A recombinant BCG vaccine generates a Th1-like response and inhibits IgE synthesis in BALB/c mice

Abstract

The tubercle vaccine, bacille Calmette-Guérin (BCG), is a strong inducer of T-helper type 1 (Th1) responsiveness, and it has been suggested that recombinant BCG (rBCG), which produces and secretes antigens, may be used to prevent allergic diseases. The effects of rBCG vaccination on allergic responses in a murine model were examined in this study. A BCG-Escherichia coli shuttle vector was developed with the promoter and signal sequence of the alpha-antigen of Mycobacterium bovis, and the vector was tested using E. coli beta-galactosidase as the model antigen and allergen. This vector enabled the expression of the E. coli beta-galactosidase gene in BCG, which was detected in its protein extract by immunoblotting analysis. Vaccination of mice with a single dose of 106 recombinant BCG generated a beta-galactosidase-specific antibody response. The splenocytes of vaccinated mice compared with controls produced significantly higher amounts of interferon-gamma (IFN-gamma) (P<0. 01) and interleukin-2 (IL-2) (P<0.05) and lower amounts of IL-5 (P<0. 01). Mice vaccinated with rBCG had significantly less (P<0.01) serum IgE compared with controls. These results together demonstrate that rBCG secreting antigens or allergens may be utilized for the induction of a Th1-like response and the down-regulation of IgE antibody response.

Figure 1

(a) Construction of the BCG–E. coli shuttle vector pBCGα. The plasmid vector pBCGα (5·870 kb) was constructed by cloning the 210-bp fragment containing the α-antigen promoter and signal sequence from M. bovis (BCG) and the 560-bp origin of replication from E. coli at the Kpn I site in the plasmid vector p16R1.²² (b) Expression of β-gal in pBCGα. The whole cell extract of control BCG (lane 1) and rBCG was subjected to 0·1% SDS–10% PAGE analysis. Lane 2 and 3 show the expression of β-gal as a 110-kDa protein. M denotes molecular weight markers ( × 10³). (c) Immunoblot analysis of the expressed protein. BCG and rBCG cell lysates were analysed for expression by Western blot analysis. Recombinant β-gal was readily identified by rabbit anti β-gal mAbs (lanes 2 and 3). Lane 1 is purified β-gal.

Figure 2

(a) Immunization with rBCG induces an anti-β-gal antibody response in mice. Vaccination of mice with 1 × 10⁶ CFU of recombinant BCG secreting β-gal generates high antibody titres against expressed protein (▴). The antibody response detectable from week 4 increased thereafter and peaked at week 10 (ELISA titre, 1:25 000). Mice vaccinated with equal numbers of non-recombinant BCG failed to generate any antibody response (▪). Experiments were performed twice and identical results were observed. (b) Vaccination of mice with rBCG stimulates IFN-γ production. Mice were vaccinated with either 1 × 10⁶ CFU rBCG (solid bars) or non-recombinant BCG (open bars). On week 12 their spleen cells were cultured in vitro (1 × 10⁶ cells) and stimulated with β-gal and the BCG vaccine (Glaxo, Greenford, UK). IFN-γ levels in supernates from 48-hr cultures were determined by ELISA. Mice vaccinated with rBCG showed nearly five times more production of IFN-γ than control mice did. Data are mean±SD (n = 6). The experiment was repeated twice with similar results. Data from a single experiment are shown.

Figure 3

Production of IFN-γ (a), IL-2 (b) and IL-5 (c) by splenocytes cultured with β-gal. BALB/c mice were vaccinated with 1 × 10⁶ CFU of either rBCG or BCG and on week 6 immunized i.p with 10 μg β-gal in 1 mg alum. Seven days later spleens were harvested from these mice and a single-cell preparation was cultured in vitro (10 × 10⁶ cells) in the presence of 10 μg/ml of β-gal. Culture supernatants were collected after 48 hr and cytokine levels were determined by ELISA. Data are mean±SD (n = 6). *P < 0·05, **P < 0·01.

Figure 4

Effect of rBCG vaccination followed by immunization on serum antibody production. Mice were vaccinated with either rBCG or BCG and immunized with β-gal in alum, as described in the Materials and Methods. On day 21 after immunization, sera were collected and analysed by ELISA for total IgE (1:50 serum dilution) (a), antigen-specific IgG2a (1:200 serum dilution) (b), IgG1 (1:200 serum dilution) (c) and IgE (1:50 serum dilution) (d). All serum dilutions were made in serum dilution buffer. Mice vaccinated with rBCG exhibited less IgE and IgG1 production and an increase in IgG2a antibody levels. Data are mean±SD (n = 6). *P < 0·05, **P < 0·01.