Three Rs Approaches in the Quality Control of Inactivated Rabies Vaccines

The Report and Recommendations of
ECVAM Workshop 481,2

Reprinted with minor amendments from ATLA 31: 429-454.

Appendix 2

The Use of Humane Endpoints in the Quality Control of Rabies Vaccines

Introduction

Rabies vaccines for human and veterinary use are important medicinal products for preventing this fatal disease. Animal tests are the basic experiments for demonstrating the efficacy and potency of rabies vaccines. Since laboratory mice are highly susceptible to rabies infection, a virulent challenge (the so-called NIH test) is used to demonstrate the protective effect of a vaccine on a batch-to-batch basis (1).

While considerable effort has been made to replace this test, and several promising in vitro tests are currently being evaluated, all the regulatory requirements still include the challenge test' Rabies challenge procedures are prescribed with precise protocols for vaccine potency testing in monographs of the European Pharmacopoeia (Ph. Eur.), the US Code of Federal Regulations, and guidelines of the World Health Organization and the Office International des Epizooties (2-6). As it is necessary to calculate the potency in international units by means of the classical multiple-dilution assay, more than 100 animals are required to test each batch of these vaccines. The assays have to be performed in such a way that more than half of the animals succumb to infection, to ensure fulfilment of the evaluation criteria.

For animal welfare reasons, such animal tests are of great concern. Due to the length of time that is necessary for the introduction and validation of alternative techniques, progress concerning the change of testing requirements has so far been limited. Therefore, it is likely that the challenge test will continue to be used to estimate the potency of rabies vaccines for the foreseeable future, for both human and veterinary applications. The large number of mice required, together with the severity of the procedure, emphasise the relevance of the rabies vaccine challenge test to the Three Rs and to animal welfare concerns. To improve the situation on behalf of the animals, much more emphasis should be placed on the refinement of vaccine testing procedures.

In general, legislation controlling animal research requires scientists to select procedures that cause the least suffering for the animals used. One possibility for refinement is through reducing the duration of the animal tests, in order to shorten the period of suffering. For example, the humane killing of animals should be permitted when typical clinical signs of infection occur. Legal requirements (for example, the Council of Europe Convention ETS 123 [7] and Directive 86/609/EEC [8]) state that, in a choice between procedures, those methods should be selected which cause the least suffering and which are most likely to provide satisfactory results. This implies the use of humane endpoints. The monographs of the Ph. Eur. usually do not specify such criteria; however, a paragraph which requires the use of humane endpoints in general has recently been included in the general monographs, Vaccines for Human Use and Vaccines for Veterinary Use (9).

The possibility of using humane endpoints largely relies on the availability of typical clinical signs and other relevant parameters such as variations in body weight and body temperature. Score sheets specifically designed for the documentation of all relevant information concerning the disease under study are necessary for all these infection models (10).

A refined test that will cause less animal suffering, and which can be considered to be sufficiently well validated to replace the traditional lethal assay with mice, is described. It is proposed that clinical signs of rabies can be used as an endpoint, instead of death. Specific behaviour and loss of body weight are suitable signs, which can be used as reliable surrogate and humane endpoints to terminate an experiment at a stage much earlier than death.

 

Rabies Vaccine Potency Test

Routine potency tests were used to perform these studies, to avoid the use of additional animals, and were carried out in accordance with the Ph. Eur. monograph for Rabies Vaccine for Human Use Prepared in Cell Cultures (2). Animals were vaccinated intraperitoneally with several vaccine dilutions, and challenged two weeks later with an intracerebral (i.e.) injection of rabies virus under anaesthesia. The animals were then allowed to recover from the anaesthetie and were monitored carefully over the next 14 days.

 

Score Sheets

Score sheets were developed for the challenge procedures (11). They included data on clinical signs, body weight and body temperature (Table 1). During the experiment, the animals were observed twice daily, between 8 a.m. and 9 a.m., and between 3 p.m. and 4 p.m. In the morning, the body temperature was recorded and the animals were weighed immediately afterwards.

Table 1: Example of the use of a score sheet in the rabies vaccine potency test

Days after infection
-1 0 1 2 3 4 5 6 7 8 9 10 11 12a
Body weight (g) 22.2 22.3 21.6 21.9 22.1 22.3 21.8 21.2 18.5 16.9 15.4 14.2 13.9
Temp (°C) 38.1 38.4 37.5 37.8 37.9 37.8 37.9 37.7 37.1 36.5 35.7 34.0 33.7
Clinical Signs (score) 0 0 0 0 0 0 0 1 2 3 4 4 5
- ruffled fur x x x x x x
- hunched back x x x x x x
- slow movements x
- circular movements x x
- trembling x x
- shaky movements x
- convulsions x x x
- paresis x x
- paralysis x
- prostration
- agony, coma x

aThe score sheet shows the parameters for an individual mouse during the course of the potency test, beginning 1 day prior to infection, and ending on day 12 with the death of the mouse.

Clinical Signs

The first signs of the disease do not usually appear before day 4 after challenge. Before that time, any clinical sign is most likely to have been caused by the i.c. injection procedure, so animals showing such early signs are normally excluded from the experiment. The clinical signs of rabies are progressive, and it may take between 2 and 6 days for an animal to die.

The following five stages can be recognised in mice infected with rabies virus, and which therefore have not been protected by the vaccine.

Stage 1: ruffled fur, hunched back (Figure 1)
Ruffled fur (sometimes referred to as a starey coat or harsh coat) indicates the first signs of clinical disease. This is a rather general sign of illness in mice and can be observed in many other diseases. The same is true for hunched back. Therefore, these clinical signs in Stage 1 are not specific indicators for rabies, but do reflect that the animal is unwell, and that its welfare is compromised.

Figure 1: Stage 1 ruffled fur, hunched back

Stage 2: slow movements, circular movements (Figure 2)
During Stage 2, animals lose their alertness. They walk more slowly than usual, and if observed carefully for a while, they sometimes show circling movements, mainly in one direction. These are the first clinical indicators of neurological disorder.

Figure 2: Stage 2 - slow movements, circular movements

Stage 3: shaky movements, trembling, convulsions (Figure 3)
In Stage 3, the neurological signs become increasingly obvious, with trembling aAd shaky movements, and convulsions appear which can often be provoked. By this time, there is a marked loss of body weight. This stage, with severe and unequivocal clinical signs, clearly indicates rabies infection.

Figure 3: Stage 3 - shaky movements, trembling, convulsions

Stage 4: signs of paralysis (Figure 4)
Lameness and paresis, usually of the hind legs, are also clear indicators of progressive infection with rabies, and are soon followed by complete paralysis. The animals become clearly dehydrated.

Figure 4: Stage 4 - signs of paralysis

Stage 5: moribund animals
In Stage 5, animals become moribund. They can be seen to be prostrate and recumbent, and they obviously do not feed or drink; yet they may still survive for 1 or 2 days.

Overall, rabies infection in mice is characterised by a slow onset of the disease, usually beginning between day 4 and day 6 after infection. In contrast to most other mammals with rabies, mice do not show any signs of aggression. The study showed that all mice which had developed Stage 3 signs did not recover and died a few days later. Clinical signs therefore offer the possibility of terminating the experiment as soon as typical signs of neurological disorder are evident (Stage 3 at the latest), without any loss of scientific data, but avoiding a slow progressive death for the animals.

 

Body Weight

Body weight was measured with electronic scales and recorded on the score sheet. Since the anaesthetic and i.c. route of infection had a negative effect on body weight for 1 day, the weight on day 2 after the infection was used as the basis for body weight loss.

Interestingly, the measurement of body weight revealed a very early decrease, even before typical clinical signs of the disease were obvious. A significant loss of body weight (> 20%) proved to be the earliest pre-lethal endpoint for rabies in laboratory mice. Figure 5 shows that body weight loss was noted, even before other clinical signs were evident. Weight loss progressed continuously and could reach 30-50% shortly before death.

Figure 5: Body weight, temperature and development of clinical signs in a mouse during rabies infection

Body weight was normalised to day 2 after infection.
= body weight; = body temperature; = score of clinical signs.

Body Temperature

To measure body temperature, temperature-sensitive transponders (incorporating a unique identifier) were implanted under the skin of-the necks of the mice, and temperatures were measured twice daily. Interestingly, hyperthermia, an early sign of infection in other mammals, especially humans, never developed in the mice; hypothermia always became marked in the final stage of the disease. However, this was much too late to be considered a suitable endpoint, and other markers were seen earlier and were more reliable.

 

Conclusions

The appearance of typical clinical signs (Stage 3), alone or Stage 2, in combination with a decrease in body weight, are suitable humane endpoints. The relatively slow progression of the disease, with an increase in severity of the clinical signs, makes the use of score sheets for rabies easy. If a decrease of body weight of more than 15% and clinical signs of neuronal dysfunction, as in Stages 2 or 3, occur in combination, a point of no return has been reached. Consideration of these two combined endpoints allows the experiment to be terminated at an even earlier phase. This reduces the duration of animal suffering by an average of 2-3 days. Figure 5 gives a typical example of the development of all three parameters.

Only a suitable balance and score sheets have to be used to identify an early humane endpoint in rabies challenge tests. The clinical signs of rabies are easy to observe, and staff can readily be trained to recognise the early stages of the disease.

A combination of clinical signs and recording of body weight are suitable humane endpoints for the rabies challenge test and would lead to a considerable reduction of animal suffering. Hopefully, many institutions and companies will rely on the use of humane endpoints instead of lethality. A video is available, which might help to change the current situation and reduce the causation of unnecessary animal suffering (12).

 

Acknowledgement

Appendix 2 is based on the results of a study (Alternative methods to replace, reduce andlor refine the use of laboratory animals in vaccine production, quality control and assessment) funded by the European Commission via ECVAM (EC contract number 11274-95-10FlED ISP NL).

 

References

  1. Wilbur, L.A. & Aubert, M.F.A. (1996). The NIH test for potency. In Laboratory Techniques in Rabies, 4th edn (ed. F-X. Meslin, M.M. Kaplan & H. Koprowski), pp. 360-368. Geneva, Switzerland: World Health Organization.
  2. Council of Europe (2002). Rabies vaccine for human use prepared in cell cultures (Monograph No. 0216). In The European Pharmacopoeia, 4th edn, pp. 2213-2214. Strasbourg, France: Council of Europe.
  3. Council of Europe (2002). Rabies vaccine (inactivated) for veterinary use (Monograph No. 0451). In The European Pharmacopoeia, 4th edn, pp. 2282-2284. Strasbourg, France: Council of Europe.
  4. CFR (1999). US Code of Federal Regulations, Title 9, Chapter I: Animal and Plant Health Inspection Service, Department of Agriculture, Subchapter E - Viruses, Serums, Toxins, and Analogous Products; Organisms and Vectors, Part 113.209, Rabies Vaccine, Killed Virus, pp. 601-603. Washington, DC, USA: US Government Printing Office.
  5. WHO (1994). Requirements for rabies vaccine (inactivated) for human use produced in continuous cell lines. Amendment 1992. WHO Technical Report Series No. 840, pp. 205-207. Geneva, Switzerland: World Health Organization.
  6. OIE (2000). Rabies. In Manual of Standards for Diagnostic Tests and Vaccines, 4th edn, pp. 276-291. Paris, France: Office International des Epizooties. Web site http://www.oie.int/eng/normes/mmanual/ A_summry.htm (Accessed 11.8.03).
  7. Council of Europe (1986). European Convention for the Protection of Vertebrate Animals Used for Experimental and Scientific Purposes ETS No. 123, 51pp. Strasbourg, France: Council of Europe.
  8. European Commission (1986). Council Directive 86/609/EEC on the approximation of laws, regulations and administrative provisions of the Member States regarding the protection of animals used for experimental and other scientific purposes. Official Journal of the European Communities L358, 1-28.
  9. Council of Europe (2002). The European Pharmacopoeia, 4th edn, 2416pp. Strasbourg, France: Council of Europe.
  10. Morton, D.B. (1995). Advances in refinement in animal experimentation over the past 25 years. ATLA 23, Suppl. 1, 812-822.
  11. Cussler, K., Morton, D.B. & Hendriksen, C.F.M. (1998). Humane endpoints for the estimation of lethality rates in the potency testing of rabies vaccines. Alternativen zu Tierexperimenten 15, Suppl. 98,40-42.
  12. The HELP Group (2001). Humane Endpoints - Lethal Parameters: Humane Endpoints Replace Lethal Parameters in Batch Potency Tests of Rabies Vaccines, 11pp. Bilthoven, The Netherlands: The HELP Group.

Back to main article