关键词: H9亚型, 禽流感病毒, mRNA疫苗, LNP, 免疫保护

Abstract:

H9 subtype of avian influenza (AI) poses a significant threat to the poultry industry, particular the broiler industry. As current inactivated vaccine cannot effectively prevent virus infection and replication in the upper respiratory tract of chickens, it is necessary to generate an mRNA vaccine targeting the hemagglutinin (HA) gene of H9 subtype avian influenza virus (AIV). Based on the HA gene sequences available on GenBank in 2022, full-length of the HA sequences (HA) and its extracellular domain (HAe) were optimized, and cloned into the pUC57 vector to construct transcription templates in vitro. After transcription in vitro, capping, and purification, the mRNAs were encapsulated in lipid nanoparticles (LNPs) and transfected into human embryonic kidney cells (293T), baby hamster kidney cells (BHK-21), and chicken embryo fibroblast cells (DF-1). HA protein expression was confirmed via Western blot. Twenty 3-week-old SPF chickens were randomly divided into four groups (n=5). Each chicken was immunized with 0.3 mL PBS, 25 μg HA mRNA LNP, 25 μg HAe mRNA LNP and 0.3 mL H9 subtype AI inactivated vaccine, respectively. Serum samples was collected 4 weeks post-immunization to assess hemagglutination inhibition (HI) antibody titers. All animals were intranasally and ocularly challenged with 0.2 mL of H9N2 subtype AIV (containing 10^6.0EID₅₀). Oral swabs were collected 5 days post-challenge to detect virus isolation, and peripheral blood mononuclear cells (PBMCs) were collected for the measurement of expression levels of IFN-γ and IL-4, alongside observing pathological changes of tracheal tissue. Western blot analysis showed that both 2 mRNA HA proteins were successfully expressed and could be effectively translated into cells following LNP encapsulation. The mean HI antibody titers (log2) post immunization with HA mRNA LNP and HAe mRNA LNP in SPF chickens were 5.6 and 3, respectively. The H9 subtype AI inactivated vaccine induced significantly higher antibody titer (9.8), compared to both two mRNA LNP groups (P < 0.01). Virus isolation rates of the challenge control, HA mRNA LNP, HAe mRNA LNP, and H9 subtype AI inactivated vaccine groups were 5/5, 1/5, 4/5, and 2/5, respectively. The HA mRNA LNP group exhibited significantly higher IFN-γ expression levels compared to other groups (P < 0.05), while the HAe mRNA LNP group showed significantly lower IL-4 expression levels (P < 0.05). The pathological changes of tracheal tissue indicated that immunization with HA mRNA LNP and H9 subtype AI inactivated vaccine effectively mitigated damage of H9 subtype AIV to tracheal epithelial cells in SPF chickens. The constructed mRNA vaccine with HA full-length of H9N2 subtype AIV could provide an effective protection to chickens on H9 subtype AIV attack, while mRNA vaccine with HA extracellular domain could not provide an effective immune protection.

Key words: H9 subtype, avian influenza virus, mRNA vaccine, LNP, immune protection