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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
A n n . Inst. P a s l e u r [ V i r o l . 1985, 136 E, 65-73
9 ELSEVIER Paris 1985
ORAL AN
WITH
IMMUNIZATION
AVIRULENT
MUTANT
EVALUATION IN AND
ITS
FOX
AGAINST
OF
ITS
(VULPES
INFECTIVITY
IN
OF
RABIES
THE
CVS
STRAIN:
EFFICACY VULPES)
SEVEN
OTHER
SPECIES
by M. P~pin Q), J. B1ancou (2), M. F. A. Aubert (2), j. Barrat (3), P. Coulon (8) and A. Flamand (8) (1) Instilut National de la Recherche Agronomique, Slation de Pathologie de la Reproduction, 37380 Nouzilly (France) (3) M!nisl~re de l'Agrieulture, Direction de la Qualild, Centre National d l~ludes sur la Rage, B P 9, 54220 Malzdville (France), and (8) Laboratoire de Gdndhque des Virus, CNRS, 91190 Gif-sur-Yvelte, (France)
SUMMARY The results of oral vaccination trials performed on foxes with the AVO1 m u t a n t strain of rabies virus selected ~with antiglycoprotein neutralizing monoclonal antibodies are reported. AVO1 was efficient when the vaccine was administered directly:into the mouths of the animals. Vaccination led to the production of virus, neutralizing antibodies in 7 out of 13 foxes and to t h e protection of 10 out of 13 after a challenge at day 90 when the titre of the vaccine exceeded 107 LDso for suckling mice. However, incorporation of the vaccine in baits (chicken heads) was not efficient. Innocuousness of AVO! was tested in foxes and in 7 non-target species, including 6 species of rodents (a total of 247 animals). The virus could be isolated from the brains of two rodents of the genus Arvicola, b u t it was absent from the salivary glands of these animals. The stability of the AVO1 strain was poor because it easily reverted to a virulent phenotype after successive passages in the brains of suckling mice. Consequently, further studies should be carried out with the aim of improving the conditions of incorporation of the virus in the baits and of increasing the genetic stability of AVO1 before testing this m u t a n t in the field. I~EY-WORDS: Rabies, Fox, Vaccine; Monoclonal antibodies, immunization, Strain AVO1.
Manuscrit regu le 29 septembre 1984, acceptd le 6 d6cembre 1984.
.Ann. Inst. Pasteur]Virol., 136 E, n ~ 1~ 1985.
Oral
66
M. P~PIN AND COLL. INTRODUCTION
Rabies in foxes has been spreading rapidly in France since 1968. At presen t, 25 (( d6partements~ ,) are actively involved in the fight against vulpine rabies. P r o p h y l a x i s to avoid contamination of domestic animals and man has mainly taken the form of programs designated to reduce the fox population in contaminated regions and bordering areas. Reduction in the fox p o p u l a t i o n is achieved through trapping, gassing of fox holes, and ,( night s h o o t i n g s , [15]. Active immunization has long been proposed as an alternative or a complement to the destruction of foxes. Numerous trials have already taken place (see review in [5]) and recently, Switzerland and the Federal Republic of Germany undertook to control rabies by this means [16, 19]. However, available modified strains of rabies virus showed numerous disadvantages: low efficiency [12] or high residual pathogenicity for non-target species [20, 24]. The use of antiglycoprotein (anti-G) neutralizing monoclonal antibodies allowed m u t a n t s of the challenge virus strain (CSV) resistant to neutralization to be selected. Among them, m u t a n t s resistant to 2 monoclonal antibodies (149-2 and 248-8) lost their pathogenic power for adult mice [8]. They induced a strong, rapid i m m u n e response and protected the adult mice against intramuscular challenge with 3 x 103 LDso of a field isolate of rabies virus [9]. The purpose of this study was to test whether one of these mutants, AV01 (avirulent Orsay), could be used efficiently to vaccinate foxes by the oral route, either by directly placing t h e vaccine in the m o u t h of t h e animal or by incorporating it in baits. In the course of this work, stability of the m u t a n t and its innocuousness for non-target species, especially for some rodents, were studied. We show t h a t (1) this strain is efficient for immunizing and protecting foxes after direct placement of the vaccine in the mouth of the animal, b u t incorporation of the vaccine in baits drastically decreases its efficiency ; (2) t h e residual virulence of this strain is very low for the 8 tested species ; and (3) this strain is unstable.
M A T E R I A L S AND METHODS Animals.
OF1 mice or suckling mice (IFFA-CREDO, 69210, St-Germain-sur-l'Arbresle, France) free of specific pathogens were used. AVO1 BHK CER CVS ERA HEP
= = = = =
A V = a v i r u l e n t ; O = O r s a y (strain). b a b y h a m s t e r k i d n e y (cell). c h i c k e m b r y o - r e l a t e d (cell). c h a l l e n g e v i r u s s t a n d a r d (strain). E. G a y n o r , R o k i t n i k i a n d A b e l s e t h (strain). = h i g h egg p a s s a g e s (strain).
IC = i n t r a e e r e b r a l ( l y ) . LDso = l e t h a l dose 5 0 % for mice. LDsosm ~ l e t h a l dose 50 % for s u c k l i n g mice. PFU = plaque-forming unit. S A D = S t r e e t A l a b a m a Dufferin. WHO = World Health Organization.
CVS MUTANT IN FOX RABIES ORAL VACCINATION
67
Foxes (Vulpes vulpes) were trapped in rabies-free areas and raised according to described conditions [12]. For the experiment, foxes ranging from 1 to 2 years of age were used. None had virus-neutralizing antibodies before the experiment. Twelve ferrets (Muslela (pulorius) [uro), 21 field-voles (Microlus spp), 37 voles (Clelhrionomys glareolus), 13 rats (Arvicola lerreslris), 56 field-mice (Apodemus sFlvalicus), 8 wild rats (Rallus norvegicus, a generous gift of Dr M. Ra,mt~aud, Laboratoire de Toxicologic, t~cole Nationale V6tdrinaire de Lyon, Marey-L Etoile, France) and 12 rats OFA (IFFA-CREDO, 69210 St-Germain-sur-l'Arbresle) were used. The wild rodents were trapped in the experimental field at Barisey-la-C6te (CNER, 54220 Malz6ville, France) as described by Spitz [18].
Viruses and cells. The mode of selection of the AVO1 mutant has been previously described [81. Briefly, this strain was isolated from a collection of mutants of the CVS strain, and had lost its sensitivity to 2 monoclonal antibodies, 194-2 and 248-8 (a generous gift of T. J. Wiktor, Wistar Institute, Philadelphia, PA, USA). This strain is nonpathogenic for 5-week-old mice or older. The virus was multiplied in baby hamster kidney cells (BHK21) and titrated either in agarose chicken-embryo-related (CER) cell plaque technique [7] or by intracerebral (IC) inoculation to several series of 10 4-day old suckling OF1 mice. The titre was expressed either in plaque-forming units per ml (PFU/ml) or in lethal doses 50% for suckling mice (LD~osm). Two viral stocks were used in our experiments. The challenge virus was a salivary gland extract from naturally rabid foxes dead found in eastern France, and prepared in conditions already described [6]. The virus was titrated by IC inoculation to batches of 10 OF~ mice (3-4 weeks old) just after use. The titre was expressed in lethal dose 50% (LDso) for mice.
Vaecinalion o[ ]oxes. Forty-eight foxes were vaccinated either by having 1 ml of the AVO1 mutant in 3 increasing dilutions placed directly in their mouth, or by being given 2 ml of vaccine incorporated in baits (chicken heads) as described by Schneider and Cox [16]. Virus suspension added with 10% egg yolk was packed in a plastic container introduced under the skin of a chicken head.
Efficiency o] oral vaccinalion o/]oxes by lhe A VO1 mulanl. This was determined by titration of virus neutralizing antibodies in sera and by the resistance to a challenge with a wild-type strain. This challenge was p.erformed at day 90 after the vaccination by injection of 1 ml of viral suspension (3 • 103 LDso) in the temporal muscle. Surviving foxes were killed 60 days later. Rabies was diagnosed for each animal according to the World Health Organization (WHO) methods [14]. Titration of virus neutralizing antibodies was performed by seroneutralization in OF1 mice following the WHO method [14]. The virus used was CVS.
Innocuousness o/A VO1. Three parameters were surveyed for testing innocuousness of AVO1 in foxes and in non-target species: (1) morbidity, (2) mortality and (3) presence of virus. Thirty-eight foxes, 12 ferrets and 197 rodents were vaccinated with the AVO1 mutant, either by the oral route (direct placement in the mouth) or by intramuscular (IM) inoculation. These animals were observed for 90 days. Autopsies were performed on days 8, 15, 30 and 60 after vaccination in order to search for the vaccinal virus in the nerve centres and salivary glands.
68
M. PIPPIN AND COLL.
Stability o[ A VO1. Successive passages in suckling mice were performed. A batch of 10 four-dayold suckling mice (OF1)was injected with AV01. Five days later, moribund animals were killed and their brains crushed. This homogenate was IC-injected into batches of adult and suckling mice, and the procedure was repeated.
RESULTS
Post-vaccinal immunity of A V01 in fox. W h e n AVO1 was placed directly in t h e m o u t h of t h e animal, it was v e r y efficient ; this oral vaccination induced 50 ~o of seroconversion at d a y 30, and 61 ~o of t h e foxes resisted t h e challenge (fig. 1) ; all controls died. If we consider only t h e 2 groups v a c c i n a t e d with the high virus dosage (1.2 • 10 e and 1.1 • 107 LDsosm), 7 6 % of t h e foxes resisted t h e challenge. If we compare the results obtained with these two groups (10 surviving foxes o u t of 13) with those of t h e third (1.1 • 10 e LDsosm ; 1 surviving fox out of 5), this suggests t h a t t h e efficiency of the A V 0 1 m u t a n t in fox b y t h e oral r o u t e depends on a high virus dosage. Incorporation of the AVO1 m u t a n t in baits did not yield successful results. Baits containing 2.5 x 107 and 5 x 10 e LDsosm, respectively, were given t o 2 groups of 10 foxes. All baits were eaten within 24 h. Only one fox receiving t h e highest concentration of virus produced virus neutralizing antibodies. All foxes, both v a c c i n a t e d and control, were killed b y t h e challenge performed 90 days after the vaccination.
Innocuousness of the A VO1 mutant. 1) Towards the fox. ~ No rabies s y m p t o m s were detected during 90 d a y s of observation in foxes vaccinated with AVO1 b y direct p l a c e m e n t of t h e vaccine in the mouth. This m u t a n t was d e m o n s t r a t e d to be avirulent in foxes b y the intramuscular route (unpublished results). 2) Towards non-target species (see t a b l e 1). - - Out of 209 animals including ferrets and 6 species of rodents, the virus was isolated from the brain of 2 rodents (genus Arvicola) at days 7 and 12 after oral administration of 3 • 10 e P F U of AVO1. One of these 2 rodents exhibited clinical s y m p toms of rabies: lack of coordination, weight loss and aggressiveness. The virus could not be isolated from salivary glands. The virus e x t r a c t e d from t h e nerve centres formed plaques in C E R cells and was neutralized b y monoclonal a n t i b o d y 248-8: these criteria allowed t h e virus to be identified as a r e v e r t a n t of AVO1 and not as a street rabies virus t h a t could have c o n t a m i n a t e d these rodents before t h e y were t r a p p e d .
Stability of A VO1. After one IC passage in suckling mice, t h e AYO1 strain recovered its pathogenicity, i. e., it killed 6-week old mice b y t h e IC r o u t e as efficiently as t h e CVS strain (7.9 • days).
CVS MUTANT IN FOX RABIES ORAL VACCINATION
69
Z H
~7_ ,-JO
3.0
r I-I---r Zr
~
A
2.0
~0r ~LXI~ bla. la12J
~ mo DA U3 . . l
1.0
b-tO ~--rn ~4
LO
H
6013_
~w OZ uJ J.~j
B
40-
L~. t Y Ct) ta-
20-
I
0
,
10
FOXES
FOXES
1.2~108 LD50sm
1.1x107
LD50sm
i-lh FOXES
1,1==106 LD50sm
5
FOXES
CONTROLS
FIe. 1. - - Efficieneg o[ oral vaccination with A V 0 1 by direct placement, in the fox mouth of 1 ml of viral suspension. Ten foxes were vaccinated w i t h 1.2 • 108 LDsosm, 3 foxes w i t h 1.1 • 107 LDsosm, 5 foxes w i t h 1.1 • l0 n LD~osm and 5 foxes non-vaccinated. E a c h fox is represented b y a rod of h i s t o g r a m . (A) The h i s t o g r a m s h o w s t h e s e r u m titres of virus-neutralizing antibodies at d a y 30 after ora v a c c i n a t i o n for each fox in the four e x p e r i m e n t a l groups. (B) The h i s t o g r a m shows t h e t i m e of s u r v i v a l of each fox in the four e x p e r i m e n t a l g r o u p s after challenge. The foxes were challenged at day 90 after oral v a c c i n a t i o n w i t h 3 • 103 LDs0 of wild-type virus. Surviving foxes were killed 60 days later.
DISCUSSION Au is an efficient vaccinal strain when the virus is placed directly in the mouth of the fox (fig. 1): 1.2 • 108 and 1.1 • 107 LD5osm per animal allows 54% of the foxes to produce virus neutralizing antibodies and 76}/0 to be protected; these paradoxical rates were already described in fox with live attenuated viruses [12, 22] and suggest that a second protective mechanism is induced by virus vaccine in immunized animals. The results
70
M. PIPPIN AND COLL. TABLE I. - - Innocuousness of AVO1 for 7 non-target species, either by direct placement in the mouth or by the intramuscular route. N u m b e r of a n i m a l s Search of vaccinal virus
Species a n d dose (PFU/animal)
Microtus s p p
vaccinated
presenting nervous symptoms
21
5
0/7
0/9
37
0
0/9
0/3
13
2
50
0
0/5
0/0
56
1
0/13
0/4
12 4 4
0 0 0
0/12 0/4 0/4
0/0 0/0 0/0
6 3 3
0 0 0
0/6 0/3 0/3
0/0 0/0 0/0
+/killed (1)
+/dead (6)
1.6 x 106
Clelhrionomys glareolus 1.6 X 10 6
Arvicola terreslris
1/5 (6)
1/4 (I)
3.0 X 10 6
Mus musculus 8.0 X 10 e
Apodemus s!tlvaticus 1.6 x 106
Rattus norvegicus 3.9 x l0 T 3.9 x 105 3.9 x 10 a
Mustela ( p u t o r i u s ) [uro 8.0 x l0 T 8.0 X 10 s 8.0 • l0 s
(1) These animals were killed at days 7, 15, 30, 60 and 90 after vaccination. (6) These animals died during t h e experiment. (a) These two positive a n i m a l s were vaccinated b y t h e oral route.
of evaluation of AVO1 efficacy are analogous to those obtained with SAD and H E P or their derive d strains used in similar con ditions [ 2, 11, 12, 13, 16]. Consequently, we u n d e r t o o k an e x p e r i m e n t of oral immunization with 2.5 x 107 and 5.0 x 10 e LDsosm of AVO1, incorporated in baits. These titres are easily obtained without a n y concentration of virus. The efficiency of AVO1 is poor when the vaccine is incorporated in baits. The incorporation of a live vaccine in a bait is often followed by a decrease in t h e initial titre due to (1) delays in preparing the baits ; (2) partial inactivation of t h e vaccine due to composition of the bait [12], and (3) a m o d e of administration which does not put t h e virus directly in c o n t a c t with buccal and lingual mucuous membranes, t h e preferred sites for virus penetration [1]. These difficulties were observed after incorporation of SAD or E R A strains in eggs [10], in chicken heads [12] and in a p a n c a k e [23]. H o w e v e r in other experiments, loss of efficiency after incorporation of vaccine in baits was not apparent [3, 22]. In our experiment, it appeared t h a t the initial titre was insufficient, and this, associated with a possible slight decrease in t h e
CVS MUTANT IN FOX RABIES ORAL VACCINATION
71
viral titre at the time of the preparation of baits, could explain the lack of efficiency of the AVO1 m u t a n t by this method. The innocuousness of AVO1 for non-target species is good: out of 247 tested animals, the rabies virus could be isolated from only 2 rodents belonging to the genus Arvicola (table I). It should be noted t h a t the salivary glands of the two rabid rodents were not infected. These results could be compared with those obtained with the other modified virus candidates for oral vaccination of foxes. Indeed, the SAD and E R A strains are pathogenic for numerous rodents species [4, 20, 24]. The Flury H E P strain also keeps a residual pathogenicity for some rodents and some carnivorous animals [5]. The 675 strain derived from H E P seems less pathogenic than the parental strain [21]. The stability of the AVO1 strain, easily identifiable with its genetic marker, is poor because it reverts to a virulent phenotype after successive passages by IC inoculation into suckling mice or after oral vaccination of rodents tested for innocuousness. This high frequency of reversion finds its cause in the genetic difference between AVO1 and its parental CVS strain. The loss of virulence is the consequence of a mutation producing the substitution of an arginine by a glutamine at position 333 of the glycoprotein [17]At present, AVO1 is clearly not fit for use in field application. A stable live vaccine must either have a deletion in the antigenic site responsible for the virulence of the CVS strain or a double mutation if a second site of virulence can be defined. Further studies are necessary for finding a means to avoid the decrease in the efficiency of the vaccine after incorporation in baits and to increase the genetic stability of these avirulent mutants, which represent a modern, original approach to obtaining a live vaccine. When these two problems are solved, the avirulent strains may be proposed as candidates for oral vaccination of foxes.
R~SUM~ VACCINATION ORALE CONTRE LA RAGE AVEC UN MUTANT AVIRULENT DE LA SOUCHE CVS IEVALUATION ET
DE
DE
SON POUVOIR
SON EFFICACIT]E INFECTANT
:
CHEZ LE RENARD
CHEZ SEPT
AUTRES
ESPI~CES
Les r6sultats d'essais de vaccination orale des renards avec la souche m u t a n t e AVO1 du virus de la rage, s61ectionn~e en pr6sence d'anticorps monoclonaux et neutralisants, dirig6s contre la glycoprot6ine, sont rapport6s. Le pouvoir immunog~ne de AVO1 a ~t6 bon lorsque le vaccin 6tait d6pos6 directement dans la gueule du renard. La vaccination a entrain~ la production d'anticorps neutralisants chez 7 renards sur 13 et la protection de 10 sur 13 apr~s une 6preuve virulente au 90~ jour, si le titre en virus vaccinal exc6dait 10' DLso souriceau. Toutefois, l'incorporation du vaccin dans les app~ts n'a pas 6t6 efficace.
72
M. PIPPIN AND COLL.
L'innocuit~ de AVO1 a 5t~ test~e chez le renard et chez 7 esp~ces non cibles, soit 247 a n i m a u x au total. Le virus de la rage n'a pu ~tre r~isol~ qu'A partir des eerveaux de 2 rongeurs du genre Arvicola; le virus ~tait absent des glandes salivaires de ces animaux. P a r passages successifs sur cerveau de souriceau; la stabilit~ de la souehe est m~diocre car AVO1 a reeouvr~ son ph~notype sauvage apr~s u n seul passage. E n consequence, les ~tudes ult~rieures devront riser h am~liorer les conditions d'incorporation d u virus dans les app~ts e t h a u g m e n t e r la stabilit~ g~n~tique de AVO1 a v a n t de tester ce m u t a n t sur le terrain. MOTS-C:L~S : Bage, R e n a r d , Vaccin ; Anticorps monoclonaux, I m m u n i sation orale, Souche AVO1.
ACKNOWLEDGMENTS We would like to thank L. Andral, C. Le Louedec, P. Pardon and M. Ptommet for their advice, M. J. Barrat, J. M, Demerson and J . George for their technical assistance and Jayne Alderson for her help in writing this paper.
REFERENCES
[1] BAER, G. M., BRODERSON, R. & YAGER, P. A., Determination of the site of oral rabies vaccination. Amer. J. Epidemiol., 1975, 101, 160-164. [2] BLACK, J. G. & LAWSON, K. F., Sylvatic rabies studies in the silver fox (Vulpes vulpes). Susceptibility and immune response. Canad. J. eomp. IVied., 1970, 34, 309-311. [3] BLACK, J. G. & LAWSON, S. F., Further studies of sylvatic rabies in the fox (Vulpes vulpes). Vaccination by the oral route. Canad. Vet. J., 1973, 14, 206-211.
[4] BLACK, J. G. & LAWSON, K. F., The safety and efficacy of immunizing foxes (Vulpes vulpes) using bait containing attenuated rabies virus vaccine. Canad. J. comp. Med., 1980, 44, 169-176. [5] BLANCOU,J., Prophylaxie m6dicale de la rage chez le renard. Rec. Mdd. vdt. (Alfort), 1979, 155, 733-741.
[6] BLANCOU,J., hUBERT, M. F. A,, ANDRAL, L. & ARTOIS, M., Rage expdrimentale du renard roux (Vulpes vulpes). - - I. Sensibilitd selon la vole d'infection et la dose infectante. Rev. Mdd. v~t., 1979, 130, 1001-1015. [7] BUSSEREAU, F., FLAMAND, A. ~: PESE-PART, D., Reproducible plaquing system for rabies virus in CER cells. J. Virol. Methods, 1982, 4, 277-282. [8] COULON, P., ROLLIN, P., hUBERT, M. F. h. & FLAMAND, A., Molecular basis of rabies virus virulence. - - I. Selection of avirulent mutants of the CVS strain with anti-G monoclonal antibodies. J. gen. Virol., 1982, 61, 97-100. [9] COULON,P., ROLLIN, P., BLANCOU, J. ~; FLAMAND, A., Avirulent mutants of the CVS strain of rabies virus. Comp, Immunol. Microbiol. in/ect. Dis., 1982, 5, 117-122. [10] DEBmE, J. G., Use of inoculated eggs as a vehicle for the oral rabies vaccination of red foxes (Vulpes [ulva). In/eet. lmmun., 1974, 9, 681-683. [11] DEBB!E, J. G., ABELSETH, K. M. & I~AER, G. M., The use of commercially available vaccines for the oral vaccination of foxes against rabies. Amer. J. Epidemiol,, t972, 96, 231-235.
CVS MUTANT IN FOX RABIES ORAL VACCINATION
73
[12] DUBREUIL, M., ANDRAL, L., AUBERT, M. F. A. & BLANCOU, J., The oral [13]
[14] [15] [16]
[17]
[18]
[19] [201
[21] [22] [23] [24]
vaccination of foxes against rabies. An experimental study. Ann. Rech. vdt., 1979, 10, 9-21. FROST, J. W., FRIEDRICH, H. & WACHENDORFER, G., Effectivity of oral vaccination of foxes against rabies with Flury HEP strain 675. Comp. Immunol. Microbiol. in~eel. Dis., 1982, 5, 181-184. KAPLAN, M. M. & KOPROWSKI, H., La rage. Techniques de laboratoire, 3e ed. (379 pp.). 0rganisation Mondiale de la Sant6, Gendve, 1974. ROBOLY,O., Application du tir de nuit des renards h la prophylaxie de la rage. Comp. Immunol. Microbiol. in/eel. Dis., 1982, 5, 389-395. SCHNEIDER, L. G. & Cox, J. H., Ein Feldversuch zur oralen Immunisierung yon Fiichsen gegen die Tollwut in der Bundesrepublik Deutschland. - I. Unschfidlichkeit Wirksamkeit und Stabilitfit der Vaksine SAD B19. Tieraerzll. Umsch., 1983, 5, 315-324. SEIF, I., PIPPIN, M., BLANCOU, J., COULON, P. & 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 ((Non-segmented negative-strand viruses )) (D. H. L. Bishop & R. W. Cornpans). Academic Press, London, New York, 1984. SPITZ, F., L 6chantillonnage des populations de petits mammifdres, in (( ProblSmes d'dcologie : l'dchantillonnage des peuplements animaux des milieux terrestres )) (M. Lamotte & F. Bourlidre) (p. 256-275). Masson, Paris, 1969. STECK, F., WANDELER, A., BlCrtSEL, P., CAPT, S. & SCHNEIDER, L. G., Oral immunization of foxes against rabies. A field study. Zbl. Med. Vet., 1982, B29, 372-396. WACHENDiJRFER, C., FARRENKOPF, R., LOHRBACH, W., FORSTER, J., FROST, J. W. & WALDER, A., Passageversuche mit einer Varianten des Tollwut. Impfstammes ERA bei wildbenden Spezies (Ondalra zibelhica und Rallus norvegicus). Ein Beitrag zur oralen Immunisierung yon Ffichsen gegen Tollwut. Dtsch. lieraerztl. Wschr., 1978, 75, 279-284. WACHENDORFER,G., KIEFFERT, C. & FROST, J. W., Safety tests with Flury HEP strain 675 in wild living European mammals. Comp. Immunol. Microbiol. in/eel. Dis., 1982, 5, 177-180. WINKLER,W. G. & BAER, G. M., Rabies immunization of red foxes (Vulpes /ulva) with vaccine in sausage baits. Amer. J. Epidemiol., 1976, 103, 408-415. WINKLER, W. G., McLEAN, R. G. & COWART, J. C., Vaccination of foxes against rabies using ingested baits. J. Wildl. Dis., 1975, 11, 382-388. WINKLER,W. G., SHADDOCK,J. H. & WILLIAMS,L. W., 0ral rabies vaccine: evaluation of its infectivity in three species of rodents. Amer. J. Epidemiol., 1976, 103, 294-298.
9 ELSEVIER Paris 1985
ORAL AN
WITH
IMMUNIZATION
AVIRULENT
MUTANT
EVALUATION IN AND
ITS
FOX
AGAINST
OF
ITS
(VULPES
INFECTIVITY
IN
OF
RABIES
THE
CVS
STRAIN:
EFFICACY VULPES)
SEVEN
OTHER
SPECIES
by M. P~pin Q), J. B1ancou (2), M. F. A. Aubert (2), j. Barrat (3), P. Coulon (8) and A. Flamand (8) (1) Instilut National de la Recherche Agronomique, Slation de Pathologie de la Reproduction, 37380 Nouzilly (France) (3) M!nisl~re de l'Agrieulture, Direction de la Qualild, Centre National d l~ludes sur la Rage, B P 9, 54220 Malzdville (France), and (8) Laboratoire de Gdndhque des Virus, CNRS, 91190 Gif-sur-Yvelte, (France)
SUMMARY The results of oral vaccination trials performed on foxes with the AVO1 m u t a n t strain of rabies virus selected ~with antiglycoprotein neutralizing monoclonal antibodies are reported. AVO1 was efficient when the vaccine was administered directly:into the mouths of the animals. Vaccination led to the production of virus, neutralizing antibodies in 7 out of 13 foxes and to t h e protection of 10 out of 13 after a challenge at day 90 when the titre of the vaccine exceeded 107 LDso for suckling mice. However, incorporation of the vaccine in baits (chicken heads) was not efficient. Innocuousness of AVO! was tested in foxes and in 7 non-target species, including 6 species of rodents (a total of 247 animals). The virus could be isolated from the brains of two rodents of the genus Arvicola, b u t it was absent from the salivary glands of these animals. The stability of the AVO1 strain was poor because it easily reverted to a virulent phenotype after successive passages in the brains of suckling mice. Consequently, further studies should be carried out with the aim of improving the conditions of incorporation of the virus in the baits and of increasing the genetic stability of AVO1 before testing this m u t a n t in the field. I~EY-WORDS: Rabies, Fox, Vaccine; Monoclonal antibodies, immunization, Strain AVO1.
Manuscrit regu le 29 septembre 1984, acceptd le 6 d6cembre 1984.
.Ann. Inst. Pasteur]Virol., 136 E, n ~ 1~ 1985.
Oral
66
M. P~PIN AND COLL. INTRODUCTION
Rabies in foxes has been spreading rapidly in France since 1968. At presen t, 25 (( d6partements~ ,) are actively involved in the fight against vulpine rabies. P r o p h y l a x i s to avoid contamination of domestic animals and man has mainly taken the form of programs designated to reduce the fox population in contaminated regions and bordering areas. Reduction in the fox p o p u l a t i o n is achieved through trapping, gassing of fox holes, and ,( night s h o o t i n g s , [15]. Active immunization has long been proposed as an alternative or a complement to the destruction of foxes. Numerous trials have already taken place (see review in [5]) and recently, Switzerland and the Federal Republic of Germany undertook to control rabies by this means [16, 19]. However, available modified strains of rabies virus showed numerous disadvantages: low efficiency [12] or high residual pathogenicity for non-target species [20, 24]. The use of antiglycoprotein (anti-G) neutralizing monoclonal antibodies allowed m u t a n t s of the challenge virus strain (CSV) resistant to neutralization to be selected. Among them, m u t a n t s resistant to 2 monoclonal antibodies (149-2 and 248-8) lost their pathogenic power for adult mice [8]. They induced a strong, rapid i m m u n e response and protected the adult mice against intramuscular challenge with 3 x 103 LDso of a field isolate of rabies virus [9]. The purpose of this study was to test whether one of these mutants, AV01 (avirulent Orsay), could be used efficiently to vaccinate foxes by the oral route, either by directly placing t h e vaccine in the m o u t h of t h e animal or by incorporating it in baits. In the course of this work, stability of the m u t a n t and its innocuousness for non-target species, especially for some rodents, were studied. We show t h a t (1) this strain is efficient for immunizing and protecting foxes after direct placement of the vaccine in the mouth of the animal, b u t incorporation of the vaccine in baits drastically decreases its efficiency ; (2) t h e residual virulence of this strain is very low for the 8 tested species ; and (3) this strain is unstable.
M A T E R I A L S AND METHODS Animals.
OF1 mice or suckling mice (IFFA-CREDO, 69210, St-Germain-sur-l'Arbresle, France) free of specific pathogens were used. AVO1 BHK CER CVS ERA HEP
= = = = =
A V = a v i r u l e n t ; O = O r s a y (strain). b a b y h a m s t e r k i d n e y (cell). c h i c k e m b r y o - r e l a t e d (cell). c h a l l e n g e v i r u s s t a n d a r d (strain). E. G a y n o r , R o k i t n i k i a n d A b e l s e t h (strain). = h i g h egg p a s s a g e s (strain).
IC = i n t r a e e r e b r a l ( l y ) . LDso = l e t h a l dose 5 0 % for mice. LDsosm ~ l e t h a l dose 50 % for s u c k l i n g mice. PFU = plaque-forming unit. S A D = S t r e e t A l a b a m a Dufferin. WHO = World Health Organization.
CVS MUTANT IN FOX RABIES ORAL VACCINATION
67
Foxes (Vulpes vulpes) were trapped in rabies-free areas and raised according to described conditions [12]. For the experiment, foxes ranging from 1 to 2 years of age were used. None had virus-neutralizing antibodies before the experiment. Twelve ferrets (Muslela (pulorius) [uro), 21 field-voles (Microlus spp), 37 voles (Clelhrionomys glareolus), 13 rats (Arvicola lerreslris), 56 field-mice (Apodemus sFlvalicus), 8 wild rats (Rallus norvegicus, a generous gift of Dr M. Ra,mt~aud, Laboratoire de Toxicologic, t~cole Nationale V6tdrinaire de Lyon, Marey-L Etoile, France) and 12 rats OFA (IFFA-CREDO, 69210 St-Germain-sur-l'Arbresle) were used. The wild rodents were trapped in the experimental field at Barisey-la-C6te (CNER, 54220 Malz6ville, France) as described by Spitz [18].
Viruses and cells. The mode of selection of the AVO1 mutant has been previously described [81. Briefly, this strain was isolated from a collection of mutants of the CVS strain, and had lost its sensitivity to 2 monoclonal antibodies, 194-2 and 248-8 (a generous gift of T. J. Wiktor, Wistar Institute, Philadelphia, PA, USA). This strain is nonpathogenic for 5-week-old mice or older. The virus was multiplied in baby hamster kidney cells (BHK21) and titrated either in agarose chicken-embryo-related (CER) cell plaque technique [7] or by intracerebral (IC) inoculation to several series of 10 4-day old suckling OF1 mice. The titre was expressed either in plaque-forming units per ml (PFU/ml) or in lethal doses 50% for suckling mice (LD~osm). Two viral stocks were used in our experiments. The challenge virus was a salivary gland extract from naturally rabid foxes dead found in eastern France, and prepared in conditions already described [6]. The virus was titrated by IC inoculation to batches of 10 OF~ mice (3-4 weeks old) just after use. The titre was expressed in lethal dose 50% (LDso) for mice.
Vaecinalion o[ ]oxes. Forty-eight foxes were vaccinated either by having 1 ml of the AVO1 mutant in 3 increasing dilutions placed directly in their mouth, or by being given 2 ml of vaccine incorporated in baits (chicken heads) as described by Schneider and Cox [16]. Virus suspension added with 10% egg yolk was packed in a plastic container introduced under the skin of a chicken head.
Efficiency o] oral vaccinalion o/]oxes by lhe A VO1 mulanl. This was determined by titration of virus neutralizing antibodies in sera and by the resistance to a challenge with a wild-type strain. This challenge was p.erformed at day 90 after the vaccination by injection of 1 ml of viral suspension (3 • 103 LDso) in the temporal muscle. Surviving foxes were killed 60 days later. Rabies was diagnosed for each animal according to the World Health Organization (WHO) methods [14]. Titration of virus neutralizing antibodies was performed by seroneutralization in OF1 mice following the WHO method [14]. The virus used was CVS.
Innocuousness o/A VO1. Three parameters were surveyed for testing innocuousness of AVO1 in foxes and in non-target species: (1) morbidity, (2) mortality and (3) presence of virus. Thirty-eight foxes, 12 ferrets and 197 rodents were vaccinated with the AVO1 mutant, either by the oral route (direct placement in the mouth) or by intramuscular (IM) inoculation. These animals were observed for 90 days. Autopsies were performed on days 8, 15, 30 and 60 after vaccination in order to search for the vaccinal virus in the nerve centres and salivary glands.
68
M. PIPPIN AND COLL.
Stability o[ A VO1. Successive passages in suckling mice were performed. A batch of 10 four-dayold suckling mice (OF1)was injected with AV01. Five days later, moribund animals were killed and their brains crushed. This homogenate was IC-injected into batches of adult and suckling mice, and the procedure was repeated.
RESULTS
Post-vaccinal immunity of A V01 in fox. W h e n AVO1 was placed directly in t h e m o u t h of t h e animal, it was v e r y efficient ; this oral vaccination induced 50 ~o of seroconversion at d a y 30, and 61 ~o of t h e foxes resisted t h e challenge (fig. 1) ; all controls died. If we consider only t h e 2 groups v a c c i n a t e d with the high virus dosage (1.2 • 10 e and 1.1 • 107 LDsosm), 7 6 % of t h e foxes resisted t h e challenge. If we compare the results obtained with these two groups (10 surviving foxes o u t of 13) with those of t h e third (1.1 • 10 e LDsosm ; 1 surviving fox out of 5), this suggests t h a t t h e efficiency of the A V 0 1 m u t a n t in fox b y t h e oral r o u t e depends on a high virus dosage. Incorporation of the AVO1 m u t a n t in baits did not yield successful results. Baits containing 2.5 x 107 and 5 x 10 e LDsosm, respectively, were given t o 2 groups of 10 foxes. All baits were eaten within 24 h. Only one fox receiving t h e highest concentration of virus produced virus neutralizing antibodies. All foxes, both v a c c i n a t e d and control, were killed b y t h e challenge performed 90 days after the vaccination.
Innocuousness of the A VO1 mutant. 1) Towards the fox. ~ No rabies s y m p t o m s were detected during 90 d a y s of observation in foxes vaccinated with AVO1 b y direct p l a c e m e n t of t h e vaccine in the mouth. This m u t a n t was d e m o n s t r a t e d to be avirulent in foxes b y the intramuscular route (unpublished results). 2) Towards non-target species (see t a b l e 1). - - Out of 209 animals including ferrets and 6 species of rodents, the virus was isolated from the brain of 2 rodents (genus Arvicola) at days 7 and 12 after oral administration of 3 • 10 e P F U of AVO1. One of these 2 rodents exhibited clinical s y m p toms of rabies: lack of coordination, weight loss and aggressiveness. The virus could not be isolated from salivary glands. The virus e x t r a c t e d from t h e nerve centres formed plaques in C E R cells and was neutralized b y monoclonal a n t i b o d y 248-8: these criteria allowed t h e virus to be identified as a r e v e r t a n t of AVO1 and not as a street rabies virus t h a t could have c o n t a m i n a t e d these rodents before t h e y were t r a p p e d .
Stability of A VO1. After one IC passage in suckling mice, t h e AYO1 strain recovered its pathogenicity, i. e., it killed 6-week old mice b y t h e IC r o u t e as efficiently as t h e CVS strain (7.9 • days).
CVS MUTANT IN FOX RABIES ORAL VACCINATION
69
Z H
~7_ ,-JO
3.0
r I-I---r Zr
~
A
2.0
~0r ~LXI~ bla. la12J
~ mo DA U3 . . l
1.0
b-tO ~--rn ~4
LO
H
6013_
~w OZ uJ J.~j
B
40-
L~. t Y Ct) ta-
20-
I
0
,
10
FOXES
FOXES
1.2~108 LD50sm
1.1x107
LD50sm
i-lh FOXES
1,1==106 LD50sm
5
FOXES
CONTROLS
FIe. 1. - - Efficieneg o[ oral vaccination with A V 0 1 by direct placement, in the fox mouth of 1 ml of viral suspension. Ten foxes were vaccinated w i t h 1.2 • 108 LDsosm, 3 foxes w i t h 1.1 • 107 LDsosm, 5 foxes w i t h 1.1 • l0 n LD~osm and 5 foxes non-vaccinated. E a c h fox is represented b y a rod of h i s t o g r a m . (A) The h i s t o g r a m s h o w s t h e s e r u m titres of virus-neutralizing antibodies at d a y 30 after ora v a c c i n a t i o n for each fox in the four e x p e r i m e n t a l groups. (B) The h i s t o g r a m shows t h e t i m e of s u r v i v a l of each fox in the four e x p e r i m e n t a l g r o u p s after challenge. The foxes were challenged at day 90 after oral v a c c i n a t i o n w i t h 3 • 103 LDs0 of wild-type virus. Surviving foxes were killed 60 days later.
DISCUSSION Au is an efficient vaccinal strain when the virus is placed directly in the mouth of the fox (fig. 1): 1.2 • 108 and 1.1 • 107 LD5osm per animal allows 54% of the foxes to produce virus neutralizing antibodies and 76}/0 to be protected; these paradoxical rates were already described in fox with live attenuated viruses [12, 22] and suggest that a second protective mechanism is induced by virus vaccine in immunized animals. The results
70
M. PIPPIN AND COLL. TABLE I. - - Innocuousness of AVO1 for 7 non-target species, either by direct placement in the mouth or by the intramuscular route. N u m b e r of a n i m a l s Search of vaccinal virus
Species a n d dose (PFU/animal)
Microtus s p p
vaccinated
presenting nervous symptoms
21
5
0/7
0/9
37
0
0/9
0/3
13
2
50
0
0/5
0/0
56
1
0/13
0/4
12 4 4
0 0 0
0/12 0/4 0/4
0/0 0/0 0/0
6 3 3
0 0 0
0/6 0/3 0/3
0/0 0/0 0/0
+/killed (1)
+/dead (6)
1.6 x 106
Clelhrionomys glareolus 1.6 X 10 6
Arvicola terreslris
1/5 (6)
1/4 (I)
3.0 X 10 6
Mus musculus 8.0 X 10 e
Apodemus s!tlvaticus 1.6 x 106
Rattus norvegicus 3.9 x l0 T 3.9 x 105 3.9 x 10 a
Mustela ( p u t o r i u s ) [uro 8.0 x l0 T 8.0 X 10 s 8.0 • l0 s
(1) These animals were killed at days 7, 15, 30, 60 and 90 after vaccination. (6) These animals died during t h e experiment. (a) These two positive a n i m a l s were vaccinated b y t h e oral route.
of evaluation of AVO1 efficacy are analogous to those obtained with SAD and H E P or their derive d strains used in similar con ditions [ 2, 11, 12, 13, 16]. Consequently, we u n d e r t o o k an e x p e r i m e n t of oral immunization with 2.5 x 107 and 5.0 x 10 e LDsosm of AVO1, incorporated in baits. These titres are easily obtained without a n y concentration of virus. The efficiency of AVO1 is poor when the vaccine is incorporated in baits. The incorporation of a live vaccine in a bait is often followed by a decrease in t h e initial titre due to (1) delays in preparing the baits ; (2) partial inactivation of t h e vaccine due to composition of the bait [12], and (3) a m o d e of administration which does not put t h e virus directly in c o n t a c t with buccal and lingual mucuous membranes, t h e preferred sites for virus penetration [1]. These difficulties were observed after incorporation of SAD or E R A strains in eggs [10], in chicken heads [12] and in a p a n c a k e [23]. H o w e v e r in other experiments, loss of efficiency after incorporation of vaccine in baits was not apparent [3, 22]. In our experiment, it appeared t h a t the initial titre was insufficient, and this, associated with a possible slight decrease in t h e
CVS MUTANT IN FOX RABIES ORAL VACCINATION
71
viral titre at the time of the preparation of baits, could explain the lack of efficiency of the AVO1 m u t a n t by this method. The innocuousness of AVO1 for non-target species is good: out of 247 tested animals, the rabies virus could be isolated from only 2 rodents belonging to the genus Arvicola (table I). It should be noted t h a t the salivary glands of the two rabid rodents were not infected. These results could be compared with those obtained with the other modified virus candidates for oral vaccination of foxes. Indeed, the SAD and E R A strains are pathogenic for numerous rodents species [4, 20, 24]. The Flury H E P strain also keeps a residual pathogenicity for some rodents and some carnivorous animals [5]. The 675 strain derived from H E P seems less pathogenic than the parental strain [21]. The stability of the AVO1 strain, easily identifiable with its genetic marker, is poor because it reverts to a virulent phenotype after successive passages by IC inoculation into suckling mice or after oral vaccination of rodents tested for innocuousness. This high frequency of reversion finds its cause in the genetic difference between AVO1 and its parental CVS strain. The loss of virulence is the consequence of a mutation producing the substitution of an arginine by a glutamine at position 333 of the glycoprotein [17]At present, AVO1 is clearly not fit for use in field application. A stable live vaccine must either have a deletion in the antigenic site responsible for the virulence of the CVS strain or a double mutation if a second site of virulence can be defined. Further studies are necessary for finding a means to avoid the decrease in the efficiency of the vaccine after incorporation in baits and to increase the genetic stability of these avirulent mutants, which represent a modern, original approach to obtaining a live vaccine. When these two problems are solved, the avirulent strains may be proposed as candidates for oral vaccination of foxes.
R~SUM~ VACCINATION ORALE CONTRE LA RAGE AVEC UN MUTANT AVIRULENT DE LA SOUCHE CVS IEVALUATION ET
DE
DE
SON POUVOIR
SON EFFICACIT]E INFECTANT
:
CHEZ LE RENARD
CHEZ SEPT
AUTRES
ESPI~CES
Les r6sultats d'essais de vaccination orale des renards avec la souche m u t a n t e AVO1 du virus de la rage, s61ectionn~e en pr6sence d'anticorps monoclonaux et neutralisants, dirig6s contre la glycoprot6ine, sont rapport6s. Le pouvoir immunog~ne de AVO1 a ~t6 bon lorsque le vaccin 6tait d6pos6 directement dans la gueule du renard. La vaccination a entrain~ la production d'anticorps neutralisants chez 7 renards sur 13 et la protection de 10 sur 13 apr~s une 6preuve virulente au 90~ jour, si le titre en virus vaccinal exc6dait 10' DLso souriceau. Toutefois, l'incorporation du vaccin dans les app~ts n'a pas 6t6 efficace.
72
M. PIPPIN AND COLL.
L'innocuit~ de AVO1 a 5t~ test~e chez le renard et chez 7 esp~ces non cibles, soit 247 a n i m a u x au total. Le virus de la rage n'a pu ~tre r~isol~ qu'A partir des eerveaux de 2 rongeurs du genre Arvicola; le virus ~tait absent des glandes salivaires de ces animaux. P a r passages successifs sur cerveau de souriceau; la stabilit~ de la souehe est m~diocre car AVO1 a reeouvr~ son ph~notype sauvage apr~s u n seul passage. E n consequence, les ~tudes ult~rieures devront riser h am~liorer les conditions d'incorporation d u virus dans les app~ts e t h a u g m e n t e r la stabilit~ g~n~tique de AVO1 a v a n t de tester ce m u t a n t sur le terrain. MOTS-C:L~S : Bage, R e n a r d , Vaccin ; Anticorps monoclonaux, I m m u n i sation orale, Souche AVO1.
ACKNOWLEDGMENTS We would like to thank L. Andral, C. Le Louedec, P. Pardon and M. Ptommet for their advice, M. J. Barrat, J. M, Demerson and J . George for their technical assistance and Jayne Alderson for her help in writing this paper.
REFERENCES
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[4] BLACK, J. G. & LAWSON, K. F., The safety and efficacy of immunizing foxes (Vulpes vulpes) using bait containing attenuated rabies virus vaccine. Canad. J. comp. Med., 1980, 44, 169-176. [5] BLANCOU,J., Prophylaxie m6dicale de la rage chez le renard. Rec. Mdd. vdt. (Alfort), 1979, 155, 733-741.
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CVS MUTANT IN FOX RABIES ORAL VACCINATION
73
[12] DUBREUIL, M., ANDRAL, L., AUBERT, M. F. A. & BLANCOU, J., The oral [13]
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[17]
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