Generation of mAbs to FMDV/A and application in a cELISA for the detection of FMDV/A

antibodies

Dr. M. Yang National Center for Foreign Animal diseases

/Canadian Food Inspection Agency

Introduction

Foot-and-mouth disease (FMD) remains one of the world’s most widespread epizootic and highly contagious disease.

FMD can cause major economic losses even in previously FMD free countries.

Over 100 countries around world are not considered FMD freeby the OIE. FMD is endemic in many areas of Asia, Africa, and South America

FMDV is recognized as 7 serotypes: O, A, C, Asia 1, SAT 1, SAT 2 and SAT 3. O and A are the most wide spread.

FMD is caused by a single-stranded RNA virus belonging to the family Picornaviridae.

FMD is difficult to control and eradicate

because of 1. rapid virus replication 2. high mutation rate 3. high levels of viral excretion 4. small doses required for infection 5. multiple forms of transmission (contact, aerosols)6. wide geographical distribution 7. broad host range (cattle, buffaloes, pigs, sheep, goats, and ~ 70 wildlife species)8. ability to establish carrier status (not in pigs)9. antigenic diversity leading to poor cross immunity among serotypes 10. relatively short duration of immunity

(Longjam et al., 2011; Maree et al, 2011)

FMDV antibody detection

FMDV specific antibody identification is very useful for:

(1) as an indicator of FMDV infection

(2) the screening of animals for the presence of antibodies before inter territorial movement, ‑

(3) testing vaccine potency and monitoring the effectiveness of vaccinations

(4) for epidemiological studies of disease in animal populations (Have and Jensen, 1983)

Virus neutralization test

VNT is routinely used to detect FMDV antibodies

1. VNT is costly and labour intensive

2. The procedure requires live virus, limiting the test to BL3

3. VNT requires 2-3 days to obtain results.

ELISAs are sensitive, specific, rapid (hours), easy to perform and scale up. cELISA is suitable for the detection of antibodies from different species.

Objectives

1. To generate and characterize FMDV/A specific mAb 2. To develop a cELISA for serological detection of FMDV A antibodies ‑ 3. To validate the cELISA

Advantages using mAbs

low cross-reactivity; easy in standardization; and less batch to batch variations

Characterization of the mAbs against FMDV serotype A

Name Isotyping VNT Epitope#1 F65-2 (F66-2) IgG2a >1:16 Con.#3 F66-4 (F65-4) IgG2a N Con.#4 F66-7 IgG3 N Con.

#5 F66-14 IgG2a >1:16 Con. cELISA#6 F67-1 IgG2a >1:16 Con.

#7 F67-18 IgG1 >1:16 Con. cELISA#8 F67-50 IgG1 N Con.#9 F67-64 IgG1 N Con.#10 F67-92 IgG2a >1:16 Con.#13 F67-25 IgG2a N Con.#14 F67-30 IgG1 N Leaner#15 F67-49 IgG2a N Con.

FMDV/A isolatesIR

N 56/99

TUR 64/11

ETH12/09

IRN 96

TUR3/12

PAK12/10

IRN8/12

ARG2/01

MAY2/11

SUD1/06

TUR7/07

BAR 18/11

PAK6/12

ETH6/00

VIT2/08

TAI5/09

IRN 36/10

NIG 38/09

IRN 1/87

IRN2/09

GHA 4/96

VIT8/09

MAU12/06

KEN&/08

ERI 2/98

EGY3/09

SAU 24/95

BKF 4/94

MAY1/07

TAW4/03

A22 IRAQ

BHU 41/03

TUR 25/07

A22 IRN/99

IRN5/03

TUR1/08

IRN 36/07

SAU 22/92

IRQ 64

COL /85

IRN1/05

AFG12/11

A24 Cruzerio

IRN 1/96

ARG /87

MAY 13/97

O.D

.

0

1

2

3

4

mAb #5 mAb #7

Reactivity of mAbs against FMDV/A 46 isolates in DAS ELISA

VP2

1

In mature virus particle, 60 copies of the four structural proteins VP1-4 associate to form a capsid which surrounds and protects the genome.

3D structure of FMDV

Localization of FMDV/A antigenic sites in capsid protein Serotype A antigenic sites

Site1 VP1 142-157Site2 VP1 200-212Site3 VP2 82-88, 196 VP3 136-139, 195Site4 VP1 169; 175-178Site5 VP3 58-61, 69-70

Maree et al, 2011

mAb resistant mutant selection for conformation epitope identification

1.FMDV/A22 Iraq and purified mAbs were incubate for 30 min at 37oC.

2. The virus/mAb mixture and controls were inoculated onto MVPK cells.

3. The flasks were incubated until 100% CPE observed and culture supernatants were collected. 1-3 steps were repeated 6 times.

4. The mutants were purified by plaque purification.

5. The selected mutants were sequenced. Pairwise Sequence Alignment is used to identify mutation regions.

ELISA results after mutant selection

Virus passage numberParental P1 P2 P3 P4 P5 P6

O.D

.

0

1

2

3

4

mAb F66A22-14 Poly-serum

Virus passage numberParental P1 P2 P3 P4 P5 P6

O.D

.0

1

2

3

4

mAb F67A22-18 Poly-serum

mAb #5 mAb #7

Identification of neutralization sites of the two mAbs in capsid protein

A22 IRQ mAb mutant VP2 VP3

F66-14 Gln79 Lys; Lys80 Thr (near site 3); Lys131 Glu Tyr192 His (near site 3)

F67-18 His77 Arg; Gln79 Glu; Lys80 Thr (near site 3); Lys131 Glu Tyr192 His (near site 3)

Percentage of Inhibition (%)

-50 -40 -30 -20 -10 0 10 20 30 40 50 60 70 80 90 100

Fre

quen

cy (

n)

0

50

100

150

200

250

Bovine (n=320)Porcine (n=475)Ovine (n=379)

Frequency distribution of the negative sera in A/cELISA

The frequencies of the PI generated from these sera were normal distributed Calculated diagnostic specificity is 99.7%

Days Post Inoculation

0 5 10 15 20 25 30

% I

nhib

itio

n

-20

-10

0

10

20

30

40

50

60

70

80

90

100

110

S25S192C140 C141 P5 P6

A/cELISA 3B cELISA

Detection of FMDV antibodies in animals inoculated with FMDV/A24

100% seroconversion at 5 dpi 0% positive at 5 dpi83% seroconversion at 7 dpi

3B cELISA is used for surveillance to monitor FMDV circulation (Chen et al., 2011)

A/cELISA 3B cELISA

Dys Post Inoculation

0 5 10 15 20 25 30 35

% I

nhib

itio

n

-20

-10

0

10

20

30

40

50

60

70

80

90

100

110

S68 S692

Detection of FMDV antibodies in animals inoculated with FMDV/A22 Iraq

Both were positive at 6 dpiBoth were seropositive at 5 dpi

Days Post Inoculation

-4 -2 0 2 4 6 8 10

% I

nhib

itio

n

-30

-20

-10

0

10

20

30

40

50

60

70

80

90

100

110Sheep #19 Sheep #20 Sheep #21 Sheep #22 Sheep #23 Sheep #24 Sheep #25 Sheep #26 Sheep #27 Sheep #28 Sheep #29 Sheep #30 Sheep #31 Sheep #32 Sheep #33 Sheep #34 Sheep #35 Sheep #36

A/cELISA 3B cELISA

Days Post Inoculation

-4 -2 0 2 4 6 8 10

% I

nhib

itio

n

-40

-30

-20

-10

0

10

20

30

40

50

60

70

80

90

100

110Sheep #19 Sheep #20 Sheep #21 Sheep #22 Sheep #23 Sheep #24 Sheep #25 Sheep #26 Sheep #27 Sheep #28 Sheep #29 Sheep #30 Sheep #31 Sheep #32 Sheep #33 Sheep #34 Sheep #35 Sheep #36

Detection of FMDV antibodies in sheep inoculated with FMDV/A Vietnam/13

#19-28 coronary band inoculated and #29-36 contact infected by Jacquelyn Horsington

100% seroconversion at 7 dpc

VNT 100% at 9 dpc

38.9% seroconversion at 7dpc100% at 10 dpc

Days Post Challenge

-10 0 10 20 30 40

VN

T T

iter

(Log

10)

0.0

0.5

1.0

1.5

2.0

2.5

3.0 Sheep #7 Sheep #8 Sheep #9 Sheep #10 Sheep #11 Sheep #12

Days Post Challenge

-10 0 10 20 30 40

Per

cent

age

of I

nhib

ition

-20

-10

0

10

20

30

40

50

60

70

80

90

100

110

Sheep #7 Sheep #8 Sheep #9 Sheep #10 Sheep #11 Sheep #12

Days Post Challenge

-10 0 10 20 30 40

Per

cent

age

of I

nhib

ition

-50

-40

-30

-20

-10

0

10

20

30

40

50

60

70

80

90

100

110

Sheep #7 Sheep #8 Sheep #9 Sheep #10 Sheep #11 Sheep #12

Detection of FMDV antibodies in vaccinated and challenged-sheep

A/cELISA VNT

Sheep were vaccinated with A22 Iraq and challenged with A Vietnam/13 after 4 days vaccination

3B cELISA

SeroconversionA/cELISA VNT 3B cELISA

8 dpc 50% 17% 17%9dpc 100% 33% 33%

Day Post Challenge

-10 -5 0 5 10 15 20 25 30

% I

nhib

itio

n

0

50

100Sheep 1Sheep 2 Sheep 3Sheep 4Sheep 5Sheep 6Sheep 7Sheep 8

O/cELISA

Day Post Challenge

-10 -5 0 5 10 15 20 25 30%

Inh

ibit

ion

0

50

100

Sheep 1Sheep 2 Sheep 3Sheep 4Sheep 5Sheep 6Sheep 7Sheep 8

A/cELISA specificity using sera from sheep vaccinated with O1 Manisa and challenged with O/SKR/10

(Jacquelyn Horsington et al., 2014) A/cELISA

Summary1. A panel of FMDV/A specific mAbs were generated. The binding epitopes of the two mAb used in this A/cELISA were well characterized.

2. One of the mAbs’ binding sites is conserved among all the tested isolates of FMDV/A.

3. The FMD A/cELISA that was developed using two mAbs and BEI inactivated FMDV/A antigen.

4. The A/cELISA exhibited comparable performance to the VNT and 3B cELISA, but more sensitive than the VNT and 3B cELISA

5. The cELISA is a simple and rapid test for the detection of FMDV/A-specific Abs.

Acknowledgements

NCFAD/CFIAHilary Bittner, Wanhong Xu, Melissa Goolia, Haben Gabir, Kate Hola, Tim Salo, Dr. Charles Nfon

Animal care staffs

Australian Animal Health LaboratoryDr. Jacquelyn Horsington