Efficient eukaryotic expression and potent antiviral activity of a long-acting recombinant feline interferon-ω2-Fc fusion protein against major feline viruses.

Abstract

Feline interferon-ω2 (FeIFN-ω2) holds potential as a therapeutic agent against feline viral infections. However, its clinical application is limited by rapid clearance and suboptimal antiviral effectiveness. Thus, in this study, an Fc-fused construct, FeIFN-ω2-Fc, was engineered to improve antiviral potency and pharmacokinetic properties both in vitro and in vivo. Three recombinant constructs-native FeIFN-ω2, FeIFN-ω2-Dimer, and FeIFN-ω2-Fc-were expressed in Chinese Hamster Ovary cells. All showed strong antiviral activity (10-10IU/mg) and effectively activated downstream interferon signaling. Functional assays, including 50 % tissue culture infectious dose assay, quantitative polymerase chain reaction, and immunofluorescence, confirmed their ability to inhibit feline calicivirus (FCV), feline herpesvirus type 1 (FHV-1), and feline parvovirus (FPV). Glycosylation analysis revealed two sites (S102 and T128) in the ω2 domain of FeIFN-ω2-Fc that contributed to its structural stability and functional enhancement. Among the three candidates, FeIFN-ω2-Fc demonstrated the best overall profile, with higher expression levels, simplified purification, and favorable pharmacokinetics. In animal models, it was well tolerated and significantly alleviated clinical symptoms, reduced viral loads, and preserved tissue integrity following FHV-1 infection. Pharmacokinetic studies showed a marked increase in plasma half-life, from 5.80 ± 1.75 h for the native protein to 34.05 ± 6.36 h for the Fc-fused form. Further extension to 40.55 ± 6.61 h was achieved by introducing YTE mutations (S250Y/S252T/T254E) within the Fc region. Based on these findings, a dosing regimen of 4 × 10IU/kg every other day is proposed, supporting FeIFN-ω2-Fc as a strong candidate for feline antiviral therapy.