Enhanced mucosal delivery of antigen with cell wall mutants of lactic acid bacteria

Abstract

The potential of recombinant lactic acid bacteria (LAB) to deliver heterologous antigens to the immune system and to induce protective immunity has been best demonstrated by using the C subunit of tetanus toxin (TTFC) as a model antigen. Two types of LAB carriers have mainly been used, Lactobacillus plantarum and Lactococcus lactis, which differ substantially in their abilities to resist passage through the stomach and to persist in the mouse gastrointestinal tract. Here we analyzed the effect of a deficiency in alanine racemase, an enzyme that participates in cell wall synthesis, in each of these bacterial carriers. Recombinant wild-type and mutant strains of L. plantarum NCIMB8826 and L. lactis MG1363 producing TTFC intracellularly were constructed and used in mouse immunization experiments. Remarkably, we observed that the two cell wall mutant strains were far more immunogenic than their wild-type counterparts when the intragastric route was used. However, intestinal TTFC-specific immunoglobulin A was induced only after immunization with the recombinant L. plantarum mutant strain. Moreover, the alanine racemase mutant of either LAB strain allowed induction of a much stronger serum TTFC-specific immune response after immunization via the vagina, which is a quite different ecosystem than the gastrointestinal tract. The design and use of these mutants thus resulted in a major improvement in the mucosal delivery of antigens exhibiting vaccine properties.

FIG. 1.

Effect of d-alanine starvation on bacterial growth and cell membrane integrity. Culture growth was measured by monitoring the OD₆₀₀ (A), and bacterial viability and membrane permeability were examined after 3 h (solid bars), 6 h (striped bars), and 24 h (cross-hatched bars) of d-alanine starvation for the WT and Alr⁻ mutant strains (B). The proportion of injured cells was determined by determining the proportion of the PI-labeled cell population.

FIG. 2.

Levels of antigen production by recombinant L. plantarum and L. lactis strains: immunoblotting of cell extracts (equivalent to 10⁸ CFU) obtained from the L. plantarum NCIMB8826/pTG2247 (lane 1) and L. lactis MG1363/pTX (lane 8) control strains and from the TTFC-producing strains L. plantarum NCIMB8826/pMEC4 (lane 2), MD007/pMEC4 (lane 3), NCIMB8826Int1/pMEC46 (lane 4), MD007Int6/pMEC46 (lane 5), NCIMB8826/pMEC127 (lane 6), and MD007/pMEC127 (lane 7) and L. lactis NZ3900/pMEC46 (lane 9) and PH3960/pMEC46 (lane 10) and immunoblotting of 50 ng of purified TTFC (lane 11).

FIG. 3.

Immune responses following intragastric immunization with recombinant L. plantarum NCIMB8826 strains. (A) Anti-TTFC serum IgG titers. Individual sera were collected 10 days after priming (solid bars) and after the first boost (diagonally striped bars), the second boost (cross-hatched bars), and the third boost (horizontally striped bars) from groups of eight mice that were immunized with buffer alone, with 10⁹ CFU of the control nonexpressing strain NCIMB8826/pTG2247, or with 10⁹ CFU of a TTFC-producing strain (WT or MD007 Alr⁻ mutant harboring an expression plasmid). The bars indicate the mean ELISA IgG titer ± standard error of the mean for each group. (B) Anti-TTFC IgA levels in intestinal lavages from groups of mice immunized intragastrically with buffer, with the control NCIMB8826/pTG2247 strain, or with the TTFC-producing MD007/pMEC127 recombinant strain. Individual lavages were performed 10 days after the last feeding. The specific IgA response was normalized by using the level of total IgA and was expressed as the specific activity (S.A.). The bars indicate the mean IgA level ± standard error of the mean for each group.

FIG. 4.

Comparative immune responses following intragastric immunization with recombinant L. plantarum and L. lactis strains. (A) Anti-TTFC serum IgG titers. Individual sera were collected 10 days after priming (solid bars) and after the first boost (striped bars) or the second boost (cross-hatched bars) from groups of eight mice immunized either with buffer alone, with 10⁹ CFU of the control nonexpressing strain NCIMB8826/pTG2247 or MG1363/pTX, or with 10⁹ CFU of the TTFC-producing strain NCIMB8826/pMEC127, MD007/pMEC127, NZ3900/pMEC46, or PH3960/pMEC46. The bars indicate the mean ELISA IgG titer ± standard error of the mean for each group. (B) Serum anti-TTFC antibody IgG1, IgG2a, IgG2b, IgG3, and IgA titers after intragastric immunization (second boost) of mice with buffer (open bars) or with 10⁹ CFU of L. plantarum NCIMB8826/pTG2247 (horizontally striped bars), NCIMB8826/pMEC127 (solid bars), or MD007/pMEC127 (bars with arrowheads) or L. lactis MG1363/pTX (diagonally striped bars), NZ3900/pMEC46 (cross-hatched bars), or PH3960/pMEC46 (bars with diamonds). The isotypic response was analyzed by using pooled sera of each group collected after the last boost.

FIG. 5.

Anti-TTFC serum IgG titers following intravaginal immunization with recombinant L. plantarum NCIMB8826 and L. lactis MG1363 strains. Individual sera were collected 10 days after priming (solid bars) and after the first boost (striped bars) and the second boost (cross-hatched bars) from groups of eight mice immunized with buffer alone, with 10⁹ CFU of the control nonexpressing strain NCIMB8826/pTG2247 or MG1363/pTX, or with 10⁹ CFU of the TTFC-producing strain NCIMB8826/pMEC127, MD007/pMEC127, NZ3900/pMEC46, or PH3960/pMEC46. The bars indicate the mean ELISA IgG titer ± standard error of the mean for each group.