Use of Synonymous Deoptimization to Derive Modified Live Attenuated Strains of Foot and Mouth Disease Virus

Figures

• 

  FIGURE 1. Plaque morphology and kinetics of growth in cell culture. (A) Plaque morphology of WT and deoptimized viruses was analyzed by plaque assay in BHK-21 cells. Cell monolayer were infected for 48 h in semisolid media followed by staining with crystal violet. Kinetics of growth in multiple cell lines: (B) BHK-21 and (C) IBRS-2 cells lines or (D) PK and (E) EBK cells were infected with FMDV A24Cru wild type (A24-WT) or deoptimized variants, A24-P2Deopt, A24-P3Deopt, and A24-P2/3Deopt at MOI = 2. Samples were taken at 1, 3, 6, and 24 hpi and virus titers were determined by plaque assay on BHK-21 cells. (F) LF-BKαVβ6 cells were mock infected or infected with either A24 WT, P2, P3, or P2/P3 deoptimized viruses and lysates were collected at 2, 4, 7, and 24 hpi. Proteins were resolved by SDS-PAGE and western blot was applied to detect FMDV VP1, eIF4G, G3BP1, and tubulin. Cleavage of eIF4G (220 kDa) generates a fragment of approximately 120 kDa (*Cp).

• 

  FIGURE 2. Deoptimized viruses are attenuated in vivo in mice. 6–7 weeks old female C57BL/6 mice (n = 6/group) were subcutaneously inoculated in the footpad with FMDV A24-deoptimized mutants, A24-P2/P3Deopt, A24-P2Deopt, or A24-P3Deopt at the indicated doses (plaque forming units –pfu-/animal). One group was inoculated with 1 × 105 pfu/animal of A24-wild type (WT) as control. (A) Survival rates determined daily post inoculation. (B) Virus titers were measured in serum samples collected for 7 days post inoculation (dpi). (C) FMDV specific antibody neutralizing titers were measured in serum samples collected at 0, 5, 7, 14, and 21 dpi and at 7 and 14 days post-challenge (dpc). ∗P < 0.05.

• 

  FIGURE 3. Clinical outcome in animals inoculated with A24 Deoptimized candidates at indicated doses. 18–23 kg castrated male Yorkshire swine (n = 3/group) were clinically monitored for 7 days after inoculation and samples of heparinized blood were collected daily. One contact animal was housed in direct contact to the animals inoculated with the different doses of indicated deoptimized candidates and subject to the same clinical evaluation and sampling regime. Average of clinical score (blue bars) and % of lymphocytes (orange line) are represented except for the contact animals that are represented individually.

• 

  FIGURE 4. Virus detection in serum and nasal swabs in animals inoculated with different viruses at indicated doses. 18–23 kg castrated male Yorkshire swine (n = 3/group) inoculated with A24-P3Deopt, A24-P2Deopt, or A24-P2/P3Deopt at indicated doses of were sampled daily. One contact animal was housed in direct contact to the animals inoculated with the different doses of A24-P2/P3Deopt and subject to the same sampling regime. The amount of virus was detected by virus isolation in serum (red line) and nasal secretions (blue line) and by qPCR and expressed as genome copy numbers (GCN) per ml of serum (red dashed line) or per ml of nasal secretions (blue dashed line). Average of viral amounts are represented except for the contact animals that are represented individually.

• 

  FIGURE 5. Determination of FMDV neutralizing antibodies or antibody isotype in the serum of animals inoculated with A24-P2, P3, or P2/P3Deopt. (A) The presence of FMDV neutralizing antibodies was evaluated in the sera of animals inoculated with A24-P2, P3, or P2/P3Deopt, by a microtiter neutralization assay on BHK-21 cells. Titers are reported as the log10 of the reciprocal of the highest dilution of serum that neutralized the virus in 50% of the wells. (B–D) Antibody isotype profiles in swine sera after inoculation. FMDV-specific IgM, IgG1, and IgG2 were detected by sandwich ELISA at 7, 14, and 21 dpi. Each data point represents the mean (±SD) of each group.

• 

  FIGURE 6. Cytokine protein profiles in serum after FMDV infection. Levels of IFN-α (A), IL-1β (B), and IL-6 (C) were detected by sandwich ELISA in the serum of inoculated animals with indicated doses, during the first 4 days after infection. Amount of protein is expressed as the relative level with respect to the amount observed at 0 dpi. Error bars represent the variation within the three animals from each group. ∗P < 0.05.

• 

  FIGURE 7. Clinical outcome and virus shedding in animals inoculated with different doses of A24-P2/P3Deopt and A24Cru wild type (A24-WT). 18–23 kg castrated male Yorkshire swine (n = 4/group) were inoculated with 102, 103, or 105 pfu/animal of FMDV A24-P2/P3Deopt or 103 pfu/animal of A24-WT as control group. Animals were monitored for 7 days. Upper graphs: average of clinical score (blue bars) and % of lymphocytes (orange line) were determined daily. Lower graphs: The amount of virus was detected by virus isolation in serum (red line) and nasal secretions (blue line) and by qPCR and expressed as genome copy numbers (GCN) per ml of serum (red dashed line) or per ml of nasal secretions (blue dashed line).

• 

  FIGURE 8. Determination of FMDV neutralizing antibodies and isotypes in the serum of animals inoculated with A24-P2/P3Deopt or A24Cru wild type (A24-WT). (A) Presence of FMDV neutralizing antibodies was evaluated by a microtiter neutralization assay on BHK-21 cells in sera of animals inoculated with different doses of A24-P2/P3Deopt or A24-WT at the indicated time points after inoculation or challenge. Titers are reported as the log10 of the reciprocal of the highest dilution of serum that neutralized the virus in 50% of the wells. (B–D) Antibody isotype profiles in swine sera after inoculation. FMDV-specific IgM, IgG1, and IgG2 were detected by sandwich ELISA at indicated time points. Each data point represents the mean (±SD) of each group.

• 

  FIGURE 9. Clinical outcome in animals challenged with A24Cru (A24-WT) after previous inoculation with A24-P2/P3Deopt. 18–23 kg castrated male Yorkshire swine (n = 4/group) were challenged with 105 plaque forming units (pfu) of FMDV A24-WT. Animals were monitored for 7 days and samples of heparinized blood were collected daily. Clinical score (blue bars) and % of lymphocytes (green line) for each animal are represented for each individual animal.