Clostridioides difficile Infection Induces an Inferior IgG Response to That Induced by Immunization and Is Associated with a Lack of T Follicular Helper Cell and Memory B Cell Expansion

The intracellularly active bacterial toxin TcdB is a major Clostridioides difficile virulence factor that contributes to inflammation and tissue damage during disease. Immunization with an inactive TcdB fragment prevents C. difficile infection (CDI)-associated pathology. The protective immune response against inactive TcdB involves development of antigen-specific memory B cells and long-lived plasma cells that encode TcdB-neutralizing antibodies. Unlike the response to inactive TcdB, very little is known about the host humoral immune response to C. difficile and TcdB during primary and recurrent infection.

ABSTRACT

The intracellularly active bacterial toxin TcdB is a major Clostridioides difficile virulence factor that contributes to inflammation and tissue damage during disease. Immunization with an inactive TcdB fragment prevents C. difficile infection (CDI)-associated pathology. The protective immune response against inactive TcdB involves development of antigen-specific memory B cells and long-lived plasma cells that encode TcdB-neutralizing antibodies. Unlike the response to inactive TcdB, very little is known about the host humoral immune response to C. difficile and TcdB during primary and recurrent infection. Here, we used a murine model of C. difficile disease recurrence to demonstrate that an initial infection induced a serum IgM and mucosal IgA response against the toxin, but a low serum IgG response, which is associated with a lack of protection against disease during reinfection. Infection induced a partial expansion of the T follicular helper cell compartment, essential for B cell memory responses, and, consistent with that, failed to significantly expand the memory B cell compartment. Further, infection failed to stimulate the memory B cell compartment in preimmunized mice, although they were protected against associated disease. These results delineate the key humoral immune events that follow primary and recurrent C. difficile infection and provide a compelling inverse correlation between B cell memory and disease recurrence.

INTRODUCTION

Clostridioides difficile is a leading cause of hospital-acquired infection worldwide and C. difficile infection (CDI) causes disease ranging in severity from mild diarrhea to fulminant colitis, sepsis, and death. Recurrence of C. difficile-associated disease significantly contributes to morbidity and mortality, with one-third of patients relapsing with progressively worsening symptoms. The frequency of recurrence increases with each subsequent episode (1, 2).

The host susceptibility to C. difficile colonization is mediated by changes in the gut microbiome, often due to broad-spectrum antibiotic treatment. After transmission, via the fecal-oral route, bacterial spores germinate and adhere to intestinal epithelial cells in the anaerobic environment of the colon (3). This process is mediated by several virulence factors, including the surface layer proteins (SLPs), the flagella, and cell wall proteins (4–6). However, disease pathogenesis is primarily caused by two large exotoxins, Toxin A (TcdA) and Toxin B (TcdB) (7). After secretion, TcdA and TcdB are translocated into the cytosol, where they target GTP-binding proteins and induce condensation of the cytoskeleton. This leads to epithelial cell death and damage of the intestinal membrane (8). The individual contributions of TcdA and TcdB to disease remain under investigation, but TcdB is reported to be the key virulence determinant in a hamster model of CDI (9, 10). Clinical isolates of TcdA-deficient C. difficile have been reported, and they demonstrate similar disease severity as wild-type strains (11, 12). Recently TcdA⁺ clinical strains that express TcdB at low levels or below the limits of detection have been reported (13).

The host immune response following C. difficile infection plays an important role in pathogenesis, but also in protection against disease. The pathogenesis of the disease is driven by activation of macrophages and polymorphonuclear leukocytes, causing severe inflammation in the colon (8). To counteract the effects of the toxins and limit inflammation and tissue damage, production of antimicrobial peptides and regulatory and anti-inflammatory cytokines are increased (7, 14). However, it is antibody (Ab)-mediated immune responses to the toxins that is critically important for protection against initial and recurrent C. difficile-associated disease (15, 16). Toxin-specific antibodies protect against disease in both patients and animal models by decreasing gastrointestinal symptoms and mortality (17). The potential role played by mucosal IgA in protection against C. difficile toxins has been examined, but systemic IgG appears to be a better determinant of clinical outcomes postinfection (18). Low titers of circulating IgG against TcdB correlate with severe disease and higher rates of recurrence (15, 19). Treatment with bezlotoxumab, a TcdB-specific monoclonal IgG1, has been associated with a lower recurrence rate in C. difficile patients (20). In a murine model of CDI, immunization with the inactive C-terminal domain of TcdB (CTD, consisting of TcdB_1651–2366 from strain VPI-10463) induces TcdB-neutralizing Abs, development of TcdB-specific memory B cells and long-lived plasma cells, and confers protection against toxin challenge and infection-associated disease (21, 22). Several clinical studies have also reported on the importance of the toxin-specific Ab response in protection against initial and recurrent disease (15, 19).

The high frequency of CDI recurrence suggests that, unlike the immune responses to inactive TcdB, patients often do not develop a protective humoral response following infection. The cellular and molecular mechanisms of the humoral immune responses to C. difficile and its toxins during infection, and the extent to which infection-induced Abs protect against future disease, are not well understood. An important component of a protective humoral response is development of plasma and memory B cells (Bmem), but whether these cells adequately develop following initial CDI is not known.

Herein we report that initial infections in mice are associated with poor primary Ab responses and lack of protection against subsequent infection. Adequate Bmem responses were not established by the initial infection, consistent with poor expansion of T follicular (Tfh) cells. Consequently, there were no detectable recall responses following a secondary infection. In contrast, immunization generated Bmem cells and bone-marrow-resident long-lived plasma cells. However, infection failed to restimulate the Bmem compartment in the preimmunized mice. This study delineates the humoral immune response to infection in the naive and immunized mouse and provides a potential explanation for recurrent C. difficile-associated disease.

RESULTS

Inadequate antibody responses to TcdB following C. difficile infection is associated with recurrent disease.

To evaluate the antibody response to TcdB after C. difficile infection (CDI) and its impact on recurrent disease, we utilized a murine model of reinfection. C57BL/6 mice were given cefoperazone in their drinking water to disrupt the gut microbiome. This was followed by antibiotic withdrawal and infection with 2 × 10⁴ CFU of C. difficile spores by oral gavage. Following a 2-month resting period, the mice were reinfected with the same dose of bacteria after a second round of antibiotic treatment. Uninfected control mice were also treated with antibiotics but were given water by oral gavage (Fig. 1A). After both the first and second infections, the mice showed significant weight loss compared to the uninfected mice. There was no difference in weight loss (Fig. 1B) and fecal bacterial counts (Fig. 1C) between the first and second infections. Following each infection, serum anti-CTD IgG1, IgG2b, and IgG2c titers were below the level of detection by enzyme-linked immunosorbent assay (ELISA) (Fig. 1D). Compared to uninfected mice, serum anti-CTD IgM titers were significantly increased in mice after the first and second infections. However, the anti-CTD IgM titers did not differ between the first and second infections (Fig. 1E). Measurable but low IgG1 titers specific for whole C. difficile were detected (Fig. 1F). C. difficile infection stimulated a toxin-specific mucosal IgA response detected in the fecal samples. However, there was no difference in the response between first and second infection showing that the second infection did not stimulate an IgA recall response and that it was not protective (Fig. 1G).

FIG 1.

Poor humoral immunity and lack of protection against recurrent disease following low-dose infection. (A) Cefoperazone-treated C57BL/6 mice were infected with 2 × 10⁴ C. difficile spores by oral gavage (first infection). Two months after the first infection, the mice were treated with antibiotic and received a second dose of bacterial spores (second infection). Uninfected control mice were gavaged with sterile water. (B) Graph depicts relative weights (mean ± standard error of the mean [SEM]) of mice following the first and second infections. The relative weight was determined based on the weight on day 0 post gavage. Data are representative of two independent experiments. (C) Graph shows CFU for C. difficile bacteria grown on TCCFA plates from fecal samples on day 3 after the first and second infections. (D and E) Serum anti-CTD IgM, IgG1, IgG2b, and IgG2c titers were measured before infection and on day 14 after the first and second infections. (F) C. difficile specific serum IgG1 titers were measured on days 0 and 14 after the second infection. (G) Fecal anti-CTD IgA was measured before infection and on day 14 after the first and second infections. Data are representative of two independent experiments and each symbol represents an individual mouse (n = 7 per group). Statistical significance is indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Next, we repeated the reinfection experiment with a dose of 5 × 10⁴ C. difficile spores per mouse. Following the first and second infections, mice showed significant weight loss compared to uninfected controls. In this experiment, there was no difference in the average or peak weight loss (Fig. S1A in the supplemental material) and the fecal bacterial counts (Fig. S1B) following the first and second infections. After the first and second infections, there were detectable anti-CTD IgM titers in the sera from all infected mice. It was observed that 50% of the mice had detectable anti-CTD IgG1 and IgG2b titers (Fig. S1C).

When mice were infected with 3 × 10⁵ spores of C. difficile, a higher dose than the previous experiments, 50% of the mice died and all surviving mice had high fecal bacterial counts (Fig. 2A). Infection induced a significant increase in the anti-CTD fecal IgA levels in surviving mice (Fig. 2B). There was a significant increase in the serum anti-CTD IgM and IgG1 titers following infection, but not serum IgG2b and IgG2c titers (Fig. 2C and D). Anti-C. difficile IgG1 titers were also significantly increased following infection (Fig. 2C). However, the serum IgG responses against CTD observed (see below) were 2 to 3 orders of magnitude lower than that typically observed following immunization with CTD (21). Neutralization assays were performed and confirmed that the serum IgG observed did not prevent toxin-associated CHO cell death (Fig. 2F).

FIG 2.

Severe disease or death and poor humoral immunity in survivors following high-dose infection. Mice were gavaged with 3 × 10⁵ C. difficile spores (n = 10) or water (n = 5). (A) Percent survival over time (left) and relative weight (mean ± SEM) (right) of mice before and after infection. The relative weight was determined based on the weight on day 0 post gavage. (B) CFU for C. difficile bacteria grown on TCCFA plates from fecal samples on day 3 after infection. (C to F) Blood and fecal samples were collected and CTD-specific IgM, IgG1, IgA, and anti-C. difficile IgG were measured by ELISA. Fecal IgA (A405) on day 14 postinfection (C); CTD-specific serum IgG1, IgG2b, and IgG2c titers measured on day 21 postinfection (D); CTD-specific serum IgM on day 14 (E); and C. difficile specific serum IgG on day 21 postinfection (F). (G) The in vitro neutralization of TcdB by sera from naive, infected, and previously immunized mice (having received a 50-μg dose of Alhydrogel-adsorbed CTD). Statistical significance is indicated as follows: **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

These data indicate that C. difficile infection induces serum IgM and mucosal IgA responses but the degree of class switch to produce TcdB-specific IgG is constrained, and is insufficient to prevent disease recurrence following reinfection.

Limited T follicular helper cell expansion following C. difficile infection.

To analyze the follicular helper response following infection, mice were treated with antibiotic as described then either administered C. difficile spores (3 × 10⁴ CFU) by oral gavage or immunized with CTD. On day 14 after gavage or immunization, lymph nodes and spleen were harvested (Fig. 3A). Cells were then stained with an antibody cocktail to detect CD4⁺CD44^hiPD1^hiCXCR5^hi germinal center (GC-Tfh) and CD4⁺CD44^hiPD1^loCXCR5^hi nongerminal center Tfh cells (non-GC Tfh) (Fig. 3B to D). Following infection, both GC and non-GC lymph node-resident Tfh cell populations were expanded compared to naive uninfected mice, but significantly less so than those observed following CTD immunization (Fig. 3B to D). In the spleen, infection stimulated expansion only of the GC-Tfh populations (Fig. 3D). These results show that infection with C. difficile induced a Tfh response but did so less efficiently than immunization.

FIG 3.

Lack of Tfh cell expansion following infection. (A) Mice were given cefoperazone in water for 5 days followed by sterile water for 2 days. A group of mice were infected with 3 × 10⁴ spores (n = 9) and another group of mice were immunized with 50 μg of Alhydrogel-adsorbed CTD (n = 5). The control (naive mice) were given sterile water by oral gavage and injected with Alhydrogel alone (n = 8). After 14 days, mesenteric lymph nodes, inguinal lymph nodes, and spleen were harvested. Mesenteric and inguinal lymph nodes were pooled. (B) Representative plots of CD4⁺CD44⁺ activated T cells, and PD1^hiCXCR5^hi and PD1^loCXCR5^hi Tfh cells from naive, infected, and immunized mice were identified after gating on lymphocyte populations. (C to D) Data from lymph nodes (C) and from spleen (D). Graphs show absolute numbers of CD4⁺CD44⁺ cells (left), PD1^loCXCR5^hi (middle) and PD1^hiCXCR5^hi (right) Tfh cells. Pooled data from two experiments are shown and each symbol represents an individual mouse. Statistical significance is indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Inefficient Bmem cell expansion following C. difficile infection.

To analyze the memory B (Bmem) cell response following infection, mice were treated with antibiotic as described then either administered C. difficile spores by oral gavage or immunized with CTD. On day 60 after gavage or immunization, spleen and lymph nodes were harvested (Fig. 4A). Cells were then stained with an antibody cocktail to detect CD19⁺IgD^lo/- Bmem cells. Compared to uninfected mice, there was no significant increase in the frequency and number of lymph node-resident Bmem cells (Fig. 4B and C). Following administration of a higher infectious dose, the splenic Bmem cell frequency, but not cell number, was increased by 2-fold compared to the naive control (Fig. 4D). This was in contrast to immunization, which led to a significant 4-fold expansion of the Bmem cell population in both lymph nodes and spleen. These results show that, unlike immunization, infection with C. difficile failed to stimulate a strong Bmem cell response.

FIG 4.

Lack of Bmem cell expansion following infection. (A) Mice were given cefoperazone in water for 5 days followed by 2 days of sterile water. The mouse groups were infected with 2 × 10⁴ spores (Inf.^lo; n = 8) or 3 × 10⁵ spores (Inf.^hi; n = 3), or immunized with 50 μg of CTD adsorbed to alum (n = 7). The control (naive mice/none; n = 6) were given sterile water by oral gavage and injected with alum. After 60 days, mesenteric lymph nodes, inguinal lymph nodes, and spleen were harvested. Mesenteric and inguinal lymph nodes were pooled. (B) CD19⁺ IgD^lo/- Bmem cells from naive, infected, and immunized mice were identified. CD19⁺ cells were selected after gating on lymphocyte populations (left). IgD^lo/- were identified from the CD19⁺ cell population (right). (C and D) Bmem data from lymph nodes (C) and spleen (D). Upper graphs show the Bmem cell frequency, lower graphs show absolute numbers. Pooled data from two experiments are shown and each symbol represents an individual mouse. Statistical significance is indicated as follows: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

C. difficile infection did not stimulate the memory B cell compartment in mice preimmunized with an inactive TcdB fragment.

To assess whether C. difficile infection stimulated the TcdB-specific memory compartment, mice were immunized with the inactive C-terminal domain of TcdB (CTD) then rested for 2 months and infected with C. difficile spores (5 × 10⁴ CFU) (Fig. 5A). At the peak of the disease on day 3, the naive-infected mice lost on average 12% of their weight, while the immunized mice lost less than 3% body weight, confirming immunization-induced protection (Fig. 5B). There were no differences in the fecal C. difficile bacterial counts between the two groups, confirming that antibodies (Ab) are targeting the secreted toxin rather than the bacterium per se. (Fig. 5C). The serum IgM, IgG1, and IgG2b titers were measured on day 0 and day 14 after infection and no increase in the antibody titers was observed (Fig. 5D).This was in contrast to the increase in Ab titers observed after administration of booster vaccines consisting of CTD (21). Therefore, infection did not induce an Ab recall response in the preimmunized mice. Moreover, CTD-specific, bone marrow-resident Ab-secreting plasma cells were observed following immunization but not infection, identifying plasma cells rather than Bmem as the source of CTD-specific IgG (Fig. 5E). Toxin-specific IgA were detected following infection but not after the subcutaneous immunization employed herein (Fig. 5F). Following immunization with CTD, there was a strong correlation between CTD-specific IgG1 titers in the serum and the fecal samples. (Fig. 5G). Thus, protection against C. difficile-associated disease correlated with long-lived plasma cell-secreted, toxin-specific IgG rather than new Abs derived from the stimulation of the Bmem cell compartment. Furthermore, the systemic CTD-specific IgG was transported to the gut.

FIG 5.

Infection fails to stimulate recall responses in immunized mice and protection is associated with plasma cell-derived IgG. (A) Experimental design of CTD immunization followed by C. difficile infection. Mice were immunized with CTD adsorbed to alum (immunized) or PBS-alum (naive) (n = 14 per group). After 2 months, they underwent antibiotic treatment (10 days cefoperazone in drinking water followed by 2 days sterile water) before being administered 5 × 10⁴ spores or sterile water (uninfected control, n = 8) by oral gavage. (B) Relative weight (mean ± SEM) after infection, compared to the initial weight. (C) CFU of C. difficile shed in the feces. (D) Serum anti-CTD antibody titers (IgM, IgG1, and IgG2b) measured by ELISA on day 0 and day 14 after infection. (E) Representative images of ELISPOT plate wells with CTD-specific spots (left) and numbers of CTD-specific spots per million cells (right). (F) Fecal IgA (A405) from the naive-infected mice group before and 14 days after infection (left) and fecal IgA from the uninfected and the immunized-infected mice groups (right). A through D shows pooled data from two experiments and each symbol represents an individual mouse. Data in E was from the second experiment and was also repeated in a further experiment. Statistical significance is indicated as follows: **, P < 0.01; ***, P < 0.001. (G) In a separate experiment, mice were immunized with Alhydrogel-adsorbed CTD and boosted on day 32. Blood and fecal samples were collected on day 32 and day 46. Fecal and serum CTD-specific IgG1 titers were measured by ELISA. Graph shows the correlation between fecal and serum anti-CTD IgG1.

DISCUSSION

The cellular and molecular characteristics of the humoral immune response following C. difficile infection are poorly understood. Here, we show that an initial C. difficile infection induced a primary serum TcdB-specific IgM and a mucosal IgA response. However, infection did not induce a strong serum isotype-switched IgG response, and there was no recall IgA or IgG response following the second infection. Further, the initial C. difficile infection did not protect against a second infection or its associated disease. These results suggest that anti-TcdB IgM and mucosal IgA are not protective against recurrence. This is consistent with the serum IgG response being the best correlate for protection against recurring disease. C. difficile disease patients with multiple episodes tend to have very low or undetectable levels of circulating anti-toxin IgG compared to patients with a single episode (17, 23). While this study focused on T cell-dependent humoral immune responses to C. difficile infection, examination of whether cytotoxic CD8⁺ T cell responses contribute to bacterial killing and clearance is warranted in future investigations, as no studies of which we are aware address this aspect of immunity to C. difficile.

During the humoral immune response, the fate of antigen-stimulated B cells depends on the interaction they establish with CD4⁺ helper T lymphocytes (Th), and the T follicular helper (Tfh) cell subset. Tfh cells provide help to the activated B cells through cognate interactions and secretion of functional cytokines (24). The nongerminal center Tfh population (non-GC Tfh) may provide help for an early B cell response in the follicle and in extra-follicular locations. The germinal center Tfh cell population (GC Tfh) is necessary for the induction of strong and long-lasting humoral immunity. Germinal center Tfh cells can be identified by their expression of the highest levels of CXCR5 and PD-1 in mice (25, 26). In this study, C. difficile infection induced a measurable but restricted expansion of Tfh cells compared to that induced by immunization with the C-terminal domain of the toxin (CTD). The poor Tfh cell expansion was consistent with the inability of the infection to induce isotype-switched antibody responses and stimulate an expansion of the memory B cell compartment.

The longevity of the Bmem cells and the strength of their response upon reactivation are the basis for restimulating protective humoral immunity following an infection or a successful immunization. We previously demonstrated that immunization with CTD induces development of Bmem cells, which encode neutralizing Abs and protect against in vivo toxin challenge (21). Others have shown that immunization with a recombinant TcdB-encapsulated particle vaccine conferred protection against C. difficile disease (22). In this study, we have also observed that infection did not induce an Ab recall response in mice preimmunized with CTD, although they were protected against disease in the presence of ongoing plasma cell responses. This showed that infection did not stimulate the memory B cell compartment, and thus protection correlated with the immunization-induced plasma cell-secreted Ab that were present prior to the infection. This observation has a tremendous implication for C. difficile vaccine design when considering that the disease is more prevalent in elderly and immunocompromised individuals. Aging is associated with inadequate germinal center responses, leading to the prevalence of low-affinity antibodies and poor survival of plasma cells (27, 28). Those patients may not be able to rely on a long-lived plasma cell response. Without the ability to induce Bmem responses by natural exposure through infection, booster vaccinations will likely be needed to maintain high levels of toxin-neutralizing Abs and prevent initial or recurrent disease.

Additionally, although immunization with CTD confers protection against disease, it did not decrease bacterial burden, suggesting that immunization conferred a nonsterilizing immunity against C. difficile. This observation has implications for the control of C. difficile infection in hospitals and the community. Individuals that receive vaccines targeting the toxin may be protected against the disease upon infection but they will still be able to spread the infection.

It is worth noting that in this study, our recurrence model of CDI consisted of an initial infection followed by recovery, incubation, antibiotic retreatment, and reinfection with C. difficile VPI 10463 rather than a spontaneous relapse (in the absence of a second gavage) following recovery. In our experience, we have not observed a spontaneous relapse in mice after recovery from the first infection or following the second cefoperazone treatment. In the clinical setting, both relapse and reinfection occur, and they may be from the same or different strains (29). A study to determine whether the observations reported here can be made using a spontaneous relapse model or using other C. difficile strains, including the hyper-virulent BI/NAP1/027, is warranted.

We observed that toxin-specific Ab responses were dependent on the infectious dose. An IgG response was only observed following oral gavage with a larger number of C. difficile spores. This suggests that the severity of the infection or the accessibility of toxin to the immune system may determine the Ab response. A study reported that cystic fibrosis (CF) patients with previous C. difficile infection appeared to have enhanced and more stable TcdA- and TcdB-specific Ab levels and circulating memory B cells compared to non-CF patients. CF is associated with increased intestinal inflammation and intestinal permeability (23, 30). This may facilitate larger amounts of TcdA and/or TcdB reaching the lymphoid organs and immunizing the host.

Immunization with the C-terminal domain of TcdB (CTD, VPI-10463 TcdB_1651–2366) induces production of high levels of toxin-neutralizing Abs (21). Here, we show that immunization with CTD induced development of long-lived plasma cells which correlated with protection against disease following infection. Also, clinical studies have reported that the titers of toxin-specific Abs and C. difficile cell-surface antigens are higher in asymptomatic carriers compared to patients who develop C. difficile-associated diarrhea (16, 31). Using a hamster model, it was shown that colonization with nontoxigenic strains prevented C. difficile infection-associated disease (32). These studies and our current study suggest that the host can respond to toxin and nontoxin C. difficile antigens, but the response is more efficient when the toxin is either inactive or absent.

In this study, it was observed that the poor humoral immune response was not restricted to TcdB. Following infection, there was also a poor IgG response to whole bacteria. TcdA and TcdB target GTP-binding proteins and disrupt cytoskeletal organization in epithelial cells to cause cell death and tissue damage. The toxins also cause severe inflammation in the colon (33). TcdA was reported to induce apoptosis of T cells, eosinophils, and macrophages in the colonic lamina propria which contribute to suppression of the colonic immune response (34). Furthermore, both TcdA and TcdB alter human T cell migration and chemotaxis by decreasing their motility (35). Thus, C. difficile toxins may impact T cell trafficking at the secondary lymphoid organs, thereby inhibiting subsequent B cell activation and production of antigen-specific antibodies. Various studies have reported similar observations with the anthrax toxin. They report that the toxin suppresses T cell activation, proliferation, and chemotaxis. Anthrax toxin also affects antigen presentation and activation of antigen-specific T cells by targeting dendritic cells (36–38). Suppression of dendritic-cell antigen presentation-dependent T cell priming, and B cell help may constitute another mechanism underlying the poor response following infection. Further research to determine whether C. difficile toxins are affecting the humoral immune response by inhibiting cellular mechanisms in B and T cell responses is warranted.

MATERIALS AND METHODS

Ethics.

This study was carried out in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All animal procedures were approved by the University of Oklahoma Health Sciences Center (OUHSC) Institutional Animal Care and Use Committee (protocols 17-078-CHI and 17-055-I).

Mice.

C57BL/6 mice were purchased from Charles River (Bethesda, MD, USA). Before experiments, all mice were kept under the same specific-pathogen-free conditions. Six- to eight-week-old mice were used for immunization and C. difficile infection.

Antibodies.

Horseradish peroxidase (HRP)-conjugated anti-mouse IgM, IgG1, IgG2b, IgG2c, and IgA were purchased from Southern Biotech (Birmingham, AL). Biotin-conjugated anti-CXCR5 (2G8), fluorescein isothiocyanate (FITC)-conjugated anti-CD4 (GK1.5) monoclonal antibodies (MAbs), and allophycocyanin (APC)-conjugated streptavidin were purchased from BD Biosciences (San Jose, CA). The R-phyco-erythrin (PE)-Cy7-conjugated anti-PD-1 (RPMI-30), PE-conjugated anti-IgD (11-26c.2a), BV421-conjugated anti-CD44 (IM7), and APC-Cy7 anti-CD19 (6D5) MAbs were purchased from Biolegend (San Diego, CA). FcR-blocking MAb 2.4G2 was purchased from BioXCell (Lebanon, NH).

C. difficile spore preparation.

C. difficile VPI 10463 spores were prepared and isolated as follows. A single colony was used to inoculate 2 ml of prereduced Columbia broth (BD) and incubated overnight at 37°C in an anaerobic chamber. The following day the overnight 2 ml was added to 40 ml of prereduced Clospore medium (20) and grown for 5 to 7 days anaerobically at 37°C. Spores were harvested via centrifugation and washed at least 3 times in ice cold sterile water. Spores were stored at 4°C in sterile water. Before infection of mice, C. difficile spores were heated for 20 min at 65°C and CFU were quantified by plating on taurocholate cycloserine cefoxitin fructose agar (TCCFA) (39).

Expression and purification of CTD.

The C-terminal domain (CTD) of the TcdB protein (TcdB_1651–2366 from strain VPI-10463) was expressed in Escherichia coli strain BL21(DE3) (Sigma-Aldrich) and purified by nickel affinity chromatography (GE Life Sciences) as previously described (21).

C. difficile infection.

Mice were housed in sterile cages with sterile food. The animals were treated with cefoperazone sodium salt (Sigma-Aldrich, St. Louis, MO) in distilled drinking water (0.5 g/liter) for five or ten days, followed by 2 days of distilled water (Thermo Fisher Scientific, Waltham, MA). Mice were infected by oral gavage with C. difficile VPI 10463 spores or distilled water to control for the gavage procedure. The number of spores used for oral gavage was determined empirically as those which resulted in a minor/moderate disease (2 × 10⁴ spores, 5 to 10% weight loss), moderate disease (5 × 10⁴ spores, 10 to 15% weight loss), or severe disease (3 × 10⁵ spores, 15 to 20% weight loss and/or death). For recurrent C. difficile infection, following a 2-month resting period, mice were rechallenged with the same dose of bacterial spores after a second round of antibiotic treatment. In this model, recurrent infection required a second treatment with cefoperazone and a second gavage with live spores, although CFU in fecal pellets remained detectable after recovery of the preinfection weight. Following both the initial and recurrent infections, C. difficile-associated pathology was assessed by monitoring daily weights and other clinical signs, such as lethargy, hunched posture, and diarrhea. Animals were euthanized if the weight loss reached 20% or the mice were moribund (39).

Fecal C. difficile enumeration.

C. difficile bacteria shedding was quantified on day 3 postgavage, unless otherwise indicated. Fecal pellets were homogenized with 1× phosphate-buffered saline (PBS), serially diluted, plated on TCCFA, and cultured under anaerobic conditions at 37°C. CFU were counted within 24 and 48 h (39).

Immunization.

Mice were anesthetized with a vaporized 4% isoflurane/96% medical air mix and immunized subcutaneously (s.c.) with 50 μg of the C-terminal domain of TcdB from C. difficile VPI 10463 (CTD) in sterile phosphate-buffered saline (PBS) adsorbed to Alhydrogel alum (Invivogen, San Diego, CA) (21).

ELISA.

To assess TcdB-specific antibodies, Immulon 4 enzyme-linked immunosorbent assay (ELISA) 96-well plates (Dynex) were coated with 10 μg/ml of CTD in phosphate coating buffer (0.1 M Na₂HPO₄ in deionized water, pH 9.0) overnight at 4°C. Wells were blocked with 1% bovine serum albumin (BSA) in PBS-T (PBS 1×, 0.05% Tween) for 2 h at room temperature, and incubated overnight at 4°C with serially diluted mouse sera or fecal supernatant in PBS-T. To confirm absence or low titers of infection-induced toxin-specific antibodies, serum samples from CTD-immunized mice were used as a positive control in each ELISA plate. Wells were washed with PBS-T and then incubated for 1h with horseradish peroxidase (HRP)-conjugated IgG1 (1:10,000), IgG2b (1:5,000), IgG2c (1:5,000), IgM (1:5,000), or IgA (1:2,500). Wells were washed and developed for 5 min at room temperature with 2,2′-azinobis(3-ethylbenzthiazolinesulfonic acid) (ABTS) substrate (KPL, Gaithersburg, MD). A 10% wt/vol SDS solution was used to stop the reaction. Endpoint Ab titers were determined by measuring the optical density at 405 nm (OD₄₀₅). For the bacterium-specific antibodies, plates were coated with oxygen-killed C. difficile bacteria in a carbonate coating buffer (Na₂CO_3, NaHCO₃, NaN) at a 1:4 dilution of the stock culture. Tris-buffered saline (TBS) was used for the washing steps.

In vitro neutralization.

TcdB neutralization by sera was assessed as previously described (21). CHO cells were cultured in 96-well plates overnight at 37°C in a 5% CO₂ incubator. Sera were diluted at 1/500 in culture media and incubated with TcdB (0.66 μg/ml) for 1 h at room temperature. The serum-TcdB mixture was added to the CHO cells and incubated for 24 h. Cell counting kit-8 (CCK-8) reagent (Sigma Chemical Co., St. Louis, MO) was added to the cells (10 μl/well) and incubated for 4 h. The absorbance at 450 nm was measured and percent survival was calculated.

ELISPOT.

Multiscreen high-throughput satellite (HTS) ELISPOT plates (Millipore, Bedford, MA) were incubated with 35% vol/vol ethanol for 30 s and washed twice with PBS. The plate were coated overnight with CTD (10 μg/ml) at 4°C. Plates were washed three times with PBS and blocked with RPMI 1640 medium containing 10% FBS for 2 h at 37°C in a 5% CO₂ incubator. Single-cell suspensions of splenocytes and bone marrow were prepared by mechanical disruption of the spleen and marrow, and red blood cells were removed by hypotonic lysis with Tris-buffered ammonium chloride. The cells were added by serial dilution into wells starting with 2 × 10⁶ cells per well and cultured in the CO₂ incubator at 37°C for 4.5 h. The cells were then removed by lysis and washing with 0.05% Tween 20 in PBS. Plates were incubated overnight at 4°C with 0.125 μg/ml HRP-goat anti-mouse IgG1 Ab (Southern Biotech, Birmingham, AL) and 5% fetal bovine serum (FBS) in PBS. The plates were then washed with PBS-Tween and colorimetric development was performed using 100 μl per well colorimetric solution (47.5 ml 0.0075 N acetic acid, 0.0175 M sodium acetate, one tablet of 3-amino-9-ethyl-carbazole [AEC; Sigma Chemical Co., St. Louis, MO] dissolved in 2.5 ml dimethylformamide and 0.0005% H₂O₂₎. Spot formation was monitored for 10 min and the reaction was stopped by washing the plates with deionized water. Spots were enumerated using an ImmunoSpot analyzer (Cellular Technology Limited, Shaker Heights, OH).

Flow cytometry.

Spleen, inguinal lymph node, and mesenteric lymph node cells were isolated by mechanical disruption and red blood cells were removed by hypotonic lysis with Tris-buffered ammonium chloride. The cells were suspended in RPMI medium with 1% FBS. The cells were incubated with anti-FcR–blocking antibody (2.4G2, 20 μg/ml) for 5 min. This was followed by staining with cocktails of fluorochrome-conjugated MAbs to detect B and T cell populations, including memory B cells and T follicular helper cells. After 30 min of incubation at room temperature, the cells were then washed with ice-cold PBS three times (200 relative centrifugal force [RCF], 5 min, 22°C) and fixed with 2% wt/vol paraformaldehyde in PBS. The cells were analyzed on a Stratedigm S1200Ex flow cytometer (Stratedigm, San Jose, CA). Data were analyzed with FlowJo software (Tree Star, Ashland, OR).

Statistics.

Data were analyzed using GraphPad Prism 8.1 (La Jolla, CA, USA). A two-tailed t test or a Mann-Whitney test, and one-way analysis of variance (ANOVA) with Tukey’s multiple-comparison test was used for statistical analysis between two and multiple experimental groups, respectively. A two-way repeated measure ANOVA with Tukey’s multiple-comparison test was used to determine statistical significance in weight loss over time.