LPS/TLR4-activated M1-polarized macrophage-derived exosomes enhance IBV vaccine efficacy in chickens

ABSTRACT Infectious bronchitis virus (IBV) imposes substantial economic losses on poultry production due to extensive serotypic diversity and limited cross-protection conferred by conventional vaccines. This study evaluated exosomes derived from M1-polarized chicken macrophages (HD11 M1 -exo) as a novel adjuvant for IBV vaccination. HD11 M1 -exo, isolated from lipopolysaccharide (LPS)-activated HD11 macrophages via ultracentrifugation, demonstrated significant immunomodulatory properties across multiple experimental systems. In vitro analyses demonstrated that HD11 M1 -exo enhances macrophage phagocytosis and promotes cellular immune activation via the LPS/TLR4 signaling pathway. In ovo analyses showed that HD11 M1 -exo pretreatment upregulates tracheal expression of IL-1β, IL-2, IL-4, IFN-γ, TNF-α, and TLR4 at different time points, thereby enhancing viral resistance and reducing pathological damage. In chickens, HD11 M1 -exo administration elevated CD80/CD86 and TGF-β4 expression in respiratory tissues and increased secretory immunoglobulin A (IgA) levels in lacrimal fluid. When co-administered with the H120 vaccine, HD11 M1 -exo significantly improved both humoral immunity (elevated serum IgY and mucosal IgA) and cellular responses (increased CD80/CD86 expression), outperforming commercial adjuvants in efficacy. Following the viral challenge, HD11 M1 -exo + H120-immunized chickens exhibited significantly reduced viral loads and attenuated histopathological lesions compared to controls. These results collectively suggest that exosome-based formulations may serve as promising adjuvants for enhancing the immunogenicity and protective efficacy of poultry vaccines. IMPORTANCE Infectious bronchitis virus (IBV) causes significant global economic losses in the poultry industry despite extensive vaccination programs. Current vaccines often fail to elicit sufficient mucosal and cellular immunity, which are critical for protection against the virus. Although commercial adjuvants have been employed to enhance vaccine efficacy, many exhibit limitations in eliciting comprehensive immune responses. In this study, we comprehensively evaluated HD11 M1 -exo as a novel adjuvant for IBV vaccines across in vitro , in ovo , and in vivo models for the first time. Our results demonstrate that HD11 M1 -exo enhances macrophage function via lipopolysaccharide (LPS)/TLR4 signaling, upregulates key cytokines and immune markers in embryonic tissues, and significantly boosts cellular, humoral, and mucosal immunity when co-administered with live-attenuated IBV vaccines, outperforming commercial adjuvants. Importantly, this adjuvant strategy significantly enhanced protective efficacy in challenged chickens. This study provides a foundation for developing exosome-based adjuvants that could advance poultry vaccination strategies against IBV and other avian respiratory pathogens.