Potential pathogenicity for rodents of vaccines intended for oral vaccination against rabies: a comparison

Potential pathogenicity for rodents of vaccines intended for oral vaccination against rabies: a comparison M. Artois *§, Caroline Guittr6*, Isabelle Thomas*, H616ne Leblois t, B. Brochier ÷ and J. Barrat* Different oral vaccines intended to control f o x rabies were administered to 271 wild rodents. Vaccines were administered orally or by the mucosal route to four different European species belonging to the genera Apodemus, Arvicola, Clethrionomys and Microtus. These rodents are likely to consume baits and to have contact with the vaccine. Two genetically engineered vaccines were tested: SAG1 (an avirulent mutant of the rabies virus) and V- R G ( vaccinia recombinant virus expressing the rabies glycoprotein gene ). Both were found to be completely innocuous when administered orally or by the mucosal route. The residual pathogenicity of conventional modified live vaccines derived from the SAD strain was confirmed. Keywords: Rabies; oral vaccination; pathogenicity; rodents

INTRODUCTION Oral vaccination of foxes against rabies is turning out to be an effective method of controlling this zoonotic disease ~. For use in the field, a product should not only be suitable for controlling rabies in the target species but should have minimal or no pathogenicity for non-target species. Despite their safety in target animals some vaccines retain residual pathogenicity for some non-target species, especially rodents. Being opportunistic feeders, rodents are likely to eat baits dispersed in the field2"3. Pathogenicity tests in wild rodents remain rare due to the large number of species and subspecies involved, and the technical difficulties involved in keeping such wild rodents in captivity for long periods of time. The results obtained following administration of live rabies vaccines to encaged small wild rodents are reported here. Species very common in eastern France and Belgium were used. After the administration of high doses of several types of currently available vaccines by the oral, mucosal or cerebral routes, mortality or pathogenic effects over the short term were investigated. MATERIALS AND METHODS Vaccines

Four vaccines were tested in this trial : two conventional modified-live, and two biotechnologically engineered. *CNEVA, LERPAS, BP 9, F54220 Malzeville, France. *Department of Virology and Immunology, Faculty of Veterinary Medicine, University of Liege, B-1070, Brussels, Belgium, ;CNRS, Laboratoire de Genetique des Virus, 91190 Gif-surYvette, France. ~To whom correspondence should be addressed. (Received 30 August 1991; revised 29 October 1991; accepted 3 January 1992) 0264-410X,,92;080524-05 ,~ 1992 Butterworth-Heinemann Ltd

524 Vaccine, Vol. 10, Issue 8, 1992

The vaccines administered were those used in actual baiting programmes or in experimental studies. They were directly extracted from vaccine blister packs (stored frozen) or from batches used for this purpose. Titres were close to 108 TCID/ml for every vaccine used in this study.

Conventional vaccines. Street Albama Dufferin (SAD) was originally isolated in 1935 from a rabid dog in Alabama1 ; that strain was then passed continuously in miscellaneous laboratory animals until 1960 when it was adapted to baby hamster kidney (BHK) cells. At that time it lost its pathogenicity for the adult dog, but remained pathogenic for some other species by the muscular or oral route; for most species, especially rodents, it remains pathogenic by the intracerebral route, permitting a titration of the virus expressed as mouse intracerebrat lethal dos%0 (MICLDso). The ERA is directly derived from the SAD strain. Two SAD strains of rabies vaccines were used: the 'standard SAD' (also called SADder, or SADswis~ in Europe) was provided to Swiss researchers (Schweizerische Tollwutzentrale der UniversitS.t, L/inggasstrasse 122, Bern, Switzerland) by American laboratories in 1974~. The "SAD-B~9'5 is a standard SAD virus cultivated in a particular clone of BHK2t cells in Germany (Bundesforschunganstalt fiir Viruskrankheiten der Tiere, Paul-Ehrlich Strasse 28, 7400 Ttibingen, Germany). Because the SAD-B~9 carries a particular marker, it can be distinguished from the standard SAD by its reactivity with monoclonal antibodies. ERA derived from the SAD, and SAD strains, grown in BHK_,~ cells have been previously tested for pathogenicity in laboratory rodents (Mus musculus, Rattus norvegicus) and wild rodents (Apodemus sylvaticus, Clethrionomys glareolus, Ondatra zibethicus, Microtus arvalis in Europe and Sigmodon hispidus in the United

Pathogenicity for rodents of rabies oral vaccines: M. Artois et al.

States) by several authors 6-L 1. Under different conditions sporadic mortalities may occur over a broad range of dilutions s. Lethal infections may even occur by cannibalism 11. By the intracerebral (i.c.) or oral routes the pathogenicity of the ERA, SAD-B19 and SADswiss strains is comparable, and is far from negligible 6. However, SADsw~ ~ virus orally passaged 12 times in suckling mice did not change its properties 9, whereas during ten passages of ERA-BHK_,t virus in muskrat and Norway rat, residual pathogenicity was suggested 10. It is generally agreed, on the basis of these results, and following intensive field studies, that there is no tendency for these vaccine strains to spread within communities of wild rodents 9. Nevertheless, mortality from vaccine-induced rabies remains a severe hazard for populations of rodents in areas in which modified-live vaccines are being used in baits to control fox rabies.

Biotechnological vaccines. Street Alabama Dufferin mutant Gif (SAG1) variants of a particular rabies virus can be isolated by selective growth in the presence of specific monoclonal antibodies. The apathogenicity has been attributed to substitution of an amino acid (arginine) at the 333 position located within the site III of the virus glycoprotein 5. t e. ~3. SAG t (manufactured by Virbac, B.P. 27, 06511 Carros), was prepared from a mutant of standard SAD. It is apathogenic by the i.c. route in adult mice 1'~'15. A further mutation in site II serves as another marker.

Table 1

Vaccinia-rabies glycoprotein recombinant ( V - R G ) vaccine (manufactured by Rh6ne M&ieux, 254, rue M. M&ieux, 69007 Lyon), was obtained by inserting the gene coding for the Standard ERA glycoprotein into the thymidine kinase (TK) region of the vaccinia virus genome (Copenhagen strain ts 2b) 16'17. Innocuity and immunogenicity of these vaccines for the red fox" ( Vulpes vulpes) were demonstrated experimentally 1s-_,o. Animals

The rodents tested belong to genera which are common in the centre of Western Europe: two murids (Apodemus flavicollis, Apodemus sylvaticus) and four microtids

(Arvicola terrestris, Clethrionom)'s glareolus, Microtus agrestis and Microtus arvalis). The rodents were caught using box traps in the vicinity of two of the laboratories involved in this study (Malzeville and Brussels). The recognition of the species belonging to the genera Apodemus and Microtus presented some difficulties, therefore only the genus name is documented in the following results. Vaccination and observation

Oral administration. Using a micropipette, 30/al (0.03 ml) of the vaccine suspension (SAD B.... SADB19, SAG 1 and V - R G ) was administered directly into the oral cavity of every rodent in a group of 125 animals ( + four controls ) ( Table 1 ).

Safety trials of oral vaccines in wild rodents

Rodents =

Day of death (postvaccination)

Observation period = (days)

Deaths vaccinat, a

Rabies pos."

4

29

0/4

014

0/4

8

45 29 43 45 35

0/28 0/5 1/4 0/4

0/1 -

22/27 4/5 2/2 3/3

4 5

29 34 34

0/20 1/4 0/5

0/20 0/4 0/5

8 6 5 4

34 41 34 44

2/8 0/6 1/5 1/4

1/8 0/6 0/5 1/4

8 5 4 5

34 41 34 43

3/8 0/5 1/4 1/5

2/8 015 0/4 0/4

No ~

FAT-neg/

FAT-pos.g

Seroconversion rate'

Control

Apo. V-RG

Apo.

20

A. terr. C. glar. Micro.

5 4 4

SAG, ( =SAD/mutant AM) Apo. 20

C. glar. Micro.

1

2/20 0/4 0/5

6

Standard-SAD

Apo. A. terr. C. glar. Micro. SAD B19 Apo.

A. terr. C. glar. Micro.

2

13

27 18 1 17

12-13

0/5 2/6 2/4 3/3 0/5 0/5 0/3 3/4

Total = 129

=Apo., Apodemus sp.; A. terr., Arvicola terrestris; C. glar, Clethrionomys glareolus; Micro., Microtus so. ~lnoculated: 10 December 1987, 3 February 1988, 8 February 1988, 18 September 1990. =Number of days of survey. "Individuals found dead in cage/total number. 'Rabies antigen detected by the fluorescent antibody test. 'Delay between administration of the vaccine and intercurrent death for unknown reason not related to the vaccine administered. gDelay between administration of the vaccine and death from rabies. "Number of seroconversions/number of samples tested. Several blood samples were not available. Titres < 0.35 EU were considered negative

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525

Pathogenicity for rodents of rabies oral vaccines: M. Artois et al.

Mucosal administration. A second group of 16 rodents was vaccinated with SAG1 or V - R G by the nasal or ocular route using a micropipette of 5 ~1. The vaccinal suspension was in contact with the eye for at least 30 s. A placebo (PBS) was given to three control rodents. Observation (oral/ mucosal administration). The inoculated animals were kept in individual cages, fed with laboratory rodent- or rabbit-food (supplemented by miscellaneous vegetables : carrots or salad) and observed for at least 4 weeks. Animals that died were necropsied and brains were examined by the rabies fluorescent antibody test (FAT), which determines the presence of nucleocapsid antigen 21. At the end of the observation period, all the surviving rodents were killed by bleeding following deep anaesthesia (methoxyflurane p.n.). Brain samples were tested for rabies by FAT. Antibodies against the rabies glycoprotein were determined by the ELISA method 22 for vaccines obtained from rabies strains (SAGB .... SADB19, SAGx). This ELISA uses a protein A (PA) and lacks sensitivity for detecting rodent antibodies. The PA-binding capacity of sera from rodents is very low 23. Antibodies in rodents immunized with V - R G were measured by the rapid fluorescent focus inhibition test ( R F F I T ) method 24 ; in all cases > 0.35 EU ml- t (equivalent units ml- t ) in ELISA or 0.35 IU ml(international units ml -~) in R F F I T were considered positive. Multiple passaging. In a third group, 29 bank voles (C. 9lareolus) were used for multiple passaging of SAG1 (14 individuals) or V - R G (15 individuals) by the i.c. or intradermal (i.d., footpad) route: four voles were inoculated i.c. and five others i.d. Two days later in the i.d. group and 4 days in the i.c. group the voles were humanely killed. Brains and feet were homogenized in PBS with gentamicin sulphate (0.2 g 1-1) to give 20% (w/vol) suspension. This homogenate was centrifuged at 2009 for 20rain at 4°C. The supernatant was then removed and inoculated into a second group of voles. The genetic stability of the vaccines was tested by i.c. inoculation of the supernatant into five adult O F 1 mice. Table 2

Oral administration (Table 1) Four bank voles (C. glareolus) died prematurely, one in each group (i.e. given V - R G , SADB .... SADBt9, SAG~); however, no pox lesions or any other lesions were discovered, and the brains analysed were rabies negative. This occurrence could only be explained by the difficulty in keeping in captivity these small wild rodents. No ground vole (A. terrestris) presented any clinical reaction, whichever vaccine was used, although several animals seroconverted. But three of 16 field mice (Apodemus sp. ) and one of nine common voles (Microtus sp.) given one of the two SAD strains (SAD B.... SAD-B~9) died (at 12, 13 [twice] and 18 days). (All dead animals were FAT-positive.) Rabies virus was not found in the four control field mice or in other analysed specimens (see Table I ). Several surviving rodents were rabies antibody-positive. Mucosal administration In the group of 12 inoculated bank voles I Clethrionomys glareolus) + three controls and four ground voles (A. terrestris), two ground voles died. one inoculated intranasally with SAGI and the other inoculated intraocularly with V - R G . No specific lesions were found and the brains were rabies-negative. During the 28 days of observation following vaccination, no rodent presented any clinical signs and no seroconversion was shown by the ELISA method.

Multiple passaging (Table 2) The V - R G remained in the supernatant until the third passage by the i.e. route, and the second passage by the i.d. route. After the first passage by i.d. route, the SAG 1

Presence of VRG virus in inoculum or supernatant

Route

Passage

V-RG

i.c.

Inoculum 1st 2nd 3rd

+ + +

Inoculum 1st 2nd

+ +

i.d.

i.c.

Inoculum 1st 2nd 3rd

Titre' of the inoculum 7.0 2.7 1.9 1.5

i.d.

Inoculum 1st

7.0 4.6

"i.c. LDso/0.2 ml in suckling mice. ~On day 15 after inoculation, FAT+

526

RESULTS

Multiple passaging in bank voles

Vaccine

SAG1

The presence of V - R G in the supernatant was tested for by incubation with Veto cells (CPE due to vaccinia virus) -'5. SAG t was titrated intracerebrally in suckling mice ( O F 1 ) ; titres were expressed as i.c. LD~0/0.02ml in suckling mice.

V a c c i n e , Vol. 10, I s s u e 8, 1992

Number of voles used

Mortality of adult mice

4 4 2

0/5 0/5 0/5 0/5

3 2

0/5 0/5 0/5

Harvest 2.7 1.9 1.5 1.0

4 4 3

O/5 1/ 5 ~ 0/5

4.6 0

3

0/5 0/5

Pathogenicity for rodents of rabies oral vaccines: M. Artois et al.

was no longer detected, but it remained during all three i.c. passages. At the second i.c. passage, one of the five laboratory mice died of rabies, 15 days after inoculation; the brain was FAT-positive. CONCLUSION The rodent sample size was unfortunately too small to measure precisely the lethality of the SAD strains (SADB . . . . SADa19). A figure of about 11% (4/45) was found in this study, whereas it was 30% (19/64) according to Winkler et al. t t . Whatever the figure, it is apparent that a significant mortality could be induced by these vaccines in natural wild rodent communities when high doses are consumed. This test confirms previously published results 6-x° on the residual lethal effect of conventional vaccines (SAD strains) for these species. This mortality was never registered during field trials in Europe because any carcasses of small animals would have been either hidden in subterranean burrows or eaten by predators. Additionally, we failed to notice any significant difference between the two SAD strains concerning their residual pathogenicity. Under a selection pressure (i.e. passage in rodents) the avirulent mutant SAGt regains pathogenicity (one mouse dead at passage 1). Attenuation results from a single mutation which is then subject to reversion. After one passage in brain of suckling mice followed by i.c. inoculation of adult mice, Tuffereau et al. ~3 isolated 41 independent revertants : 39 were distinguishable from the SAD parental strains. These were not more pathogenic than the oral vaccine currently used over large areas in western Europe. The remaining two revertants had a lysine instead of an arginine in position 333 of the glycoprotein and were 30 times less virulent than the parental strain by the i.c. route. It appears most likely that a SAGt-revertant will remain within the vaccinated animal and will not be transmitted to any others. Despite the fact that the ELISA method probably underestimates the real level of antibodies in nonhomologous sera, seroconversion was found in some animals. This indicates that there is some replication o f the vaccine virus in rodents when orally administered. The results of this study provide further evidence for the innocuity for wild, non-target species of new vaccines produced by biotechnology, i.e. the SAGx mutant as well as V - R G , no matter which external route was used for inoculation (i.e. oral or mucosa126'2v). ACKNOWLEDGEMENTS The authors are grateful to Dr Anne Flamand (CNRS, France), Dr B. Languet (Rh6ne M6rieux, France), Dr Martel (Virbac, France), Professor L.G. Schneider (Bundesforschunganstalt ffir Viruskrankheiten, Germany) and Professor A. Wandeler (Bern University, Switzerland) for providing the vaccines and to Dr A. Aubert (Virbac, France), Dr Ph. Desmesttre and G. Chappuis (Rh6ne M6rieux, France), Dr J. Blancou (CNEVA, France) and Professor P. P. Pastoret (Liege University, Belgium) for encouraging the study. B. Favier (CNEVA, France) checked the serology results. Janet Armstrong, A. Webster and Dr K. Charlton (ADRI, Canada) made valuable criticisms and helped in the translation of an early draft of the manuscript. The authors are also grateful to Professor J. Campbell

(University of T o r o n t o ) who reviewed the manuscript, as well as to the two referees for their very useful comments on former versions of this paper. The technical assistance of J. Ambert, E. Cain, J.M. Demerson, N. Lemaire, C. Patron and M. Selve (CNEVA) was greatly appreciated. This work was carried out under Contract CEE (Bridge) BAP0382F. REFERENCES 1 Wandeler, A.I. Oral immunization of wildlife. In: The Natural History of Rabies 2nd edn (Ed. Baer, G.M.), CRC Press, Boca Raton, 1991, pp 485-503 2 Brochier, B. Iokem, A., Ginter, A., Lejeune, E., Costy, F., Marchal, A. et al. Premiere campagne de vaccination antirabique du renard par voie oral menee en Belgique. Contr61es d'efficacite et d'innocuite chez le renard roux (Vulpes vulpes L.). Ann. M~d. Vet. 1987, 131,463-472 3 Pastoret, P.P., Frisch, R., Blancou, J., Woff, F., Brochier, B. and Schneider, L.G. Campagne internationale de vaccination antirabique du renard par vole orale menee au grand-duch~ de Luxembourg, en Belgique et en France. Ann. M~d. V~t. 1987, 131, 441-447 4 Steck, F., Wandeler, A., Bichsel, P., Capt, S. and Schneider, L.G. Oral immunization of foxes against rabies. A field study. Zentralbl. Vet. Med. 1982, 29, 372-396 5 Schneider, L.G., Cox, J.H., Wandeler, A.I., Blancou, J. and Meyer, S. Oral rabies vaccine. In: Rabies in the Tropics (Eds Kuwert, E., M~rieux, C., Koprowski, H. and B6gel, K.) Springer Verlag, Berlin, 1985, pp 53-59 6 Leblois, J. and Flamand, A. Studies on pathogenicity in mice of rabies virus strains used for oral vaccination of foxes in Europe. In Vaccination to Control Rabies in Foxes (Eds Pastoret, P.P., Brochier, B., Thomas, I. and Blancou, J.), Office for Official Publications of the European Communities, Luxembourg, 1988, pp 101-104 7 Schneider, L.G. and Cox, J.H. Ein Fetdversuch zur oralen Immunisierung von Ffichsen gegen Tollwut in der Bundesrepublik Deutschland. Tier~rtzl. Umsch. 1983, 5, 315-324 8 Steck, F., Wandeler, A., Bichsel, P., Capt, S., H&fliger, U. and Schneider, L. Oral immunization of foxes against rabies. Laboratory and field studies. Comp. Immun. Microbiol. Infect. Dis. 1982, 5, 165-171 9 Wandeler, A., Bauder, W., Prochaska, S. and Steck, F. Small mammal studies in a SAD baiting area. Comp. Immun. Microbiol. Infect. Dis. 1982, 5, 173-176 10 Wachend6rfer, G., Farrenkopf, P., Lohrbach, W., F6rster, U., Frost, J.W. and Valder, W.A. Passage-versuche mit einer Varianten des Tollwut-lmpfstammes ERA bei wildlebenden Spezies. Ein Beitrag zur oralen Immunisierung yon F0chsen gegen Tollwut. Dtsch. Tier~rtzl. Wochenschr. 1978, 85, 273-308 11 Winkler, W.G., Shaddock, J.H. and Williams, LW. Oral rabies vaccine: evaluation of its infectivity in three species of rodents. Am. J. Epidemiol. 1976, 104, 294-298 12 Ftamand, A., Coulon, P., Pepin, M., Blancou, J., Rollin, P. and Portnoi, D. Immunogenic and protective power of avirulent mutants of rabies virus selected with neutralizing monoclonal antibodies. In: Modern Approaches to Vaccines (Eds Chanock, R. and Lerner, R.), Cold Spring Harbor Symposium, 1984, pp 289-294 13 Tuffereau, C., Leblois, H., Benejean, J., Coulon, P., Lafay, F. and Flamand, A. Arginine or lysine in position 333 of ERA and CVS glycoprotein is necessary for rabies virulence in adult mice. Virology 1989, 172, 206 14 Pg~pin,M., Blancou, J., Aubert, M.F.A., Barrat, J., Coulon, P. and Flamand, A. Oral immunization against rabies with an avirulent mutant of the CVS strain: evaluation of its efficacy in fox (Vulpes vulpes) and its infectivity in seven other species. Ann. Inst. Pasteur/Virol. 1985, 136E, 65-73 15 Self, I., P~pin, M., Blancou, J., Coulon, P. and Flamand, A. Change in pathogenicity and amino acid substitution in the glycoprotein of several spontaneous and induced mutants of the CVS strain of rabies virus. In: Nonsegmented Negative Strand Viruses (Eds Bishop, D.H.L. and Compans, R.W.), 1984, pp 295-300 16 Wiktor, T., MacFarlan, R.I., Reagan, K.J., Dietzschold, B., Curtis, P.J., Wunner, W.H. et al. Protection from rabies by a vaccinia virus recombinant containing the rabies virus glycoprotein gene. Proc. Natl Acad. Sci. 1984, 81, 7194-7198 17 Kieny, M.P., Lathe, R., Drillien, R., Spehner, D., Skory, S., Schmitt, D. et al. Expression of rabies virus glycoprotein from a recombinant vaccinia virus. Nature 1984, 312, 163-166

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P a t h o g e n i c i t y for rodents of r a b i e s o r a l vaccines: M. Artois et al. 18

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