Chen Et Al., 2011 - IBDV DNA Vaccination

7/31/2019 Chen Et Al., 2011 - IBDV DNA Vaccination

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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 theC-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 pro-

tected by DNA vaccination prior to and after virus chal-

lenge [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 immunofluorescent antibody assay(IFA) at 10 days post-challenge [4, 5, 13]. These findings

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 vacci-

nation followed by IBDV challenge.

Materials and methods

Chickens

Specific-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 waterad 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.

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 (DNA-CC). 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 vaccina-

tion regimen were as described previously [4, 5, 13, 15].

Briefly, one-day-old SPF chickens were injected intra-

muscularly 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]. Briefly, 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

defined 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 immunofluorescent 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 immunofluorescent antibody assay

was performed as described previously [4, 5, 13, 14].

Briefly, frozen sections of bursa and spleen collected from

six different time points were cut into 6-lm-thick sections,

transferred to glass slides and fixed with acetone at room

temperature for 5 minutes. After fixation, sections were

incubated with anti-VP2 monoclonal antibody R63

(ATCC, Manassas, VA, USA) followed by goat anti-mouse

IgG secondary antibody labeled with fluorescein isothio-

cyanate (FITC) (KPL, Gaithersburg, MD, USA).

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sections were incubated with Poly HRP Reagent (BioGenex)

in a humidified 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 theAmericas, Center Valley, PA, USA). Positive CD4?- or

CD8?-staining cells in the bursa were counted at 4009

magnification. Eight fields 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 at37C for one hour (Promega, Madison, WI, USA), fol-

lowed by inactivation at 72C for 15 minutes before cDNA

synthesis. Chicken spleen IFNc, IL-4 and GADPH mRNA

expression levels were quantified by real-time RT-PCR

using the method described [1, 18] with the modified PCR

reaction mixture described above for IBDV RNA detec-

tion. The cycle profile 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-3 0),

IFNc probe (FAM-50-TGGCCAAGCTCCCGATGAACGA

-30TAMRA), IL-4 -F (50-AACATGCGTCAGCTCCTGA

AT-30), IL-4-R (50-TCTGCTAGGAACTTCTCCATTGA

A-30), IL-4 probe (FAM50-AGCAGCACCTCCCTCAAG

GCACC-30TAMRA), GAPDH-F (50-CCCCAATGTCT

CTGTTGTTGAC-30), GAPDH-R (50-CAGCCTTCACTA

CCCTCTTGAT-3 0), GAPDH probe (FAM50-CTTGG

CTGGTTTCTCC-30TARMA). 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 com-

parison, Students t test was applied. Statistical significance

was set at p\ 0.05.

Results

DNA vaccine protection efficacy 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 grosslesion 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-vacci-

nated 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 immu-

nofluorescent 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 quantification

by real-time RT-PCR in bursa, spleen, cecal tonsil

and PBMC

The results of IBDV viral RNA detection and quantification

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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six time points (Table 3). Viral RNA was detected at eachof the five time points from 24 HPI to 10 DPI in the

chickens of the CC group (Table 3). The level of viral

RNA was significantly 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 significant difference (p[0.05) in ELISA

antibody titer to IBDV between the DNA-CC and DNA-

NC 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 significantly lower

(p\ 0.05) VN antibody titers were observed at 12 HPI in

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 12 HPId 24 HPI 3 DPId 5 DPI 7 DPI 10 DPI

Gross lesion scoreb

NC 1.0 0 1.0 0 1.0 0 1.0 0 1.0 0 1.0 0

DNA-NC 1.0 0 1.0 0 1.0 0 1.0 0 1.0 0 1.0 0

DNA-CC 1.0 0 1.0 0 1.0 0 1.0 0 1.0 0 1.0 0CC 1.0 0 1.0 0 1.0 0 2.6 0.6* 3.8 0.5* 4.0 0*

B/B ratioc

NC 6.1 1.2 7.2 2.1 6.0 0.5 5.8 1.2 6.8 0.9 6.9 1.8

DNA-NC 6.9 0.6 6.9 1.4 6.0 0.9 6.0 1.8 6.3 0.8 6.6 1.7

DNA-CC 6.5 0.9 7.5 0.9 6.7 2.1 6.9 2.0 5.8 0.8 5.8 0.7

CC 7.5 2.0 6.8 1.9 6.4 1.1 2.8 0.7* 1.8 0.3* 1.6 0.3*

* Indicates statistical difference among different groups (p\ 0.05) at each time pointa 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)b 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

c The B/B ratio was calculated as (bursal weight)/(body weight) x 1000 and presented as the mean S.D. for each groupd HPI: hours postinfection; DPI: days postinfection

Table 2 IBDV antigen in bursa and spleen detected by immunofluorescence antibody assay (IFA) a

Groupc No. positive/no. of chickensb

12 HPI 24 HPI 3 DPI 5 DPI 7 DPI 10 DPI

Bursa

NC 0/5 0/5 0/5 0/5 0/5 0/5

DNA-NC 0/5 0/4 0/5 0/5 0/5 0/5

DNA-CC 0/5 0/5 0/5 0/4 0/5 0/5

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

Spleen

NC 0/5 0/5 0/5 0/5 0/5 0/5

DNA-NC 0/5 0/4 0/5 0/5 0/5 0/5

DNA-CC 0/5 0/5 0/5 0/4 0/5 0/5

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

a Photomicrographs of IFA results are not shown. They were consistent with those of previous publications [1315]b 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 groupc The definition of each group is given in Table 1

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while significantly more cells (p\ 0.05) infiltrated 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 spleenby 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 significantly higher (p\ 0.05) when com-

pared 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 exper-

imental groups, and there was no significant difference

(p[ 0.05) in chicken IL-4 mRNA levels among the DNA-NC, DNA-CC and CC groups at 12 HPI and 10 DPI

(Table 6).

Discussion

The present study was carried out to elucidate how DNA-

vaccinated chickens with low ELISA antibody titers are

protected against virus challenge. In addition to the 10 DPI

time point, five earlier time points were included in the

study. DNA vaccination with a three-dose regimen proved

to be effective in providing protection against IBDV

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 sen-

sitive 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 quanti-

tative RT-PCR at 3 and 5 DPI in the bursae and 5 and 7DPI 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 findings confirmed 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 inthe bursae, spleens and cecal tonsils in the DNA-vaccinated

chickens is likely due to a combination of DNA-vaccine-

induced humoral and cellular immune responses, which

neutralize extracellular viral particles and destroy virus-

infected 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 significantly higher than those in the

DNA-vaccinated chickens at 5, 7 and 10 DPI. Such find-

ings are consistent with those from previous studies

Table 5 Chicken T cell number in bursa of Fabricius by IHCa

Groupb Average number of CD4- or CD8-positive cells/field (400x)

12 HPI 24 HPI 3 DPI 5 DPI 7 DPI 10 DPI

CD41

T cells

NC 18 2* 19 2* 23 7* 25 4* 23 5* 28 2

DNA-NC 12 4* 24 4* 26 5* 23 11* 21 5* 18 2*

DNA-CC 19 6* 15 8* 21 5* 24 5* 22 1* 28 2

CC 16 5* 16 6* 93 34 182 11 165 6 112 2

CD81 T cells

NC 23 6* 22 4* 16 3* 25 5* 25 4* 23 2*

DNA-NC 22 5* 32 13* 22 7* 25 8* 23 2* 19 1*

DNA-CC 21 4* 21 3* 29 6 22 7* 25 3* 24 2*

CC 15 7* 20 8* 116 9 202 22 188 14 168 7

*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 fields were

examined for positive cell counts [27]. Data are presented as mean SD for each groupb The definition of each group is given in Table 1

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Chen Et Al., 2011 - IBDV DNA Vaccination