Baculovirus-based production of virus-like particles for avian influenza: development, immunogenicity, and cross-protection against H5 and H7 AIVs

The persistent threat of H5 and H7 highly pathogenic avian influenza (HPAI) necessitates effective broad-spectrum vaccines. This study systematically evaluated the immunogenicity and protective efficacy of H5N6 virus-like particle (H5N6-VLP) vaccine and subsequently developed a trivalent VLP vaccine (H5N6-H5N8-H7N9-VLP) using the baculovirus expression system (BEVS). Production of H5N6-VLP was optimized at a multiplicity of infection (MOI) of 0.1 to achieve efficient assembly. The H5N6-VLP vaccine induced potent immune responses in specific-pathogen-free (SPF) chickens, providing 100% protection against homologous H5N6-GD154 virus when the HA titer reached 9 log2. Compared with traditional inactivated vaccines, the H5N6-VLP vaccine induced faster humoral immune responses and higher CD8⁺ T cell proportions. The HI seroconversion rate was 30% on day 7 post-immunization (0% for inactivated vaccine), reached 100% by day 10, while it was only 80% on day 14 for the inactivated vaccine. Furthermore, the protective efficacy of the H5N6-VLP vaccine was associated with increased IFN‑γ expression and elevated proportions of B cells and CD8⁺ T cells. Although both vaccines provided complete homologous protection, the H5N6-VLP vaccine showed better cross-protection against the heterologous H5N1-D889 strain (70% vs. 40%). A delayed challenge experiment further demonstrated that the H5N6-VLP vaccine induced durable immunity, with HI titers remaining above the protective threshold and 100% protection against homologous challenge at 8 weeks post-immunization. Based on these findings, a trivalent H5N6-H5N8-H7N9-VLP vaccine was developed. The trivalent vaccine induced substantial antibody responses and provided complete protection against parental and circulating strains, with efficacy comparable to a commercial inactivated vaccine and no detectable antigenic interference between components. Our findings highlight the potential of the BEVS-derived H5N6-VLP vaccine, particularly its early antibody induction, which allows rapid flock protection during acute HPAI outbreaks, and its cross-reactivity against heterologous strains. Furthermore, the trivalent vaccine formulation extends the potential application of this VLP platform for broad-spectrum HPAI control.