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

Abstract

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.

摘要

目 的

探讨杨树菇血凝素(AAL)作为疫苗佐剂的作用机理, 为有囊膜病毒疫苗佐剂的研究提供新思路和数据。

创新点

首次研究AAL 作为禽流感疫苗佐剂的作用机理。

方 法

大肠杆菌重组表达、纯化野生型的AAL(AAL-wt)和糖识别结构域( CRD ) 突变型的AAL(AAL-mutR63H)(图1); 动物试验证实AAL-wt具有免疫佐剂活性, AAL-mutR63H 失去免疫佐剂活性(图2); 免疫胶体金电镜(immunogoldelectron microscopy, IEM)试验发现AAL-wt 能吸附H₉N₂ 病毒粒子, 而AAL-mutR63H 不能吸附病毒粒子(图4); 凝集素印迹(lectin blot)、免疫共沉淀(co-immunoprecipitation, Co-IP)试验证明了CRD 区与H₉N₂ 病毒中血球凝集素(HA)和神经氨酸酶(NA)这两种糖蛋白的相互作用(图5)。

结 论

AAL 的CRD 区是发挥佐剂作用的关键区域, CRD区与禽流感病毒表面的HA 糖蛋白结合, 可能暴露病毒的免疫识别位点, 或者将病毒粒子连接在一起, 形成大的病毒复合体, 增加病毒的抗原性(图6)。