Evidence of residual neutralisation after removal of five neutralising antigenic sites in serotype O FMDV

Transcription

1 Evidence of residual neutralisation after removal of five neutralising antigenic sites in serotype O FMDV By Amin Asfor, S. Upadhyaya, D. Paton, D. King, N. Knowles and M. Mahapatra Transmission Biology Group Pirbright Laboratory United Kingdom

2 Conclusion FMDV O1K 5-site mutant virus has been produced for the first time using reverse genetics Mutation of the amino acid residues of the neutralising antigenic sites result in rescue of a viable virus not critical for viability of the virus Residual neutralisation observed after mutations of all the 5 known antigenic sites indicates presence of yet unidentified epitopes Two Novel Residues (VP2-74 and VP2-191) could be part of Site 2. proved to be significantly important in reduction of virus neutralization titre

3 Genome orgnization + sense ss RNA genome ~8300nt RNA 5' 1300nt AUG OPEN READING FRAME 3'UTR (VPg) STRUCTURAL NONSTRUCTURAL NH2 myristoylation Polyprotein Lpro VP4 VP2 1A 1B 1C 1D VP3 VP1 2A 2B B 2C 1 B 3B B 3A 3 C 3 D 2 B 3 B AAA (n) AAA (n) protease Selfcleavage Capsid protease RNA polymerase Heparin sulphate receptor binding G-H Loop Receptor binding VPg

4 Antigenic sites of type O Red- VP1, Blue- VP2, Light green- VP3, Green-VP4. Site 5 is not shown, but located on the GH loop of VP1 within site 1.

5 Project Background Dunn et al (1998) reported that a mutant virus (5 sites mutant) which is resistant to neutralising murine monoclonal antibodies (Mabs) also resists neutralisation by bovine polyclonal sera Guinea pigs were protected in cross-challenge studies from virulent wild-type and mutant viruses using either wild-type or mutant 146S antigen as inactivated whole virus vaccine. The relative importance of different epitopes has not been ascertained. This project is aiming to : Define viral determinants of antibody mediated protection Identifying new surface exposed neutralizing or non neutralising epitope/s using reverse genetic technique This will contribute to the development of sequence-based vaccine selection methods and novel broadly cross-reactive vaccines

6 6 AIM To identify the contribution of different epitopes present on the surface of foot-and-mouth disease virus (FMDV) towards antibody mediated protection Infectious clone(ic) O1 Kaufbeuren CHALLENGE VIRUS Addition of more mutations Design of neutral virus in vitro neutralization using O1K antiserum in-vivo experiment to check the lack of protection against challenge virus Final construct Reconstitution of individual epitops in vitro neutralization using O1K antiserum selected mutants antisera Assessment of protection Disease generalization Body temperature

7 Neutral virus design and preparation (1) Order a synthetic gene containing all the required mutations (26 amino acid changes): sub-cloned into the full length FMDV plasmid No viable virus was recovered- too many mutations to be tolerated G67 and O 1 K-WT G67: quadruple mab escape mutant of O 1 K that bears point mutations conferring resistance to neutralization by mabs, specific for each of the four major antigenic sites (site 1-4) defined previously O 1 Kaüfbeuren (O 1 K-wt) parent of G67

8 Preparation of G67 mab mutant neutral virus (2) pt7s3/o1k (11.2 Kbp) AflII Primer 1 5` 3` Primer 2 SpeI restriction site Inverse PCR Inverse PCR (16 cycle) with primer1 and primer2 AflII SpeI 5` 3` AflII G67 capsid (4-site mutant)+ VP1 149 cloned wt capsid genes (2.5kb) containing all the required mutations in an intermediate vector WT capsid from which G67 was derived SpeI

9 9 Results In-vitro transcription of pt7s3-wt pt7s3-g67+ VP1 149 (5-site mutant) pt7s3-o1 Italy (Control) RNA was electroporated in to BHK 21 IB-RS 5,2 5,1 4,9 5 4,8 4,7 4,6 4,5 4,4 4,3 Passage to BHK3 Passage to BTY3 Viable virus was recovered from all construct

10 In vitro assessment of the mutant viruses using virus neutralisation test GP post-vaccinal sera against WT Bovine post-vaccinal sera M O1Italy 0 5M O1Italy Percent (%) reduction in neutralising antibody titre by mutant viruses

11 How can we increase the percentage of reduction in neutralisation?

12 Antigeni c sites/mut ants Viral protein position s VP1 133 VP1 138 Site 1 VP1 148 Additional mutations VP1 150 Site 2 VP2 72 Site 3 VP1 43 Site 4 VP3 58 Site 5 VP1 149 VP3 85 Additional mutations O1K-wt K R L V S T E Q H S T 5M E K R A N K V H H S T M1 E K R A N K V H Q S T M2 E K R A N K V H H P T M3 E K R A N K V H H A T M4 E K R A N K V H H S N M5 E K R A N K V H H S A DM1 E K R A N K V H Q P T DM2 E K R A N K V H Q A T DM3 E K R A N K V H H P A VP2 74 VP2 191 VP2-74 VP2-191 VP3-85 O1 K reduced structure showing critical residues of 5 neut. antigenic sites (blue) and additional mutations (green/pink/red)

13 Virus titre in log 10 TCID 50 /ml Growth- kinetics of single and double mutant viruses O1K WT 5M M1 Phenotypic characterisation 2 M2 1 M3 M4 0 M5 0 Hr 3 hr 6 hr 9 hr 12 hr 24 hr 6 WT M2 M O1K WT 5M DM1 5M DM1 DM3 1 DM2 DM3 0 0 Hr 3 hr 6 hr 9 hr 12 hr 24 hr Hours post-infection

14 Optical density mabs binding activity of WT versus Mutant viruses Technique used: Indirect ELISA Murine mabs specific for the five known antigenic sites Mab Site Critical residues B2 1 VP1-144, 148, 154 D9 1 VP1-144 C6 2 VP2-73 C9 2 VP2 71, 72, 75, 77 C8 3 VP1-43,44 14EH9 4 VP3-58 OC3 5 VP1-149 D9, C8, C6, 14EH9, OC3 All (O1K) VP1-43, VP1-138, VP1-133-VP1-148, VP1-149, VP1-150, VP2-72, VP3-58 Critical residues of type O mabs (Crowther et al., 1993) Viruses

15 In vitro assessment of the mutant viruses using virus neutralisation test GP post-vaccinal sera Bovine O1BFS post-vaccinal sera * * * * * * M M1 M2 M3 M4 M5 DM1 DM2 DM3 Percent (%) reduction in mutant viruses neutralising antibody titre Neutral virus 5 M + VP2.191(N-T)= M4 5 M +VP2.191(N-A)=M5 5 M +M5+M2 +DM3 5 M + VP2.74(S-P) = M2

16 Threshold of reduction in neutralizing antibody titre of GP sera To determine the threshold of reduction in neutralizing antibody titre of GP sera using different FMDV topotypes and serotypes (A 22 IRQ, Asia1) To confirm the specificity of the antibody and its homologous epitopes Topotypes Serotypes ME-SA AFRICA CATHAY % reduction in virus neutralisation antibody titre of GP post-vaccinal sera

17 Conclusion FMDV O 1 K 5-site mutant virus has been produced for the first time using reverse genetics The amino acids residues of the neutralising antigenic sites (mutated so far) - not critical for viability of the virus Residual neutralisation indicates presence of yet unidentified epitopes Residues VP2-74 and VP2-191 could be part of Site 2. They are in a close proximity to VP2-72 and VP2-188 and proved be significantly important in reduction of virus neutralization titre VP2-74 VP2-188 VP2-191

18 Future work Production of hyper immune sera against the neutral virus (M5) in GPs Reconstitution of individual epitopes recover and characterise mutant viruses In vitro and in-vivo studies: to determine the relative importance of different epitopes

19 Thank You Dr. Mana Mahapatra Prof. David Paton Dr. Donald King Mr. Nick Knowles Dr. Paul Barnett Sasmita Upadhyaya Daryl Borley Fufa Bari D. J. Kalita CODA-CERVA-VAR (Belgium) D. Lefebvre A. De Vleeschauwer K. De Clercq Funding: