Construction of recombinant baculovirus vaccines for Newcastle disease virus and an assessment of their immunogenicity

Accepted Manuscript Title: Construction of recombinant baculovirus vaccines for Newcastle Disease Virus and an assessment of their immunogenicity Author: Jingping Ge Ying Liu Liying Jin Dongni Gao Chengle Bai Wenxiang Ping PII: DOI: Reference:

S0168-1656(16)30144-4 http://dx.doi.org/doi:10.1016/j.jbiotec.2016.03.037 BIOTEC 7468

To appear in:

Journal of Biotechnology

Received date: Revised date: Accepted date:

25-7-2015 18-3-2016 21-3-2016

Please cite this article as: Ge, Jingping, Liu, Ying, Jin, Liying, Gao, Dongni, Bai, Chengle, Ping, Wenxiang, Construction of recombinant baculovirus vaccines for Newcastle Disease Virus and an assessment of their immunogenicity.Journal of Biotechnology http://dx.doi.org/10.1016/j.jbiotec.2016.03.037 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

*Highlights (for review)

Highlights 

Newcastle Disease Virus (NDV) is an infectious poultry disease with high mortality.



Baculovirus vaccines were engineered expressing the NDV F and HN proteins.



The F-series was more immunogenic and offered better protection than the HN-series.



WPRE and VSV-GED elements increased vaccine immunogenicity and antigen expression.



Internal terminal repeats (ITRs) increased the duration of the cytokine response.

*Manuscript

1

Construction of recombinant baculovirus vaccines for Newcastle Disease Virus

2

and an assessment of their immunogenicity

3

Jingping Ge, Ying Liu, Liying Jin, Dongni Gao, Chengle Bai, Wenxiang Ping*

4

Key Laboratory of Microbiology, College of Life Science, Heilongjiang University,

5

Harbin 150080, China

6 7

*

8

[email protected].

Author

for

correspondence:

Fax:

9

1

+86-0451-86608046;

Email:

10

Abstract

11

Newcastle disease (ND) is a lethal avian infectious disease caused by Newcastle

12

disease virus (NDV) which poses a substantial threat to China's poultry industry.

13

Conventional live vaccines against NDV are available, but they can revert to virulent

14

strains and do not protect against mutant strains of the virus. Therefore, there is a

15

critical unmet need for a novel vaccine that is safe, efficacious, and cost effective.

16

Here, we designed novel recombinant baculovirus vaccines expressing the NDV F or

17

HN genes. To optimize antigen expression, we tested the incorporation of multiple

18

regulatory elements including: (1) truncated vesicular stomatitis virus G protein

19

(VSV-GED), (2) woodchuck hepatitis virus post-transcriptional regulatory element

20

(WPRE), (3) inverted terminal repeats (ITRs) of adeno-associated virus (AAV

21

Serotype Ⅱ), and (4) the cytomegalovirus (CMV) promoter. To test the in vivo

22

efficacy of the viruses, we vaccinated chickens with each construct and characterized

23

the cellular and humoral immune response to challenge with virulent NDV (F48E9).

24

All of the vaccine constructs provided some level of protection (62.5-100%

25

protection). The F-series of vaccines provided a greater degree of protection

26

(87.5-100%) than the HN-series (62.5-87.5%). While all of the vaccines elicited a

27

robust cellular and humoral response subtle differences in efficacy were observed.

28

The combination of the WPRE and VSV-GED regulatory elements enhanced the

29

immune response and increased antigen expression. The ITRs effectively increased

30

the length of time IFN-γ, IL-2, and IL-4 were expressed in the plasma. The F-series

31

elicited higher titers of neutralizing antibody and NDV-specific IgG. The baculovirus 2

32

system is a promising platform for NDV vaccine development that combines the

33

immunostimulatory benefits of a recombinant virus vector with the non-replicating

34

benefits of a DNA vaccine.

35

Keywords: Newcastle disease virus; F gene; HN gene; Baculovirus expression

36

vector system; Immunogenicity

37

3

38

1. Introduction

39

Infectious diseases, including Newcastle disease (ND), cause major economic

40

hardship in the poultry industry. ND is caused by Newcastle disease virus (NDV;

41

Maas et al., 2003), and is characterized by acute morbidity and high mortality (Lam

42

et al., 2011). A means of controlling the spread of NDV is an urgent unmet need in

43

the global poultry industry.

44

Current strategies for preventing ND utilize inactivated and attenuated vaccines.

45

However, it is always possible that immune failure can occur and there will be a

46

resurgence in virulent NDV (Kattenbelt et al., 2006). The F protein is one of the

47

major protective antigens in NDV (White et al., 2008; Yin et al., 2006). It acts as a

48

fusion protein and contributes to viral adsorption (Lamb et al., 2007). The HN protein

49

is the other major protective antigen in NDV. The HN protein combines

50

hemagglutinin (HA) and neuraminidase (NA) activities (Takimoto et al., 2002).

51

Previous studies have achieved a 100% rate of protection by immunizing chickens

52

using a recombinant NDV vaccine containing the F and HN gene using the avian

53

paramyxovirus type III virus (APMV 3) as the vector (Kumar et al., 2011). Similarly,

54

a subunit vaccine developed by Lee et al. using recombinant F and HN protein

55

elicited a good immune response, and the protection rates were 100% and 80%,

56

respectively (Lee et al., 2008). However, it is likely that conventional vaccines will

57

be replaced by genetically engineered vaccines.

58

Compared with other recombinant expression systems, the baculovirus system has

59

distinct advantages. For example, it can accommodate large fragments of exogenous 4

60

genes (Sakaguchi et al., 1998) and post-translationally modify products without

61

causing cytotoxic effects (Li et al., 2009). In addition, baculovirus systems can

62

express multiple genes simultaneously at high levels (Mahonen et al., 2007). The

63

expressed products also retain their biological activity (Hu, 2008). Most notably, the

64

baculovirus expression system is generally considered a very safe way to express

65

exogenous genes.

66

The baculovirus system has been modified in many different ways to optimize the

67

expression of exogenous genes. For example, mammalian cell promoters such as

68

simian

69

enhancer/chickenβactin promoter (CAG) are utilized to optimize the efficiency of

70

exogenous gene expression (Hu, 2006). The CMV promoter is a particularly strong

71

promoter that controls expression from recombinant baculovirus expression

72

platforms in mammalian and poultry cells (Krishnan, 2000). The woodchuck hepatitis

73

virus post-transcriptional regulatory element (WPRE) added to the 3' untranslated

74

region of the expressed gene can also improve the expression efficiency of target

75

gene expression (Donello et al., 1998; Mahonen et al., 2007). The transduction

76

efficiency of the baculovirus system can be increased by displaying a truncated

77

vesicular stomatitis virus G protein (VSV-GED) (Kaikkonen, 2006) on the surface of

78

the baculovirus. Finally, inverted terminal repeats (ITRs) from AAV extend the length

79

of time that target genes are expressed in vivo. Sustained expression has been

80

observed for up to 90 days in vivo from a CMV expression cassette containing

81

adenovirus ITRs (Wang et al., 2006). Thus modified baculovirus systems have the

virus

40

(SV40),

cytomegalovirus

5

(CMV),

and

CMV

early

82

potential to produce large amounts of recombinant proteins in a sustained manner,

83

suggesting they could be an ideal platform for NDV vaccine development.

84

The aim of this study was to investigate the effects of the VSV-GED, WPRE, and

85

ITRs regulatory elements on expression of the NDV F and HN genes controlled by

86

the CMV promoter in a recombinant baculovirus vaccine for NDV. To assess the

87

efficacy of the vaccine we assessed the humoral and cellular immune response in

88

vitro to the F and HN proteins in the presence and absence of each regulatory element.

89

We also assessed the level of target protein expression. Finally, the in vivo efficacies

90

of the constructs were tested in vivo, and the humoral and cellular immune response

91

to vaccination was characterized.

92 93

2. Materials and methods

94

2.1. Ethics Statement

95

All animal experiments were carried out in accordance with the Guidelines for

96

Animal Experiments of the National Institute of Infectious Diseases (NIID, Japan).

97

Experimental protocols were reviewed and approved by the Animal Ethics

98

Committee of Harbin Veterinary Research Institute of the Chinese Academy of

99

Agricultural Sciences (CAAS) and the Animal Ethics Committee of Heilongjiang

100

Province (SYXK (H) 2006-032).

101

2.2. Virus, plasmids, and cells

102

The virulent NDV strain F48E9 was purchased from the China Veterinary

103

Microbiology Culture Collection. The plasmids pLM(-), pLM, pLM-ITRs, 6

104

pTYL-HA-F, and pNDV-HN were obtained from the Key Laboratory of

105

Microbiology in the College of Life Science at Heilongjiang University. The F and

106

HN genes were obtained from plasmids pTYL-HA-F and pNDV-HN using Xho I /

107

Sal I and Xho I / Sph I, respectively. Plasmid pLM(-) contains the CMV promoter

108

and simian virus 40 (SV40) poly(A) ; Plasmid pLM contains the elements of WPRE,

109

VSV-GED, CMV promoter, SV40 poly(A) and gp64 signal peptide (gp64sp)

110

sequence; Plasmid pLM-ITRs contains the elements of ITRs, WPRE, VSV-GED,

111

CMV promoter, SV40 poly(A) and gp64sp sequence. The chicken embryo fibroblast

112

cells and Sf9 insect cells were maintained in our laboratory.

113

2.3. Construction of baculovirus vectors

114

Six (6) plasmids were constructed: (1) pLM(-)-F, (2) pLM-F, (3) pLM-ITRs-F, (4)

115

pLM(-)-HN, (5) pLM-HN and (6) pLM-ITRs-HN. The F or HN genes were

116

individually amplified with primers that also inserted a the His tag (The forward

117

primer for the F gene was 5′-ATCCTCGAGATGGGCTCCAGACCTTCTACC-3′.

118

The

119

5′-GGCGTCGACTCAATGATGATGATGATGAT

120

GCATTTTTGTAGTGGCTCTCATC-3′.

121

5′-ATCCTCGAGATGGACCGCGCAGTTAGC-3′ and the reverse primer for HN

122

was: 5′-CGGGCATGCCTAATGATGATGATGATGATGACCAGACCTGGCTTATCT

123

AACCTAT-3′). F and HN genes were inserted into the plasimids pLM(-), pLM and

124

pLM-ITRs with Xho I / Sal I and Xho I / Sph I endonuclease, respectively.

125

The E.coli DH10 Bac competent cells were prepared using the SEM method (Rong et

reverse

primer

for

The

7

the

F

gene

forward

primer

for

was:

HN

was:

126

al., 2002). The plasmids (pLM(-)-F, pLM-F, pLM-ITRs-F, pLM(-)-HN, pLM-HN and

127

pLM-ITRs-HN, pLM(-), pLM, and pLM-ITRs) were transformed into E.coli DH10

128

Bac competent cells. Positive colonies were identified by blue-white screening and

129

the recombinant Bacmid (rBac) DNA was extracted using the alkaline lysis method.

130

The recombinants were identified as rBac-LM(-)-F, rBac-LM-F, rBac-LM-ITRs-F,

131

rBac-LM(-)-HN, rBac-LM-HN, and rBac-LM-ITRs-HN, rBac-LM(-), rBac-LM, and

132

rBac-LM-ITRs (Fig. 1) by PCR amplification with the M13 primer (CGCCAGGGTT

133

TTCCCAGTCACGAC).

134

2.4. Cell culture

135

Sf9 insect cells were cultured in suspension at 27°C in Sf900II SFM medium

136

containing 10% FBS (Gibco, CA, USA) and 1% antibiotics (100 U/mL penicillin and

137

100 μg/mL streptomycin) in a 0 mL volumes. Primary avian cells were cultured in a

138

6-well plate at 37°C in 11-day-old embryonated specific-pathogen-free (SPF) chicken

139

eggs. The primary avian cells were prepared according to a standard protocol

140

(Spector et al., 1988) and were maintained in Dulbecco’s modified Eagle medium

141

(DMEM, Hyclone, Logan, USA) supplemented with 10% FBS (Gibco, CA, USA), 2

142

mM glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin.

143

A third passage (P3) baculovirus (section 2.5) was used to infect chicken embryo

144

fibroblasts (CEFs) at 80% confluence using a multiplicity of infection (MOI) of 100

145

in the presence of 10mM sodium butyrate in 2 mL of DMEM for 12h. The media

146

containing virus was replaced by fresh DMEM containing 10% FBS and the cells

147

were incubated for an additional 48 h. The cells were pelleted by centrifugation and 8

148

washed three times with PBS then lysed using 200 µL/well cell lysates buffer

149

(Beyotime, Shanghai, China) in a 6-well culture plate.

150

2.5. Generation and titering of recombinant baculovirus

151

Sf9 cells were transfected with the individual baculovirus transfer vectors, such as

152

rBac-LM(-)-F, rBac-LM-F, rBac-LM-ITRs-F, rBac-LM(-)-HN, rBac-LM-HN, and

153

rBac-LM-ITRs-HN, rBac-LM-ITRs, rBac-LM(-), rBac-LM and rBac-LM-ITRs using

154

the liposome-mediated method (Whitt et al., 2001). The co-transfection supernatants

155

were collected after 72 h culture. The viruses were passaged 3 times in Sf9 cells to

156

obtain high titer viral stocks. The viruses were allowed to infect the Sf9 cells

157

(2×106/mL) at room temperature for 30 min, then cultured at 27°C with agitation (70

158

r/min).The culture supernatants from infected cells were collected once 80% of the

159

cells had been infected The same process were repeated to obtain second and third

160

passage virus. The viral genome was extracted and amplified with the M13 universal

161

primer, and each reverse primer, to confirm that the target genes were correctly

162

inserted into the recombinant baculoviruses. The recombinant baculoviruses were

163

named BV-LM(-)-F, BV-LM-F, BV-LM-ITRs-F, BV-LM(-)-HN, BV-LM-HN,

164

BV-LM-ITRs-HN,

165

respectively. The titre of the baculovirus stocks were measured by plaque assay

166

(Burleson etal., 1992). Briefly, Sf9 cells were plated in 6-well plates, and 10-fold

167

serial dilutions of the virus stocks were added to the cells. Viruses and cells were

168

allowed to interact for 1 h before the viruses were removed, and the cell monalayers

169

were overlaid with plaquing medium. The cells were incubated for 8 days, stained

BV-LM-ITRs,

BV-LM(-),

9

BV-LM

and

BV-LM-ITRs,

170

with neutral red, and the numbers of plaques present at each dilution were counted.

171

Viral titers were shown in Table 1.

172

2.6. Expression of the F and HN proteins from chicken embryo fibroblasts

173

The recombinant proteins were detected by SDS-PAGE using a 4% stacking gel and

174

12% separation gel. The expression of recombinant proteins was determined by

175

Western blot. An equivalent volume (30μL) of recombinant protein was loaded in

176

each well of the SDS-PAGE gel. A rabbit anti-His tag antibody (1/100 dilution) was

177

the primary antibody, and a horseradish peroxidase (HRP) conjugated goat anti-rabbit

178

IgG (1/500 dilution) was the secondary antibody (Bioss, Beijing). The Western blot

179

strips were analyzed using a Biology Software Gel-Pro analyzer 4.5 (Media

180

Cybernetics, US). BV-LM(-), BV-LM and BV-LM-ITRs were used as controls. The

181

expression levels of the target protein were compared between baculoviruses.

182

2.7. Chicken immunization

183

The SPF chickens were bred and immunized at the Harbin Veterinary Research

184

Institute. Chickens were housed separately in sterile isolators and provided with

185

standard food and water. The health of the chickens was monitored daily. Fourteen

186

(14)-day old chickens were randomly divided into ten groups containing eight

187

chickens in each group (80 total chickens). The chickens were randomly assigned to

188

be immunized with baculoviruses containing the F protein (cohort A: BV-LM(-)-F, B:

189

BV-LM-F, and C: BV-LM-ITRs-F), the HN protein (D: BV-LM(-)-HN, E:

190

BV-LM-HN,

191

BV-LM-ITRs-F+BV-LM-ITRs-HN). The empty vector (H: BV-LM-ITRs) was used

and

F:

BV-LM-ITRs-

10

HN),

or

a

combination

(G:

192

as the vector control. Additional controls were the Lasota commercial vaccine (cohort

193

J; vaccinated control) and PBS (cohort I; unvaccinated control). For the vaccinated

194

groups: the cohorts vaccinated with baculovirus (A-H) received 109pfu recombinant

195

baculovirus and the vaccinated control group (J) received 0.2 mL of the commercial

196

vaccine. The same doses were administered as a boost 14 days after the first

197

immunization. Blood was drawn from each chicken group at days 14, 28, 42, 56 and

198

70 (Figure 2) to detect their immune response. The timeline was shown in Figure 2.

199

The chickens were challenged with 104 TCID50 of virulent NDV F48E9 14 days after

200

the second immunization. The chickens were monitored for clinical symptoms and

201

physiological changes, and the rate of protection was determined.

202

2.8. Lymphoproliferation assay

203

Lymphoproliferative responses in peripheral blood mononuclear cells (PBMCs) from

204

vaccinated

205

(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay to assess the

206

cellular immune response to stimulation with NDV F48E9 at 49 days. The peripheral

207

bloods of immunized chickens were collected regularly to prepare chicken peripheral

208

T lymphocytes. ConA and NDV F48E9 were used to stimulate lymphocyte

209

proliferation. 100 μL of peripheral blood lymphocytes were inoculated into each well

210

of a 96-well plate. RPMI1640 medium (Hyclone, Logan, USA) containing 50 μL of

211

NDV F48E9 (TCID50=10-4.33/100 μL) was added to the experimental groups.

212

RPMI1640 medium containing ConA (5 μg/mL ) was added to the positive controls,

213

while only RPMI1640 medium was added to the negative control wells. Each group

chickens

were

measured

11

using

the

MTT

214

was done in triplicate. Cells were cultured at 37℃ with 5% of CO2 for 44h. At 44h,

215

10 μL of 5 mg/mL CellTiter 96® Aqueous One Solution (Promega) was added to

216

each well and incubated in the dark for 4h. The OD490 values were obtained using a

217

microplate reader (Gene, US) and the stimulation index (SI) of each group was

218

calculated. The calculated stimulation index (SI) was defined as the ratio of the mean

219

counts per minute (cpm) from the wells incubated with NDV F48E9 to the mean cpm

220

of wells incubated with medium alone. Medium alone was used as the negative

221

control and 5 μg/mL concanavalin A (ConA, Sigma) was used as the positive control.

222

2.9. Serological assays

223

Blood samples were obtained from six randomly selected chickens in each of the

224

immunized groups 0, 7, 14, 21, 28, 35, 42, 49 and 56 days post the first vaccination.

225

The serum was collected by centrifugation for analysis (Kosaka et al., 1998).

226

A serum neutralization test was conducted to determine whether the serum was able

227

to neutralize NDV infection in vitro. The serum collected from the vaccinated

228

chickens was inactivated at 56°C for 30 min and then serially diluted with PBS in

229

96-well plates. For the assay, 70 μL of each diluted serum sample was mixed with an

230

equal amount of F48E9 virus suspension (100 TCID50) in a 96-well plate at 37°C in

231

5% CO2 for l h. The virus/serum mixture was then used to infect 96-well plates of

232

CEFs at 1×106 cells/well at 37°C in 5% CO2 for l h. The controls for the

233

neutralization assay were serum alone and F48E9 virus suspension (100 TCID50)

234

alone. After 1 h, the virus/serum suspension was replaced with DMEM containing

235

10% FBS and the cells were incubated at 37°C for 96 h in 5% CO2. The cellular 12

236

morphology and cytopathic effects were observed every 12 h. The median protective

237

dose (PD50) was determined using the Reed-Muench method (Tukamoto et al., 2002).

238

The PD50 of each serum sample was calculated, as well as the geometric mean titers

239

(GMT) in each group, and the neutralizing antibody titer. NDV-specific serum IgG

240

levels were determined by ELISA (Abcam, UK). The levels of Interleukin (IL)-2,

241

IL-4, and Interferon-γ (IFN-γ) were determined by ELISA (Abcam, UK), and the

242

concentration was calculated using standard linear regression curves (Meng et al.,

243

2001).

244

2.10. Statistical analysis

245

The mean differences between groups were compared and presented as X ± SD using

246

SPSS Statistics19 software (SPSS, US). Statistical significance was assessed by

247

one-way analysis of variance, P < 0.05 was considered significant.

248 249

3. Results

250

3.1. Expression of the NDV F and HN recombinant proteins in CEFs with different

251

regulatory elements

252

We first assessed whether the F and HN proteins were successfully expressed from

253

the baculovirus expression systems, and compared the effect of different regulatory

254

elements (WBRE, ITRs, VSV-GED) on expression levels of the NDV proteins. The

255

cell lysates from the BV-F-series of recombinant baculovirus transduced primary

256

CEF cells (BV-LM(-)-F, BV-LM-F, and BV-LM-ITRs-F) contained a single band of

257

approximately 55 KDa (Fig. 3A (A)). Similarly, the cell lysates from the BV-HN 13

258

(BV-LM(-)-HN, BV-LM-HN, and BV-LM-ITRs-HN) series also contained a single

259

band at approximately 74 KDa (Fig. 3A (B)). The sizes of the bands were consistent

260

with the target proteins F and HN, indicating successful expression from the

261

baculovirus constructs. The protein expression levels from each baculovirus were

262

quantified based on their intensity (Fig. 3B). Compared to the control group

263

BV-LM(-)- F, the expression levels of the F protein from the BV-LM-F and

264

BV-LM-ITRs-F baculoviruses were 3.45 and 3.62 higher, respectively. Similarly,

265

compared to the control group BV-LM(-)-HN the expression levels of the HN protein

266

were 3.99 and 4.60 fold higher from the BV-LM-HN and BV-LM-ITRs-F

267

baculoviruses, respectively. These results indicated that the VSV-GED and WPRE

268

regulatory elements markedly improved the expression of the target protein.

269

3.2. Testing the in vivo efficacy of the F and HN expressing baculovirus vaccines

270

To test the in vivo protective efficacy of the baculovirus constructs, ten groups

271

(n=8/group) of 14-day old chicks were immunized with the F-series, the HN-series,

272

the combined F-ITRs and HN-ITRs constructs, a commercial vaccine, PBS, or

273

BV-LM-ITRs (as a control); and then challenged with a virulent strain of NDV

274

(F48E9). The vaccine and booster (section 2.7) did not elicit any clinical symptoms

275

in any group.

276

Following F48E9 challenge, the chickens in groups H (BV-LM-ITRs; empty vector)

277

and I (PBS) appeared depressed, had a suppressed appetite, and passed white stool. In

278

each group, 4 chickens died 3 days post-challenge. In groups A (BV-LM(-)-F), E

279

(BV-LM-HN), and F (BV-LM-ITRs- HN) 1 chicken/group exhibited signs of 14

280

depression and eventually died. The same symptoms were also observed in two

281

chickens from group D (BV-LM(-)-HN). There were no obvious clinical symptoms

282

following

283

(BV-LM-ITRs-F+BV-LM-ITRs-HN) and J (commercial vaccine) as seen in the

284

survival plot was shown (Figure 4).

285

Five (5) days after the challenge (47 days after chicken birth), the protection rate in

286

each experimental group was calculated (Table 2). The BV-LM-F, BV-LM-ITRs-F,

287

BV-LM-ITRs-F+BV-LM-ITRs-HN, and commercial vaccine groups all achieved

288

100% protection, which was markedly higher than the control group of PBS (12.5%)

289

and empty vector group BV-LM-ITRs (12.5%) (P < 0.05). Taken together, the results

290

indicated that the recombinant baculovirus vaccines could protect against challenge

291

with a virulent NDV strain. In addition, the protection rate of the F-series immunized

292

groups (mean 95.83%) were higher than the HN-series (mean 75%), suggesting that

293

the F protein elicited stronger protective immunity than the HN protein.

294

3.3. Assessing lymphoproliferative responses to NDV F48E9 following vaccination

295

To better understand the mechanism of protection induced by the baculovirus

296

vaccines, we examined the cellular and humoral immune response elicited following

297

challenge. Cellular immunity was measured by assessing the lymphoproliferative

298

responses to F48E9 stimulation in PBMCs collected from vaccinated chickens at 21,

299

36, 49 and 56 days after the first vaccination. ConA was used as the positive control.

300

The proliferative response to F48E9 and ConA was increased in all of the vaccinated

301

chickens following challenge indicating that the recombinant baculovirus vaccine did

challenge

in

groups

B

(BV-LM-F),

15

C

(BV-LM-ITRs-F),

G

302

elicit a cellular immune response. Seven (7) days after challenge (49 days after

303

chicken birth) with F48E9, the SI values were highest in the BV-LM-ITRs-F (2.94),

304

BV-LM-ITRs-F+BV-LM-ITRs-HN (2.830), and commercial vaccine (2.73) groups.

305

The SI index in these groups was significantly higher than the BV-LM(-)-F (2.14)

306

and BV-LM(-)-HN (2.190) groups (P < 0.05; Fig. 5). Thus, recombinant baculovirus

307

vaccines containing the WPRE and VSV-GED regulatory elements significantly

308

improved the level of cellular immunity.

309

To determine whether the increased cellular immunity was persistent, we repeated the

310

lymphoproliferative response assay 42 days post the first vaccination (56 days after

311

the chicken birth). The SI values for groups vaccinated with baculovirus constructs

312

containing ITRs (BV-LM-ITRs-F = 2.910; BV-LM-ITRs-HN = 2.212) and the

313

commercial vaccine (2.78) remained significantly higher than the SI values without

314

ITRs (BV-LM-F= 2.06; BV-LM-HN= 1.78; P < 0.05). These results indicated that

315

ITRs effectively extended the duration of the cellular immune response to the vaccine.

316

The SI values in the BV-LM-ITRs-F+BV-LM-ITRs-HN group, which provided 100%

317

protection and had one of the highest SI values 42 days post the first vaccination, was

318

slightly lower than the BV-LM-ITRs-F group, but significantly higher than the

319

BV-LM-ITRs-HN group (P < 0.05; Fig. 5).

320

3.4 Neutralizing titer assays

321

The ability of serum to neutralize virus infection is an important indicator of humoral

322

immunity. We used a serum neutralization assay to determine the residual infectivity

323

of NDV F48E9 after exposure to immune sera to assess the effects of the F or HN 16

324

antigen, and the different regulatory elements on humoral immunity. As shown in

325

Figure 6, all of the immunizations elicited peak levels of neutralizing antibodies at 28

326

days post the first vaccination (42 days after the chicken birth). However, different

327

vector and antigen combinations did not elicit the same neutralizing antibody titer.

328

For example, constructs containing the VSV-GED and WPRE regulatory elements

329

and expressing the same antigen produced higher titers of neutralizing antibodies

330

than the plasmid without the regulatory elements (pLM (-)); for the F-series, the

331

difference in titer of serum neutralizing antibody (GMT) was 1159.64 compared to

332

413.06, and for the HN-series the difference was 720.17 compared to 383.68 28 days

333

post the first vaccination. Thus, including the VSV-GED and WPRE regulatory

334

elements significantly improved the induction of neutralizing antibodies, consistent

335

with better protection observed in the in vivo challenge.

336

42 days post the first vaccination (56 days after the chicken birth), the levels of

337

neutralizing antibodies from the vectors without ITRs were significantly reduced (P <

338

0.05) compared to the vectors containing the ITRs regardless of F or HN antigen

339

expression. In contrast, vaccination with vectors that contained ITRs elicited

340

neutralizing antibody titers that did not decline significantly 42 days post the first

341

vaccination suggesting that the neutralizing titers elicited by ITRs persisted to 14

342

days.

343

Finally, in general the F antigen vaccine series tended to induce a greater humoral

344

immune response in immunized chickens than the HN antigen series, which was

345

consistent with better in vivo protection in the F48E9 challenge in chickens 17

346

vaccinated with the F-series vaccines.

347

3.5. Detection of NDV-specific IgG titers in immune serum

348

The IgG titer is as an important index of humoral immunity in immunized chickens

349

for a response specific to NDV. The BV-LM-F constructs containing the WPRE and

350

VSV-GED regulatory elements had significantly higher NDV-specific IgG titers

351

(1.673) than constructs without the regulatory elements (1.372; P < 0.05) 42 days

352

post the first vaccination (56 days after the chicken birth). Similarly, the BV-LM-HN

353

constructs containing the WPRE and VSV-GED elements elicited significantly higher

354

NDV-specific IgG titers than constructs without the regulatory elements (1.265 vs.

355

1.131; P < 0.05; Fig. 7). In addition, ITRs elicited high titers of NDV-specific IgG 56

356

days post the first vaccination (70 days after the chicken birth) compared to

357

constructs without ITRs (BV-LM-ITRs-F= 1.560; BV-LM-ITRs-HN= 1.173; P <

358

0.05). When the baculovirus constructs containing the same regulatory elements but

359

expressing different antigen (F or HN) were compared to each other, vaccines

360

containing the F antigen elicited more NDV-specific IgG antibodies than the HN

361

gene.

362

3.6. Cytokine levels in immune serum

363

The cytokine levels in immune sera can be used to measure the immune state of the

364

host. IFN-γ and IL-2 are secreted by T-helper type 1 (Th1) cells and play important

365

roles in regulating the cellular (T cell) immune response. IL-4 is secreted by Th2 cells

366

and can stimulate B cell proliferation, and antibody production involved in the

367

humoral immune response. Thus, by comparing the levels of cytokine production it is 18

368

possible to determine whether the baculovirus constructs are eliciting a

369

predominately Th1 or Th2 response.

370

The concentration of IFN-γ was significantly (P < 0.05) increased at 14 and 28 days

371

post the first vaccination (28 and 42 days after the chicken birth) in the immunized

372

groups compared to the control groups (PBS and BV-LM-ITRs; Fig. 8). The

373

concentration of IFN-γ peaked at 42 days post the first vaccination (56 days after the

374

chicken birth) in all of the immunized groups. The highest concentrations of IFN-γ

375

were

376

BV-LM-ITRs-F+BV-LM-ITRs-HN (69.65 ng/mL) groups.

377

As shown in Figure 9, the mean level of IL-2 increased significantly in all of the

378

groups, except the control groups (P < 0.05); providing further evidence that the

379

baculovirus constructs elicited a robust cellular immune response. The levels of IL-2

380

were significantly higher than the control groups in the commercial vaccine (74.65

381

ng/mL), BV-LM-ITRs-F (71.26 ng/mL), and BV-LM-ITRs-F+BV-LM-ITRs-HN

382

(63.08 ng/mL) groups 28 days post the first vaccination (42 days after the chicken

383

birth). Different combinations of antigen genes and regulatory elements elicited

384

different levels of IL-2 production. In constructs expressing the same antigen, the

385

levels of IL-2 production were greater when constructs contained the WPRE and

386

VSV-GED regulatory elements than without (F-series: 61.62 ng/mL vs. 33.34 ng/mL;

387

HN-series: 37.69 ng/mL vs. 28.13 ng/mL).

388

With regard to IL-4, the highest levels of IL-4 were observed in the commercial

observed

in

commercial

vaccine

19

(72.57

ng/mL)

and

the

389

vaccine

group

(P<0.05);

followed

by

the

F

antigen

gene

series,

390

BV-LM-ITRs-F+BV-LM-ITRs-HN, the HN antigen gene series, and the controls

391

(PBS, BV-LM-ITRs; Fig. 10A). Similar to IFN-γ and IL-2, the levels of IL-4 were

392

highest in the groups vaccinated with baculovirus constructs containing the WPRE

393

and VSV-GED elements.

394

We also assessed the cytokine levels at 70 days to determine whether the vaccines

395

had any lasting effects on cytokine production. The IFN-γ, IL-2, and IL-4 levels in

396

the BV-LM-ITRs-F (55.02ng/mL, 44.72 ng/mL, 106.84 ng/mL, respectively) and

397

BV-LM-ITRs-HN (42.03ng/mL, 21.40ng/mL, 66.74ng/mL, respectively) were

398

significantly (P < 0.05) elevated compared to the groups without ITRs (BV-LM-F:

399

24.53ng/mL, 17.50ng/mL, 27.21ng/mL; BV-LM-HN: 20.48ng/mL, 13.85ng/mL,

400

16.54ng/mL).

401

Taken together, the cytokine results provided further evidence that the baculovirus

402

constructs elicited a robust cellular and humoral response. In addition, the ITRs

403

consistently improved the magnitude and duration of the cellular and humoral

404

immune response.

405 406

4. Discussion

407

NDV has no effective treatment in part due to the reemergence of virulent strains

408

(Alexander, 2011). Vaccination is the best strategy for preventing and controlling

409

NDV spread (Wu et al., 2006).

410

vaccine series using the F and HN proteins of NDV that efficiently express the

Here, we have developed a novel baculovirus

20

411

antigen target and elicit a robust immune response. Baculovirus constructs containing

412

the WPRE, VSV-GED, and ITR elements can be directly injected into chickens. The

413

high neutralizing antibody titer, increased IL-4 levels, and increased IFN-γ, and IL-2

414

levels indicated that the baculovirus vaccine had the dual advantages of recombinant

415

viral vector vaccines and DNA vaccines. The vaccines effectively delivered

416

exogenous antigen to the poultry cells and stimulated the production of humoral and

417

cellular immune responses.

418

In terms of optimizing the vaccine constructs, the regulatory elements WPRE and

419

VSV-GED increased the amount of antigen protein (F or HN) expressed compared to

420

constructs without these elements. Vaccines containing ITRs also appeared to elicit a

421

longer lasting immunity (up to 70 days) than constructs without ITRs. The SI values

422

(BV-LM-ITRs-F: 2.910

423

neutralizing antibodies (BV-LM-ITRs-F: 1337.74

424

56 days, and the IgG titer (BV-LM-ITRs-F: 1.560

425

70 days indicated that the ITR elements were helpful to maintain the immune level.

426

In addition, the results of the cytokine levels also demonstrated that the ITRs aided in

427

long-lasting cytokine production. The IFN-γ, IL-2, and IL-4 levels in the

428

BV-LM-ITRs-F (55.02ng/mL, 44.72 ng/mL, and 106.84 ng/mL, respectively) and

429

BV-LM-ITRs-HN (42.03ng/mL, 21.40ng/mL, and 66.74ng/mL, respectively) were

430

significantly (P < 0.05) elevated compared to the groups without ITRs (BV-LM-F:

431

24.53ng/mL, 17.50ng/mL, and 27.21ng/mL; BV-LM-HN: 20.48ng/mL, 13.85ng/mL,

432

and 16.54ng/mL) at 70 days. In other studies, AAV-ITRs have been shown to reduce

VS

BV-LM-F: 2.056; P < 0.05) at 56 days, the level of

21

VS

BV-LM-F: 274.30; P < 0.05) at

VS

BV-LM-F: 0.635; P < 0.05) at

433

expression heterogeneity (Hsiao et al., 2001). Our work supports the benefits of using

434

ITRs. Finally, the F antigen elicited a better immune response and provided a greater

435

degree of protection in the in vivo challenge than the HN antigen, which was

436

consistent with previous findings (Kumar et al., 2011). The HN protein series

437

provided the lowest average rate of protection in the in vivo challenge (75.0%). Both

438

the F-series (95.8%) and the combination of the F and HN vaccines (100%) provided

439

superior protection. The protection rate of the combination was similar to the live

440

vaccine (Colman et al., 2003).

441 442

5. Conclusions

443

The baculovirus vectors described here have the dual advantages of mimicking

444

natural virus infection by a recombinant viral vector vaccine and the non-replicating

445

characteristic of a DNA vaccine. Therefore, development of genetically engineered

446

vaccines will play an important role in ND prevention and treatment.

447 448

Competing interests

449

The authors declare that they have no competing interests.

450 451

Acknowledgments

452

This work was supported by grants from the National Natural Science

453

Foundation of China (31470537), the National Natural Science Foundation of China

454

(31270534), the National Natural Science Foundation of China (31270143), and the 22

455

National Science Foundation for Distinguished Young Scholars of China (31570492),

456

the Innovation Team in Science and Technology of Heilongjiang Province (the

457

Fermentation Technology of Agricultural Microbiology, 2012td009).

458

23

459

References

460

Alexander, D.J., 2011. Newcastle disease in the European Union 2000 to 2009. Avian

461 462 463

Pathology 40, 547-558. Burleson, F.G., Chambers, T.M., Wiedbrauk, D.L., 1992. Plaque Assays. Virology, 16, 74-84.

464

Donello, J.E., Loeb, J.E., Hope, T.J., 1998. Woodchuck hepatitis virus contains a

465

tri-partite post-transcriptional regulatory element. Journal of Virology 72,

466

5085-5092.

467

Hsiao, C.D., Hsieh, F.J., Tsai, H.J., 2001. Enhanced expression and stable

468

transmission of transgenes flanked by inverted terminal repeats from

469

adeno-associated virus in zebrafish. Journal Virology 323, 220-232.

470 471

472 473

474 475

Hu, Y.C., 2006. Bzculovirus vectors for gene therapy. Advances in Viurs Research 68, 287-320. Hu, Y.C., 2008. Baculoviral Vectors for Gene Delivery: A Review. Current Gene Therapy 8, 54-65. Kattenbelt, J.A., Stevens, M.P., Gould, A.R, 2006. Sequence variation in the Newcastle disease virus genome. Virus Research 116, 168-184.

476

Kosaka, T., Maeda, T., Nakada, Y., Yukawa, M., Tanaka, S., 1998. Effect of Balillus

477

subtilis spore administration on activation of macrophages and natural killer

478

cells in mice. Veterinary Microbiology 60, 215-225.

24

479

Kumar, S., Nayak, B., Collins, P.L., Samal, S.K., 2011. Evaluation of the Newcastle

480

disease virus F and HN proteins in protective immunity by using a recombinant

481

avian paramyxovirus type 3 vector in chickens. Journal of Virology 85,

482

6521-6534.

483 484

Krishnan, B.R., 2000. Current status of DNA vaccines in veterinary medicine. Advanced Drug Delivery Review 43, 3-11.

485

Kaikkonen, M.U., Raty, J.K., Airenne, K.J., Wirth, T., Heikura, T., Yia-Herttuala, S.,

486

2006. Truncated vesicular stomatitis virus G protein improves baculovirus

487

transduction efficiency in vitro and in vivo. Gene Therapy 13, 304-312.

488

Kumar, S., Nayak, B., Collins, P.L., Samal, S.K., 2011. Evaluation of the Newcastle

489

disease virus F and HN proteins in protective immunity by using a recombinant

490

avian paramyxovirus type 3 vector in chickens. Journal of Virology 85,

491

6521-6534.

492 493

Lamb, R.A., Jardetzky, T.S., 2007. Structural basis of viral invasion: lessons from paramyxovirus F. Current Opinion in Structural Biology 17, 427-436.

494

Lam, H.Y., Yeap, S.K., Rasoli, M., Rasoli, M., Omar, A.R., Yusoff, K., Suraini, A.A.,

495

Alitheen, N.B, 2011. Safety and clinical usage of Newcastle disease virus in

496

cancer therapy. Journal of Biomedicine and Biotechnology 2011, 1-13.

497

Lee, Y.J., Sung, H.W., Choi, J.G., Lee, E.K., Yoon, H., Kim, J.H., Song, C.S., 2008.

498

Protection of chickens from Newcastle disease with a recombinant baculovirus

499

subunit vaccine expressing the fusion and hemagglutinin-neuraminidase proteins. 25

500

Journal of Veterinary Science 9, 301-308.

501

Li, Y.M., Ye, J., Cao, S.B., Xiao, S.B., Zhao, Q., Liu, X.Q., Meilin, J., Chen, H.C.,

502

2009. Immunization with pseudotype baculovirus expressing envelope protein

503

of Japanese encephalitis virus elicits protective immunity in mice. The Journal

504

of Gene Medicine 11, 57-65.

505

Maas, R.A., Komen, M., Van, Diepen, M., Oei, H.L., Classen, I.J.T.M., 2003.

506

Correlation of haemagglutinin- neuraminidase and fusion protein content with

507

protective antibody response after immunisation with inactivated Newcastle

508

disease vaccines. Vaccine 21, 3137-3142.

509

Mahonen, A.J., Airenne, K.J., Purola, S., Peltomaa, E., Kaikkonen, M.U., Riekkinen,

510

M.S., Heikura, T., Kinnunen, K., Roschier, M.M., Wirth, T., Yia-Herttuala, S.,

511

2007. Post-transcriptional regulatory element boosts baculovirus-mediated gene

512

expression in vertebrate cells. Biotechnol 131, 1-8.

513

Meng, Y., Huang M, Y., Wang, S.L., 2001. The overview of Newcastle disease

514

detection technology research. Animal husbandry and veterinary in guizhou 25,

515

12-14.

516

Rong, J., Cheng, T., Liu, X., Jiang, T., Gu, H., Zou, G., 2002. Development of

517

recombinant VP2 vaccine for the prevention of infectious bursal disease of

518

chickens. Vaccine 23, 4822-4851.

519

Sakaguchi, M., Nakamura, H., Sonoda, K., Okamura, H., Yokogawa, K., Matsuo, K.,

520

Hira, K., 1998. Protection of chickens with or without maternal antibodies 26

521

against both Marek’s and Newcastle diseases by one time vaccination with

522

recombinant vaccine of Marek’ s disease virus type 1. Vaccine 16, 472-479.

523

Spector, D.L., Goldman, R.D., Leinwand, L.A., 1988. Cells. Cold Spring Harbor

524

Laboratory Press. New York.

525

Tukamoto, K., Salto, S., Saeki, S., 2002. Complete long-lasting protection against

526

lethal inefetious bursal disease virus challenge by a single vaccination with an

527

avian herpesvirus vector expressing VP2 antigens. Virology 76, 5637-5645.

528

Takimoto, T., Taylor, G.L., Connaris, H.C., Crennell, S.J., Portner, A., 2002. Role of

529

the

hemagglutinin-

neuraminidase

protein

in

the

mechanism

of

530

paramyxovirus-cell membrane fusion. Journal of Virology 76, 13028-13033.

531

Wang, C.Y., Li, F., Yang, Y., Guo, H.Y., Wu, C.X., Wang, S., 2006. Recombinant

532

baculovirus containing the diphtheria toxin A gene for malignant glioma therapy.

533

Cancer Research 66, 5798-5806.

534

White, J.M., Delos, S.E., Brecher, M., Schornberg, K., 2008. Structures and

535

mechanisms of viral membrane fusion proteins: multiple variations on a

536

common theme. Critical Reviews in Biochemistry and Molecular Biology 43,

537

189-219.

538 539

540

Whitt, M., Buonocore, L., Rose, J.K., 2001. Liposome-mediated transfection. Current protocols in immunology 10:10.16.1-10.16.4. Wu, M.H., Ling, W.Z., 2006. The research of Newcastle disease. The technology 27

541

information of animal husbandry and veterinary 3, 17-18.

542

Yin, H.S., Wen, X., Paterson, R.G., Lamb, R.A., Jardetzky, T.S., 2006. Structure of

543

the parainfluenza virus 5 F protein in its metastable, prefusion conformation.

544

Nature 439, 38-44.

545

546

547

548

549

550

551

552

553

28

554

Figure Legends

555

Fig 1. Plasmid constructions. The F or HN genes were individually inserted under

556

the control of the CMV promoter. The gp64SP and VSV-GED expression cassettes

557

were inserted under the polyhedrin promoter. WPRE expression cassettes including

558

the F or HN genes were controlled by the CMV promoter. ITRs: Adeno-associated

559

virus inverted terminal repeats are from the plasmid of pAAV-LacZ (AAV Serotype

560

Ⅱ); SV40 polyA: Simian virus 40 polyA; WPRE: Woodchuck hepatitis virus

561

post-transcriptional regulatory element; PPH: Polyhedrin promoter; gp64SP: gp64

562

signal peptide; VSV-GED: Truncated vesicular stomatitis virus G protein; polyhedrin

563

locus is based on the pFastbac[PH] baculovirus vector of the Bac-to-Bac system.

564

Plasmid pLM(-) contains the CMV promoter and the simian virus 40 (SV40) poly(A).

565

Plasmid pLM contains the WPRE, VSV-GED, CMV promoter, SV40 poly(A) and

566

gp64 signal peptide (gp64sp) sequences. Plasmid pLM-ITRs contains the AAV ITRs,

567

WPRE, VSV-GED, CMV promoter, SV40 poly(A) and gp64sp sequences..

568

569

Fig. 2 Schematic of the Experimental Timeline (since the chicken birth). 14-day

570

old chickens were administered recombinant baculovirus vaccine. The same doses

571

were administered as a boost 14 days after the first immunization. Chickens were

572

challenged with NDV F48E9 14 days after the second immunization.

573

29

574

Fig. 3 Detection of F and HN proteins by Western blot from Cells Transduced

575

with BV-F and BV-HN. Recombinant proteins were detected using a rabbit anti-His

576

tag antibody (1/100 dilution) as the primary antibody and a horseradish peroxidase

577

(HRP) labeled goat anti-rabbit IgG (1/500 dilution) as the secondary antibody. (A)

578

Protein expression using recombinant baculovirus BV-F-series; (B) Protein

579

expression using recombinant baculovirus of BV-HN-series. β-actin was used as

580

control and the molecular weight of F and HN protein were about 55 and 74 KDa. In

581

Fig 3B, the level of protein expression was quantified using the Biology Software

582

Gel-Pro analyzer 4.5 to analyze Western blotting strips. *** indicates a significant

583

difference (P<0.001) between the constructs without regulatory elements

584

(BV-LM(-)-F or BV-LM(-)-HN) and the constructs with regulatory elements

585

(BV-LM-F, BV-LM-ITRs-F or BV-LM-HN, BV-LM-ITRs-HN).

586

587

Fig. 4 Survival plot of the immunized chickens post challenge with F48E9. The

588

number of survivors post-challenge vaccinated with: A, BV-LM(-)-F; B, BV-LM-F; C,

589

BV-LM-ITRs-F; D, BV-LM(-)-HN; E, BV-LM-HN; F, BV-LM-ITRs- HN; G,

590

BV-LM-ITRs-F+BV-LM-ITRs-HN; H, BV-LM-ITRs; J, vaccinated control; I,

591

unvaccinated control.

592

593

Fig. 5 Lymphocyte proliferation at 49 and 56 days. PBMCs were isolated from the

594

blood of chickens one week after infection with NDV F48E9. T cell proliferation in

595

vaccinated chickens was measured in response to F48E9 or concanavlin A (ConA).

596

Bars indicate the mean (±SEM) stimulation index calculated for each group of 30

597

animals. The bar with slash is NDV while the black bar is ConA.

598

599

Fig. 6 Induction of neutralizing antibodies in the serum following vaccination

600

with recombinant baculovirus vaccines. Serum was isolated from the blood of

601

chickens every two weeks following the initial vaccination with recombinant

602

baculovirus vaccine. (A) The neutralizing antibody titers elicited by the F-series of

603

recombinant baculovirus vaccines; (B) The neutralizing antibody titers elicited by the

604

HN-series of recombinant baculovirus vaccines. Line chart represents the mean

605

(±SEM) neutralizing antibody titers calculated for each group of animals (n=8). A)

606

hollow circle: BV-LM(-)-F; solid circle: BV-LM-F; hollow diamond: BV-LM-ITRs-F;

607

solid diamond: Lasota vaccine; hollow triangle: BV-LM-ITRs; solid triangle: PBS. B)

608

hollow circle: BV-LM(-)-HN; solid circle: BV-LM-HN; hollow diamond:

609

BV-LM-ITRs-HN; solid diamond: BV-LM-ITRs-F+BV-LM-ITRs-HN; hollow

610

triangle: PBS; solid triangle: BV-LM-ITRs.

611

612

Fig. 7 Serum IgG antibody titers following vaccination with recombinant

613

baculovirus vaccines. IgG antibodies were detected in individual chickens by

614

ELISA. Data are reported as the OD450nm for each sample of immunized serum

615

collected every two weeks. (A) IgG antibody level elicited by the F-series of

616

baculovirus vaccines and the control groups; (B) IgG antibody level elicited by the

617

HN-series baculovirus vaccines and control groups. A) hollow circle: BV-LM(-)-F; 31

618

solid circle: BV-LM-F; hollow diamond: BV-LM-ITRs-F; solid diamond: Lasota

619

vaccine; hollow triangle: BV-LM-ITRs; solid triangle: PBS. B) hollow circle:

620

BV-LM(-)-HN; solid circle: BV-LM-HN; hollow diamond: BV-LM-ITRs-HN; solid

621

diamond: BV-LM-ITRs-F+BV-LM-ITRs-HN; hollow square: BV-LM-ITRs; solid

622

triangle: PBS.

623

624

Fig. 8 Serum IFN-γ concentration following vaccination with recombinant

625

baculovirus vaccines. The IFN-γ concentration present in the blood of chickens was

626

measured by IFN-γ ELISA assay at each time point. (A) IFN-γ concentration in

627

immune serum from chickens immunized with the F-series baculovirus vaccines and

628

the control groups; (B) IFN-γ concentration in immune serum from chickens

629

immunized with the HN-series of baculovirus vaccines and control groups. A) solid

630

circle: BV-LM(-)-F; hollow diamond: BV-LM-F; hollow circle: BV-LM-ITRs-F;

631

solid diamond: Lasota vaccine; solid triangle: BV-LM-ITRs; hollow square: PBS. B)

632

solid circle: BV-LM(-)-HN; hollow circle: BV-LM-HN; hollow diamond:

633

BV-LM-ITRs-HN; solid diamond: BV-LM-ITRs-F+BV-LM-ITRs-HN; solid triangle:

634

BV-LM-ITRs; hollow square: PBS.

635

636

Fig. 9 Serum IL-2 concentration following vaccination with recombinant

637

baculovirus vaccines. The IL-2 concentration present in the blood of chickens was

638

measured by IL-2 ELISA assay at each time point. (A) IL-2 concentration in immune 32

639

serum from chickens immunized with the F-series baculovirus vaccines and the

640

control groups; (B) IL-2 concentration in immune serum from chickens immunized

641

with the HN-series of baculovirus vaccines and control groups. A) hollow diamond:

642

BV-LM(-)-F; solid circle: BV-LM-F; hollow circle: BV-LM-ITRs-F; solid diamond:

643

Lasota vaccine; solid triangle: BV-LM-ITRs; hollow square: PBS. B) hollow circle:

644

BV-LM(-)-HN; hollow square: BV-LM-HN; solid square: BV-LM-ITRs-HN; solid

645

square: BV-LM-ITRs-F+BV-LM-ITRs-HN; solid triangle: BV-LM-ITRs; hollow

646

triangle: PBS.

647

648

Fig. 10 Serum IL-4 concentration following vaccination with recombinant

649

baculovirus vaccines. The IL-4 concentration present in the blood of chickens was

650

measured by IL-4 ELISA assay at each time point. (A) IL-4 concentration in immune

651

serum from chickens immunized with the F-series baculovirus vaccines and the

652

control groups; (B) IL-4 concentration in immune serum from chickens immunized

653

with the HN-series of baculovirus vaccines and control groups. A) solid cirlcle:

654

BV-LM(-)-F; hollow triangle: BV-LM-F; hollow circle: BV-LM-ITRs-F; solid square:

655

Lasota vaccine; hollow square: BV-LM-ITRs; solid triangle: PBS. B) hollow circle:

656

BV-LM(-)-HN; solid circle: BV-LM-HN; hollow diamond: BV-LM-ITRs-HN; solid

657

diamond: BV-LM-ITRs-F+BV-LM-ITRs-HN; solid triangle: BV-LM-ITRs; hollow

658

square: PBS.

33

Figure

Fig 1. Plasmid constructions. The F or HN genes were individually inserted under the control of the CMV promoter. The gp64SP and VSV-GED expression cassettes were inserted under the polyhedrin promoter. WPRE expression cassettes including the F or HN genes were controlled by the CMV promoter. ITRs: Adeno-associated virus inverted terminal repeats are from the plasmid of pAAV-LacZ (AAV Serotype Ⅱ); SV40 polyA: Simian virus 40 polyA; WPRE: Woodchuck hepatitis virus post-transcriptional regulatory element; PPH: Polyhedrin promoter; gp64SP: gp64 signal peptide; VSV-GED: Truncated vesicular stomatitis virus G protein; polyhedrin locus is based on the pFastbac[PH] baculovirus vector of the Bac-to-Bac system. Plasmid pLM(-) contains the CMV promoter and the simian virus 40 (SV40) poly(A). Plasmid pLM contains the WPRE, VSV-GED, CMV promoter, SV40 poly(A) and gp64 signal peptide (gp64sp) sequences. Plasmid pLM-ITRs contains the AAV ITRs, WPRE, VSV-GED, CMV promoter, SV40 poly(A) and gp64sp sequences.

Figure

Fig. 2 Timeline of the chicken immunization. 14-day old chickens were administered recombinant baculovirus vaccine. The same doses were administered as a boost 14 days after the first immunization. Chickens were challenged with NDV F48E9 14 days after the second immunization.

Figure

Fig. 3 Detection of F and HN proteins by Western blot from Cells Transduced with BV-F and BV-HN. Recombinant proteins were detected using a rabbit anti-His tag antibody (1/100 dilution) as the primary antibody and a horseradish peroxidase (HRP) labeled goat anti-rabbit IgG (1/500 dilution) as the secondary antibody. (A) Protein expression using recombinant baculovirus BV-F-series; (B) Protein expression using recombinant baculovirus of BV-HN-series. -actin was used as control and the molecular weight of F and HN protein were about 55 and 74 KDa. In Fig 3B, The level of protein expression was quantified using the Biology Software Gel-Pro analyzer 4.5 to analyze Western blotting strips. *** indicates a significant difference

(P<0.001)

between

the

constructs

without

regulatory

elements

(BV-LM(-)-F or BV-LM(-)-HN) and the constructs with regulatory elements (BV-LM-F, BV-LM-ITRs-F or BV-LM-HN, BV-LM-ITRs-HN).

Figure

Fig. 4 Survival plot of the immune chicken post challenge with F48E9. The number of survivors post-challenge vaccinated with: A, BV-LM(-)-F; B, BV-LM-F; C, BV-LM-ITRs-F; D, BV-LM(-)-HN; E, BV-LM-HN; F, BV-LM-ITRs- HN; G, BV-LM-ITRs-F+BV-LM-ITRs-HN; H, BV-LM-ITRs; J, vaccinated control; I, unvaccinated control.

Figure

Fig. 5 Lymphocyte proliferation at 49 and 56 days. PBMCs were isolated from the blood of chickens one week after infection with NDV F48E9. T cell proliferation in vaccinated chickens was measured in response to F48E9 or concavalin A (ConA). Bars indicate the mean (±SEM) stimulation index calculated for each group of animals. The bar with slash is NDV while the black bar is ConA.

Figure

Fig. 6 Induction of neutralizing antibodies in the serum following vaccination with recombinant baculovirus vaccines. Serum was isolated from the blood of chickens every two weeks following the initial vaccination with recombinant baculovirus vaccine. (A) The neutralizing antibody titers elicited by the F-series of recombinant baculovirus vaccines; (B) The neutralizing antibody titers elicited by the HN series of recombinant baculovirus vaccines. Line chart represents the mean (±SEM) neutralizing antibody titers calculated for each group of animals (n=8). A) hollow circle: BV-LM(-)-F; solid circle: BV-LM-F; hollow diamond: BV-LM-ITRs-F; solid diamond: Lasota vaccine; hollow triangle: BV-LM-ITRs; solid triangle: PBS. B) hollow circle: BV-LM(-)-HN; solid circle: BV-LM-HN; hollow diamond: BV-LM-ITRs-HN;

solid

diamond:

BV-LM-ITRs-F+BV-LM-ITRs-HN;

hollow

triangle: PBS; solid triangle: BV-LM-ITRs.

Figure

Fig. 7 Serum IgG antibody titers following vaccination with recombinant baculovirus vaccines. IgG antibodies were detected in individual chickens by ELISA. Data are reported as the OD450nm for each sample of immunized serum collected every two weeks. (A) IgG antibody level elicited by the F-series of baculovirus vaccines and the control groups; (B) IgG antibody level elicited by the HN-series baculovirus vaccines and control groups. A) hollow circle: BV-LM(-)-F; solid circle: BV-LM-F; hollow diamond: BV-LM-ITRs-F; solid diamond: Lasota vaccine; hollow triangle: BV-LM-ITRs; solid triangle: PBS. B) hollow circle: BV-LM(-)-HN; solid circle: BV-LM-HN;

hollow

diamond:

BV-LM-ITRs-HN;

solid

diamond:

BV-LM-ITRs-F+BV-LM-ITRs-HN; hollow square: BV-LM-ITRs; solid triangle: PBS.

Figure

Fig. 8 Serum IFN-γ concentration following vaccination with recombinant baculovirus vaccines. The IFN-γ concentration present in the blood of chickens was measured by IFN-γ ELISA assay at each time point. (A) IFN-γ concentration in immune serum from chickens immunized with the F-series baculovirus vaccines and the control groups; (B) IFN-γ concentration in immune serum from chickens immunized with the HN-series of baculovirus vaccines and control groups. A) solid circle: BV-LM(-)-F; hollow diamond: BV-LM-F; hollow circle: BV-LM-ITRs-F; solid diamond: Lasota vaccine; solid triangle: BV-LM-ITRs; hollow square: PBS. B) solid circle:

BV-LM(-)-HN;

hollow

circle:

BV-LM-HN;

hollow

diamond:

BV-LM-ITRs-HN; solid diamond: BV-LM-ITRs-F+BV-LM-ITRs-HN; solid triangle:

BV-LM-ITRs; hollow square: PBS. .

Figure

Fig. 9 Serum IL-2 concentration following vaccination with recombinant baculovirus vaccines. The IL-2 concentration present in the blood of chickens was measured by IL-2 ELISA assay at each time point. (A) IL-2 concentration in immune serum from chickens immunized with the F-series baculovirus vaccines and the control groups; (B) IL-2 concentration in immune serum from chickens immunized with the HN-series of baculovirus vaccines and control groups. A) hollow diamond: BV-LM(-)-F; solid circle: BV-LM-F; hollow circle: BV-LM-ITRs-F; solid diamond: Lasota vaccine; solid triangle: BV-LM-ITRs; hollow square: PBS. B) hollow circle: BV-LM(-)-HN; hollow square: BV-LM-HN; solid square: BV-LM-ITRs-HN; solid square: BV-LM-ITRs-F+BV-LM-ITRs-HN; solid triangle: BV-LM-ITRs; hollow triangle: PBS.

Figure

Fig. 10 Serum IL-4 concentration following vaccination with recombinant baculovirus vaccines. The IL-4 concentration present in the blood of chickens was measured by IL-4 ELISA assay at each time point. (A) IL-4 concentration in immune serum from chickens immunized with the F-series baculovirus vaccines and the control groups; (B) IL-4 concentration in immune serum from chickens immunized with the HN-series of baculovirus vaccines and control groups. A) solid cirlcle: BV-LM(-)-F; hollow triangle: BV-LM-F; hollow circle: BV-LM-ITRs-F; solid square: Lasota vaccine; hollow square: BV-LM-ITRs; solid triangle: PBS. B) hollow circle: BV-LM(-)-HN; solid circle: BV-LM-HN; hollow diamond: BV-LM-ITRs-HN; solid diamond: BV-LM-ITRs-F+BV-LM-ITRs-HN; solid triangle: BV-LM-ITRs; hollow square: PBS.

Table 1: Titers of P3 recombinant baculovirus vaccine strains in Sf9 cells Number of Virus name-P3

Virus titer (pfu/mL)

Dilution plaques

BV-LM(-)-F

1.90 ±0.26 ×108

10-7

19

BV-LM-F

1.11 ±0.82×109

10-7

111

BV-LM-ITRs-F

4.90 ±0.41×108

10-7

49

BV-LM(-)-HN

8.60 ±0.97×108

10-7

86

BV-LM-HN

1.00 ±0.34×109

10-7

100

BV-LM-ITRs-HN

6.10 ±0.23×108

10-7

61

Table. 2 Rate of protection following baculovirus immunization and challenge with F48E9 No. deaths after challenge with F48E9 Protection Cohorts

Vaccines

No. deaths / 2d

3d

4d

rate

5d total number

A

BV-LM(-)-F

0

0

0

1

1/8

87.5%

B

BV-LM-F

0

0

0

0

0/8

100%

C

BV-LM-ITRs-F

0

0

0

0

0/8

100%

D

BV-LM(-)-HN

0

0

1

2

3/8

62.5%

E

BV-LM-HN

0

0

1

1

2/8

75.0%

F

BV-LM-ITRs-HN

0

0

0

1

1/8

87.5%

0

0

0

0

0/8

100%

BV-LM-ITRs-F+ G BV-LM-ITRs-HN J

Lasota attenuated

0

0

0

0

0/8

100%

H

BV-LM-ITRs

1

4

2

0

7/8

12.5%

I

PBS

2

4

1

0

7/8

12.5%