Regulatory Acceptance and Use of 3R Models: a Multilevel Perspective

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Regulatory Acceptance and Use of Serology for Inactivated Veterinary Rabies Vaccines

In April 2013 the mouse antibody serum neutralization test (SNT) was formally incorporated into European Pharmacopoeia monograph 0451 for potency testing of inactivated veterinary rabies vaccines. The SNT is designed to replace the highly variable and pain and distress causing NIH mouse rabies challenge assay. The adoption of the SNT meets the European ambition (i.e., EC and CoE) to replace, reduce and/or refine laboratory animal testing. However, regulatory acceptance and use of 3R models, such as the SNT, remains challenging. This paper aims at clarifying the process of acceptance and use of the SNT. For this purpose it reconstructs the process and reveals barriers and drivers that have been observed by involved stakeholders to have played a role. In addition it extracts lessons to stimulate regulatory acceptance in similar future processes. The incorporation of the SNT into the monographs went relatively quickly due to a thorough test development and pre-validation phase, commitment and cooperation of relevant stakeholders and a strong project coordination of the international validation study. The test was developed by the Paul Ehrlich Institut, a leading European OMCL. This facilitated its European regulatory use. The use by industry is in a critical phase. At this stage product specific validation and the question whether the SNT will be accepted outside Europe are important influencing factors

Evaluation of Two Serological Methods for Potency Testing of Whole Cell Pertussis Vaccines - Potency Testing of Whole Cell Pertussis Vaccines

The European Pharmacopoeia (Ph. Eur.) and the World Health Organization (WHO) require the performance of extensive quality control testing including a potency test before a vaccine batch is released for human use. Whole cell pertussis (wP) vaccine potency is assessed by a mouse protection test (MPT) based on the Kendrick test. This test compares the vaccine dose necessary to protect 50% of mice against the effect of a lethal intracerebral dose of Bordetella pertussis and the dose of a suitable reference vaccine needed to give the same protection level. Due to the large variability in the results of this test and the severe distress which is inflicted on the many animals involved, its replacement by an alternative method is highly desirable. At the initiative of the European Directorate for the Quality of Medicines and HealthCare (EDQM) of the Council of Europe, in collaboration with the WHO and the In-vitro toxicology Unit/European Centre for the Validation of Alternative Methods (ECVAM) of the European Commission (EC) Joint Research Centre-Institute for Health and Consumer Protection (JRC-IHCP), wP vaccine specialists from all over the world were invited to present an overview of candidate alternatives at a symposium organized in Geneva (CH) in March 2005. Although no alternative method was found suitable for immediate implementation of batch potency control, the Pertussis Serological Potency Test (PSPT), initially developed in mice and recently transferred to guinea pigs (gps), was identified as a model of interest. Using the PSPT in gps to test several components of combined vaccines such as Diphtheria-Tetanus-wP vaccines in the same animal series would allow further implementation of the European 3Rs policy to batch potency control, by additional method refinement and reduction of animal use. The present study evaluated 2 features of the serological response to wP vaccination: 1) the overall antibody response as measured by a ¿whole cell¿ ELISA (PSPT-wC-ELISA) which uses the B. pertussis 18323 challenge strain prescribed for the MPT to coat the assay plates and 2) the functional neutralising antibodies to pertussis toxin (PT, one of the main virulence factors of B. pertussis), as measured by the Chinese Hamster Ovary (CHO) cell assay. The results showed that 1) the gp model can be used for wP vaccine potency testing; 2) despite good repeatability and precision, the CHO cell assay did not generate results comparable to the MPT. Moreover, the CHO cell assay showed significant differences in the ability of wP vaccines to induce neutralising anti-PT antibodies, which did not correlate to the overall antibody response evaluated by PSPT-wC-ELISA; 3) comparable potencies were obtained in the MPT and the PSPT-wC-ELISA.JRC.I.3-In-vitro method

Vaccine-Mediated Activation of Human TLR4 Is Affected by Modulation of Culture Conditions during Whole-Cell Pertussis Vaccine Preparation

<div><p>The potency of whole-cell pertussis (wP) vaccines is still determined by an intracerebral mouse protection test. To allow development of suitable <i>in vitro</i> alternatives to this test, insight into relevant parameters to monitor the consistency of vaccine quality is essential. To this end, a panel of experimental wP vaccines of varying quality was prepared by sulfate-mediated suppression of the BvgASR master virulence regulatory system of <i>Bordetella pertussis</i> during cultivation. This system regulates the transcription of a range of virulence proteins, many of which are considered important for the induction of effective host immunity. The protein compositions and <i>in vivo</i> potencies of the vaccines were BvgASR dependent, with the vaccine containing the highest amount of virulence proteins having the highest <i>in vivo</i> potency. Here, the capacities of these vaccines to stimulate human Toll-like receptors (hTLR) 2 and 4 and the role these receptors play in wP vaccine-mediated activation of antigen-presenting cells <i>in vitro</i> were studied. Prolonged BvgASR suppression was associated with a decreased capacity of vaccines to activate hTLR4. In contrast, no significant differences in hTLR2 activation were observed. Similarly, vaccine-induced activation of MonoMac-6 and monocyte-derived dendritic cells was strongest with the highest potency vaccine. Blocking of TLR2 and TLR4 showed that differences in antigen-presenting cell activation could be largely attributed to vaccine-dependent variation in hTLR4 signalling. Interestingly, this BvgASR-dependent decrease in hTLR4 activation coincided with a reduction in GlcN-modified lipopolysaccharides in these vaccines. Accordingly, expression of the <i>lgmA-C</i> genes, required for this glucosamine modification, was significantly reduced in bacteria exposed to sulfate. Together, these findings demonstrate that the BvgASR status of bacteria during wP vaccine preparation is critical for their hTLR4 activation capacity and suggest that including such parameters to assess consistency of newly produced vaccines could bring <i>in vitro</i> testing of vaccine quality a step closer.</p></div

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

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