Fig 1.
Schematic representation of the FMDV genome and the rSFV plasmids used in this study.
The P1-2A, P1-2A-3CC142S and P1-2A-mIRES-3C FMDV cDNA cassettes have been described elsewhere [13,26] while the pSFV3 and the split helper plasmids have also been described previously [23]. Positions of relevant restriction enzyme sites used are shown. Abbreviations: P1-2A: capsid precursor protein; 3C: 3Cpro wild-type or C142S mutant; mIRES: internal ribosome entry site GTTA mutant; SP6: SP6 promotor; nsP1-P4: SFV non-structural proteins 1–4; PS: packaging signal; 26S: SFV 26S subgenomic promotor; C: SFV capsid; p62, 6K and E1: SFV spike proteins.
Fig 2.
Expression of FMDV capsid proteins using the SFV split-helper system.
Uninfected BHK cells (A) or cells infected with rSFV (B), rSFV-FMDV-P1-2A (C), rSFV-FMDV-P1-2A-3CC142S (D) or rSFV-FMDV-P1-2A-mIRES-3C (E), at an MOI of 20, were immunostained at 16 h post infection. FMDV proteins were detected with an anti-FMDV O1 Manisa polyclonal antibody and a secondary antibody labeled with Alexa Fluor 568 (red). The cellular nuclei were visualized with DAPI (blue). Bar, 100 μm. The results shown are representative of three independent experiments.
Fig 3.
Characterization of expressed FMDV capsid proteins.
(A) Uninfected BHK cells (control, lane 1) or cells infected with rSFV (lane 2), rSFV-FMDV-P1-2A (lane 3), rSFV-FMDV-P1-2A-3CC142S (lane 4) or rSFV-FMDV-P1-2A-mIRES-3C (lane 5) were harvested and the cell lysates were fractionated by SDS–PAGE. The proteins were transferred to PVDF membrane and probed with antibodies specific for FMDV capsid proteins (top), FMDV 2A (second), FMDV 3Cpro (third) and β-actin (bottom) as indicated. Detection of β-actin was used as a control for equal protein loading. The results shown are representative of three independent experiments. Molecular mass markers (kDa) are indicated on the left. (B) Cell lysates, as used in panel A, were diluted (10-fold initially and then 2-fold dilutions) and analysed with a FMDV serotype O-specific antigen ELISAs. The results shown are representative of two independent experiments. AU, absorbance units.
Fig 4.
Assembly and properties of FMDV protomers, pentamers and empty capsids expressed from rSFV vectors.
(A) BHK cells were infected as described in Fig 2 using the indicated rSFV-FMDVs. Cytoplasmic extracts were prepared (16 h post infection) and sedimented through sucrose gradients (10–30%) and fractionated. FMDV proteins from each fraction were detected using a serotype-specific antigen ELISA. The location of protomers, pentamers and empty capsids are indicated. (B) Fractions (fr) from the rSFV-FMDV-P1-2A-mIRES-3C infected BHK cells containing protomers (fr. 3), pentamers (fr. 7) and empty capsids (fr. 15) were assayed to detect FMDV antigen binding, in the presence or absence of EDTA as indicated, to the integrin αvβ6 coated directly onto plates. Binding buffer was used as the control. Results are presented as mean ± SEM of triplicate samples.
Fig 5.
Vaccination of cattle with rSFV-FMDV particles and response to FMDV challenge.
The indicated animals (in experiment 1) were unvaccinated (controls, C1, C2) or vaccinated on post-vaccination day (PVD) 0 with either the rSFV-FMDV-P1-2A (calves C3-C5) or the rSFV-FMDV-P1-2A-mIRES-3C (calves C6-C8) vectors (indicated by vertical dotted line, marked rSFV) and challenged with FMDV on PVD 21 (indicated by vertical line, marked FMDV). (A) Body (rectal) temperatures in each animal were monitored on a daily basis. (B) Serum samples collected at each indicated day were assayed for anti-FMDV antibodies in a serotype O-specific blocking ELISA. The diagnostic cut-off level (50%) in the ELISA is indicated by the horizontal line. (C) The number of FMDV RNA copies present in the serum was determined by RT-qPCRs by reference to a dilution series of a known concentration of FMDV RNA. The results are presented as copies of FMDV RNA/ml of serum. A level of 107 copies/ml is indicated by a horizontal line.
Table 1.
Reciprocal titres of anti-FMDV antibodies (serotype O) in sera from unvaccinated and rSFV-FMDV vaccinated calves in experiment 1.
Table 2.
Reciprocal titres of anti-FMDV antibodies (serotype O) in sera from unvaccinated and rSFV-FMDV vaccinated calves in experiment 2.
Fig 6.
Prime-boost vaccination strategy effectively induces anti-FMDV antibodies in cattle and blocks virus circulation post-challenge.
In experiment 3, calves in group 1 (panels A, D, G) were unvaccinated. Calves in group 2 (panels B, E, H) were vaccinated with rSFV-FMDV-P1-2A-mIRES-3C on PVD 0 (dotted vertical line, marked rSFV) and then with O1 Manisa empty capsid particles on PVD 14 (dotted vertical line, marked ECs). Calves in group 3 (panels C, F, I) were vaccinated with O1 Manisa empty capsid particles on PVD 0 (dotted vertical line, marked ECs) and then with rSFV-FMDV-P1-2A-mIRES-3C on PVD 14 (dotted vertical line, marked rSFV). All animals were challenged with FMDV (O UKG/34/2001) on PVD 28 (indicated by solid vertical line, marked FMDV). Body temperatures in each animal were monitored on a daily basis (panels A-C). Sera were collected from each calf on the indicated days and assayed in the serotype O blocking ELISA (panels D-F). Results are presented as ODP (%) with a cut-off value of 50% (indicated by horizontal line). The presence of FMDV RNA circulating in serum was assayed by RT-qPCR (panels G-I). The results are presented as copies of FMDV RNA/ml of serum as in Fig 5. A level of 107 copies/ml is indicated by a horizontal line.
Table 3.
Reciprocal titres of anti-FMDV antibodies (serotype O) in sera from calves in experiment 3.
Fig 7.
Production of neutralizing anti-FMDV antibodies in cattle.
Sera from the calves in group 1 (C1-C3, black bars, control), group 2 (C4-C6, red bars, vaccinated with rSFV-FMDV-P1-2A-mIRES-3C on PVD 0 followed by empty capsids on PVD 14) and group 3 (C7-C9, blue bars, vaccinated with empty capsids on PVD 0 followed by rSFV-FMDV-P1-2A-mIRES-3C on PVD 14) were collected during the experiment 3. All animals were challenged with FMDV on PVD 28. Samples from PVD 14 (A), PVD 28 (B) (prior to challenge) and PVD 36 (C) (post challenge) were assayed for the presence of neutralizing anti-FMDV antibodies using VNTs. Results were calculated as reciprocal VNT titres and are displayed as log2 values. VNT titres ≤ 11 (log2 11 = 3.459) are considered negative while titres ≥45 (log2 45 = 5.492) are considered positive. Intermediate titres are considered as inconclusive (indicated within grey bar).