cGAMP-Loaded M2e Nanovaccine Elicits Cross-Reactive Immunity and Mitigates H6N1 Avian Influenza Infection in Chickens.

Introduction: Avian influenza virus (AIV) is a rapidly evolving zoonotic pathogen that causes significant economic loss in the poultry industry and threatens public health. In the development of a broadly protective influenza vaccine, the extracellular domain of matrix protein 2 (M2e) is a promising antigen candidate as it is highly conserved across most influenza A subtypes. M2e-induced immune protection is primarily mediated through antibody-dependent cellular cytotoxicity (ADCC), which enables the clearance of infected cells before viral release. However, the immunogenicity of M2e is limited due to its small size and low abundance on the viral surface, necessitating effective antigen delivery systems to enhance its presentation. Methods: We developed a PLGA-based nanoshell vaccine co-encapsulating M2e peptide and a STING agonist and evaluated its immunogenicity and protective efficacy in specific-pathogen-free (SPF) chickens. Results: Immunization of SPF chickens with the M2e nanoshell vaccine significantly enhanced immune-related gene expression and B-cell expansion, leading to a robust and durable M2e-specific antibody response. In addition, the induced antibodies demonstrated cross-reactivity by effectively recognizing the M2e protein expressed on MDCK cells infected with seven different strains of influenza viruses. Following H6N1 challenge, vaccinated chickens showed attenuated lesions in the kidneys and decreased viral load and viral antigen in the respiratory and intestinal lymphoid tissues, suggesting reduced viral replication. Furthermore, to explore potential Fc-mediated effector functions, an in vitro killing assay demonstrated that chicken splenic natural killer cells were capable of lysing influenza-infected target cells in the presence of serum from M2e nanoshell-immunized chickens, providing initial functional evidence of ADCC activity in the avian system. Conclusion: Collectively, these results demonstrate that the M2e nanoshell vaccine elicits durable and cross-reactive immune responses and reduces tissue damage, potentially involving ADCC-mediated effector mechanisms. The M2e nanoshell vaccine shows potential as a broadly protective avian influenza vaccine and holds value for controlling AIV outbreaks in poultry.