[Optimisation and establishing of serological methods for the potency testing of immunglobulins against Clostridium tetani-toxin]

The quality control of human tetanus immunglobulin requires animal experiments according to European Pharmacopoeia monograph 398. The potency estimation has to be done in a toxin neutralisation test in mice (MNT) or guinea pigs. Immunoassays could also be used if they show a suitable sensitivity and specificity. The first results of our study verify that an indirect enzyme linked immunosorbent assay (ELISA), a rocket immunelectrophoresis (RIE) and a toxin binding inhibition test (ToBI) could be used as serological alternativ methods to the MNT. Studies on the reproducibility of the in vitro methods resulted inter-assay coefficients of variation between 2 and 27%. The ELISA is more sensitive (limit of detectability: 0,005 IE/ml) than the ToBI (0,04 IE/ml) and the RIE (5 IE/ml). The transferability of the ELISA to other labs is proofed. The transferability of the RIE and the ToBI will be tested in the near future.

[Humane endpoints as a replacement for the estimation of lethality rates in the potency testing of rabies vaccines]

Vaccines are successfully used to prevent rabies infections in humans and animals. The quality requirements for these products are high. After licensing it is still necessary for batch release to perform animal challenge tests to demonstrate the potency of the vaccines. In these experiments it is registered how many animals in the immunised groups die or show symptoms of rabies within a defined time frame. An ECVAM working group has examined whether clinical signs can be used in challenge tests for vaccine control to replace lethality as criterion. The severe distress and the typical signs of the disease made the rabies infection a promising example for this study. Clinical signs, body temperature and body weight were the criteria used to define humane endpoints. It could be shown, that the reduction of body weight and the appearance of typical clinical signs are suitable to define humane endpoints. Pain and distress could be considerably reduced by the implementation of these criteria in to the legal requirements for potency tests of rabies vaccines.

[Detection of extraneous virus in live vaccines]

In the study in vitro alternatives to a non-validated and harmful animal test for the absence of extraneous virus in live vaccines were investigated. For evaluation of a suitable in vitro method the porcine herpesvirus (Aujeszkyvirus, Pseudorabiesvirus) was used as a model virus. In artificially contaminated live vaccines the aujeszkyvirus could be detected by moleculargenetical and cellular methods. Regarding the threshold values of virus detection in vitro tests showed to be more efficacious than animal testing. Meanwhile the European Pharmacopoeia Commission deleted the animal test for extraneous virus from two monographs. The discussion, if respective animal testing can be cancelled for the other live vaccines as well, is still ongoing. The study was supported by the German Ministry of Education, Science, Research and Technology.

[How to prove complete virus inactivation in rabies vaccines. A comparison of an in vivo to an in vitro method]

At present, the complete inactivation of rabies virus in rabies vaccines ad us. vet. is proven by an animal experiment which causes severe suffering, the intracerebral injection of mice. This animal experiment yet is not validated. We have quantified the sensitivity of the mouse test and examined whether the animal experiment may be replaced by the immunofluorescence assay (IFT) as an in vitro method. Detection limits of both assays were determined depending on the examined product, i.e. prior to and after the addition of adjuvans and preservative, respectively. Furthermore, symptoms of the rabies desease were recorded and their severity was classified on a range of 1-5. Symptoms of rabies-infected mice were clear and highly specific. Symptoms classified as >/= 2 in context with a loss of >/= 15% of the initial weight were defined as humane endpoints of the desease. The quantitative detection of active virus was not inhibited in the presence of even high concentrations of inactivated virus. The detection limit of the mouse test was 10 viruses ml-1 independent of the examined product. The detection limit of the IFT prior to the addition of adjuvans and preservative was 10 viruses ml-1 as well. After the addition of these substances the detection limit rose to 103 viruses ml-1. Advantages and disadvantages of the mouse test and IFT are discussed.

[Development and prevalidation of alternative methods for the potency testing of C. perfringens vaccines]

The requirements for the quality control of C. perfringens vaccines for veterinary use are described in the monograph 363 of the European Pharmacopoeia (Ph. Eur.). In the current used potency test neutralising antibodies against C. perfringens beta- and epsilontoxin are determined in a mouse neutralisation test (MNT). Two ELISA methods were developed for the replacement of the MNT. Both methods use monoclonal antibodies to determine the quantity of specific antibodies against beta toxin (Capture-ELISA) and epsilon toxin (Competitive-ELISA) in vitro. In parallel to the routine batch potency test in mice, the beta- and epsilonantitoxin levels in 523 samples were estimated in the ELISA procedures. A high specificity and a good reproducibility are evident for both test systems. An interlaboratory prevalidation study was carried out to evaluate the relevance and the transferability of the ELISA procedures. It is concluded that both ELISA systems seems to be suitable alternative methods for assessing the potency of beta- and epsilontoxoid in batches of vaccines for veterinary use.

[The establishment of endotoxin limits which satisfy animal welfare considerations in the testing of porcine vaccines]

Endotoxin (lipopolysaccharide, LPS) is a constituent of the cell walls of gram-negative bacteria and is found in many vaccines produced from these bacteria. High levels of endotoxin can give rise to a range of pathophysiological reactions, and adverse reactions tend to be seen in animals following vaccination. In this study, pigs of various ages and weights were vaccinated with licensed porcine vaccines and the endotoxin content of the vaccines was determined with the LAL, conducted according to DAB 10. The experiments followed the DAB guidelines relating to the safety testing of veterinary vaccines. The animals were monitored for 48 h after vaccination, their body temperatures were measured, and blood samples were taken for analysis and for the determination of plasma endotoxin levels. There was a clear relationship between vaccine endotoxin content and changes in blood cell counts and in the clinical picture. Elevated plasma endotoxin levels correlated with the occurrence of initial leucopenia followed by leucocytosis as well as with clinical symptoms ranging from refusal of food and depression to shock-like symptoms. After 24 h, normal physiological values were regained. Young animals weighing between 10 and 40 kg were found to be very sensitive to elevated endotoxin content in vaccines. The differences in individual reactions could be due not only to differences in vaccine endotoxin content, but also to differences in the reactivity of the organism, and in the type of bacteria used or in the composition of the vaccine.

[Evaluation of the relevance of the target animal safety test for the quality control of veterinary immunological medicinal products]

The legal requirements for veterinary vaccines require the testing of each new vaccine batch for safety. In the European Pharmacopoeia a target animal safety test (TAST) is regularly required as a final product test for almost all vaccine batches. This test is also required in the "General requirements for the production and control of live mammalian viral and bacterial vaccines (GRLMV)" and the "General requirements for the production and control of inactivated mammalian viral and bacterial vaccines (GRIMV)" by the European Union. In several publications the relevance of this TAST has been questioned. Particularly, it is questionable whether this animal test is able to give a significant contribution to the safety of immunologicals considering today"s standards of the quality control of animal vaccines, e.g. Good manufacturing practice and Good laboratory practice. However, due to a lack of sufficient amount of data the relevance of this animal test is discussed contradictorily. 1934 batch release protocols were sent to the Paul-Ehrlich-Institut between January of 1994 and December of 1997 for bacterial vaccines including combinations with viral antigens, vaccines against parasitic or fungal diseases and sera. During that period the manufacturers needed 5250 animals to perform the TAST. Obviously, the requirement for the TAST has a great impact on the animal numbers needed for the quality control of veterinary vaccines. The deletion of the TAST as a routine test could reduce the number of animals considerably. Preliminary results of a survey on this problem are presented and discussed.

[: A comparison of commercially available adjuvants in BALB/c-mice immunised with a weekly immunogenic peptide]

The antibody-stimulating activities and side effects of commercially available adjuvants were compared in BALB/c mice immunised with the immunosuppressive (ISU) peptide of HIV I. Clinical and pathological-histological parameters as well as behavioural changes, were used to assess the distress and pain caused to the animals. Complete Freund's Adjuvant (FAk) was used as the positive control and PBS as the negative control. The oil-based adjuvants Montanide ISA 51(R) (M 51), Specol(R), and Hunter's TiterMax Gold(R) (HTMG) were used with the ISU-peptide conjugated to KLH. The water-soluble Gerbu Adjuvant(R) was administered together with the antigen KLH conjugate and also with the ISU-peptide conjugated to cholera toxin B subunit (ChTxB). The Dutch "Code of Practice" was used as a guideline for all immunisations. No changes in animal activity or behaviour was observed in any of the groups. All the oil-based adjuvants gave rise to swellings and encapsulations, which were most pronounced in the HTMG-group. Although body weight increased throughout the study, no increase was seen in any adjuvant group for a short period after each booster immunisation, nor after the first immunisation in the HTMG group. FAk induced a light fever after all immunisations. FAk, Specol and M 51 as well as Gerbu-ChTxB induced antibody titres which were detectable in the ELISA, but no detectable antibody the water-soluble adjuvants or following administration of HTMG applied with the ISU-peptide-KLH conjugate.