Safety testing of acellular pertussis vaccines: Use of animals and 3Rs alternatives

Abstract

The current test of acellular Bordetella pertussis (aP) vaccines for residual pertussis toxin (PTx) is the Histamine Sensitization test (HIST), based on the empirical finding that PTx sensitizes mice to histamine. Although HIST has ensured the safety of aP vaccines for years, it is criticized for the limited understanding of how it works, its technical difficulty, and for animal welfare reasons. To estimate the number of mice used worldwide for HIST, we surveyed major aP manufacturers and organizations performing, requiring, or recommending the test. The survey revealed marked regional differences in regulatory guidelines, including the number of animals used for a single test. Based on information provided by the parties surveyed, we estimated the worldwide number of mice used for testing to be 65,000 per year: ∼48,000 by manufacturers and ∼17,000 by national control laboratories, although the latter number is more affected by uncertainty, due to confidentiality policies. These animals covered the release of approximately 850 final lots and 250 in-process lots of aP vaccines yearly. Although there are several approaches for HIST refinement and reduction, we discuss why the efforts needed for validation and implementation of these interim alternatives may not be worthwhile, when there are several in vitro alternatives in various stages of development, some already fairly advanced. Upon implementation, one or more of these replacement alternatives can substantially reduce the number of animals currently used for the HIST, although careful evaluation of each alternative's mechanism and its suitable validation will be necessary in the path to implementation.

Assays for Determining Pertussis Toxin Activity in Acellular Pertussis Vaccines

“…This method provides a quantitative estimate of the PTx activity of a test vaccine relative to a PTx reference standard which has been assigned histamine sensitization units (HSU) or International Units (IU). The accepted level of PTx in test vaccines also varies by region with 0.4 HSU the level in Japan and 0.8 HSU in China (Table 1) [90,100,101,102]. The animals can then be culled once the temperature reading has taken place and thus reduce the severity of the procedure.…”

“…Both forms of the HIST assay can be used to measure the levels of PTx activity in samples by comparing the drop in temperature, or deaths per group, for a range of doses to the responses in a reference preparation [103,104,105]. While both death and changes in body temperature have been found to be dose dependent they do not seem to be linked and the modes of action may not be the same [99,102].…”

“…Also, the fact that a single batch going to multiple markets must pass the various versions of HIST relevant to each market substantially increases the number of animals used. It has recently been estimated that approximately 65,000 mice are used globally in HIST assays per year [102]. On top of this the large number of variations in HIST make it difficult to standardize [4].…”

“…Finally, there are ethical concerns about animal welfare as the test can cause severe pain and distress, especially for mice receiving a PTx reference standard. For these reasons HIST has long been regarded as a priority for replacement with in vitro alternative assays [4,67,93,102,110,111,112]. However, following consultations between vaccine manufacturers and national control laboratories, the requirement for testing for residual and reversion to toxicity in final formulated vaccines will be removed from the European Pharmacopoeia in 2020 once the detoxification process has been demonstrated to be robust and stable [106].…”

iGIST—A Kinetic Bioassay for Pertussis Toxin Based on Its Effect on Inhibitory GPCR Signaling

“…However, there are major regional differences in the regulatory guidelines. 17 , 18 No internationally agreed upper limit for active PTX in pertussis ACVs exists. Only China and Japan have these kinds of defined figures, of 0.8 and 0.4 histamine sensitization units/mL (HSU/mL), respectively, based on the mouse histamine sensitization test (HIST).…”