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transcript
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
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
The problem: vaccine integrity
Intact antigen Dissociated subunits
● protective response ● reduced immunogenicity
● 146S ● altered antigenicity
● 12S
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
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
Fundamental research on picornavirus uncoating and membrane penetration
Receptor
binding ?
Poliovirus‘Altered’
particle
Empty
particle
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
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.
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
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
…. 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
VP4 is exposed at the capsid surface but absent from dissociated subunits
VP4
(VP4 epitope exposed) (VP4 lost upon dissociation)
+ VP4 - VP4
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)
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
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
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
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