Enhanced Protection with Inactivated Foot-

and-Mouth Disease Virus Purified by a Tagged-Marker Expressing on Virus Surface

Animal and Plant Quarantine Agency (QIA), Republic of Korea

Jong-Hyeon Park, D.V.M, Ph.D

GFRA 2015, HANOI , VIETNAM, OCTOBER 20-22, 2015

Background and Objectives

1. Antigen purification for vaccine preparation by classical methods for differentiation with infected and vaccinated animals (DIVA) is necessary, but expensive and technically difficult. – inserted a hexa-histidine tag (6xHIS) to the VP1 C-

terminus for easy purification

2. Needs of a modification of antigen structure for maintaining stable immunity in the field.1. replaced two amino acids of VP1/VP2 to enhance the

stability on the capsid of the FMD virus (FMDV) Asia1/MOG/05.

• Checking a protection by lethal challenge in immunized mice with the purified FMDV antigen.

VP1 ~EIIAPEHHHHHHKQ~ 2A 6xHIS Cleavage site

VP4VP4 VP2VP2LL VP3VP3 VP1VP1 2B2B 2C2C 3A3A 3B3B 3C3C 3D3D2A2A5’UTR 3’UTR

O1 Manisa (O/Manisa/Turkey/69)

VP4VP4 VP2VP2LL VP3VP3 VP1VP1 2B2B 2C2C 3A3A 3B3B 3C3C 3D3D2A2A5’UTR 3’UTR

N17DH145Y

Asia1/MOG/05 O/manisa/Turkey/69

Materials and MethodsStrategy of Virus for Stable and Easy Purification

O1M-AsM-P1

VP1 ~EIIAPEHHHHHHKQ~ 2A 6xHIS Cleavage site

To evaluate protection using challenge model in mice

Inside view

outside view

VP2 H145Y

VP1 N17D

VP1

6xHIS

N17D

6xHIS

Plaque assay

O1m AsM P1

O1m AsM P1_ N17D H145Y

O1m AsM P1_ N17D H145Y_6H

Results

Stabilized (S)Naïve (N) Stabilized+ Tagged (S, T)

in ZZ-R 145 cell

IF DAPI MERGE

6xHIS-FMDV (S,T)

Non-6xHIS-FMDV (S)

Immunofluorescence for 6X HIS

37

25

kDa

37

25

Anti-Asia1 VP1

Anti-6x histidine

Western Blot

O1m AsM P1

O1m AsM P1_ N17D H145Y-6H

N S, T

Purification using Histidine-tagged protein purification column

(PrepEase ®Histidine Purification Kit)

Eluted fraction of 6XHIS tag protein

PBM FMDV antigen detection kit

1 2 3 4 5

Harvest the infected cell and the sup.↓

BEI inactivation↓

PEG 6000 ppt↓

PrepEase ®Histidine Purification (≒1 hrs)

100nm

TEM & IEM (S+T form)

Virus Growth after acid (pH5.5, 6.0, 7.4) treatment for confirmation of stable antigen

O1m_A

sM P1

O1m_A

sM P1_

N17D H14

5Y

O1m_A

sM P1_

N17D H14

5Y_6

H0

1

2

3

4

5

6pH5.5pH6.0pH7.4

Log1

0(TI

CD

50/m

l)30 min treatment

N S ST

Challenge after mice immunization with stable non-tagged (S) antigen treated with acid

treatment (pH5.5, pH6.0)

0 1 2 3 4 5 6 7 80

10

20

30

40

50

60

70

80

90

100

O1m_AsM P1

Tris-NaCl

O1m_AsM N17D H145Y

Days post infection

Perc

ent s

urvi

val (

%)

pH6.0

0 1 2 3 4 5 6 7 80

10

20

30

40

50

60

70

80

90

100O1m_AsM P1

Tris-NaCl

O1m_AsM N17D H145Y

Days post infection

Perc

ent s

urvi

val (

%)

pH5.5

S

N

S

N

• 0.2 ug antigen with oil adjuvant, ISA 201 • 1 week immunization and challenged with 50 LD50 of Asia1 Shamir

0 1 2 3 4 5 6 7 80

10

20

30

40

50

60

70

80

90

100

O1m_AsM P1 N17D H145Y_6HO1m_AsM P1Tris-NaCl

Days post infection

Perc

ent s

urvi

val (

%)

Challenge after mice immunization with stable tagged (S,T) antigen treated with acid

treatment (pH 6.0)

0 1 2 3 4 5 6 7 80

10

20

30

40

50

60

70

80

90

100

O1m_AsM P1 N17D H145Y_6H

Commercial vaccineTris-NaCl

Days post infection

Perc

ent s

urvi

val (

%)

Non-treatment pH6.0-treatmentSTSTV

C C

N

ZZ-R

LF-BKBHK21ZZ-R

4P

6p

8p

Sequence variation of 6XHIS through serial passages (O1m_AsM P1_N17D H145Y_6H, ST form )

Z4

Z3

Z4B2

Z4B4

Z4L2

Z4L4

Z6

Z8

Z: ZZRB: BHK21L: LF-BK

P P P

R PP P

3 passages

Different positions in VP1 for easy purification

VP4VP4 VP2VP2LL VP3VP3 VP1VP1 2B2B 2C2C 3A3A 3B3B 3C3C 3D3D2A2A5’UTR 3’UTR

Asia1/MOG/05 O/manisa/Turkey/69

N17DH145YAsia1 Shamir

VP1(150/151) ~ GETTSRRGDMAALA GHHHHHHG QRLSARLPTSFNY

VP1(139/140) ~ GET GHHHHHHG TSRRGDMAALAQRLSARLPTSFNY

VP1(153/154) ~ GETTSRRGDMAALAQRL GHHHHHHG SARLPTSFNY

6H-1396H-1506H-153

VP1 (211/212) -EIIAPEHHHHHHKQ~ 2A

O1 AsMS

O1 AsMS6H-211

Sequences of 6XHIS in viruses through consecutive passages

O1 AsMS- 6H-211(10p, Z5B5)

O1 AsMS 6H-139 (11p, Z5B6)

O1 AsMS 6H-153 (11p, Z5B6)

O1 AsMS 6H-150 (11p, Z5B6)

Z: ZZRB: BHK21

Conclusions1. Possiblity to easy and simple purification of FMDV antigen

within 1 hrs using a commercial metal-affinity column.

2. Obtaining stable 6xHIS-tagged FMDVs sustained under acid conditions (pH 6.0).

3. Vaccination by the purified stable-tagged antigen confer higher protection rate compared to naïve antigen under disassociated condition.

4. The 6XHIS sites would be mutated after serial passages, but the mutation formation depends on the VP1 sequences.

5. The stabilized and tagged antigen offers an alternative to the current methods of antigen purification and enhanced immunity.