Factors influencing the antibody response to vaccination against rabies.

Preventive vaccination against rabies virus is a highly effective method for preventing rabies in humans and animals. For travel purposes, vaccination of domestic carnivores is obligatory. In addition, some countries require testing for neutralizing antibodies against rabies. The minimal threshold level accepted by WHO/OIE is 0.5 IU/ml. Despite proper vaccination some animals do not reach the threshold. The objective of this study was to identify specific risk factors in dogs and cats for post-vaccination rabies antibody titres below 0.5 IU/ml by FAVN test. Rabies vaccination protocols and recommendations were reviewed with regard to travel regulations. Comprehensive data was collected on animals tested for rabies antibodies via a questionnaire sent to veterinarians who submitted sera for rabies titration. The questionnaire included data on species, age, sex, breed, vaccine used, date of last vaccination and blood sampling, vaccination history and further medical treatments at time of vaccination. Data on around 1,200 animals was analysed. Most animals older than one year had already received more than one rabies vaccination. The influence of breed and sex on antibody titre seems to be insignificant. Young dogs have a high risk of results below 0.5 IU/ml after their first vaccination. This risk can be minimised by the application of a second vaccination and blood sampling according to the manufacturer's recommendations. An important factor for the test outcome might be the virus strain used in the vaccine.

Development of a cell culture assay for the quantitative determination of vaccination-induced antibodies in rabbit sera against Clostridium perfringens epsilon toxin and Clostridium novyi alpha toxin.

Cell culture assays are possible alternatives to replace in vivo neutralization tests currently required for potency testing of clostridial vaccines. Cell culture assays based on the MDCK cell line and the Vero cell line which are sensitive to the Clostridium (C.) perfringens type D epsilon toxin and Clostridium novyi type B alpha toxin, respectively, were developed, and the test conditions were standardized. The antibody titres of vaccinated rabbits measured in vitro were compared with the results of current test procedures recommended by European Pharmacopoeia. The correlation coefficients calculated were significant for all sera tested. The cell culture assays proved to be sensitive, specific, reproducible and reliable. Therefore, these cell culture assays could be suitable in vitro alternatives to the in vivo mouse neutralization experiments required for potency tests of clostridial vaccines, but further validation studies are necessary.

[Progress in the replacement of animal experiments in the quality control of clostridial vaccines]. / Fortschritte beim Ersatz von Tierversuchen in der Qualitätskontrolle von Clostridien-Impfstoffen.

Animal experiments still play a central role in the quality control of vaccines. Generally, performance of these experiments is provided by law and laid down in the European Pharmacopoeia. Classical vaccines, the efficacy of which is calculated in International Units, require a very high number of experimental animals for quality control testing. The testing mainly consists of infection and intoxication experiments causing extreme suffering of the animals involved. This classical product group includes clostridial vaccines which are used to a great extent in veterinary medicine in particular. Within the last years, considerable efforts have been made to reduce the number of animal experiments in this field, lower the number of animals, and decrease the suffering of the animals during testing. Several research projects for the development and validation of alternative methods have been initiated. Furthermore, the 3R Concept (Replacement, Reduction and Refinement) is increasingly taken into consideration when developing or revising legal provisions. This led to various improvements regarding animal welfare in the quality control of clostridial vaccines.

[The importance of allergic skin test with Johnin, antibody ELISA, cultural fecal test as well as vaccination for the sanitation of three chronically paratuberculosis-infected dairy herds in Rhineland-Palatinate]. / Zur Bedeutung von allergischem Hauttest mit Johnin, Antikörper-ELISA, kultureller Kotuntersuchung sowie der Impfung für die Sanierung dreier chronisch Paratuberkulose-infizierter Milchviehherden in Rheinland-Pfalz.

Three chronically paratuberculosis infected herds were tested for six years twice a year (intradermal Johnin test, antibody ELISA (IDEXX Corp.), microbial culture) according to a sanitary program. Culling of shedding animals and vaccination of calves with NEOPARASEC (Merial Corp.) were part of the program. In course of experiment, 1015 samples of 228 non vaccinated cows and 1502 samples of 293 vaccinated cattle have been tested. 3.8% of the vaccinated animals proved positive in microbial culture. Nearly all vaccinated calves developed granulomas sized from hazelnut to loaf at the injection site. Positive reactions in intradermal test as well as in antibody ELISA were found in very young calves. 24.3%, 33.7%, 25.9%, respectively of the non vaccinated animals were identified as shedders of M. avium subsp. paratuberculosis (MAP) by microbial culture. In the first and in the second herd most shedders of MAP were found in the first herd examination (66.7%, 42.9%, respectively), whereas in the third herd they were detected in the fifth examination (31.0%). At the beginning, 17.9% of non vaccinated animals proved positive in intradermal test, 14.4% in antibody ELISA. Afterwards, the number of positive test results decreased but increased again towards the end of the experiment. 48.5% of the 66 shedders showed positive reactions in intradermal test, 57.6% in antibody ELISA, 77.3% in at least one of these both tests. Antibodies in ELISA were found in rising frequency from two years before the time of shedding. 50.0% of the shedders reacted positive in ELISA at the time of shedding. In selected shedders first positive results were found at the age of about two years. Unfortunately, only incomplete hygienic measures were realized by the farmers. Under field conditions the realisation of attending sanitary programs is difficult. MAP is spread mainly by buying of animals, therefore a certification program for paratuberculosis free herds is urgently necessary as well as an improvement of diagnostic methods.

Batch potency testing of inactivated erysipelas vaccines by ELISA--development, validation and implementation.

Inactivated erysipelas vaccines are widely used to protect pigs against erysipelas disease caused by the bacterium Erysipelothrix (E.) rhusiopathiae. Quality control tests for this vaccine are laid down in the European Pharmacopoeia (Ph.Eur.) Monograph No. 64. A laboratory animal model using a vaccination-challenge procedure is currently required as batch potency test. More than 10 years ago we initiated the first studies to develop an alternative ELISA potency model to replace this regulatory challenge test in mice. A short retrospective outline of the various steps from the development of the method until implementation into the regulatory requirements is described.

Potency testing of inactivated erysipelas vaccines by ELISA--influence of the adjuvant on antibody development.

The humoral immune response after vaccination may be greatly influenced by the type of adjuvant used. In this study we investigated the influence of three different adjuvants on the serological response in laboratory mice after immunisation with commercial erysipelas vaccines.

Collaborative study for the establishment of erysipelas ELISA coating antigen. European Biological Reference Preparation batch no. 1.

The development and validation of suitable alternatives for the replacement of in vivo challenge testing in the evaluation of vaccines is an important goal for national authorities and manufacturers involved in the assessment of quality, safety and efficacy of such products. To that end, 13 laboratories from 9 European countries, including 5 manufacturers, 7 authorities and EDQM, have taken part in a collaborative study to evaluate the suitability of a candidate reference preparation of erysipelas coating antigen for ELISA as a European Pharmacopoeia Biological Reference Preparation (Ph. Eur. BRP No. 1). The new Ph. Eur. BRP is intended for use in a serological assay, which would significantly reduce the suffering of animals in the potency assays of inactivated erysipelas vaccines. Participants were provided with sufficient study material, including the candidate coating antigen, and a panel of test sera from mice which had been immunised with vaccines representative of products on the European market, in order to evaluate the performance of the coating antigen in an enzyme-linked immunosorbent assay (ELISA) which had previously performed successfully in a prevalidation study [1] and in an international validation study [2]. Results of the collaborative study indicate that the candidate batch of erysipelas ELISA coating antigen is suitable to act as a Ph. Eur. biological reference preparation. The final study report was presented at the 110th session of the Ph. Eur. Commission (June 19-21, 2001) and the material was duly adopted as Erysipelas ELISA Coating Antigen Ph. Eur. BRP No. 1 for use in the enzyme-linked immunosorbent assay in the context of the serological potency assay for inactivated erysipelas vaccines.

[100 years of erysipelas prophylaxis: significance and reduction of animal experiments]. / 100 Jahre Rotlaufprophylaxe: Bedeutung und Reduzierung der Tierversuche.

The history of erysipelas prophylaxis began in 1882 when Pasteur first discovered the attenuating effect of rabbit passages on the erysipelas bacterium. Ten years later, the German veterinarian Lorenz demonstrated the protecting effect of erysipelas antiserum. He developed a method of serovaccination which was successfully used in Germany for more than 50 years. Both scientists employed laboratory animals for the development of their live vaccines. Lorenz additionally recommended an animal model with grey mice to control the potency of erysipelas sera. In 1944, Fortner and Dinter published the results of their investigation on a skin scarification test in swine. This modus of infection was the basis of the first reliable model for efficacy testing of erysipelas vaccines in domestic animals. Shortly after World War II, the first inactivated erysipelas vaccines were being developed. At that time, also a strict quality control was introduced for this product group which required extensive animal experiments in laboratory mice and pigs for the determination of efficacy. WHO established International Standards for erysipelas vaccines and antisera concerning potency testing in mice. These animal models were finally incorporated in pharmacopoeia monographs. Animal experiments have played an important role in the development and quality control of erysipelas vaccines. And the success of this quality control based on animal experiments has had a significant impact on the quality control systems for veterinary vaccines in general. Today, we have a far more detailed knowledge about pathogenesis and immunology of swine erysipelas. This knowledge now allows the introduction of alternative methods according to the 3R concept. With these new methods, animal numbers can be decreased and suffering caused by challenge infection can be reduced. The ultimate goal, i.e. quality control of erysipelas vaccines carried out without routine performance of animal experiments, should be achieved in the near future.

[Clinical endpoints during rabies vaccine control tests]. / Klinische Endpunkte bei der Tollwutimpfstoffprüfung.

All warm-blooded animals, including humans, are susceptible to rabies. The infection with the virus leads inevitably to the death of the recipient. Vaccines and anti-serums are at present the only possibility to prevent rabies infections in the human and veterinary medicine. In order to be able to guarantee the production of a reliably safe and effective vaccine, each batch has to be tested. These tests contain animal experiments. Alone for efficacy tests of rabies vaccines done for each batch far over one hundred mice are used. In a defined time period in challenge experiments the number of deaths rates of immunised animals are compared with non vaccinated control animals. At present several replacement methods for this test are in development. However, so far none of them has been validated in an international multicenter study. The modifications necessary for example in the WHO (World Health Organization) or O.I.E. (Office International d"Epizooties) guidelines will need several more years. Therefore, this test method will be internationally used in the foreseeable future. In order to avoid unnecessary suffering of the animals, we looked for signs, which can be used to replace lethality as criterion. For this purpose score-sheets were developed, on which the observed clinical signs were recorded. The decrease of body-temperature, which was measured with transplanted transponders, occurred too late to be usable. A clear reduction of the body weight is the earliest sign of an illness. Slow and circular movements, followed by cramps and paralyses, are the first neurological symptoms of rabies. The combination of these parameters can serve as a reliable indicator for humane endpoints. This saves the mice on average between four and five days of the most stressful phase of the experiment and is a clear "refinement" of the test conditions in the sense of the 3R. A video which shows the clinical signs is available.

Quality control of inactivated erysipelas vaccines: results of an international collaborative study to establish a new regulatory test.

According to the specifications of the European Pharmacopoeia (Ph. Eur.) monograph (Swine Erysipelas Vaccine (Inactivated), Monograph no. 64, European Pharmacopoeia, 3rd edn., 1997) on erysipelas vaccines for veterinary use, batch potency is estimated in a multi-dilution assay after immunisation and infection of mice. Recently, we described a serological assay system (ELISA) which has the potential to replace this challenge-based model (Beckmann R, Cussler K. Wirksamkeitsprüfung von Rotlaufimpfstoffen an der Labormaus. ELISA kontra Infektionsversuch. ALTEX 1994;Suppl. 1:39-45; Rosskopf-Streicher U, Johannes S, Hausleithner D, Gyra H, Cussler K. Suitability of an ELISA for the batch potency test in laboratory mice. Pharmeuropa BIO 1998;1:65-70). The humoral immune response is quantified in pooled sera of ten mice three weeks after immunisation. The results are expressed as relative potency (RP) in comparison to a reference serum. After a pre-validation study had been performed with success (Rosskopf-Streicher U, Johannes S, Wilhelm M, Gyra H, Cussler K. Potency testing of swine erysipelas vaccines by serology - results of a pre-validation study. ALTEX 1999;16:123-8), we initiated an international collaborative study with five European manufacturers and seven regulatory authorities to validate the assay and model. All participants were provided with blind-coded erysipelas vaccines of different potencies, the ELISA kit and test instructions. The participants had to immunise mice, to prepare serum samples and to perform the ELISA. Inter-laboratory reproducibility was reported by the pass/fail criteria of the vaccines under test. Intra-laboratory precision was assessed by comparing repeated measurements on three consecutive days. Day-to-day variation within the laboratories was statistically analysed by comparing pairs of RPs using Lin's concordance correlation coefficient. The results show that the ELISA is indeed a suitable alternative to replace the vaccination-challenge test. Furthermore, this new model reduces the number of animals required for the potency test by approximately 80%.

A 4R concept for the safety testing of immunobiologicals.

Safety tests in animals are an important part of the licensing procedures for human and veterinary medicines. Pharmacopoeial and other legal regulations require immunobiologicals to be approved for safety on a batch-to-batch basis. A large number of animals is needed to perform these general and specific safety tests. The search for alternatives according to the famous 3R concept proposed by Russel & Burch [1] is also relevant to safety tests. This session of the conference will highlight recent progress in the application of this concept. However, before considering potential alternatives to a test, it is advisable to re-evaluate the necessity of the safety test in question. Many animal-based safety test procedures were introduced because the production of biologicals was difficult and methods to control this production were limited. Nowadays much progress has been achieved due to a better knowledge of the prophylaxis of infectious diseases and the standardisation of methods of production and control. Therefore, some animal tests may no longer be necessary. A good example of this is the deletion of the abnormal toxicity test from the European Pharmacopoeia. Efforts to limit the use of animals in the quality control of immunobiologicals should therefore include a reassessment of the value of the safety test. Consequently, reassessment should be the first R used to evaluate whether an animal test can be removed. The 3R concept of Russel & Burch [1] to replace, reduce and refine animal tests has to be considered if that does not prove to be possible.

Development and prevalidation of two different ELISA systems for the potency testing of Clostridium perfringens beta and epsilon-toxoid containing veterinary vaccines.

The potency testing of Clostridium perfringens mono- and multicomponent veterinary vaccines is currently performed with the mouse neutralisation test (MNT) to estimate levels of C. perfringens beta- and epsilon-antitoxin levels in the sera of rabbits immunised with the vaccine. Two in vitro methods based on monoclonal antibodies (mAb) have been developed for the determination of specific antibodies against C. perfringens beta-toxin (capture ELISA) and epsilon-toxin (competitive ELISA) in these sera. Both test systems show high specificity and good reproducibility. These ELISA procedures were used in addition to the routine batch potency test in mice (MNT) to determine beta- and epsilon-antitoxin levels in 523 samples of rabbit serum. There was good agreement between the rank order of sera determined in vivo and the rank order determined in vitro. Linear regression analysis gave correlation coefficients of 0.88 for the capture ELISA and 0.41 for the competitive ELISA, with a significance level of P < 0.01 in both cases. Furthermore, a prevalidation study was carried out in four laboratories to evaluate the transferability of the ELISA procedures and the interlaboratory reproducibility of the results. Three coded serum samples were tested several times. The results indicated that both ELISA systems are suitable candidates to replace the MNT used for the potency testing of beta- and epsilon-toxoid in mono- and multicomponent veterinary vaccines. However, these assays still need to be validated in an international collaborative study.

Potency Testing of Swine Erysipelas Vaccines by Serology Results of a Pre-validation Study.

The European Pharmacopoeia (Ph. Eur.) monograph on Swine Erysipelas Vaccine (inactivated) (Ph. Eur., 1997) requires the potency of each batch to be demonstrated in a mouse protection test. In this parallel-line bioassay, mice are challenged with a virulent strain of Erysipelothrix (E.) rhusiopathiae after immunisation with different doses of either the standard preparation or of the test vaccine. More than one hundred animals are necessary for the routine testing of a single batch. In previous studies (Beckmann und Cubetaler, 1994; Rosskopf-Streicher et al., 1998), we have shown that an indirect enzyme-linked immunosorbent assay (ELISA) may be used to quantify the humoral response of mice. This method can replace the challenge model for the purposes of potency testing in the following manner: Ten mice are immunised subcutaneously with 1/10 of the vaccine dose required for pig vaccination. After three weeks, the mice are bled under anaesthesia. Serum samples are pooled and the antibody content is compared to that of a reference serum. In view of animal welfare the advantages of the alternative model are obvious: A highly reduced number of animals and the replacement of challenge exposure. This includes the discontinuation of the control group with a mortality rate of 100%. A pre-validation study was initiated to evaluate the performance of the serological method. Eight laboratories used the test kits to evaluate pooled serum samples from vaccinated mice. Both the reagents and the test protocol were shown to be satisfactory. The intra- and inter-laboratory reproducibility that was achieved indicates that the method is a strong candidate for validation.

Characterization of a protective protein antigen of Erysipelothrix rhusiopathiae.

Although vaccination is widely practiced against infection by Erysipelothrix rhusiopathiae in pigs and turkeys, the protective antigen(s) involved have not been fully characterized or purified to homogeneity. Antigens of E. rhusiopathiae strain T28, serotype 2b, and of FRANKFURT XI, serotype N, in culture supernatant and in extracts made with hot acid, 10 mM NaOH, ultrasound or EDTA were compared by SDS-PAGE and immunoblotting and in a mouse protection test. EDTA and 10 mM NaOH yielded highly protective extracts; culture supernatant was less protective and ultrasonic or hot acid extracts stimulated little or no protection in mice. Protective antisera from swine, horses and mice recognized prominent bands of molecular mass (m.m.) of 66-64 and 40-39 kDa in EDTA and 10 mM NaOH extracts. Mice immunized with preparations of the 66-64 kDa band purified by preparative electrophoresis were protected. Both antigens were trypsin sensitive, contained no detectable polysaccharide, and showed a marked tendency to aggregate in the absence of SDS.

Intrathymic tolerance induction: determination of tolerance to class II major histocompatibility complex antigens in maturing T lymphocytes by a bone marrow-derived non-lymphoid thymus cell.

Two new experimental approaches were established to analyse the influence of the thymus on tolerance induction to major histocompatibility complex (MHC) antigens: The aim of the first experiment was to perform successful transplantation of adult allogeneic thymus tissue into nude mice, an attempt that has been unsuccessful in the past. Tolerance for the MHC genotype of a prospective thymus graft recipient (A) was induced in mice of strain B by injection of (A X B) splenocytes during the neonatal period. Adult thymic tissue obtained from these allogeneic donors (B) were grafted into the nude mice of strain A. The allogeneic thymus was accepted by the nude mice and immunoreconstitution was achieved. Subsequently the recipients developed tolerance to the MHC antigens of the allogeneic thymus donor as proved by mixed lymphocyte cultures and the acceptance of skin grafts. The second experiment was designed to determine which Ia-positive thymic compartment participates in conferring tolerance to MHC antigens in maturing T lymphocytes. Chimaeric thymus grafts were created by transplantation of neonatal thymus (A) into allogeneic nude mice (B) for a period of 8 weeks. The graft was populated with host bone marrow-derived Ia antigen-positive cells. The chimaeric thymuses consisting of type A epithelium but populated with both type A and B lymphocytes and non-lymphoid cells (i.e. Ia-positive macrophages and dendritic cells), were newly transplanted into nude mice of strain A. The engraftment lead to immunological reconstitution and the nude mice acquired tolerance to the MHC antigens expressed by the allogeneic Ia-positive cells populating the chimaeric graft. Irradiation of the chimaeric thymus prior to transplantation allowed transplantation of chimaeric thymus devoid of living thymocytes but still populated with functionally intact Ia-positive non-lymphoid cells. Transplantation of irradiated chimaeric thymuses resulted in immunoreconstitution and induced exactly the same allotolerance pattern as described above. The results demonstrate that not thymus epithelial cells but a bone-marrow-derived non-lymphoid thymus cell, most likely the Ia-antigen-positive thymic macrophage of dendritic cell, is responsible for the induction of tolerance to MHC antigens in developing T lymphocytes.