Use of Protein Pegylation to Prolong the Antiviral Effect of IFN Against FMDV

FIGURE 1. In vitro antiviral activity of PEGpoIFNα. Porcine IB-RS2 cells were treated with two-fold serial dilutions of Ad5-poIFNα expressed poIFNα or PEGpoIFNα (starting material 2– 1 to 2– 12 dilutions or ∼10 μg/ml, respectively), and incubated for up to 7 days post-treatment (dpt). At 1 day intervals, supernatants were collected from individual plates and stored at –70°C. Fresh IB-RS2 cells were treated with the stored supernatants samples containing Ad5-poIFNα expressed poIFNα or PEGpoIFNα. Twenty-four hours post-treatment, IFN containing supernatants were removed and cells were infected with FMDV at MOI-0.1. Cell viability after challenge was measured by MTT assay, 72 h post-infection. OD values of one represent 100% viability, or absence of cytotoxicity. OD values of 0.4 represent maximum cytotoxicity. Results represent the mean of four independent experiments ± SD.

FIGURE 2. Systemic antiviral response induced by treatment. Bioactivity against FMDV assayed by inhibition of virus growth (A) or by MxCAT ELISA (B) was determined in sera of treated animals with PEGpoIFNα or with Ad5-poIFNα after 1- or 5-days post-treatment (dpt) right before the animals were challenged (0 dpc). A group of PBS-treated animals was included as a negative control. For all groups, samples taken before any treatment (0 dpt) were used as baseline. Graph represents group average (bar) and individual values of each animal in the group at indicated time points. ∗∗∗P ≤ 0.001. (C) Gene expression in PBMCs of treated animals with PEGpoIFNα or with Ad5-poIFNα after 1 or 5 dpt [right before the animals were challenged (0 dpc)] was measured by qRT-PCR. A group of PBS-treated animals was included as negative control. Results are expressed as relative fold induction values with respect to 0 dpt (ΔΔCT). GAPDH expression was used as normalizer.

FIGURE 3. Clinical outcome in animals after challenged with FMDV A24Cru. 18–23 kg castrated male Yorkshire swine (n = 4/group) were clinically monitored for 7 days after challenge intradermally (ID) in the heel bulb with FMDV A24Cru and samples of heparinized blood were collected daily. Clinical score (blue bars) and % of lymphocytes (orange line) are represented for each animal 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 = 4/group) challenged with FMDV A24Cru after indicated treatments (groups) were sampled for 7 days. The amount of virus was detected by virus isolation in serum (orange line) and nasal secretions (blue line) and by qPCR and expressed as RNA copy numbers per ml of serum (gray bars) or per ml of nasal secretions (black bars) are represented for each animal individually.

FIGURE 5. Determination of FMDV neutralizing antibodies. The presence of FMDV neutralizing antibodies was evaluated in sera of animals treated with PEGpoIFNα or with Ad5-poIFNα and challenged after 1 or 5 dpt, 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.