Bacillus subtilis spores as adjuvants against avian influenza H9N2 induce antigen-specific antibody and T cell responses in White Leghorn chickens

Abstract

Low-pathogenicity avian influenza H9N2 remains an endemic disease worldwide despite continuous vaccination, indicating the need for an improved vaccine strategy. Bacillus subtilis (B. subtilis), a gram-positive and endospore-forming bacterium, is a non-pathogenic species that has been used in probiotic formulations for both animals and humans. The objective of the present study was to elucidate the effect of B. subtilis spores as adjuvants in chickens administered inactivated avian influenza virus H9N2. Herein, the adjuvanticity of B. subtilis spores in chickens was demonstrated by enhancement of H9N2 virus-specific IgG responses. B. subtilis spores enhanced the proportion of B cells and the innate cell population in splenocytes from chickens administered both inactivated H9N2 and B. subtilis spores (Spore + H9N2). Furthermore, the H9N2 and spore administration induced significantly increased expression of the pro-inflammatory cytokines IL-1β and IL-6 compared to that in the H9N2 only group. Additionally, total splenocytes from chickens immunized with inactivated H9N2 in the presence or absence of B. subtilis spores were re-stimulated with inactivated H9N2. The subsequent results showed that the extent of antigen-specific CD4⁺ and CD8⁺ T cell proliferation was higher in the Spore + H9N2 group than in the group administered only H9N2. Taken together, these data demonstrate that B. subtilis spores, as adjuvants, enhance not only H9N2 virus-specific IgG but also CD4⁺ and CD8⁺ T cell responses, with an increase in pro-inflammatory cytokine production. This approach to vaccination with inactivated H9N2 together with a B. subtilis spore adjuvant in chickens produces a significant effect on antigen-specific antibody and T cell responses against avian influenza virus.

Figure 1

Antigen-specific IgG response and HI titre in chickens with intramuscular administration of inactivated H9N2 and/orB. subtilisspores. A Scheme of the immunization schedule. One-week-old White Leghorn chickens (N = 5) were administered with PBS as a negative control, 2 × 10⁹ CFU of B. subtilis spores, or inactivated H9N2 without or with B. subtilis spores. B The antigen-specific IgG antibody response in serum was measured and expressed as arbitrary units at 3 and 4 weeks. C HI assays with sera against H9N2 virus at 4 weeks. To determine the significance, one-way ANOVA followed by a Friedman test corrected by Dunn’s multiple comparison test was performed. Data are expressed as the mean values ± SDs. Different letters on each group denote a significant difference at P ≤ 0.05.

Figure 2

Analysis of splenic B cells and innate immune cells in chickens administered inactivated H9N2 andB. subtilisspores. Seven-day-old chickens (N = 10) were immunized twice with inactivated H9N2 and/or B. subtilis spores at one-week intervals. Single cells from splenocytes were stained with the proper combination of anti-chicken CD3, Bu-1, and KUL01 antibodies, and flow cytometric analysis was performed. A Total splenocytes, B the percentage and C absolute number of KUL01⁺ monocytes/macrophages cells, and D the percentage and E absolute number of Bu-1⁺ cells were examined by using flow cytometry. To determine the significance, one-way ANOVA followed by a Friedman test corrected by Dunn’s multiple comparison test was performed. Data are expressed as the mean values ± SDs. Different letters on each group denote a significant difference at P ≤ 0.05.

Figure 3

mRNA expression patterns of pro-inflammatory cytokines in monocytes/macrophages and B cell proliferation/survival-related genes in chicken splenocytes treated with inactivated H9N2 andB. subtilisspores. Total splenocytes and monocytes/macrophages were isolated from 3-week-old chickens (N = 5) and stimulated with inactivated H9N2 and/or B. subtilis spores for 3 h. A The expression patterns of the IL-1 and IL-6 genes, as pro-inflammatory cytokines, in monocytes/macrophages. B The mRNA expression levels of the B cell proliferation-related genes BAFF, BAFF-R, TACI, CD40, and CD40L in total splenocytes. C The expression patterns of the IL-4 and IL-15 genes in total splenocytes by qRT-PCR. To determine the significance, one-way ANOVA followed by a Friedman test corrected by Dunn’s multiple comparison test was performed. Data are expressed as the mean values ± SDs. Different letters on each group denote a significant difference at P ≤ 0.05.

Figure 4

CD4⁺and CD8⁺T cell proliferation after TCR stimulation co-treatment with the supernatant from splenocytes or monocytes/macrophages stimulated with inactivated H9N2 andB. subtilisspores. A CTV-labelled purified CD3⁺ T cells were cultured in anti-CD3 and anti-CD28 antibody-coated plates with supernatants from splenocytes or monocytes/macrophages stimulated with inactivated H9N2 and/or B. subtilis and analysed by using flow cytometry. B–E The proliferative populations of CD4⁺ T cells (B and C) and CD8⁺ T cells (D and E) were measured by CTV histograms and division index scores. To determine the significance, one-way ANOVA followed by a Friedman test corrected by Dunn’s multiple comparison test was performed. Data are expressed as the mean values ± SDs. Different letters on each group denote a significant difference at P ≤ 0.05.

Figure 5

Comparison of the immune cell proportion after administration of H9N2 withB. subtilisspores or a commercial vaccine. Seven-day-old chickens (N = 5) were immunized twice with inactivated H9N2 and/or B. subtilis spores at one-week intervals. At one week post second immunization, single cells from splenocytes were stained with anti-KUL01, anti-Bu-1, anti-CD4 and anti-CD8 antibodies, and flow cytometry analysis was performed. The percentages and absolute numbers of A KUL01⁺ monocytes/macrophage cells, B Bu-1⁺ B cells, and C CD4⁺ and D CD8⁺ T cells were examined. To determine the significance, one-way ANOVA followed by a Friedman test corrected by Dunn’s multiple comparison test was performed. Data are expressed as the mean values ± SDs. Different letters on each group denote a significant difference at P ≤ 0.05.

Figure 6

mRNA expression of cytokines in T cells in spleen from chickens vaccinated with inactivated H9N2 andB. subtilisspores. Chickens (N = 5) were administered H9N2 oil vaccine (commercially available), inactivated H9N2 or inactivated H9N2 and B. subtilis spores twice at one-week interval. Seven days after the last administration, splenocytes were re-stimulated with inactivated H9N2 for 24 h; then, T cells were isolated by magnetic bead sorting, and mRNA expression of A IFN-γ, B IL-17 and C IL-4 was examined by qRT-PCR. To determine the significance, one-way ANOVA followed by a Friedman test corrected by Dunn’s multiple comparison test was performed. Data are expressed as the mean values ± SDs. Different letters on each group denote a significant difference at P ≤ 0.05.