Presentación de PowerPoint...Hyang-Sim Lee, Young-Joon Ko, Byounghan Kim, Seo-Yong Lee, Soo-Jeong Jung EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT . Title: Presentación de PowerPoint

Appendix 22

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 1

Development of a bovine enterovirus-based vector that expresses multi-epitope of FMDV

Jong-Hyeon Park [email protected] FMDV lab, FMD Division Animal Plant and Fisheries Quarantine and Inspection Agency (QIA),

480 Anyang 6 dong, Anyang, Republic of Korea

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Conclusions and Recommendations • A recombinant BEV vectors carrying B and T cell epitopes of

FMDV were constructed and recovered as live viruses • BEV-based viral vector is able to deliver approximately 720 bps of foreign genes • The rBEVs show a proper growth without affecting of replication

kinetics and plaque sizes compared to the parent virus. • The rBEVs were detected and isolated in feces and serum samples

after administration to cattle. • The calves inoculated with rBEVs showed no clinical signs • The rBEVs show a possibility as live vaccine vector for FMD - Experiment for the immune and protective effect against FMDV is still undertaken.

- Expression of larger sized FMDV molecule with molecular adjuvant should be tried

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Objectives

• To verify the possibility of BEV as live and

delivery vaccine vector

• To investigate the multiplication and safety as

a live vaccine candidate for FMD in vivo

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Why use BEV-1 (LC R4 strain) as a vaccine vector?

• Safe to animals (isolated from healthy dairy cattle, Kunin, 1957) - BEV infections are ubiquitous and are normally without significant or severe clinical

symptoms • The same Picornaviridae family with FMDV

• Thermostable - maintenance of the cold chain is not always guaranteed from manufacturing to

delivery)

• pH-stable - can be administered orally

• Very wide tissue tropism for cell types in vitro • Replicable in cattle (or other animals) • Generating high titers in various cell lines.

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

FMDV and BEV genome

P1

P2

P3

Poly(C)

L VP4 VP2 VP3 VP1 2A 2B

2C

3A 3B 3C

3D

FMDV VPg

VP4 VP2 VP3 VP1

2A 2B

2C

3A 3B 3C

3D BEV

VPg

(Kbps)

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Strategy for rBEV construction

Cloning of full-length cDNA of BEV and recovery

• BEV Type 1 strain LCR4 (ATCC® Number: VR-248TM) • Cloned into pBluescript II SK (+) vector

T7

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Appendix 22

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 2

EGFP and synthetic FMDV epitopes

• EGFP for marker gene: 720 bps

• O/Andong/SKR/2010 (O-SEA topotype, Mya-98 lineage isolated from epidemic of Andong region, Korea in Nov. 2010)

• O1 Manisa (ME-SA topotype, PanAsian lineage, vaccine strain) - FMDV VP1 (a.a 141-160, 200-213)

- FMDV VP4 (a.a 20-34) - FMDV 3A (a.a 21-35) • T-helper epitope

- Pan-HLA-DR (PADRE): a universal DR-restricted T-helper epitope

(Hung CF et al. 2007; Alexander J et al. 1994)

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Construction of rBEV containing foreign genes 2A Cleavage site (TSY/G)

BEV_EGFP EGFP (720 bps)

VP4

VP2

VP3

VP1

EGFP

2A

2B

2C

3A

3B

3C

3D

(A)16

BEV_O-AD-multi-epi/ O-Manisa-multi-epi

VP4

VP2

VP3

VP1

2A

2B

2C

3A 3B

3C

3D

(A)16

2A Cleavage site

(TSY/G)

(GGSGG) linker

FMDV_VP1200-213

327 bps(109aa)

linker linker

2A Cleavage site

(TSY/G)

linker linker

VP420-34

PADRE FMDV_VP1141-160 3A21-35

Hisx6

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

rBEV-EGFP

rBEV-AD rBEV-Manisa

12

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

EGFP expression from rBEV-EGFP ( 6 hours after infection in MDBK cells)

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Indirect immunofluorescence assay

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Appendix 22

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 3

Growth of rBEVs in MDBK cells

10 BEV-full clone

8

BEV-EGFP BEV-AnDong multiepi 6

BEV-Manisa multiepi

4

2

0

Hour post-infection

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Search for pre-existing antibody against BEV-1 type in

the field

Distribution of BEV-1 (LC-R4 strain) VN titers in Korea

60 50 40

BEV-1 Cattle

BEV-1 Pigs

(N=172) (N=160)

• Positive rate of cattle is 48.3%

30 20 10 0

<2 2 2.5 3

• Positive rate of pigs is 70.6%

3.5 4 4.5 5 5.5 6 6.5 7 >7.0 BEV antibody titers (Log2)

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Infection of rBEVs to calves

Virus titer Calf No.

Inoculation virus

Inoculation route (TCID50/ml)

1180 9362 0522 0564

rBEV-EGFP

rBEV-AnDong- Multi-epi

107.29

107.44

Intramuscular 2ml,

Intranasal 2ml,

Oral 2ml

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

No clinical sign during 14 days after inoculation

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

RT-PCR for detection of rBEVs from infected calves

BEV in feces samples (DPI) Groups

Calf No.

0

1

2

3

4

5

6

1180

-

+

-

-

-

+

+

rBEV-EGFP 9362

-

+

-

-

-

+

+

rBEV-AD

multi

0522 - + 0564 - +

- - - + + - - - + +

BEV in serum samples (DPI) Groups

Calf No.

0

1

2

3

4

5

6

rBEV-AD

1180

-

nd

-

nd

-

nd

+ multi

9362

-

nd

-

nd

-

nd

+ 0522

-

nd

-

nd

-

nd

+

rBEV-EGFP 0564

-

nd

-

nd

-

nd

+ nd : not done

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Virus isolation of rBEV in feces of inoculated calves Bright-field

Fluorescence

CPE

rBEV-EGFP

No CPE

CPE rBEV-AnDong

-Multi-epi

CPE

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Appendix 22

Open Session of the EuFMD: 2012, Jerez de la Frontera, Spain 4

Virus neutralizing antibody against BEV and FMDV

Serum Neutralization Titers using BEV-1 LCR4

Calf No.

Inoculation

virus

0dpi 2dpi 4dpi

6dpi 8dpi 10dpi 12dpi 13dpi

1180

<8

<8

<8

<8

<8

<8

<8

<8 BEV-EGFP

9362

<8

<8

<8

<8

<8

<8

<8

<8

0522

BEV-AD-

<8 <8 <8

<8 <8 <8 <8 <8

0564

multi-epi <8

<8 <8 <8

<8 <8 <8 <8

Serum Neutralization Titers using O/AnDong/SKR/2010

Calf No.

Inoculation

virus

0dpi 2dpi 8dpi

10dpi 12dpi 13dpi

1180

<8

<8

<8

<8

<8

<8 BEV-EGFP

9362

<8

<8

<8

<8

<8

<8

0522

BEV-AD-

<8 <8 <8

<8 <8 <8

0564

multi-epi

<8

<8 <8 <8 <8 22

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Conclusions and Recommendations • A recombinant BEV vectors carrying B and T cell epitopes of

FMDV were constructed and recovered as live viruses • BEV-based viral vector is able to deliver approximately 720 bps of foreign genes • The rBEVs show a proper growth without affecting of replication

kinetics and plaque sizes compared to the parent virus. • The rBEVs were detected and isolated in feces and serum samples

after administration to cattle. • The calves inoculated with rBEVs showed no clinical signs • The rBEVs show a possibility as live vaccine vector for FMD - Experiment for the immune and protective effect against FMDV is still undertaken.

- Expression of larger sized FMDV molecule with molecular adjuvant should be tried

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT

Acknowledgements

Members of FMDV Lab, FMD Division, QIA. Korea. Jia-Qi Chu, Jeong-Nam Park, Yeo-Joo Lee, Rae-Hyung Kim,

Su-Mi Kim, Kwang-Nyeong Lee,

Hyang-Sim Lee, Young-Joon Ko, Byounghan Kim,

Seo-Yong Lee, Soo-Jeong Jung

EuFMD 2012 JEREZ DE FRONTERA SPAIN 29-31 OCT