Mucosal Antibodies to the C Terminus of Toxin A Prevent Colonization of Clostridium difficile

Abstract

Mucosal immunity is considered important for protection against Clostridium difficile infection (CDI). We show that in hamsters immunized with Bacillus subtilis spores expressing a carboxy-terminal segment (TcdA_26-39) of C. difficile toxin A, no colonization occurs in protected animals when challenged with C. difficile strain 630. In contrast, animals immunized with toxoids showed no protection and remained fully colonized. Along with neutralizing toxins, antibodies to TcdA_26-39 (but not to toxoids), whether raised to the recombinant protein or to TcdA_26-39 expressed on the B. subtilis spore surface, cross-react with a number of seemingly unrelated proteins expressed on the vegetative cell surface or spore coat of C. difficile These include two dehydrogenases, AdhE1 and LdhA, as well as the CdeC protein that is present on the spore. Anti-TcdA_26-39 mucosal antibodies obtained following immunization with recombinant B. subtilis spores were able to reduce the adhesion of C. difficile to mucus-producing intestinal cells. This cross-reaction is intriguing yet important since it illustrates the importance of mucosal immunity for complete protection against CDI.

Keywords: Clostridium difficile; colonization; immune exclusion; mucosal immunity; oral vaccines.

FIG 1

Mucosal vaccination with PP108 spores. Hamsters (6/group) were immunized by the s.o. route with live (PP108-L) or formalin-inactivated (PP108-F) PP108 spores, live PY79 spores, or the rTcdA_26–39 protein by injection (i.m.). (A) Kaplan-Meier survival estimates after oral challenge of these animals with 100 spores of C. difficile 630. (B) Anti-TcdA_26–39 IgG titers in individual animals from each group 2 days before challenge. *, P < 0.05. (C) Counts of 630 spores in feces at 24 h postchallenge in individual animals. (D and E) Levels of toxin A (D) and toxin B (E) detected in hamster feces from individual groups. A capture ELISA was used to determine toxin levels, and the presence of functional toxins was also confirmed by using a cell cytotoxicity assay to measure toxin A (HT29 cells) and toxin B (Vero cells) levels (data not shown). (F) Analysis of fecal IgA and serum IgG obtained from a separate hamster experiment. In this study, groups (n = 5) were orally dosed with PP108 spores or by injection (i.m.) with rTcdA_26–39, with 3/5 hamsters being protected in the PP108-dosed group. i.p., intraperitoneal.

FIG 2

Immune responses in immunized CD-1 mice. Groups (n = 6) of CD-1 mice were immunized i.g. with live spores of PP108 (PP108-L), formaldehyde-inactivated PP108 spores (PP108-F), or live PY79 spores or parenterally (i.m.) with rTcdA_26–39 (5 μg/dose). Oral dosing was done by using 5 × 10¹⁰ CFU/dose on days 0, 1, 2, 14, 15, 16, 28, 29, 30, 55, 56, and 57. Parenteral dosing was done on days 0, 14, and 28. Fecal IgA was collected 10 to 15 days after the final immunization, serum IgG was collected 21 days after the final immunization (A), and anti-TcdA_26–39 titers were determined (B). *, P < 0.05. Neutralization endpoint titers are shown above the bars and were determined as described previously (22).

FIG 3

Parenteral vaccination with toxoids. Hamsters (6/group) were immunized with toxoids A and B using three injections (i.m.) and then challenged with 100 spores of C. difficile 630. (A) Survival of animals compared to the naive groups. (B) Anti-toxoid A and anti-toxoid B IgG in immunized groups 2 days before challenge. (C) Counts of 630 spores in feces at 24 h postchallenge in immunized and naive groups. This experiment was repeated once. (D and E) Levels of toxins A (D) and B (E) detected in cecal samples as determined by a capture ELISA, together with samples from nonimmunized animals (naive infected). Cell cytotoxicity assays were also used to confirm the presence of toxins.

FIG 4

Anti-TcdA_26–39 antibodies recognize C. difficile. (A) Protein extracts were run on SDS-PAGE gels and probed with rabbit PAbs (1/6,000 dilution) raised against B. subtilis PY79 spores. Lane 1, rTcdA_26–39 protein (∼0.7 to 1 μg); lane 2, spore coat proteins extracted from PY79 spores; lane 3, proteins extracted from C. difficile 630 vegetative cells; lane 4, extracts of vegetative cells of a toxin A-negative, toxin B-negative mutant; lane 5, spore coat extracts from 630 spores. (B) Cross-reaction to vegetative cells (vc) of 630 or an isogenic 630 mutant carrying insertions in the tcdA and tcdB genes (toxin A negative and toxin B negative) by an ELISA. Three murine antisera were used: naive, anti-TcdA_26–39, and anti-C. difficile 630. **, P < 0.0018. (C) Identification of cross-reactions of different murine antibodies to spores of C. difficile 630 by an ELISA. Wells of 96-well plates were coated with spores (∼10⁸ spores/well), and sera (all diluted 1/300) from immunized mice were used for detection by an ELISA. α-rA26-39, anti-TcdA_26–39; α-CDsp, anti-C. difficile 630 spores. **, P = 0.008; *, P < 0.0112. (D) Identification of cross-reactions of different murine antibodies to vegetative cells of C. difficile 630 by an ELISA. Wells of 96-well plates were coated with spores (∼10⁸ spores/well), and sera (all diluted 1/300) from immunized mice were used for detection by an ELISA. α-rA26-39, anti-TcdA_26–39; α-CDvc, anti-C. difficile 630 vegetative cells. **, P < 0.002.

FIG 5

C. difficile proteins recognized by anti-TcdA_26–39. Shown are Western blots of proteins probed with rabbit anti-rTcdA_26–39 IgG (1/3,000 dilution) (A), mouse anti-toxoid A IgG (1/5,000 dilution) (B), mouse anti-PP108 IgA (1/200 dilution) (C), and mouse anti-PY79 IgA (1/200 dilution) (D). SDS-PAGE gels were loaded as follows: lane 1, rTcdA_26–39 protein (∼1 μg); lane 2, extracts of B. subtilis (Bs) PY79 spore coats; lane 3, extracts of C. difficile (Cd) 630 vegetative cells; lane 4, extracts of vegetative cells of a 630 strain deficient in toxin A and toxin B; lane 5, extracts of vegetative cells of a 630 strain unable to produce the sporulation transcription factor σ^K; lane 6, spore coat protein extracts from 630 spores; lane 7, spore coat protein extracts from spores deficient in the production of toxin A and toxin B. Molecular mass markers (in kilodaltons) are shown, together with the identity of known proteins or proteins referred to in the text.

FIG 6

Verification of cross-reacting proteins. (A) Recombinant proteins identified by anti-TcdA_26–39. Approximately 1 μg of purified protein was run per lane and probed with antibodies at a dilution of 1/4,000. (B) Bands recognized in proteins extracted from spores of the wild type (630Δerm) and a ΔcdeC mutant probed with anti-TcdA_26–39 (1/4,000 dilution). Molecular masses (in kilodaltons) are indicated.

FIG 7

Mucosal antibodies to TcdA_26–39 reduce adhesion of vegetative cells to mucus-producing HT29-MTX cells. HT29-MTX cells were grown for 14 days, and the production of mucus was confirmed. Vegetative cells (A) or spores (B) of C. difficile 630 were pretreated with either serum IgG or fecal IgA (1/300 dilution) obtained from CD-1 mice immunized s.o. with live PP108 spores or live PY79 spores, as shown in Fig. 2. Adhesion to cells was expressed as a percentage of untreated cells or spores run in parallel. Adhesion of 100% equates to the number of cells or spores adhering to HT29-MTX cells without pretreatment and in parallel. The experiment was repeated two times, with similar findings. *, P < 0.05.