Protection of chickens, with or without maternal antibodies, against IBDV infection by a recombinant IBDV-VP2 protein

Vaccine 28 (2010) 3990–3996

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Protection of chickens, with or without maternal antibodies, against IBDV infection by a recombinant IBDV-VP2 protein Xuemei Zhou a,c,1 , Decheng Wang b,1 , Jinmao Xiong a , Peijun Zhang c , Yongqing Li c,∗ , Ruiping She a,∗ a

College of Veterinary Medicine, China Agricultural University, Beijing 100193, China Key Laboratory of Medical Molecular Virology, Shanghai Medical College, Fudan University, Shanghai 200032, China Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences, No. 9 Shuguanghuayuan Middle Road, Beijing 100097, China b c

a r t i c l e

i n f o

Article history: Received 3 November 2009 Received in revised form 25 February 2010 Accepted 10 March 2010 Available online 23 March 2010 Keywords: Infectious bursal disease virus VP2 gene Recombinant MDV

a b s t r a c t The use of avian herpesviruses (Marek’s disease virus, MDV) as vectors to express the capsid protein of infectious bursal disease virus (IBDV) was well established, and its protection against IBDV challenge has been evaluated previously. However, there is little data about rMDV1 expressing the VP2 protein of IBDV protecting SPF and commercial chickens against virulent IBDV (vIBDV) challenge. In this study, we constructed a stable rMDV1 expressing the VP2 protein of IBDV by inserting the coding sequence within the US10 gene of MDVl by homologous recombination and designated this as rMDVl-US10L, and evaluated effectiveness of the recombinant VP2 protein with SPF chickens and commercial chickens with maternal antibodies in vIBDV challenge. The results can be summarized as follows: (1) We constructed a rMDV1 expressing IBDV-VP2 under the control of the MDV1 glycoprotein B (gB) promoter [rMDV1-US10L]. (2) rMDV-VP2 protein was readily expressed and induced 53% protection against a vIBDV challenge in SPF chickens with 103 PFU/chicken, whereas 104 PFU induced 73% protection. (3) Vaccination of commercial chickens having maternal antibodies to rMDV1-VP2 induced 87% protection in vIBDV challenge, which was similar to results using the live vaccine, BJ87 IBDV strain, in commercial chickens. These results demonstrate that the VP2 antigen expressed in the MDV vector was an effective and stable vaccine in correlation with the vaccine efficacy against lethal IBDV challenge, and can provide a better protective effect that is likely to persist for the life of the chickens. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Infectious bursal disease virus (IBDV), the prototype member of the Avibirnavirus genus of the Birnaviridae family [1], is the causative agent of a highly infectious disease affecting young chickens. Chickens infected with IBDV develop immunodeficiency due to depletion of B lymphocytes, increasing their susceptibility to opportunistic pathogens and reducing the growth rate of surviving birds [2]. The virus genome consists of two segments, A and B. Segment A (3.2 kb) contains two partial overlapping open reading frames (ORFs). The large ORF encodes a 110 kDa polyprotein, pVP2-VP4-VP3, which is cleaved by autoproteolysis to form the viral proteins VP2, VP3 and VP4 [3,4]. It has been reported that the VP2 protein is the major protective antigen of the virus and contains antigenic epitopes responsible for the induction of neu-

∗ Corresponding authors. E-mail addresses: [email protected] (Y. Li), [email protected], [email protected] (R. She). 1 Both authors contributed equally to this paper. 0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2010.03.021

tralizing antibodies [5]. Protection against IBDV could be induced by using VP2 or polyprotein subunit vaccines [6]. Commercial attenuated live IBDV vaccines are usually used to prevent IBDV infection [7,8]. However, it is difficult to protect field chickens with maternal antibodies by using live vaccines. Some IBDV live vaccines cause moderate bursal atrophy, which may promote the infection of opportunistic pathogens. Additionally, live virus vaccines are unstable in their antigenic and pathogenic characteristics often rendering them harmful to the immunized animal [9]. Thus, it is necessary to develop safer and more efficacious vaccines against IBDV in the field. In recent years, many investigators have used recombinant technology to express structural proteins of IBDV [6,10,11]. Live viral vectors that serve as a polyvalent vaccine, with their ability to express foreign genes, have the potential to be used in vaccination against many diseases [12–14]. Marek’s disease (MD) is a highly contagious malignant Tlymphomatosis of chickens caused by the highly infectious cell-associated alphaherpesvirus MD virus serotype 1 (MDV1) [15,16]. MD is the first cancer to be prevented and controlled by use of live attenuated or naturally avirulent vaccines since the early 1970s [17]. Three serotypes of MDV have been completely

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identified and sequenced [18–20]. The MDV vaccine strains are apathogenic MDV1, naturally apathogenic MDV serotype 2 (MDV2), and MDV3 (herpesvirus of turkeys [HVT]). The viruses can induce lifetime protection against MD in chickens and have a natural host range limited to avian species [21,22]. MDV strains are safe to other domestic animals and people working in the poultry industry, and techniques for generating recombinant MDVs have been well established [23], therefore MDV vaccine strains are considered as the most potent vector for expressing foreign genes that could provide effective protection against poultry disease [24]. Among the vaccine viruses, HVT has been used worldwide both as a live vaccine and polyvalent vaccine vector [14,25,26]. However, attenuated MDV1 strains such as CVI988c/R6 and R2/23 [27] elicit a better effect than HVT [28] because MDV1 vaccine has the closest antigenic similarity to the very virulent (vv) MDV1, including virulent strains isolated from field. Among the three serotypes of MDV vaccine viruses, attenuated MDV1 is the best choice for the prevention of MD. Laboratory and field experience has clearly demonstrated that its protection against MD is greater than 95% [27]. Previous studies have demonstrated that MDV1 can be used as a live virus vector for expressing foreign genes [12,13,22,29]. Thus, attenuated MDV1 is suitable for construction of a recombinant vaccine against avian diseases. In an earlier study a recombinant HVT vector to express the VP2 protein of IBDV was constructed. The VP2 coding sequence was inserted into the ribonucleotide reductase (RR) small subunit or gI genes of HVT [26]. However, these recombinant HVTs provides very low protection against both IBD (60%) and MD (10%) in SPF chickens. The IBDV-VP2 antigen was also inserted into the US2 site of the MDV1 vaccine [12]; so the partial protection against very virulent IBDV (55%) and full protection against vvMDV were achieved in SPF chickens. But the efficacy of the rMDV against IBDV was not evaluated in commercial chickens with high titers of maternal antibodies. In the present study, we constructed a recombinant MDV expressing the VP2 protein of IBDV and the LacZ gene of Escherichia coli. We examined the protective efficacy against virulent IBDV challenge by one-time vaccination of SPF and commercial chickens with maternal antibodies. 2. Materials and methods 2.1. Virus and cells The IBDV LX strain used in our study for plasmid construction is a very virulent (vv) strain (vvIBDV), which was isolated from a broiler chicken factory in 2000 [30]. The standard challenge (STC) virulent stain of IBDV CJ801 and IBDV BJ836 attenuated strain were obtained from the China Institute of Veterinary Drug Control (Beijing, China), and used to evaluate the protective efficacy of rMDV. A commercial live vaccine IBDV-A was an IBDV BJ836 isolate. MDVCVI988/Risepens and its recombinant form were propagated in chicken embryo fibroblasts (CEFs), which were grown as a monolayer in Eagle’s minimum essential medium supplemented with 5% fetal calf serum. 2.2. Construction of the transfer vector pUS-VP2 The IBDV LX strain propagated in SPF eggs was used for plasmid construction. The primer sequences for VP2 amplification were: P1 5 -CCGAATTCATGACAAACCTGCAAGATCAAA CC C-3 and P2 5 -TTCTAGACTACCTTATGGCCCGGATTATGT-3 . The cDNA corresponding to the VP2 polypeptide was synthesized with a cDNA synthesis kit (Takara Co., China) according to the manufacturer’s instruction. The VP2 gene was inserted into the EcoRI-XholI sites

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Fig. 1. Schematic representation of transfer vector pUS2-VP2. The relative positions of the MDV US2 gene, hCMV promoter, E. coli LacZ gene, IBDV VP2 gene and SV40 polyA are indicated.

of the pcDNA4.0 vector (Invitrogen, USA) under the control of the hCMV immediate early promoter to create plasmid pcDNA-VP2. This plasmid was digested with AvrII and BglII and the resulting 5.3 kb DNA expression cassette containing the VP2 gene of IBDV, the LacZ gene of E. coli, the hCMV early promoter and the SV40 poly-adenylation signal, was inserted into the AvrII and BglII site in the US2 gene of MDV to create the transfer vector pUS2-VP2. In this plasmid, the LacZ and VP2 genes were flanked by 0.7 and 3.7 kb of MDV1 sequences designated for homologous recombination. The plasmid map is shown in Fig. 1. 2.3. Transfection and isolation of recombinant MDV Primary CEFs were seeded at 1.2 × 106 cells per well in 24-well plates. They were infected with 50 PFU MDV when monolayer cells were approximately 80% confluent. A complex of pUS2-VP2 and LipofectamineTM 2000 (Invitrogen, Beijing, China) was transfected into CEFs infected with MDV according to the manufacturer’s instruction. Four days after transfection, plaques were observed. The culture medium was renewed and 5-bromo-4-chloro-3-indolyl-␤-d-galactoside (X-gal) added as a ␤galactosidase indicator at a final concentration of 0.2 mg/mL. Blue plaques were picked and plated on fresh primary CEFs in 96-well tissue culture plates. The resulting plaques were again selected by staining with X-gal. The recloning process was repeated until all plaques were positive. 2.4. Detection of VP2 gene expression 2.4.1. Immunostaining When clones were stably expressing the LacZ gene, we detected expression of the IBDV-VP2 with an anti-VP2 antibody. Briefly, rMDV-infected cells were grown in 96-well tissue culture plates. The plates were stained with 0.2 mg/mL X-gal. Once blue plaques were visible, immunostaining was performed using standard procedures as reported previously with an anti-IBDV chicken antiserum (Chinese Institute of Veterinary Drug Control, Beijing, China) [31]. 2.4.2. Western-blot analysis CEFs infected with rMDV or MDV were harvested 4 days postinfection. The protein was separated and identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE)

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Fig. 2. Blue plaque of CEFs infected with rMDV expressing the LacZ gene. (A) CEFs infected with rMDV (400×); (B) CEFs infected with MDV (400×).

based on a previous study [32]. Separated proteins were transferred to nitrocellulose membrane and incubated with SPF chicken antiIBDV antiserum followed by horseradish peroxidase-conjugated mouse anti-chicken IgG. A 3,3 -diaminobenzidine substrate was used to detect the VP2 band. 2.5. PCR analysis The procedures used for DNA extraction were described previously [33]. The PCR products were used to determine the occurrence of recombination in a specific site. A PCR amplification that covered the partial VP2 gene, 850 bp US2 region and partial genome of MDV was performed on DNA extracted from rMDV-infected cells with two primers, PVP2-MD1 (5 -AAGGAAGCCTGA GTGAACTGACAGA-3 ) and PVP2-MD2 (5 ACTATAGCGACACGCCCATACCAAAC-3 ). Using primers PVP1 and PVP2 described previously, another PCR on the VP2 gene was carried out on extracted DNA from rMDV-infected cells. 2.6. Animals 2.6.1. Protection against virulent IBDV (CJ801 strain) in SPF chickens Fifty-five 1-day-old SPF white leghorn chickens were obtained from the Beijing laboratory animal center in China (Merial Co. Ltd., Beijing, China). These chickens were negative to Newcastle disease virus and IBDV. Chickens were maintained in negativepressure isolation units. All birds were randomly divided into four groups: 15 birds in the 103 PFU (I) and 104 PFU (II) vaccinated group with rMDV, respectively; 15 birds were unvaccinated (III) and 10 birds were in the control group (C). Blood samples were collected from 1 to 5 weeks post-vaccination. Five weeks later, each chicken in groups I, II and III were challenged with 0.2 mL vIBDV CJ801, where the median embryo lethal dose (ELD50) of vIBDV was 106.5 /0.2 mL, by means of nasal and eye drops. In the control group, chickens received the equivalent volume of saline solution. Each group was separated into individual negative pressure isolators. All chickens were provided feed and water ad libitum. Animals were visually examined daily, clinical signs and mortality were recorded, euthanasia of all chickens was performed by intravenous injection of sodium pentobarbital in a brachial vein, and necropsies performed immediately postmortem. Parameter of bursa/body weight ratio, gross bursal lesions, mortality and protection were recorded to evaluate the results of challenge studies.

2.6.2. Protective effect against virulent IBDV in commercial chickens To evaluate the protective efficacy of recombinant MDV vaccine against the vIBDV in commercial chickens having maternal antibodies, a total of 55 one-day-old commercial chickens were obtained from Huadu Broiler chicken company (Beijing, China), raised in isolation and maternal antibody titers determined by ELISA. All chickens were divided into four groups: 15 birds were in the rMDV (104 PFU/chick; I) and IBDV BJ836 vaccine vaccinated groups (II), respectively; they were vaccinated at 14 days old. Group III contained 15 chickens unvaccinated with IBDV BJ836 and rMDV, but infected with vIBDV CJ801 strain (NC), and 10 chickens were used as controls (C). Each chicken was challenged with the vIBDV CJ801 strain as described previously. Animals were visually examined daily, clinical signs and mortality were recorded as well as other related parameters as per the procedures employed in the SPF chicken challenge. The SPF and commercial challenge study was approved by the Institutional Animal Care and Use Committee of the Beijing Academy of Agriculture and Forestry Science.

2.7. Detection of antibody to IBDV by ELISA The collected serum was tested for antibody levels to IBDV by ELISA (IDEXX, FlockChek standard; Fort Dodge Animal Health, Beijing, China) according to the manufacturer’s instructions. Statistical analysis of the mean ELISA titers was performed by ANOVA oneway of SAS (Copyright 1999–2001 by SAS Institute Inc., Cary, NC, USA) statistical program. Differences were considered significant at p < 0.05.

2.8. Antibody titers of IBDV by virus neutralization Blood samples were taken from 10 chickens selected randomly from each group before challenge. Serum samples were prepared, inactivated for 30 min at 56 ◦ C, and stored at −20 ◦ C. The virus neutralization (VN) assay was carried out with varying serum levels and a constant virus concentration on primary SPF CEFs as previously described [34]. The VN titer was determined from the cytopathic effect (CPE) using the system described by Reed and Muench [35]. The geometric mean titer (GMT) of antibody was calculated for each group.

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Fig. 3. Identification of recombinant MDV by PCR. (1) DL2000 Maker; (2) Negative control (primers PVP1 and PVP2); (3) VP2 amplification product; (4) Negative control (primers PVP2-MD1 and PVP2-MD2); (5) Amplification of a segment that covers the partial VP2 gene, US2 region and partial genome of MDV.

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Fig. 5. Western-blot analysis of VP2 (IBDV) expressed in rMDV. (1) CEFs infected with parent MDV; (2) CEFs infected with rMDV.

2.9. Histopathologic examination of bursas

3.2. Detection and identification of rMDV

Birds were euthanized and their bursas were taken for histological examination. Bursas (BFs) were fixed in 2.5% (v/v) glutaraldehyde–polyoxymethylene solution immediately after chickens were weighed and killed. The tissue samples were dehydrated and embedded in paraffin. Serial paraffin sections (5 mm) were obtained and stained with hematoxylin–eosin (HE) according to a routine method. The histopathological changes of BFs were counted under a light microscope. Sampling of the sections was unbiased.

PCR was performed to determine the presence of the IBDV VP2 gene in rMDV. A 1.7 kb PCR segment that covered the partial VP2 gene, 850 bp of the US2 region and partial genome of MDV and the whole VP2 gene was obtained from DNA extracted from rMDVinfected cells by different primers (Fig. 3).

3. Results 3.1. Transfection and isolation of recombinant MDV CEFs infected with MDVCVI988 were transfected with plasmid pUS2-VP2. Four days later, blue plaques were visible. The blue plaques were picked and serially passaged on CEFs in 96-well tissue culture plates. This process was repeated until all plaques were blue (Fig. 2).

3.3. Expression of the VP2 gene in MDV infected CEF cells We have shown that IBDV-VP2 protein was expressed in rMDV-infected CEFs by immunostaining of the blue plaques with anti-IBDV chicken antibody (Fig. 4). CEFs infected with MDV were negative when tested by immunostaining. Western-blot analysis demonstrated the VP2 protein (approximately 42 kDa) of IBDV in rMDV-infected CEFs (Fig. 5). 3.4. Protective efficacy against virulent IBDV in SPF chickens and in commercial chickens To examine the protective efficacy of rMDV in SPF chickens and commercial chickens with maternal antibody, 55 one-day-old SPF

Fig. 4. Detection of VP2 gene expression with anti-IBDV antibody in CEFs infected with rMDV and MDV. (A) Blue plaques showing expression of the VP2 gene and detected with an anti-IBDV antibody. (B) CEFs infected with MDV presented no plaques.

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Table 1 Protection efficacy of rMDV against vIBDV CJ801 strain in SPF chickens and in commercial chickens. SPF chickens

Vaccine Challenged with Mortality Gross BF lesions Bursa/body weight (×103 ) Histopathologic BF lesion scores Protection

Commercial chickens

C

rMDV(I)

rMDV(II)

III

C

rMDV

BJ836

NC

– PBS 0/10 (0%) 0/10 (0%) 5.32a 0.0 100%

103 PFU/chicken CJ801 2/15 (13%) 7/15 (47%) 4.38a 3.56 53%

104 PFU/chicken CJ801 0/15 (0%) 4/15 (27%) 4.79a 2.73 73%

– CJ801 6/10 (60%) 10/10 (100%) 3.24b 5.0 0%

– PBS 0/10 (0%) 0/10 (0%) 7.23a 0.0 100%

104 PFU/chicken CJ801 0/15 (0%) 2/15 (13%) 6.71a 2.1 87%

BJ836 CJ801 0/15 (0%) 0/15 (0%) 6.78a 1.7 95%

– CJ801 3/15 (20%) 15/15 (100%) 5.17b 4.65 0%

SPF chickens and commercial chickens were vaccinated with or without rMDV, and then challenged with vIBDV CJ801 strains at 5 weeks. A IBDV live vaccine was inoculated into 14-day-old commercial chickens. The protection criteria meant that chickens with BF lesion scores of 0–4 were considered protected histopathologically. C, the control group; III, SPF chickens nonvaccinated with rMDV, but infected with vIBDV CJ801; NC, commercial chickens nonvaccinated with rMDV and BJ836, but infected with vIBDV CJ801. a Indicates significant difference from the unvaccinated chickens of bursal/body weight at p > 0.05. b Indicates significant difference from the unvaccinated chickens of bursal/body weight at p < 0.05.

and commercial chickens were used. The results were summarized in Table 1. In SPF chickens, the gross BF lesions and histopathologic BF lesion scores indicated that none of the groups were fully protected against the virulent IBDV challenge. The percentage of protected chickens vaccinated with 104 PFU of rMDV (73%) was higher than that of chickens vaccinated with 103 PFU of rMDV (53%). By 5 days post challenge, the unvaccinated and unchallenged control group remained healthy and showed no gross bursal lesions. In contrast, onset of disease or death occurred in all of the unvaccinated but challenged chickens with a mortality rate of 60%. In commercial chickens, more than 87% of chickens vaccinated with rMDV and 95% chickens vaccinated with attenuated live vaccine were protected against virulent IBDV. The onset of the disease was observed in 1 of 15 chickens vaccinated with rMDV, and the chickens had 13% gross lesions. Onset of disease or death occurred in the unvaccinated but challenged chickens and the mortality rate was approximately 20%. 3.5. Detection of antibody titers of IBDV The serum samples were examined by ELISA for IBDV antibody titers from weeks 0 to 5 post-immunization. In SPF chickens, the IBDV antibody titers in chickens vaccinated with rMDV increased from weeks 0 to 5 post-immunization, but titers were very low. Five weeks following vaccination, the antibody titers of chickens vaccinated with 103 PFU and 104 PFU were 1:356 and 1:498,

respectively. All the commercial chickens contained high levels of maternal antibody up to the age of 1 week. In the control and unvaccinated groups, the antibody titers dropped rapidly from 1 to 5 weeks. At 4 weeks, the antibody titer was only 1:35. The antibody titers of chickens vaccinated with attenuated IBD vaccine was lower than that of the rMDV group 1 week after vaccination but increased from week 4 to week 5. At 5 weeks, antibody titers up to 1:986 were observed. Antibody titers of chickens immunized with rMDV decreased slowly before week 2 and then gradually increased slowly from weeks 2 to 5. At 5 weeks of age, the antibody titer was at 1:574 which was significantly lower than those vaccinated with live vaccine (p < 0.05; data not shown). 3.6. IBDV neutralizing antibodies In SPF chickens, following vaccination with 103 PFU of rMDV, chickens developed low titer-neutralizing antibodies before challenge (titer 4–16), and chickens in the 104 PFU group also developed low titer-neutralizing antibodies (titer 16–32). In commercial chickens, all chickens immunized with rMDV developed slightly higher titer-neutralizing antibodies (titer 32–64). Chickens vaccinated with live attenuated vaccine developed even higher antibodies (titer ≥ 64). Neutralizing antibodies were not demonstrable in challenged control (CC) or unvaccinated chickens (titer < 2). The neutralizing antibody titer of IBDV was low prior to challenge in protected chickens receiving rMDV, suggesting that a small amount

Fig. 6. Pathological examination of BFs in SPF chickens (A–D) and commercial chickens (E–H). (A and E) Bursa from the control group. (B and C) Depletion of bursal lymphocytes in folliculus lymphaticus from the rMDV (104 PFU and 103 PFU) vaccinated group; bursal lymphocytes were severely depleted and “satellite-like vacuity” in the 103 PFU rMDV vaccinated chickens. (D and H) Degeneration and necrosis of lymphocytes in the medullary area of bursal follicles in vIBDV infected chickens, lymphocytes missing and connective tissues among lymphoid nodules dissolved and vanished. (F and G) Slight similar changes observed in the bursa of 104 PFU rMDV and BJ836 vaccinated chickens (20×).

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of antibody against VP2 was sufficient to protect chickens against clinical IBD, corresponding to a previous study [12]. 3.7. Histopathological examination of BFs in SPF and commercial chickens The health and behavior of chickens in groups II and III as well as the control group were consistently normal throughout the experiment. Chickens showed no symptoms consistent with depression, decreased appetite, diarrhea, dehydration and behavioral changes, such as fluffing plumage. Gross pathological findings showed light hemorrhage and swelling in the bursa of Fabricius in SPF chickens. However, gross examination and histopathological examination showed that the BF did not contain any abnormalities (Fig. 6). 4. Discussion Since attenuated MDV1 vaccine has the closest antigenic similarity to the vvMDV1, the MDV1 vaccine appears to be the best choice for prevention of vvMDV infection. It was shown that rMDV expressing NDV conferred full protection against vvMDV challenge [29]. The rMDV was constructed to express the IBDV-VP2 antigen, which provided 100% protection against vvMDV [12]. In the present study, the growth curve of the rMDV was similar to that of MDV in infected CEFs with blue plaques observed over 15 passages. The results demonstrated that the marker gene and host protective antigen were stably integrated into the predicted site of the MDV genome by homologous recombination. This suggests that rMDV might be effective as a MD vaccine, although the efficacy of rMDV against vvMDV has not been evaluated in this study. IBDV is a pathogen of major economic importance to the poultry industry worldwide. Maternal antibodies protect chickens up to the age of 3 weeks, but reduce their immune response to active immunization. Our results showed that the rMDV vaccine not only protected mature birds at the age of 5 weeks, but avoided maternal antibodies when administered to chickens that were 1-day old. The MD live vaccine viruses are known to persistently infect chickens despite the presence of neutralizing antibodies and induce a high titer of antibody against MDV. The recombinant MDV expressing the NDV F gene demonstrated a good vaccine efficacy in SPF chickens without maternal antibodies. Vaccine efficacy was decreased in commercial chickens with maternal antibodies [13]. The previous MDV-based polyvalent vaccines used heterologous promoters, such as the SV40 late promoter and the Rous sarcoma virus long terminal repeat, to express NDV antigens. These promoters are known to show very strong activity, resulting in high expression levels of NDV antigens in chickens given the vaccines. Another study showed that an MDV1 vector expressing NDV F glycoprotein under the control of a gB late promoter at the US10 site, was more efficacious than a rMDV employing the SV40 late promoter, although the gB promoter was weaker than the SV40 promoter in CEFs [29]. Another group reported that construction of rHVT expressing the IBDV-VP2 antigen under the control of a Pec promoter, conferred full protection against vvIBDV challenge [36]. The Pec promoter consists of a CMV enhancer and a ␤-actin promoter and has exhibited promoter activity in CEFs approximately three times stronger than that of the CMV promoter. As a result, the rHVT-pecVP2 induced high-level antibody titers to IBDV-VP2, approximately 10 times higher than baseline levels. This protection persisted for the lifetime of the SPF chickens. There are several differences between rMDV-US10P (F) and rHVT-pecVP2, such as vector strains, gene sites and promoters. More importantly, the IBDV-VP2 antigen accumulates in the cytoplasm, whereas NDV F glycoprotein is expressed on the cell surface. We speculate that the IBDV-VP2 antigen accumulated in the cytoplasm but NDV F protein was expressed on the cell sur-

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face, and that may be the major reason to explain the different results. rMDV expressing IBDV-VP2 infects chickens persistently from cell to cell similar to normal MDV. The VP2 antigen accumulates in the cytoplasm, therefore the antigen can escape from the host immune system and establish a persistent infection in chickens. Continuous stimulation of host immune systems by expressed antigens was demonstrated by findings that immune responses to IBDV-VP2 increased for 16 weeks in chickens after vaccination with rHVT-pecVP2. The results of our study correspond with these findings [36]. The antibody titers against IBDV increased gradually before challenge in SPF chickens. The reason for higher protection in commercial chickens with maternal antibodies compared with SPF chickens is not known. However, this difference may be attributed to chickens being maintained in different environments. Commercial chickens were maintained in an environment containing a variety of antigens after hatching. All these antigens may continuously stimulate host immune systems and boost cellular immunity that plays an important role in commercial chickens vaccinated with rMDV against vIBDV. Our study also indicated that the mobility and mortality of the challenged control group was much higher in SPF chickens than in commercial chickens. In summary, our data demonstrated the protective efficacy of the IBDV-VP2 antigen expressed by an MDV vector; and that this protein has the different protective effects in SPF and commercial chickens against vIBDV infection. The present results showed that the expressed VP2 protein demonstrates better protection in commercial chickens and only a limited effect in SPF chickens. Compared with the commercial live vaccine, rMDV expressing the IBDV VP2 gene was much safer and more stable than the attenuated live vaccine. It does not cause side effects in the BF and produce virulent IBDV. In addition, the efficacy of rMDV against virulent IBDV in commercial chickens (87%) is high enough for it to be considered competitive with the attenuated live vaccine (95%). Acknowledgements This work was supported by the National Natural Science Foundation of China (Grant. 30440011). We thank Dr. Kenji Tsukamoto for providing the plasmid pUS2 and Dr. Xiaoling Chen for her advice and assistance in the preparation of this manuscript. References [1] Dobos P, Hill BJ, Hallett R, Kells DT, Becht H, Teninges D. Biophysichal and biochemical characterization of five animal viruses with bisegmented doublestranded RNA genomes. J Virol 1979;32(2):593–605. [2] Becht H, Muller H. Infectious bursal disease: B cell-dependent immunodeficiency syndrome in chickens. Behring Inst Mitt 1991;68:217–25. [3] Mundt E, Beyer J, Müller H. Identification of a novel viral protein in infectious bursal disease virus-infected cells. J Gen Virol 1995;176:437–43. [4] Bayliss CD, Spies U, Shaw K, Papageorgiou A, Müller H, Boursnell MEG. A comparison of the sequences of segment A of four infectious bursal disease virus strains and identification of a variable region in VP2. J Gen Virol 1990;71:1303–12. [5] Becht H, Müller H, Müller HK. Comparative studies on structural and antigenic properties of two serotypes of infectious bursal disease virus. J Gen Virol 1998;69:631–40. [6] Rong J, Cheng TP, Liu XN, Jiang TZ, Gu H, Zou GL. Development of recombinant VP2 vaccine for the prevention of infectious bursal disease of chickens. Vaccine 2005;23(40):4844–51. [7] Rautenschlein S, Kraeme C, Vanmarcke J, Montiel E. Protective efficacy of intermediate and intermediate plus infectious bursal disease virus (IBDV) vaccines against very virulent IBDV in commercial broilers. Avian Dis 2005;49(2): 231–7. [8] Giambrone JJ, Closser J. Efficacy of live vaccines against serologic subtypes of infectious bursal disease virus. Avian Dis 1990;34:7–11. [9] Snyder DB. Changes in the field status of infectious bursal disease virus. Avian Pathol 1990;19(3):419–23. [10] Pitcovski J, Gutter B, Gallilib G, Goldwaya M, Perelmanb B, Grossa G, et al. Development and large-scale use of recombinant VP2 vaccine for the prevention of infectious bursal disease of chickens. Vaccine 2003;21:4736–43. [11] Tsukamotoa K, Satob T, Saitob S, Tanimuraa N, Hamazakia N, Masea M, et al. Dual-viral vector approach induced strong and long-lasting protec-

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