Safety and Efficacy of a Turkey Herpesvirus Vector Laryngotracheitis Vaccine for Chickens Author(s): Motoyuki Esaki, Lauren Noland, Tim Eddins, Alecia Godoy, Sakiko Saeki, Shuji Saitoh, Atsushi Yasuda, and Kristi Moore Dorsey Source: Avian Diseases, 57(2):192-198. 2013. Published By: American Association of Avian Pathologists DOI: http://dx.doi.org/10.1637/10383-092412-Reg.1 URL: http://www.bioone.org/doi/full/10.1637/10383-092412-Reg.1

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AVIAN DISEASES 57:192–198, 2013

Safety and Efficacy of a Turkey Herpesvirus Vector Laryngotracheitis Vaccine for Chickens Motoyuki Esaki,AB Lauren Noland,A Tim Eddins,A Alecia Godoy,A Sakiko Saeki,B Shuji Saitoh,B Atsushi Yasuda,B and Kristi Moore DorseyAC A B

Ceva Animal Health (Biomune Campus), 8901 Rosehill Road, Lenexa, KS 66215 Ceva Animal Health (Japan Campus), 1-6 Suehiro-cho, Yokohama, 230-0045 Japan

Received 1 October 2012; Accepted 21 January 2013; Published ahead of print 25 January 2013 SUMMARY. Turkey herpesvirus vector laryngotracheitis vaccine (HVT/LT) expressing the glycoprotein B gene of laryngotracheitis virus (LTV) has been developed. In vitro growth kinetics of HVT/LT were similar to those of parental turkey herpesvirus (HVT), FC-126 strain. Genetic and phenotypic stabilities of HVT/LT after in vitro (in cell culture) or in vivo (in chickens) passage were confirmed by various assays, including Southern blot analysis, western blot analysis, and an indirect immunofluorescence assay. Safety of HVT/LT was assessed by an overdose study as well as by a backpassage study in specificpathogen-free (SPF) chickens. The overdose study indicated that HVT/LT did not cause any adverse effects in chickens. The backpassage study confirmed that HVT/LT does not revert to virulence after five passages in chickens. The vaccine did not transmit laterally from vaccinated chickens to commingled nonvaccinated chickens. Efficacy of HVT/LT was evaluated in SPF layer chickens after vaccination by the subcutaneous route at 1 day of age. The majority of the vaccinated chickens (92%–100%) were protected against challenge with virulent LTV at 7 wk of age. Efficacy of HVT/LT was further evaluated in broiler chickens from a commercial source after in ovo vaccination to embryos at 18 days of incubation. After challenge with virulent LTV at 21 and 35 days of age, 67% and 87% of HVT/LT-vaccinated chickens did not develop LT clinical signs, respectively, while 100% (21 days of age) and 73% (35 days of age) of the challenge control chickens showed clinical signs of LT. These results suggest that HVT/LT is a safe and efficacious vaccine for control of laryngotracheitis (LT). RESUMEN. Seguridad y eficacia en pollos de una vacuna contra laringotraqueı´tis utilizando un herpesvirus de los pavos como vector. Se desarrollo´ una vacuna contra la laringotraqueı´tis con un herpesvirus de los pavos como vector (HVT/LT) que expresaba el gene de la glicoproteı´na B del virus de la laringotraqueitis. La cine´tica de crecimiento in vitro de esta vacuna recombinante fue similar a las observadas con el herpesvirus de pavo de origen, la cepa FC-126. Se confirmo´ la estabilidad gene´tica y fenotı´pica de esta vacuna recombinante despue´s de los pasajes in vitro (en cultivo celular) o in vivo (en pollos) por diversos ensayos, incluyendo el ana´lisis de transferencia Southern, el ana´lisis de inmunotransferencia y un ensayo de inmunofluorescencia indirecta. La seguridad de esta vacuna recombinante HVT/LT se evaluo´ mediante un estudio de sobredosis, ası´ como por un estudio en pasajes regresivos en aves libres de pato´genos especı´ficos. El estudio de sobredosis de la vacuna recombinante HVT/LT no causo´ efectos adversos en los pollos. El estudio confirmo´ que el pasaje regresivo de la vacuna recombinante no indujo la reversio´n a la virulencia despue´s de cinco pases en pollos. La vacuna no se transmitio´ horizontalmente de pollos vacunados a pollos no vacunados. La eficacia de la vacuna recombinante se evaluo´ en pollos libres de pato´genos especı´ficos despue´s de la vacunacio´n por vı´a subcuta´nea al dı´a de edad. La mayorı´a de los pollos vacunados (92% a 100%) estuvieron protegidos contra el desafı´o virulento con el virus de laringotraqueı´tis a las 7 semanas de edad. La eficacia de la vacuna recombinante HVT/LT se evaluo´ adicionalmente en pollos de engorde de una fuente comercial despue´s de la vacunacio´n in ovo en embriones de 18 dı´as de incubacio´n. Despue´s del desafı´o virulento con laringotraqueı´tis a los 21 y 35 dı´as de edad, el 67% y el 87% de los pollos vacunados no desarrollaron signos clı´nicos, respectivamente, mientras que el 100% (21 dı´as de edad) y el 73% (35 dı´as de edad) de los pollos controles desafiados mostraron signos clı´nicos de laringotraqueı´tis. Estos resultados sugieren que la vacuna recombinante HVT / LT es una vacuna segura y eficaz para el control de la laringotraqueı´tis. Key words: laryngotracheitis, turkey herpesvirus, vector vaccine, genetic stability, safety, protection Abbreviations: BCIP/NBT 5 5-bromo-4-chloro-3-indolyl-phosphate/nitro blue tetrazolium; CEF 5 chicken embryo fibroblasts; CEO 5 chicken embryo-origin; CPE 5 cytopathic effect; DIG 5 digoxigenin; EID50 5 50% egg infective dose; FITC 5 fluorescein isothiocyanate; gB 5 glycoprotein B; HVT 5 turkey herpesvirus; HVT/LT 5 turkey herpesvirus vector laryngotracheitis vaccine; IFA 5 indirect immunofluorescence assay; kb 5 kilobase; kDa 5 kilodalton; LT 5 laryngotracheitis; LTV 5 laryngotracheitis virus; MD 5 Marek’s disease; MDV 5 Marek’s disease virus; ML 5 modified live; PBMC 5 peripheral blood mononuclear cells; pfu 5 plaque forming units; SPF 5 specific pathogen free; SQ 5 subcutaneous; USDA 5 U.S. Department of Agriculture

Laryngotracheitis (LT; also called infectious laryngotracheitis or ILT) is an acute respiratory disease of chickens characterized by signs of respiratory depression, gasping, swollen heads, and expectoration of bloody exudates (15). Laryngotracheitis distributes worldwide and causes significant economic losses due to mortality and decreased egg production (15). The etiologic agent of LT is laryngotracheitis virus C

Corresponding author. E-mail: [email protected]

(LTV; also called infectious laryngotracheitis virus or ILTV) or gallid herpesvirus 1, which belongs to the genus Iltovirus, the family Herpesviridae and subfamily Alphaherpesvirinae. Laryngotracheitis has been a concern mainly to the layer industry; however, in recent years, there have been an increasing number of cases in which broiler flocks are affected by the disease (6,9,35). Two types of modified live (ML) vaccines, a chicken embryo-origin (CEO) virus and a tissue cultureorigin virus, have been used successfully for years to control LT. However, these ML vaccine viruses are capable of spreading from

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vaccinated chickens to nonvaccinated chickens (1,17,31), and it has been shown that their inherent residual virulence increases after birdto-bird passages (14). In fact, a majority of viruses that are isolated from recent LT outbreaks are indistinguishable from the CEO virus (9,12,13,26). The ability of ML vaccine viruses to go latent in chickens and to sporadically reactivate, which leads to shedding of virus, is also a matter of concern for disease control (18). It has also been suggested recently that ML LT vaccines recombine with another ML LT vaccine to form virulent strains in the field (22). Therefore, a need exists for developing new-generation vaccines that are capable of inducing protective immunity while not introducing live LTV in the field (2). The glycoproteins of LTV are important protective antigens that can elicit both humoral and cell-mediated immunity (42). A subunit vaccine with the 205 kilodalton (kDa) glycoprotein complex of LTV containing glycoprotein B (gB) provided excellent protection against challenge (43). Glycoprotein B is highly conserved within all the members of the Herpesviridae and is essential for herpesvirus infectivity because of its involvement in virus attachment and penetration (27,36). It has also been shown that herpesvirus gB protein elicits neutralizing antibodies (5,23) as well as cell-mediated immune responses (32,44); therefore, it is considered to be an excellent candidate antigen for subunit or recombinant vaccines (5,32). The LTV gB possesses characteristics that are common to herpesvirus gB including conserved 10 cysteine residues on the surface of the molecule and conserved positions of N-linked glycosylation sites (11). It has been shown that the LTV gB protein assembles into homodimers that are rapidly cleaved to form two disulphide-linked species (28). Recombinant fowlpox virus–vector vaccines expressing the LTV gB gene, and a DNA vaccine containing the gB gene, have also been developed and have been shown to provide protective immunity in chickens (3,8,37,38). However, no turkey herpesvirus (HVT) vector vaccines expressing the LTV gB gene have been described to date. Turkey herpesvirus, or Meleagrid herpesvirus 1, is classified in the family Herpesviridae in the subfamily Alphaherpesvirinae. Nononcogenic HVT is part of the Marek’s disease virus (MDV) group and is designated as serotype 3 MDV. The virus was originally isolated from normal turkeys (21,40) and has been used extensively as a safe and efficacious vaccine against Marek’s disease (MD) (25,34). Turkey herpesvirus is administered either to 1 day-of-age chicks by the subcutaneous (SQ) route or in ovo to chicken embryos at 18– 19 days of incubation. The HVT establishes infection in lymphocytes and can be isolated from blood lymphocytes of infected chickens as early as 5 days postinfection (41). Owing to the large DNA genome, HVT has been evaluated for use as a viral vector carrying protective antigen gene(s) of various poultry pathogens (7,24,29,30,39). For LT, recombinant HVT containing glycoprotein D and glycoprotein I genes has been developed (10). In an effort to produce a safe LT vaccine that is efficacious while not introducing live LTV, we have developed a HVT vector vaccine expressing the extracellular region of the LTV gB gene. To our knowledge, this is the first HVT vector vaccine expressing the LTV gB gene. The objectives of the present study were to evaluate genetic stability, safety, and efficacy of this vaccine. For stability and safety, a backpassage study to confirm a lack of reversion to virulence and an overdose study, respectively, were conducted. The ability of turkey herpesvirus vector laryngotracheitis vaccine (HVT/LT) to transmit horizontally from vaccinated chickens to unvaccinated chickens was also examined. Efficacy was initially evaluated after administration of the vaccine into specific-pathogen-free (SPF) chicks at 1 day of age and then evaluated in broiler chickens after vaccination into embryos at 18 days of incubation.

MATERIALS AND METHODS Viruses. Turkey herpesvirus vector laryngotracheitis vaccine, VectormuneH LT (CEVA Biomune, Lenexa, KS), contains the extracellular region of the gB gene of LTV. The HVT/LT virus was propagated in chicken embryo fibroblasts (CEFs). Parental HVT FC-126 strain (40) was obtained from Dr. R. L. Witter at the Avian Disease and Oncology Laboratory, East Lansing, MI, propagated in CEF and used as a control in various assays. The very virulent RB1B strain of serotype 1 MDV (33) was obtained from Dr. K. A. Schat, Cornell University, Ithaca, NY, propagated in SPF chickens, and lymphocyte cells prepared from the inoculated chickens were used in an overdose safety study to show that chickens from the same flock and hatch as the vaccinates were susceptible to MDV. The U.S. Department of Agriculture (USDA) LTV challenge strain was used as a challenge virus in immunogenicity studies, obtained from the National Veterinary Services Laboratories, USDA/Animal and Plant Health Inspection Service, Ames, IA, and propagated on the chorioallantoic membrane of 9-to-11-day-old SPF chicken embryos. Preparation of antiserum against LTV gB protein. Antiserum against the LTV gB protein was produced in goat by immunization with purified gB protein produced in Escherichia coli. The antiserum was used in various assays, including a western blot assay and an indirect immunofluorescence assay (IFA), to confirm the expression of the LTV gB protein in CEF infected with HVT/LT. Confirmation of gene structure. To confirm the gene structure of HVT/LT, Southern blot analysis was conducted. Briefly, genomic DNA of HVT/LT was digested with restriction enzymes, XhoI and PstI; separated by agarose gel electrophoresis; and blotted on a nylon membrane (Pall Corporation, Port Washington, NY). The membrane was incubated with either digoxigenin (DIG)-labeled LTV gB probe or DIG-labeled HVT insertion site (UL45/46 region) probe. DNA fragments that bound to the probes were visualized after incubation with alkaline phosphatase–labeled anti-DIG antibody (Roche Applied Science, Indianapolis, IN) and developed with 5-bromo-4-chloro-3indolyl-phosphate/nitro blue tetrazolium (BCIP/NBT; Bio-Rad Laboratories, Hercules, CA). Expression of LTV antigenic protein. Expression of the LTV gB protein was confirmed by western blot analysis and an IFA, both using the goat anti-LTV gB serum. Briefly, for western blot analysis, lysates of CEF infected with HVT/LT for 3 days were separated on 10% acrylamide gel and transferred on polyvinylidene difluoride membrane (Millipore, Billerica, MA). The membrane was incubated with the goat anti-LTV gB serum and then alkaline phosphatase-labeled anti-goat IgG antibody (Bethyl Laboratories, Montgomery, TX) and developed with BCIP/NBT (Bio-Rad Laboratories). For the IFA, monolayers of CEF infected with HVT/LT for 5 days were fixed with a mixture of methanol and acetone (volume ratio 5 1:2). The fixed monolayer was treated with the goat anti-LTV gB serum and then fluorescein isothiocyanate (FITC)-labeled anti-goat IgG antibody (Sigma-Aldrich, St. Louis, MO) and observed under an inverted fluorescence microscope. In vitro growth characterization of HVT/LT. Monolayers of CEF in 100-mm tissue culture plates were inoculated with approximately 2000 plaque forming units (pfu) per plate of either HVT/LT or parental HVT. At 10 different time points (16, 24, 40, 44, 48, 52, 64, 69, 72, and 88 hr) until 88 hr after inoculation, cells in the plates were harvested with trypsin and titrated on CEF. Safety of HVT/LT. An overdose study and a backpassage study were conducted to investigate safety of HVT/LT. For the overdose study, HVT/LT at 30,000 pfu/chicken was inoculated either by the in ovo route into 18-day-old SPF embryos (Charles River Laboratories, Wilmington, CT) or subcutaneously into 1 day-of-age SPF chicks. A group of chickens was inoculated subcutaneously with 200 pfu/chicken of the very virulent RB1B strain of MDV at 5 days of age to demonstrate that chickens used in this study were susceptible to MDV. For the RB1B-inoculated group, chickens were observed until 7 wk of age. Surviving chickens in this group were necropsied at 7 wk of age and observed for gross lesions of MD. For other groups, chickens were observed closely until 18 wk of age for any adverse vaccine reactions and

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Fig. 1. Expression of LTV gB protein by HVT/LT. (A) Western blot analysis. Cell lysates prepared from CEF infected with (lane 1) the fifth in vivo passage of HVT/LT, (lane 2) the fifth in vitro passage of HVT/LT, (lane 3) HVT/LT before passage, (lane 4) parental HVT FC126, or (lane 5) a mock were subjected to western blot using anti-LTV gB serum. An arrow indicates the 60-kDa expressed gB protein. M: Precision Plus Protein Standards (Bio-Rad Laboratories). (B) Indirect immunofluorescence assay. A CEF monolayer infected with HVT/LT was incubated for 5 days and fixed with methanol:acetone. The monolayer with HVT/LT plaques was reacted with goat anti-LTV gB serum and then with FITC-labeled anti-goat IgG antibody and observed under an inverted fluorescence microscope. for any clinical signs associated with MD or LT. At 18 wk of age, chickens were necropsied and observed for grossly observable lesions. For the backpassage study, 1 day-of-age SPF chicks were inoculated subcutaneously with HVT/LT at 30,000 pfu/chicken. After 7 days, blood was taken from the chicks using heparin, and 1 ml of the blood/ chicken was inoculated to another set of 1 day-of-age SPF chicks by the SQ route. This process was repeated four more times. At the last (fifth) passage, chickens were observed for any clinical signs associated with MD or LT for 45 days. After 45 days, chickens were necropsied and observed for grossly observable lesions. Also at the fifth passage, virus isolation was attempted by inoculating peripheral blood mononuclear cells (PBMCs) prepared from blood of inoculated chicks using HistopaqueH-1077 (Sigma-Aldrich) on a CEF monolayer. Recovered virus was further propagated and used for various assays to confirm genetic and phenotypic stability of HVT/LT. Contact transmission study of HVT/LT. Specific-pathogen-free chicken embryos (Charles River, CT) at 18 days of incubation were inoculated by the in ovo route either with HVT/LT or parental HVT at 30,000 pfu/egg. Another group of SPF embryos remained unvaccinated. After hatch, 25 chicks selected randomly from the group of chicks vaccinated with HVT/LT were commingled with 15 nonvaccinated contact chicks. Similarly, 25 chicks vaccinated with parental HVT were commingled with 15 nonvaccinated contact chicks. Five nonvaccinated chickens were kept separately as negative controls. All chickens from each group were bled at 10, 14, and 21 days of age. Heparinized blood samples from five chickens within each group were pooled, and PBMCs were purified from the blood after centrifugation at 400 3 g for 15 min on Histopaque-1077 (Sigma-Aldrich). The PBMCs were inoculated on CEF in six-well plates at 1 3 106 cells per well. Each sample was inoculated into two wells. Inoculated CEF was incubated at 37 C for 5 to 7 days and observed for cytopathic effects (CPE) typical of HVT. Efficacy of HVT/LT against LT in SPF chickens. Specificpathogen-free chicks (Nissei-ken, Japan) were divided into four groups. Each group contained 10–14 chickens. Two groups (Vaccine Group 1 and Vaccine Group 2) were vaccinated subcutaneously with two unique formulations of HVT/LT at 1 day of age. Another group was vaccinated ocularly at 4 wk of age with a commercial LTV live attenuated vaccine (‘‘Kaketsuken’’ CE strain, Kaketsuken, Japan). One group was left unvaccinated and served as a challenge control. The chickens were housed separately in isolators. At 7 wk of age, chickens were challenged

by the infraorbital sinus route with 103.0 50% egg infective dose (EID50) of the USDA LTV challenge strain. After challenge, chickens were observed for 8 days for clinical signs typical of LT, such as nasal exudates, conjunctivitis, and watery eyes. Efficacy of HVT/LT in commercial broilers. Broiler chickens from commercial sources were used in this study. In the vaccinated group, each embryo at 18 days of incubation was inoculated in ovo with one commercial dose (in 0.05 ml) of HVT/LT. A group of chickens were kept unvaccinated and served as a challenge control. After hatch, chickens were housed in isolators. At 21 (Trial 1) or 35 (Trial 2) days of age, groups of chickens were challenged by the infraorbital sinus route with 103.5 EID50 of the USDA LTV challenge strain. After challenge, chickens were observed for 10 days for clinical signs typical of LT, such as nasal exudates, conjunctivitis, and watery eyes.

RESULTS

Expression of LTV gB protein by HVT/LT. Western blot analysis using goat anti-LTV gB serum confirmed that the gB gene insert was expressed as expected (approximately 60 kDa) in CEF infected with HVT/LT (Fig. 1A). Lane 3 contains the contents of CEF infected with HVT/LT and has a band near 60 kDa, whereas Lane 4, which contains the contents of CEF infected with the parent strain of HVT, does not have a band near 60 kDa. Additionally, IFA using goat anti-LTV gB serum confirmed that gB protein was expressed in HVT/LT plaques formed on a CEF monolayer (Fig. 1B); fluorescence from FITC-labeled anti-goat IgG antibody was observed under an inverted fluorescence microscope. In vitro growth characterization of HVT/LT. Growth of HVT/ LT in CEF was compared with that of the parental HVT FC126 strain. Inoculated CEF monolayers were harvested at 10 different time points until 88 hr after inoculation and titrated. Growth kinetics of HVT/LT were very similar to those of parental HVT (Fig. 2). This result indicates that HVT/LT replicates at a similar rate to that of the parental HVT in CEF. Genetic and phenotypic stabilities of HVT/LT. Genetic stability of HVT/LT was examined after both in vivo and in vitro

Turkey herpesvirus vector vaccines

Fig. 2. In vitro growth kinetics of HVT/LT. Approximately 2000 pfu of HVT/LT or parental HVT FC126 was inoculated on a CEF monolayer in 100-mm tissue culture plates. Infected cells were harvested various times until 88 hr postinoculation and titrated on CEF.

passages. For the in vivo stability, HVT/LT was passed five times in SPF chickens. The DNA from virus isolated from the fifth passage was analyzed by Southern blot analysis. The LTV gB probe detected a 3.3-kilobase (kb) XhoI-PstI fragment in both the HVT/LT before the in vivo passage (Lane 3, Fig. 3A) and the HVT/LT isolated from the fifth in vivo passage (Lane 4, Fig. 3A), while no band was detected in parental HVT (Lane 2, Fig. 3A). The HVT insertion site probe bound to 4.6-kb and 3.3-kb XhoI-PstI fragments in both of these same HVT/LT viruses (Lanes 3 and 4, Fig. 3B), while a 2.0-kb fragment was detected in parental HVT (Lane 2, Fig. 3B). Therefore, it was confirmed that the HVT/LT virus isolated from the fifth in vivo passage possessed the same gene structure as the HVT/LT before the in vivo passage. Sequencing of the insert region also confirmed the genetic stability of HVT/LT after in vivo passages (data not shown). Additionally, the western blot analysis shown in Fig. 1A confirmed that the fifth in vivo passage of HVT/LT expressed the gB protein (Lane 1, Fig. 1A). In vitro genetic stability of HVT/LT was verified similarly after passing the virus up to 20 times in CEF (data not shown). Safety of HVT/LT. Safety of HVT/LT was evaluated by an overdose study and a backpassage study. For the overdose study, SPF embryos at 18 days of incubation or 1 day-of-age SPF chicks

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received HVT/LT at 30,000 pfu. In ovo application of the HVT/LT overdose did not affect hatchability (Table 1). Chickens were observed closely until 18 wk of age for any clinical signs or adverse reactions. No chickens that received the HVT/LT had any clinical signs or adverse reactions (Table 1). One chicken in the SQ group died at 7 days of age as a result of yolk sac infection. One chicken in the in ovo group died at 20 days of age and the other at 123 days of age because of cannibalism. No gross lesions were observed with these chickens. Remaining chickens were necropsied at 18 wk of age and observed for grossly observable lesions. No gross lesions were observed in any of the vaccinated chickens (Table 1). In the RB1Binoculated group, most of chickens died of MD or developed grossly observable lesions of MD, confirming that the chickens used in this study were susceptible to MD (Table 1). For the backpassage study, to confirm that HVT/LT will not revert to virulence, HVT/LT was passed five times in SPF chickens by using heparinized blood from chickens in the previous passage to inoculate a new set of SPF chicks. Chickens inoculated with the virus at the fifth passage, i.e., inoculated with the heparinized blood from the fourth passage, were observed closely for any clinical signs for 45 days. Chickens were then necropsied and observed for grossly observable lesions. No chickens had any clinical signs, adverse reactions, or gross lesions. Therefore, the HVT/LT did not revert to virulence after backpassages in chickens. Lack of HVT/LT transmission by contact. One day-of-age SPF chickens vaccinated in ovo with either HVT/LT or parental HVT were commingled in isolators with nonvaccinated contact chickens. Virus was isolated from the PBMCs collected from chickens vaccinated with HVT/LT or parental HVT at all time points tested, i.e., 10, 14, and 21 days of age (Table 2). No virus was isolated from any of the contact chickens at any time points tested between 10 and 21 days, indicating that neither HVT/LT nor parental HVT spread from vaccinated chickens to nonvaccinated contact chickens during that period. Efficacy of HVT/LT against LT in SPF chickens. Efficacy of HVT/LT against LT was evaluated in SPF chickens, which were vaccinated with HVT/LT subcutaneously at 1 day of age. Two groups were vaccinated with two unique formulations. Each bird received one dose (in 0.2 ml) of the vaccine diluted with the vaccine

Fig. 3. Genetic stability of HVT/LT after in vivo passages was confirmed by Southern blot analysis using (A) the LTV gB probe and (B) the HVT insertion site probe. Lane 1, CEF DNA; Lane 2, parental HVT-infected CEF DNA; Lane 3, HVT/LT-infected CEF DNA; Lane 4, fifth in vivo passage HVT/LT-infected CEF DNA; Lane 5, plasmid control DNA; M, DNA Molecular Weight Marker II, DIG-labeled (Roche).

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Table 1. Safety of HVT/LT in the overdose study.

Group

1. 2. 3. 4.

No. hatched/No. embryos set (% hatchability)

Vaccine route

HVT/LT HVT/LT RB1B controls Negative controls

In ovo SQ Not applicable Sham in ovo

61/65 224/244B 224/244 60/65

No. chicks set

(94) (92) (92) (92)

Mortality (No. with gross MD lesions)

2A 1 35 0

61 50 50 60

(0) (0) (33) (0)

No. chickens with adverse reactions or clinical signs

No. chickens at necropsy (No. with gross MD lesions)

0 0 Not applicable 0

59 49 15 60

(0) (0) (15) (0)

A

Two chickens in the in ovo group and one chicken in the SQ group died before the end of the observation period, but causes of deaths were determined to be unrelated to the vaccine. B Hatchability for all noninoculated embryos was calculated together and chicks were divided into groups after hatch.

diluent. After challenge with the LTV USDA standard challenge strain at 7 wk of age, none of the chickens in Vaccine Group 1 showed clinical signs (100% protection) and only 1 out of 13 chickens (92% protection) showed clinical signs in Vaccine Group 2 (Table 3). All of the challenge control chickens showed clinical signs typical of LT such as nasal exudates and watery eyes. Chickens vaccinated with the live LTV vaccine did not show any clinical signs of LT after challenge. Efficacy of HVT/LT against LT in commercial broilers. Efficacy of HVT/LT was evaluated in commercial broiler chickens. One commercial dose of HVT/LT was administered in ovo to each of broiler chicken embryos at 18 days of incubation. In Trial 1, chickens were challenged by the infraorbital sinus route with the USDA challenge strain of LTV at 21 days of age and observed for clinical signs of LT until 10 days postchallenge. In Trial 2, chickens were challenged at 35 days of age and observed for clinical signs of LT until 10 days postchallenge. After challenge at 21 days of age, 67% (10/15) of chickens in the group that received HVT/LT were free from clinical signs of LT, while all of chickens in the challenge control group developed clinical signs of LT (Table 4). After challenge at 35 days of age, 87% (13/15) of chickens in the group vaccinated with HVT/LT did not develop clinical signs of LT, while 73% of the challenge control chickens showed clinical signs of LT (Table 4). DISCUSSION

A HVT vector vaccine expressing the extracellular region of the LTV gB gene, HVT/LT, was characterized by the assays described here. Specifically, growth kinetics, genetic and phenotypic stabilities, safety, and immunogenicity of the vaccine were examined. In vitro growth kinetics of HVT/LT were similar to those of parental HVT, FC126 strain, indicating that the insertion of the LTV gB gene did not affect the replication capability of the virus. Genetic and phenotypic stabilities of HVT/LT after passages in cell culture, as well as passages in chickens, were demonstrated as well.

Safety of HVT/LT was demonstrated by an overdose study and a backpassage study. In the overdose study, HVT/LT inoculated either by the in ovo route to SPF embryos at 18 days of incubation or subcutaneously into 1 day-of-age SPF chicks did not cause any adverse reactions or MD tumors. Furthermore, in ovo application of an overdose of HVT/LT did not affect hatchability. In the backpassage study, HVT/LT did not increase virulence after five passages in chickens. This result was expected because HVT has been used extensively as a vaccine against MD for 40 yr without causing any problems in the field, and the LTV gB gene is not considered to be a virulence factor. However, existing live LT vaccines have been shown to become virulent after bird-to-bird passages (14), so the HVT/LT vaccine presents quite a significant advantage over the ML LT vaccines. In summary, HVT/LT was found to be safe for use as a vaccine in chickens. Turkey herpesvirus has been shown to replicate in feather follicle epithelium (16,45), and HVT DNA has been detected in chicken dander using quantitative real time PCR (19,20). Islam et al. (20) detected HVT DNA in chicken dander as early as 9 days postinoculation. However, interestingly, HVT is not thought to readily spread horizontally between chickens (4,20). In our contact transmission study, no transmission of HVT/LT or parental HVT from vaccinated SPF chickens to nonvaccinated contact chickens occurred during the 10 to 21 days of testing, supporting the assertion that the ability of HVT to transmit horizontally is very limited. Efficacy of HVT/LT against LTV challenge was demonstrated in both SPF layer chickens and commercial broiler chickens. In the SPF layers, day-of-age chicks were vaccinated subcutaneously with HVT/ LT. This one shot vaccination with HVT/LT at 1 day of age was sufficient to provide excellent protection against LTV challenge at 7 wk of age. In the commercial broiler study, vaccination was conducted by the in ovo route to chicken embryos at 18 days of incubation. Significant protection against LTV challenge was observed as early as 21 days of age, suggesting early onset of immunity due to early replication of HVT/LT, which was detected as early as 10 days of age.

Table 2. Lack of horizontal transmission of HVT/LT. Table 3.

Virus isolationA Group

10 days of age

14 days of age

21 days of age

1. HVT/LT 2. Contact chickens for HVT/LT 3. Parental HVT 4. Contact chickens for parental HVT 5. Nonvaccinated controls

5/5 0/3 5/5 0/3 0/1

5/5 0/3 5/5 0/3 0/1

5/5 0/3 5/5 0/3 0/1

A

No. of pools positive for CPE/No. of total pools in the group. Blood from five chickens were pooled and inoculated on CEF.

Efficacy of HVT/LT in SPF chickens. Group

1. 2. 3. 4.

HVT/LT Vaccine Group 1 HVT/LT Vaccine Group 2 LTV live vaccine Nonvaccinated challenge controls A

Positive/totalA

% LTB

0/14a 1/13a 0/10a 14/14b

0 8 0 100

Lowercase letters indicate a significant difference among groups by Fisher exact test (P , 0.05). B Percentage LT based on the number of chickens that showed clinical signs of LT divided by the number of challenged chickens 3 100.

Turkey herpesvirus vector vaccines

Table 4. Onset of efficacy of HVT/LT in commercial broiler chickens. Challenge at 21 days of age (Trial 1) Group

1. HVT/LT 2. Nonvaccinated controls

Challenge at 35 days of age (Trial 2)

Positive/totalA % LTB Positive/total % LT

5/15a 15/15b

33 100

2/15a 11/15b

13 73

A Lowercase letters indicate a significant difference among groups by Fisher exact test (P , 0.05). B Percentage LT based on the number of chickens that showed clinical signs of LT divided by the number of challenged chickens 3 100.

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ACKNOWLEDGMENTS We thank Peter Flegg for his great contribution to animal trials and Dr. Stacy Overman for her assistance with technical writing.