Adjuvant effects mediated by the carbohydrate recognition domain of Agrocybe aegerita lectin interacting with avian influenza H₉N₂ viral surface glycosylated proteins

Objective: To evaluate the potential adjuvant effect of Agrocybe aegerita lectin (AAL), which was isolated from mushroom, against a virulent H₉N₂ strain in vivo and in vitro.

Methods: In trial 1, 50 BALB/c male mice (8 weeks old) were divided into five groups (n=10 each group) which received a subcutaneous injection of inactivated H₉N₂ (control), inactivated H₉N₂+0.2% (w/w) alum, inactivated H₉N₂+0.5 mg recombinant AAL/kg body weight (BW), inactivated H₉N₂+1.0 mg AAL/kg BW, and inactivated H₉N₂+2.5 mg AAL/kg BW, respectively, four times at 7-d intervals. In trial 2, 30 BALB/c male mice (8 weeks old) were divided into three groups (n=10 each group) which received a subcutaneous injection of inactivated H₉N₂ (control), inactivated H₉N₂+2.5 mg recombinant wild-type AAL (AAL-wt)/kg BW, and inactivated H₉N₂+2.5 mg carbohydrate recognition domain (CRD) mutant AAL (AAL-mutR63H)/kg BW, respectively, four times at 7-d intervals. Seven days after the final immunization, serum samples were collected from each group for analysis. Hemagglutination assay, immunogold electron microscope, lectin blotting, and co-immunoprecipitation were used to study the interaction between AAL and H₉N₂ in vitro.

Results: IgG, IgG1, and IgG2a antibody levels were significantly increased in the sera of mice co-immunized with inactivated H₉N₂ and AAL when compared to mice immunized with inactivated H₉N₂ alone. No significant increase of the IgG antibody level was detected in the sera of the mice co-immunized with inactivated H₉N₂ and AAL-mutR63H. Moreover, AAL-wt, but not mutant AAL-mutR63H, adhered to the surface of H₉N₂ virus. The interaction between AAL and the H₉N₂ virus was further demonstrated to be associated with the CRD of AAL binding to the surface glycosylated proteins, hemagglutinin and neuraminidase.

Conclusions: Our findings indicated that AAL could be a safe and effective adjuvant capable of boosting humoral immunity against H₉N₂ viruses in mice through its interaction with the viral surface glycosylated proteins, hemagglutinin and neuraminidase.