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Immunization of dogs with a DNA vaccine induces protection against rabies virus
Vaccine 18 (2000) 479±486
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Immunization of dogs with a DNA vaccine induces protection against rabies virus P. Perrin a,*, Y. Jacob a, A. Aguilar-SeÂtien b, E. Loza-Rubio c, C. Jallet a, E. DesmeÂzieÁres a, M. Aubert d, F. Cliquet d, N. Tordo a a
Laboratoire des Lyssavirus; Institut Pasteur 25, rue du Dr. Roux 75724, Paris Cedex 15, France Unidad de Investigacion MeÂdica en Immunologia, Coordinacion de investigacion, Intituto Mexicano del Seguro Social, Apartado Postal 73-032, 03020 Mexico D.F., Mexico c National Institute of Veterinary Microbiology Researches, INIFAP, Carretera Federal Mexico Toluca Km 15.5, CP 05110 Mexico City, Mexico d Laboratoire d'Etudes sur la Rage et la Pathologie des Animaux Sauvages, Centre National d'Etudes VeÂteÂrinaires et Alimentaires, CNEVA Nancy, Domaine de PixeÂreÂcourt, BP 9, F-54220 Malzeville, France b
Received 22 March 1999; received in revised form 10 May 1999; accepted 10 May 1999
Abstract Rabies is a fatal encephalomyelitis which is transmitted to man, mostly by dogs in developing countries. This zoonosis can be prevented by vaccination of humans before or after exposure. However, a more radical approach is possible, involving the elimination of the principal vector/reservoir by vaccinating dogs. The vaccine must be eective, safe and inexpensive. Mass production of plasmids is possible and DNA-based immunization with a plasmid encoding the antigen responsible for inducing protection seems to be more cost-eective than classical techniques involving cell culture. Beagles were immunized by intramuscular (i.m.) injection with a plasmid encoding the rabies virus (PV strain) glycoprotein. Neutralizing antibodies against both wild-type rabies virus and European Bat Lyssaviruses (EBL1 and EBL2) were detected after a single injection and a boost, but levels of neutralizing antibodies against EBL1 were low. Moreover, all vaccinated dogs were protected against a lethal challenge with a wild-type dog rabies strain. This is one of the ®rst studies to demonstrate that dogs can be protected by DNA vaccines, and opens important perspectives for rabies control. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Vaccine; DNA immunization; Rabies; Lyssavirus; Dog
1. Introduction Rabies is a zoonosis that can be transmitted to man by contact with infected animals. Dogs have been implicated in the transmission of rabies to humans, since at least the 23rd century BC [23]. They are still the major vector for rabies transmission throughout the world, and are responsible for 94% of the estimated 40,000 human deaths from rabies per year. * Corresponding author: Tel.: +33-1-40-68-87-56; fax.: +33-1-4061-32-56. E-mail address: [email protected] (P. Perrin)
Ninety eight percent of these human cases occur in the developing countries of Africa, Asia and Latin America [7,13,24]. As for any zoonosis, humans can be protected directly or indirectly, by vaccinating humans or dogs, respectively. Ecient post-exposure vaccination of humans is possible but being expensive, its cost severely limits its availability in many developing countries. Mass campaigns of preventive vaccination for dogs has the advantage of breaking the natural cycle of the disease by rendering the principal vector inecient. Only 34 million of the 500 million dogs that are potentially exposed to rabies [16] have been vaccinated.
0264-410X/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 9 9 ) 0 0 2 4 7 - 9
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Table 1 Dog characteristics and injection protocol.a Group
Dog number
Sexa
Age (years)
A
1 2 3 4 5 6 7 8 9 10b 11 12
M F F F M F M F F M M M
8 6 6 6 6 8 4 7 4 4 4 4
B C D
a b
Injection Product injected
Number of sites
Plasmid Plasmid Plasmid Plasmid Plasmid Plasmid Plasmid Plasmid Plasmid PBS PBS PBS
1 1 1 3 3 3 1 1 1 1 1 1
Day 0, 21, 42 and 0, 21, 42 and 0, 21, 42 and 0, 21, 42 and 0, 21, 42 and 0, 21, 42 and 0 and 175 0 and 175 0 and 175 0, 21, 42 and 0, 21, 42 and 0, 21, 42 and
175 175 175 175 175 175
175 175 175
M: male; F: female. Discarded on day 160 (sick).
Thirty percent of these are given traditional vaccines consisting of more or less inactivated viral suspensions from the brains of experimentally infected animals (suckling mouse, sheep or goat). These traditional vaccines are only weakly immunogenic but are still produced and used in developing countries because they are less expensive than vaccines produced by tissue culture, despite attempts to reduce the cost of tissue culture vaccines [18]. Rabies vaccines currently available (genotype 1) give better protection against the European Bat Lyssavirus 2 (EBL2: genotype 6) than against EBL1 (genotype 5) which was recently implicated in infection of a sheep in Denmark [14,15]. Inoculation of mice with plasmids containing the gene encoding the rabies virus glycoprotein eciently induces humoral and cellular immune responses resulting in protection against intracerebral challenge with the virus [25]. DNA immunization also protects nonhuman primates against rabies [12]. The spectrum of protection against lyssaviruses (including rabies and rabies-related viruses) has been broadened by using chimaeric lyssavirus glycoproteins in mouse models [4,9]. DNA vaccines, which are based on versatile and less expensive technology and oer many other advantages [3], may therefore be a valuable alternative for immunizing dogs, thereby protecting humans against rabies. However, there have been very few reports of DNA-based immunization of dogs [10] and none of them reports protection against rabies. We immunized dogs with a plasmid containing the gene encoding the rabies virus (PV strain genotype 1) glycoprotein and then challenged the dogs with a lethal dose of a wildtype canine rabies virus. Further, we evaluated the production of neutralizing antibodies against both rabies and European bat lyssaviruses.
2. Materials and methods 2.1. Viruses and cells BHK-21 and BSR [21] cells were grown in Eagle's minimal essential medium (MEM) containing 5% fetal bovine serum (FBS) and 5% new-born calf serum or 10% FBS respectively [20]. Fixed rabies (genotype 1) PV-Paris/BHK-21 (Pasteur virus) and CVS (challenge virus standard) strains were maintained by serial passages in BHK-21 cells as previously described [20]. Fox wild-type rabies virus (FWRV) from France (genotype 1), EBL1b (8916FRA) (genotype 5) and EBL2 (9007FIN) (genotype 6) strains were propagated on BSR cells [9,19]. As cell passages caused no change in the nucleotide sequence of the FWRV nucleoprotein gene, FWRV was considered to be a `wild-type' virus [19]. The wild-type canine rabies strain (Ariana 1009) was selected in Tunisia by Haddad and Chappuis (1993 personal communication). From the ground salivary glands of nine naturally infected dogs, they selected the isolate that produced the highest titer in mice when injected intracerebrally. This strain has been used to challenge dogs in four previous vaccine potency trials. When administrated intramuscularly (1 ml, diluted 1 in 50), this strain transmitted rabies to 26 of a total of 27 non-vaccinated dogs after 18 to 94 days (Haddad N. and Chappuis G., 1993; ld Sidi Yahia S. 1995; Hammami S. 1996, personal communications and personal results). 2.2. Dogs The beagles used, came from a pack established since 1970 at the Instituto National de Investigaciones
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Fig. 1. Kinetics of virus neutralizing antibody production in dogs injected with the plasmid encoding the rabies virus glycoprotein. Animals received a single injection (V: 100 mg) or three injections (vvv: 3 33 mg) on various days. Virus neutralizing antibodies were assayed against: (A) the Pasteur virus (PV); (B) the challenge virus standard (CVS); and (C) the wild-type fox rabies virus (FWRV).
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Fig. 2. Immunisation of dogs with the plasmid encoding the rabies virus glycoprotein: individual virus neutralizing antibody response against the wild-type fox rabies virus (FWRV) and survival to the challenge carried out on day 231 by injecting intramuscularly the wild-type rabies virus isolated from rabid Tunisian dogs.
Forestales y Agropecuarias in Mexico. They had previously been used in the study of a leptospira vaccine. However, they had not previously been vaccinated against rabies, and none had rabies virus neutralizing antibodies in their serum. The dogs were 4±8 years old and were assigned to four experimental groups (A, B, C and D: see Table 1). Dog 10 was excluded from the study on day 160 because he contracted diarrhoea due to infection with Coccidia sp. Each dog was housed in a separate cage with a cement ¯oor (3 1.5 m outdoor surface and a covered construction 1.5 1 m in area) and fed daily with 500 g of a commercial balanced food for adult dogs (Pedigree r). Water was provided ad libitum. 2.3. Plasmid construction The gene encoding the rabies virus (PV strain) glycoprotein (G) was inserted in the pClneo vector (Promega) (pGPV) under the control of the cytomegalovirus, an immediate early promoter and enhancer, as described elsewhere [4]. 2.4. Immunization and bleeding All animals received injections of plasmid pGPV (groups A, B and C) or phosphate-buered saline (PBS: group D) in the tibialis muscle of the right back leg. Animals from groups A and D received a single injection (900 ml containing 100 mg of plasmid or 900
ml PBS respectively) on days 0, 21, 42 and 175. Animals from group B received three injections (3 300 ml each containing 33 mg of plasmid) on days 0, 21, 42 and 175. Animals from group C received a single injection (900 ml containing 100 mg plasmid) on days 0 and 175. Blood samples were collected from the humeral vein of all dogs on days 0, 28, 49, 70, 120, 175, 189 and 231.
2.5. Virus neutralizing antibody assay Lyssavirus neutralizing antibodies (VNAb) were titrated by the rapid ¯uorescent focus inhibition test (RFFIT) [22] with the modi®cations described elsewhere [17]. BHK-21 were used for VNAb assay against both rabies PV and CVS strains, whereas BSR cells were used for VNAb assay against FWRV, EBL1b and EBL-2 [9]. Anti-lyssavirus VNAb titers are expressed in international units per ml (IU/ml) using the Second International Standard (Statens Seruminstitut, Copenhagen, Denmark) as the reference or as the reciprocal serum dilution (rd) that inhibited 50% of the ¯uorescent focus. Under these conditions, a titer of 40, 70 or 60 (reciprocal serum dilution = rd) was equivalent to 0.5 IU/ml against PV, CVS, or FWRV respectively.
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2.6. Sera analysis by western blot Dog sera were analyzed by western blot using puri®ed rabies virus (PV strain). After SDS-PAGE electrophoresis, rabies polypeptides were transferred onto nitrocellulose membrane. Membrane was incubated for 2 h, with 2% new born bovine serum diluted in `blocking reagent' (Boehrringer Mannheim) washed, then incubated overnight at 48C in the presence of rabbit antirabies (diluted 1/1000) or dog sera (diluted 1/25). After washing, peroxidase conjugated anti-rabbit (Nordic Immunological Labs) or anti-dog (Biosys) antibodies were added and incubated at room temperature for 1 h. Peroxidase conjugates were revealed by chemiluninescence (ECL: Amersharn Pharmacia Biotech). 2.7. Challenge of dogs Beagles injected with the plasmid or PBS were challenged on day 231 by inoculating 1 ml of the Ariana 1009 canine rabies strain (diluted 1/50) into the temporal muscle. After inoculation of the last control dog, the challenge virus suspension was immediately titrated in mice by intracerebral injection. This titration demonstrated that each dog had received 40,000 times the mouse intracerebral lethal dose50 and that the virus titer was similar to previous determinations (personal observation). We tested for the presence of rabies virus antigens in the brain impressions of dead dogs by assaying with an antinucleocapsid antibody ¯uorescein conjugate [20]. 3. Results 3.1. VNAb production against PV and CVS viruses On day 0, none of the 12 dogs used in this experiment were seropositive (VNAb titer: rd < 40; <0.5 IU/ml), indicating that they had never been in contact with rabies antigens (Figs. 1 and 2). A single intramuscular injection into dogs of the plasmid, pGPV (100 mg), encoding the rabies virus (PV strain) glycoprotein (group C), induced virus neutralizing antibodies (VNAb) against the parental PV strain (Fig. 1A). Signi®cant VNAb titers were obtained as early as day 28 (rd = 95). They then decreased until day 70 and stabilised or slightly increased until day 175. If a boost with the same amount of plasmid was given on day 21 at a single site (group A), very high VNAb titers were obtained on day 28 (rd = 410). If a second similar boost was given on day 42, VNAb titers on day 49 were not signi®cantly higher (rd = 830) (group A). As for the dogs of group C that received a single injection, there was then a progressive decrease
483
in VNAb titers in dogs of group A from day 70 to day 120 (rd = 100), followed by a signi®cant increase on day 175 (rd = 300). If the same amount of plasmid was injected at three sites (group B; 3 33 mg per injection), the kinetics were similar to those with injection at a single site (group A; 100 mg), although VNAb titers were slightly higher, except on day 28. If a boost was given on day 175 to all 3 groups of dogs, VNAb titers were much higher on day 189 (rd = 2000, 4000 and 600 for group A, B and C, respectively). Thus, the various DNA vaccination protocols induced high levels of VNAb against the parental virus, but injection of the plasmid at several sites did not signi®cantly increase VNAb production. If the rabies virus used for antibody titration was changed from the PV strain (Fig. 1A) to which the glycoprotein gene was homologous, to heterologous virus, such as the CVS strain (Fig. 1B), the kinetics of VNAb production was very similar. Even with only a single i.m. injection of plasmid (group C), a mean VNAb titer >0.5 IU/ml (classically recognised as ecient seroconversion) was obtained as early as day 28 and was maintained until the boost on day 175. After this boost, dramatic increases in mean VNAb titers of 27, 30 and 7 IU/ml were recorded for groups A, B and C, respectively (Fig. 1B). This clearly indicates that DNA-based immunization of dogs with plasmid encoding the PV glycoprotein induced very high levels of VNAb against viruses of the genotype 1.
3.2. VNAb production against the wild-type fox rabies virus VNAb titers against the wild-type rabies virus were also analyzed and the individual responses of dogs are reported in Fig. 2. None of the control group D (dogs nos. 10, 11 and 12) were found to be seropositive at any time during the experiment until the challenge. All the dogs in groups A and B (nos. 1±6) given several injections, seroconverted, and were found to be seropositive whenever tested between days 28 and 189 (dog 3 was not tested on day 120). Only the 3 dogs of group C given a single injection of 100 mg of plasmid gave heterogeneous results: dog 9 was clearly seropositive from day 28 until the boost on day 175; dogs 7 and 8 did not seroconvert (always 0.5 IU/ml), although they generally scored higher than the control dogs of group D. However, all the dogs in group C clearly seroconverted after receiving a boost on day 175. This shows that a single injection followed by a boost on day 21 or 175 were enough to induce high levels of VNAb against a wild-type rabies virus of the genotype 1.
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FWRV, they remained high, but decreased comparatively to day 189. Against EBL1B, they were low. Against EBL2 they were high. Thus the DNA plasmid encoding the pV glycoprotein has induced VNAb against European lyssaviruses but to a smaller extent against EBL1b. 3.5. Protection against a challenge with the wild-type dog rabies virus
Fig. 3. Western blot of sera from dogs immunized with pGPV. Sera blotted on puri®ed rabies virus corresponded to: rabbit anti-rabies serum (lane 1), pool of sera (day 189) of dogs from group B (lane 2) and pool of sera (day 189) of control dogs from group D (lane 3).
3.3. Western blot analysis of sera from immunized dogs Sera from dogs bled on day 189 were analyzed by western blot on puri®ed rabies virus (Fig. 3). Pooled sera from dogs of group B clearly reacted with the rabies virus glycoprotein (lane 2), whereas pooled sera from dogs of group D did not (lane 3). This indicated that beside VNAb recognizing conformational epitopes, antibodies recognizing non conformational epitopes were also induced. 3.4. VNAb level against European lyssaviruses before challenge Antibody levels before challenge (day 231) were also controlled against European lyssaviruses (genotypes 1, 5 and 6). As reported in Table 2, neutralizing antibodies against FWRV, EBL1b and EBL2 were induced in dogs after DNA-based immunization. Against Table 2 Neutralizing antibody induced against wild-type rabies virus and European Bat Lyssaviruses measured before challenge of dogs.a VNAb level (rda) against:
Group Group Group Group a
A B C D
FWRV
EBL1b
EBL2
354 (289) 221 (158) 77 (17) < 15
115 (30) 92 (42) 50 (30) < 15
1000 (355) 833 (531) 156 (66) < 15
rd: reciprocal dilution.
We studied the eectiveness of plasmid injection, by challenging all dogs with a lethal dose of a wild-type rabies virus isolated from rabid dogs in Tunisia. We challenged with a dog-speci®c strain to avoid the problems resulting from the survival of non-vaccinated animals: animal mortality is lower if dogs (or foxes) are challenged with very high concentrations of a heterologous rabies virus strain from fox (or dog, respectively) [5,6]. Dogs immunized with a single (group C) or several (groups A and B) injections of plasmid plus a boost were protected against the viral challenge (Fig. 2). The nonimmunized dogs, 11 and 12 rapidly developed rabies. Dog 12 began to have characteristic symptoms (anorexia, change in tone, salivation), 12 days after the challenge and died 8 days later. For dog 11, the symptoms started 15 days after challenge and death occurred 4 days later. After the death of these animals, rabies virus nucleocapsids were detected on brain impressions by direct immuno¯uorescence. The use of only two control dogs (dog 10 died before challenge), to prevent unnecessary animal suering, was possible due to the accurate calibration of the challenge dose required by previous trials in dogs. Titration of the challenge virus in mice immediately after use demonstrated that the virus dose used for the challenge exceeded the 100% lethal i.m. dose for dogs (see Section 2). All surviving dogs after challenge have a VNAb titer r0.5 IU/ml. Under these conditions, a very weak level of VNAb was enough to obtain protection against a lethal challenge as shown for dogs 4, 7 and 8 having about 0.5 IU/ml. 4. Discussion Previous studies have shown that DNA-based immunization with plasmids encoding the rabies virus glycoprotein (CVS, ERA and PV strains) protects mice against rabies [4,9,11,25]. A comparative study of DNA vaccination (G of CVS strain) versus a classical vaccine prepared using human diploid cells has been carried out in non-human primates (Macaca fascicularis ) [12]. This study indicates the potential for DNA vaccination of humans. However, this approach would encounter several obstacles. The use of DNA immunization in humans is controversial due to the potential
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risks of DNA integration, particularly for diseases like rabies against which alternative safe and ecient vaccines are already available. In addition, DNA immunization has only been demonstrated to be ecient for preventive vaccination, a situation which is exceptional for rabies in humans, which are primarily treated, post-exposure. Thus, we investigated the potential of DNA immunization for the preventive vaccination of dogs against rabies and European Bat Lyssaviruses because post-exposure treatments are required chie¯y, following possible contamination via dogs. We decide to use i.m. injection for several reasons: (1) needle injection seems more appropriate than gene gun delivery for mass vaccination campaigns, for dogs, in developing countries; (2) i.m. injection of DNA vaccines has been demonstrated to be ecient in both rodents and monkeys, whereas intradermal injection was not ecient in monkeys. We compared various protocols (2±4 i.m. injections, one or three sites) and found that two i.m. injections, each of 100 mg of plasmid (PClneo encoding the rabies virus glycoprotein), on days 0 and 28 or 0 and 175, were sucient to induce high VNAb titers against the parental virus (PV strain), the standard virus (CVS strain) and a wild-type rabies virus isolated from a French fox (FWRV). We also found that high levels of neutralizing antibodies were elicited against EBL2 but few against EBL1b. Moreover, vaccinated dogs survived a lethal challenge with a wild-type rabies virus isolated from rabid Tunisian dogs. This clearly demonstrates, for the ®rst time, that preventive DNA-based immunization of dogs would be eective against canine rabies, with considerable bene®ts for human health. DNA immunization has great potential for improving the strategy for vaccination against lyssaviruses. It would be most useful if a strong enough immune response could be obtained after a single shot, because it is dicult to recover dogs for booster injections in developing countries. In all three groups of dogs, mean VNAb titers signi®cantly increased from day 70/120 to day 175. The reason for this is unclear but it may be that there was constant stimulation of the immune system due to low but persistent levels of glycoprotein production by the injected plasmid. The VNAb titers against FWRV measured after a single i.m. injection of 100 mg (group C) of plasmid were variable (only 1 dog in 3 clearly seroconverted), but were slightly higher than those of the control dogs. This is why a boost was given to all dogs on day 175 before challenge. Under these conditions, levels of antibody dramatically increased as evidenced on day 189 (Fig. 2 ; lowest titer for dog 8: 1.6 IU/ml; highest titer for dog 3: 64 IU/ml). These high levels of VNAb were not maintained and in particular, for dogs 4, 7 and 8 which have about 0.5 IU/ml. Nevertheless, all immunized dogs were protected against the challenge.
485
Consequently, as all dogs, including those receiving a single injection, have VNAb titer higher than 0.5 IU/ ml on day 175 before boosting, we can imagine that the boost would not be necessary, because there are several reports of protection in dogs with VNAb titers above 0.1 IU/ml [2]. Thus, dogs are as sensitive to i.m. injection of DNA vaccine as monkey [12] and 100 mg of plasmid (here) was sucient to generate an eective immune response in dogs. It is well known that rabies encephalomyelitis is caused by diverse lyssaviruses (genotype 1 for rabies viruses, 6 other genotypes of rabies-related viruses). In a mouse model the rabies vaccines currently available (genotype 1), give protection against genotype 1, 6 (EBL2) and 7 (Australian pteroid bat), no protection against the more divergent genotypes 2, 3 and 4 or weak protection against EBL1 [4,8,9]. Both EBL1 and EBL2 viruses were responsible for human cases (see [1]. We showed that the plasmid encoding the PV glycoprotein induced in dogs the production of VNAb, against not only the wild-type rabies viruses that naturally infect red foxes in Western Europe (FWRV), but also against EBL2 and in less extent against EBL1. The versatility of DNA recombinant methods makes it possible to generate chimeric lyssavirus glycoproteins with a broadened spectrum of protection against lyssaviruses. Indeed, as recently demonstrated, mice immunized with the chimeric lyssavirus plasmid pGEBL1PV were protected against an intracerebral challenge with European lyssaviruses including EBL1 [9]. The use of such a chimeric glycoprotein for immunizing dogs would be bene®cial. The results for dog protection demonstrate that DNA immunization is of great potential value for protecting humans against rabies and rabies-related viruses by immunizing the canine reservoir against the disease.
Acknowledgements We would like to thank Dr. D. Drocourt (CAYLA, 5, rue Jean Rodier, Z1 Montauban F-31400 Toulouse) for plasmid preparation and Drs O.De Paz and E.L. Espinosa for excellent technical assistance.
References [1] Amengal B, Whitby J, Cobo S, Bourhy H. Evolution of european bat lyssaviruses. J Gen Virol 1997;78:2319±28. [2] Aubert MFA. Can vaccination validated by the titration of rabies antibodies in serum of cats and dogs be an alternative to quarantine measures? Abstract of Hygiene and Communicable Diseases 1993;68:R1±R22. [3] Babiuk L, Lewis P, Drunen S, Tikoo S, Liang X. Nucleic acid vaccines: veterinary applications. In: Compans RW, et al., edi-
486
[4] [5] [6] [7] [8] [9] [10] [11]
[12]
[13] [14] [15]
P. Perrin et al. / Vaccine 18 (2000) 479±486 tors. Current topics microbio-logy and immunology, vol. 226. Berlin: Springer-Verlag, 1998. p. 90. Bahloul C, Jacob Y, Tordo N, Perrin P. DNA-based immunization for exploring the enlargement of immunological crossreactivity against the lyssaviruses. Vaccine 1998;16:417±25. Blancou J, Aubert MFA, Soulebot JP. Dierence dans le pouvoir pathogeÁne de souches de virus rabiques adapteÂes au renard et au chien. Ann Virol 1983;134E:523±31. Blancou J, Aubert MFA. Transmission of the rabies virus: importance of the species barrier. Bull Acad Natl Med 1997;181:301±12. Dreesen D. A global review of rabies vaccines for human use. Vaccine 1997;15:S2±S6. Gould A, Hyatt A, Lunt R, Kattenbelt J, Hengstberger S, Blacksell S. Characterisation of a novel lyssavirus isolated from Pteroid bats in Australia. Virus Research 1998;54:165±87. Jallet C, Jacob Y, Bahloul C, Drings A, DesmeÂzieÁres E, Tordo N, Perrin P. Chimeric lyssavirus glycoproteins with increased immunological potential. J Virol 1999;73:225±33. Jiang W, Baker H, Swango L, Schorr J, Self M, Smith B. Nucleic acid immunization protects dogs against challenge with virulent canine parvovirus. Vaccine 1998;16:601±7. Lodmell D, Ray N, Ewalt R, Hanlon D, Shaddock J, Sanderlin D, Rupprecht C. Gene gun particle-mediated vaccination with plasmid DNA confers protective immunity against rabies virus infection. Vaccine 1998;16:115±8. Lodmell D, Ray N, Parnell M, Ewalt R, Hanlon D, Shaddock J, Sanderlin D, Rupprecht C. DNA immunization protects nonhuman primates against rabies virus. Nature Med 1998;4:949± 52. Meslin F-X, Fishbein D, Matter H. Rationale and prospects for rabies elimination in developing countries. In: Rupprecht, et al., editors. Lyssaviruses. Berlin: Springer-Verlag, 1994. p. 1. OIE. Rabies in Denmark in sheep. Disease information 1998;11(34). OIE. Rabies in Denmark. Disease information 1998;11(37).
[16] Pastoret P-P, Brochier B, Aguilar-SetieÂn A, Blancou J. Vaccination against rabies. In: Pastoret P-P, et al., editors. Veterinary vaccinology. Amsterdam: Elsevier, 1997. p. 616. [17] Perrin P, Versmisse P, Delagneau JF, Lucas G, Rollin P, Sureau P. The in¯uence of the type of immunosorbent on rabies antibody EIA; advantages of puri®ed glycoprotein over whole virus. J Biol Standard 1986;14:95±102. [18] Perrin P, Madhusudana S, Jallet C, Petres S, Tordo N, Merten O-W. An experimental rabies vaccine produced with a new BHK-21 suspension cell culture process: use of serum-free medium and perfusion system. Vaccine 1995;13:1244±50. [19] Perrin P, De Franco M, Jallet C, Fouque F, Morgeaux S, Tordo N, Colle J-H. The antigen-speci®c cell-mediated immune response in mice is suppressed by infection with pathogenic lyssaviruses. Res Virol 1996;147:289±99. [20] Perrin P. Techniques for the preparation of rabies conjugates. In: Meslin F-X, Kaplan M, Koprowski H, editors. Laboratory techniques in rabies, 4th ed. Geneva: WHO, 1996. p. 433. [21] Sato M, Maeda N, Yoshida H, Urade M, Saito S, Miyazaki T, Shibata T, Watanabe M. Plaque formation of herpes virus Hominis type 2 and rubella virus in variants isolated from the colonies of BHK-21/WI-2 cells formed in soft agar. Arch Virol 1977;53:269±73. [22] Smith J, Yager P, Baer G. A rapid ¯uorescent focus inhibition test (RFFIT) for determining virus-neutralizing antibody. In: Meslin F-X, Kaplan M, Koprowski H, editors. Laboratory techniques in rabies, 4th ed. Geneva: WHO, 1996. p. 181. [23] Theodorides J. In: Histoire de la rage. Paris: Masson, 1986. p. 17. [24] WHO. World Survey of Rabies. World Health Organization, Geneva 1994; 210. [25] Xiang Z, Spitalnik S, Tran M, Wunner W, Cheng J, Ertl H. Vaccination with a plasmid vector carrying the rabies virus glycoprotein gene induces protective immunity against rabies virus. Virol 1994;199:132±40.
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Immunization of dogs with a DNA vaccine induces protection against rabies virus P. Perrin a,*, Y. Jacob a, A. Aguilar-SeÂtien b, E. Loza-Rubio c, C. Jallet a, E. DesmeÂzieÁres a, M. Aubert d, F. Cliquet d, N. Tordo a a
Laboratoire des Lyssavirus; Institut Pasteur 25, rue du Dr. Roux 75724, Paris Cedex 15, France Unidad de Investigacion MeÂdica en Immunologia, Coordinacion de investigacion, Intituto Mexicano del Seguro Social, Apartado Postal 73-032, 03020 Mexico D.F., Mexico c National Institute of Veterinary Microbiology Researches, INIFAP, Carretera Federal Mexico Toluca Km 15.5, CP 05110 Mexico City, Mexico d Laboratoire d'Etudes sur la Rage et la Pathologie des Animaux Sauvages, Centre National d'Etudes VeÂteÂrinaires et Alimentaires, CNEVA Nancy, Domaine de PixeÂreÂcourt, BP 9, F-54220 Malzeville, France b
Received 22 March 1999; received in revised form 10 May 1999; accepted 10 May 1999
Abstract Rabies is a fatal encephalomyelitis which is transmitted to man, mostly by dogs in developing countries. This zoonosis can be prevented by vaccination of humans before or after exposure. However, a more radical approach is possible, involving the elimination of the principal vector/reservoir by vaccinating dogs. The vaccine must be eective, safe and inexpensive. Mass production of plasmids is possible and DNA-based immunization with a plasmid encoding the antigen responsible for inducing protection seems to be more cost-eective than classical techniques involving cell culture. Beagles were immunized by intramuscular (i.m.) injection with a plasmid encoding the rabies virus (PV strain) glycoprotein. Neutralizing antibodies against both wild-type rabies virus and European Bat Lyssaviruses (EBL1 and EBL2) were detected after a single injection and a boost, but levels of neutralizing antibodies against EBL1 were low. Moreover, all vaccinated dogs were protected against a lethal challenge with a wild-type dog rabies strain. This is one of the ®rst studies to demonstrate that dogs can be protected by DNA vaccines, and opens important perspectives for rabies control. # 1999 Elsevier Science Ltd. All rights reserved. Keywords: Vaccine; DNA immunization; Rabies; Lyssavirus; Dog
1. Introduction Rabies is a zoonosis that can be transmitted to man by contact with infected animals. Dogs have been implicated in the transmission of rabies to humans, since at least the 23rd century BC [23]. They are still the major vector for rabies transmission throughout the world, and are responsible for 94% of the estimated 40,000 human deaths from rabies per year. * Corresponding author: Tel.: +33-1-40-68-87-56; fax.: +33-1-4061-32-56. E-mail address: [email protected] (P. Perrin)
Ninety eight percent of these human cases occur in the developing countries of Africa, Asia and Latin America [7,13,24]. As for any zoonosis, humans can be protected directly or indirectly, by vaccinating humans or dogs, respectively. Ecient post-exposure vaccination of humans is possible but being expensive, its cost severely limits its availability in many developing countries. Mass campaigns of preventive vaccination for dogs has the advantage of breaking the natural cycle of the disease by rendering the principal vector inecient. Only 34 million of the 500 million dogs that are potentially exposed to rabies [16] have been vaccinated.
0264-410X/99/$ - see front matter # 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 9 9 ) 0 0 2 4 7 - 9
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Table 1 Dog characteristics and injection protocol.a Group
Dog number
Sexa
Age (years)
A
1 2 3 4 5 6 7 8 9 10b 11 12
M F F F M F M F F M M M
8 6 6 6 6 8 4 7 4 4 4 4
B C D
a b
Injection Product injected
Number of sites
Plasmid Plasmid Plasmid Plasmid Plasmid Plasmid Plasmid Plasmid Plasmid PBS PBS PBS
1 1 1 3 3 3 1 1 1 1 1 1
Day 0, 21, 42 and 0, 21, 42 and 0, 21, 42 and 0, 21, 42 and 0, 21, 42 and 0, 21, 42 and 0 and 175 0 and 175 0 and 175 0, 21, 42 and 0, 21, 42 and 0, 21, 42 and
175 175 175 175 175 175
175 175 175
M: male; F: female. Discarded on day 160 (sick).
Thirty percent of these are given traditional vaccines consisting of more or less inactivated viral suspensions from the brains of experimentally infected animals (suckling mouse, sheep or goat). These traditional vaccines are only weakly immunogenic but are still produced and used in developing countries because they are less expensive than vaccines produced by tissue culture, despite attempts to reduce the cost of tissue culture vaccines [18]. Rabies vaccines currently available (genotype 1) give better protection against the European Bat Lyssavirus 2 (EBL2: genotype 6) than against EBL1 (genotype 5) which was recently implicated in infection of a sheep in Denmark [14,15]. Inoculation of mice with plasmids containing the gene encoding the rabies virus glycoprotein eciently induces humoral and cellular immune responses resulting in protection against intracerebral challenge with the virus [25]. DNA immunization also protects nonhuman primates against rabies [12]. The spectrum of protection against lyssaviruses (including rabies and rabies-related viruses) has been broadened by using chimaeric lyssavirus glycoproteins in mouse models [4,9]. DNA vaccines, which are based on versatile and less expensive technology and oer many other advantages [3], may therefore be a valuable alternative for immunizing dogs, thereby protecting humans against rabies. However, there have been very few reports of DNA-based immunization of dogs [10] and none of them reports protection against rabies. We immunized dogs with a plasmid containing the gene encoding the rabies virus (PV strain genotype 1) glycoprotein and then challenged the dogs with a lethal dose of a wildtype canine rabies virus. Further, we evaluated the production of neutralizing antibodies against both rabies and European bat lyssaviruses.
2. Materials and methods 2.1. Viruses and cells BHK-21 and BSR [21] cells were grown in Eagle's minimal essential medium (MEM) containing 5% fetal bovine serum (FBS) and 5% new-born calf serum or 10% FBS respectively [20]. Fixed rabies (genotype 1) PV-Paris/BHK-21 (Pasteur virus) and CVS (challenge virus standard) strains were maintained by serial passages in BHK-21 cells as previously described [20]. Fox wild-type rabies virus (FWRV) from France (genotype 1), EBL1b (8916FRA) (genotype 5) and EBL2 (9007FIN) (genotype 6) strains were propagated on BSR cells [9,19]. As cell passages caused no change in the nucleotide sequence of the FWRV nucleoprotein gene, FWRV was considered to be a `wild-type' virus [19]. The wild-type canine rabies strain (Ariana 1009) was selected in Tunisia by Haddad and Chappuis (1993 personal communication). From the ground salivary glands of nine naturally infected dogs, they selected the isolate that produced the highest titer in mice when injected intracerebrally. This strain has been used to challenge dogs in four previous vaccine potency trials. When administrated intramuscularly (1 ml, diluted 1 in 50), this strain transmitted rabies to 26 of a total of 27 non-vaccinated dogs after 18 to 94 days (Haddad N. and Chappuis G., 1993; ld Sidi Yahia S. 1995; Hammami S. 1996, personal communications and personal results). 2.2. Dogs The beagles used, came from a pack established since 1970 at the Instituto National de Investigaciones
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Fig. 1. Kinetics of virus neutralizing antibody production in dogs injected with the plasmid encoding the rabies virus glycoprotein. Animals received a single injection (V: 100 mg) or three injections (vvv: 3 33 mg) on various days. Virus neutralizing antibodies were assayed against: (A) the Pasteur virus (PV); (B) the challenge virus standard (CVS); and (C) the wild-type fox rabies virus (FWRV).
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Fig. 2. Immunisation of dogs with the plasmid encoding the rabies virus glycoprotein: individual virus neutralizing antibody response against the wild-type fox rabies virus (FWRV) and survival to the challenge carried out on day 231 by injecting intramuscularly the wild-type rabies virus isolated from rabid Tunisian dogs.
Forestales y Agropecuarias in Mexico. They had previously been used in the study of a leptospira vaccine. However, they had not previously been vaccinated against rabies, and none had rabies virus neutralizing antibodies in their serum. The dogs were 4±8 years old and were assigned to four experimental groups (A, B, C and D: see Table 1). Dog 10 was excluded from the study on day 160 because he contracted diarrhoea due to infection with Coccidia sp. Each dog was housed in a separate cage with a cement ¯oor (3 1.5 m outdoor surface and a covered construction 1.5 1 m in area) and fed daily with 500 g of a commercial balanced food for adult dogs (Pedigree r). Water was provided ad libitum. 2.3. Plasmid construction The gene encoding the rabies virus (PV strain) glycoprotein (G) was inserted in the pClneo vector (Promega) (pGPV) under the control of the cytomegalovirus, an immediate early promoter and enhancer, as described elsewhere [4]. 2.4. Immunization and bleeding All animals received injections of plasmid pGPV (groups A, B and C) or phosphate-buered saline (PBS: group D) in the tibialis muscle of the right back leg. Animals from groups A and D received a single injection (900 ml containing 100 mg of plasmid or 900
ml PBS respectively) on days 0, 21, 42 and 175. Animals from group B received three injections (3 300 ml each containing 33 mg of plasmid) on days 0, 21, 42 and 175. Animals from group C received a single injection (900 ml containing 100 mg plasmid) on days 0 and 175. Blood samples were collected from the humeral vein of all dogs on days 0, 28, 49, 70, 120, 175, 189 and 231.
2.5. Virus neutralizing antibody assay Lyssavirus neutralizing antibodies (VNAb) were titrated by the rapid ¯uorescent focus inhibition test (RFFIT) [22] with the modi®cations described elsewhere [17]. BHK-21 were used for VNAb assay against both rabies PV and CVS strains, whereas BSR cells were used for VNAb assay against FWRV, EBL1b and EBL-2 [9]. Anti-lyssavirus VNAb titers are expressed in international units per ml (IU/ml) using the Second International Standard (Statens Seruminstitut, Copenhagen, Denmark) as the reference or as the reciprocal serum dilution (rd) that inhibited 50% of the ¯uorescent focus. Under these conditions, a titer of 40, 70 or 60 (reciprocal serum dilution = rd) was equivalent to 0.5 IU/ml against PV, CVS, or FWRV respectively.
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2.6. Sera analysis by western blot Dog sera were analyzed by western blot using puri®ed rabies virus (PV strain). After SDS-PAGE electrophoresis, rabies polypeptides were transferred onto nitrocellulose membrane. Membrane was incubated for 2 h, with 2% new born bovine serum diluted in `blocking reagent' (Boehrringer Mannheim) washed, then incubated overnight at 48C in the presence of rabbit antirabies (diluted 1/1000) or dog sera (diluted 1/25). After washing, peroxidase conjugated anti-rabbit (Nordic Immunological Labs) or anti-dog (Biosys) antibodies were added and incubated at room temperature for 1 h. Peroxidase conjugates were revealed by chemiluninescence (ECL: Amersharn Pharmacia Biotech). 2.7. Challenge of dogs Beagles injected with the plasmid or PBS were challenged on day 231 by inoculating 1 ml of the Ariana 1009 canine rabies strain (diluted 1/50) into the temporal muscle. After inoculation of the last control dog, the challenge virus suspension was immediately titrated in mice by intracerebral injection. This titration demonstrated that each dog had received 40,000 times the mouse intracerebral lethal dose50 and that the virus titer was similar to previous determinations (personal observation). We tested for the presence of rabies virus antigens in the brain impressions of dead dogs by assaying with an antinucleocapsid antibody ¯uorescein conjugate [20]. 3. Results 3.1. VNAb production against PV and CVS viruses On day 0, none of the 12 dogs used in this experiment were seropositive (VNAb titer: rd < 40; <0.5 IU/ml), indicating that they had never been in contact with rabies antigens (Figs. 1 and 2). A single intramuscular injection into dogs of the plasmid, pGPV (100 mg), encoding the rabies virus (PV strain) glycoprotein (group C), induced virus neutralizing antibodies (VNAb) against the parental PV strain (Fig. 1A). Signi®cant VNAb titers were obtained as early as day 28 (rd = 95). They then decreased until day 70 and stabilised or slightly increased until day 175. If a boost with the same amount of plasmid was given on day 21 at a single site (group A), very high VNAb titers were obtained on day 28 (rd = 410). If a second similar boost was given on day 42, VNAb titers on day 49 were not signi®cantly higher (rd = 830) (group A). As for the dogs of group C that received a single injection, there was then a progressive decrease
483
in VNAb titers in dogs of group A from day 70 to day 120 (rd = 100), followed by a signi®cant increase on day 175 (rd = 300). If the same amount of plasmid was injected at three sites (group B; 3 33 mg per injection), the kinetics were similar to those with injection at a single site (group A; 100 mg), although VNAb titers were slightly higher, except on day 28. If a boost was given on day 175 to all 3 groups of dogs, VNAb titers were much higher on day 189 (rd = 2000, 4000 and 600 for group A, B and C, respectively). Thus, the various DNA vaccination protocols induced high levels of VNAb against the parental virus, but injection of the plasmid at several sites did not signi®cantly increase VNAb production. If the rabies virus used for antibody titration was changed from the PV strain (Fig. 1A) to which the glycoprotein gene was homologous, to heterologous virus, such as the CVS strain (Fig. 1B), the kinetics of VNAb production was very similar. Even with only a single i.m. injection of plasmid (group C), a mean VNAb titer >0.5 IU/ml (classically recognised as ecient seroconversion) was obtained as early as day 28 and was maintained until the boost on day 175. After this boost, dramatic increases in mean VNAb titers of 27, 30 and 7 IU/ml were recorded for groups A, B and C, respectively (Fig. 1B). This clearly indicates that DNA-based immunization of dogs with plasmid encoding the PV glycoprotein induced very high levels of VNAb against viruses of the genotype 1.
3.2. VNAb production against the wild-type fox rabies virus VNAb titers against the wild-type rabies virus were also analyzed and the individual responses of dogs are reported in Fig. 2. None of the control group D (dogs nos. 10, 11 and 12) were found to be seropositive at any time during the experiment until the challenge. All the dogs in groups A and B (nos. 1±6) given several injections, seroconverted, and were found to be seropositive whenever tested between days 28 and 189 (dog 3 was not tested on day 120). Only the 3 dogs of group C given a single injection of 100 mg of plasmid gave heterogeneous results: dog 9 was clearly seropositive from day 28 until the boost on day 175; dogs 7 and 8 did not seroconvert (always 0.5 IU/ml), although they generally scored higher than the control dogs of group D. However, all the dogs in group C clearly seroconverted after receiving a boost on day 175. This shows that a single injection followed by a boost on day 21 or 175 were enough to induce high levels of VNAb against a wild-type rabies virus of the genotype 1.
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FWRV, they remained high, but decreased comparatively to day 189. Against EBL1B, they were low. Against EBL2 they were high. Thus the DNA plasmid encoding the pV glycoprotein has induced VNAb against European lyssaviruses but to a smaller extent against EBL1b. 3.5. Protection against a challenge with the wild-type dog rabies virus
Fig. 3. Western blot of sera from dogs immunized with pGPV. Sera blotted on puri®ed rabies virus corresponded to: rabbit anti-rabies serum (lane 1), pool of sera (day 189) of dogs from group B (lane 2) and pool of sera (day 189) of control dogs from group D (lane 3).
3.3. Western blot analysis of sera from immunized dogs Sera from dogs bled on day 189 were analyzed by western blot on puri®ed rabies virus (Fig. 3). Pooled sera from dogs of group B clearly reacted with the rabies virus glycoprotein (lane 2), whereas pooled sera from dogs of group D did not (lane 3). This indicated that beside VNAb recognizing conformational epitopes, antibodies recognizing non conformational epitopes were also induced. 3.4. VNAb level against European lyssaviruses before challenge Antibody levels before challenge (day 231) were also controlled against European lyssaviruses (genotypes 1, 5 and 6). As reported in Table 2, neutralizing antibodies against FWRV, EBL1b and EBL2 were induced in dogs after DNA-based immunization. Against Table 2 Neutralizing antibody induced against wild-type rabies virus and European Bat Lyssaviruses measured before challenge of dogs.a VNAb level (rda) against:
Group Group Group Group a
A B C D
FWRV
EBL1b
EBL2
354 (289) 221 (158) 77 (17) < 15
115 (30) 92 (42) 50 (30) < 15
1000 (355) 833 (531) 156 (66) < 15
rd: reciprocal dilution.
We studied the eectiveness of plasmid injection, by challenging all dogs with a lethal dose of a wild-type rabies virus isolated from rabid dogs in Tunisia. We challenged with a dog-speci®c strain to avoid the problems resulting from the survival of non-vaccinated animals: animal mortality is lower if dogs (or foxes) are challenged with very high concentrations of a heterologous rabies virus strain from fox (or dog, respectively) [5,6]. Dogs immunized with a single (group C) or several (groups A and B) injections of plasmid plus a boost were protected against the viral challenge (Fig. 2). The nonimmunized dogs, 11 and 12 rapidly developed rabies. Dog 12 began to have characteristic symptoms (anorexia, change in tone, salivation), 12 days after the challenge and died 8 days later. For dog 11, the symptoms started 15 days after challenge and death occurred 4 days later. After the death of these animals, rabies virus nucleocapsids were detected on brain impressions by direct immuno¯uorescence. The use of only two control dogs (dog 10 died before challenge), to prevent unnecessary animal suering, was possible due to the accurate calibration of the challenge dose required by previous trials in dogs. Titration of the challenge virus in mice immediately after use demonstrated that the virus dose used for the challenge exceeded the 100% lethal i.m. dose for dogs (see Section 2). All surviving dogs after challenge have a VNAb titer r0.5 IU/ml. Under these conditions, a very weak level of VNAb was enough to obtain protection against a lethal challenge as shown for dogs 4, 7 and 8 having about 0.5 IU/ml. 4. Discussion Previous studies have shown that DNA-based immunization with plasmids encoding the rabies virus glycoprotein (CVS, ERA and PV strains) protects mice against rabies [4,9,11,25]. A comparative study of DNA vaccination (G of CVS strain) versus a classical vaccine prepared using human diploid cells has been carried out in non-human primates (Macaca fascicularis ) [12]. This study indicates the potential for DNA vaccination of humans. However, this approach would encounter several obstacles. The use of DNA immunization in humans is controversial due to the potential
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risks of DNA integration, particularly for diseases like rabies against which alternative safe and ecient vaccines are already available. In addition, DNA immunization has only been demonstrated to be ecient for preventive vaccination, a situation which is exceptional for rabies in humans, which are primarily treated, post-exposure. Thus, we investigated the potential of DNA immunization for the preventive vaccination of dogs against rabies and European Bat Lyssaviruses because post-exposure treatments are required chie¯y, following possible contamination via dogs. We decide to use i.m. injection for several reasons: (1) needle injection seems more appropriate than gene gun delivery for mass vaccination campaigns, for dogs, in developing countries; (2) i.m. injection of DNA vaccines has been demonstrated to be ecient in both rodents and monkeys, whereas intradermal injection was not ecient in monkeys. We compared various protocols (2±4 i.m. injections, one or three sites) and found that two i.m. injections, each of 100 mg of plasmid (PClneo encoding the rabies virus glycoprotein), on days 0 and 28 or 0 and 175, were sucient to induce high VNAb titers against the parental virus (PV strain), the standard virus (CVS strain) and a wild-type rabies virus isolated from a French fox (FWRV). We also found that high levels of neutralizing antibodies were elicited against EBL2 but few against EBL1b. Moreover, vaccinated dogs survived a lethal challenge with a wild-type rabies virus isolated from rabid Tunisian dogs. This clearly demonstrates, for the ®rst time, that preventive DNA-based immunization of dogs would be eective against canine rabies, with considerable bene®ts for human health. DNA immunization has great potential for improving the strategy for vaccination against lyssaviruses. It would be most useful if a strong enough immune response could be obtained after a single shot, because it is dicult to recover dogs for booster injections in developing countries. In all three groups of dogs, mean VNAb titers signi®cantly increased from day 70/120 to day 175. The reason for this is unclear but it may be that there was constant stimulation of the immune system due to low but persistent levels of glycoprotein production by the injected plasmid. The VNAb titers against FWRV measured after a single i.m. injection of 100 mg (group C) of plasmid were variable (only 1 dog in 3 clearly seroconverted), but were slightly higher than those of the control dogs. This is why a boost was given to all dogs on day 175 before challenge. Under these conditions, levels of antibody dramatically increased as evidenced on day 189 (Fig. 2 ; lowest titer for dog 8: 1.6 IU/ml; highest titer for dog 3: 64 IU/ml). These high levels of VNAb were not maintained and in particular, for dogs 4, 7 and 8 which have about 0.5 IU/ml. Nevertheless, all immunized dogs were protected against the challenge.
485
Consequently, as all dogs, including those receiving a single injection, have VNAb titer higher than 0.5 IU/ ml on day 175 before boosting, we can imagine that the boost would not be necessary, because there are several reports of protection in dogs with VNAb titers above 0.1 IU/ml [2]. Thus, dogs are as sensitive to i.m. injection of DNA vaccine as monkey [12] and 100 mg of plasmid (here) was sucient to generate an eective immune response in dogs. It is well known that rabies encephalomyelitis is caused by diverse lyssaviruses (genotype 1 for rabies viruses, 6 other genotypes of rabies-related viruses). In a mouse model the rabies vaccines currently available (genotype 1), give protection against genotype 1, 6 (EBL2) and 7 (Australian pteroid bat), no protection against the more divergent genotypes 2, 3 and 4 or weak protection against EBL1 [4,8,9]. Both EBL1 and EBL2 viruses were responsible for human cases (see [1]. We showed that the plasmid encoding the PV glycoprotein induced in dogs the production of VNAb, against not only the wild-type rabies viruses that naturally infect red foxes in Western Europe (FWRV), but also against EBL2 and in less extent against EBL1. The versatility of DNA recombinant methods makes it possible to generate chimeric lyssavirus glycoproteins with a broadened spectrum of protection against lyssaviruses. Indeed, as recently demonstrated, mice immunized with the chimeric lyssavirus plasmid pGEBL1PV were protected against an intracerebral challenge with European lyssaviruses including EBL1 [9]. The use of such a chimeric glycoprotein for immunizing dogs would be bene®cial. The results for dog protection demonstrate that DNA immunization is of great potential value for protecting humans against rabies and rabies-related viruses by immunizing the canine reservoir against the disease.
Acknowledgements We would like to thank Dr. D. Drocourt (CAYLA, 5, rue Jean Rodier, Z1 Montauban F-31400 Toulouse) for plasmid preparation and Drs O.De Paz and E.L. Espinosa for excellent technical assistance.
References [1] Amengal B, Whitby J, Cobo S, Bourhy H. Evolution of european bat lyssaviruses. J Gen Virol 1997;78:2319±28. [2] Aubert MFA. Can vaccination validated by the titration of rabies antibodies in serum of cats and dogs be an alternative to quarantine measures? Abstract of Hygiene and Communicable Diseases 1993;68:R1±R22. [3] Babiuk L, Lewis P, Drunen S, Tikoo S, Liang X. Nucleic acid vaccines: veterinary applications. In: Compans RW, et al., edi-
486
[4] [5] [6] [7] [8] [9] [10] [11]
[12]
[13] [14] [15]
P. Perrin et al. / Vaccine 18 (2000) 479±486 tors. Current topics microbio-logy and immunology, vol. 226. Berlin: Springer-Verlag, 1998. p. 90. Bahloul C, Jacob Y, Tordo N, Perrin P. DNA-based immunization for exploring the enlargement of immunological crossreactivity against the lyssaviruses. Vaccine 1998;16:417±25. Blancou J, Aubert MFA, Soulebot JP. Dierence dans le pouvoir pathogeÁne de souches de virus rabiques adapteÂes au renard et au chien. Ann Virol 1983;134E:523±31. Blancou J, Aubert MFA. Transmission of the rabies virus: importance of the species barrier. Bull Acad Natl Med 1997;181:301±12. Dreesen D. A global review of rabies vaccines for human use. Vaccine 1997;15:S2±S6. Gould A, Hyatt A, Lunt R, Kattenbelt J, Hengstberger S, Blacksell S. Characterisation of a novel lyssavirus isolated from Pteroid bats in Australia. Virus Research 1998;54:165±87. Jallet C, Jacob Y, Bahloul C, Drings A, DesmeÂzieÁres E, Tordo N, Perrin P. Chimeric lyssavirus glycoproteins with increased immunological potential. J Virol 1999;73:225±33. Jiang W, Baker H, Swango L, Schorr J, Self M, Smith B. Nucleic acid immunization protects dogs against challenge with virulent canine parvovirus. Vaccine 1998;16:601±7. Lodmell D, Ray N, Ewalt R, Hanlon D, Shaddock J, Sanderlin D, Rupprecht C. Gene gun particle-mediated vaccination with plasmid DNA confers protective immunity against rabies virus infection. Vaccine 1998;16:115±8. Lodmell D, Ray N, Parnell M, Ewalt R, Hanlon D, Shaddock J, Sanderlin D, Rupprecht C. DNA immunization protects nonhuman primates against rabies virus. Nature Med 1998;4:949± 52. Meslin F-X, Fishbein D, Matter H. Rationale and prospects for rabies elimination in developing countries. In: Rupprecht, et al., editors. Lyssaviruses. Berlin: Springer-Verlag, 1994. p. 1. OIE. Rabies in Denmark in sheep. Disease information 1998;11(34). OIE. Rabies in Denmark. Disease information 1998;11(37).
[16] Pastoret P-P, Brochier B, Aguilar-SetieÂn A, Blancou J. Vaccination against rabies. In: Pastoret P-P, et al., editors. Veterinary vaccinology. Amsterdam: Elsevier, 1997. p. 616. [17] Perrin P, Versmisse P, Delagneau JF, Lucas G, Rollin P, Sureau P. The in¯uence of the type of immunosorbent on rabies antibody EIA; advantages of puri®ed glycoprotein over whole virus. J Biol Standard 1986;14:95±102. [18] Perrin P, Madhusudana S, Jallet C, Petres S, Tordo N, Merten O-W. An experimental rabies vaccine produced with a new BHK-21 suspension cell culture process: use of serum-free medium and perfusion system. Vaccine 1995;13:1244±50. [19] Perrin P, De Franco M, Jallet C, Fouque F, Morgeaux S, Tordo N, Colle J-H. The antigen-speci®c cell-mediated immune response in mice is suppressed by infection with pathogenic lyssaviruses. Res Virol 1996;147:289±99. [20] Perrin P. Techniques for the preparation of rabies conjugates. In: Meslin F-X, Kaplan M, Koprowski H, editors. Laboratory techniques in rabies, 4th ed. Geneva: WHO, 1996. p. 433. [21] Sato M, Maeda N, Yoshida H, Urade M, Saito S, Miyazaki T, Shibata T, Watanabe M. Plaque formation of herpes virus Hominis type 2 and rubella virus in variants isolated from the colonies of BHK-21/WI-2 cells formed in soft agar. Arch Virol 1977;53:269±73. [22] Smith J, Yager P, Baer G. A rapid ¯uorescent focus inhibition test (RFFIT) for determining virus-neutralizing antibody. In: Meslin F-X, Kaplan M, Koprowski H, editors. Laboratory techniques in rabies, 4th ed. Geneva: WHO, 1996. p. 181. [23] Theodorides J. In: Histoire de la rage. Paris: Masson, 1986. p. 17. [24] WHO. World Survey of Rabies. World Health Organization, Geneva 1994; 210. [25] Xiang Z, Spitalnik S, Tran M, Wunner W, Cheng J, Ertl H. Vaccination with a plasmid vector carrying the rabies virus glycoprotein gene induces protective immunity against rabies virus. Virol 1994;199:132±40.