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A universal test to quantitate
protective antigen during production
of foot-and-mouth disease vaccines
EuFMD
Open Session 2018
Amin Asfor, Nathalie Howe, Santina Grazioli, Emiliano Brocchi and Toby Tuthill
Pirbright and ISZLER
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Foot-and-mouth disease virus (FMDV)
Picornavirus family, related to poliovirus and rhinovirus
• Seven serotypes
• High levels of antigenic variation within serotypes
• Capsid protein VP4: ‘internal’ and highly conserved
• Vaccine produced by chemical inactivation
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The problem: vaccine integrity
Intact antigen Dissociated subunits
● protective response ● reduced immunogenicity
● 146S ● altered antigenicity
● 12S
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Existing approaches to measure vaccine integrity
Virus particles separated by
ultracentrifugation
in sucrose gradients
Use of 146S-specific
antibodies
Y
Laborious, low throughput
epitope?
specificity?
Virus serotype/strain
specific
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Existing approaches to measure vaccine integrity
Virus particles separated by
ultracentrifugation
in sucrose gradients
Use of 146S-specific
antibodies
Laborious, low throughput
Biologicals. 1990 Oct;18(4):315-9.
Quantification of intact 146S foot-and-mouth disease antigen for vaccine production by a double antibody sandwich ELISA using monoclonal antibodies.
Van Maanen C1, Terpstra C.
1990
2017
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Fundamental research on picornavirus uncoating and membrane penetration
Receptor
binding ?
Poliovirus‘Altered’
particle
Empty
particle
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VP4 multimerises to form a size-selective membrane pore
RFU
Time (min)
PV VP4 Dye Release
Liposomes only
DE3 cells + liposomes
PV VP4 + liposomes
1µm mellitin + liposomes
VP4
Mellitin
Controls
Time
Hogle Lab, Harvard Medical School
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How can the inside of a virus capsid contain targets for antibodies?
The picornavirus capsid is a dynamic structure. Internal components that exit from the particle during cell entry are transiently exposed at the capsid surface under normal physiological conditions.
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10 20 30 40 50 60
....|....|....|....|....|....|....|....|....|....|....|....|
Type O1 Manisa-AAT01766.1 O1 M GAGQSSPATGSQNQSGNTGSIINNYYMQQYQNSMDTQLGDNATSGGSNEGSTDTTSTHTT
Type AA22/IRQ24/64 -.........................................I.................
type C ACO40496.1 ..........................................I.................
Type Asia 1 ABF74751.2 ..........................................I................N
type SAT 1 ADI24382.1 ..........................................I................N
Type SAT 2 AGZ15285.1 ..........................................I................N
70 80 90 100 110 120
....|....|....|....|....|....|....|....|....|....|....|....|
Type O1 Manisa-AAT01766.1 O1 M NTQNNDWFSKLASSAFSGLFGALLADKKTEETTLLEDRILTTRNGHTTSTTQSSVGVTYG
Type AA22/IRQ24/64 ............................................................
type C ACO40496.1 ..........................................................F.
Type Asia 1 ABF74751.2 T........R......T..............A............................
type SAT 1 ADI24382.1 ............Q......V......................SH.T..........I...
Type SAT 2 AGZ15285.1 ............Q..I...........................H.T............F.
130 140 150 160 170 180
....|....|....|....|....|....|....|....|....|....|....|....|
Type O1 Manisa-AAT01766.1 O1 M YATAEDFVSGPNTSGLETRVAQAERFFKTHLFDWVTSDPFGRCHLLELPTDHKGVYGSLT
Type AA22/IRQ24/64 .S.Q..H.............V.......K.....TPDKA..HLEK............H.V
type C ACO40496.1 ......ST............H.......MA.....P.QN..HM.KVV..HEP.....G.V
Type Asia 1 ABF74751.2 ..V...A.............T.......K.....TPNLA..H.YY....SE........M
type SAT 1 ADI24382.1 ..SSDK..P....N......E.......HK....TLEQK..TT.V.........I..Q.V
Type SAT 2 AGZ15285.1 ..D.DS.RP...........Q.......EK....TSDK...TLYV....K....I..K..
VP4
VP4 N terminus is very highly conserved
among all FMDV serotypes
VP2
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A broadly cross-reactive antibody….
ELISA
Asia
1
A C O
SA
T1
SA
T2
SA
T3
neg
ati
ve
0 .0
0 .5
1 .0
OD
49
0n
m
IF
Microscopy by Stephen Berryman
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…. recognizes an epitope in VP4
V P 4 N -1 5 V P 4 N -3 0 V P 4 N -4 5 V P 4 C -1 5 V P 4 C -4 5 V P 2 N -1 5 V P 2 N -3 0 V P 2 N -4 5
0 .0
0 .5
1 .0
1 .5
2 .0
p e p t id e s 1 u g /m l
OD
49
0
KD - Asia 1 A C O SAT1 SAT2 SAT3
5037
25
2015
10
VP0
VP4
Western Blot
Peptide ELISA
VP2
peptides
VP4
peptides
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VP4 is exposed at the capsid surface but absent from dissociated subunits
VP4
(VP4 epitope exposed) (VP4 lost upon dissociation)
+ VP4 - VP4
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A universal ELISA for intact antigen
SampleHeated (dissociates all antigen)
Capture with recombinant integrinDetect with VP4 –specific antibody
Not-Heated (preserves all antigen)
A1061
O IR
AN
SA
T1
SA
T2
SA
T3 C
0 .0
0 .2
0 .4
0 .6
0 .8
1 .0
1 0 u l o f v iru s ly s a te p e r w e ll-5 B 6 1 in 1 0 0 - in te g r in 1 in 7 5
OD
(4
90
nm
)
1 2 S
1 4 6 S
(heated)
(not heated)
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A universal ELISA for intact antigen
O1M
an
isa
A1061
0 .0
0 .5
1 .0
1 .5
OD
(4
90
nm
)
5 B 6 -1 4 6 S
M 1 7 0 -1 4 6 S
VP4 antibody sample heated
M170 existing 146S specific antibody 3 2 6 4 1 2 8 2 5 6
0 .0
0 .5
1 .0
1 .5
OD
49
0n
m
M 1 7 0 -1 2 S
M 1 7 0 -1 4 6 S
5 B 6 -1 2 S
5 B 6 -1 4 6 S
v iru s d ilu tio n
A serotype
Concentration of capsid material
VP4 antibody sample not heated
A O
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Summary
• Capsid dissociation is a major problem for vaccine
antigen
• Capsid ‘breathing’ allows internal conserved sequences
(VP4) to form epitopes at the capsid surface
• Dissociated pentamers do not contain VP4
• A VP4 specific monoclonal antibody can recognize all
FMDV serotypes and has specificity for intact capsid
• A potential universal test for vaccine antigen• Further work/test validation ongoing
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Acknowledgements
• Pirbright
• Stephen Berryman
• Julian Seago
• Don King and WRLFMD
• EuFMD
• Keith Sumption
• BI
• Jose Coco-Martin
• Genomia Fund & BBSRC Impact Acceleration Award
• Continued development of a validated test
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A universal test to quantitate
protective antigen during production
of foot-and-mouth disease vaccines
EuFMD
Open Session 2018
Amin Asfor, Nathalie Howe, Santina Grazioli, Emiliano Brocchi and Toby Tuthill
Pirbright and ISZLER