Expression of FMDV Expression of FMDV CapsidCapsid Proteins in Proteins in SilkwormSilkworm--BaculovirusBaculovirus Expression system Expression system

and Its Utilization as a Subunit Vaccineand Its Utilization as a Subunit Vaccine

Biotechnology Research Institute, Lanzhou Veterinary Research Institute,

Chinese Academy of Agricultural Sciences, 12 Zhongguancun South Street, Beijing, China

Post Code: 100081, Tel: +86-10-68919854, Fax: +86-10-68975136

E-mail: zhangzf@caas.net.cn

Naming of The Naming of The BaculovirusBaculovirus The name of the Baculovirus comes from Greek.

“baculo” means stick in Greek. The Baculoviridae includes two genus:

Nucleopolyhedrovirus The another genus: Granulovirus. The genomic DNA of the Baculovirus was

supercoiled double strands circular DNA, about 80~180Kb in size. So, It can be expressed large foreign DNA fragment(~10kb) in this system.

1. Accommodate the large foreign gene fragment (~10Kb) .

2. ploh and p10 are both unnecessary genes of the Baculovirus. But they both have strong promoter activities.

3. ploh is a very late gene, so the product would be able to expression, even it is a cytotoxic .

4. It is a safe expression system to human and entironment.

Characteristic of BES-Silkworm:

5. Even the recombinant virus has a strong infecting activity on both the cells and the larva. So mass product might be expected .

6. Unlike prokaryotic expression system, product expressed by BES has a good way of after-processing.

7. Expression of fusion protein and polyprotein are permitted.

8. BES can express foreign genes from virus, bacteria, archaeon, fungi, plant or animal.

9. The cost of Bm-BES is very cheap.

hGM-CSF HCV-core antigen EGF: Gastricism IGF-I rabies virus: Mice were vaccinated orally (by bait),

gastrically (by stomach tube) with a recombinant virus expressing the rabies glycoprotein and nucleoprotein, IgAantibodies against rabies virus were detected at two weeks postvaccination. After challenge at one month postvaccination with CVS rabies virus, all mice that had developed antibodies were protected. It suggests that this recombinant virus might be a suitable vaccine for oral immunization against rabies.

10. The product could be taken orally: Edible

The Schematic protocol of Baculovirus-Silkworm Expression System

Silkworm rearing with artificial feedSilkworm rearing with artificial feed

Construction of Different FMDV Gene’s Combination for Expression in Silkworm

P12A3C

Expression of FMDV polypeptides in Expression of FMDV polypeptides in BmBm--N cells was N cells was analysedanalysed by IFATby IFAT

Bm-P12A3C BmBacPAK-6

Produced FMDV Virus Like Particles in Silkworm

Expression of FMDV polypeptides in silkworm larvae was estimated by the sandwich-ELISA

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1/2

1/4

1/8

1/16

1/32

1/64

1/128

1/256

1/512

1/1024

1/2048

1/4096

1/8192

1/16384

1/32768

1/65536

OD(492nm)

Expression of FMDV polypeptides in silkworm larvae was estimated by the sandwich-ELISA.

0.070

0.682

0.0540

1:16384

0.084

0.821

0.063

1:8192

0.087

0.995

0.085

1:4096

0.0630.0560.1500.1040.1130.1150.148

0.1710.241Negative control

1.1141.0831.2751.2841.3311.3261.245

1.3731.404Haemoly-mph with Bm-P12A3C

0.0830.1210.2270.3430.5560.7430.964

1.2041.315FMDV antigen

1：2048

1：1024

1：5121：2561：1281：641：321：161：8

Expression of FMDV polypeptides in silkworm larvae was estimated by the sandwich-ELISA

The expression time-course of FMDV polypeptides in Silkworm

The screening of high expression The screening of high expression viral clones viral clones

N OD(492nm)

N OD(492nm)

N OD(492nm)

N OD(492nm)

1 0.78 6 1.57 11 1.39 16 0.98

2 0.45 7 1.53 12 1.46 17 1.40

3 1.01 8 1.43 13 1.48 18 1.36

4 0.68 9 1.02 14 0.55 19 1.03

5 1.34 10 0.75 15 0.66 20 1.46

<8<8<8<8<8BmBacPAK-611

<8<8<8<8<8BmBacPAK-62

36036036045<8Bm-P12A3C50

90909020<8Bm-P12A3C45

45454520<8Bm-P12A3C122

90909020<8Bm-P12A3C124

36036018032<8Bm-P12A3C33

2821147-７number

dpvcLBPE-antibodyb

VaccineaAnimal #

FMDV-specific antibody response after vaccination with Bm-P12A3C’s

-4+mouth3Day 2BmBacPAK-611

-4+mouth3Day 2BmBacPAK-62

+---Bm-P12A3C50

-22Day 6Bm-P12A3C45

+---Bm-P12A3C122

+---Bm-P12A3C124

+---Bm-P12A3C33

ProtectioncLesion scoresb

Duration ofPyrexia(day)

Days of onset of pyrexiaa

vaccineAnimalnumber #

Protection and clinical signs in cattle after challenge with FMDV Asia I/HNK/CHA/05 strain

+1：45 1：901-15-1：451：20 1-14-1：81：8 1-13-1：451：20 1-12+1：451：20 1-11

1/9

+1：1801：1801-10+1：1801：1801-9-1：16 1：45 1-8+1：901：901-7

+++++

6.34

PD50

1/3

1：180 1：360 1-61：180 1：180 1-51：2561：90 1-41：45 1：32 1-31：128 1：180 1-2

Animalnumber

+

Protection

1：512 1：3601-1

1

LPBE-Antibody

(21)

LPBE-Antibody

(14)Immunize

dose

The result of PD50 test -1

-1：451：641-15-1：901：641-14-1：901：90 1-13+1：1801：1281-12+1：1801：1281-11

1/9

+1：1801：1281-10-1：1801：1281-9+1：1801：256 1-8+1：3601：2561-7

++++-

7.05

PD50

1/3

1：90 1：90 1-61：720 1：5121-51：901：45 1-41：3601：1801-31：512 1：64 1-2

Animal number +

Protection

1：512 1：3601-1

1

LPBE-Antibody

(21)

LPBE-Antibody

(14)Immunize

dose

The result of PD50 test-2

+1：901：902-15-1：451：452-14+1：1281：90 2-13+1：901：452-12-1：1801：902-11

1/9

+1：901：452-10+1：1801：902-9+1：901：45 2-8+1：3601：902-7

++++-

9.0

PD50

1/3

1：45 1：122-61：1801：2562-51：3601：5122-41：3601：3602-31：128 1：256 2-2

Animalnumber

+

Protection

1：1801：1802-1

1

LPBE-Antibody

(21)

LPBE-Antibody

(14)Immunize

dose

The result of PD50 test-3 15 cattlesvaccines from one larvasproduction

There are two different strains of Asia I FMDV prevalence in China, which are Asia 1/HNK/CHA/05 and JSL/06 isolate. When vaccinated with inactivated vaccine from Asia 1 /HNK/CHA/05, it can’t get the protection after challenge with JSL/06 strain and vice versa. So we designed an experiment to verify whether this produced antigen can get the protection against the two strains.All vaccinated cattle were challenged with 10,000 BID50 of AsiaI/HNK/CHA/05 at 21 dpv. The result showed the subunit vaccine potency could get 8.06、9.0、6.47 PD50. The vaccinated cattle were challenged with 10,000 BID50 of JSL/06 at 21 dpv. The result showed the subunit vaccine potency could get 6.47、5.20、6.47 PD50.

Cross-protection

ACKNOWLEDGEMENTS

Liu Jixing(Lanzhou VRI) Chen Yin Lin Xu’ai Yi Yongzhu He Jialu Shen Guifang Li Yinv Li Zhiyong and Yin Xiangping (Lanzhou

VRI) Zhang Zhidong and Li Yanmin (Institute for

Animal Health, Pirbright )

Thank you！