Genes of HA and M1 were codon-optimized for a high level of expression in High Five cells, and a 6His epitope tag was simultaneously fused to the C-terminal end of the optimized gene. vaccine dose could further enhance the cross-protective efficacy of H5N1 VLP vaccine and confer completely sterilizing protection against antigenically divergent H5N1 computer virus challenge, which was mediated by neutralizing antibodies. Our results suggest that the H5N1 VLP vaccine can provide broad-spectrum protection against divergent H5N1 influenza viruses as determined by adjuvant and vaccine dose. KEYWORDS:H5N1, virus-like particle, adjuvant, vaccine dose, neutralizing antibodies, cross-protection == Introduction == H5N1 HPAIV has been widely circulating in domestic poultry and wild birds around the world and is responsible for millions of domestic poultry deaths [1,2]. In addition to causing huge economic losses to the poultry industry worldwide, H5N1 HPAIV also poses a threat to public health. The H5N1 avian influenza computer virus (AIV) causes human infection without an intermediate host [3]. H5N1 AIV has continued to infect people worldwide since 2003, as of 14 July 2023, a total of 878 laboratory-confirmed cases of H5N1 AIV contamination were reported to WHO, including 458 deaths [4]. Currently, H5N1 subtype HPAIV revealed a novel propensity after continuous evolution in nature, such as reassort with other influenza computer virus neuraminidase (NA) subtypes, including H5N6 and H5N8 [5,6]. This unquestionably increases the risk of the H5 subtype HPAIV outbreak; thus preventing the H5N1 subtype HPAIV pandemic is usually imperative. Vaccination is considered one of the current strategies to prevent and control the spread of H5N1 HPAIV in China [7]. However, the current commercial whole computer virus inactivated vaccines do not provide optimal protection against contamination by novel H5N1 variants due to antigenic mismatch between vaccine and circulating computer virus staining. Besides, the production of the inactivated influenza vaccine relies on embryonated chicken eggs (ECEs) with many shortfalls including endogenous computer virus contamination, insufficient supply of ECEs during pandemic outbreaks, and biohazardous waste materials generation [7]. In the mean time, H5N1 strains exhibit high variability and rapidly mutate making any vaccine strain unlikely Aliskiren (CGP 60536) to retain close identity with circulating staining. Therefore, it is particularly important to develop preferable alternatives to the traditional inactivated vaccine with optimal immunization strategies to provide cross-protection against H5N1 variants. Virus-like particles (VLP) show high plasticity and Aliskiren (CGP 60536) scalability to develop multiple subtypes of influenza vaccines, including H5 [8], H6 [9], H7 [10], and H9 [11] subtypes, which are produced in numerous expression systems, such as insect cell-baculovirus [10], herb cells [12], and mammalian cells [13]. Furthermore, the innocuous VLP retains the structural and immunological properties of a native computer virus. Influenza VLP vaccines can induce comprehensive immune responses to provide cross-protection against homologous and heterologous influenza computer virus challenges by numerous administration routes [14,15]. Therefore, influenza VLP vaccine is considered one of the most encouraging alternatives to the traditional inactivated vaccine. Generally, influenza VLP constructs contain hemagglutinin (HA) and matrix protein M1. HA is the main target antigen for eliciting serological immunity to the influenza computer virus [16]. HA is used to develop influenza subunit vaccines and has been shown to induce excellent protection against influenza computer virus infection in addition to the H5 serotype because H5 HA is a poor antigen [17,18]. Influenza vaccine LEG8 antibody efficacy is not only associated with HA similarity between vaccine and circulating computer virus strains but also involved antigen structure, adjuvant selection, vaccine antigen dose, and immunization Aliskiren (CGP 60536) strategy. Benefiting from the scalability of VLP, studies have shown that incorporating additional immunological proteins adjuvants such as nucleoprotein (NP),Escherichia coliheat-labile enterotoxin B subunit protein (LTB), and flagellin into influenza VLP induces better cross-protection against heterologous H5N1 difficulties than VLP alone [19,20]. The strategies of multiple immunizations, sequential immunization, and combining with appropriate adjuvants are very major measures to improve the cross-protective efficacy of influenza vaccines against homologous, heterologous, and heterosubtypic influenza computer virus challenges [21-23]. Therefore, H5 influenza vaccines with optimal immunization strategies are particularly important for enhancing cross-protection efficacy. In this study, we developed an H5N1 subtype VLP using a baculovirus expression vector system (BEVS). The immunogenicity of H5N1 VLP was evaluated by combining with two adjuvants, including MONTANIDETM ISA 201 VG (ISA 201) and MONTANIDETM ISA 71 VG (ISA 71), and using numerous antigen doses. Low-dose immunization of chickens with H5N1 VLP vaccines elicited strong HI antibody titres and induced protection against homologous H5N1 HPAIV challenge. ISA 71 adjuvanted VLP vaccine induced higher Th1-type and Th2-type immune responses and provided better cross-protection Aliskiren (CGP 60536) against antigenically divergent H5N1 HPAIV than ISA 201 adjuvanted VLP vaccine at the same dose. Enhancing doses.