Animal testing for vaccines. Implementing replacement, reduction and refinement: challenges and priorities.

Transition to in vitro alternative methods from in vivo in vaccine release testing and characterization, the implementation of the consistency approach, and a drive towards international harmonization of regulatory requirements are most pressing needs in the field of vaccines. It is critical for global vaccine community to work together to secure effective progress towards animal welfare and to ensure that vaccines of ever higher quality can reach the populations in need in the shortest possible timeframe. Advancements in the field, case studies, and experiences from Low and Middle Income Countries (LMIC) were the topics discussed by an international gathering of experts during a recent conference titled "Animal Testing for Vaccines - Implementing Replacement, Reduction and Refinement: Challenges and Priorities". This conference was organized by the International Alliance for Biological Standardization (IABS), and held in Bangkok, Thailand on December 3 and 4 2019. Participants comprised stakeholders from many parts of the world, including vaccine developers, manufacturers and regulators from Asia, Europe, North America, Australia and New Zealand. In interactive workshops and vibrant panel discussions, the attendees worked together to identify the remaining barriers to validation, acceptance and implementation of alternative methods, and how harmonization could be promoted, especially for LMICs.

Antigenic fingerprinting of diphtheria toxoid adsorbed to aluminium phosphate.

The antigenicity of alum-adsorbed diphtheria toxoid (DTd) was determined in combination vaccines, containing DTd, tetanus toxoid and inactivated poliovirus. A panel of monoclonal antibodies was used, covering five epitopes, distributed over the antigen. The resulting antigenic fingerprint of DTd demonstrates consistency of adsorption at antigen level in final product combination vaccines. The antigenic quality of DTd alone, adsorbed to aluminium phosphate, was also determined and compared with pre-adsorbed toxoid (starting material as well as toxoid desorbed from aluminium phosphate). Some epitopes became less accessible after adsorption, while others became relatively better exposed. Some epitopes disappeared almost completely upon adsorption, but were re-established after desorption of the antigen. The results indicate that DTd is adsorbed to aluminium phosphate in a preferred orientation and not randomly.

The Isolated Chicken Eye test to replace the Draize test in rabbits.

In 1944, Draize et al., published a paper entitled "Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes". The Organization for Economic Co-operation and Development published their first guideline on eye irritation in 1981, using rabbits. In the early eighties the development of alternative non-animal tests to replace the Draize eye test started. The first attempts to validate alternative tests for eye irritation were considered to be relatively simple by comparing in vitro and in vivo irritation index scores. In the early nineteen-eighties, we introduced the use of isolated eyes as an alternative test for the Draize eye irritation test. What was expected to be a process of several years, however, turned out to be a decades spanning process still not fully completed. For a large part, this can be attributed to the nature of the in vivo test in rabbits, which is more complicated and compromised than originally believed. This paper describes, most chronologically, the development, performance, validation and application of the Isolated Eye Test and, in broader perspective, the international validation and acceptance of this alternative test by regulatory authorities and agencies.

Regulatory acceptance and use of the Extended One Generation Reproductive Toxicity Study within Europe.

The two-generation study (OECD TG 416) is the standard requirement within REACH to test reproductive toxicity effects of chemicals with production volumes >100 tonnes. This test is criticized in terms of scientific relevance and animal welfare. The Extended One Generation Reproductive Toxicity Study (EOGRTS), incorporated into the OECD test guidelines in 2011 (OECD TG 443) has the potential to replace TG 416, while using only one generation of rats and being more informative. However, its regulatory acceptance proved challenging. This article reconstructs the process of regulatory acceptance and use of the EOGRTS and describes drivers and barriers influencing the process. The findings derive from literature research and expert interviews. A distinction is made between three sub-stages; The stage of Formal Incorporation of the EOGRTS into OECD test guidelines was stimulated by retrospective analyses on the value of the second generation (F2), strong EOGRTS advocates, animal welfare concern and changing US and EU chemicals legislation; the stage of Actual Regulatory Acceptance within REACH was challenged by legal factors and ongoing scientific disputes, while the stage of Use by Industry is influenced by uncertainty of registrants about regulatory acceptance, high costs, the risk of false positives and the manageability of the EOGRTS.

Replacing the NIH test for rabies vaccine potency testing: a synopsis of drivers and barriers.

Approximately 70% of animal use is utilized to demonstrate quality control of vaccines. Especially rabies vaccine potency testing, using the NIH challenge test, involves objections in terms of scientific relevance, animal welfare concern and costs. Several 3R models have been proposed to refine, reduce or replace this test. Some are formally incorporated into regulatory requirements, but actual regulatory acceptance and use by industry lags behind, raising the question concerning which factors influence this process. This question is answered by a combination of literature review, interviews and a survey among 50 rabies vaccine experts. The findings are analyzed using the multilevel perspective on technology transition, which distinguishes 3 levels of factors influencing innovation acceptance. At the micro level (where 3R models are developed and validated) the dis-advantages of, and fractional experience with, 3R models, scarce data sharing and demanding validation processes exist. The meso level (existing regulatory regime) encloses the barriers of the 'gold standard', the lack of harmonization and the driving force of legislation stimulating 3Rs use. The macro level (the societal context) combines risk aversion and increased concern for animal welfare. Regulatory acceptance and use of 3R models requires dedicated stakeholder communication, cooperation and coordination at all three levels.

Regulatory acceptance and use of 3R models for pharmaceuticals and chemicals: expert opinions on the state of affairs and the way forward.

Pharmaceuticals and chemicals are subjected to regulatory safety testing accounting for approximately 25% of laboratory animal use in Europe. This testing meets various objections and has led to the development of a range of 3R models to Replace, Reduce or Refine the animal models. However, these models must overcome many barriers before being accepted for regulatory risk management purposes. This paper describes the barriers and drivers and options to optimize this acceptance process as identified by two expert panels, one on pharmaceuticals and one on chemicals. To untangle the complex acceptance process, the multilevel perspective on technology transitions is applied. This perspective defines influences at the micro-, meso- and macro level which need alignment to induce regulatory acceptance of a 3R model. This paper displays that there are many similar mechanisms within both sectors that prevent 3R models from becoming accepted for regulatory risk assessment and management. Shared barriers include the uncertainty about the value of the new 3R models (micro level), the lack of harmonization of regulatory requirements and acceptance criteria (meso level) and the high levels of risk aversion (macro level). In optimizing the process commitment, communication, cooperation and coordination are identified as critical drivers.

Physicochemical and immunochemical assays for monitoring consistent production of tetanus toxoid.

The detoxification of tetanus toxin by formaldehyde is a crucial step in the production of tetanus toxoid. The inactivation results in chemically modified proteins and it determines largely the ultimate efficacy and safety of the vaccine. Currently, the quality of tetanus toxoid lots is evaluated in potency and safety tests performed in animals. As a possible alternative, this article describes a panel of in vitro methods, which provides detailed information about the quality of tetanus toxoid. Ten experimental lots of tetanus toxoid were prepared using increasing concentrations of formaldehyde and glycine to obtain tetanus toxoids having differences in antigenicity, immunogenicity, residual toxicity and protein structure. The structural properties of each individual toxoid were determined using immunochemical and physicochemical methods, including biosensor analysis, ELISA, circular dichroism, TNBS assay, differential scanning calorimetry, fluorescence and SDS-PAGE. The quality of a tetanus toxoid lot can be assessed by these set of analytical techniques. Based on antigenicity, immunogenicity and residual toxicity data, criteria are formulated that tetanus toxoids lot have to meet in order to have a high quality. The in vitro methods are a valuable selection of techniques for monitoring consistency of production of tetanus toxoid, especially for the detoxification process of tetanus toxin.

Workshop on Animal free Detection of Pertussis Toxin in Vaccines--Alternatives to the Histamine Sensitisation Test.

The Paul-Ehrlich-Institut (PEI), the Nederlands Vaccin Instituut (NVI) and the European Directorate for the Quality of Medicines & HealthCare (EDQM) organised the international scientific workshop "Animal free Detection of Pertussis Toxin in Vaccines--Alternatives to the Histamine Sensitisation Test" at the PEI in Langen (Germany) on 09-10 June 2011. Twenty-seven experts (regulators, representatives from national control laboratories, vaccine manufacturers and academia) from 7 countries participated in this workshop. The meeting was triggered by the lack of satisfaction with the current safety testing for acellular pertussis vaccines, the "Histamine Sensitisation Test" (HIST) in mice, and the growing attention for the alternatives under development. The workshop objectives were: a) to review the current status of available alternative methods, b) to discuss the sensitivity that an alternative test needs, c) to plan experiments that allow for comparison of the alternative tests. The results of the workshop are summarised in this meeting report.

The consistency approach for quality control of vaccines - a strategy to improve quality control and implement 3Rs.

Current batch release testing of established vaccines emphasizes quality control of the final product and is often characterized by extensive use of animals. This report summarises the discussions of a joint ECVAM/EPAA workshop on the applicability of the consistency approach for routine release of human and veterinary vaccines and its potential to reduce animal use. The consistency approach is based upon thorough characterization of the vaccine during development and the principle that the quality of subsequent batches is the consequence of the strict application of a quality system and of a consistent production of batches. The concept of consistency of production is state-of-the-art for new-generation vaccines, where batch release is mainly based on non-animal methods. There is now the opportunity to introduce the approach into established vaccine production, where it has the potential to replace in vivo tests with non-animal tests designed to demonstrate batch quality while maintaining the highest quality standards. The report indicates how this approach may be further developed for application to established human and veterinary vaccines and emphasizes the continuing need for co-ordination and harmonization. It also gives recommendations for work to be undertaken in order to encourage acceptance and implementation of the consistency approach.

Three Rs Approaches in the Production and Quality Control of Fish Vaccines.

The workshop on Three Rs Approaches in the Production and Quality Control of Fish Vaccines aimed a) to identify animal tests currently stipulated for the production and quality control of fish vaccines and to highlight animal welfare concerns associated with these tests; b) to identify viable options to replace, reduce, and refine animal use for fish vaccine testing; and c) to discuss the way forward and set out how the Three Rs may be implemented without jeopardizing the quality of the vaccines. The workshop participants - experts from academia, regulatory authorities, a scientific animal welfare organization, and the fish vaccine industry - agreed that efforts should be undertaken to replace the vaccination-challenge batch potency testing with tests based on antigen quantification or antibody response tests. Regulatory requirements of questionable scientific value and relevance for the quality of fish vaccines, such as the re-testing of batches produced outside Europe, or the double-dose batch safety test, should be re-considered. As an immediate measure the design of the current animal tests should be evaluated and modified in the light of refinement and reduction, for example, the number of unprotected control fish in vaccination-challenge tests should be reduced to the minimum.

Overcoming scientific barriers in the transition from in vivo to non-animal batch testing of human and veterinary vaccines.

INTRODUCTION: Before release, vaccine batches are assessed for quality to evaluate whether they meet the product specifications. Vaccine batch tests, in particular of inactivated and toxoid vaccines, still largely rely on in vivo methods. Improved vaccine production processes, ethical concerns, and suboptimal performance of some in vivo tests have led to the development of in vitro alternatives. AREAS COVERED: This review describes the scientific constraints that need to be overcome for replacement of in vivo batch tests, as well as potential solutions. Topics include the critical quality attributes of vaccines that require testing, the use of cell-based assays to mimic aspects of in vivo vaccine-induced immune responses, how difficulties with testing adjuvanted vaccines in vitro can be overcome, the use of altered batches to validate new in vitro test methods, and how cooperation between different stakeholders is key to moving the transition forward. EXPERT OPINION: For safety testing, many in vitro alternatives are already available or at an advanced level of development. For potency testing, in vitro alternatives largely comprise immunochemical methods that assess several, but not all critical vaccine properties. One-to-one replacement by in vitro alternatives is not always possible and a combination of methods may be required.

Variability of in vivo potency tests of Diphtheria, Tetanus and acellular Pertussis (DTaP) vaccines.

For batch release of legacy vaccines such as DTaP, in vivo potency release assays are required. We quantified the variability of in vivo potency release assays for four DTaP (Diphtheria, Tetanus, acellular Pertussis) products of different manufacturers. With their large CV (Coefficients of Variance) ranging from 16% to 132%, these in vivo assays are of limited value to ensure their potency is consistent and similar to the clinical batches used for the marketing authorisation. Our data show that, although individual potency test results show high variability, the DTaP batches are manufactured with great consistency, because repeated potency testing yields similar averages for the different batches. The economic impact of variability of in vivo tests is significant since it may result in the need for greater amount of antigen than may be required or for repeating a test. For monitoring the consistency of potency, in vitro assays are superior to in vivo assays. Animal-free potency determination is common practice for newly developed vaccines under modern GMP quality systems. However, replacement of in vivo potency tests for legacy vaccines like DTaP is challenging and would require a 'reverse characterisation' strategy in which the antigens are further characterised at the level of drug substance and drug product to identify critical quality attributes (CQA) that can be tested with in vitro assays. Based on these an updated set of release tests without animal tests can be proposed. Our data can serve as benchmark for the innovative methods.

Comparison of clastogen-induced gene expression profiles in wild-type and DNA repair-deficient Rad54/Rad54B cells.

BACKGROUND: Previously we found that Rad54/Rad54B cells are more sensitive towards mitomycin C (MMC) as compared to wild-type (WT) cells. This difference in sensitivity was absent upon exposure to other clastogens like bleomycin (BLM) and gamma-radiation. In order to get further insight into possible underlying mechanisms, gene expression changes in WT and Rad54/Rad54B MEFs (mouse embryonic fibroblasts) after exposure to the clastogens MMC and BLM were investigated. Exposures of these cells to mutagens (N-ac-AAF and ENU) and vehicle were taken as controls. RESULTS: Most exposures resulted in an induction of DNA damage signaling and apoptosis genes and a reduced expression of cell division genes in cells of both genotypes. As expected, responses to N-ac-AAF were very similar in both genotypes. ENU exposure did not lead to significant gene expression changes in cells of both genotypes, presumably due to its short half-life. Gene expression responses to clastogens, however, showed a genotype-dependent effect for BLM and MMC. MMC treated Rad54/Rad54B MEFs showed no induction of p53-signaling, DNA damage response and apoptosis as seen for all the other treatments. CONCLUSION: These data support our finding that different types of clastogens exist and that responses to these types depend on the DNA repair status of the cells.

Application of the Three Rs to challenge assays used in vaccine testing: tenth report of the BVAAWF/FRAME/RSPCA/UFAW Joint Working Group on Refinement.

This report aims to facilitate the implementation of the Three Rs (reduction, refinement and replacement) in the testing of vaccines for regulatory and other purposes. The focus is predominantly on identification of reduction and refinement opportunities in batch potency testing but the principles described are widely applicable to other situations that involve experimental infections of animals. The report should also help to interpret the requirements of the European Pharmacopoeia with regard to the use of alternative tests, humane endpoints and other refinements. Two specific worked examples, for batch potency testing of Clostridium chauvoei and canine leptospira, with recommendations for harmonisation of international test requirements for these and other vaccines, are provided as appendices online.

lacZ mouse embryonic fibroblasts detect both clastogens and mutagens.

The clastogenic effects of MMC and BLM and the mutagenic effects of B[a]P, N-ac-AAF and ENU were studied in mouse embryonic fibroblasts derived from wild-type (WT) and Rad54/Rad54B-deficient mice. Clastogens as well as mutagens showed a statistically significant induction of mutations in the lacZ reporter gene both in a WT and Rad54/Rad54B-deficient genetic background. Rad54/Rad54B MEFs appeared equally sensitive to the clastogens compared to WT MEFs, except for MMC. The type of mutations induced by the different compounds was investigated further by hybridizing the mutant colonies with total mouse DNA. An obvious increased number of mouse DNA positive clones was observed after BLM and MMC exposure, indicating that after these treatments genome rearrangements/translocations had occurred. In this hybridization assay, Rad54/Rad54B MEFs did not show more rearrangements/translocations than WT MEFs. As expected, the mutagens used showed no increase in chromosomal rearrangements or transloctions in MEFs derived from both genotypes. These results show that WT MEFs carrying the lacZ reporter gene on a plasmid are capable to detect both clastogenic as well as mutagenic effects of compounds in vitro. Deletion of the Rad54 and Rad54B genes did not further enhance the sensitivity of MEFs towards clastogens.

Reducing variation in a rabbit vaccine safety study with particular emphasis on housing conditions and handling.

This paper describes the results of a study of the effects of modified housing conditions, conditioning and habituation on humans using a rabbit model for monitoring whole-cell pertussis vaccine (pWCV)-induced adverse effects. The study has been performed with reference to previous vaccine safety studies of pWCV in rabbits in which results were difficult to interpret due to the large variation in experimental outcome, especially in the key parameter deep-body temperature (T(b)). Certain stressful laboratory conditions, as well as procedures involving humans, e.g. blood sampling, inoculation and cage-cleaning, were hypothesized to cause this large variation. The results of this study show that under modified housing conditions rabbits have normal circadian body temperatures. This allowed discrimination of pWCV-induced adverse effects in which handled rabbits tended to show a dose-related increase in temperature after inoculation with little variance, whereas non-handled rabbits did not. Effects of experimental and routine procedures on body temperature were significantly reduced under modified conditions and were within the normal T(b) range. Handled animals reacted less strongly and with less variance to experimental procedures, such as blood sampling, injection and cage-cleaning, than non-handled rabbits. Overall, handling had a positive effect on the behaviour of the animals. Data show that the housing modifications have provided a more robust model for monitoring pWCV adverse effects. Furthermore, conditioning and habituation of rabbits to humans reduce the variation in experimental outcome, which might allow for a reduction in the number of animals used. In addition, this also reduces distress and thus contributes to refining this animal model.

DNA-repair-deficient Rad54/Rad54B mice are more sensitive to clastogens than wild-type mice.

The sensitivity of DNA-repair-deficient Rad54/Rad54B mice for clastogens was studied and compared to that of wild-type mice. LacZ mutant frequencies (MF) in Rad54/Rad54B mice, after treatment with mitomycin C (MMC), bleomycin (BLM) and gamma-irradiation, were compared to those of the wild-type mice following the same treatments. While none of the clastogens showed an induction of the lacZ MF in the wild-type mice, there was a significant increase of the lacZ MF in the bone marrow of the Rad54/Rad54B mice after treatment with BLM and gamma-irradiation and in the spleen after MMC treatment. As expected, the positive control ENU showed a significant increase in the lacZ MF in all tested organs in wild-type mice. Mutant colonies were hybridized with total mouse DNA in order to discriminate between small gene mutations and large DNA rearrangements and translocations (size-change mutations). The hybridization studies showed a significant increase in mouse DNA positive clones 4 days after treatment with MMC and BLM in the bone marrow of the wild-type mice, which is indicative for chromosomal rearrangements and translocations to occur. An even more pronounced increase was seen 28 days after treatment with the same compounds in the Rad54/Rad54B mice.