1.
NOVEL HIS-TAG MARKER FOOT-AND-MOUTH DISEASE
VIRUS VACCINE BOUND TO NANOLIPOPROTEIN
ADJUVANT VIA METAL IONS: UTILITY ON VACCINE AND
DIAGNOSTIC DEVELOPMENTS
Elizabeth Rieder PhD.
Foreign Animal Disease
Research Unit, USDA-ARS
Plum Island Animal Disease Center, New York, USA.
EUFMD 2016 October 27, 2016

2.
6xHis
A24 FMDV 2A6H
3Cpro cleavage
3B1235’UTR
1AL 1B 1C 1D
2A
2B 2C 3A 3C 3D
3'UTR
A24 FMD P16H
3Cpro cleavage
3B1235’UTR
1AL 1B 1C 1D
2A
2B 2C 3A 3C 3D
3'UTR
PfIMI (R1) NheI (R2)
An
S
PKs
Cn
VP1 2A
TTAT....….EVSSSQDRHKQKIIAPAKQ↓LLNFDLL
TTAT............................................KIIAPAKQHHHHHHKQLLNFDLL
TTAT....KQKIIAPAKHHHHHHIIAPAKQ↓LLNFDLL
ViableA24 FMDV WT
A24 FMDV 2A6H
A24 FMD P16H
↓
K210 VP1 Q211 VP1
Viable
Viable
3B123
IRES
1AL 1B 1C 1D
2A
2B 2C 3A 3C 3D
3'UTR
Structural Nonstructural
Design of 6XHis-FMDV A24 clones

3.
2A
6H
KQ-VP1 6H
IIPAKQ-VP1
2A6H tag
P16H tag
VP1
VP2
VP3
A24 FMDV P16H
VP1
VP2
VP3
GH loop
residues
138-155
Site of tag insertion
A24 FMDV 2A6H
Ni++
/Co+
+
Affinitycolumn
orbead
Ni++
NLP-MPLA
6XHistag
A
Anti-Histagantibody
Modeling of FMDV A24 capsid for selecting the site of insertion and schematic
representation of different components of virus-adjuvant complex
HHHHHHKQLLNFD
LLKLAGDVESNPG
HHHHHHIIAPAKQ

4.
TCID50/ml
Time (hr post infection)
1.1 X 107
0.8 X 107
2.0 X 107
A24 FMD P16H
A24 FMDV WT
A24 FMDV 2A6H
0
1
2
3
4
5
6
7
8
0 4 8 12 16 20 24
A24FMDV WT
A24FMDV 2A6H
A24FMDV P16H
BHK-21
24 hours
Titer
PFU/ml
Growth curve and phenotype of 6xHis-FMDVs
Both A24 FMDV2A6H and A24 FMDVP16H follow WT FMDV 24 like growth
kinetics and produce Plaque phenotypes similar to the later.
A B

5.
Determination of specific neutralizing antibody response of parental
and mutant viruses to cattle FMDV A24Cru antisera
Virus Bovine anti A24WT sera
Neutralization Titersa
r1-valuesb
A24WT 3.15 1.00
A24FMDV 2A6H 3.45 1.99
A24FMDV P16H 3.00 0.7
Virus neutralizing titers (log10 of reciprocal of the last serum dilution to neutralize 100 TCID50 of
virus in 50% of the wells).
Sensitivity > 0.9. Sera was produced in a bovine infected by intradermolingual inoculation of 4
log10 bovine tongue infectious doses of FMDV A24WT and collecting blood 21 days post-infection.
bThe mean r1-values as measured against the reference strain A24WT.
R1 vallue from virus neutralization titer experiiment proves that the 6xHis
FMDVs retain antigenicity of parental FMDV A24 virus.

6.
A24 FMDV WT
NiNLP-MPLA
A24 FMDV WT+
NiNLP-MPLA
A24 FMDV 2A6H +
NiNLP-MPLA
A24 FMDV P16H +
NiNLP-MPLA
A24 FMDV 2A6H A24 FMDV P16H
NiNLP-MPLA No. of NiNLP-
MPLA bound
A24 FMDV WT A24 FMDV
P16H
0 124 15
1 9 23
2 0 16
3 0 22
4 0 14
5 0 9
A
B
C D
EM shows that 6xHis tagged FMDVs form complex with
MPLA-NiNLP

7.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
PBS
Alone
0 ng 1 ng 2.5ng 5 ng 10 ng 25 ng 50 ng 100 ng
O.D.(630nm)
NiNLP-MLPA Concentration
Ni
Ni
NiNi
Ni Ni
Ni
Ni
Ni
Ni Ni
Ni
Detection
with Anti-His
antibody
Virus binding
Ni
Ni
Ni
Ni Ni
Ni
Ni-NLP-MPLA
Competition
Ni
Ni
NiNi
Ni Ni
Wash
ELISA assay further confirmed a specific interaction between 6xHis FMDV
capsid and MPLA-NiNLP.
Determination of 6xHistag mediated binding of FMDV capsid to MPLA-
NiNLP using Ni-NTA coated plate ELISA plate: implication in diagnostic
assay development

8.
V FT W E1 E2 E3 E4
Percentrecoveryofinfectiousvirus
Fraction number
V FT W E1 E2 E3 E4
Percentrecoveryofinfectiousvirus
Fraction number
Imidazole Elution
VP1
3Dpol
6xHisFMDV
antigen
Cellular proteins
FMDV Non-
structural proteins
FMDV-infected cells
A24FMDV P16H
Affinity purification
(Co++ or Ni ++columns)
Purified antigen
A24FMDV P16H
Clarified FMDV
cell lysate
EDTA Elution
VP1
3Dpol
0
20
40
60
80
100
V FT W E1 E2 E3 E4 0
20
40
60
80
100
V FT W E1 E2 E3 E4
Co-NTA column purification of A24 FMDV P16H

9.
2A
6H
KQ-VP1
6H
IIPAKQ-VP1
2A6H tag P16H tag
VP1
VP2
VP3
A24 FMDV P16H
VP1
VP2
VP3
GH loop
residues
138-155
Site of tag insertion
A24 FMDV 2A6H
Ni++
/Co+
+
Affinitycolumn
orbead
Ni++
NLP-MPLA
6XHistag
A
B
Anti-Histagantibody
Modeling of FMDV A24 capsid for selecting the site of insertion and schematic
representation of different components of virus-adjuvant complex
HHHHHHKQLLNFD
LLKLAGDVESNPG
HHHHHHIIAPAKQ
Ni-NLP-MPLA
Lipid bi layer disc
MPLA adjuvant
Ni++ chelating lipids
Lawrence P, Pacheco JM, Uddowla S, Hollister J, Kotecha A, Fry E, Rieder E.
Foot-and-mouth disease virus (FMDV) with a stable FLAG epitope in the VP1
G-H loop as a new tool for studying FMDV pathogenesis. Virology. 2013 Feb
5;436(1):150-61.

10.
Days post-challenge
Percentsurvival
0 1 2 3 4 5 6 7
0
20
40
60
80
100
0.01 A24His
0.025 A24His
0.05 A24His
0.1 A24His
0.01 A24His+NLP
0.025 A24His+NLP
0.05 A24His+NLP
0.1 A24His+NLP
NLP-control
PBS-control
Survival curve
pfu/mlserum
1.0E+00
1.0E+01
1.0E+02
1.0E+03
1.0E+04
1.0E+05
1.0E+06
1.0E+07
1.0E+08
1.0E+09
0dpc 1dpc 3dpc 5dpc 7dpc
Viremia
Log10TCID50/mlserum
0
0.5
1
1.5
2
2.5
3
3.5
0dpv 0dpc 7dpc 14dpc
Seroneutralization
+ NiNLP-MPLA
A24FMDV P16H
A24FMDV P16H:
NiNLP-MPLA
Mice inoculation with inactivated A24 FMDV P16H :MPLA-NiNLP
adjuvanted vaccine and subsequent challenge with A24 FMDV WT
Vaccine:adjuvant complex
prevented the disease in mice in
a dose dependent manner.

11.
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1 2 30 5 10
Nanograms of 6xHis tagged PCBP
A24 FMDV WT infected
Serum at 7 days post infection
A24 FMDV P16H vaccinated
Serum 14 days post vaccination
Absorbanceat630nM
Determination of anti-6xHis TAG antibody in A24 FMDV
P16xH treated mice serum
Successful generation of anti-6H response in vaccinated animals can lead
us to further design assays to differentiate vaccinated from infect animals.

12.
• Engineered FMD viruses with histidine residues inserted into or fused to the
FMDV capsid. 6xHis FMDVs exhibited growth kinetics, plaque
morphologies and antigenic characteristics similar to wild-type virus.
• Electron microscopy and biochemical assays revealed that the 6xHis
FMDVs readily assembled into antigen: adjuvant complexes with Ni2+
chelated nanolipoprotein and monophosphoryl lipid A adjuvant
(MPLA:NiNLP).
• The 6xHis tag allowed one-step purification of the mutant virions by Co2+
affinity columns.
• Animals Immunized with the inactivated 6xHisFMDV:MPLA:NiNLP vaccine
acquired enhanced protective immunity against FMDV challenge compared
to virions alone.
• Induction of anti-6xHis in the immunized animals could be exploited in the
differentiation of vaccinated from infected animals needed for the
improvement of FMD control measures.
SUMMARY

13.
Acknowledgements
Rieder Laboratory
Devendra Rai
Anna Kloc,
Beth Schafer
Zaheer Ahmed
Collaborators at PIADC
Dr. Teresa de los Santos lab
Dr.Fayna Diaz San-Segundo
Dr. Luis Rodriguez.
Dr.Tom Burrage, Ben Clark - DHS
Dr. Paul D. Hoeprich
Lawrence Livermore National
Laboratory, Livermore, CA

14.
Days post-challenge
Percentsurvival
0 1 2 3 4 5 6 7
0
20
40
60
80
100
0.025ug BEI A24
0.05ug BEI A24
0.1ug BEI A24
0.025ug BEI A24+ISA206
0.05ug BEI A24+ISA206
0.1ug BEI A24+ISA206
control
Survival curve
1.0E+00
1.0E+01
1.0E+02
1.0E+03
1.0E+04
1.0E+05
1.0E+06
1.0E+07
1.0E+08
1.0E+09
0dpc 1dpc 3dpc 5dpc 7dpc
Viremia
pfu/mlserum
0
0.5
1
1.5
2
2.5
3
3.5
0dpv 0dpc 7dpc 14dpc
PRN50/mlserum
Seroneutralization
Mice inoculation with inactivated A24 FMDV WT and ISA206
adjuvant and challenge with A24 FMDV WT
Virus caused lethal disease
was prevented by vaccine
adjuvant preps in mice in a
dose dependent manner.