Virus Research 111 (2005) 101–105

Oral vaccination of dogs with recombinant rabies virus vaccines

Charles E. Rupprechta,∗, Cathleen A. Hanlona, Jesse Blantona, Jamie Manangana,Patricia Morrilla, Staci Murphya, Michael Niezgodaa, Lillian A. Orciari a,

Carolin L. Schumacherb, Bernhard Dietzscholdc

a Centers for Disease Control and Prevention, Division of Viral and Rickettsial Diseases, Viral and Rickettsial Zoonoses Branch,Rabies Unit, Mailstop G-33, Atlanta, GA 30333, USA

b Merial SAS, Lyon 69007, Francec Thomas Jefferson University, Philadelphia, PA 19107, USA

Available online 8 April 2005

Abstract

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 oralr rpose-bredb ither diluento day 0,1v ation wered als within1 All controla preliminaryd ion that thesevP

K

1

t1aNbtcv

pro-cen-(RV)ori-ll,

mil-onal

15etactic ani-ibuted

0d

oute, compared to a commercial vaccinia-rabies glycoprotein (V-RG) recombinant virus vaccine. Animals consisted of naive pueagles of both sexes, and were 6 months of age or older. Dogs were randomly assigned to one of six groups, and received er vaccine (PBS; V-RG; RV SN10-333; RV SPBN-Cyto c; RV SPBNGA; RV SPBNGAGA), with at least six animals per group. Onml of each vaccine (or PBS) was administered to the oral cavity of each dog, at an approximate concentration of 108 to 109 TCID50. Afteraccination, dogs were observed daily and bled weekly, for 5 weeks, prior to RV challenge. No signs of illness related to vaccinetected during the observation period. Excluding the controls, RV neutralizing antibodies were detected in the majority of anim–2 weeks of primary vaccination. Thereafter, all dogs were inoculated in the masseter muscle with a street virus of canine origin.nimals developed rabies, but no vaccinates succumbed, with the exception of a single dog in the V-RG group. Review of theseata demonstrates the non-inferiority of recombinant RV products, as concerns both safety and efficacy, and supports the suggestaccines may hold promise for future development as oral immunogens for important carnivore species, such as dogs.ublished by Elsevier B.V.

eywords:Rabies; Rabies virus; Rabies vaccination; Oral vaccination; Canine vaccination

. Introduction

During the last 30 years, great progress has been made inhe development of oral vaccines against rabies (Wandeler,991). The primary focus of these efforts has been towardspplication in control against wildlife rabies in Europe andorth America, by the strategic distribution of vaccine-ladenaits (Stohr and Meslin, 1996; MacInnes et al., 2001). For

hese activities, self-replicating virus vaccines are needed toontact the oral mucosa of a diversity of mammalian carni-ores because large amounts of inactivated antigens are re-

∗ Corresponding author. Tel.: +1 404 639 1050; fax: +1 404 639 1564.E-mail address:cyr5@cdc.gov (C.E. Rupprecht).

quired for minimal protection (Rupprecht et al., 1992). Cur-rent vaccine production methods are cost-prohibitive toduce these products, which may require milligram contrations of purified antigens, such as the rabies virusglycoprotein (G), at both high density and in a similarentation as intact viral particles (Dietzschold and Schne2002).

Depending upon the host species of interest, tens oflions of vaccine doses may be distributed annually in naticampaigns by hand or via aircraft, at bait densities fromto 75 baits or more per km2 (Aubert et al., 1994; Slateal., 2002). Considering the opportunity for potential contbetween non-target species, such as humans, domestmals, endangered species, etc., and RV vaccines distr

168-1702/$ – see front matter. Published by Elsevier B.V.oi:10.1016/j.virusres.2005.03.017

102 C.E. Rupprecht et al. / Virus Research 111 (2005) 101–105

in the environment, safety concerns have been paramount inthe conceptual design of these biologicals (Wandeler, 2000).Historically, residual neurovirulence was assessed by the ex-perimental inoculation of RV vaccine candidates into thebrain of laboratory animals (Koprowski, 1996). Moreover,the first generation of RV vaccines intended for oral vacci-nation retained the opportunity to cause occasional disease,by the parenteral or oral routes (Winkler et al., 1976; Wan-deler, 1988; Bingham et al., 1992; Vos et al., 1999). To min-imize these drawbacks, additional research efforts concen-trated upon other more attenuated RV and recombinant vac-cines that would retain potency, but not induce rabies by theoral, peripheral or intracerebral routes in immune competentadult animals (Dietzschold et al., 1983; Wiktor et al., 1984;Preve et al., 1990; Schumacher et al., 1993; Xiang et al.,2003).

Over the past decade, significant insights have appearedinto the function and mechanisms of action of individual viralgenes in pathogenesis and immunity, after direct use of RVas an expression vector system (Conzelmann and Schnell,1994; Schnell et al., 1994; Morimoto et al., 2001). To furthertest the applied feasibility of the reverse genetics approachin the development of new vaccines, the objective of thiscurrent work was to investigate both the safety and effec-tiveness of a variety of novel recombinant RVs (Dietzscholdand Schnell, 2002). Preliminary research with these viruses inl ty ande ;P ,n con-c cies,s

vel-o -o are ani ity toc e peop nimab ei ion ofn om-b rcialr andc

2

2

ainstr mer-c nti-fi umo tiono ures

were performed under an approved protocol in compliancewith the Centers for Disease Control and Prevention Institu-tional Animal Care and Use Guidelines.

2.2. Vaccination

Dogs were assigned randomly to one of six groups. Ofthe 42 dogs in the study, 12 were assigned as controls. Ofthe remaining animals, six each were assigned to one offive vaccination groups, A–E (Table 1). Briefly, group Areceived a commercial vaccinia rabies-glycoprotein (V-RG)recombinant virus vaccine (Wiktor et al., 1984). Group Breceived RV SN10-333, generated from RV SN10, a non-pathogenic derivative of the RV vaccine strain SAD B19,as described (Schnell et al., 1994; Morimoto et al., 2001).The RV SN10-333, which contained an intact psi (�) non-translated sequence, was constructed by site-directed muta-genesis, with the replacement of an arginine to a glutaminemutation (AGA→ GAG) at RV G position 333 (Morimotoet al., 2001). Group C received RV SPBN-Cyto c, derivedfrom RV SPBN (having a deleted�), with the human cy-tochrome c gene introduced between the RV G and L genes,as described (Pulmanausahakul et al., 2001). Group D re-ceived RV SPBNGA, derived from RV SPBN, having a RVG with an arginine to a glutamine exchange (AGA→ GAG)aG GAg at− r os1 ) pH7 ml oft ringe.

2

wasa d cen-t f RVn flu-oa da ingd inen orh e in

TR

G s

ABCDEC

aboratory rodents has demonstrated comparable safeffectiveness to other RV vaccines (Morimoto et al., 2001ulmanausahakul et al., 2001; Faber et al., 2002). Howevero comparative data are available for proof of concepterning effects after oral vaccination of more relevant speuch as dogs or other carnivores.

Dogs remain the primary reservoir for rabies in deping countries (Meslin et al., 1994). In addition, in develped countries that have eliminated canine rabies, dogs

mportant non-target species because of their opportunonsume vaccine-laden baits and subsequently exposle, as an obvious consequence of the close human–aond (Rupprecht et al., 2001). Specifically, in this study, w

nvestigate the occurrence of adverse events, the inducteutralizing antibody and the protective efficacy of recinant RVs in captive beagles, compared to a commeecombinant poxvirus vaccine used for rabies preventionontrol in Europe and North America.

. Materials and method

.1. Animals

Forty-two purpose-bred beagles (not vaccinated agabies), of mixed age and sex, were obtained from comial sources. All dogs were individually housed, and ideed by a unique tattoo. Dogs were quarantined a minimf 30 days for general health observations, prior to initiaf this study. All animal care and experimental proced

-l

t position 333 (termed GA), as described (Faber et al., 2002).roup E received RV SPBNGA-GA, which contains twoenes in tandem (Faber et al., 2002). Vaccines were stored80◦C, prior to use. On day 0, control dogs received pe.0 ml of sterile 0.01 M phosphate buffered saline (PBS.5, whereas dogs in the vaccination groups received 1

hawed vaccine per os, administered via needle-less sy

.3. Rabies virus neutralizing antibody determination

After vaccination, dogs were bled weekly. The bloodllowed to clot and the serum was separated by low spee

rifugation. Serum samples were tested for evidence oeutralizing antibodies (RVNA), determined by the rapidrescent focus inhibition test (RFFIT), as described (Smith etl., 1996). A minimum positive RVNA result was defines the neutralization of approximately 50 focus-formoses50 per 0.1 ml of RV (strain CVS-11, produced on mureuroblastoma cells) at an initial serum dilution of 1:5igher. Once antibodies were detected, a four-fold ris

able 1abies vaccines used in this study

roup Vaccine Concentrationa Number of dog

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 6

ontrols None None 12a Tissue culture infectious doses50 per ml.

C.E. Rupprecht et al. / Virus Research 111 (2005) 101–105 103

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 ap value of 0.05 for significance.

2.4. Rabies virus challenge

The challenge virus was prepared from the submandibu-lar salivary glands of a naturally infected dog from Texas,USA. The salivary glands were homogenized (10%, wt/vol)in 2% horse serum as diluent. The resulting homogenate wasclarified by low-speed centrifugation, and the supernatantwas stored at−80◦C prior to use. The suspension yieldeda concentration of approximately 107.4 median mouse in-tracerebral lethal doses50 per ml. This isolate was used asrepresentative of a RV variant that was prevalent among dogsand coyotes (Canis latrans) along the Texas–Mexico border(

eres ineh kg).D .5 mls 2%h anda iplet stiveo axia,p allys nizedW rmedw urates pleso dog

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/DFAdiagnosis/DFA protocol-b.htm]. Surviving dogs were observed for aminimum of 90 days post-challenge.

3. Results

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 7–14 days post-vaccination in the majority ofanimals. By inspection of the GMT by group, RVNA hadpeaked and stabilized by 3–4 weeks (Table 2). Once a dogdeveloped a primary response, these RVNA persisted throughthe time of challenge. If a dog did not develop detectableRVNA within the 2 weeks after vaccination, none were founduntil after RV challenge.

No significant differences were observed in GMT be-tween vaccine groups regardless of the day post-vaccination(ANOVA, p> 0.05), but the temporal development of RVNAv 7 ing B,a ogsi ofs hadd alsb NAo ay7 enceo FFITm e-t 12c

RGv biesd is of

TD ed dog

G

2

ABCDEC <

mined

Rohde et al., 1997).Approximately 5 weeks after vaccination, all dogs w

edated by the intramuscular administration of tiletamydrochloride and zolazepam hydrochloride (0.5 mg/ogs were inoculated in the masseter muscles with a 0uspension of the canine challenge RV diluted 1/100 inorse serum as diluent. From the time of vaccinationfter RV inoculation, dogs were observed daily at mult

imes for any adverse clinical signs, especially suggef rabies, such as lethargy, anorexia, agitation, ataresis, paralysis, cranial nerve deficits, etc. Any clinicuspect animals were restrained, sedated and euthahile under sedation, euthanasia of animals was perfoith an intravenous overdose of a concentrated barbitolution (approximately 2 ml/kg). Representative samf brain tissue were collected from each euthanized

able 2evelopment of rabies virus neutralizing antibody titersa in orally vaccinat

roup Days post-vaccination

7 14 21

1.4 (0.6) 1.7 (1.0) 2.2 (1.3)1.2 (0.4) 1.5 (0.3) 1.6 (0.3)

<0.7 1.2 (0.5) 1.1 (0.4)0.7 (0.2) 1.1 (0.5) 1.3 (0.5)1.2 (0.6) 1.6 (0.5) 1.7 (0.6)

ontrols <0.7 <0.7 <0.7a Geometric mean titers (standard deviation), log base 10, as deterb Number of animals surviving rabies virus challenge per group.c Day of rabies virus challenge.d Seven days after rabies virus challenge.

.

aried in terms of seroprevalence. For example, by dayroup A, four of six dogs had developed RVNA. In groupll dogs had detectable RVNA within the first week. No d

n group C had detectable RVNA until day 14, when fiveix dogs had seroconverted. In group D, only a single dogetectable RVNA on day 7, compared to four of six animy day 14. In group E, half of the dogs had detectable RVn day 7, with five of six seroconverting by day 14. By dpost-RV challenge, each vaccine group displayed evidf an anamnestic response, compared to the previous Reasurements (Table 2). In contrast, no control dogs had d

ectable RVNA before RV challenge. At least 7 of theseontrols had developed antibodies a week later.

All control dogs, and one of the six vaccinates in the V-accine group, displayed compatible clinical signs of rauring the 11–12 days after RV challenge. The diagnos

s and survivorship after rabies virus challenge

Survivorshipb

8 35c 42d

2.2 (1.3) 2.2 (1.1) 3.3 (1.1) 5/61.7 (0.4) 1.7 (0.3) 3.1 (0.4) 6/61.3 (0.5) 1.4 (0.6) 3.5 (0.3) 6/61.3 (0.5) 1.4 (0.6) 3.1 (0.1) 6/61.8 (0.4) 1.8 (0.3) 3.2 (0.2) 6/60.7 <0.7 1.3 (0.9) 0/12

by the rapid fluorescent focus inhibition test.

104 C.E. Rupprecht et al. / Virus Research 111 (2005) 101–105

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.

4. Discussion

The results of this short-term experiment compare fa-vorably with other research that has generated initial im-munogenicity and efficacy data after oral vaccination of dogsagainst rabies (Blancou et al., 1989; Svrcek et al., 1995;Fekadu et al., 1996; Rupprecht et al., 1998; Hammami etal., 1999). In addition, the current results were superior todata generated after the administration of other modern ra-bies recombinant viruses to dogs, such as an E1-deleted hu-man adenovirus type 5, which appeared ineffective (Vos etal., 2001). Unlike previous considerations for oral vaccina-tion, the use of novel recombinant RVs would exploit thebasic immunological value of a self-replicating agent withantigens most akin to the pathogen in question, but with theproven utility of attenuation by molecular alteration of viralgenes at loci known to result in attenuation, and the addedbenefits of foreign gene inclusion, to enhance overt efficacywithout compromising upon safety (Pulmanausahakul et al.,2001; Faber et al., 2002).

The induction of neutralizing antibodies to RV G antigensi pro-d ,1 pedr noth chal-l 50 int og’st trola s af-t le tot vac-c ogi 250a thers rs ine ationsi fterv timeo

ina-t sug-g otherc ntrol( 03B oxes,r uld bet du-r her fetya tion

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-ranging animals, as an adjunct public health tool in both thedeveloping and developed world (Haddad et al., 1994; Pereraet al., 2000; Estrada et al., 2001; Corn et al., 2003).

Acknowledgments

The authors thank personnel in the Viral and RickettsialZoonoses Branch and the Animal Resources Branch, CDCas well as Dr. Joanne Maki and staff, Merial Inc. for assis-tance and technical expertise related to this research. Thiswork was supported in part by Phase II SBIR Grant 2 R44CI0081-02 and by Public Health Service Grants AI45097-6and AI09706-32.

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