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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
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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.
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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
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Inside view
outside view
VP2 H145Y
VP1 N17D
VP1
6xHIS
N17D
6xHIS
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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
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IF DAPI MERGE
6xHIS-FMDV (S,T)
Non-6xHIS-FMDV (S)
Immunofluorescence for 6X HIS
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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
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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)
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100nm
TEM & IEM (S+T form)
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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
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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
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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
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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
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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
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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
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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.