Systemic and mucosal immune responses after intranasal administration of recombinant Mycobacterium bovis bacillus Calmette-Guérin expressing glutathione S-transferase from Schistosoma haematobium

Abstract

A major goal of current vaccine development is the induction of strong immune responses against protective antigens delivered by mucosal routes. One of the most promising approaches in that respect relies on the use of live recombinant vaccine carriers. In this study, Mycobacterium bovis BCG was engineered to produce an intracellular glutathione S-transferase from Schistosoma haematobium (Sh28GST). The gene encoding Sh28GST was placed under the control of the mycobacterial hsp60 promoter on a replicative shuttle plasmid containing a mercury resistance operon as the only selectable marker. The recombinant Sh28GST produced in BCG bound glutathione and expressed enzymatic activity, indicating that its active site was properly folded. Both intraperitoneal and intranasal immunizations of BALB/c mice with the recombinant BCG resulted in strong anti-Sh28GST antibody responses, which were enhanced by a boost. Mice immunized intranasally produced a mixed response with the production of Sh28GST-specific immunoglobulin G1 (IgG1), IgG2a, IgG2b, and IgA in the serum. In addition, high levels of anti-Sh28GST IgA were also found in the bronchoalveolar lavage fluids, demonstrating that intranasal delivery of the recombinant BCG was able to induce long-lasting secretory and systemic immune responses to antigens expressed intracellularly. Surprisingly, intranasal immunization with the BCG producing the Sh28GST induced a much stronger specific humoral response than intranasal immunization with BCG producing the glutathione S-transferase from Schistosoma mansoni, although the two antigens have over 90% identity. This difference was not observed after intraperitoneal administration.

FIG. 1

Production, purification, and enzymatic activity of Sh28GST produced in recombinant BCG. (A) Immunoblot analysis of GST production in BCG. Crude lysates of BCG producing either Sm28GST (lane 4) or Sh28GST (lane 3) were compared to a lysate of untransformed BCG (lane 2) by immunoblot analysis using a rat polyclonal serum directed against peptide 190-211 of the Sm28GST. Lane 1 contains 100 ng of purified recombinant Sm28GST. (B) SDS-PAGE analysis after single-step affinity chromatography on a GSH-agarose column. The Sh28GST-containing fractions were pooled, concentrated by ultrafiltration, dialyzed against PBS, and then subjected to SDS-PAGE followed by Coomassie blue staining. Lane 1 contains whole-cell extracts of the recombinant BCG strain, lane 2 contains 1 μg of purified Sh28GST produced in yeast, and lane 3 contains purified Sh28GST produced by the recombinant BCG. Sizes of molecular mass markers, indicated in kilodaltons, are shown on the left. (C) Measurement of the GST activity catalyzed by Sh28GST purified either from recombinant BCG (open circles) or from yeast (closed squares) over time. OD, optical density.

FIG. 2

Antibody responses induced after i.p. immunization. BALB/c mice were immunized i.p. with either 10⁸, 5 × 10⁶, or 5 × 10⁵ untransformed BCG or BCG producing Sh28GST (BCG/Sh28GST) and boosted 8 weeks later in the same way. The sera from each group were collected 2 weeks after the first immunization (black bars) or 2 (gray bars), 10 (white bars), or 19 (hatched bars) weeks after the boost, pooled, and analyzed by ELISA using BCG total soluble proteins (A) or purified Sh28GST produced in yeast (B).

FIG. 3

Comparison of anti-GST antibody responses induced after i.p. immunization with BCG producing either Sm28GST or Sh28GST. BALB/c mice were immunized i.p. with 5 × 10⁶ untransformed BCG, BCG producing Sm28GST (BCG/Sm28GST), or BCG producing Sh28GST (BCG/Sh28GST) and boosted 8 weeks later in the same way. The sera from each group were collected 2 weeks after the boost, pooled, and analyzed by ELISA using purified Sm28GST produced in yeast (left) or purified Sh28GST produced in yeast (right).

FIG. 4

Antibody responses induced after i.n. immunization. BALB/c mice were immunized i.n. with 10⁷ untransformed BCG or BCG producing Sh28GST (BCG/Sh28GST) and boosted 16 weeks later in the same way. The sera from each group were collected 4 (black bars) or 16 (gray bars) weeks after the first immunization or 6 (white bars) or 12 (hatched bars) weeks after the boost, pooled, and analyzed by ELISA using BCG total soluble proteins (A) or purified Sh28GST produced in yeast (B).

FIG. 5

Anti-Sh28GST antibody isotype profiles elicited after i.n. immunization with BCG producing Sh28GST. BALB/c mice were immunized i.n. with 10⁷ BCG organisms producing Sh28GST (BCG/Sh28GST) and boosted 16 weeks later in the same way or left unimmunized (control). The sera from each group were collected 4 (black bars) or 16 (gray bars) weeks after the first immunization or 6 (white bars) or 12 (hatched bars) weeks after the boost, pooled, and analyzed by ELISA for the presence of specific anti-Sh28GST IgG1, IgG2a, IgG2b, and IgA isotypes.

FIG. 6

Mucosal immune responses elicited after i.n. immunization. BALB/c mice were immunized i.n. with 10⁷ untransformed BCG or BCG producing Sh28GST (BCG/Sh28GST) and boosted 16 weeks later in the same way or left unimmunized (control). The BALF from each group were collected 4 (black bars) or 16 (gray bars) weeks after the first immunization or 6 (white bars) or 12 (hatched bars) weeks after the boost, pooled, and analyzed by ELISA for the presence of total IgA (A), anti-BCG IgA (B), and anti-Sh28GST IgA (C).

FIG. 7

Comparisons of specific antibody responses and cross-reactivities after i.n. immunization. Groups of 10 BALB/c mice were immunized i.n. with 5 × 10⁶ untransformed BCG, BCG producing Sm28GST (BCG/Sm28GST), or BCG producing Sh28GST (BCG/Sh28GST) and boosted 8 weeks later in the same way. The sera from each group were collected 8 weeks (black bars) after the first immunization or 6 weeks (gray bars) after the boost, pooled, and analyzed by ELISA using BCG total soluble proteins (right), purified Sh28GST produced in yeast (middle), or purified Sm28GST produced in yeast (left).

FIG. 8

Neutralization of the GST activity by the anti-Sh28GST antisera elicited in mice after i.n. immunization with recombinant BCG. The neutralizing activity was analyzed for sera from BALB/c mice obtained 6 weeks after the boost and immunized i.n. with either untransformed BCG (open circles) or BCG(pENSh28) (closed squares). The catalytic inhibition was measured in the absence or presence of increasing concentrations of antisera.