Progress and challenges in the replacement of the Histamine Sensitisation Test (HIST)

Pertussis is a highly contagious disease, particularly in small children, and is characterised by severe coughing spells that typically include a whooping sound.

There are now two vaccines on the market to protect against whooping cough: the classical whole cell vaccine based on inactivated pertussis bacteria and a newer vaccine, which is called the acellular pertussis vaccine. The latter vaccine only includes the parts of the vaccines that are considered to have protective properties. The cheaper whole cell vaccine is still widely used in non-industrialised countries, but is occasionally associated with severe side effects. The acellular vaccine, which is associated with fewer side effects but is more expensive, is predominantly used in industrialised countries.

All acellular vaccines contain pertussis toxoid (PTd); one of the factors of Bordetella pertussis that is assumed to be important for the effectiveness of the vaccine. Pertussis toxoid is produced by inactivating its toxic counterpart; pertussis toxin (PTx). This toxin is assumed to contribute to the side effects of whole cell vaccines. Since acellular pertussis vaccines contain high quantities of PTd, the success of inactivation is monitored during production and in the final product. Globally, there is a requirement to test each batch of vaccine that is produced for residual PTx by the in vivo Histamine Sensitisation Test (HIST).

In this test, mice are injected with the vaccine and subsequently challenged with a high dose of histamine. Although histamine alone is unpleasant for the animals, the mice will only die if the vaccine contains pertussis toxin. When pertussis toxin is present, the mice will die within 24 hours due to an anaphylactic shock. This makes the test a matter of concern in terms of animal welfare. The need for an alternative for this in vivo test has been acknowledged from the regulatory as well as the industrial perspective, owing to ethical concerns, the lack of scientific relevance and the poor reproducibility.  

Several newer models have been developed. One of these models measures a drop in body temperature in the 30 minutes following histamine challenge rather than death of the animals, but there are still concerns that the test is unpleasant for the animals involved. Non-animal models are based on cell cultures or on physicochemical/ immunochemical assays and are currently considered to be the way forward.

In 2011 groups working on alternative methods, manufacturers and representatives of regulatory bodies came together at the Paul Ehrlich Institute in Germany to discuss future steps.  One of the outcomes was the establishment of a working group to facilitate the evaluation of alternative in vitro  methods for  PTx measurement. The working group has met twice since then. As a satellite meeting to the 9^th World Congress on Alternatives in Prague, the working group convened again to discuss the progress and challenges of implementing in vitro assays and the necessary regulatory framework. The meeting was set up by an impressive list of organisations: NICEATM and ICCVAM from the US, EURL ECVAM from Europe, Health Canada and EDQM of the Council of Europe. This broad support underlines the need and willingness to move away from the HIST.

The workshop was sponsored by NC3Rs and was attended by stakeholders from academia, regulatory agencies and the industry. The presentations during the workshop covered a wide range of topics associated with the HIST, including the resurgence of the Bordetella pertussis, vaccination programmes, a survey of the number of animals used for the HIST, an overview of the alternatives and a roadmap of the development of the alternatives so far.

We presented an overview of different specifications in HIST requirements around the world and discussed the opportunity for mutual recognition of test results. We also showed the results of a survey on numbers of animals being used, and concluded that the global number must be somewhere around 100,000. Several questions were addressed and the discussion revealed that, for most of the issues, the stakeholders have reached consensus. It was concluded that it was preferable for one method that measures clinically relevant effects of PTx for toxicity to be adopted and implemented in the regulation, rather than several methods each of which assesses one aspect of toxicity.

One of the in vitro methods – the modified CHO cell clustering test – measures all of the relevant aspects for toxicity: binding as well as enzymatic activity. In a transfection study it was demonstrated that cell clustering depends on the enzymatic activity of the toxin. The application of the CHO cell clustering test for the final vaccine will be determined in a collaborative study (including 13 laboratories) in the autumn of this year. The aim of the collaborative study is to test the in vitro CHO cell clustering assay for sensitivity and inter-laboratory variability using two new protocols. In this study, four vaccines that have proven to be safe will be spiked with PTx, in order to mimic vaccines that contain PTx. The results will be discussed and reviewed during a workshop hosted by the NC3Rs in London, in March next year with the aim to reach a consensus for global acceptance of a replacement for the HIST.

Coauthored by Marieke Hoonakker, IntraVacc, Netherlands

Read the report of the international workshop: 'Alternatives to HIST for acellular pertussis vaccines: progress and challenges in replacement'.