Immunogenicity of the B monomer of Escherichia coli heat-labile toxin expressed on the surface of Streptococcus gordonii

Abstract

The B monomer of the Escherichia coli heat-labile toxin (LTB) was expressed on the surface of the human oral commensal bacterium Streptococcus gordonii. Recombinant bacteria expressing LTB were used to immunize BALB/c mice subcutaneously and intragastrically. The LTB monomer expressed on the streptococcal surface proved to be highly immunogenic, as LTB-specific immunoglobulin G (IgG) serum titers of 140,000 were induced after systemic immunization. Most significantly, these antibodies were capable of neutralizing the enterotoxin in a cell neutralization assay. Following mucosal delivery, antigen-specific IgA antibodies were found in feces and antigen-specific IgG antibodies were found in sera. Analysis of serum IgG subclasses showed a clear predominance of IgG1 when recombinant bacteria were inoculated subcutaneously, while a prevalence of IgG2a was observed upon intragastric delivery, suggesting, in this case, the recruitment of a Th1 type of immune response.

FIG. 1

Immunoblot analysis of the surface proteins of recombinant S. gordonii. The envelope fractions of recombinant strain GP1246, expressing the M6-LTB fusion protein (lanes 1), and of control strain GP1221.1 (lanes 2) were analyzed by Western blotting. The arrows indicate a major protein band of 44 kDa (the expected molecular mass of the recombinant protein was 41 kDa) that was found to be reactive with both anti-M6 (panel A) and anti-LTB (panel B) polyclonal antibodies. The positions of the molecular weight standards are indicated on the left (in kDa).

FIG. 2

ELISA titers of serum LTB-specific total IgG following s.c. (A) and i.g. (B) immunizations with recombinant S. gordonii. Two groups of eight BALB/c mice each were inoculated s.c. at weeks 0, 3, and 6 with 10⁹ CFU of S. gordonii vaccine strain GP1246 expressing LTB (solid bars) or with the control strain S. gordonii GP246 expressing an unrelated antigen (E7 of HPV-16) (open bars). For the i.g. inoculum, two groups of eight BALB/c mice each were inoculated with live recombinant S. gordonii GP1246 (solid bars) or with control strain GP246 (open bars). Mice were immunized twice (weeks 0 and 4), and each immunization consisted of two doses (24 h apart) of 10¹⁰ CFU of live bacteria. Serum samples were collected from animals immunized s.c. and i.g. at weeks 0, 5, and 8 and 0, 4, and 7, respectively. Samples from each animal were titrated in twofold dilutions and analyzed by ELISA for the presence of LTB-specific IgG. Antibody titers are expressed as the reciprocal of the highest serum dilution with an absorbance per hour value of >0.2 after subtraction of the background value. The titer of preimmune sera is also reported (gray bars). Arrows indicate the times of inoculations. Results are expressed as the mean ± SD. Significant differences between vaccine and control animals inoculated s.c. and i.g. were calculated (P = 0.002 and P = 0.004, respectively).

FIG. 3

LTB-specific serum antibodies after s.c. (A) and i.g. (B) immunizations with recombinant S. gordonii. The concentrations of total and LTB-specific IgG1 and IgG2a antibodies were determined by ELISA using standard curves of mouse myeloma IgG1 and IgG2a. The amounts of LTB-specific IgG1 and IgG2a were normalized to the concentrations of total IgG1 and IgG2a, and results were expressed in micrograms of LTB-specific IgG1 or IgG2a per milligram of total IgG1 or IgG2a, respectively. Serum samples were collected at weeks 0, 5, and 8 (s.c.) and 0, 4, and 7 (i.g.). On the x axes, arrows indicate the times of inoculations.

FIG. 4

LTB-specific IgA in fecal samples of mice inoculated i.g. Two groups of eight BALB/c mice each were immunized with either the vaccine strain GP1246 (solid bars) or the control strain GP246 (open bars) as described for Fig. 2. Inoculations were performed at weeks 0 and 4. Feces were collected at weeks 0, 2, 4, 5, 6, 7, and 8. To calculate the concentrations of total and LTB-specific IgA, a standard curve of mouse myeloma IgA was used. The concentration of LTB-specific IgA was determined in pools of fecal samples containing 10 μg of total IgA from each sample per ml. The amount of LTB-specific IgA was normalized to the concentration of total IgA and expressed in micrograms of specific IgA per milligram of total IgA. Antigen-specific IgA in pooled preimmune fecal samples is also reported (gray bar).