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Technical Requirements for the Licensing of Pseudorabies (Aujeszky's Disease) Vaccines in the European Union
ADVANCES IN VETERINARY MEDICINE, VOL. 41
Technical Requirements for the Licensing of Pseudorabies (Aujeszky's Disease) Vaccines in the European Union E VANNIER Cneva Zoop6le Les Croix, BP 53 22400 Ploufragan, France
I. Introduction II. Safety Testing A. Principles B. Laboratory Testing C. Field Testing III. EfficacyTesting A. Laboratory Trials B. Field Trials IV. Batch Release Controls V. Summary References
I. I n t r o d u c t i o n In general, vaccine properties cannot be evaluated by a few simple tests. Many series of experiments have to be done in order to provide adequate information on the biological properties of a vaccine. These properties cannot be established without a review of all tests done on the vaccine and have to be determined by objective and quantifiable criteria. This approach is totally applicable to Aujeszky's disease (AD) vaccines. So, testing of AD vaccines is founded on several trials to define the properties of this kind of vaccine. But, in a second step and particularly in the case of a marketing authorization, it is necessary to define an 615 Copyright 9 1999 by Academic Press All rights of reproduction in any form reserved. 1093-975X/99 $25.00
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acceptability threshold for safety as well as for efficacy. This problem is not easy to solve. How do we determine this threshold? What criteria can be taken into consideration? Different options have been choosen in the existing regulatory and technical texts which are mainly the 92/18 E. U. directive and the European Pharmacopoeia (EP) monographs about live and inactivated AD vaccines used in pigs. In that text, because the quality part of the file is not specific to AD vaccines, it is not particularly developed, contrary to the safety and efficacy ones.
II. Safety Testing For a vaccine, local and general reactions have to be determined. When a live vaccine is used, it is necessary to differentiate the exact safety properties of the vaccinal strain from those of the finished product including the adjuvant. A. PRINCIPLES
In general, safety is tested initially under experimental conditions. When the results of these preliminary tests are known, it is necessary to enlarge the number of animals vaccinated in order to evaluate, under practical conditions, the safety of the vaccine. In all the cases, safety testing must be done on the pigs under the usual conditions of use or under abnormal conditions in order to reveal any reactions not discovered previously. In a first step, laboratory trials have to be performed and in a second step only, field trials can be done. These trials supplement, on a greater scale, the laboratory tests. B. LABORATORYTESTING General and local reactions must be examined. 1. General Effects Assessment a. Live Vaccines. The definition of the properties of an AD viral strain depends on the characteristics of this virus; so specific tests need to be performed to better understand the behavior of the vaccinal strain. Because the AD virus is neurotropic and is particularly pathogenic for young piglets, intracerebral tests and vaccination of 3-day-old piglets are very useful for determining the degree of safety of a strain. So in the EP monograph, five piglets, 3 - 5 days old each, received 104.5 TCIDso of the vaccine virus intracerebrally. None of the piglets should die or show signs of neurologic disorders.
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All of the information about intrinsic properties of AD strains cannot be provided because they are part of confidential marketing authorization applications files. But, as examples, the following data will illustrate this text. Some strains such as Alfort 26, which is thermosensitive, and ADV Omnimark have no effect on 2-day-old piglets inoculated by the intracerebral route (Toma et al., 1979; Kit, 1989). These strains as the 783 (TK-, gI-) and the Begonia ones do not provoke nervous signs when they are injected by the intramuscular route to very young piglets (2-4 days old) even if the 783 strain can induce slight depression and fever (Van Oirschot et al., 1990; Visser and Lfitticken, 1989). But, nervous signs and mortality depend on a dose-effect law: When the Begonia strain is injected by the intracerebral route to 2-day-old piglets (titer: 106.3 TCID5o/0.1 ml), five out of six piglets die, whereas no mortality is observed with a 105.3 TCID5o dose. This means that even TK- strains remain pathogenic particularly for the central nervous system when high doses of virus are used (Visser and Lfitticken, 1989). It is essential, too, to assess the properties of a vaccine and specifically of live ones in the target animals (e.g., in normal conditions of use for fattening pigs), generally vaccinated when they are between 9 and 12 weeks old and for pregnant sows when it is claimed by the manufacturer and authorized. Assays have been performed in pigs (4-10 weeks old) with Alfort 26, Bartha, and Begonia strains (Toma, 1979; McFerran and Dow, 1975; Visser and Lfitticken, 1989). No clinical signs including thermic reactions were observed after vaccination. So, in the EP monograph the animals used in the test for immunogenicity are also used to evaluate safety. The rectal temperature of the vaccinated animal is measured at the time of vaccination and 6, 24, and 48 hours later. No animal shows a temperature rise greater than 1.5~ and the number of animal showing a temperature greater than 41~ does not exceed 10% of the group. At slaughter, the injection site is examined for local reactions. No abnormal local reactions attributable to the vaccine are produced. The animals used for field trials are also used to evaluate safety. A test is carried out in each category of animals for which the vaccine is intended (sows, fattening pigs). Not fewer than three groups each of not fewer than 20 animals are used with corresponding groups of not fewer than 10 controls. The rectal temperature of each animal is measured at the time of vaccination and 6, 24, and 48 hours later. No animal shows a temperature rise greater than 1.5~ and the number of animals showing a temperature greater than 41~ does not exceed 25% of the group. Again, at slaughter, the injection site is examined for local reactions. No abnormal local reactions attributable to the vaccine are produced.
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In addition, 10 piglets, 3 - 4 weeks old that do not have antibodies against AD virus or against a fraction of the virus, each receive by a recommended route a quantity of virus corresponding to 10 doses of vaccines. Ten piglets of the same origin and age are kept as controls. The animals are observed for 21 days. The piglets have to remain in good health. The weight curve of the vaccinated piglets does not differ significantly from that of the controls. Ten piglets, 3 - 5 days old, which do not have antibodies against AD virus or against a fraction of the virus, each receive by the intranasal route a quantity of virus corresponding to 10 doses of vaccine. The animals are observed for 21 days. None of the piglets dies or shows signs of neurologic disorder attributable to the vaccine virus. Ten piglets, 3 - 4 weeks old, and which do not have antibodies against AD or against a fraction of the virus, each receive a daily injection of 2 mg of prednisolone per kilogram of body mass for 5 consecutive days. On the third day, each piglet receives a quantity of virus corresponding to one dose of vaccine by a recommended route. The animals are observed for 21 days following administration of the virus. The piglets m u s t remain in good health. To complement these studies in the target species, the real degree of attenuation of an AD viral strain can be evaluated by inoculation into other species such as chickens, dogs, cats, mice, etc. Reversion to virulence following serial passage has to be examined. Primary vaccination is done by the recommended route of administration, which is most likely to be followed by reversion to increased virulence. A series of at least five passages in piglets are made. The objective of these assays is to test the genetic stability of live vaccinal strains. They seem to be less necessary or unuseful when a genetically modified live strain is involved, especially when it was developed by gene deletion. However, it may be necessary to examine the possibility of recombination or genomic rearrangement with strains existing in the field or with other strains (Henderson et al., 1991). The virus excretion of a vaccinal strain by a vaccinated pig is interesting. Obviously the more the vaccine strain is disseminated throughout the body of a vaccinated animal, the greater the risk of spreading and shedding. Vaccinal strains such as Alfort 26 and Bartha are, in most cases, only recovered from the site of inoculation and the satellite lymph nodes (Toma et al., 1979; McFerran and Dow, 1975). In the EP monograph, 14 pigs, 3 - 4 weeks old and which do not have antibodies against AD virus or against a fraction of the virus, are vaccinated with one dose of vaccine by the recommended route and at the recommended site. Four pigs are kept as contact controls. Nasal and oral swabs
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are collected daily from the day before vaccination until 10 days after vaccination. The vaccine is acceptable if the virus is not isolated from the secretions collected. The ability of the AD vaccine strain to spread from a vaccinated pig to unvaccinated ones (transmissibility) must be tested by using the recommended route of administration. In the EP monograph, the same test is carried out on four separate occasions. Each time, four piglets of the same age (without any AD antibody) are kept together with them. Antibodies are not detected in any group of contact controls (5 weeks later). Until recently, it appeared interesting to know more about the possibility t h a t a t t e n u a t e d AD vaccinal strains can also become latent with the initiation of eradication programs in different countries (Mengeling, 1991). Studies demonstrated t h a t an a t t e n u a t e d TK negative strain of AD virus can establish a reactivatable latent infection in pigs (Mengeling, 1991). No reactivation was observed after vaccination of pigs with vaccinal strains t h a t had a naturally occurring gene deletion for viral glycoprotein I (gI) (Mengeling, 1991; Van Oirschot and Gielkens, 1984). If live vaccines are used on pregnant sows, the effects on the progeny have to be studied. The born piglets should not become infected by the vaccinal strain. b. Inactivated Vaccines. As for live ones, it is essential to test the inactivated vaccines in the target animals in normal conditions of use for fattening pigs and for sows when it is claimed by the m a n u f a c t u r e r and authorized in the different countries. As described previously, it is fundamental to use objective and quantifiable criteria to detect and to measure adverse reactions such as t e m p e r a t u r e before and after the vaccinations on vaccinated and control groups, weight performances, litter size, and reproductive performance. So the tests have to be performed by administering the vaccine in the recommended dose and at each recommended route of administration to the pigs for which it is intended. The pigs or sows are usually kept under observation and submitted to examinations until any reaction has disappeared and the period of observation m u s t not be less t h a n 14 days after administration. This period has to be extended when, for example, the vaccine is used in pregnant sows and it is necessary to assess the putative effects of the vaccine on the reproductive performance, which means the period of observation is, for those conditions, the duration of pregnancy and lasts until the farrowing. Moreover, it is generally requested to vaccinate with a double dose to have a better opportunity to detect adverse reactions which could be at the limit of a detectable level when a single dose is administered.
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Local reactions are often associated with the use of inactivated vaccines as these side effects can be induced by adjuvants and particularly oil adjuvants. But some AD live vaccines are mixed with different adjuvants which modify what was observed up to now. The local reactions are of two types" allergic or inflammatory. In the case of AD vaccines, local reactions are mainly inflammatory and can be more or less complicated (necrotic or suppurative) depending on the nature of the adjuvants used and the aseptic conditions of the vaccination. Oil adjuvants can induce a great variety of effects ranging from muscular degeneration to granuloma, fibrosis, and abscessation. In addition to the nature of the oil used (the intensity of the reaction is reduced when metabolizable oils are used in the vaccine), it is the type of the emulsion (water/oil, oil/water, water/oil/water) which induces these reactions to a greater or lesser extent (Hall et al., 1989; Vannier, 1986). In consequence, it is necessary to observe not only from the outside the site of injection, but also by dissection when slaughtering the pigs and particularly the finishing ones. C. FIELD TESTING
Field trials are necessary to assess the safety of an AD vaccine in a high number of pigs or sows. In Europe, tests are carried out in each category of animals for which the vaccine is intended (sows, fattening pigs). Not fewer than three groups each of not fewer than 20 animals are used with corresponding groups of not fewer than 10 controls. The rectal temperature of each animal is measured at the time of vaccination and 6, 24, and 48 hours later. At slaughter, the injection site has to be examined for local reactions. If the vaccine is intended to be used in sows, reproductive performances have to be recorded. [Editor's note: The extensive battery of safety tests described here for vaccines licensed by the European Union is not required by the USDA for licensing of similar vaccines in the U.S.]
III. Efficacy Testing A. LABORATORYTRIALS
The biological properties of vaccines are generally based on the clinical protection they confer through passive immunity by vaccinating the dams or by actively immunizing the growing pigs.
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1. Assessment of Passive Immunity To test the efficacy of vaccines, it is important to mimic the natural infection conditions. AD infection provokes important losses in young piglets from nonimmune sows. So, when vaccinating sows, the main goal is to protect the young piglets through passive immunity confered by the colostrum ingested immediately after birth. To measure this passive immunity and the protection induced by vaccinating the sows, experimental models were carried out. Eight sows are vaccinated according to the vaccinal scheme and by the recommended route during pregnancy and, when the piglets are between 6 and 10 days old they are given an intranasal challenge exposure with a virulent AD strain (Andries et al., 1978; Vannier et al., 1976). Different values of virulent virus titers were used in such assays: 2 x 105 TCID5o/2 ml or 103 to 104 UFP/ml. It is better to use a strain titrated in lethal dose 50. It is recommended to inoculate by the nasal route, 102 pig LD 50 per pig in 1 ml. The efficacy of the vaccine is assessed by comparing clinical signs, mainly mortality on piglets from unvaccinated dams (minimum of four in EP monographs) with the ones observed on piglets from vaccinated sows. The vaccine is satisfactory if not less than 80% protection against mortality is found in the piglets from the vaccinated sows compared to those from the control sows. The test is not valid if the average number of piglets per litter for each group is less than six. 2. Assessment of Active Immunity a. Clinical protection. Several criteria can be taken under consideration to measure the active immunity induced by vaccinating the pigs. Generally, the pigs are vaccinated at the beginning of the growing period, which means when they are between 9 and 12 weeks old. The laboratory trials are performed by challenging the pigs at the end of the finishing period when they weigh between 80 and 90 kg. In general, at least three criteria such as rectal temperature, weight loss, and clinical signs with mortality are used to measure the clinical protection of pigs after vaccination and challenge. The antibody titers have little predictive value for the efficacy of the vaccines (de Leeuw and Van Oirschot, 1985). Weight loss compared between the vaccinated and control groups is certainly the parameter that is the most reproducible and quantifiable when the challenge conditions are well standardized. The measure of the difference of weight gain or loss between the two groups of pigs and in the interval of time between challenge (DO) and D7 (7 days later) has a very good predictive value for the efficacy of the vaccines (de Leeuw and Van Oirschot, 1985). In the EP monograph, it is indicated that each animal (10 vaccinated pigs, 5
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controls) is weighed and then challenged by the intranasal route with a suitable quantity of a virulent strain of AD virus (at least 106 TCID5o of a virulent strain having undergone not more than three passages and administered in not less than 4 ml of diluent). Each animal is weighed 7 days after challenge or at the time of death if this occurs earlier and the average daily gain is calculated as a percentage. The vaccine complies with the test if: 9 All the vaccinated pigs survive and the difference between the averages of the daily gains for the two groups is not less than 1.5 9 The geometrical mean titers and the duration of excretion of the challenge virus are significantly lower in vaccinates than in controls. The test is not valid unless all the control pigs display signs of AD and the average of their daily gains is less than -0.5. Mean titers and the duration of excretion of the challenge virus are determined in swabs taken from the nasal cavity of each animal daily from the day before challenge until virus is no longer detected. This method to evaluate the efficacy of AD vaccine is now well tested, which allows us to lay down an objective index that provides the opportunity to determine the level of efficacy of a vaccine (Stellmann et al., 1989). This index, which compares the relative weight losses between vaccinated pigs and control ones, can also be used in releasing batch controls (potency testing) as in efficacy testing. But the acceptable value of the index is different in the two tests (1.5 for the efficacy testing, 1 for potency testing). Figure 1 shows the mean virus titers excreted by pigs from different vaccinated and control groups. Different synthetic index can be used to express the quantity of virulent virus excreted by pigs taking into consideration the duration and the level of viral excretion, as the number of pigs excreting virulent virus. Differences between vaccines can be observed using similar protocols (Pensaert et al., 1990; Vannier et al., 1991). The effects of vaccine with regard to viral shedding were compared when the vaccines where used in the presence or absence of passive immunity. The comparison suggested that the clinical protection provided by the vaccines was relatively lower when the pigs were first vaccinated while posessing passive antibodies, which is a well-known phenomenon. Likewise, viral excretion appeared to be elevated when passive antibodies were present, but the relative position of the vaccines was the same when they were compared in the presence or absence of maternal immunity. Thus, it may be prudent to compare vaccines without passive antibodies to better standardize the assay.
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Control batch 8Vaccine
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A
Vaccine B
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Days FIG. 1. Mean titers of virus excreted by pigs vaccinated with three different vaccines compared with the level of virus shedding by control pigs.
B. F I E L D TRIALS
In general terms, it is extremely difficult to assess vaccine efficacy in animal populations. To do this, it would be necessary to vaccinate the animals in the absence of the pathogen that the vaccines protects against, then to await the moment of infection and to compare the effects of infection in vaccinated animals (or the offspring of vaccinated dams) with the effects in the unvaccinated animals of the same age, in the same building, and in the same batch as the vaccinated animals (or those protected passively). All of these conditions are difficult to realize in the field. That is why field trials are certainly more appropriate to safety testing than to efficacy testing. In Directive 92/18 for efficacy, field trials are not absolutely necessary if good experimental data are provided.
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P. VANNIER IV. Batch Release Controls
It is essential to differentiate the tests that are carried out on a routine basis to release the produced batches from those performed to define the biological properties of a vaccine. The trials carried out for batch releasing are not the same as the ones carried out once to determine safety and efficacy of a vaccine. The batch release controls are always short-term trials, as cheap as possible, and not systematically carried out in the pigs. Their purpose is mainly to attest to the consistency of the quality of the finished product, which has to be in conformity with the quality initially defined in the marketing authorization application. For safety, these batch release trials can be performed on guinea pigs, pigs, rabbits, or other species depending on whether the vaccine is inactivated or live. For potency, if good correlation was established between trials carried out on a routine basis and the efficacy ones done once, it can be used in vitro tests (titration, etc.) in vivo trials by challenging pigs or other susceptible species (mice, rabbits, etc.), by measuring the antibody response after vaccination. In t h a t kind of control, the most difficult point is to determine an acceptability threshold to accept or to reject the batch according to the results obtained. [Editor's note: Batch release controls involving animals are not required by USDA, but many of my colleagues feel they should be required with vaccines used in food animals and companion animals. A small number of animals should be used to assess the continued immunogenicity of the vaccine.]
V. S u m m a r y Under the light of current scientific knowledge, particularly with the progress of molecular biology and of the definition of assays to be performed, it is possible to know, as accurately as possible, the biological properties of a vaccine. Most requirements of EP monographs and Directive 92/18 are founded on t h a t concept. It is clear that there is a balance between safety and efficacy in the case of a live attenuated viral strain t h a t means the more efficient a strain, the less safe it can be. Nevertheless, the problem is more complex; considerable progress has been done to set up new finished products and particularly with the adjuvants which are used now even in combination with live attenuated AD strains. The efficacy of a vaccine can be greatly enhanced, maintaining good local and general safety.
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B u t a d e b a t e a l w a y s occurs w h e n it is n e c e s s a r y to d e t e r m i n e t h e a c c e p t a b i l i t y t h r e s h o l d of a v a c c i n e w i t h r e g a r d to its s a f e t y a n d efficacy. T h e p o i n t s of v i e w a r e o f t e n v e r y d i v e r g e n t . B u t , in a n y case, t h i s t h r e s h o l d d e p e n d s on t h e local c o n d i t i o n s in t h e d i f f e r e n t c o u n t r i e s . I t is c l e a r t h a t o b j e c t i v e s of a v a c c i n a t i o n p r o g r a m a n d t h e r e q u i r e m e n t s a b o u t a v a c c i n e c a n n o t be t h e s a m e in h e a v i l y i n f e c t e d c o u n t r i e s w i t h a c o m p u l s o r y v a c c i n a t i o n p r o g r a m as in c o u n t r i e s or r e g i o n s w i t h a low p r e v a l e n c e of AD i n f e c t i o n or w i t h a n a b s e n c e of a n y infection. M o r e over, it m u s t also be c o n s i d e r e d t h a t v a c c i n e s c o n s t i t u t e only one elem e n t of a c o n t r o l or e r a d i c a t i o n p r o g r a m t a r g e t e d a g a i n s t A u j e s z k y ' s disease virus.
REFERENCES Andries, K., Pensaert, M. B., and Vandeputte, J. (1978). Effect of experimental infection with pseudorabies (Aujeszky's Disease) virus on pigs with maternal immunity from vaccinated sows. Am. J. Vet. Res. 39(8), 1282-1285. de Leeuw, P. W., and Van Oirschot, J. T. (1985). Vaccines against Aujeszky's disease: Evaluation of their efficacy under standardized laboratory conditions. Vet. Q. 7, 780786. Hall, W., Molitor, T. W., Joo, H. S., and Pijoan, C. (1989). Comparison of protective immunity and inflammatory responses of pigs following immunization with different Actinobacillus pleuropneumoniae preparations with and without adjuvants. Vet. Immunol. Immunopathol. 22, 175-186. Henderson, L. M., Randall, L. L., David, A. J., and Stutz, D. R. (1991). Recombination of pseudorabies virus vaccine strains in swine. Am. J. Vet. Res. 52(6), 820-825. Kit, S. (1989). Safety and efficacy of genetically engineered Aujeszky's disease vaccines. In "Vaccination and Control of Aujeszky's Disease," (J. T. Van Oirschot, ed.), pp. 4555. McFerran, J. B., and Dow, C. (1975). Studies on immunisation of pigs with the Bartha strain of Aujeszky's disease virus. Res. Vet. Sci. 19, 17-22. Mengeling, W. L. (1991). Virus reactivation in pigs latently infected with a thymidine kinase negative vaccine strain of pseudorabies virus. Arch. Virol. 120, 57-70. Pensaert, M. B., de Smet, K., and de Waele, K. (1990). Extent and duration of virulent virus excretion upon challenge of pigs vaccinated with different Glycoprotein-Deleted Aujeszky's disease vaccines. Vet. Microbiol. 22, 107-117. Stellmann, C., Vannier, P., Chappuis, G., Brun, A., Dauvergne, M., Fargeaud, D., Bugaud, M., and Colson, X. (1989). The potency testing of pseudorabies vaccines in pigs. A proposal for a quantitative criterion and a minimum requirement. J. Biol. Stand. 17, 17-27. Toma, B. (1979). Obtention et caracterisation d'une souche thermosensible de virus de la maladie d'Aujeszky (souche ALFORT 26). Recl. Med. Vet. 155(2), 131-137. Toma, B., Brun, A., Chappuis, G., and Terre, J. (1979). Propri6t6s biologiques d'une souche thermosensible (ALFORT 26) de virus de la maladie d'Aujeszky. Recl. Med. Vet. 155(3), 245-252. Vannier, P. (1986). Immunisation de porcs charcutiers contre la maladie d'Aujeszky avec deux vaccins ~ adjuvants huileux. Etude des r6actions locales. Recl. Med. Vet. 162(1), 37-44.
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Vannier, P., Tillon, J. P., Toma, B., Delagneau, J. F., Loquerie, R., and Prunet, P. (1976). Protection conf6r~e au porc par un nouveau vaccin huileux h virus inactiv~ contre la maladie d'Aujeszky. Consequences pratiques. J. Rech. Porc Fr. pp. 281-290. Vannier, P., Hutet, E., Bourgueil, E., and Cariolet, R. (1991). Level of virulent virus excreted by infected pigs previously vaccinated with different glycoprotein deleted Aujeszky's diseases vaccines. Vet. Microbiol. 29, 213-223. Van Oirschot, J. T., and Gielkens, A. L. J. (1984). Intranasal vaccination of pigs against pseudorabies: Absence of vaccinal virus latency and failure to prevent latency of virulent virus. Am. J. Vet. Res. 45(10), 2099-2103. Van Oirschot, J. T., Terpstra, C., Moormann, R. J. M., Berns, A. J. M., and Gielkens, A. L. J. (1990). Safety of an Aujeszky's disease vaccine based on deletion mutant strain 783 which does not express thymidine kinase and glycoprotein I. Vet. Rec. 127, 443-446. Visser, N., and Ltitticken, D. (1989). Experiences with a gl-/TK- modified live pseudorabies virus vaccine: Strain Begonia. In 'Vaccination and Control of Aujeszky's Disease" (J. T. Van Oirschot, ed.), 37-44.
Technical Requirements for the Licensing of Pseudorabies (Aujeszky's Disease) Vaccines in the European Union E VANNIER Cneva Zoop6le Les Croix, BP 53 22400 Ploufragan, France
I. Introduction II. Safety Testing A. Principles B. Laboratory Testing C. Field Testing III. EfficacyTesting A. Laboratory Trials B. Field Trials IV. Batch Release Controls V. Summary References
I. I n t r o d u c t i o n In general, vaccine properties cannot be evaluated by a few simple tests. Many series of experiments have to be done in order to provide adequate information on the biological properties of a vaccine. These properties cannot be established without a review of all tests done on the vaccine and have to be determined by objective and quantifiable criteria. This approach is totally applicable to Aujeszky's disease (AD) vaccines. So, testing of AD vaccines is founded on several trials to define the properties of this kind of vaccine. But, in a second step and particularly in the case of a marketing authorization, it is necessary to define an 615 Copyright 9 1999 by Academic Press All rights of reproduction in any form reserved. 1093-975X/99 $25.00
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acceptability threshold for safety as well as for efficacy. This problem is not easy to solve. How do we determine this threshold? What criteria can be taken into consideration? Different options have been choosen in the existing regulatory and technical texts which are mainly the 92/18 E. U. directive and the European Pharmacopoeia (EP) monographs about live and inactivated AD vaccines used in pigs. In that text, because the quality part of the file is not specific to AD vaccines, it is not particularly developed, contrary to the safety and efficacy ones.
II. Safety Testing For a vaccine, local and general reactions have to be determined. When a live vaccine is used, it is necessary to differentiate the exact safety properties of the vaccinal strain from those of the finished product including the adjuvant. A. PRINCIPLES
In general, safety is tested initially under experimental conditions. When the results of these preliminary tests are known, it is necessary to enlarge the number of animals vaccinated in order to evaluate, under practical conditions, the safety of the vaccine. In all the cases, safety testing must be done on the pigs under the usual conditions of use or under abnormal conditions in order to reveal any reactions not discovered previously. In a first step, laboratory trials have to be performed and in a second step only, field trials can be done. These trials supplement, on a greater scale, the laboratory tests. B. LABORATORYTESTING General and local reactions must be examined. 1. General Effects Assessment a. Live Vaccines. The definition of the properties of an AD viral strain depends on the characteristics of this virus; so specific tests need to be performed to better understand the behavior of the vaccinal strain. Because the AD virus is neurotropic and is particularly pathogenic for young piglets, intracerebral tests and vaccination of 3-day-old piglets are very useful for determining the degree of safety of a strain. So in the EP monograph, five piglets, 3 - 5 days old each, received 104.5 TCIDso of the vaccine virus intracerebrally. None of the piglets should die or show signs of neurologic disorders.
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All of the information about intrinsic properties of AD strains cannot be provided because they are part of confidential marketing authorization applications files. But, as examples, the following data will illustrate this text. Some strains such as Alfort 26, which is thermosensitive, and ADV Omnimark have no effect on 2-day-old piglets inoculated by the intracerebral route (Toma et al., 1979; Kit, 1989). These strains as the 783 (TK-, gI-) and the Begonia ones do not provoke nervous signs when they are injected by the intramuscular route to very young piglets (2-4 days old) even if the 783 strain can induce slight depression and fever (Van Oirschot et al., 1990; Visser and Lfitticken, 1989). But, nervous signs and mortality depend on a dose-effect law: When the Begonia strain is injected by the intracerebral route to 2-day-old piglets (titer: 106.3 TCID5o/0.1 ml), five out of six piglets die, whereas no mortality is observed with a 105.3 TCID5o dose. This means that even TK- strains remain pathogenic particularly for the central nervous system when high doses of virus are used (Visser and Lfitticken, 1989). It is essential, too, to assess the properties of a vaccine and specifically of live ones in the target animals (e.g., in normal conditions of use for fattening pigs), generally vaccinated when they are between 9 and 12 weeks old and for pregnant sows when it is claimed by the manufacturer and authorized. Assays have been performed in pigs (4-10 weeks old) with Alfort 26, Bartha, and Begonia strains (Toma, 1979; McFerran and Dow, 1975; Visser and Lfitticken, 1989). No clinical signs including thermic reactions were observed after vaccination. So, in the EP monograph the animals used in the test for immunogenicity are also used to evaluate safety. The rectal temperature of the vaccinated animal is measured at the time of vaccination and 6, 24, and 48 hours later. No animal shows a temperature rise greater than 1.5~ and the number of animal showing a temperature greater than 41~ does not exceed 10% of the group. At slaughter, the injection site is examined for local reactions. No abnormal local reactions attributable to the vaccine are produced. The animals used for field trials are also used to evaluate safety. A test is carried out in each category of animals for which the vaccine is intended (sows, fattening pigs). Not fewer than three groups each of not fewer than 20 animals are used with corresponding groups of not fewer than 10 controls. The rectal temperature of each animal is measured at the time of vaccination and 6, 24, and 48 hours later. No animal shows a temperature rise greater than 1.5~ and the number of animals showing a temperature greater than 41~ does not exceed 25% of the group. Again, at slaughter, the injection site is examined for local reactions. No abnormal local reactions attributable to the vaccine are produced.
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In addition, 10 piglets, 3 - 4 weeks old that do not have antibodies against AD virus or against a fraction of the virus, each receive by a recommended route a quantity of virus corresponding to 10 doses of vaccines. Ten piglets of the same origin and age are kept as controls. The animals are observed for 21 days. The piglets have to remain in good health. The weight curve of the vaccinated piglets does not differ significantly from that of the controls. Ten piglets, 3 - 5 days old, which do not have antibodies against AD virus or against a fraction of the virus, each receive by the intranasal route a quantity of virus corresponding to 10 doses of vaccine. The animals are observed for 21 days. None of the piglets dies or shows signs of neurologic disorder attributable to the vaccine virus. Ten piglets, 3 - 4 weeks old, and which do not have antibodies against AD or against a fraction of the virus, each receive a daily injection of 2 mg of prednisolone per kilogram of body mass for 5 consecutive days. On the third day, each piglet receives a quantity of virus corresponding to one dose of vaccine by a recommended route. The animals are observed for 21 days following administration of the virus. The piglets m u s t remain in good health. To complement these studies in the target species, the real degree of attenuation of an AD viral strain can be evaluated by inoculation into other species such as chickens, dogs, cats, mice, etc. Reversion to virulence following serial passage has to be examined. Primary vaccination is done by the recommended route of administration, which is most likely to be followed by reversion to increased virulence. A series of at least five passages in piglets are made. The objective of these assays is to test the genetic stability of live vaccinal strains. They seem to be less necessary or unuseful when a genetically modified live strain is involved, especially when it was developed by gene deletion. However, it may be necessary to examine the possibility of recombination or genomic rearrangement with strains existing in the field or with other strains (Henderson et al., 1991). The virus excretion of a vaccinal strain by a vaccinated pig is interesting. Obviously the more the vaccine strain is disseminated throughout the body of a vaccinated animal, the greater the risk of spreading and shedding. Vaccinal strains such as Alfort 26 and Bartha are, in most cases, only recovered from the site of inoculation and the satellite lymph nodes (Toma et al., 1979; McFerran and Dow, 1975). In the EP monograph, 14 pigs, 3 - 4 weeks old and which do not have antibodies against AD virus or against a fraction of the virus, are vaccinated with one dose of vaccine by the recommended route and at the recommended site. Four pigs are kept as contact controls. Nasal and oral swabs
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are collected daily from the day before vaccination until 10 days after vaccination. The vaccine is acceptable if the virus is not isolated from the secretions collected. The ability of the AD vaccine strain to spread from a vaccinated pig to unvaccinated ones (transmissibility) must be tested by using the recommended route of administration. In the EP monograph, the same test is carried out on four separate occasions. Each time, four piglets of the same age (without any AD antibody) are kept together with them. Antibodies are not detected in any group of contact controls (5 weeks later). Until recently, it appeared interesting to know more about the possibility t h a t a t t e n u a t e d AD vaccinal strains can also become latent with the initiation of eradication programs in different countries (Mengeling, 1991). Studies demonstrated t h a t an a t t e n u a t e d TK negative strain of AD virus can establish a reactivatable latent infection in pigs (Mengeling, 1991). No reactivation was observed after vaccination of pigs with vaccinal strains t h a t had a naturally occurring gene deletion for viral glycoprotein I (gI) (Mengeling, 1991; Van Oirschot and Gielkens, 1984). If live vaccines are used on pregnant sows, the effects on the progeny have to be studied. The born piglets should not become infected by the vaccinal strain. b. Inactivated Vaccines. As for live ones, it is essential to test the inactivated vaccines in the target animals in normal conditions of use for fattening pigs and for sows when it is claimed by the m a n u f a c t u r e r and authorized in the different countries. As described previously, it is fundamental to use objective and quantifiable criteria to detect and to measure adverse reactions such as t e m p e r a t u r e before and after the vaccinations on vaccinated and control groups, weight performances, litter size, and reproductive performance. So the tests have to be performed by administering the vaccine in the recommended dose and at each recommended route of administration to the pigs for which it is intended. The pigs or sows are usually kept under observation and submitted to examinations until any reaction has disappeared and the period of observation m u s t not be less t h a n 14 days after administration. This period has to be extended when, for example, the vaccine is used in pregnant sows and it is necessary to assess the putative effects of the vaccine on the reproductive performance, which means the period of observation is, for those conditions, the duration of pregnancy and lasts until the farrowing. Moreover, it is generally requested to vaccinate with a double dose to have a better opportunity to detect adverse reactions which could be at the limit of a detectable level when a single dose is administered.
620
P. VANNIER 2. Local Reactions
Local reactions are often associated with the use of inactivated vaccines as these side effects can be induced by adjuvants and particularly oil adjuvants. But some AD live vaccines are mixed with different adjuvants which modify what was observed up to now. The local reactions are of two types" allergic or inflammatory. In the case of AD vaccines, local reactions are mainly inflammatory and can be more or less complicated (necrotic or suppurative) depending on the nature of the adjuvants used and the aseptic conditions of the vaccination. Oil adjuvants can induce a great variety of effects ranging from muscular degeneration to granuloma, fibrosis, and abscessation. In addition to the nature of the oil used (the intensity of the reaction is reduced when metabolizable oils are used in the vaccine), it is the type of the emulsion (water/oil, oil/water, water/oil/water) which induces these reactions to a greater or lesser extent (Hall et al., 1989; Vannier, 1986). In consequence, it is necessary to observe not only from the outside the site of injection, but also by dissection when slaughtering the pigs and particularly the finishing ones. C. FIELD TESTING
Field trials are necessary to assess the safety of an AD vaccine in a high number of pigs or sows. In Europe, tests are carried out in each category of animals for which the vaccine is intended (sows, fattening pigs). Not fewer than three groups each of not fewer than 20 animals are used with corresponding groups of not fewer than 10 controls. The rectal temperature of each animal is measured at the time of vaccination and 6, 24, and 48 hours later. At slaughter, the injection site has to be examined for local reactions. If the vaccine is intended to be used in sows, reproductive performances have to be recorded. [Editor's note: The extensive battery of safety tests described here for vaccines licensed by the European Union is not required by the USDA for licensing of similar vaccines in the U.S.]
III. Efficacy Testing A. LABORATORYTRIALS
The biological properties of vaccines are generally based on the clinical protection they confer through passive immunity by vaccinating the dams or by actively immunizing the growing pigs.
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1. Assessment of Passive Immunity To test the efficacy of vaccines, it is important to mimic the natural infection conditions. AD infection provokes important losses in young piglets from nonimmune sows. So, when vaccinating sows, the main goal is to protect the young piglets through passive immunity confered by the colostrum ingested immediately after birth. To measure this passive immunity and the protection induced by vaccinating the sows, experimental models were carried out. Eight sows are vaccinated according to the vaccinal scheme and by the recommended route during pregnancy and, when the piglets are between 6 and 10 days old they are given an intranasal challenge exposure with a virulent AD strain (Andries et al., 1978; Vannier et al., 1976). Different values of virulent virus titers were used in such assays: 2 x 105 TCID5o/2 ml or 103 to 104 UFP/ml. It is better to use a strain titrated in lethal dose 50. It is recommended to inoculate by the nasal route, 102 pig LD 50 per pig in 1 ml. The efficacy of the vaccine is assessed by comparing clinical signs, mainly mortality on piglets from unvaccinated dams (minimum of four in EP monographs) with the ones observed on piglets from vaccinated sows. The vaccine is satisfactory if not less than 80% protection against mortality is found in the piglets from the vaccinated sows compared to those from the control sows. The test is not valid if the average number of piglets per litter for each group is less than six. 2. Assessment of Active Immunity a. Clinical protection. Several criteria can be taken under consideration to measure the active immunity induced by vaccinating the pigs. Generally, the pigs are vaccinated at the beginning of the growing period, which means when they are between 9 and 12 weeks old. The laboratory trials are performed by challenging the pigs at the end of the finishing period when they weigh between 80 and 90 kg. In general, at least three criteria such as rectal temperature, weight loss, and clinical signs with mortality are used to measure the clinical protection of pigs after vaccination and challenge. The antibody titers have little predictive value for the efficacy of the vaccines (de Leeuw and Van Oirschot, 1985). Weight loss compared between the vaccinated and control groups is certainly the parameter that is the most reproducible and quantifiable when the challenge conditions are well standardized. The measure of the difference of weight gain or loss between the two groups of pigs and in the interval of time between challenge (DO) and D7 (7 days later) has a very good predictive value for the efficacy of the vaccines (de Leeuw and Van Oirschot, 1985). In the EP monograph, it is indicated that each animal (10 vaccinated pigs, 5
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controls) is weighed and then challenged by the intranasal route with a suitable quantity of a virulent strain of AD virus (at least 106 TCID5o of a virulent strain having undergone not more than three passages and administered in not less than 4 ml of diluent). Each animal is weighed 7 days after challenge or at the time of death if this occurs earlier and the average daily gain is calculated as a percentage. The vaccine complies with the test if: 9 All the vaccinated pigs survive and the difference between the averages of the daily gains for the two groups is not less than 1.5 9 The geometrical mean titers and the duration of excretion of the challenge virus are significantly lower in vaccinates than in controls. The test is not valid unless all the control pigs display signs of AD and the average of their daily gains is less than -0.5. Mean titers and the duration of excretion of the challenge virus are determined in swabs taken from the nasal cavity of each animal daily from the day before challenge until virus is no longer detected. This method to evaluate the efficacy of AD vaccine is now well tested, which allows us to lay down an objective index that provides the opportunity to determine the level of efficacy of a vaccine (Stellmann et al., 1989). This index, which compares the relative weight losses between vaccinated pigs and control ones, can also be used in releasing batch controls (potency testing) as in efficacy testing. But the acceptable value of the index is different in the two tests (1.5 for the efficacy testing, 1 for potency testing). Figure 1 shows the mean virus titers excreted by pigs from different vaccinated and control groups. Different synthetic index can be used to express the quantity of virulent virus excreted by pigs taking into consideration the duration and the level of viral excretion, as the number of pigs excreting virulent virus. Differences between vaccines can be observed using similar protocols (Pensaert et al., 1990; Vannier et al., 1991). The effects of vaccine with regard to viral shedding were compared when the vaccines where used in the presence or absence of passive immunity. The comparison suggested that the clinical protection provided by the vaccines was relatively lower when the pigs were first vaccinated while posessing passive antibodies, which is a well-known phenomenon. Likewise, viral excretion appeared to be elevated when passive antibodies were present, but the relative position of the vaccines was the same when they were compared in the presence or absence of maternal immunity. Thus, it may be prudent to compare vaccines without passive antibodies to better standardize the assay.
623
LICENSING OF PSEUDORABIES VACCINES IN THE E . U .
Control batch 8Vaccine
7
~
~
/ 6
A
Vaccine B
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-
'
9
.... VaccineC
.--. 5 4 o.)
3 2 1 0 DO
D+I
D+2
D+3
D+4
D+5
D+6
D+7
D+8
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Days FIG. 1. Mean titers of virus excreted by pigs vaccinated with three different vaccines compared with the level of virus shedding by control pigs.
B. F I E L D TRIALS
In general terms, it is extremely difficult to assess vaccine efficacy in animal populations. To do this, it would be necessary to vaccinate the animals in the absence of the pathogen that the vaccines protects against, then to await the moment of infection and to compare the effects of infection in vaccinated animals (or the offspring of vaccinated dams) with the effects in the unvaccinated animals of the same age, in the same building, and in the same batch as the vaccinated animals (or those protected passively). All of these conditions are difficult to realize in the field. That is why field trials are certainly more appropriate to safety testing than to efficacy testing. In Directive 92/18 for efficacy, field trials are not absolutely necessary if good experimental data are provided.
624
P. VANNIER IV. Batch Release Controls
It is essential to differentiate the tests that are carried out on a routine basis to release the produced batches from those performed to define the biological properties of a vaccine. The trials carried out for batch releasing are not the same as the ones carried out once to determine safety and efficacy of a vaccine. The batch release controls are always short-term trials, as cheap as possible, and not systematically carried out in the pigs. Their purpose is mainly to attest to the consistency of the quality of the finished product, which has to be in conformity with the quality initially defined in the marketing authorization application. For safety, these batch release trials can be performed on guinea pigs, pigs, rabbits, or other species depending on whether the vaccine is inactivated or live. For potency, if good correlation was established between trials carried out on a routine basis and the efficacy ones done once, it can be used in vitro tests (titration, etc.) in vivo trials by challenging pigs or other susceptible species (mice, rabbits, etc.), by measuring the antibody response after vaccination. In t h a t kind of control, the most difficult point is to determine an acceptability threshold to accept or to reject the batch according to the results obtained. [Editor's note: Batch release controls involving animals are not required by USDA, but many of my colleagues feel they should be required with vaccines used in food animals and companion animals. A small number of animals should be used to assess the continued immunogenicity of the vaccine.]
V. S u m m a r y Under the light of current scientific knowledge, particularly with the progress of molecular biology and of the definition of assays to be performed, it is possible to know, as accurately as possible, the biological properties of a vaccine. Most requirements of EP monographs and Directive 92/18 are founded on t h a t concept. It is clear that there is a balance between safety and efficacy in the case of a live attenuated viral strain t h a t means the more efficient a strain, the less safe it can be. Nevertheless, the problem is more complex; considerable progress has been done to set up new finished products and particularly with the adjuvants which are used now even in combination with live attenuated AD strains. The efficacy of a vaccine can be greatly enhanced, maintaining good local and general safety.
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B u t a d e b a t e a l w a y s occurs w h e n it is n e c e s s a r y to d e t e r m i n e t h e a c c e p t a b i l i t y t h r e s h o l d of a v a c c i n e w i t h r e g a r d to its s a f e t y a n d efficacy. T h e p o i n t s of v i e w a r e o f t e n v e r y d i v e r g e n t . B u t , in a n y case, t h i s t h r e s h o l d d e p e n d s on t h e local c o n d i t i o n s in t h e d i f f e r e n t c o u n t r i e s . I t is c l e a r t h a t o b j e c t i v e s of a v a c c i n a t i o n p r o g r a m a n d t h e r e q u i r e m e n t s a b o u t a v a c c i n e c a n n o t be t h e s a m e in h e a v i l y i n f e c t e d c o u n t r i e s w i t h a c o m p u l s o r y v a c c i n a t i o n p r o g r a m as in c o u n t r i e s or r e g i o n s w i t h a low p r e v a l e n c e of AD i n f e c t i o n or w i t h a n a b s e n c e of a n y infection. M o r e over, it m u s t also be c o n s i d e r e d t h a t v a c c i n e s c o n s t i t u t e only one elem e n t of a c o n t r o l or e r a d i c a t i o n p r o g r a m t a r g e t e d a g a i n s t A u j e s z k y ' s disease virus.
REFERENCES Andries, K., Pensaert, M. B., and Vandeputte, J. (1978). Effect of experimental infection with pseudorabies (Aujeszky's Disease) virus on pigs with maternal immunity from vaccinated sows. Am. J. Vet. Res. 39(8), 1282-1285. de Leeuw, P. W., and Van Oirschot, J. T. (1985). Vaccines against Aujeszky's disease: Evaluation of their efficacy under standardized laboratory conditions. Vet. Q. 7, 780786. Hall, W., Molitor, T. W., Joo, H. S., and Pijoan, C. (1989). Comparison of protective immunity and inflammatory responses of pigs following immunization with different Actinobacillus pleuropneumoniae preparations with and without adjuvants. Vet. Immunol. Immunopathol. 22, 175-186. Henderson, L. M., Randall, L. L., David, A. J., and Stutz, D. R. (1991). Recombination of pseudorabies virus vaccine strains in swine. Am. J. Vet. Res. 52(6), 820-825. Kit, S. (1989). Safety and efficacy of genetically engineered Aujeszky's disease vaccines. In "Vaccination and Control of Aujeszky's Disease," (J. T. Van Oirschot, ed.), pp. 4555. McFerran, J. B., and Dow, C. (1975). Studies on immunisation of pigs with the Bartha strain of Aujeszky's disease virus. Res. Vet. Sci. 19, 17-22. Mengeling, W. L. (1991). Virus reactivation in pigs latently infected with a thymidine kinase negative vaccine strain of pseudorabies virus. Arch. Virol. 120, 57-70. Pensaert, M. B., de Smet, K., and de Waele, K. (1990). Extent and duration of virulent virus excretion upon challenge of pigs vaccinated with different Glycoprotein-Deleted Aujeszky's disease vaccines. Vet. Microbiol. 22, 107-117. Stellmann, C., Vannier, P., Chappuis, G., Brun, A., Dauvergne, M., Fargeaud, D., Bugaud, M., and Colson, X. (1989). The potency testing of pseudorabies vaccines in pigs. A proposal for a quantitative criterion and a minimum requirement. J. Biol. Stand. 17, 17-27. Toma, B. (1979). Obtention et caracterisation d'une souche thermosensible de virus de la maladie d'Aujeszky (souche ALFORT 26). Recl. Med. Vet. 155(2), 131-137. Toma, B., Brun, A., Chappuis, G., and Terre, J. (1979). Propri6t6s biologiques d'une souche thermosensible (ALFORT 26) de virus de la maladie d'Aujeszky. Recl. Med. Vet. 155(3), 245-252. Vannier, P. (1986). Immunisation de porcs charcutiers contre la maladie d'Aujeszky avec deux vaccins ~ adjuvants huileux. Etude des r6actions locales. Recl. Med. Vet. 162(1), 37-44.
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Vannier, P., Tillon, J. P., Toma, B., Delagneau, J. F., Loquerie, R., and Prunet, P. (1976). Protection conf6r~e au porc par un nouveau vaccin huileux h virus inactiv~ contre la maladie d'Aujeszky. Consequences pratiques. J. Rech. Porc Fr. pp. 281-290. Vannier, P., Hutet, E., Bourgueil, E., and Cariolet, R. (1991). Level of virulent virus excreted by infected pigs previously vaccinated with different glycoprotein deleted Aujeszky's diseases vaccines. Vet. Microbiol. 29, 213-223. Van Oirschot, J. T., and Gielkens, A. L. J. (1984). Intranasal vaccination of pigs against pseudorabies: Absence of vaccinal virus latency and failure to prevent latency of virulent virus. Am. J. Vet. Res. 45(10), 2099-2103. Van Oirschot, J. T., Terpstra, C., Moormann, R. J. M., Berns, A. J. M., and Gielkens, A. L. J. (1990). Safety of an Aujeszky's disease vaccine based on deletion mutant strain 783 which does not express thymidine kinase and glycoprotein I. Vet. Rec. 127, 443-446. Visser, N., and Ltitticken, D. (1989). Experiences with a gl-/TK- modified live pseudorabies virus vaccine: Strain Begonia. In 'Vaccination and Control of Aujeszky's Disease" (J. T. Van Oirschot, ed.), 37-44.