Arch Virol (2011) 156:22412250 DOI 10.

1007/s00705-011-1127-4

ORIGINAL ARTICLE

Infectious bursal disease DNA vaccination conferring protection by delayed appearance and rapid clearance of invading viruses
Yung-Yi Chen Ming Kun Hsieh Chun-Yu Tung Ching Ching Wu Tsang Long Lin

Received: 10 June 2011 / Accepted: 17 September 2011 / Published online: 9 October 2011 Springer-Verlag 2011

Abstract The present study was undertaken to determine the kinetics of viral load and immune response in protection against infectious bursal disease virus (IBDV) by DNA vaccination. Chickens were DNA-vaccinated and challenged with IBDV one week after the third vaccination. Tissues were collected at 12 hours postinfection (HPI), 1 day postinfection (DPI), 3, 5, 7 and 10 DPI. The vaccinated chickens had less viral RNA, with delayed appearance and shorter duration in the bursa of Fabricius, spleen, and cecal tonsil than the challenged control chickens. Their ELISA and neutralizing antibody titers were decreased at 12 HPI and signicantly lower (P \ 0.05) than those in the challenged control chickens at later time points. Their spleen IFNc expression was up-regulated compared to that in the DNA-vaccinated chickens without IBDV challenge. These results indicate that DNA vaccination confers protection against IBDV challenge by delayed appearance and rapid clearance of the invading viruses.

Introduction Infectious bursal disease (IBD) virus (IBDV) infects chickens and belongs to the family Birnaviridae, genus
Y.-Y. Chen M. K. Hsieh C.-Y. Tung C. C. Wu T. L. Lin (&) Department of Comparative Pathobiology, School of Veterinary Medicine, Purdue University, 406 South University Street, West Lafayette, Indiana 47907, USA e-mail: tllin@purdue.edu Present Address: M. K. Hsieh Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, Taichung 40227, Taiwan

Avibirnavirus. Infection outcomes can be lethal, clinically diseased, or immunosuppressed, depending on the host and virus virulence interactions [2], but all lead to atrophy of the viral target organ, the bursa of Fabricius. The form of acute disease is most prominent in the age between 3-6 weeks [2, 19]. Although IBDV persists in chickens for a short period of time, the lesions in the bursa can last for at least 10 weeks [34], resulting in immunosuppression in infected chickens. The suppressed immune status can increase susceptibility to secondary bacterial or viral infection [28] and reduce the immune response to vaccination against Newcastle disease, resulting in signicant economic loss in the poultry industry [2, 23, 30]. Furthermore, there is growing evidence that IBDV-infected chickens are better vessels than healthy chickens in adaptation of water fowl avian inuenza virus (AIV) in domestic poultry, a process that can lead to pathogenic AIV generation [26]. Thus, prevention of IBD by vaccination is critical to poultry health and well-being. IBDV has non-enveloped, icosahedral particles (T = 13). The genome contains two segments of doublestranded RNAs, which encode ve mature proteins. Segment A (3.3 kb) has two partially overlapping open reading frames (ORFs). The small ORF encodes the nonstructural protein, viral protein (VP) 5 (17 kDa), which may play a role in virus release and virulence [22, 35]. The large ORF encodes the polyprotein VP243 (109 kDa) which is co-translationally processed through VP4 (28 kDa), the viral protease, to generate pVP2, VP4 and VP3 [17]. pVP2 (48 kDa) is further processed at the C-terminus by VP4 and its autoproteolytic activity to generated three small peptides (7-11 amino acids) and one large peptide (46 amino acids), respectively [8, 31]. These four peptides are found to be associated with viral particles [8]. The largest peptide, pep46, is able to permeate biological membranes and may

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play a role in the cell entry process [6, 11]. VP2 (37 kDa) forms homotrimers and is the only component of the viral capsid [7]. It is also the only viral protein that induces virus-neutralizing antibodies in the host [10]. However, when expressing VP2 or pVP2 alone, subviral particles (T = 1) and hexagon tubules, respectively, are formed instead of virus-like particles (VLPs) [3]. Correct capsid assembly is associated with the interaction between the C-termini of pVP2 and VP3 and also pVP2 maturation to VP2 [16, 24]. An IBD DNA vaccine coding for the polyprotein VP243 has been shown to confer protection of chickens against IBDV challenge [4, 5, 13, 15]. Unlike conventional live attenuated or subunit vaccines, low or undetectable ELISA antibody titers to IBDV were detected in chickens protected by DNA vaccination prior to and after virus challenge [4, 5]. In addition, the chickens protected by DNA vaccination had intact bursae of Fabricius with no visible gross or microscopic lesions, and no detectable IBDV antigens were found by immunouorescent antibody assay (IFA) at 10 days post-challenge [4, 5, 13]. These ndings raise questions with regard to the mechanism by which the chickens are protected against IBD by DNA vaccination. Thus, the purpose of the present study was to elucidate the mechanism by which protection against IBDV challenge is conferred by DNA vaccination, by determining the kinetics of viral load in the bursa, spleen, cecal tonsil and peripheral blood mononuclear cells (PBMC) of vaccinated chickens. Antibody titers to IBDV, CD4? and CD8? T cells in the bursae, and IFNc and IL-4 mRNA expression in the spleen were also assessed as immune responses to DNA vaccination followed by IBDV challenge.

DNA vaccination and virus challenge One hundred eighteen hatched chicks were randomly assigned to four groups (29 or 30 chicks per group). The four groups were as follows: unvaccinated, unchallenged negative control group (NC), unvaccinated, challenged control group (CC), DNA-vaccinated, unchallenged group (DNA-NC) and DNA-vaccinated, challenged group (DNACC). The DNA vaccine was constructed with pCR3.1 vector as described previously [4, 13]. For the two groups that received DNA vaccination, the vaccine and vaccination regimen were as described previously [4, 5, 13, 15]. Briey, one-day-old SPF chickens were injected intramuscularly three times at 1-week intervals with 400 lg of IBDV DNA vaccine containing the IBDV polyprotein gene (VP243 gene). Chickens in the challenged groups were inoculated orally with 0.2 ml of 4.1 9 103 EID50/ml embryo infective dose (EID50) of IBDV strain VE. Evaluation of protection After virus challenge, blood, bursa of Fabricius, spleen, and cecal tonsil were collected at 12 hours postinfection (HPI), 1 day post-infection (DPI), 3 DPI, 5 DPI, 7 DPI and 10 DPI. Protection was evaluated at each time point as described previously [4, 5, 1315]. Briey, chickens and bursae were weighed, and the bursal/body weight ratio (B/B ratio) was calculated as (bursal weight)/(body weight) 9 1000. Bursae were also subjected to gross pathology examination, and the degree of bursal atrophy was determined. Bursal lesions were scored from 1 to 4 based on the severity of bursal atrophy (1: 0-10%; 2: 10-30%; 3: 30-70%; 4: more than 70%) compared to negative controls. Protection against IBDV challenge was dened by a gross lesion score of 1 and a B/B ratio of no less than two standard deviations (SD) below the average ratio of the NC group. Detection of IBDV in bursa and spleen by immunouorescent antibody assay (IFA) Bursa of Fabricius and spleen from each chicken were collected at each time point, chilled in dry ice, and stored at -80C until use. An immunouorescent antibody assay was performed as described previously [4, 5, 13, 14]. Briey, frozen sections of bursa and spleen collected from six different time points were cut into 6-lm-thick sections, transferred to glass slides and xed with acetone at room temperature for 5 minutes. After xation, sections were incubated with anti-VP2 monoclonal antibody R63 (ATCC, Manassas, VA, USA) followed by goat anti-mouse IgG secondary antibody labeled with uorescein isothiocyanate (FITC) (KPL, Gaithersburg, MD, USA).

Materials and methods Chickens Specic-pathogen-free (SPF) embryonated chicken eggs were obtained from Charles River Laboratories (North Franklin, CT, USA), and the chicks hatched out after 21 days of incubation. One-day-old SPF chickens were kept in Horsfall-Bauer isolators with food and water ad libitum. The protocol used in the animal study was approved by the Purdue University Animal Care and Use Committee. Virus Infectious bursal disease virus strain variant E (VE) was used as the template for generation of the DNA vaccine and as the challenge virus in the animal study.

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Detection and quantitation of IBDV RNA in various tissues by real-time RT-PCR Chicken tissues, including bursa, spleen and cecal tonsil, were collected at each time point and preserved in RNAlater (QIAGEN, Valencia, CA, USA). Peripheral blood mononuclear cells (PBMC) were puried as described previously [14] and pelleted and stored in RNAlater until use. RNA from each tissue was extracted using an RNeasy Mini Kit (QIAGEN, Valencia, CA, USA) following the manufacturers instructions. The RNA concentration of each sample was determined using a GeneQuantTM 1300 Spectrophotometer (GE Healthcare, Piscataway, NJ, USA). Viral cDNA synthesis was performed from 2-2.5 lg of RNA of each sample with SuperScript III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA) as described in the manufacturers instructions with 200 ng of random primer (N6) per reaction (Sigma-Genosys, The Woodlands, Texas, USA). The viral cDNA was amplied by real-time PCR with specic primers and a probe for the variant IBDV strain and normalized using cDNA of 28S rRNA as described previously [25], with the modication that Platinum Quantitative PCR Supermix-UDG (Invitrogen, Carlsbad, CA, USA) was used in a Rotor Gene thermal cycling system (RG-3000, Corbett Research, Mortlake, Australia). The PCR reaction mixture was set up to contain 200 nM forward and reverse primers, 100 nM probe, a 29 concentration of Platinum Taq DNA polymerase, Tris-HCl, KCl, 6 mM MgCl2, 400 lM dGTP, 400 lM dATP, 400 lM dCTP, 800 lM dGTP, uracil DNA glycosylase and stabilizers. The cycle prole was as follows: 50C hold for 2 min; 95C hold for 2 min; 45 cycles at 95C for 20 sec, and 60C for 60 sec for virus detection; and 40 cycles at 95C for 20 sec and 59C for 60 sec for the 28S RNA internal control. The forward primer, reverse primer and dual-labeled probe sequences were as follows: VE2026-F (50 -CCCATACCTC CTATTGTGGG-30 ), VE2161-R (50 -GTGGCGAGCTTGG TGCTTCT-30 ), VE probe (FAM50 -CAACGCTTGTGGC GAGATTGAGAAAATAAG-30 TAMRA), 28S-F (50 -GG CGAAGCCAGAGGAAACT-30 ), 28S-R (50 -GACGACCG ATTTGCACTGC-30 ), and 28S probe (FAM50 -AGGACC GCTACGGACCTCCACCA-30 TAMRA). The viral RNA concentration (copy number/ll) was calculated from a standard curve, which was generated by serial 10-fold dilution of a plasmid containing the IBDV VP243 gene and normalized using 28S rRNA as an internal control, which was also calculated from a standard curve generated by serial 10-fold dilution of a plasmid containing a chicken 28S rRNA gene. The viral RNA concentration for each group is presented as mean standard deviation (SD).

Antibody titers to IBDV by enzyme-linked immunosorbent assay (ELISA) Blood from each chicken was drawn through the wing vein before challenge and through the heart at each time point after challenge. The serum antibody titer to IBDV was determined using a commercial antibody-capture enzymelinked immunosorbent assay (ELISA) kit: FlockChek Infectious Bursal Disease Antibody Test Kit (IDEXX, Westbrook, ME, USA). Antibody titers were calculated from S/P ratios following the instructions provided by the manufacturer. Neutralizing antibody titers to IBDV The virus-neutralizing (VN) antibody titer in the serum was detected by a method described previously [13, 14]. Briey, serum collected at each time point was serially diluted 4-fold in 96-well plates and incubated with 1000 plaque-forming units of IBDV strain PBG98 in the presence of chicken embryo broblasts (CEF) for 5 days. To read the results, CEFs were xed with 10% buffered formalin for 5 minutes and stained with 1% crystal violet for 3 minutes. The VN antibody titer was dened as the highest serum dilution at which CEFs were completely intact, and titers are presented as log4 values. Detection of CD4? and CD8? T cells in bursa by immunohistochemistry (IHC) The bursa of Fabricius from each chicken was collected, frozen, cut into 4-lm-thick sections, and xed in acetone at room temperature for 5 minutes. Before staining, the sections were re-hydrated in Tris-buffered saline Tween-20 (TBST) for 10 minutes. Immunohistochemistry (IHC) staining was done with the Super SensitiveTM PolymerHRP Detection System (BioGenex Laboratories, San Ramon, CA, USA) according to the manufacturers instructions. Briey, endogenous peroxidase activity was blocked using Peroxide Block (BioGenex) for 10 minutes at room temperature, and the samples were washed in TBST for three 5-minute treatments. This was followed by incubation with Power BlockTM Reagent (BioGenex) for 10 minutes at room temperature without washing afterwards. The primary antibody was 1:100-PBS-diluted mouse anti-chicken CD4 antibody or mouse anti-chicken CD8 antibody (Southern Biotechnology, Birmingham, AL, USA), and immunostaining was done in a humidied chamber at 4C overnight. After being washed three times in TBST for 5 minutes, Super EnhancerTM reagent (BioGenex) was applied to the sections in humidied chamber for 20 minutes at room temperature and rinsed with TBST. The

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sections were incubated with Poly HRP Reagent (BioGenex) in a humidied chamber for 30 minutes at room temperature and rinsed in TBST. After the sections were incubated with AEC solution (BioGenex) for 5 minutes, Mayers hematoxylin (BioGenex) counterstaining was applied for 5 minutes. The sections were mounted with SuperMount (BioGenex), covered with cover slips and examined under a light microscope (BX40, Olympus Corporation of the Americas, Center Valley, PA, USA). Positive CD4?- or CD8?-staining cells in the bursa were counted at 4009 magnication. Eight elds per bursal section were counted, and the average count was calculated and presented as mean SD for each group [27]. Chicken IFNc and IL-4 mRNA expression in spleen by real-time RT-PCR Total RNA extraction and cDNA synthesis from spleen were done as described previously with an additional treatment with 2 units of RNase-free DNase per reaction at 37C for one hour (Promega, Madison, WI, USA), followed by inactivation at 72C for 15 minutes before cDNA synthesis. Chicken spleen IFNc, IL-4 and GADPH mRNA expression levels were quantied by real-time RT-PCR using the method described [1, 18] with the modied PCR reaction mixture described above for IBDV RNA detection. The cycle prole was as follows: hold at 50C for 2 min and 95C for 2 min, followed by 40 cycles at 95C for 20 sec and 59C for 60 sec. The forward primer, reverse primer and dual-labeled probe sequences are as follows: IFNc-F (50 -GTGAAGAAGGTGAAAGATATCA TGGA-30 ), IFNc-R (50 -GCTTTGCGCTGGATTCTCA-30 ), IFNc probe (FAM-50 -TGGCCAAGCTCCCGATGAACGA -30 TAMRA), IL-4 -F (50 -AACATGCGTCAGCTCCTGA AT-30 ), IL-4-R (50 -TCTGCTAGGAACTTCTCCATTGA A-30 ), IL-4 probe (FAM50 -AGCAGCACCTCCCTCAAG GCACC-30 TAMRA), GAPDH-F (50 -CCCCAATGTCT CTGTTGTTGAC-30 ), GAPDH-R (50 -CAGCCTTCACTA CCCTCTTGAT-30 ), GAPDH probe (FAM50 -CTTGG CTGGTTTCTCC-30 TARMA). The IFNc and IL-4 mRNA levels in each group were corrected using the Ct value obtained for GAPDH mRNA as an internal control by the -DDCt value method [12] and presented as the average fold change relative to the NC group (mean SD). Statistical analysis All data are presented as mean standard deviation (SD) for each group and were analyzed using SPSS 18 (IBM, Armonk, NY, USA) by one-way ANOVA followed by Tukeys test. When there were only two groups for comparison, Students t test was applied. Statistical signicance was set at p \ 0.05.

Results DNA vaccine protection efcacy against IBDV challenge Severe bursal atrophy was observed after challenge with IBDV strain VE in unvaccinated chickens (CC group). Protection against IBDV challenge was assessed by gross lesion score and B/B ratio value. Therefore, the higher lesion score and lower B/B ratio indicate bursal atrophy after virus challenge. The bursal lesion scores for the CC group gradually increased, and the B/B ratios decreased from 5 to 10 DPI, indicating bursal atrophy (Table 1). At 10 DPI, unvaccinated chickens in the CC group showed the most severe lesions (Table 1). Conversely, DNA-vaccinated chickens challenged with IBDV strain VE (DNA-CC group) showed no difference in lesion score and B/B ratio when compared to groups without virus challenge (NC and DNA-NC groups) at six time points (Table 1), indicating protection against virus challenge by DNA vaccination. IBDV antigen detection by IFA in bursa and spleen Monoclonal antibodies against the capsid protein VP2 were used to detect IBDV antigen by IFA. There was no detectable viral antigen in the bursae of chickens in the DNA-CC group at six time points, while 80 to 100% of the chickens in the CC group showed positive green immunouorescent staining in the bursae from 3 to 10 DPI (Table 2). There were no detectable IBDV antigens in the bursae of chickens in the DNA-NC and NC groups at any of the six time points (Table 2). There were no detectable IBDV antigens in the spleens of chickens in the DNA-CC, DNA-NC and NC groups at any of the six time points (Table 2), but for unprotected chickens in the CC group, IBDV viral antigens were detected in the spleens at 3 and 5 DPI, with a 60% positive rate (Table 2). IBDV viral RNA detection and quantication by real-time RT-PCR in bursa, spleen, cecal tonsil and PBMC The results of IBDV viral RNA detection and quantication in bursa, spleen, cecal tonsil and PBMC from each group are summarized in Table 3. Chickens in the DNA-CC group had detectable IBDV viral RNA in the bursae only at 3 and 5 DPI, while those in the CC group had detectable viral RNA starting from 24 HPI until 10 DPI. At 3 and 5 DPI, the viral RNA level in the bursae of chickens in the DNA-CC group was lower than those in the CC group. IBDV viral RNA was detected in the spleens and cecal tonsils of chickens in the DNA-CC group at 5 and 7 DPI, but viral RNA was not detected in PBMCs at any of the

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Table 1 Protection by DNA vaccination against infectious bursal disease virus (IBDV) challenge as determined by bursal gross lesion score and bursal/body weight (B/B) ratio Groupa Gross lesion scoreb NC DNA-NC DNA-CC CC B/B ratioc NC DNA-NC DNA-CC CC
a

12 HPId

24 HPI

3 DPId

5 DPI

7 DPI

10 DPI

1.0 0 1.0 0 1.0 0 1.0 0 6.1 1.2 6.9 0.6 6.5 0.9 7.5 2.0

1.0 0 1.0 0 1.0 0 1.0 0 7.2 2.1 6.9 1.4 7.5 0.9 6.8 1.9

1.0 0 1.0 0 1.0 0 1.0 0 6.0 0.5 6.0 0.9 6.7 2.1 6.4 1.1

1.0 0 1.0 0 1.0 0 2.6 0.6* 5.8 1.2 6.0 1.8 6.9 2.0 2.8 0.7*

1.0 0 1.0 0 1.0 0 3.8 0.5* 6.8 0.9 6.3 0.8 5.8 0.8 1.8 0.3*

1.0 0 1.0 0 1.0 0 4.0 0* 6.9 1.8 6.6 1.7 5.8 0.7 1.6 0.3*

* Indicates statistical difference among different groups (p \ 0.05) at each time point NC: negative control (without DNA vaccination and without challenge); DNA-NC: DNA vaccine control (with DNA vaccination but without challenge); DNA-CC: with DNA vaccination and with challenge; CC: challenge control (without DNA vaccination but with challenge)

The bursal gross lesion was scored from 1 to 4 based on the increasing severity of bursal atrophy (1: 0-10%; 2: 10-30%; 3: 30-70%; 4: more than 70% bursal size reduction) as compared to those in NC group The B/B ratio was calculated as (bursal weight)/(body weight) x 1000 and presented as the mean S.D. for each group HPI: hours postinfection; DPI: days postinfection

c d

Table 2 IBDV antigen in bursa and spleen detected by immunouorescence antibody assay (IFA)a Groupc No. positive/no. of chickensb 12 HPI Bursa NC DNA-NC DNA-CC CC Spleen NC DNA-NC DNA-CC CC
a b

24 HPI

3 DPI

5 DPI

7 DPI

10 DPI

0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5

0/5 0/4 0/5 0/5 0/5 0/4 0/5 0/5

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

0/5 0/5 0/4 5/5 0/5 0/5 0/4 3/5

0/5 0/5 0/5 4/5 0/5 0/5 0/5 0/5

0/5 0/5 0/5 4/5 0/5 0/5 0/5 0/5

Photomicrographs of IFA results are not shown. They were consistent with those of previous publications [1315]

Bursa and spleen were collected at each of 6 time points, frozen sectioned and subjected to IFA with a monoclonal antibody against IBDV VP2 protein. Results were presented as the number of chickens positive by IBDV staining/total number of chickens examined in the group The denition of each group is given in Table 1

six time points (Table 3). Viral RNA was detected at each of the ve time points from 24 HPI to 10 DPI in the chickens of the CC group (Table 3). The level of viral RNA was signicantly lower (p \ 0.05) in the DNA-CC group at 5 and 7 DPI in the spleens and at 5 DPI in the cecal tonsils when compared to that in the CC group (Table 3). There was no detectable viral RNA in the bursae, spleens, cecal tonsils and PBMCs of chickens in the DNA-NC and NC groups at any of the six time points (data not shown).

ELISA and VN antibody titers to IBDV There was no signicant difference (p [ 0.05) in ELISA antibody titer to IBDV between the DNA-CC and DNANC groups (Table 4). Their titers remained low and were less than 600 in ELISA for both DNA-vaccinated groups. On the other hand, the VN antibody titer increased from 12 HPI onward and maintained within log43-5 throughout the six time points. Lower ELISA titers and signicantly lower (p \ 0.05) VN antibody titers were observed at 12 HPI in

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2246 Table 3 Quantication of IBDV RNA in bursa, spleen, CT and PBMC by real-time RT-PCRa, Groupe,
f b

Y.-Y. Chen et al.

IBDV RNA concentration (copy number/ll)c, 12 HPI 24 HPI 3 DPI

5 DPI

7 DPI

10 DPI

Bursa DNA-CC CC Spleen DNA-CC CC CT DNA-CC CC PBMC DNA-CC CC

a

0 0 0 0 0 0 0 0

0 (5.6 7.6) 9 104 0 (6.6 6.6) 9 10 0 (3.1 6.8) 9 10 0 (3.6 1.1) 9 104
4 4

(2.6 5.8) 9 103 (3.0 3.5) 9 107 0 (7.5 5.9) 9 10 * 0 (1.6 1.9) 9 10 0 (1.2 0.3) 9 105
6 5

(1.1 1.3) 9 106 (3.1 5.8) 9 107 (1.2 1.4) 9 103 (2.8 1.5) 9 10 * (1.4 2.5) 9 104 (2.2 1.2) 9 10 * 0 (2.7 0.4) 9 104*
5 5

0 (9.3 9.3) 9 106 (5.2 11.6) 9 103 (1.5 1.0) 9 105* (1.2 2.7) 9 104 (6.2 4.5) 9 10 0 (3.0 0.8) 9 104
4

0 (6.4 3.7) 9 106* 0 (6.9 6.9) 9 104 0 (3.3 3.7) 9 104 0 (1.5 0.7) 9 104

* Indicates statistical difference among different groups (p \ 0.05) at each time point Total RNA was extracted using an RNeasy Mini Kit (QIAGEN, Valencia, CA,USA). Two-step RT-PCR was followed by Superscript III reverse transcriptase and platinum quantitative PCR supermix-UDG (Invitrogen, Carlsbad, CA, USA) in an RG-3000 real-time thermal cycling system (Corbett Research, Mortlake, Australia). The RNA concentration was normalized by 28S RNA in the same sample There was no detectable viral RNA for the NC and DNA-NC groups at the 6 time points (data not shown) 0 indicates no detectable IBDV RNA Viral RNA concentration (copy number/ll) is presented as meanSD for each group The denition of each group is given in Table 1 The number of samples was 4 or 5 per group at each time point. Please see the detailed information in Table 2

b c d e f

Table 4 ELISA and virus neutralizing antibody titer to IBDV Groupa Before 12 HPI 24 HPI 3 DPI 5 DPI 7 DPI 10 DPI

ELISA antibody titerb NC DNA-NC DNA-CC CC NC DNA-NC DNA-CC CC 1 0* 141 142* 242 159 1 0* 0* 2.0 1.87* 1.4 1.34* 0* 1 0* 371 493* 158 72* 1 0*
c

1 0* 397 328* 458 490* 1 0* 0* 3.75 1.26 3.8 0.45 0*

1 0* 449 356 304 153* 1 0* 0* 3.4 0.55 3.6 0.55 0*

1 0* 229 113* 165 115* 1251 539 0* 3.0 0 3.25 0.5 4.2 0.45

1 0* 344 306* 586 307* 2429 580 0* 3.4 0.55 3.4 0.55 4.0 0

1 0* 217 86* 331 16* 3606 607 0* 3.4 0.55 3.2 0.45 5.0 0.71

Virus neutralizing (VN) antibody titer 0*

3.8 0.45 3.2 0.45 0*

* or or Indicates statistical difference among different groups (p \ 0.05) at each time point. When the groups have the same symbol in the superscript, there is no statistical difference among the groups (p [ 0.05)
a b

The denition of each group is given in Table 1

The ELISA titer was determined using an ELISA kit (IDEXX, Westbrook, ME, USA) and was calculated as log10titer = 1.09 (log10 S/P) ? 3.36
c

The unit of VN antibody titer is log4

the DNA-CC group than in the DNA-NC group, suggesting the consumption of the serum antibody by the virus. The chickens in the CC group started to show detectable ELISA and VN antibody titers beginning at 5 DPI, and their titers were signicantly higher (p \ 0.05) than those in the DNA-vaccinated groups at 5, 7 and 10 DPI by ELISA and at 5 and 10 DPI by VN (Table 4).

Chicken CD4- and CD8-positive T cells in bursa Chicken CD4? and CD8? T cells detected by IHC in bursae after challenge with IBDV strain VE are summarized in Table 5. For CD4? T cells, no signicant change (p [ 0.05) was observed in the bursae of chickens in the DNA-CC group throughout the entire experimental period,

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while signicantly more cells (p \ 0.05) inltrated the bursae in the CC group from 3 DPI to 10 DPI. The number peaked at 5 DPI (Table 5). The numbers of CD8? T cells in the bursae in the CC group also increased from 3 DPI to 10 DPI and peaked at 5 DPI (Table 5). Chicken IFNc and IL-4 mRNA expression in spleen by real-time RT-PCR Chicken IFNc mRNA levels in the spleens increased in both the DNA-NC and DNA-CC groups at 24 HPI and 3 DPI, but the level was higher in the DNA-CC group (Table 6). Chicken IFNc expression in the spleens of the CC group was signicantly higher (p \ 0.05) when compared to DNA-vaccinated chickens and reached the peak at 3 DPI (Table 6). On the other hand, the expression levels of chicken IL-4 in spleens were much lower in all experimental groups, and there was no signicant difference (p [ 0.05) in chicken IL-4 mRNA levels among the DNANC, DNA-CC and CC groups at 12 HPI and 10 DPI (Table 6).

Discussion The present study was carried out to elucidate how DNAvaccinated chickens with low ELISA antibody titers are protected against virus challenge. In addition to the 10 DPI time point, ve earlier time points were included in the study. DNA vaccination with a three-dose regimen proved to be effective in providing protection against IBDV
Table 5 Chicken T cell number in bursa of Fabricius by IHCa Groupb

challenge at six sequential time points, including 12 HPI, 24 HPI, 3 DPI, 5 DPI, 7 DPI and 10 DPI in the 10-day period. Compared to IFA, quantitative RT-PCR was more sensitive for IBDV detection in bursa and spleen. While no viral antigen was detected by IFA for the DNA-CC group at the six time points, viral RNA was detected by quantitative RT-PCR at 3 and 5 DPI in the bursae and 5 and 7 DPI in the spleens (Table 2, 3). The highest IBDV RNA load for the CC group was consistently seen at 3 DPI, while for the DNA-CC group, the peak of IBDV RNA load was at 5 DPI in the bursa, and bursae had the highest viral RNA levels in both the CC and DNA-CC groups. Detectable viral RNA in the DNA-CC group was delayed by one time point in the bursa and two time points in the spleen and cecal tonsil. These ndings conrmed that the bursa is the target and primary organ for IBDV replication and also indicated that IBDV can be distributed to and replicate in other immune tissues such as spleen and cecal tonsil. The delayed appearance and quick clearance of viral RNA in the bursae, spleens and cecal tonsils in the DNA-vaccinated chickens is likely due to a combination of DNA-vaccineinduced humoral and cellular immune responses, which neutralize extracellular viral particles and destroy virusinfected cells, respectively. Both the DNA-NC and DNA-CC groups showed low ELISA antibody titers before and after virus challenge in the present study (Table 4). The unvaccinated challenged chickens (CC group) started to show ELISA titers at 5 DPI, and the titers were signicantly higher than those in the DNA-vaccinated chickens at 5, 7 and 10 DPI. Such ndings are consistent with those from previous studies

Average number of CD4- or CD8-positive cells/eld (400x) 12 HPI 24 HPI 3 DPI 5 DPI 7 DPI 10 DPI

CD41 T cells NC DNA-NC DNA-CC CC CD81 T cells NC DNA-NC DNA-CC CC 23 6* 22 5* 21 4* 15 7* 22 4* 32 13* 21 3* 20 8* 16 3* 22 7* 29 6 116 9 25 5* 25 8* 22 7* 202 22 25 4* 23 2* 25 3* 188 14 23 2* 19 1* 24 2* 168 7 18 2* 12 4* 19 6* 16 5* 19 2* 24 4* 15 8* 16 6* 23 7* 26 5* 21 5* 93 34 25 4* 23 11* 24 5* 182 11 23 5* 21 5* 22 1* 165 6 28 2 18 2* 28 2 112 2

* or or Indicates statistical difference among different groups (p \ 0.05) at each time point. When the groups have the same symbol in the superscript, there is no statistical difference among the groups (p [ 0.05) a Bursal frozen sections were stained by immunohistochemistry (IHC) for chicken CD4- or CD8-positive T cells. Eight microscopic elds were examined for positive cell counts [27]. Data are presented as mean SD for each group
b

The denition of each group is given in Table 1

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2248 Table 6 Chicken IFNc and IL-4 mRNA expression levels in spleen by real-time RT-PCR Groupb,
e

Y.-Y. Chen et al.

Fold change relative to the NC groupa 12 HPI 24 HPI 3 DPI 5 DPI 7 DPI 10 DPI

IFNc DNA-NC DNA-CC CC IL-4 DNA-NC DNA-CC CC 0.74 0.25* 0.82 0.47* 1.02 0.04* 0.70c 0.91 1.70 1.82
c

1.34 0.83* 1.01 0.63* 0.90 0.71*

5.00 3.27* 7.31 2.16* 10.78 8.05*

5.47 2.28* 7.43 3.16* 18.08 8.43 NDd NDd 3.68 1.78

1.18 0.56* 1.93 1.50* 11.11 7.58 0.55 0.51 0.25c 1.10 0.24

0.45 0.20* 1.71 1.30* 2.31 1.26 0.38 0.12 1.92 0.84 NDd

0.85 0.44* 1.01 0.63* 3.31 0.84 0.95 0.40* 0.81 0.30* 1.25 1.04*

* or Indicates statistical difference among different groups (p \ 0.05) at each time point. When the groups have the same symbol in the superscript, there is no statistical difference among the groups (p [ 0.05)
a

The GADPH Ct value of each sample and the average Ct value of the NC group were used to calculate the cytokine expression level by the -DDCt method b The denition of each group is given in Table 1
c d e

Only one sample had a detectable Ct value for IL-4 in the group; thus, standard deviation cannot be calculated ND: No detectable Ct value for IL-4 in the whole group The number of samples was 4 or 5 per group at each time point. Please see the detailed information in Table 2

showing that chickens protected from IBDV challenge by DNA vaccination had a low or undetectable ELISA antibody titer against IBDV before and after challenge using the cutoff titer of 396 as dened by the ELISA kit [4, 5, 15]. On the other hand, the VN titer seems to be more representative than the ELISA titer since DNA-vaccinated chickens, both unchallenged (DNA-NC) and challenged (DNA-CC), showed a 12-fold increase in VN titer at 12 HPI and maintained high VN titers within the experimental period (Table 4), indicating that DNA vaccination can induce a high level of humoral immunity to IBDV. After virus challenge, decreased ELISA and VN antibody titers were observed in the DNA-CC group at 12 HPI when compared to those of the DNA-NC group (Table 4), suggesting that the consumption of antibodies in the serum by the invading IBDV contributed to the delayed appearance of viral RNA in the bursa, spleen and cecal tonsil. It also suggests that since IBDV could not be completely neutralized by antibodies induced by DNA vaccination, antigen-specic CD8? T cells may play a role in IBDV clearance and challenge protection. There are only a few T cells in the bursa of Fabricius in normal healthy chickens, and thus the inux of a large quantity of both CD4? and CD8? T cells into the bursa of Fabricius after IBDV infection is signicant [32, 33]. Not only T cells with anti-virus activities but also T cells with suppressive features were found in the viral target organ, the bursa of Fabricius [20, 21]. While most of the intrabursal T cells are believed to help eliminate the viral infection, suppressive T cells may be present to control extensive inammation, since CD4? CD25? T cells with

regulatory properties have been identied recently [29]. In the present study, large numbers of CD4? and CD8? T cells appeared in the bursae from 3 to 10 DPI in the unvaccinated challenged chickens (CC group) but not in the chickens protected by DNA vaccination (DNA-CC) (Table 5). The reasons could be as follows: (1) a small number of effector T cells is enough to control the infection; (2) antiviral T cells may arrive at the infection sites, destroy infected cells and leave the bursa within a very short time frame, between the time points we used in this experiment causing us not to observe it; and (3) most incoming IBDV had been neutralized by antibodies. Nevertheless, rapid viral clearance in the bursa is still considered to be helped by CD4? T cells and executed by antigen-specic CD8? T cells triggered by DNA vaccination. Th1/Th2 polarization has been found to be conserved among avian species and mammals to resolve intracellular infection and extracellular pathogens, respectively [9]. A Th1-biased response is characterized by production of IFNc, and a Th2 response is characterized by IL-4, IL-5, IL-10 and IL-13 release. Low or undetectable IL-4 and upregulated IFNc expression in the spleen suggest that both natural viral infection (CC group) and DNA vaccination (DNA-NC and DNA-CC groups) induced a Th1-biased immune response (Table 6). The up-regulation of IFNc mRNA in the spleen of DNA-NC chickens at 24 HPI and 3 DPI suggests the boosting effect from IBD DNA vaccination may take about 11 to 13 days to reactivate memory Th1 helper CD4? and cytotoxic CD8? T cells. When vaccinated chickens were challenged with IBDV (DNA-CC

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IBDV DNA vaccine protection

2249 virus, a non-enveloped virus, possesses a capsid-associated peptide that deforms and perforates biological membranes. J Biol Chem 282:2077420784 Giulietti A, Overbergh L, Valckx D, Decallonne B, Bouillon R, Mathieu C (2001) An overview of real-time quantitative PCR: applications to quantify cytokine gene expression. Methods 25:386401 Hsieh MK, Wu CC, Lin TL (2006) The effect of co-administration of DNA carrying chicken interferon-gamma gene on protection of chickens against infectious bursal disease by DNAmediated vaccination. Vaccine 24:69556965 Hsieh MK, Wu CC, Lin TL (2007) Priming with DNA vaccine and boosting with killed vaccine conferring protection of chickens against infectious bursal disease. Vaccine 25:54175427 Hsieh MK, Wu CC, Lin TL (2010) DNA-mediated vaccination conferring protection against infectious bursal disease in broiler chickens in the presence of maternal antibody. Vaccine 28:39363943 Irigoyen N, Garriga D, Navarro A, Verdaguer N, Rodriguez JF, Caston JR (2009) Autoproteolytic activity derived from the infectious bursal disease virus capsid protein. J Biol Chem 284:80648072 Jagadish MN, Staton VJ, Hudson PJ, Azad AA (1988) Birnavirus precursor polyprotein is processed in Escherichia coli by its own virus-encoded polypeptide. J Virol 62:10841087 Kaiser P, Rothwell L, Galyov EE, Barrow PA, Burnside J, Wigley P (2000) Differential cytokine expression in avian cells in response to invasion by Salmonella typhimurium, Salmonella enteritidis and Salmonella gallinarum. Microbiology 146(Pt 12): 32173226 Kibenge FS, Dhillon AS, Russell RG (1988) Biochemistry and immunology of infectious bursal disease virus. J Gen Virol 69(Pt 8):17571775 Kim IJ, Sharma JM (2000) IBDV-induced bursal T lymphocytes inhibit mitogenic response of normal splenocytes. Vet Immunol Immunopathol 74:4757 Kim IJ, You SK, Kim H, Yeh HY, Sharma JM (2000) Characteristics of bursal T lymphocytes induced by infectious bursal disease virus. J Virol 74:88848892 Lombardo E, Maraver A, Espinosa I, Fernandez-Arias A, Rodriguez JF (2000) VP5, the nonstructural polypeptide of infectious bursal disease virus, accumulates within the host plasma membrane and induces cell lysis. Virology 277:345357 Muller H, Islam MR, Raue R (2003) Research on infectious bursal diseasethe past, the present and the future. Vet Microbiol 97:153165 Ona A, Luque D, Abaitua F, Maraver A, Caston JR, Rodrguez JF (2004) The C-terminal domain of the pVP2 precursor is essential for the interaction between VP2 and VP3, the capsid polypeptides of infectious bursal disease virus. Virology 322:135142 Peters MA, Lin TL, Wu CC (2005) Real-time RT-PCR differentiation and quantitation of infectious bursal disease virus strains using dual-labeled uorescent probes. J Virol Methods 127:8795 Ramirez-Nieto G, Shivaprasad HL, Kim CH, Lillehoj HS, Song H, Osorio IG, Perez DR (2010) Adaptation of a mallard H5N2 low pathogenicity inuenza virus in chickens with prior history of infection with infectious bursal disease virus. Avian Dis 54:513521 Rautenschlein S, Yeh HY, Njenga MK, Sharma JM (2002) Role of intrabursal T cells in infectious bursal disease virus (IBDV) infection: T cells promote viral clearance but delay follicular recovery. Arch Virol 147:285304 Rosenberger JK, Cloud SS (1989) The effects of age, route of exposure, and coinfection with infectious bursal disease virus on the pathogenicity and transmissibility of chicken anemia agent (CAA). Avian Dis 33:753759

group), the spleen IFNc mRNA expression level had increased only slightly above the level in the DNA-NC group, suggesting that the invading IBDV was neutralized by the antibody and/or a small number of memory CD8? T cells was activated to clear virus infection. This corresponds to the nding that the bursae were not inltrated by large number of T cells. In summary, the ndings from the present study indicate that although DNA-mediated vaccination in chickens does not completely inhibit IBDV replication in bursae, spleens and cecal tonsils, it provides protection against IBDV challenge by delayed occurrence and rapid clearance of infecting viruses in these tissues.
Acknowledgments We are grateful for the excellent animal care provided for the experimental chickens by the Purdue University veterinary laboratory animal care staff.

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References
1. Avery S, Rothwell L, Degen WD, Schijns VE, Young J, Kaufman J, Kaiser P (2004) Characterization of the rst nonmammalian T2 cytokine gene cluster: the cluster contains functional single-copy genes for IL-3, IL-4, IL-13, and GM-CSF, a gene for IL-5 that appears to be a pseudogene, and a gene encoding another cytokine like transcript, KK34. J Interferon Cytokine Res 24:600610 2. Balamurugan V, Kataria JM (2006) Economically important nononcogenic immunosuppressive viral diseases of chickencurrent status. Vet Res Commun 30:541566 3. Caston JR, Martinez-Torrecuadrada JL, Maraver A, Lombardo E, Rodriguez JF, Casal JI, Carrascosa JL (2001) C terminus of infectious bursal disease virus major capsid protein VP2 is involved in denition of the T number for capsid assembly. J Virol 75:1081510828 4. Chang HC, Lin TL, Wu CC (2001) DNA-mediated vaccination against infectious bursal disease in chickens. Vaccine 20:328 335 5. Chang HC, Lin TL, Wu CC (2003) DNA vaccination with plasmids containing various fragments of large segment genome of infectious bursal disease virus. Vaccine 21:507513 6. Chevalier C, Galloux M, Pous J, Henry C, Denis J, Da Costa B, Navaza J, Lepault J, Delmas B (2005) Structural peptides of a nonenveloped virus are involved in assembly and membrane translocation. J Virol 79:1225312263 7. Coulibaly F, Chevalier C, Gutsche I, Pous J, Navaza J, Bressanelli S, Delmas B, Rey FA (2005) The birnavirus crystal structure reveals structural relationships among icosahedral viruses. Cell 120:761772 8. Da Costa B, Chevalier C, Henry C, Huet JC, Petit S, Lepault J, Boot H, Delmas B (2002) The capsid of infectious bursal disease virus contains several small peptides arising from the maturation process of pVP2. J Virol 76:23932402 9. Degen WG, Daal N, Rothwell L, Kaiser P, Schijns VE (2005) Th1/Th2 polarization by viral and helminth infection in birds. Vet Microbiol 105:163167 10. Fahey KJ, Erny K, Crooks J (1989) A conformational immunogen on VP-2 of infectious bursal disease virus that induces virusneutralizing antibodies that passively protect chickens. J Gen Virol 70(Pt 6):14731481 11. Galloux M, Libersou S, Morellet N, Bouaziz S, Da Costa B, Ouldali M, Lepault J, Delmas B (2007) Infectious bursal disease

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

28.

123

2250 29. Shanmugasundaram R, Selvaraj RK (2011) Regulatory T cell properties of chicken CD4?CD25? cells. J Immunol 186:1997 2002 30. Sharma JM, Kim IJ, Rautenschlein S, Yeh HY (2000) Infectious bursal disease virus of chickens: pathogenesis and immunosuppression. Dev Comp Immunol 24:223235 31. Sanchez AB, Rodriguez JF (1999) Proteolytic processing in infectious bursal disease virus: identication of the polyprotein cleavage sites by site-directed mutagenesis. Virology 262:190 199 32. Tanimura N, Sharma JM (1997) Appearance of T cells in the bursa of Fabricius and cecal tonsils during the acute phase of

Y.-Y. Chen et al. infectious bursal disease virus infection in chickens. Avian Dis 41:638645 33. Vervelde L, Davison TF (1997) Comparison of the in situ changes in lymphoid cells during infection with infectious bursal disease virus in chickens of different ages. Avian Pathol 26:803821 34. Wintereld RW, Fadly AM, Bickford A (1972) Infectivity and distribution of infectious bursal disease virus in the chicken. Persistence of the virus and lesions. Avian Dis 16:622632 35. Yao K, Vakharia VN (2001) Induction of apoptosis in vitro by the 17-kDa nonstructural protein of infectious bursal disease virus: possible role in viral pathogenesis. Virology 285:5058

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