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

Adjuvant effects mediated by the carbohydrate recognition domain of Agrocybe aegerita lectin interacting with avian influenza H 9 N 2 viral surface glycosylated proteins 杨树菇血凝素糖识别域与禽流感病毒H 9 N 2 表面糖蛋白结合介导的佐剂效果 1 first-author 1 1 2 1 1 2 2 1 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:heqigai@yahoo.comsunhui@whu.edu.cn †E-mail:heqigai@yahoo.comsunhui@whu.edu.cn heqigai@yahoo.com 2 3 4 corresp ‡Corresponding Authors ‡Corresponding Authors †E-mail:heqigai@yahoo.comsunhui@whu.edu.cn †E-mail:heqigai@yahoo.comsunhui@whu.edu.cn heqigai@yahoo.com College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan 430070, China College of Animal Sciences and Technology, Huazhong Agricultural University, Wuhan 430070, China College of Life Sciences, Wuhan University, Wuhan 430072, China College of Life Sciences, Wuhan University, Wuhan 430072, China Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, Wuhan 430071, China Key Laboratory of Combinatorial Biosynthesis and Drug Discovery, Ministry of Education, Wuhan 430071, China State Key Laboratory of Virology, Wuhan University, Wuhan 430072, China State Key Laboratory of Virology, Wuhan University, Wuhan 430072, China Objective: 2 To evaluate the potential adjuvant effect of Agrocybe aegerita lectin (AAL), which was isolated from mushroom, against a virulent H 9 N 2 strain in vivo and in vitro. Methods: 2 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 H 9 N 2 (control), inactivated H 9 N 2 +0.2% (w/w) alum, inactivated H 9 N 2 +0.5 mg recombinant AAL/kg body weight (BW), inactivated H 9 N 2 +1.0 mg AAL/kg BW, and inactivated H 9 N 2 +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 H 9 N 2 (control), inactivated H 9 N 2 +2.5 mg recombinant wild-type AAL (AAL-wt)/kg BW, and inactivated H 9 N 2 +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 H 9 N 2 in vitro. Results: 2 IgG, IgG1, and IgG2a antibody levels were significantly increased in the sera of mice co-immunized with inactivated H 9 N 2 and AAL when compared to mice immunized with inactivated H 9 N 2 alone. No significant increase of the IgG antibody level was detected in the sera of the mice co-immunized with inactivated H 9 N 2 and AAL-mutR63H. Moreover, AAL-wt, but not mutant AAL-mutR63H, adhered to the surface of H 9 N 2 virus. The interaction between AAL and the H 9 N 2 virus was further demonstrated to be associated with the CRD of AAL binding to the surface glycosylated proteins, hemagglutinin and neuraminidase. Conclusions: 2 Our findings indicated that AAL could be a safe and effective adjuvant capable of boosting humoral immunity against H 9 N 2 viruses in mice through its interaction with the viral surface glycosylated proteins, hemagglutinin and neuraminidase. 目的 2 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 2 首次研究AAL作为禽流感疫苗佐剂的作用机理。 方法 2 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（immunogold electron microscopy，IEM）试验发现AAL-wt能吸附H 9 N 2 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H 9 N 2 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 2 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）。 Adjuvant Agrocybe aegerita lectin Carbohydrate recognition domain Glycosylated protein Avian influenza H 9 N 2 virus 杨树菇凝集素 禽流感病毒H 9 N 2 糖识别结构域（CRD） 血球凝集素（HA） 神经氨酸酶（NA） Introduction 1 In many countries, avian influenza H 9 N 2 virus is still a major threat to the poultry industry. It causes a huge economic burden when it leads to mild respiratory abnormality, reduced egg production, immune suppressive disorders, and even mortality (Li et al., 2011 2011 ). Development of effective and safe vaccines is a priority in preparation for an influenza pandemic. Although vaccine has proved highly immunogenic in laboratory trials and prevents clinical diseases and reduces viral shedding in field conditions (Choi et al., 2008 2008 ), it cannot prevent vaccinated poultry from becoming infected and from shedding wild viruses in farm settings (Rahman et al., 2012 2012 ; Dabaghian et al., 2014 2014 ). Hence, it is important to improve vaccination strategies to control H 9 N 2 outbreaks in poultry farms. The immune-promoting activity of any given vaccination strategy is determined not only by the presence of the relevant antigenic components in the vaccine formulation, but also by the addition of suitable adjuvants which activate, promote, and extend the magnitude of a specific immune response (Liu, 1998 1998 ; Li et al., 2011 2011 ). Emphasis is therefore being placed on the development of effective adjuvants to improve the efficiency of vaccines, especially inactivated viral and subunit vaccines. Many substances have been used as adjuvants in experimental vaccines, such as aluminum, Freund’s complete adjuvant (FCA), immune stimulating complexes (ISCOMs), liposomes, polymeric microparticles, and saponins (Rajput et al., 2007 2007 ). Most of them have major disadvantages, including toxicity, allergy, poor activity, and potential neurotoxicity, which limit their practical application (Sivakumar et al., 2011 2011 ). Since numerous Chinese herbal medicines have been shown to have immune-enhancing effects (Yang et al., 2008 2008 ), and also have many other advantages such as extensive availability, low cost, reliable efficacy, and decreased risk of side-effects and toxicity (Kong et al., 2004 2004 ; 2006 2006 ; Ung et al., 2007 2007 ), their potential use as new adjuvants for humans or animals has aroused much research interest in recent years. Agrocybe aegerita lectin (AAL) is a 15.8-kD homodimeric protein, isolated from the medicinal mushroom A. aegerita by our laboratory, which shows anti-tumor activity by inducing apoptosis in cancer cells (Zhao et al., 2003 2003 ). We demonstrated that AAL belongs to a galectin family with a unique carbohydrate recognition domain (CRD) that specifically recognizes β-galactose (Yang et al., 2005 2005 ; Feng et al., 2010 2010 ). AAL has antifungal, antibacterial, antiviral (Sun et al., 2003 2003 ), and antitumor functions and can improve immune function, and it is recognized that these functions are associated with the ability of its CRD to bind to glycan on glycosylated proteins (Rabinovich and Croci, 2012 2012 ). We therefore hypothesized that AAL has adjuvant activity against the H 9 N 2 virus by interacting with the glycosylated proteins, hemagglutinin (HA) and neuraminidase (NA) on the surface of the virus (Liu et al., 2003 2003 ). Materials and methods 1 Vaccine preparation of AAL, AAL-wt, and AAL-mutR63H 2 Avian influenza H 9 N 2 virus (Academy of Veterinary Medicine, Huazhong Agricultural University, Wuhan, China) was cultured in 9-d-old embryonic eggs at 37 °C for 48 h. The allantoic fluid containing the viruses was harvested and purified by sucrose density gradient centrifugation. Purified vaccine viruses were inactivated by incubating at 75 °C for 15 min. The AAL from A . aegerita was extracted and purified by the chromatography method as previously described (Zhao et al., 2003 2003 ). Arginine in the 63th locus of the CRD region of AAL (Arg 63 ) is involved in the determination of the binding affinity of AAL to antigens (Feng et al., 2010 2010 ). Wild-type AAL (AAL-wt) and CRD mutant AAL (AAL-mutR63H in which histidine replaced arginine in the 63th locus of the CRD region) with a C-terminal 6× His-tag were expressed in Escherichia coli BL21 (DE3) and purified as previously described (Liang et al., 2009 2009 ). The recombinant AALs (with His-tag) were confirmed by anti-His antibody (Bioss, Beijing, China). Mice, treatments, and sample collection 2 The Institutional Animal Care and Use Committee of Huazhong Agricultural University, China approved our animal protocol. In experiment 1, 50 BALB/c male mice (8 weeks old; Experimental Animal Center, Wuhan University, Wuhan, China) were divided into five groups ( n =10 each group). All mice were allowed free access to commercial rodent diets (Yamaguchi et al., 2005 2005 ) and water. After a week of acclimatization, the control group received a subcutaneous injection of 0.1 ml inactivated H 9 N 2 , and the alum group received a subcutaneous injection of 0.1 ml inactivated H 9 N 2 with adjuvants of 0.2% (w/w) alum (Thermo, USA). The three other treated groups received a subcutaneous injection of 0.1 ml inactivated H 9 N 2 with 0.5, 1.0, or 2.5 mg AAL/kg body weight (BW), respectively, four times at 7 d intervals (Huang et al., 2008 2008 ; Kim et al., 2011 2011 ). The inactivated viruses were mixed with the recombinant protein just prior to injection. Seven days after the final immunization the sera were harvested from the immunized mice (Kim et al., 2011 2011 ). The sera were prepared by centrifugation of the whole blood at 1000 g for 15 min at 4 °C and stored at −80 °C (Sun et al., 2014 2014 ). In experiment 2, 30 BALB/c male mice (8 weeks old) were divided into three groups ( n =10 each group) and received the same housing conditions as in experiment 1. The control group received a subcutaneous injection of 0.1 ml inactivated H 9 N 2 , while the two treated groups received a subcutaneous injection of 0.1 ml inactivated H 9 N 2 with adjuvants of 2.5 mg AAL-wt/kg BW and 2.5 mg AAL-mutR63H/kg BW, respectively, four times at 7 d intervals (Huang et al., 2008 2008 ; Kim et al., 2011 2011 ). Seven days after the final immunization, serum samples were collected following the method described by Kim et al. ( 2011 2011 ). No negative effects on the experimental animals were observed. Measurement of antigen-specific antibodies 2 The H 9 N 2 -specific IgG, IgG1, and IgG2a antibodies in the sera were analyzed by enzyme-linked immunosorbent assay (ELISA) as described previously with minor modifications (Sun and Liu, 2008 2008 ). Briefly, the 96-well microtiter plates were coated with 100 μl of H 9 N 2 solution (50 μg/ml in 50 mmol/L carbonate buffer, pH 9.6) for 24 h at 4 °C. The wells were washed three times with 200 μl of phosphate buffer saline (PBS) containing 0.05% (v/v) Tween 20 (PBST), and then blocked with 100 μl of PBST within 0.5% (v/v) bovine serum albumin (BSA) at 37 °C for 1 h. One hundred microliters of diluted serum samples were then added to triplicate wells, and then incubated at 37 °C for 1 h, followed by washing three times. Aliquots of 100 μl of horseradish peroxidase-conjugated goat anti-mouse IgG, IgG1, or IgG2a (SouthernBiotech, China) were added and incubated for 1 h at 37 °C. An assay was developed using 3,3',5,5'-tetramethylbenzidine (TMB) (Beytime Biotechnology, China), and the reaction was stopped by adding 0.5 mol/L H 2 SO 4 . The absorbance at 450 nm was measured by an ELISA plate reader. Hemagglutinating assays 2 The hemagglutinating assays were performed as described previously with minor modifications (Yang et al., 2005 2005 ). Briefly, a serial two-fold dilution of AAL-wt or AAL-mutR63H solution in microtiter U-plates (50 µl) was mixed with 50 µl of 1.0% suspension of mouse erythrocytes in PBS (pH 7.2) at 20 °C. The initial concentration was 1 mg/ml. The results were read after about 1 h, when the control (no AAL added) had fully precipitated. The hemagglutination titer, defined as the reciprocal of the highest dilution exhibiting hemagglutination, was defined as one hemagglutination unit. Specific activity was the number of hemagglutination units per mg of protein. Immunogold electron microscope assays 2 Immunogold electron microscope assays were performed as described previously (Pena et al., 2013 2013 ). Briefly, 10 μl purified H 9 N 2 viruses were adsorbed onto formvar-coated and carbon-vaporized 300-mesh copper grids (Electron Microscopy Sciences, Hatfield, PA, USA). The grids were blocked in Tris-buffered saline (TBS) containing 0.2% (v/v) BSA at room temperature (RT) for 1 h, and washed three times with TBS with 0.3% Tween 20 (TBST). They were then incubated with TBS (control), 10 μl AAL-wt (500 ng/ml), or AAL-mutR63H (500 ng/ml) at RT for 1 h. Subsequently, the grids were washed in TBS and incubated with rabbit anti-His (anti-AAL) at RT for 1 h, and then incubated with goat anti-rabbit IgG antibody conjugated to 15-nm gold beads (Bioss, Beijing, China) at RT for 1 h. Finally, the grids were negatively stained with 2% (v/v) phosphotungstic acid (PTA) for 30 min, dried, and examined under a transmission electron microscope (Hitachi H-7000FA). Lectin blot assays 2 Lectin blot assays were performed as described previously with minor modifications (Matsumura et al., 2007 2007 ). Briefly, the protein extracts of the H 9 N 2 virus were isolated by methanol and chloroform, and 60 μg of the extracted protein was then subjected to 15% (0.15 g/ml) sodium dodecyl sulfate-polyarylamide gel electrophoresis (SDS-PAGE). One of the samples was treated with PNGase F (New England BioLabs, MA, USA) to remove the glycan on glycosylated protein following the manufacturer’s instructions. After electrophoresis, the gels were blotted onto polyvinylidene difluoride (PVDF) membranes which were incubated overnight with 5% (v/v) BSA in TBST (50 mmol/L Tis-HCl, 150 nmol/L NaCl, 0.1% Tween 20, pH 7.4) at 4 °C, and then for 100 min with 500 ng/ml of AAL-wt or AAL-mutR63H in TBST. After being washed with TBST, the membranes were incubated with anti-His (anti-AAL) antibody (Bioss, Beijing, China) at RT for 100 min. Staining was then performed with ECL Western blot detection reagents as previously described (Sun et al., 2013 2013 ). Co-immunoprecipitation assays 2 Co-immunoprecipitation (Co-IP) assays were performed as described previously (Rajik et al., 2009 2009 ). Briefly, the extracted protein of H 9 N 2 virus was diluted to a concentration of 1–2 μg/μl in 50 μl Co-IP buffer, and was then incubated overnight with 50 μl of 100 μmol/L AAL-wt at 4 °C. Anti-His (anti-AAL) antibody, anti-HA antibody (Sino Biological Inc., Beijing, China), and IgG antibody were added to the mixture, respectively, and rotated gently for 3.5 h at 4 °C. Subsequently, protein G plus-agarose beads were added, and the mixture was rotated gently for 3 h at 4 °C. The beads were collected by centrifugation at 100 g for 3 min at 4 °C, and then washed three times with ice-cold NP40 lysis buffer. Finally, the proteins were eluted from the beads by boiling in SDS loading buffer for 5 min, and analyzed by Western blot assay using the anti-His (anti-AAL) antibody and anti-HA antibody with appropriate secondary antibodies. Statistical analysis 2 Data generated from animal studies were analyzed using Student’s t- test by using SAS 8.2 (SAS Institute). Data are presented as mean±standard error (SE) with the significance level set at P< 0.05. Results 1 Adjuvant activity of AAL on antibody titers against H 9 N 2 in mice 2 AAL was successfully extracted and purified by chromatography and confirmed by anti-AAL for Western blot assay (Figs. 1a and 1d 1a and 1d ). In the first animal experiment, after 4 weeks of treatment, compared to mice immunized with H 9 N 2 alone (control), IgG antibody levels in the sera were increased ( P < 0.05) by 97%, 147%, and 93% when mice were co-immunized with inactivated H 9 N 2 and alum, AAL (1.0 mg/kg BW), and AAL (2.5 mg/kg BW), respectively (Fig. 2a 2a ). IgG1 antibody levels in the sera were increased ( P < 0.05) by 237%, 340%, and 372% when mice were co-immunized with inactivated H 9 N 2 and AAL at the doses of 0.5, 1.0, and 2.5 mg/kg BW, respectively (Fig. 2b 2b ). In addition, IgG2a antibody levels in the sera were increased ( P < 0.05) by 60%, 133%, and 145% when mice were co-immunized with inactivated H 9 N 2 and AAL at the doses of 0.5, 1.0, and 2.5 mg/kg BW, respectively (Fig. 2c 2c ). M, sizes (kD) of molecular weight markers; Lane 1, purified AAL, AAL-wt, or AAL-mutR63H; Land 2, flow-through; Land 3, crude extract AAL Values are expressed as mean±SE, n =10 each group. Values without sharing a common letter differ significantly ( P < 0.05). 0.5AAL: 0.5 mg AAL/kg BW; 1.0AAL: 1.0 mg AAL/kg BW; 2.5AAL: 2.5 mg AAL/kg BW Hemagglutinating and adjuvant activity assays of AAL-wt and AAL-mutR63H 2 To analyze the role of the CRD of AAL by site-directed mutagenesis, Arg 63 at the domain was replaced with His 63 based on the three-dimensional structures of AAL to produce the mutated AAL (AAL-mutR63H) which was then purified and confirmed by Western blot. This showed that Arg 63 is a key function site of AAL (Figs. 1c and 1f 1c and 1f ) (Feng et al., 2010 2010 ). We also produced and purified the recombinant AAL-wt and confirmed it by Western blot (Figs. 1b and 1e 1b and 1e ). The hemagglutinating assay showed that AAL-wt has hemagglutinating activity (1:2–1:128), while no activity was found in AAL-mutR63H (Fig. 3a 3a ). The second animal experiment showed that AAL-wt significantly enhanced ( P < 0.05) H 9 N 2 -specific IgG levels in the sera of mice, but that AAL-mutR63H had no significant effect ( P > 0.05) (Fig. 3b 3b ). Values are expressed as mean±SE, n =10 each group. Values without sharing a common letter differ significantly ( P < 0.05) Immunogold electron microscope, lectin blot, and co-immunoprecipitation assays 2 The immunogold electron microscope results showed that, compared with the control group, AAL-wt accumulated intensively on the surface of the H 9 N 2 virus, but only a few of the mutant AAL-mutR63H did (Fig. 4 4 ). To test whether the two glycosylated proteins, HA and NA, on the surface of the H 9 N 2 virus could be the binding targets of AAL’s CRD, we preformed lectin blot analysis (Fig. 5a 5a ). The results showed that both glycoproteins NA (55 kD) and HA (26 kD) were displayed on the AAL-wt-incubated PVDF membrane (Land 1); however, these two bands disappeared after the N-linked glycan of virus was removed by PNGase F (Land 2) or in the AAL-mutR63H-incubated PVDF membrane (Land 3). These data indicated that AAL interacted with the glycosylated proteins HA and NA, while the mutant AAL-mutR63H lost its ability to interact with these two proteins. The interaction of AAL with HA was further confirmed by Co-IP assay (Fig. 5b 5b ). Protein extracts of H 9 N 2 virus were incubated with AAL-wt/AAL-mutR63H, and then with anti-HA, anti-AAL, and anti-IgG antibodies. Finally, the total extracts and the precipitates were Western-blotted against anti-HA and anti-AAL (Fig. 5b 5b ). HA/AAL-wt was detected in AAL-wt/HA immunoprecipitates with H 9 N 2 virus protein extracts, indicating that the HA/AAL-wt was captured by the anti-AAL-wt/HA antibody. The Co-IP results further indicated that AAL interacted with the glycosylated proteins HA. In contrast, no interaction was found between HA and AAL-mutR63H. Black arrow: H 9 N 2 virus; White arrow: gold particle Input is mixed HA with AAL-wt or AAL-mutR63H Discussion 1 The two novel findings from the present study are: (1) AAL has an adjuvant activity against avian influenza H 9 N 2 virus in mice; (2) the mechanism of AAL’s adjuvant activity is associated with the interaction between the CRD of AAL with the H 9 N 2 virus surface glycosylated proteins HA and NA. Levels of total IgG, IgG1, and IgG2a in serum have been described as valuable parameters of humoral immunity (Li et al., 2011 2011 ). The first animal study showed that co-injecting inactivated H 9 N 2 virus with ≥1.0 mg AAL/kg BW induced higher levels of antigen-specific IgG, IgG1, and IgG2a antibody titers in mice than injecting inactivated H 9 N 2 avian influenza virus (AIV) vaccine alone. Furthermore, the levels of IgG, IgG1, and IgG2a were similar in the mice co-injected with inactivated H 9 N 2 virus with AAL or alum. These outcomes indicated that AAL, like alum, has an effective adjuvant activity that enhances antigen-specific humoral immune responses. Similar results were obtained from recent studies in which Korean mistletoe lectin was found to have the ability to serve as a mucosal adjuvant (Jung et al., 2011 2011 ) and mushroom lectin as an adjuvant of hepatitis B virus (HBV) DNA vaccine (Gao et al., 2013 2013 ). AAL is attractive as a new adjuvant for animal H 9 N 2 vaccines, since it can be isolated from mushroom A. aegerita , which is extensively available and has reliable efficacy but lower risks of side-effects and toxicity than alum salts or other adjuvants (Aguilar and Rodríguez, 2007 2007 ; Sivakumar et al., 2011 2011 ). To validate our hypothesis that the CRD plays a vital role in AAL’s adjuvant activity, we obtained AAL-mutR63H, by replacing Arg 63 at CRD with His 63 (Feng et al., 2010 2010 ), and studied its biological function. The in vitro study showed that AAL-wt had haemagglutination activity, but that AAL-mutR63H lost this activity. The second animal study showed that AAL-wt enhanced the levels of IgG in the sera of mice, while AAL-mutR63H had no significant effect. Taken together, these outcomes indicated that the CRD of AAL plays a key role in the adjuvant effect of AAL against the H 9 N 2 virus in mice. Similar results were obtained by Dodson et al. ( 1999 1999 ), who reported that the CRD of the Entamoeba histolytica Gal/GalNAc lectin played key roles in infection and immunity. Since lectin’s antiviral function and ability to improve immune function are associated with the ability of its CRD to bind to glycan on glycosylated protein (Rabinovich and Croci, 2012 2012 ), we carried out immunogold electron microscope, lectin blot, and Co-IP assays to determine whether AAL’s adjuvant activity against the H 9 N 2 virus was associated with the ability of its CRD to bind to the surface glycosylated proteins HA and NA. The immunogold electron microscope results showed that AAL-wt bound strongly to the surface of the H 9 N 2 virus, but the mutant AAL-mutR63H had lost this ability. Since HA and NA are the two major glycosylated proteins on the surface (Liu et al., 2003 2003 ), lectin blot and Co-IP were done to determine AAL’s ability to bind to the surface through its CRD interacting with these two glycosylated proteins. The lectin blot results showed that AAL-wt interacted with both HA and NA, but AAL-mutR63H lost its ability to interact with these two proteins. The interaction between AAL and the HA protein was further confirmed by Co-IP assay. Overall, the immunogold electron microscope, lectin blot, and Co-IP assays demonstrated that the CRD of AAL plays a vital role in interacting with the glycosylated proteins HA and NA on the surface of the H 9 N 2 virus. The glycosylation of viral envelope proteins plays an important role in viral immune evasion by masking antigenic sites from immune recognition (Kreisman and Cobb, 2012 2012 ). Previous reports have shown that N-linked glycosylation can limit the neutralizing antibody response to simian immunodeficiency virus (SIV) and shield the virus from immune recognition (Reitter et al., 1998 1998 ), and that the glycosylations of HA and NA are significant for influenza virus to evade the host immune system (Klenk et al., 2001 2001 ; Tsuchiya et al., 2002a 2002a ; 2002b 2002b ). Therefore, we speculated that the enhancement of antigenicity of the H 9 N 2 virus by AAL may be related to the interaction of AAL with glycosylated proteins on the virus surface by exposing antigenic sites to immune recognition (Fig. 6a 6a ). Since AAL is a homodimeric protein with two CRDs (Zhao et al., 2003 2003 ), which may link two viral particles, an antigen depot is formed (Fig. 6b 6b ). The antigen depot can provide a slow release of antigen, which can continue the stimulation of the immune system (Lindblad, 2004 2004 ). (a) The enhancement of antigenicity of the H 9 N 2 virus by AAL may be related to the interaction of AAL with the glycosylated proteins on the virus surface, resulting in the exposure of the antigenic sites to immune recognition. (b) AAL as a homodimeric protein with two CRDs may link two viral particles and form an antigen depot. The antigen depot can provide a slow release of antigen, continuing the stimulation of the immune system Conclusions 1 In summary, AAL purified from mushroom A. aegerita and AAL-wt both exhibited adjuvant effects against avian influenza H 9 N 2 virus. The site-directed mutant AAL-mutR63H (Arg 63 at the CRD of AAL replaced with His 63 ) lost haemagglutination activity in vitro and adjuvant activity in vivo, indicating that the CRD of AAL plays a vital role in the adjuvant effect against the H 9 N 2 virus in mice. 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An antitumour lectin from the edible mushroom Agrocybe aegerita Compliance with ethics guidelines Li-bao MA, Bao-yang XU, Min HUANG, Lv-hui SUN, Qing YANG, Yi-jie CHEN, Ya-lin YIN, Qi-gai HE, and Hui SUN declare that they have no conflict of interest. All institutional and national guidelines for the care and use of laboratory animals were followed. 10.1631/jzus.B1600106 Project supported by the National Natural Science Foundation of China (Nos. 30771501 and 81102850), the National Basic Research Program (973) of China (No. 2011CB811302), the National Mega Project on Major Drug Development (No. 2009ZX09301-014-1), the Chinese 111 Project (No. B06018), and the Wuhan Municipal Project (No. 201160923296), China 2016-03-05 2016-09-30 2022-01-05 Copyright © Zhejiang University and Springer-Verlag Berlin Heidelberg 2017 2017 Journal of Zhejiang University-SCIENCE B (Biomedicine and Biotechnology) 08 18