oral vaccination of dogs with recombinant rabies virus vaccines
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Virus Research 111 (2005) 101105
Oral vaccination of dogs with recombinaC Jesse
ttsial Di, GA 30Francehia, PA
Oral rabies virus (RV) vaccines are used to immunize a diversity of mammalian carnivores, but no single biological is effective for all majorspecies. Recently, advances in reverse genetics have allowed the design of recombinant RV for consideration as new vaccines. The objective ofthis experiment was to examine the safety, immunogenicity and efficacy of recombinant RV vaccines administered to captive dogs by the oralroute, compbeagles of bor vaccine (1 ml of eachvaccination,detected dur12 weeks oanimals devdata demonsvaccines maPublished by
Duringthe develop1991). TheapplicationNorth Amebaits (Stohthese activicontact thevores beca
0168-1702/$doi:10.1016/jared to a commercial vaccinia-rabies glycoprotein (V-RG) recombinant virus vaccine. Animals consisted of naive purpose-bredoth sexes, and were 6 months of age or older. Dogs were randomly assigned to one of six groups, and received either diluentPBS; V-RG; RV SN10-333; RV SPBN-Cyto c; RV SPBNGA; RV SPBNGAGA), with at least six animals per group. On day 0,vaccine (or PBS) was administered to the oral cavity of each dog, at an approximate concentration of 108 to 109 TCID50. Afterdogs were observed daily and bled weekly, for 5 weeks, prior to RV challenge. No signs of illness related to vaccination wereing the observation period. Excluding the controls, RV neutralizing antibodies were detected in the majority of animals withinf primary vaccination. Thereafter, all dogs were inoculated in the masseter muscle with a street virus of canine origin. All controleloped rabies, but no vaccinates succumbed, with the exception of a single dog in the V-RG group. Review of these preliminarytrates the non-inferiority of recombinant RV products, as concerns both safety and efficacy, and supports the suggestion that thesey hold promise for future development as oral immunogens for important carnivore species, such as dogs.
abies; Rabies virus; Rabies vaccination; Oral vaccination; Canine vaccination
the last 30 years, great progress has been made inment of oral vaccines against rabies (Wandeler,primary focus of these efforts has been towardsin control against wildlife rabies in Europe and
rica, by the strategic distribution of vaccine-ladenr and Meslin, 1996; MacInnes et al., 2001). Forties, self-replicating virus vaccines are needed tooral mucosa of a diversity of mammalian carni-
use large amounts of inactivated antigens are re-
ding author. Tel.: +1 404 639 1050; fax: +1 404 639 1564.dress: email@example.com (C.E. Rupprecht).
quired for minimal protection (Rupprecht et al., 1992). Cur-rent vaccine production methods are cost-prohibitive to pro-duce these products, which may require milligram concen-trations of purified antigens, such as the rabies virus (RV)glycoprotein (G), at both high density and in a similar ori-entation as intact viral particles (Dietzschold and Schnell,2002).
Depending upon the host species of interest, tens of mil-lions of vaccine doses may be distributed annually in nationalcampaigns by hand or via aircraft, at bait densities from 15to 75 baits or more per km2 (Aubert et al., 1994; Slate etal., 2002). Considering the opportunity for potential contactbetween non-target species, such as humans, domestic ani-mals, endangered species, etc., and RV vaccines distributed
see front matter. Published by Elsevier B.V..virusres.2005.03.017harles E. Rupprecht a,, Cathleen A. Hanlon a,Patricia Morrill a, Staci Murphy a, Michael N
Carolin L. Schumacher b, Bernha Centers for Disease Control and Prevention, Division of Viral and Ricke
Rabies Unit, Mailstop G-33, Atlantab Merial SAS, Lyon 69007,
c Thomas Jefferson University, PhiladelpAvailable online 8 Aprilnt rabies virus vaccinesBlanton a, Jamie Manangan a,oda a, Lillian A. Orciari a,
seases, Viral and Rickettsial Zoonoses Branch,333, USA
102 C.E. Rupprecht et al. / Virus Research 111 (2005) 101105
in the environment, safety concerns have been paramount inthe conceptual design of these biologicals (Wandeler, 2000).Historically, residual neurovirulence was assessed by the ex-perimentalbrain of lathe first genation retaby the paredeler, 1988imize thesetrated uponcines that woral, periphadult animPreve et al2003).
Over thinto the fungenes in paas an expr1994; Schntest the appin the devecurrent wotiveness ofand SchnellaboratoryeffectivenePulmanausno comparcerning effesuch as dog
Dogs reoping counoped countimportant nconsume v
ple, as an obond (Rupinvestigateneutralizinbinant RVsrecombinancontrol in E
Forty-twrabies), ofcial sourcefied by a uof 30 daysof this stud
were performed under an approved protocol in compliancewith the Centers for Disease Control and Prevention Institu-tional Animal Care and Use Guidelines.
ogs were assigned randomly to one of six groups. Of2 dogs in the study, 12 were assigned as controls. Ofemaining animals, six each were assigned to one ofvaccination groups, AE (Table 1). Briefly, group Aved a commercial vaccinia rabies-glycoprotein (V-RG)
binant virus vaccine (Wiktor et al., 1984). Group Bved RV SN10-333, generated from RV SN10, a non-genicscribe
RV SNlated ssis, wition (A, 2001RV Some cscribed RVth an asitionp E res in tanC, prl of stherea
fter vaed to cation.alizinent fo996).e neu
s50 perblastor. On
ssue culinoculation of RV vaccine candidates into theboratory animals (Koprowski, 1996). Moreover,neration of RV vaccines intended for oral vacci-ined the opportunity to cause occasional disease,nteral or oral routes (Winkler et al., 1976; Wan-; Bingham et al., 1992; Vos et al., 1999). To min-
drawbacks, additional research efforts concen-other more attenuated RV and recombinant vac-ould retain potency, but not induce rabies by theeral or intracerebral routes in immune competent
als (Dietzschold et al., 1983; Wiktor et al., 1984;., 1990; Schumacher et al., 1993; Xiang et al.,
e past decade, significant insights have appearedction and mechanisms of action of individual viralthogenesis and immunity, after direct use of RV
ession vector system (Conzelmann and Schnell,ell et al., 1994; Morimoto et al., 2001). To furtherlied feasibility of the reverse genetics approachlopment of new vaccines, the objective of thisrk was to investigate both the safety and effec-a variety of novel recombinant RVs (Dietzscholdl, 2002). Preliminary research with these viruses inrodents has demonstrated comparable safety andss to other RV vaccines (Morimoto et al., 2001;ahakul et al., 2001; Faber et al., 2002). However,ative data are available for proof of concept con-cts after oral vaccination of more relevant species,s or other carnivores.main the primary reservoir for rabies in devel-tries (Meslin et al., 1994). In addition, in devel-ries that have eliminated canine rabies, dogs are anon-target species because of their opportunity to
accine-laden baits and subsequently expose peo-bvious consequence of the close humananimal
precht et al., 2001). Specifically, in this study, wethe occurrence of adverse events, the induction ofg antibody and the protective efficacy of recom-
in captive beagles, compared to a commercialt poxvirus vaccine used for rabies prevention andurope and North America.
ls and method
o purpose-bred beagles (not vaccinated againstmixed age and sex, were obtained from commer-s. All dogs were individually housed, and identi-nique tattoo. Dogs were quarantined a minimumfor general health observations, prior to initiationy. All animal care and experimental procedures
Dthe 4the rfivereceirecom
receipathoas deThetransgenemutaet al.fromtochras deceiveG wiat poGrougene801.0 m7.5, wthaw
al., 1as thdoseneuro
a Tiderivative of the RV vaccine strain SAD B19,d (Schnell et al., 1994; Morimoto et al., 2001).10-333, which contained an intact psi () non-equence, was constructed by site-directed muta-th the replacement of an arginine to a glutamineGAGAG) at RV G position 333 (Morimoto). Group C received RV SPBN-Cyto c, derivedPBN (having a deleted ), with the human cy-gene introduced between the RV G and L genes,d (Pulmanausahakul et al., 2001). Group D re-SPBNGA, derived from RV SPBN, having a RVrginine to a glutamine exchange (AGAGAG)
333 (termed GA), as described (Faber et al., 2002).ceived RV SPBNGA-GA, which contains two GAdem (Faber et al., 2002). Vaccines were stored at
ior to use. On day 0, control dogs received per oserile 0.01 M phosphate buffered saline (PBS) pHs dogs in the vaccination groups received 1 ml ofcine per os, administered via needle-less syringe.
s virus neutralizing antibody determination
ccination, dogs were bled weekly. The blood waslot and the serum was separated by low speed cen-Serum samples were tested for evidence of RV
g antibodies (RVNA), determined by the rapid flu-cus inhibition test (RFFIT), as described (Smith etA minimum positive RVNA result was definedtralization of approximately 50 focus-forming0.1 ml of RV (strain CVS-11, produced on murinema cells) at an initial serum dilution of 1:5 or
ce antibodies were detected, a four-fold rise in
es used in this study
Vaccine Concentrationa Number of dogs
V-RG 1 108.9 6SN10-333 1 108.6 6SPBN-Cyto c 1 108.4 6SPBNGA 1 108.2 6SPBNGA-GA 1 108.6 6None None 12
ture infectious doses50 per ml.
C.E. Rupprecht et