1. 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

2. 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. –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.

3. VP1 ~EIIAPEHHHHHHKQ~ 2A 6xHIS Cleavage site VP4 VP2 L VP3 VP1 2B 2C 3A 3B 3C 3D 2A 5’UTR3’UTR O1 Manisa (O/Manisa/Turkey/69) VP4 VP2LVP3VP12B2C3A3B3C3D 2A 5’UTR3’UTR N17DH145Y Asia1/MOG/05 O/manisa/Turkey/69 Materials and Methods Strategy of Virus for Stable and Easy Purification O1M-AsM-P1 VP1 ~EIIAPEHHHHHHKQ~ 2A 6xHIS Cleavage site To evaluate protection using challenge model in mice

4. Inside view outside view VP2 H145Y VP1 N17D VP1 6xHIS N17D 6xHIS

5. 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

6. IFDAPIMERGE 6xHIS- FMDV (S,T) Non-6xHIS- FMDV (S) Immunofluorescence for 6X HIS

7. 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

8. 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)

9. 100nm TEM & IEM (S+T form)

10. Virus Growth after acid (pH5.5, 6.0, 7.4) treatment for confirmation of stable antigen 30 min treatment N S ST

11. Challenge after mice immunization with stable non- tagged (S) antigen treated with acid treatment (pH5.5, pH6.0) pH6.0pH5.5 S N S N 0.2 ug antigen with oil adjuvant, ISA 201 1 week immunization and challenged with 50 LD 50 of Asia1 Shamir

12. Challenge after mice immunization with stable tagged (S,T) antigen treated with acid treatment (pH 6.0) Non-treatmentpH6.0-treatment ST V C C N

13. ZZ-R LF-BK BHK21 ZZ-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: ZZR B: BHK21 L: LF-BK PPP R P P P 3 passages

14. Different positions in VP1 for easy purification VP4 VP2 L VP3VP12B2C3A3B3C3D 2A 5’UTR3’UTR Asia1/MOG/05 O/manisa/Turkey/69 N17DH145Y Asia1 Shamir VP1(150/151) ~ GETTSRRGDMAALA GHHHHHHG QRLSARLPTSFNY VP1(139/140) ~ GET GHHHHHHG TSRRGDMAALAQRLSARLPTSFNY VP1(153/154) ~ GETTSRRGDMAALAQRL GHHHHHHG SARLPTSFNY 6H-139 6H-150 6H-153 VP1 (211/212) -EIIAPEHHHHHHKQ~ 2A O1 AsMS 6H-211

15. 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: ZZR B: BHK21

16. Conclusions 1.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.