BCG Cell Wall Skeleton As a Vaccine Adjuvant Protects Both Infant and Old-Aged Mice from Influenza Virus Infection

Abstract

Bacillus Calmette-Guerin (BCG) and the cell wall skeleton (CWS) derived from BCG are known to enhance nonspecific immune activation and anti-cancer immunity; however, their roles as a vaccine adjuvant are largely unknown. Here, we report that BCG-CWS acts as a strong immune adjuvant by promoting the protective immune responses in mouse models with influenza vaccination. The different aged mice immunized with inactivated split vaccine with or without BCG-CWS were challenged with an influenza pandemic virus. When protective immune responses were compared, even a single immunization of adult mice with a BCG-CWS-adjuvanted vaccine showed significantly enhanced humoral immune responses with increased IgG1 and IgG2a isotype antibodies. Importantly, the protective effects by the BCG-CWS adjuvant for influenza vaccination upon humoral and cellular immunogenicity were comparable between infants (6 days and 2 weeks old) and aged (20 months old) mice. Moreover, BCG-CWS dramatically augmented vaccine-mediated protective responses, including decreased viral loads, lung damage, and airway resistance, as well as increased mouse survival, amelioration of weight loss, and proinflammatory cytokine expression in all experimental groups including infant, adults, and old aged mice. We further provided the evidence that the BCG-CWS adjuvant effects were mediated through Toll-like receptors (TLR) 2 and TLR4 signaling pathways. Together, these data suggest that BCG-CWS can be promising as a potential influenza vaccine adjuvant in both young and old aged population through TLR2/4-mediated immune-boosting activities.

Keywords: cell wall skeleton (CWS) adjuvant; immunological protective immunity; influenza virus; split vaccine.

Figure 1

BCG-CWS exhibits significant adjuvant effects on enhancing IgG, HAI, and protective efficacy in adult mice after single dose influenza vaccination. (A) Immunization schedule (n = 20). 6-week-old (6 W) BALB/c mice were intramuscular (i.m.) immunized either with sCal (S) or S+CWS. CWS: BCG-CWS. Blood samples were collected at 2 weeks after prime. (B–D) Antibody responses specific for inactivated A/Californial/04/2009 virus (iA/Cal) in prime immune sera. € Hemagglutinin inhibition titers (HAI) to A/California/04/2009 (A/Cal) were determined in prime immune sera. (F–H) Immunized mice were infected with A/Cal virus at a dose of 2 × LD₅₀ (50% mouse lethal dose) equivalent to 3 × 10³ EID₅₀ (50% embryo infectious dose) at 4 weeks after prime. PenH (F) and body weight changes or survival rates (G) were monitored for 7 days and 14 days (n = 10), respectively. (H) Lung viral titers were determined by an egg inoculation assay at 7 days post infection (n = 10). Mock: PBS control and no infected, S: split sCal vaccine only, S+CWS: split plus CWS, CWS: BCG-CWS adjuvant only, Mock inf: PBS and infected with A/Cal virus. Statistical significance was calculated by using one- or two-way ANOVA and a Bonferroni’s multiple-comparison test. Error bars indicate the mean ± SEM. *; p < 0.05, **; p < 0.01, ***; p < 0.001.

Figure 2

IgG antibody responses and HAI titers in BCG-CWS adjuvanted vaccination in neonatal (6 D) or infant (2W) age mice. (A) Immunization schedule. BALB/c mouse pups (n = 18) at 6 days (6 D) and 2 weeks (2 W) age were i.p./i.m. or i.m./i.m. immunized either with sCal (S) vaccine or S+CWS (CWS: BCG-CWS) at a 3-week interval. (B–D) Serum IgG, IgG1, and IgG2a specific for virus antigen in 6 D age mice, and (F–H) 2 W age mice. (E,I) HAI titers against A/Cal in boost sera of 6 D and 2 W age mice. Statistical significance was calculated by using one- or two-way ANOVA and a Bonferroni’s multiple-comparison test. Error bars indicate the mean ± SEM. *; p < 0.05, **; p < 0.01, ***; p < 0.001.

Figure 3

BCG-CWS adjuvant effects on enhancing protective efficacy in vaccinated 6 D and 2 W age mice. (A) Challenge plan of immunized mice. Vaccinated 6 D and 2 W groups were challenged with A/Cal virus (2 × LD₅₀) at 3 weeks after boost. (B,C) Body weight changes and survival rates in 6 D age mice (n = 8). (E,F) Body weight changes and survival rates in 2 W age mice (n = 8). (D,G) Lung viral titers were determined by an egg inoculation assay at 7 dpi (n = 10). Statistical significance was calculated by using one- or two-way ANOVA and a Bonferroni’s multiple-comparison test. Error bars indicate the mean ± SEM. *; p < 0.05, **; p < 0.01, ***; p < 0.001.

Figure 4

BCG-CWS-adjuvanted 6 D and 2 W mice protect against excessive cellular infiltration into the lung after challenge. Cellular phenotypes were determined by flow cytometry in BAL and lung samples collected at 7 dpi with A/Cal virus. (A,F) Monocytes (⁺CD11b⁺Ly6c^hiF4/80⁺) and (B,G) Eosinophils (⁺CD11b⁺CD11c^-SiglecF⁺) were gated from CD11b⁺ cells. CD11c⁺ dendritic cells (DCs, CD45⁺F4/80^-CD11c⁺ MHCII^hi), CD103⁺ DCs (CD45⁺F4/80^-CD11c⁺MHCII^hiCD11b^-CD103⁺), and CD11b⁺ DCs (CD45⁺F4/80^-CD11c⁺MHCII^hiCD11b⁺CD103^-) were gated from CD45⁺F4/8^- cells (C,H) and CD45⁺F4/80^-CD11c⁺MHCII^hi cells (D–J), respectively, in BAL and Lung of 6 D and 2 W age mice (n = 10 each age mice). The low cell numbers of eosinophils were observed to be 1030 and 3292 in the BAL and lungs from mock infected mice. Statistical significance was calculated by using two-way ANOVA and a Bonferroni’s multiple-comparison test. Error bars indicate the mean ± SEM. *; p < 0.05, **; p < 0.01, ***; p < 0.001.

Figure 5

BCG-CWS adjuvant effects on enhancing IgG, HAI, and protective efficacy in vaccinated aged (20 M) mice. (A) Immunization and challenge schedule. The groups of aged (20 M) mice (n = 10) were i.m. twice immunized with sCal (S) or S+CWS (CWS: BCG-CWS) at a 3-week interval. (B–D) Serum IgG, IgG1, IgG2a specific for virus antigen in boost sera. (E) HAI titers against A/Cal virus in boost sera. (F,G) Weight changes and survival rates after lethal dose A/Cal virus infection in aged (20 M) mice (n = 5). (H) Lung viral titers (n = 5) at 7 dpi. Statistical significance was calculated by using one- or two-way ANOVA and a Bonferroni’s multiple-comparison test. Error bars indicate the mean ± SEM. *; p < 0.05, **; p < 0.01, ***; p < 0.001.

Figure 6

Inflammatory cytokines and virus specific IgG antibody responses in lung samples from vaccinated mice after infection. BALF and lung lysates were collected at 7dpi after challenging of immunized mice (n = 10 for 6 D, 2 W, and 6 W mice, n = 5 for 20 M mice) to evaluate protective efficacy. The levels of cytokine and antibody were determined by ELISA. (A) Inflammatory cytokines (IL-6, TNF-α, and IFN-γ) in lung samples collected from 6 D or 2 W mice with prime-boost. (B) Lung inflammatory cytokines from 6 W adult mice with prime. (C) Lung inflammatory cytokines from 20 M aged mice with prime-boost. (D–G) Virus specific IgG antibody levels in BALF and lung samples collected at 7 dpi from the 6 D, 2 W, 6 W, and 20 M mouse groups. Statistical significance was calculated by using one-way ANOVA and a Dunnett’s multiple-comparison test. Error bars indicate the mean ± SEM. *; p < 0.05, **; p < 0.01, ***; p < 0.001.

Figure 7

IFN-γ secreting cellular responses in lung and spleens at early time after challenge. IFN-γ secreting cells were determined in lung and spleen samples collected from the 6 D, 2 W, 6 W, and 20 M groups at 7 dpi with A/Cal virus challenge. (A–C) Virus antigen-specific IFN-γ secreting cells in the lung or splenocytes from the different age vaccinated groups as determined by ELISpot assay. (D–H) The IFN-γ-secreting CD4⁺ T cells (IFN-γ⁺CD4⁺ T cells) in the lung, airway (BAL), or spleens from the different age vaccinated groups as determined by intracellular cytokine staining and analyzed by flow cytometry. Statistical significance was calculated by using one-way ANOVA and a Dunnett’s multiple-comparison test. Data are presented from all animals. Error bars indicate the mean ± SEM. *; p < 0.05, **; p < 0.01, ***; p < 0.001.

Figure 8

BCG-CWS stimulate antigen presenting cells to secrete cytokines via TLR-2 and TLR-4 pathways. Primary BMDCs isolated from WT (C57BL/6), TLR-2 KO, or TLR-4 KO mice were stimulated with BCG-CWS (10 µg/mL) or medium control for 24 h. (A,B) The data represent the concentrations of TNF-α and IL-6 in the BMDCs culture supernatants. Error bars indicate the mean ± SEM. ***; p < 0.001.