologics license application or a license supplement when an alternative method is included in the application.

Participants also emphasized the need for funding opportunities to further research and development and validation efforts, referring specifically to the NICEATM-ICCVAM nominations and submissions process as an example.

8. Discussion

This was the first international workshop in the United States to focus on the reduction, refinement, and replacement of animal use for potency and safety release testing of human and veterinary vaccines. A key accomplishment of the workshop was bringing together experts from industry, academia, and government in the areas of potency and safety testing for both human and animal vaccines. There was broad recognition among vaccine manufacturers, regulatory authorities, and participants that these workshops vastly improve information exchange not only between global regions but also between regulatory authorities in the same country. This interaction can accelerate the development of alternative methods once priorities are firmly established.

Workshop participants addressed the state of the science of reduction, refinement, and replacement alternative methods for human vaccine post-licensing safety testing. They recommended priority research and other activities necessary to advance the development and/or implementation of these alternative methods. Participants highlighted the need for basic scientific research, product-specific research and validation efforts, and regulatory harmonization to advance the development, validation, and implementation of post-licensing vaccine safety test methods that reduce, refine (less or no pain and distress), and replace the use of animals. Similar to those identified at other session breakout groups, criteria for prioritization included assays with lethal endpoints, tests that use the largest number of animals and have the highest yearly lot production, tests that are highly variable, and those that cause the greatest pain and distress. Reducing the use of nonhuman primates in vaccine safety testing was highlighted as particularly important, and participants called for focused research on finding and implementing alternatives, not only for the MNVT for OPV, but to replace neurovirulence tests for mumps and measles.

Advances have been recognized in the application of alternative vaccine safety tests, but participants identified specific vaccine safety tests that could attain wider acceptance and use by manufacturers with additional focused research, including the Vero cell assay for diphtheria toxin, the HST/HSA assay with body temperature endpoint for aP, and the transgenic mouse test for OPV.

Specific research was considered necessary to address scientific knowledge gaps in our basic understanding of the mode of action of toxins and their interaction with other vaccine components. Participants recommended research to expand understanding of how the in vivc response in animals correlates with the in vitrc assay and specific research to develop a fully functional in vitrc assay for tetanus toxin. Recurring themes included the need for the following:

• More open access to methods and information such as pharmacopoeial monographs

• Increased sharing and dissemination of information among regulators, manufacturers, and the scientific community

• International harmonization of requirements and specifications for test methods

• Recommendation that the regulatory authorities consider various means to encourage the use of 3Rs approaches, including waiving user fees, direct funding of research, facilitation of collaborations among industry and the academic community, and organization of international collaborative studies

These general themes highlight the international consensus that exists for applying the 3Rs to vaccine safety testing. By building on and reaffirming these important conclusions and recommendations, the outcome of this international workshop will advance alternative methods for vaccine safety testing that will reduce, refine, and replace the use of animals while ensuring the quality and safety of human vaccines and the protection of human health, animal health, and the environment.

9. Conclusions

This workshop session reviewed the current status of human vaccine post-licensing safety testing. Participants identified research, development, and validation activities necessary to further advance testing methods and strategies that would eliminate or reduce the use of animals or result in more humane treatment of animals. Workshop participants strongly agreed that increasing our understanding of the mode of action of toxins and focusing on efforts to replace the use of nonhuman primates in post-licensing vaccine safety testing will have the greatest impact toward advancing the use of alternative methods in this area. It was recommended that experts at the WHO, FDA, and Ph. Eur. could play a role in guiding wider acceptance and use of the transgenic mouse neurovirulence test for OPV.

Workshop participants stressed the need for increased international harmonization, acknowledging that it is the key to advancing the use of 3Rs alternatives in vaccine potency and safety testing. Workshop participants also agreed that the continued interaction of the human and veterinary global vaccine communities could facilitate and expedite the unified goal of replacing animals for vaccine post-licensing safety testing.

Acknowledgements

The authors extend their sincere appreciation to all participants of the international workshop for their enthusiastic contributions leading to the workshop recommendations and conclusions. The members of the ICCVAM Interagency Biologics Working Group and NICEATM staff are acknowledged for their contributions to the planning of the workshop, and the many invited experts are acknowledged for their contributions to breakout group discussions and workshop proceedings. Finally, the authors thank Brett Jones and Vivian Doelling for their assistance in the preparation of this manuscript.

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