Oral vaccination of dogs with recombinant rabies virus vaccines

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<ul><li><p>Virus Research 111 (2005) 101105</p><p>Oral vaccination of dogs with recombinaC Jesse</p><p>iezgard</p><p>ttsial Di, GA 30Francehia, PA</p><p>2005</p><p>Abstract</p><p>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</p><p>Keywords: R</p><p>1. Introdu</p><p>Duringthe develop1991). TheapplicationNorth Amebaits (Stohthese activicontact thevores beca</p><p> CorresponE-mail ad</p><p>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.</p><p>Elsevier B.V.</p><p>abies; Rabies virus; Rabies vaccination; Oral vaccination; Canine vaccination</p><p>ction</p><p>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</p><p>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-</p><p>use large amounts of inactivated antigens are re-</p><p>ding author. Tel.: +1 404 639 1050; fax: +1 404 639 1564.dress: cyr5@cdc.gov (C.E. Rupprecht).</p><p>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).</p><p>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</p><p> 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</p><p>Carolin L. Schumacher b, Bernha Centers for Disease Control and Prevention, Division of Viral and Ricke</p><p>Rabies Unit, Mailstop G-33, Atlantab Merial SAS, Lyon 69007,</p><p>c Thomas Jefferson University, PhiladelpAvailable online 8 Aprilnt rabies virus vaccinesBlanton a, Jamie Manangan a,oda a, Lillian A. Orciari a,</p><p>Dietzschold c</p><p>seases, Viral and Rickettsial Zoonoses Branch,333, USA</p><p>19107, USA</p></li><li><p>102 C.E. Rupprecht et al. / Virus Research 111 (2005) 101105</p><p>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).</p><p>Over thinto the fungenes in paas an expr1994; Schntest the appin the devecurrent wotiveness ofand SchnellaboratoryeffectivenePulmanausno comparcerning effesuch as dog</p><p>Dogs reoping counoped countimportant nconsume v</p><p>ple, as an obond (Rupinvestigateneutralizinbinant RVsrecombinancontrol in E</p><p>2. Materia</p><p>2.1. Anima</p><p>Forty-twrabies), ofcial sourcefied by a uof 30 daysof this stud</p><p>were performed under an approved protocol in compliancewith the Centers for Disease Control and Prevention Institu-tional Animal Care and Use Guidelines.</p><p>Vaccination</p><p>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)</p><p>binant virus vaccine (Wiktor et al., 1984). Group Bved RV SN10-333, generated from RV SN10, a non-genicscribe</p><p>RV SNlated ssis, wition (A, 2001RV Some cscribed RVth an asitionp E res in tanC, prl of stherea</p><p>ed vac</p><p>Rabie</p><p>fter vaed to cation.alizinent fo996).e neu</p><p>s50 perblastor. On</p><p>1s vaccin</p><p>ls</p><p>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-</p><p>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</p><p>als (Dietzschold et al., 1983; Wiktor et al., 1984;., 1990; Schumacher et al., 1993; Xiang et al.,</p><p>e past decade, significant insights have appearedction and mechanisms of action of individual viralthogenesis and immunity, after direct use of RV</p><p>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</p><p>accine-laden baits and subsequently expose peo-bvious consequence of the close humananimal</p><p>precht et al., 2001). Specifically, in this study, wethe occurrence of adverse events, the induction ofg antibody and the protective efficacy of recom-</p><p>in captive beagles, compared to a commercialt poxvirus vaccine used for rabies prevention andurope and North America.</p><p>ls and method</p><p>ls</p><p>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</p><p>2.2.</p><p>Dthe 4the rfivereceirecom</p><p>receipathoas deThetransgenemutaet al.fromtochras deceiveG wiat poGrougene801.0 m7.5, wthaw</p><p>2.3.</p><p>Aallowtrifugneutroresc</p><p>al., 1as thdoseneuro</p><p>highe</p><p>TableRabie</p><p>Group</p><p>ABCDEContro</p><p>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)</p><p>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</p><p>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.</p><p>s virus neutralizing antibody determination</p><p>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</p><p>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</p><p>ce antibodies were detected, a four-fold rise in</p><p>es used in this study</p><p>Vaccine Concentrationa Number of dogs</p><p>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</p><p>ture infectious doses50 per ml.</p></li><li><p>C.E. Rupprecht et al. / Virus Research 111 (2005) 101105 103</p><p>titer between paired sera was used as supportive evidence ofan apparent anamnestic response. Serum samples were con-sidered negative if no neutralization was observed at a serumdilution of less than 1:5. The RVNA titers obtained werecompared against a national reference serum (U.S. StandardRabies Immune Globulin lot R-3) obtained from the Office ofBiologics Research and Review, Food and Drug Administra-tion, Bethesda, MD 20205, USA, diluted to a concentrationof 2.0 international units (IU)/ml. Results obtained in the RF-FIT were transformed to geometric mean titers (GMT). Thecalculated GMT were compared between groups by bleed-ing date using a one-tailed analysis of variance (ANOVA),selecting a p value of 0.05 for significance.</p><p>2.4. Rabies virus challenge</p><p>The challenge virus was prepared from the submandibu-lar salivaryUSA. Thein 2% horsclarified bywas storeda concentrtracerebralrepresentatand coyote(Rohde et a</p><p>Approxsedated byhydrochlorDogs weresuspensionhorse serumafter RV intimes for aof rabies,paresis, pasuspect anWhile undewith an intsolution (aof brain ti</p><p>and were kept frozen at 80 C. Brain tissue samples wereexamined for the presence of RV antigen by use of thedirect fluorescent antibody (DFA) test [http://www.cdc.gov/ncidod/dvrd/rabies/professional/publications/DFA diagnosis/DFA protocol-b.htm]. Surviving dogs were observed for aminimum of 90 days post-challenge.</p><p>3. Results</p><p>After RV challenge administration, no adverse signs wereobserved in any vaccinated dogs, in excess of a combined1000 dog days of observation. Antibodies to RV were notdetected in any initial baseline canine sera on day 0, prior tovaccination. Analysis of serum samples by the RFFIT fromthe vaccinated dogs demonstrated the development of RVNAtiters within 714 days post-vaccination in the majority of</p><p>als. Bed andloped aime ofA withafter Ro signn vacc</p><p>VA, pd in te</p><p>A, fogs had</p><p>oup Cogs hatabley 14.y 7, wt-RVanam</p><p>ureme</p><p>ble RVols hall contne grog the</p><p>Table 2Development urvivors</p><p>Group</p><p>A .3)B .4)C .5)D .5)E .4)Controls</p><p>a Geometric uoresceb Number oc Day of rabd Seven dayglands of a naturally infected dog from Texas,salivary glands were homogenized (10%, wt/vol)e serum as diluent. The resulting homogenate was</p><p>low-speed centrifugation, and the supernatantat 80 C prior to use. The suspension yielded</p><p>ation of approximately 107.4 median mouse in-lethal doses50 per ml. This isolate was used as</p><p>ive of a RV variant that was prevalent among dogss (Canis latrans) along the TexasMexico borderl., 1997).</p><p>imately 5 weeks after vaccination, all dogs werethe intramuscular administration of tiletamine</p><p>ide and zolazepam hydrochloride (0.5 mg/kg).inoculated in the masseter muscles with a 0.5 mlof the canine challenge RV diluted 1/100 in 2%</p><p>as diluent. From the time of vaccination andoculation, dogs were observed daily at multipleny adverse clinical signs, especially suggestivesuch as lethargy, anorexia, agitation, ataxia,</p><p>ralysis, cranial nerve deficits, etc. Any clinicallyimals were restrained, sedated and euthanized.r sedation, euthanasia of animals was performedravenous overdose of a concentrated barbituratepproximately 2 ml/kg). Representative samplesssue were collected from each euthanized dog</p><p>animpeakdevethe tRVNuntil</p><p>Ntwee(ANOvariegroupall doin grsix ddetecby daon da7 posof anmeas</p><p>tectacontr</p><p>Avaccidurin</p><p>of rabies virus neutralizing antibody titersa in orally vaccinated dogs and s</p><p>Days post-vaccination</p><p>7 14 21 28</p><p>1.4 (0.6) 1.7 (1.0) 2.2 (1.3) 2.2 (11.2 (0.4) 1.5 (0.3) 1.6 (0.3) 1.7 (0</p></li><li><p>104 C.E. Rupprecht et al. / Virus Research 111 (2005) 101105</p><p>rabies was confirmed in suspect animals by the DFA test,whereas survivors were negative for evidence of RV antigenin the brain when euthanized at the conclusion of the study.</p><p>4. Discuss</p><p>The resvorably wimunogenicagainst rabFekadu etal., 1999).data generabies recomman adenoal., 2001).tion, the ubasic immantigens mproven utilgenes at lobenefits ofwithout co2001; Fabe</p><p>The induis one of thductive vir1977). In trabies washave an applenge. Antithis particutiter was sianimals thater infectiothe overallcinated doin group Aafter RV chsero-negatiexcess of 1in dogs, thvaccine priof exposure</p><p>In toto,tion trial, agest that recommercia(DietzschoBesides extraccoons, s</p><p>targeted toration of imrapidity ofand genetic</p><p>with these attenuated RVs. Such background data are criti-cal before the further development of these products and anyconsideration of field trials involving oral vaccination of free-</p><p>ng anloping, 2000</p><p>owled</p><p>e autoses Bell as</p><p>andwas s</p><p>81-02I097</p><p>rence</p><p>t, M.F.ccinatiopprechrrent Trlin, ppam, J.,A., Perccine g1, 555ou, J., ALangue</p><p>ccine cally tolmann,alogs o3719.J.L., M</p><p>livery oed. Hyg.H., Din. II. B, 7547chold,ith, C.</p><p>terminaies virchold,live attchold,eventiona, R., Vccinatios. 1, 23M., Pu</p><p>an, J.Prabies</p><p>tiviral iu, M., Nnderlinies vacd, N.,andeler,ion</p><p>ults of this short-term experiment compare fa-th other research that has generated initial im-ity and efficacy data after oral vaccination of dogsies (Blancou et al., 1989; Svrcek et al., 1995;al., 1996; Rupprecht et al., 1998; Hammami etIn addition, the current results were superior toted after the administration of other modern ra-</p><p>binant viruses to dogs, such as an E1-deleted hu-virus type 5, which appeared ineffective (Vos etUnlike previous considerations for oral vaccina-se of novel recombinant RVs would exploit theunological value of a self-replicating agent withost akin to the pathogen in question, but with theity...</p></li></ul>