Identification Of Adjuvant Component From Medical Fungus Agrocybe Aegerita

Our lab has previously purified an anti-tumor component Yt from the medicinal fungus Agrocybe aegerita. In tumor-bearing mouse model, the result showed 70% of the tumors failed to grow when an intratumoral inject was given. Yt played the anti-tumor activity through killing tumor cells directly and stimulating immune system indirectly. Thus, we hypothesis Yt could regulate immune activity and then enhance the role of vaccine immunogenicity as an immune adjuvant. In this article, we studied effect of Yt as an adjuvant of the inactivated avian influenza virus H9N2.We next identified the sub-component Yp and the final purified AAL and found they all had the functional role as an adjuvant.In the Yt treated experiment, compared to the control group mice(no adjuvant treated), the average P/N value of IgG in 0.2mg/mL Yt treated group was significantly elevated (P<0.01), increasing 36.2%; The average P/N value of IgG in 0.5mg/mL Yt treated group also increased 32.4%(P<0.01). These results suggested that Yt had the function as an adjuvant of inactivated H9N2 avian influenza virus. We nexted detected the expression level of IgG subtypes:IgGl and IgG2a, the results showed that the average P/N value of IgGl in lmg/mL Yt,0.5mg/mL Yt,0.2mg/mL Yt treated group was significantly different from the control (P<0.01), increasing 144.1%,167.2% and 196.9% respectively. But for IgG2a, there was no difference. In the cytokine test, we pretreated the mice with Yt and inactivated H9N2, then isolated the lymphocytes from the mice spleen, stimulated them with H9N2 in vitro. The result showed that, compared to the control, the IFN-r expression in the pretreatment of Yt group was 244.53pg/mL (P<0.01). In vitro proliferation results also showed that,0.625μg/mL Yt,1.25μg/mL Yt together with the ConA could stimulate the lymphocytes proliferation by 210.8% and 187.5% respectively. These results showed the Yt could be an adjuvant by stimulating the spleen lymphocyte proliferation and promoting Th2 cell activity.Further we separated and identified the effective components of mixture Yt. Yt are divided into the protein components Yp and small molecular components of Ys, Yp and Ys in Yt respectively account of 60% and 40%. In the Ys treated experiment, compared to the control group mice (no adjuvant treated), the average P/N value of IgG was no different. The results indicate Ys isn’t effective adjuvant components of Yt. In the Yp treated experiment, compared to the control group mice(no adjuvant treated), the average P/N value of IgG in 0.5mg/mL Yp,0.2mg/mL Yp, 0.1mg/mL Yp was significantly elevated (P<0.01), increasing 39.2%,47.2%,24.1% respectively. These results suggested that Yp had the function as an adjuvant of inactivated H9N2 avian influenza virus. We nexted detected the expression level of IgG subtypes:IgG1 and IgG2a, the results showed that the average P/N value of IgG1 in 0.5mg/mL Yp, 0.1mg/mL Yp treated group was significantly different from the control (P<0.01), increased 201.1%,274.7% respectively. But for IgG2a, there was no difference. In the cytokine test, we pretreated the mice with Yp and inactivated H9N2, then isolated the lymphocytes from the mice spleen, stimulated them with H9N2 in vitro. The result showed that, compared to the control, the IFN-r expression in the pretreatment of Yp group was 146.21pg/mL (P<0.01). In vitro proliferation results also showed that,0.625μg/mL Yp,1.25μg/mL Yp,2.5μg/mL Yp together with the ConA could stimulate the lymphocytes proliferation by 265.4%,321.1%, 347.1%, respectively. These results showed the Yp could be an adjuvant by stimulating the spleen lymphocyte proliferation and promoting Th2 cell activity.Our Previous studies show the galectin-AAL is the main component in Yp. So we identified immune adjuvant activity of AAL. In the AAL treated experiment, compared to the control group mice (no adjuvant treated), the average P/N value of IgG in 0.2mg/mL AAL, 0.1mg/mL AALwas significantly elevated (P<0.01), increasing 98.01%,157.5% respectively. These results suggested that AAL had the function as an adjuvant of inactivated H9N2 avian influenza virus. We nexted detected the expression level of IgG subtypes:IgG1 and IgG2a, the results showed that the average P/N value of IgGl in 0.5mg/mL AAL,0.2mg/mL AAL, O.lmg/mL AAL treated group was significantly different from the control (P<0.01), increased 399.1%,362.3%,251.6%. But for IgG2a, there was no difference. In the cytokine test, we pretreated the mice with AAL and inactivated H9N2, then isolated the lymphocytes from the mice spleen, stimulated them with H9N2, in vitro. The result showed that, compared to the control, the IFN-r expression in the pretreatment of AAL group was 174.64pg/mL (P<0.01). In vitro proliferation results also showed that,12.5μg/mLAAL together with the ConA could stimulate the lymphocytes proliferation by 134.1%(P<0.01); 6.5μg/mLAAL together with the ConA could stimulate the lymphocytes proliferation by 131.2%(P<0.05). These results showed the AAL could be an adjuvant by stimulating the spleen lymphocyte proliferation and promoting Th2 cell activity.In summary,we identified Agrocybe aegerita extraction Yt, protein component Yp, single-component AAL, Yt, Yp and AAL has adjuvant activity, they enhanced antigen immunogenicity, increased antigen antibody titer by stimulating lymphocytes proliferation and promoting Th2 cell activity.