| The transmission of avian influenza viruses (AIV) is mainly through the respiratory routes. Intranasal immunization can indirectly cut off the main entering route of ATV and be comfirmed as an effective method for preventing influenza viral infection. However, mucosal immunization by intranasal delivery with inactivated virus alone is often insufficiently effective, and suitable mucosal adjuvants can effectively enhance the efficiency of viral antigens. Bacillus subtilis spores (B. subtilis spores), Polyriboinosinieaeid (Poly(I:C) and CpG oligodeoxynucleotides (CpG-ODN) have been proved to be potent and safe vaccine adjuvants in mammals. However, much less are investigated in chicken. The aim of our study is to investigate the effects of B.subtilis spores, Poly(I:C) and CpG-ODN on inactivated avian influenza virus (IAIV) vaccine in chicken in vivo and in vitro test, and finally filter a more affordable and efficacious adjuvant for IAIV. In vitro test:the immunostimulatory activity of B.subtilis spores, Poly(I:C) or CpG-ODN on chicken spleen lymphocytes and chicken bone marrow-derived dendritic cells (chBM-DCs) were investigated. In vivo test:240 3-day-old hyline chickens were randomly divided into eight groups and were immunized twice (at 10-day-old and 17-day-old). Chickens from group 1 to group 4 were intranasally immunized with phosphate-buffered saline (PBS) and adjuvants only. Chickens from group 5 were intranasally immunized with IAIV only. Chickens from group 6 and group 8 were intranasally immunized with IAIV respective together with B.subtilis spores (5 x10, Poly (I:C) (50 μg/each bird) or CpG-ODN (50 μg/ each bird). Firstly the expression levels of IFN-y mRNA, IL-6 mRNA and IL-12 mRNA in nasal cavity and trachea were measured to investigate the effects of B.subtilis spores, Poly(I:C) and CpG-ODN on local cell immunity. The levels of AIV specific sIgA antibody both in nasal cavity and trachea were measured to investigate the effects of B.subtilis spores, Poly(I:C) and CpG-ODN on the local humoral immunity. The levels of HI antibody and AIV-specific IgG in serum were measured to investigate the effects of B.subtilis spores, Poly(I:C) and CpG-ODN on systemic immunity. Finally TLRs mRNA expression levels in the nasal cavity and trachea were measured to investigate immunostimulatory activities of B.subtilis spores, Poly(I:C) and CpG-ODN on chicken.1 The immunostimulatory activity of B.subtilis spores, Poly(I:C) or CpG-ODN on chicken spleen lymphocytesSpleen lymphocytes were cultured in 96-well culture plates and stimulated by different consistences of B.subtilis spores, Poly(I:C) and CpG-ODN. The immunostimulatory active-ties of these adjuvants on the chicken spleen lymphocytes were analyzed using a WST-8 Cell Counting Kit-8 (CCK-8). The results showed that co-incubation of naive splenocytes with B.subtilis spores and Poly(I:C) could led to signification level of cellular proliferation (P< 0.01) in a dose-related manner. CpG-ODN could stimulate spleen lymphocytes proliferation in low concentrations, but lost its stimulatory potential at high concentrations (60 and 100 μg/mL). These results demonstrated that the designed CpG-ODN had good immunostimulatory effects on chicken spleen lymphocytes.2 The immunostimulatory activity of B.subtilis spores, Poly(I:C) or CpG-ODN on chBM-DCsThe chBM-DCs were cultured in complete medium for 6 days and stimulated respectively by B.subtilis spores, Poly(I:C) and CpG-ODN for 24 hours. Cell surface costimulatory molecules were detected by flow cytometry and their ability to drive naive T proliferation was analyzed by mix lymphocyte responses (MLR). The results showed that B.subtilis spores, Poly(I:C) and CpG-ODN showed the up-regulation surface expression levels of CD40 and CD86 molecules, with spores showing the strongest up-regulating activity for CD40 and CD86 cell surface molecules. MRL showed that the chBM-DCs stimulated B.subtilis spores, Poly(I:C) and CpG-ODN showed the strong ablity to induce the T-cell proliferative response, with B.subtilis spores being the most efficient. The results demonstrated that B.subtilis spores, Poly(I:C) and CpG-OD could drive chBM-DCs to become phonotypical and functional maturation, with B.subtilis spores had the strongest ability to stimulate chBM-DCs maturation.3 The effect of B.subtilis spores, Poly(I:C) or CpG-ODN on local mucosal immunity of chickensThe expression levels of IFN-γ mRNA, IL-6 mRNA and IL-12 mRNA in nasal cavity and trachea were detected at 24h after final immunization by the real time PCR. The results showed that the expression levels of IFN-y mRNA, IL-6 mRNA and IL-12 mRNA in nasal cavity and trachea from chikens immunized with IAIV plus B.subtilis spores, Poly(I:C) or CpG-ODN were significantly increased compared with that immunized with IAIV only (P< 0.05 or P< 0.01). Beside, the expression levels of IL-6 mRNA and IL-12 mRNA in nasal cavity and trachea were much higher after immunization with IAIV plus spores.The AIV-specific IgA levels in nasal cavity and trachea were measured at 3rd,5th and 7th weeks after the first immunization by indirect ELISA. The result showed that the AIV-specific IgA levels in nasal cavity and trachea from chikens immunized with IAIV plus B.subtilis spores, Poly(I:C) or CpG-ODN were significantly increased compared with that immunized with IAIV only (P< 0.05 or P<0.01). In addition, the level of specific IgA antibody was a little higher after immunization with IAIV plus CpG-ODN.The results of two essays above prompted that intranasal immunization with IAIV and B.subtilis spores, Poly(I:C) or CpG-ODN could significantly increase the local mucosal immunity on chicken.4 The effects of B.subtilis spores, Poly(I:C) and CpG-ODN on the systemic immunity of chickensThe antibody titer in serum was measured by the HI assay. The serum titers from the chickens that immunized intranasal with IAIV plus our adjuvants increased by 3rd weeks and peaked at 4th weeks after the first immunization. Beside, the combine of IAIV with CpG-ODN had highest titer at 4th weeks, whereas groups without adjuvants were always very low after the first immunization.The IgG antibody levels in the serum were detected at 1st,3rd,5th and 7th weeks after the first immunization by indirect ELISA. The result showed that the AIV-specific IgG levels serum from chikens immunized with IAIV plus B.subtilis spores, Poly(I:C) or CpG-ODN were significantly increased compared with that immunized with IAIV only(P < 0.05 or P< 0.01). In addition, the level of AIV-specific IgG antibody was a little higher after immunization with IAIV plus CpG-ODN.The results of two essays above remindered that intranasal immunization with IAIV and B.subtilis spores, Poly(I:C) or CpG-ODN could improve the systemic immunity level in some degree.5 The effect of B.subtilis spores, Poly(I:C) or CpG-ODN on the expression level of chicken TLRs mRNAThe expression levels of TLRs in nasal cavity and trachea were measured at 24h after the final immunization by the real time PCR. The results showed that the expression level of TLR3 mRNA or TLR21 mRNA in nasal cavity and trachea from the group immunized with IAIV plus Poly(I:C) or CpG-ODN were significantly increased compared with that immunized with IAIV alone (P< 0.05 or P< 0.01). However, the addition of spores just influenced the expression of TLR2 mRNA in the nasal cavity. The results indicated that the B.subtilis spores, Poly(I:C) and CpG-ODN could activate their corresponding TLRs and up-regulate the expression of the related TLRs mRNA in chicken. |
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