MEDICINE—INFECTIOUS DISEASES

Evaluation of the Efficacy Provided by a Recombinant -Vectored Equine West Nile Against an Experimental West Nile Virus Intrathecal Challenge in Horses

Leonardo Siger, DVM, MS; Richard A. Bowen, DVM, PhD; Kemal Karaca, DVM, PhD; and Jules M. Minke, DVM, PhD

The efficacy of a recombinant canarypox-vectored equine West Nile Virus (WNV) vaccinea was evaluated in a WNV disease model in horses by intrathecal administration of virulent WNV. Ten horses received two doses of a commercial serial of vaccine by the intramuscular route on days 0 and 35, and ten horses were held as unvaccinated controls. Horses were challenged with virulent WNV by intrathecal administration under general anesthesia on day 49. After challenge, eight of ten controls developed clinical signs of encephalomyelitis, whereas one vaccinate exhibited muscle fas- ciculation in one observation. Fever was present in nine controls and one vaccinate. Histopathol- ogy revealed mild to moderate non-suppurative encephalitis in eight controls and one vaccinate. All of the controls and none of the vaccinates had detectable post-challenge WNV viremia. These and the results of previous studies show the efficacy of this vaccine in horses against WNV-induced clinical disease and natural challenge with WNV-infected mosquitoes. Authors’ addresses: Merial Lim- ited, 115 Transtech Drive, Athens, GA 30601 (Siger, Karaca); College of Veterinary Medicine and Biomedical Sciences, Colorado State University, 3801 Rampart Road, Fort Collins, CO 80552 (Bo- wen); and Merial SAS, 254 rue Marcel Me´rieux, Lyon 69007, France (Minke); e-mail: [email protected]. © 2007 AAEP.

1. Introduction Histologically, brains of affected horses show a non-suppurative polio encephalitis characterized by West Nile is an arthropod-born disease caused by a infiltration of T lymphocytes and to a lesser degree, member of the Japanese encephalitis virus com- 2 1 by infiltration of macrophages. At present, there plex. The disease is endemic to Africa and the is no effective treatment protocol for West Nile Virus Middle East. In recent years, the disease has (WNV) infection in horses, and control of the disease emerged in Europe, the Far East, and North Amer- relies on vaccination and management. ica where it has become an important public, veter- Recently, a disease model that induced clinical inary, and wildlife health threat. Most equine disease of WNV in horses by intrathecal administra- infections will result in transient mild disease char- tion of virulent WNV into the cisternal space of the acterized by fever, but sometimes, the virus causes atlanto-occipital joint has been reported.3 This ar- neurological disease with a fatal outcome. ticle describes the results and conclusions from a

NOTES

AAEP PROCEEDINGS ր Vol. 53 ր 2007 225 MEDICINE—INFECTIOUS DISEASES recent experimental study conducted to assess the ples were stored frozen at Ϫ80°C until virus isola- efficacy of the canarypox-vectored recombinant tion was performed. WNV vaccine in this clinical disease model. A complete physical examination was performed on each horse twice daily on days 50–60 and once 2. Materials and Methods daily on days 48, 49 (before challenge), and 70. Examinations included measurement of rectal tem- Animals perature and observations of signs of anorexia, de- Twenty mixed-breed horses of mixed sex from a pression, muscle fasciculations, lip twitching, head commercial herd that were between the ages of 14 shaking, agitation, hyperactivity, aggression, inces- and 18 mo were included in this study. None of the sant circling behavior, ataxia, weakness, or reluc- horses had detectable antibody against WNV or St. tance to move; each behavior was recorded as Louis encephalitis virus as shown by plaque-reduc- present or absent. Fever was defined as a rectal tion neutralization testing at the beginning of the temperature Ͼ102°F (38.8°C). trial. Horses were housed in a single pasture at a Horses that developed severe neurological signs research facility in Montana until day 42 after the as a result of WNV infection were euthanized for initial vaccination; at that time, they were trans- humane reasons. At the end of the trial, all re- ported to Colorado State University. At this loca- maining horses were humanely euthanized, and a tion, horses were housed in a biosecurity level-3 gross and histological evaluation of the brainstem containment building (2–3 horses/room) until the was conducted. Cerebrospinal fluid and two re- end of the trial. At both sites, horses were fed a gions of the brainstem (medulla and pons) were col- diet of pelleted concentrates and maintained in ac- lected for WNV-virus isolation, and the remainder of cordance with animal use and care guidelines of the brain was fixed in buffered formalin. A section of Merial and Colorado State University Institutional brainstem through the pons was stained with Animal Care and Use Committees. haematoxylin and eosin and evaluated histologically by a board-certified veterinary pathologist who was Vaccine Against WNV blinded as to the treatment group of the samples. A commercial serial of a recombinant canarypox- The persons performing laboratory analyses, clinical vectored equine WNV vaccinea that contains recom- observations, and gross and histopathological eval- binant canarypox virus expressing WNV prM/E uations were blinded to the group assignment. genes derived from a 1999 New York isolate of WNV was used. Virus Isolation Serum and cerebrospinal fluid (CSF) samples were Experimental Design assessed for live virus through titration in a plaque Horses were randomly assigned to one of two groups assay, as previously described.4 Briefly, duplicate (ten horses/group). On day 0 and day 35, horses in 0.1-ml samples were inoculated onto Vero-cell mono- group 1 received a single dose (1 ml) of recombinant layers in six-well culture plates and incubated for canarypox virus-WNV vaccine by intramuscular ad- 1 h at 37°C in an atmosphere containing 5% carbon ministration in the neck. Group 2 horses were not dioxide. Cells were overlaid with 2 ml of 0.5% aga- vaccinated and served as controls. On day 49, all rose in Dulbecco’s modified Eagle medium (without horses (ten treated and ten control horses) in the phenol red) that was supplemented with 5% fetal study were sedated with xylazineb (1.1 mg/kg body bovine serum and antimicrobials. After 48 h of ad- weight, IV) and then anesthetized with ketaminec ditional incubation, a second 2-ml overlay of the (2.2 mg/kg body weight, IV). Each horse was chal- same solution with 0.004% neutral red was added to lenged by intrathecal administration into the cister- the cells. Plaques were counted on days 3, 4, and 5 nal space of the atlanto-occipital joint of 1 ml of virus of incubation, and results were expressed as pfu/ml suspension containing 105 plaque-forming units serum. Brain samples were homogenized in bo- (pfu) of NY99–4132 WNV originally isolated from vine-albumin medium to a concentration of 10% wt/ an infected crow. After challenge, unused chal- vol, clarified by centrifugation, and then inoculated lenge material was submitted to the laboratory for as described for sera. subsequent back titration. Blood samples for assessment of serum antibodies Determination of Antibody Responses against WNV were collected from all horses on days Antibody response against WNV was evaluated by 0, 7, 14, 35, 49, 56, 63, and 70. On days 0, 35, and use of a plaque-reduction neutralization test. Ti- 49, samples were obtained before both vaccination tration of sera was conducted in blinded fashion as and challenge. Sera were prepared from the blood described previously4 at Colorado State University. samples and stored at Ϫ80°C until analyzed to de- Briefly, sera were heat-inactivated at 56°C for 30 termine antibody responses. In addition, blood min, and serial two-fold dilutions of those sera were samples were obtained twice daily from all horses mixed with an equal volume (0.1 ml) of a prepara- for 7 days after the challenge (days 50–56) and once tion containing 200–300 pfu of the NY99–4132 daily on days 49 (before the challenge), 57–63, and strain of WNV. After incubation at 4°C for 16–20 70 to provide sera for virus isolation. Serum sam- h, 0.1 ml of each mixture was laid on Vero-cell mono-

226 2007 ր Vol. 53 ր AAEP PROCEEDINGS MEDICINE—INFECTIOUS DISEASES layers in six-well plates and processed as for the Clinical Signs plaque assay. Neutralization endpoints were re- Eight of ten unvaccinated control horses developed corded as the highest dilution of serum with which clinical encephalomyelitis characterized by one or there was a 50% reduction of plaques relative to more of the following clinical signs: depression, re- control wells. A titer of 1:10 or higher was consid- luctance to move, agitation, weakness, ataxia, ered to represent a positive antibody response tremor, muscle fasciculation, head shaking, and lip against WNV. twitching. Encephalomyelitis developed primarily Statistical Analyses between post-challenge days 8 and 13 and had a Comparison of the incidence of fever and neurologi- mean duration of 3.9 days. One control horse be- cal disease between groups was analyzed using came recumbent on post-challenge day 13 and had to Fisher exact test. The average number of days be euthanized; another control horse showed persis- with clinical signs in the control and vaccinated tent signs of WNV disease for 12 days but then groups was determined. A repeated-measures recovered. The two horses that did not show any mixed-effects analysis of variance with fixed effects neurological signs developed fever after challenge; of group, day, and group-day interaction was used to duration of the fever was 1 day in one horse and 2 compare body temperatures between groups over days in the other horse. Of the vaccinated horses, time. Observations belonging to the same animal one horse exhibited one of the clinical signs (muscle were recognized by the model. Pre-challenge aver- fasciculation) at one observation point (post-chal- age baseline temperature was used as a covariate in lenge day 12). All vaccinated horses survived until the model. Average baseline temperature was cal- the end of the trial. culated using the mean body temperatures on days The proportion of control horses with neurological 48 and 49. All statistical analyses were conducted disease (eight of ten horses) was significantly higher d compared with the vaccinated horses (one of ten using SAS software. Statistical significance was ϭ based on two-tailed tests of the null hypothesis re- horses; p 0.0055; Fisher’s exact test). The me- sulting in p values Յ 0.05. dian duration was 3.5 days in the control group and 0 days in the vaccinate group. 3. Results Virus Isolation by Plaque Assay Antibody Responses WNV was not isolated from the blood of any vacci- Antibody to WNV was not detected in any pre-im- nated horse. All ten control horses developed de- munization serum. Although there was an anti- tectable viremia 1–2 days after WNV challenge; body response starting at 7 days after the first peak virus titers were between 100.6 and 101.9 pfu/ml vaccination, none of the vaccinated horses developed of serum, and the mean duration was 3.2 days an apparent anamnestic response after the first vac- (range ϭ 2–4 days). There was a statistically sig- cination. At the time of challenge (2 wk after the nificant difference in the incidence of viremia be- second vaccination), all ten vaccinated horses had tween vaccinates and controls after challenge (p Ͻ developed detectable antibodies against WNV with a 0.05; Fisher’s exact test). ϭ geometric mean titer of 61 (range 31–118; 95% CI WNV was not isolated from the brainstem (medulla ϭ interval and range 20–160). None of the control and pons) or from CSF samples of vaccinates or con- horses seroconverted to WNV before the challenge. trols (samples collected at the end of the study). After the intrathecal challenge, all horses serocon- verted, and titers of vaccinated horses increased Post-Mortem Examination relative to the day of challenge. No gross changes were observed in the brain at Clinical Signs to the WNV Challenge necropsy. There was a significantly higher incidence (p ϭ Rectal Temperatures 0.0055; Fisher’s exact test) of brain lesions compat- Nine of ten control horses became pyretic (rectal ible with encephalitis in the control horses (eight of temperature Ͼ102.0°F; maximum temperature ϭ ten horses) versus the vaccinated horses (one of ten 104.2°F) during the post-challenge observation pe- horses). Histopathological changes in the brains of riod, whereas only one of ten vaccinated horses de- eight control horses included mild (n ϭ 7) to moder- veloped a single episode of fever (102.2°F) on post- ate (n ϭ 1) non-suppurative encephalitis character- challenge day 13. Fever lasted for an average of 2.1 ized by mild to moderate perivascular cuffing with days (range ϭ 1–3 days) for the control horses that lymphocytes, plasma cells, and multifocal gliosis. became pyrectic. Overall, the rectal temperatures Moderate lesions were found in the horse that be- of the vaccinated horses were significantly lower came recumbent on post-challenge day 13 and had to than those of the control animals on post-challenge be euthanized. Two controls did not show any his- days 9, 10, 12, and 14 (p Ͻ 0.05; F test), and the topathological lesions. One vaccinated horse that incidence of fever was significantly higher in the had shown clinical signs of WNV disease on post- controls versus vaccinates (p ϭ 0.0011; Fisher’s ex- challenge day 12 showed mild non-suppurative en- act test). cephalitis of the brain. None of the brains of the

AAEP PROCEEDINGS ր Vol. 53 ր 2007 227 MEDICINE—INFECTIOUS DISEASES other nine vaccinated horses showed histopatholog- All vaccinated horses developed detectable anti- ical lesions compatible with encephalitis. bodies to WNV as soon as 7 days after the first vaccination. After the challenge, all control horses 4. Discussion seroconverted, whereas vaccinated animals mounted Merial has developed a canarypox-vectored recom- an apparent anamnestic response. binant WNV vaccine expressing the prM and E The lack of abnormal gross central nervous lesions genes of WNV. Previously, the efficacy of this vac- that was seen in this study is not uncommon; horses cine was assessed in response to a challenge with naturally exposed to WNV usually have few or no WNV-infected mosquitoes. The results showed gross lesions.2,11,12 that the vaccine provided protection against the de- There was an association between neurological velopment of West Nile viremia as early as 26 days signs and histopathological changes seen in the con- after a single dose.5 The protective immunity of trol horses. However, one control horse had clinical the product was also proven to last for 1 yr after a signs but no detectable lesions in the brainstem, and primary course of two injections.6 another horse had histopathological lesions in the The results of the present study clearly show that brainstem but no clinical signs. Similar lesions in the vaccine significantly protected horses in a WNV- the brainstem have been reported in horses after disease model. Nine of ten vaccinated horses met natural exposure and after using this challenge the criteria for satisfactory clinical protection model.2,3,13 against a WNV infection that induced neurological The results of this study and previous studies5,6 disease in eight of ten control horses. Only one show significant protection data from a single dose vaccinated horse showed mild neurological signs of vaccine and protective immunity lasting for 1 yr during one observation on one day. Although it has after a course of two injections. Therefore, we con- been recently reported that a live chimera vaccine cluded that this canarypox-vectored WNV vaccine induced similar levels of protection using a similar may provide with an important tool in challenge model,3 the recombinant canarypox-vec- preventing WNV infection and disease under high- tored equine WNV vaccine is, to the authors’ knowl- risk field conditions. edge, the only vaccine to have shown that it provides protection under both sets of challenge conditions. Data from this study was originally published in The clinical signs observed in this experimental Veterinary Therapeutics Journal, 2006;7:249–256, model are in line with those seen after natural ex- and used with permission from Veterinary Learning posure2,7–9 and were primarily characterized by Systems. Funding for this study was provided by mentation changes (depression and agitation), mus- Merial Limited, Duluth, GA. cle fasciculation, tremors, and gait abnormalities with varying degrees of weakness and ataxia. In all but one affected horse, these signs were not pro- References and Footnotes gressive; they suggested a diffuse central nervous 1. Hubalek Z, Halouzka J. West Nile fever—a remerging mos- system disease involving the brain and spinal cord. quito-borne disease in Europe. Emerg Infect Dis Although fever has not been a consistent finding 1999;5:643–659. in natural outbreak cases,8–10 nearly all control 2. Cantille C, Di Guardo G, Eleni C, et al. Clinical and neuro- horses in the present study developed fever lasting pathological features of West Nile virus equine encephalomy- elitis in Italy. Equine Vet J 2000;32:31–35. from 1 to 3 days. This difference may be attributed 3. Long MT, Gibbs EP, Seino KK, et al. Safety and efficacy of to a low transient fever that may be missed under a live attenuated West Nile chimera vaccine in horses with field conditions. In this study, horses were ob- experimentally induced West Nile virus clinical disease, in served twice daily after the experimental challenge. Proceedings. Am Assoc Equine Pract 2005;51:177–179. 4. Bunning ML, Bowen RA, Cropp CB, et al. Experimental After challenge, WNV was isolated from the blood of infection of horses with West Nile virus. Emerg Infect Dis all control horses as early as 24 h after inoculation, 2002;8:380–386. which indicates that the blood-brain barrier is dis- 5. Siger L, Bowen RA, Karaca K, et al. Assessment of the rupted by the inoculation of the virus. Similar find- efficacy of a single dose of a recombinant vaccine against West ings have been reported in previous studies.3 Nile virus in response to a natural challenge with West Nile virus-infected mosquitoes in horses. Am J Vet Res 2004;65: Interestingly, both magnitude and duration of 1459–1462. viremia were comparable with those obtained after 6. Minke JM, Siger L, Karaca K, et al. Recombinant canary- challenge with WNV-infected mosquitoes4–6; how- poxvirus vaccine carrying the prM/E genes of West Nile virus ever, clinical signs were not observed until 7 days protects horses against a West Nile virus-mosquito challenge. later. Therefore, it takes some time for the virus to Arch Virol 2004;18(Suppl):221–230. 7. Snook CS, Hyamn SS, Del Piero F, et al. West Nile virus induce the changes in the central nervous system encephalitis in eight horses. J Am Vet Med Assoc 2001;218: that will result in detectable clinical signs. Virus 1576–1579. was not isolated from samples collected at the end of 8. Porter MB, Long Mt, Getman SG, et al. West Nile Virus the study from the medulla, pons, and CSF. This encephalomyelitis in horses: 46 cases (2001). JAmVet Med Assoc 2003;9:1241–1247. was not unexpected, because these samples were 9. Shuler LA, Khaitsa ML, Dyer NW, et al. Evaluation of an collected 21 days after the challenge; at that point, outbreak of West Nile virus infection in horses: 569 cases the clinical signs in most animals had subsided. (2002). J Am Vet Med Assoc 2004;225:1084–1089.

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10. Ward MP, Levy M, Thacker HL, et al. Investigation of an aRECOMBITEK® Equine West Nile virus vaccine, Merial, Du- outbreak of encephalomyelitis caused by West Nile virus in luth, GA, 30096. (RECOMBITEK® is a registered trademark of 136 horses. J Am Vet Med Assoc 2004;225:84–89. Merial in the United States and elsewhere.) 11. Ostlund EN, Andresen JE, Andresen M. West Nile enceph- bTRANQUIVED™, Vedco, Inc., St. Joseph, MO, 64507. (TRAN- alitis. Vet Clin North Am [Equine Pract] 2000;16:427–441. QUIVED™ is a trademark of Vedco, Inc. in the United States.) 12. Guillon JC, Joubert OJ, Hannoun C, et al. Histological le- cKETAVED®, Vedco, Inc., St. Joseph, MO, 64507. (KETAVED® sions of the nervous system in West Nile virus infection in is a registered trademark of Vedco, Inc. in the United States.) horses. Annals De L’Institute pasteur 1968;114:539. dSAS® v.8, SAS Institute Inc., Cary, NC, 27513. (SAS® is a 13. Castillo-Olivares J, Wood J. West Nile virus infection of registered trademark of SAS Institute, Inc. in the United States horses. Vet Res 2004;35:468–483. and elsewhere.)

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