Induction and antiviral activity of human β-defensin 3 against adenovirus and influenza A virus in vitro infection of human airway epithelial cells

Background: Respiratory pathogens such as adenoviruses (AdV) and influenza A viruses (IAV) can cause serious upper and lower respiratory infections in infants, the elderly, and immunocompromised individuals. However, options for antiviral drugs targeting respiratory viruses are limited. Human beta-defensins (hBDs) are disulfide-rich peptides that demonstrate broad antimicrobial activity against bacteria, fungi, and some viruses, while also playing immunomodulatory roles. Among defensins, human beta-defensins (hBDs) are the most prevalent, with six peptides identified: hBD-1 through hBD-6. Nonetheless, the production of hBD-3 during respiratory virus infections has not been extensively studied, nor has the effect of hBD-3 on these infections. Objective: This study investigated whether infection with AdV-5 or IAV induces hBD-3 expression in human airway epithelial cells and whether a recombinant form of hBD-3 (rhBD-3) can inhibit in vitro infection by these viruses, one DNA virus and one RNA virus. Methods: In vitro models of human airway epithelial cells (A549 and HEp-2) infected with AdV-5 and IAV were established. In vitro expression of hBD-3 mRNA was assessed by RT-PCR; hBD-3 protein was examined using immunofluorescence and Western blot. The inhibition of viral infection by hBD-3 was quantified using cytopathic effect and plaque reduction assays. Additionally, epithelial cells of animal origin, MDCK and MDBK, were similarly tested for rHBD-3 antiviral activity against IAV infection. Results: IAV and AdV infections significantly increased hBD-3 mRNA levels in A549 and HEp-2 cells. Immunofluorescence and Western blotting confirmed the presence of hBD-3 protein on the surface of IAV-infected cells. Cells infected with AdV-5 produced less hBD-3 protein. Additionally, rhBD-3 inhibited both AdV-5 and IAV infections in both cell lines at concentrations of 7.5-50 μg/mL, with the strongest effect at the highest concentration. Lastly, rhBD-3 also inhibited IAV infection in MDBK and MDCK cells within the same range. Conclusions: In vitro infection of human airway epithelial cells with IAV and AdV induces hBD-3 mRNA and protein expression. In turn, rhBD-3 inhibits viral infection in a dose-dependent manner in HEp-2 and A549 cells against these viruses, an RNA virus and a DNA virus. hBD-3 is expressed and localized on the surface of infected cells. rHBD-3 also inhibits IAV infection in bovine and canine epithelial cells. Our findings suggest that hBD-3 plays a broad-spectrum role in defense against respiratory viruses in humans and animals and modulates the innate immune responses. Perspective: This work supports the use of hBD-3 as a natural, broad-spectrum antiviral agent, either alone or in combination, for treating viral infections, particularly emerging respiratory agents such as IAV and coronaviruses, for viral pandemic preparedness and response.