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