Generation of mabs to FMDV/A and application in a celisa for the detection of FMDV/A antibodies

Transcription

1 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

2 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 1 countries around world are not considered FMD free by 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.

3 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 ~ 7 wildlife species) 8. ability to establish carrier status (not in pigs) 9. antigenic diversity leading to poor cross immunity among serotypes 1. relatively short duration of immunity (Longjam et al., 211; Maree et al, 211)

4 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)

5 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.

6 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

7 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 F67-5 IgG1 N Con. #9 F67-64 IgG1 N Con. #1 F67-92 IgG2a >1:16 Con. #13 F67-25 IgG2a N Con. #14 F67-3 IgG1 N Leaner #15 F67-49 IgG2a N Con.

8 Reactivity of mabs against FMDV/A 46 isolates in DAS ELISA 4 mab #5 mab #7 3 O.D. 2 1 IRN 56/99 TUR 64/11 ETH12/9 IRN 96 TUR3/12 PAK12/1 IRN8/12 ARG2/1 MAY2/11 SUD1/6 TUR7/7 BAR 18/11 PAK6/12 ETH6/ VIT2/8 TAI5/9 IRN 36/1 NIG 38/9 FMDV/A isolates IRN 1/87 IRN2/9 GHA 4/96 VIT8/9 MAU12/6 KEN&/8 ERI 2/98 EGY3/9 SAU 24/95 BKF 4/94 MAY1/7 TAW4/3 A22 IRAQ BHU 41/3 TUR 25/7 A22 IRN/99 IRN5/3 TUR1/8 IRN 36/7 SAU 22/92 IRQ 64 COL /85 IRN1/5 AFG12/11 A24 Cruzerio IRN 1/96 ARG /87 MAY 13/97

9 3D structure of FMDV VP2 1 In mature virus particle, 6 copies of the four structural proteins VP1-4 associate to form a capsid which surrounds and protects the genome.

10 Localization of FMDV/A antigenic sites in capsid protein Serotype A antigenic sites Site1 VP Site2 VP Site3 VP , 196 VP , 195 Site4 VP1 169; Site5 VP , 69-7 Maree et al, 211

11 mab resistant mutant selection for conformation epitope identification 1.FMDV/A22 Iraq and purified mabs were incubate for 3 min at 37 o C. 2. The virus/mab mixture and controls were inoculated onto MVPK cells. 3. The flasks were incubated until 1% 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.

12 ELISA results after mutant selection 4 4 mab F66A22-14 Poly-serum mab F67A22-18 Poly-serum 3 3 O.D. 2 O.D Parental P1 P2 P3 P4 P5 P6 Virus passage number Parental P1 P2 P3 P4 P5 P6 Virus passage number mab #5 mab #7

13 Identification of neutralization sites of the two mabs in capsid protein A22 IRQ mab mutant VP2 VP3 F66-14 Gln79 Lys; Lys8 Thr (near site 3); Lys131 Glu Tyr192 His (near site 3) F67-18 His77 Arg; Gln79 Glu; Lys8 Thr (near site 3); Lys131 Glu Tyr192 His (near site 3)

14 Frequency distribution of the negative sera in A/cELISA 25 Frequency (n) Bovine (n=32) Porcine (n=475) Ovine (n=379) Percentage of Inhibition (%) The frequencies of the PI generated from these sera were normal distributed Calculated diagnostic specificity is 99.7%

15 % Inhibition Detection of FMDV antibodies in animals inoculated with FMDV/A24 A/cELISA Days Post Inoculation S25 S192 C14 C141 P5 P6 3B celisa 3B celisa is used for surveillance to monitor FMDV circulation (Chen et al., 211) 1% seroconversion at 5 dpi % positive at 5 dpi 83% seroconversion at 7 dpi

16 Detection of FMDV antibodies in animals inoculated with FMDV/A22 Iraq % Inhibition Dys Post Inoculation S68 S692 A/cELISA Both were seropositive at 5 dpi 3B celisa Both were positive at 6 dpi

17 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 % Inhibition Sheep #19 Sheep #2 Sheep #21 Sheep #22 Sheep #23 Sheep #24 Sheep #25 Sheep #26 Sheep #27 Sheep #28 Sheep #29 Sheep #3 Sheep #31 Sheep #32 Sheep #33 Sheep #34 Sheep #35 Sheep #36 % Inhibition Sheep #19 Sheep #2 Sheep #21 Sheep #22 Sheep #23 Sheep #24 Sheep #25 Sheep #26 Sheep #27 Sheep #28 Sheep #29 Sheep #3 Sheep #31 Sheep #32 Sheep #33 Sheep #34 Sheep #35 Sheep # Days Post Inoculation Days Post Inoculation A/cELISA 3B celisa 1% seroconversion at 7 dpc VNT 1% at 9 dpc 38.9% seroconversion at 7dpc 1% at 1 dpc

18 Detection of FMDV antibodies in vaccinated and challenged-sheep Percentage of Inhibition A/cELISA Days Post Challenge Sheep #7 Sheep #8 Sheep #9 Sheep #1 Sheep #11 Sheep #12 VNT Titer (Log 1) VNT Days Post Challenge Sheep #7 Sheep #8 Sheep #9 Sheep #1 Sheep #11 Sheep #12 Percentage of Inhibition B celisa Days Post Challenge Sheep #7 Sheep #8 Sheep #9 Sheep #1 Sheep #11 Sheep #12 Sheep were vaccinated with A22 Iraq and challenged with A Vietnam/13 after 4 days vaccination Seroconversion A/cELISA VNT 3B celisa 8 dpc 5% 17% 17% 9dpc 1% 33% 33%

19 A/cELISA specificity using sera from sheep vaccinated with O1 Manisa and challenged with O/SKR/1 % Inhibition 1 5 Sheep 1 Sheep 2 Sheep 3 Sheep 4 Sheep 5 Sheep 6 Sheep 7 Sheep 8 % Inhibition 1 5 Sheep 1 Sheep 2 Sheep 3 Sheep 4 Sheep 5 Sheep 6 Sheep 7 Sheep Day Post Challenge A/cELISA Day Post Challenge O/cELISA (Jacquelyn Horsington et al., 214)

20 Summary 1. 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.

21 Acknowledgements NCFAD/CFIA Hilary Bittner, Wanhong Xu, Melissa Goolia, Haben Gabir, Kate Hola, Tim Salo, Dr. Charles Nfon Animal care staffs Australian Animal Health Laboratory Dr. Jacquelyn Horsington