DNA-mediated vaccination conferring protection against infectious bursal disease in broiler chickens in the presence of maternal antibody

Vaccine 28 (2010) 3936–3943

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DNA-mediated vaccination conferring protection against infectious bursal disease in broiler chickens in the presence of maternal antibody Ming Kun Hsieh a,b , Ching Ching Wu a , Tsang Long Lin a,∗ a b

Department of Comparative Pathobiology, School of Veterinary Medicine, Purdue University, 406 South University Street, West Lafayette, IN 47907-2065, USA Graduate Institute of Microbiology and Public Health, College of Veterinary Medicine, National Chung Hsing University, Taichung 40227, Taiwan, ROC

a r t i c l e

i n f o

Article history: Received 21 October 2009 Received in revised form 8 March 2010 Accepted 26 March 2010 Available online 13 April 2010 Keywords: Infectious bursal disease Infectious bursal disease virus DNA vaccine Maternal antibody

a b s t r a c t The objective of the present study was to determine if a DNA vaccine carrying large segment gene of infectious bursal disease virus (IBDV) could confer protection against infectious bursal disease (IBD) in broiler chickens in the presence of maternal antibody. Broiler chickens with maternal antibody titers to IBDV were intramuscularly injected with a DNA plasmid coding for VP2, VP3, and VP4 genes of IBDV strain variant E (VE) (P/VP243/E) at 1-day, 1-week, and/or 2 weeks old. The dose of P/VP243/E used ranging from 400 ␮g to 10 mg. Broiler chickens at 3 weeks old were orally challenged with IBDV strain (VE) and observed for 10 days. Only broiler chickens vaccinated with 7.5 or 10 mg of P/VP243/E 3 times had 90 or 100% protection against challenge by IBDV strain VE and protected broiler chickens had significantly higher (P < 0.05) bursa weight/body weight (B/B) ratios, significantly lower (P < 0.05) bursal lesion scores, and the absence of IBDV antigens in bursae determined by immunofluorescent antibody assay (IFA). Antibody titers to IBDV as determined by enzyme-linked immunosorbent assay (ELISA) or virus neutralization (VN) assay in chickens of each group in each trial were gradually decreased prior to challenge. There was no significant difference (P > 0.05) in ELISA or VN titers to IBDV among all groups of broiler chickens or among the groups of broiler chickens vaccinated with various dose of P/VP243/E before challenge. Broiler chickens in the groups receiving 7.5 or 10 mg of P/VP243/E had significantly lower (P < 0.05) ELISA or VN titers to IBDV than those in the challenge control (CC) groups or the other groups vaccinated with various dose of P/VP243/E after challenge. Broiler chickens in the groups vaccinated with 10 mg of P/VP243/E 3 times had significantly higher (P < 0.05) stimulation indices for IBDV-stimulated lymphocyte proliferation response than those in the vector control (VC) or CC group at 14, 21, 24, or 31 days after first DNA vaccination. The results indicated that DNA vaccination with DNA encoding large segment gene of IBDV confers protection against challenge by IBDV in broiler chickens with maternal antibody to IBDV. © 2010 Elsevier Ltd. All rights reserved.

1. Introduction Infectious bursal disease (IBD) caused by infectious bursal disease virus (IBDV) is an acute and highly contagious disease in young chickens with mortality ranging from 0 to 90% depending on the strains involved [1]. Chickens less than 3 weeks old infected with IBDV have no or mild clinical signs and no mortality, but they are severely immunosuppressed [2,3]. This is because IBDV attacks the bursa of Fabricius and infects B lymphocytes in the bursal follicles, resulting in lymphocytolysis, lymphoid depletion, and eventually bursal atrophy. Immunosuppressed chickens are highly susceptible to secondary bacterial or viral infections and have poor immune response to vaccination, a major concern in the poultry industry [4,5].

∗ Corresponding author. Tel.: +1 765 494 7440; fax: +1 765 494 9181. E-mail address: [email protected] (T.L. Lin). 0264-410X/$ – see front matter © 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2010.03.066

Infectious bursal disease is largely controlled by vaccination. Newly hatched chickens are protected by maternal antibodies (Mab) to IBDV, transferred from hyper-immunized hens for the first several days after hatching, followed by vaccination with attenuated IBD vaccines. There are disadvantages to the use of conventional vaccines, such as cost and production complexity. In the case of live attenuated vaccines, selection pressure has contributed to the generation of variant strains, resulting in failure of immunoprotection by current vaccines and continuing outbreaks of diseases [6]. Conventional vaccines can also be inhibited by Mab making the timing of vaccination difficult. The titers of Mab present at the time of vaccination can also determine antibody response in the neonate. If the attenuated vaccine is given too early, Mab may interfere and reduce vaccination efficacy by causing antigen–antibody complexes to form between the pre-existing Mab and the introduced vaccine antigen [7,8]. Specific B cell epitopes for the vaccine can bind to Mab preventing the interaction of neonatal B cells with such epitopes [7,8]. A very high Mab-tovaccine antigen ratio can completely neutralize live attenuated

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vaccine, inhibiting replication and therefore, the effective dose for adequate immune response is decreased below the threshold required for activation of CD4/CD8 response [8]. If attenuated vaccines are given later to avoid high Mab titers, chickens would most likely be left unprotected for a period. DNA vaccination is an alternative approach for the prevention and control of disease. DNA vaccination involves the injection of plasmid DNA carrying a gene that codes for an antigen, the vaccine does not contain intact virus, preventing problems associated with conventional vaccines such as reverted virulence, divergent mutants, and environmental contamination. DNA vaccination has been shown to induce a broad spectrum of immune response including humoral and/or cellular immunity and has been shown to confer protection against infectious diseases in multiple animal species [9]. An IBDV STC strain VP243 protein-expressing plasmid constructed in our laboratory has shown to generate humoral immunity and possibly cellular immunity and protect 80–100% of specific-pathogen-free (SPF) chickens against challenge by IBDV [10,11]. A plasmid coding for the nucleoprotein (NP) of influenza virus was able to induce CTL, resulting in the clearance of influenza virus in neonatal mice [12]. A study involving DNA vaccination with hemagglutinin (HA)-expressing plasmids in neonatal mice revealed a mixed TH1/TH2 immune response [13]. Although high titers of Mab may negate induction of humoral immunity by DNA vaccination in neonatal animals, DNA vaccination is able to elicit prolonged in vivo antigen production and immune stimulation, allowing immune maturation and activation in the presence of Mab [8]. In addition, the immunostimulatory CpG motif associated with plasmid DNA can activate antigen presenting cells (APCs) and induce TH1 and CTL cells [14,15]. Studies have been performed to determine the efficacy of DNA vaccination in the presence of Mab. When neonatal mice, containing passively acquired anti-lymphocytic choriomeningitis virus (LCMV) antibodies, were vaccinated with plasmid DNA encoding LCMV nucleoprotein gene, Mab did not significantly inhibit the immune response [16]. DNA immunization effectively induced immunity to herpes simplex virus (HSV) in neonatal mice containing Mab to HSV [17]. Siegrist et al. showed that Mab inhibited antibody response but not T cell response in DNA vaccination against measles hemagglutinin [18]. The objective of the present study was to determine if a DNA vaccine carrying large segment gene of IBDV could confer protection against IBD in broiler chickens in the presence of maternal antibody. 2. Materials and methods 2.1. Chickens One-day-old broiler chickens with maternal antibody were obtained from a commercial hatchery. All chickens were raised in Horsefall-Bauer isolators with free access to feed and water. All animal studies were supervised by the Purdue University Animal Care and Use Committee. 2.2. Virus Infectious bursal disease virus standard challenge (STC) or variant E (VE) strains were used for plasmid construction and challenge study. Virus was prepared from bursal homogenate collected form 24-day-old specific-pathogen-free (SPF) chickens 3 days after challenge by IBDV. 2.3. Plasmid construction Plasmid carrying VP243 gene of IBDV strain VE (P/VP243/E) or STC (P/VP243/STC) was constructed with pCR3.1 vector in the previous studies [11,19].

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Table 1 Definition of experimental groups in animal studies. Groupsa

Group definitions

NC

Negative control (injected with saline at 1-day, 1-week and 2 weeks old) Challenge control (injected with saline at 1-day, 1-week, and 2 weeks old) Vector control (injected with pCR3.1 vector at 1-day, 1-week and 2 weeks old) Injected with P/VP243/STC at 1-day old Injected with P/VP243/STC at 1-day, and 1-week old; Injected with P/VP243/STC at 1-day, 1-week, and 2 weeks old Injected with P/VP243/STC at 1-week old Injected with P/VP243/STC at 1-week, and 2 weeks old Injected with 1 mg of P/VP243/E 3 times Injected with 2 mg of P/VP243/E 3 times Injected with 4 mg of P/VP243/E 3 times Injected with 5 mg of P/VP243/E 3 times Injected with 7.5 mg of P/VP243/E 3 times Injected with 10 mg of P/VP243/E 3 times Injected with 10 mg of pCR3.1 vector 3 times

CC

VCx3

VPx1 (1 day)a VPx2 (1-day, 1 week)a VPx3 (1-day, 1 week, 2 weeks)a VPx1 (1 week)a VPx2 (1 week, 2 weeks)a VPx3 (1 mg)b VPx3 (2 mg)b VPx3 (4 mg)b VPx3 (5 mg)b VPx3 (7.5 mg)b VPx3 (10 mg)b VCx3 (10 mg)b

a Injected with 400 ␮g of DNA plasmid encoding large segment gene of IBDV strain STC (P/VP243/STC) with various schedule. b Injected with various doses of plasmid carrying large segment gene of IBDV strain VE (P/VP243/E) or pCR3.1 vector at 1-day, 1-week, and 2 weeks old.

2.4. Characterization of the constructed plasmid in vitro and in vivo The plasmid P/VP243/E was characterized in vitro by immunofluorescent detection of expressed VP243 protein in COS-7 cells transfected with P/VP243/E in the previous study [11,19]. For characterizing the constructed P/VP243/E in vivo, 1-day-old broiler chickens were intramuscularly injected with plasmid P/VP243/STC or pCR3.1 vector in to thigh muscles and thigh muscles were collected 1 week after injection. Frozen sections of thigh muscles at 6 ␮m thickness were fixed in acetone at room temperature for 5 min. Protein expression was detected by immunofluorescent antibody assay (IFA) with monoclonal anti-IBDV VP2 antibody R63 (ATCC, Manassas, VA) as primary antibody and FITC conjugated goat anti-mouse IgG antibody (KPL, Gaithesburg, MD) as secondary antibody. 2.5. Vaccination and challenge Four trials were conducted in this study. The groups, the numbers of chickens, the doses, and times of injection in each trial are shown in Table 1. Chickens in negative control (NC) groups or challenge control (CC) groups were injected with saline at 1-day, 1-week, and 2 weeks old. In the trial 1, chickens were injected with 400 ␮g of P/VP243/STC at 1-day, 1-week or 2 weeks old. All chickens except those in the NC groups were challenged by 2 × 103.8 embryo infective dose (EID)50 /ml of IBDV strain STC at 3 weeks old. In the trial 2, chickens were injected with 1, 2, 4, 5, 7.5 or 10 mg of P/VP243/E at 1-day, 1-week, and 2 weeks old. In the trials 3 and 4, chickens injected with 10 mg of P/VP243/E or 10 mg of vector

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at 1-day, 1-week and 2 weeks old. Chickens except those in the NC groups in trials 2, 3, and 4 were challenged by 6.25 × 102.8 EID50 /ml of IBDV strain VE at 3 weeks old. 2.6. Protection efficacy against challenge with IBDV Ten days after challenge, chickens and bursae were weighed. Bursa weight/body weight (B/B) ratio was calculated as (bursa weight/body weight) × 1000. A gross bursal lesion score was given to each chicken from 1 to 4 according to the increasing severity of bursal atrophy (1: 0–10%; 2: 10–30%; 3: 30–70%; 4: 70–100% decrease in bursal size). A chicken with gross lesion score of 1 and B/B ratio of not less than 2 standard deviation (S.D.) below the average B/B ratio of the NC group was defined as protected against IBDV challenge. 2.7. Detection of IBDV antigen in bursa of Fabricius The presence of IBDV antigen in bursae was examined by IFA 10 days after challenge by IBDV. Frozen bursae were cut in 6-␮m section and fixed in acetone at room temperature for 5 min. The fixed sections were incubated with undiluted monoclonal antibody R63 to IBDC (ATCC) for 30 min and followed by incubating with FITC conjugated goat anti-mouse IgG at 1:400 dilution for 30 min. All sections were examined under a fluorescent microscope. 2.8. Antibody titers to IBDV by enzyme-linked immunosorbent assay (ELISA) Blood was collected from the wing vein of chicken at 1-day, 1-week, 2 weeks and 3 weeks old and from heart 10 days after challenge. A commercial antibody-capture ELISA kit for IBD (IDEXX, Portland, ME) was used to determine the serum anti-IBDV antibody titer. The relative level antibody titer in the unknown was determined by calculating the sample to positive (S/P) ratio. Endpoints titers were calculated by the equation: log 10 titer = 1.09 (log 10 S/P) + 3.36 (FlockCheck program, IDEXX). 2.9. Virus neutralization (VN) titers to IBDV by VN assay The VN assay was performed using the procedures reported previously [20]. Briefly, infectious bursal disease virus strain PBG98 grown in chicken embryo fibroblasts (CEF) was titrated and diluted in M199 complete media (Invitrogen, Carlsbad, CA) (2 mM l-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 0.04 mM ␤-mercaptoethanol, 10,000 U penicillin/ml, 10 mg streptomycin/ml) with 10% FBS (Atlanta Biologicals, Norcross, GA) to yield 1000 plaque forming units (PFU) per 25 ␮l of diluted virus. Serum from each chicken was 4-fold serially diluted from 1:4 to 1:65,536 in a final volume of 75 ␮l of M199 complete medium and added to a 96-well plates containing 24-h-old confluent monolayers of CEF. Twenty-five microliters of virus with 1000 PFU were added to each well in the plates and incubated at 37 ◦ C for 5 days. The cells were fixed with 10% buffered formalin for 5 min and stained with 1% crystal violet for 3 min. The VN titer for each serum was recorded as the reciprocal of the highest dilution where monolayers remained 100% intact as compared with the control wells.

2.11. Lymphocyte proliferation The proliferation assays of spleen or blood lymphocytes collected from chickens at 17, 21, 24, and 31 days post-priming (DPP) with P/VP243/E in the trial 4 were carried out as described previously [21]. Briefly, spleens from each chicken were macerated within Hanks’ balanced salt solution (HBSS) (Sigma, St. Louis, MO). Single splenocytes were obtained by passing through a mesh with pore size of 60 ␮m and span down by centrifugation at 200 × g for 5 min. Blood lymphocytes were obtained from whole blood collected by syringes with heparin (Sigma) (500 units of heparin/10 ml of blood). Heparined blood was diluted 1:1 in HBSS buffer. Six microliters of blood–HBSS mixture were layered on the top of 6 ml of Ficoll–paque (Amersham Biosciences, Piscataway, NJ) in a 15 ml tube and this tube was centrifuged at 600 × g for 15 min. Blood lymphocytes were collected from the interface layer. Spleen or blood lymphocytes were washed with 5 ml of HBSS and centrifuged at 200 × g for 5 min. Pelleted cells were resuspended in 5 ml ACK buffer (0.15 M NH4 Cl, 1 M K2 CO3 , 0.01 M EDTA, pH 7.2) with gentle vortex and kept at room temperature for 5 min. Cells were centrifuged at 200 × g for 5 min, washed with 5 ml of HBSS and centrifuged at 200 × g for 5 min again. Pelleted cells were resuspended in RPMI-1640 complete medium (Invitrogen) (2 mM l-glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 0.04 mM ␤-mercaptoethanol, 10,000 U penicillin/ml, 10 mg streptomycin/ml) with 5% fetal bovine serum (FBS) (Atlanta Biologicals, Norcross, GA). Cells were counted in a hemocytometer and viability was determined by the trypan blue exclusion method. Each cell suspension was adjusted to a concentration of 5 × 106 cells per ml. Cells were added into 96-well flat-bottomed tissue culture plates at 5 × 105 cells/well and purified inactivated IBDV was added to each well at final concentration of 25 ␮g/ml. Plates were incubated at 41 ◦ C for 48 h in a humidified incubator with 5% CO2. After incubation for 43 h, thymidine (3 H) (Amersham Biosciences) was added to each well at 1 ␮Ci/well and incubated for another 5 h. After incubation, lymphocytes were harvested using by cell harvester (Packard Bioscience, Meriton, CT, USA) onto 96-unifilter plates (Packard Bioscience) and let them air-dry. Thirty microliters of Microscint 20 scintillation liquid (Packard Bioscience) were added to filter plates and plates were counted in a 1205 Betaplate counter (Packard Bioscience). The counts per minute (cpm) was determined for each stimulated and unstimulated cells, and the stimulation indices (S.I.) were calculated from the ratio mean cpm stimulated/mean cpm unstimulated for each group. 2.12. Statistical analysis All results were presented as mean ± S.D. Data were analyzed using one-way analysis of variance (ANOVA) and means of treatments were compared using Duncan’s multiple range test by SPSS 12.00 (SPSS Inc., Chicago, IL). P < 0.05 was considered statistically significant. 3. Results 3.1. Characterization of the constructed plasmid in vivo Protein expression of P/VP243/E in thigh muscles was detected by IFA. Positive green fluorescence was seen in the frozen sections from chickens injected with P/VP243/E (Fig. 1A) but not in the sections from chickens injected with pCR3.1 vector (Fig. 1B).

2.10. Purification of IBDV antigen 3.2. Protection efficacy against IBDV challenge Infectious bursal disease virus was purified from chicken embryo fibroblasts (CEF) infected with IBDV strain PBG-98 by CsCl purification as previously described [21].

The groups used in the animal studies were defined in Table 1. The results of bursal lesion scores, B/B ratios, and protection in each

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Fig. 1. Expression of encoded protein of P/VP243/E in the thigh muscles of broiler chickens with maternal antibodies. One-day-old broiler chickens were intramuscularly injected with 400 ␮g of P/VP243/E (A) or pCR3.1vector (B). Thigh muscles injected with plasmid were collected 1 week after injection. Protein expression was determined by immunofluorescent antibody assay (IFA).

trial are summarized in Table 2. Severe bursal atrophy was seen in the challenge control (CC) groups (Fig. 2A) and vector control (VCx3) groups or other groups not protected by DNA vaccination. Chickens in the groups injected with 7.5 or 10 mg of P/VP243/E at 1-day, 1-week, and 2 weeks old had a bursa comparable to chickens

Table 2 Protection efficacy of DNA vaccine determined by bursal lesion score, bursa weight/body weight (B/B) ratio, and protection in chickens 10 days after challenge with infectious bursal disease virus (IBDV) strain variant E (VE) or standard challenge (STC). Groupsa

Lesion scoreb

Trial 1 NC CC VCx3 VPx1 (1 day) VPx2 (1 day, 1 week) VPx3 (1 day, 1 week, 2 weeks) VPx1 (1 week) VPx2 (1 week, 2 week)

1.0 3.7 3.5 3.1 2.3 2.5 2.6 2.7

± ± ± ± ± ± ± ±

01 e 1.03 0.53 1.12,3 0.52 1.02 1.12 0.72

2.4 0.9 0.8 1.1 1.0 1.0 1.1 0.9

± ± ± ± ± ± ± ±

0.82 0.91 0.31 0.61 0.31 0.51 0.91 0.11

7/7 0/7 0/7 0/7 0/7 0/7 0/7 0/7

Trial 2 NC CC VPx3 (1 mg) VPx3 (2 mg) VPx3 (4 mg) VPx3 (5 mg) VPx3 (7.5 mg) VPx3 (10 mg)

1.0 4.0 4.0 4.0 3.8 4.0 1.0 1.0

± ± ± ± ± ± ± ±

01 02 02 02 0.52 02 01 01

3.2 0.9 1.0 0.8 1.1 0.8 3.2 2.7

± ± ± ± ± ± ± ±

0.33 0.21 0.21 0.21 0.21 0.11 0.53 0.42

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

Trial 3 NC CC VCx3 (10 mg) VPx3 (10 mg)

1.0 3.4 3.6 1.1

± ± ± ±

01 0.52 0.52 0.31

3.0 1.1 1.0 3.1

± ± ± ±

1.02 0.21 0.21 0.82

5/5 0/5 0/10 9/10

Trial 4 NC CC VCx3 (10 mg) VPx3 (10 mg)

1.0 3.8 4.0 1.3

± ± ± ±

01 0.42 02 0.61

2.0 0.7 0.7 1.9

± ± ± ±

0.522 0.171 0.131 0.962

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

a

B/B ratiosc

Protectiond

Definition of each group is described in Table 1. Bursal lesion score from 1 to 4 was given to each bursa 10 days after challenge based on increasing severity of bursal atrophy (1: 0–10%; 2: 10–30%; 3: 30–70%; 4: 70–100% decrease in bursal size). c Bursa/body weight (B/B) ratio was calculated by (bursa weight/body weight) × 1000. d Protection was presented by the number of protected chickens/the number of total chickens in a group for each trial. A protected chicken was defined by the absence of gross bursal lesions and the B/B ratios no less than 2 standard deviation (S.D.) below the average B/B ratios of NC group. e Bursal lesion score and B/B ratio were presented as mean ± S.D. from each group in each trial. Groups labeled with the same number in each trial indicate no statistical significance (P > 0.05) among these groups. The results were determined by one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test. b

in the negative control (NC) groups (Fig. 2A). Chickens in the groups injected with 400 ␮g, 1, 2, 4, or 5 mg of P/VP243/E at 1-day, 1-week, and 2 weeks old had 0% protection against challenge by IBDV strain STC or VE. Chickens in the groups injected with 7.5 or 10 mg of P/VP243/E at 1-day, 1-week, and 2 weeks old had 90–100% protection against challenge by IBDV strain VE and significantly higher (P < 0.05) B/B ratios and significantly lower (P < 0.05) bursal lesion scores than chickens in other groups challenged by IBDV. 3.3. IBDV antigen in bursa of Fabricius The presence of IBDV antigen in the bursae from each trial detected by IFA 10 days after challenge is summarized in Table 3. Chickens in the groups NC or groups injected with 7.5 or 10 mg of P/VP243/E at 1-day, 1-week and 2 weeks did not have detectable IBDV antigen in bursae (Figs. 2B-a and B-c). Large amount of IBDV antigen was seen in the groups CC, VCx3 (Figs. 2B-b and B-d), and groups not protected by DNA vaccination. 3.4. Anti-IBDV enzyme-linked immunosorbent assay (ELISA) titers The results of serum antibody titers to IBDV measured by ELISA are summarized in Table 3. The averages of Mab titers to IBDV in 1-day-old broiler chickens in each trial ranged from 3200 to 5541. Prior to challenge, Mab titers of chickens in each trial were gradually decreased. Chickens in the groups injected with 7.5 or 10 mg of P/VP243/E had higher or significantly higher (P < 0.05) ELISA titers than chickens in other groups at 3 weeks old. Chickens in the groups injected with 7.5 or 10 mg of P/VP243/E had lower or significantly lower (P < 0.05) ELISA titers than chickens in other groups injected with P/VP243/E or vector 10 days after challenge. 3.5. Virus neutralization (VN) titers to IBDV The results of VN titers to IBDV in trials 2, 3, and 4 are summarized in Table 4. Similar to the results of ELISA titers to IBDV, chickens in the groups injected with 7.5 or 10 mg of P/VP243/E at 1-day, 1-week, and 2 weeks old had higher or significantly higher (P < 0.05) VN titers at 3 weeks old and lower or significantly lower (P < 0.05) VN titers 10 days after challenge than chickens in other groups. 3.6. Lymphocyte proliferation assay The results of proliferation assays of lymphocytes prepared from spleen or blood are summarized in Tables 5 and 6. Spleen lymphocytes of chickens in the group injected with 10 mg of P/VP243/E had significantly higher (P < 0.05) S.I. than that of chickens in the groups

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Fig. 2. Bursal gross morphology and IBDV antigen detection in bursa from the representative bursal specimens in animal studies. (A) Morphology and size of bursae from broiler chickens in the groups of negative control (NC), challenge control (CC), or chickens injected with 10 mg of plasmid encoding large segment gene of IBDV 3 times at 1-day, 1-week, and 2 weeks old (VPx3 (10 mg)) 10 days after challenge with IBDV. (B) IBDV antigen in bursa of Fabricius detected by immunofluorescent antibody assay (IFA). Frozen bursal sections were obtained from chickens in (a) negative control (NC), (b) challenge control (CC), (c) group injected with 10 mg of P/VP243/E at 1-day, 1-week, and 2 weeks old (VPx3 (10 mg)), and (d) group injected with 10 mg of pCR3.1 vector at 1-day, 1-week, and 2 weeks old (VCx3 (10 mg)) 10 days after challenge with IBDV.

CC and VCx3 at 17, 21, 24, and 31 DPP. Blood lymphocytes of chickens in the group injected with 10 mg of P/VP243/E at 1-day, 1-week, and 2 weeks old had significantly higher (P < 0.05) S.I. than that of chickens in the groups CC and VCx3 at 21 and 24 DPP (Table 7).

Table 3 IBDV antigen in bursa of Fabricius determined by immunofluorescent antibody assay (IFA) in chickens 10 days after challenge with IBDV. Groupa

NC CC VCx3 (0.4 mg) VCx3 (10 mg) VPx1 (1 day) VPx1 (1 week) VPx2 (1 day, 1 week) VPx2 (1 week, 2 weeks) VPx3 (1 day, 1 week, 2 weeks) VPx3 (1 mg) VPx3 (2 mg) VPx3 (4 mg) VPx3 (5 mg) VPx3 (7.5 mg) VPx3 (10 mg) a

No. of positive/no. of chickensb Trial 1

Trial 2

Trial 3

Trial 4

0/7 7/7 7/7

0/3 3/3

0/5 5/5

0/5 5/5

10/10

5/5

1/10

3/3

7/7 7/7 7/7 7/7 7/7 4/4 4/4 4/4 3/3 0/3 0/4

Definition of each group is described in Table 1. The results were presented as the number of bursae with IBDV antigen/the total number of bursae in each group. b

4. Discussion The expression of IBDV VP243 protein in the thigh muscles of broiler chickens injected with P/VP243/E was detected by IFA in the present of Mab to IBDV was similar to that in SPF chickens [19]. The results indicated that Mab did not inhibit expression of VP243 protein from DNA vaccine. In addition, the expressed VP243 in the thigh muscles was reacted with monoclonal antibody R63 that is specific for a conformational epitope on VP2, indicating that the epitope in the expressed VP243 recognized by R63 was not blocked by Mab. A major problem with vaccination in the presence of Mab is that Mab may bind to the surface epitopes of vaccine antigens and rapidly clear the vaccine [7]. In order to avoid the interference of Mab in IBD vaccination, a vectored vaccine using turkey herpesvirus as the vector carrying classical IBDV VP2 gene had demonstrated 100% protection against IBDV challenge when vaccines were given in the face of high-titerd Mab [22]. In our previous studies, a DNA vaccine using 400 ␮g of P/VP243/STC in SPF chickens provided 80–100% protection against IBD [10,11]. However, the DNA vaccine with 400 ␮g of P/VP243/STC did not provide any protection against IBDV challenge in the broiler chickens with high titers of Mab. The reason may be due to the neutralization of IBDV VP243 protein expressed by P/VP243/E by Mab to IBDV in broiler chickens. Inhibition of neonatal immune responses to a live vaccine in the presence of Mab depends upon the relative ratios of Mab titers at the time of vaccination and of viral vaccine load/replicating capac-

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Table 4 Antibody titers to IBDV determined by enzyme-linked immunosorbent assay (ELISA) in chickens at 1-day old (D1), 1-week old (W1), 2-week old (W2), before challenge (pre-C) at 3 weeks old, and 10 days after challenge (post-C) with IBDV. ELISA titers (mean ± S.D.)b

Groupa

D1

W1

W2

Pre-C

Post-C

Trial 1 NC CC VCx3 VPx1 (1 day) VPx2 (1 day, 1 week) VPx3 (1 day, 1 week, 2 weeks) VPx1 (1 week) VPx2 (1 week, weeks)

4198 3916 4894 4818 5233 4345 4091 4784

± ± ± ± ± ± ± ±

22851 c 25821 14111 16271 19211 26701 19041 29961

1956 2536 1928 2889 2783 2179 2167 2563

± ± ± ± ± ± ± ±

19971 14371 8431 16381 11081 14141 15501 24881

672 721 344 864 789 552 576 835

± ± ± ± ± ± ± ±

10091 6911 3301 7981 5571 3891 5491 8951

293 146 59 304 218 191 96 344

± ± ± ± ± ± ± ±

5761 1961 841 4251 2841 1621 1451 3061

179 1987 2301 1428 2768 3784 2339 3120

± ± ± ± ± ± ± ±

2341 9722,3 7722,3 9332 15563,4 17393,4 18032,3 15634

Trial 2 NC CC VPx3 (1 mg) VPx3 (2 mg) VPx3 (4 mg) VPx3 (5 mg) VPx3 (7.5 mg) VPx3 (10 mg)

3200 5541 5043 4416 4702 4006 4204 5309

± ± ± ± ± ± ± ±

22641 23671 19621 20401 10431 16331 14891 23081

2314 2942 1959 2351 1997 1915 1554 2574

± ± ± ± ± ± ± ±

26651 9631 8541 8641 4841 10271 8161 11321

917 534 421 701 429 564 827 738

± ± ± ± ± ± ± ±

11161 2351 4371 3521 2321 2431 3991 2571

364 99 226 395 408 501 820 728

± ± ± ± ± ± ± ±

5561,2 951 2111,2 681,2 1521,2 3921,2 7492 4591,2

105 1866 6245 5010 5240 8516 1327 1439

± ± ± ± ± ± ± ±

1641 3481 18712 10822 10802 29563 5341 5971

Trial 3 NC CC VCx3 (10 mg) VPx3 (10 mg)

3496 3310 4577 5182

± ± ± ±

13471,2 12001,2 26931,2 16162

1516 2000 1936 2684

± ± ± ±

8131 8381,2 11771,2 9662

341 388 531 708

± ± ± ±

3621 2001 5661 3891

68.8 114.6 134.3 347.3

± ± ± ±

46.31 72.91 131.71 90.42

4.8 2470 3796 1218.5

± ± ± ±

8.51 11703 13654 141.12

64.8 1060 1367 1286

± ± ± ±

63.71 2782 1022 6432

Trial 4 NC CC VCx3 (10 mg) VPx3 (10 mg)

3540.2 ± 10411

2986 ± 11471

461 ± 5041

192 ± 1211

4172 ± 11421 5126 ± 13322

2039 ± 7901,2 3497 ± 4552

528 ± 4811 705 ± 3201

294 ± 1131 673 ± 1932

a

Definition of each group is described in Table 1. ELISA titers were determined by IDEXX ELISA kit and carried out by the procedures recommended by the manufacturer. c Groups labeled with the same number in each trial indicate no statistical significance (P > 0.05) among these groups. The results were determined by ANOVA followed by Duncan’s multiple range test. b

Table 5 Virus neutralization (VN) titers to IBDV in chickens at 1-week old (W1), 2 weeks old (W2), before challenge (Pre-C) at 3 weeks old, and 10 days after challenge (post-C) with IBDV. Groupa

VN titers (mean ± S.D.)b W1

W2

Pre-C

Post-C

Trial 2 NC CC VPx3 (1 mg) VPx3 (2 mg) VPx3 (4 mg) VPx3 (5 mg) VPx3 (7.5 mg) VPx3 (10 mg)

5.7 5.7 6.0 6.0 5.0 5.3 5.3 6.5

± ± ± ± ± ± ± ±

1.11 c 0.61 01 1.41 0.81 0.61 0.61 0.61

4.0 4.3 3.5 4.3 4.3 4.3 4.3 4.3

± ± ± ± ± ± ± ±

1.01 1.21 1.71 1.31 0.51 0.61 0.61 1.01

3.7 4.3 3.3 4.0 3.8 3.3 3.3 3.8

± ± ± ± ± ± ± ±

1.21 0.61 1.01 0.81 1.01 0.61 0.61 1.01

2.0 5.0 5.3 5.5 4.8 6.3 2.0 2.3

± ± ± ± ± ± ± ±

1.01 1.02 1.32 1.32 1.52 1.52 01 0.51

Trial 3 NC CC VCx3 (10 mg) VPx3 (10 mg)

4.0 4.6 4.6 5.0

± ± ± ±

0.711 0.551 0.841 1.251

3.8 4.0 4.0 4.2

± ± ± ±

0.451 1.001 1.331 0.791

2.6 3.6 3.3 4.0

± ± ± ±

0.551 0.552 1.341,2 0.672

2.2 5.2 5.8 2.7

± ± ± ±

0.451 0.842 0.632 0.821

3.2 5.2 4.8 4.7

± ± ± ±

0.451 1.302 0.962 0.582

Trial 4 NC CC VCx3 (10 mg) VPx3 (10 mg) a

5.4 ± 0.551

3.7 ± 0.581

3.4 ± 0.541

5.8 ± 0.451 5.6 ± 0.551

4.6 ± 1.341 4.6 ± 0.551

3.6 ± 0.541 4.4 ± 0.542

Definition of each group is described in Table 1. VN titers were presented by geometric mean. c Groups labeled with the same number in each trial indicate no statistical significance (P > 0.05) among these groups. The results were determined by ANOVA followed by Duncan’s multiple range test. b

ity [23]. Under the condition of high titers of Mab and low viral load, the viral replication and possibly B and T cell responses could be inhibited. Under the condition of low Mab titers and high viral load, the viral replication and B and T cell response remain unaffected. A “Deventer formula” has been used in the poultry industry to estimate the optimal time for vaccination with attenuated IBDV strain by considering the level of Mab, variation of Mab, genetic background of chickens, and the vaccine strains. A large scale of field study had been conducted to test the Deventer formula and found that all flocks vaccinated on the optimal time calculated by Deventer formula were seroconverted within 14 days of IBDV vaccination [24]. The flocks vaccinated at earlier than the optimal vaccination time faced the high titers of Mab and showed delayed or prevented

Table 6 Lymphocyte proliferations of spleen cells stimulated by IBDV antigen were measured at each timepoint. Groupsa

NC CC VCx3 (10 mg) VPx3 (10 mg) a

S.I. of lymphocyte proliferation (mean ±S.D.)b by IBDV antigen 14 DPPc

21 DPP

24 DPP

0.9 ± 0.161,2 d

0.7 ± 0.061

0.8 ± 0.111 1.0 ± 0.162

0.6 ± 0.091 0.9 ± 0.122

0.8 0.7 0.8 1.0

± ± ± ±

0.081 0.091 0.151 0.072

31 DPP 0.8 0.8 0.7 1.2

± ± ± ±

0.111 0.121 0.061 0.492

Definition of each group is described in Table 1. Stimulation indices (S.I.) were calculated from the ratio mean counts per minutes (cpm) stimulated/mean cpm unstimulated for each group. c Days post-first priming (DPP) indicated that days after the first priming with DNA vaccine. d Values followed by the same numerical superscripts within the column do not differ significantly (P > 0.05) as determined by one-way ANOVA followed by multiple rank comparisons with Duncan’s test. b

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Table 7 Lymphocyte proliferations of blood lymphocytes stimulated by IBDV antigen were measured at each timepoint. Groupsa

NC CC VCx3 (10 mg) VPx3 (10 mg)

S.I. of lymphocyte proliferation (mean ±S.D.)b by IBDV antigen 14 DPPc

21 DPP

24 DPP

1.1 ± 0.131 d

1.0 ± 0.162

1.1 ± 0.111 1.1 ± 0.211

0.8 ± 0.231 1.4 ± 0.203

0.9 0.8 0.8 1.3

± ± ± ±

0.141 0.151 0.081 0.142

31 DPP 1.0 1.3 1.1 1.2

± ± ± ±

0.271 0.141 0.221 0.201

a

Definition of each group is described in Table 1. Stimulation indices (S.I.) were calculated from the ratio mean counts per minutes (cpm) stimulated/mean cpm unstimulated for each group. c Days post-first priming (DPP) indicated that days after the first priming with DNA vaccine. d Values followed by the same numerical superscripts within the column do not differ significantly (P > 0.05) as determined by one-way ANOVA followed by multiple rank comparisons with Duncan’s test. b

induction of humoral immunity [24]. However, no matter the vaccination was given before or at the optimal time, the induction of humoral immunity usually correlated with the induction of bursal lesions caused by the attenuated IBD vaccine [24]. The interference of Mab may also occur in the case of targeted antigenic protein expressed by DNA vaccination against IBD. However, adequate levels of targeted antigenic protein expressed by DNA vaccine should overcome the interference by Mab. Thus, various doses ranging from 1 to 10 mg of P/VP243/E DNA were used to evaluate protection efficacy of DNA vaccine in broiler chickens with Mab. Only 10 mg of P/VP243/E showed 100% protection against challenge by IBDV. In addition to the high titer of Mab, broiler chickens are larger and about 5 times heavier than SPF chickens at 21 days old before challenge. Therefore, additional or a large amount of DNA was necessary to induce immune response and protection. The ELISA and VN titers to IBDV gradually decreased from 1day old to 3 weeks old. Prior to challenge at 3 weeks old, ELISA or VN titers of broiler chickens in the groups without protection against challenge by IBDV were similar to those of chickens injected with P/VP243/STC or P/VP243/E 3 times with 80–100% protection against IBD in the previous studies [11,19], suggesting humoral immune response may not be the only immune response induced by DNA vaccination. With conventional vaccines, high titers of antibody have been associated with protection. However, chickens that had functional T cells removed by neonatal thymectomy and Cyclosporin A treatment showed insufficient protection against challenge by IBDV with vaccination by inactivated vaccines, but chickens with intact T cells had about 90% protection against IBD [25]. One-day-old chickens treated with cyclophosphamide to compromise antibody-producing ability could induce protective immune responses against IBDV challenge in the absence of antibody [26]. Therefore, cellular immunity may also play an important role in the protection of chickens against challenge by IBDV. After challenge, ELISA titers of protected chickens had significantly lower (P < 0.05) titers than that in other challenged groups. Similar finding were also seen in the previous study with DNA vaccination against challenge by IBDV in SPF chickens [10,11]. The lower ELISA titers to IBDV in chickens protected by P/VP243/E or P/VP243/STC were associated with no detectable IBDV antigen in bursae. Without a sufficient amount of virus present in the bursae, high antibody titers to IBDV were not seen, as opposed to the higher antibody titers to IBDV in the unprotected chickens after challenge. Memory B cells were induced by DNA vaccine in piglets against cgallenge by pseudorabies virus (PRV) [27]. Piglets with Mab to PRV were primed with plasmid encoding PRV gB, gC or gD and boosted with the same plasmid. The ELISA antibody to PRV was only induced after priming with the plasmid when Mab titers was not too high, but the ELISA titers to PRV were induced after boosting with the plasmid in the presence of any titers of Mab. The results indicated

that B memory cells could be induced by priming with DNA vaccine even in the presence of high titers of Mab. In the present study, broiler chickens injected with 10 mg of P/VP243/E 3 times had low ELISA titers to IBDV but the titers were significantly higher (P < 0.05) than those in chickens in the other groups prior to challenge in trials 2, 3, and 4, indicating that memory B cells were induced in the presence of Mab and might contribute to protection against challenge by IBDV in DNA vaccination. The mechanisms in which DNA vaccination induces cellular immune responses can occur in two ways. One pathway is that DNA is taken up by somatic cells (such as muscle cells) and vaccine antigens are produced and released by these cells and phagocytized by antigen presenting cells (APCs) to be presented by major histocompatibilty complex (MHC) II molecules to activate T cell to become TH cells for helping differentiation and proliferation of CTL or B cells [28]. The other pathway is that DNA is taken up directly by APCs such as dendritic cells or macrophages and the viral antigens are presented by MHC I molecules to activate T cells to become CTL [29,30]. By cross-priming, APCs uptake vaccine antigens produced by somatic cells and can also present the exogenous peptides by MHC I molecule to activate cytotoxic T cells [31–33]. In the presence of Mab, the induction of cellular immunity by DNA vaccination is not inhibited when the DNA is directly taken up by APCs. In addition, the immune complex formed between Mab and protein expressed by DNA increases the chance that the protein will be taken up by APCs and to activate TH cells or CTL by crosspriming [7]. The unmethylated CpG motif in the bacterial vector used to clone DNA vaccine can bind to the Toll-like receptor (TLR)9 on the surface of APCs or nature killer (NK) cells to produce IL-12 that stimulates NK cells to produce interferon (IFN)-␥ and favor the development of TH1 subset [14,15]. Studies have shown that Mab blocks humoral but not T cell responses [18,34]. In the present study, cellular immune response was induced by DNA vaccination as indicated by increased lymphocyte proliferation response of spleen or blood lymphocytes stimulated by IBDV antigens, demonstrating that cellular immune response could be induced by DNA vaccination and was not blocked by Mab. Due to neutralization with vaccine antigens, maternal antibody titers usually drop rapidly in neonatal animals when receiving conventional vaccines [35]. The extremely high doses of DNA vaccines needed for inducing protective immune responses in the present study overcame the large size of the broiler chickens and neutralization of vaccine antigens with Mab. In order to inject the large amount of DNA plasmid, more injections were needed and therefore, more APCs could be directly transfected with DNA vaccines resulting in an increased amount of protein expressed from DNA for antigen presentation by APCs. To the best of our knowledge, this is the first paper to show that DNA vaccine can confer protection of chickens against challenge by IBDV in the presence of Mab. Although the extremely high dose of DNA required to induce immune response and provide protection is not practical in the field, improvement of transfection and expression efficiency from DNA plasmids in the somatic cells and/or APCs by different constructs, delivery vehicles, and delivery routes may be achieved to overcome this obstacle. References [1] Chettle N, Stuart JC, Wyeth PJ. Outbreak of virulent infectious bursal disease in East Anglia. Vet Rec 1989;125(10):271–2. [2] Allan WH, Faragher JT, Cullen GA. Immunosuppression by the infectious bursal agent in chickens immunised against Newcastle disease. Vet Rec 1972;90(18):511–2. [3] Faragher JT, Allan WH, Cullen GA. Immunosuppressive effect of the infectious bursal agent in the chicken. Nat New Biol 1972;237(73):118–9. [4] Cho BR. Experimental dual infections of chickens with infectious bursal and Marek’s disease agents. I. Preliminary observation on the effect of infectious bursal agent on Marek’s disease. Avian Dis 1970;14(4):665–75.

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