Clostridium difficile strains were cultured anaerobically on brain-heart infusion (BHI) agar or in BHI broth (Thermo Scientific, Waltham, MA, United States) supplemented with 0.05% L-cysteine. Anaerobic experiments were conducted inside a Don Whitley DG250 anaerobic workstation (Don Whitley Scientific Ltd, West Yorkshire, United Kingdom). E. coli strains were grown at 37°C in LB (Luria Broth, Thermo Scientific, Waltham, MA, United States).

Spores were prepared by plating a 1:100 dilution of overnight culture onto BHIS agar plates and then incubated for 10 days at 37°C under anaerobic conditions. Spores were harvested with ice-cold sterile distilled water and purified with 50% Nicodenz (Axis Shield, Oslo, Norway) as previously described (Sorg and Sonenshein, 2008). Spores were purified to >99% purity as determined by phase contrast microscopy and the number of spores/ml was quantified by visual enumeration using a Neubauer Chamber (Sigma–Aldrich, St. Louis, MO, United States). Spores were stored at -80°C and viability were confirmed by plating onto BHI agar containing the germinant sodium taurocholate prior to use.

Recombinant TcdB antigen was constructed based on part of the TcdB RBD (amino acids 1852-2363). Genomic DNA of C. difficile strain R20291 was used in a PCR reaction with primers tcdB-F and tcdB-R (Table 1). Restriction endonuclease sites (EcoRI at the 3′-end and HindIII at the 5′-end) were designed into the primers. The PCR-amplified product was subsequently cloned into the pET21B expression vector (Merck Millipore, Darmstadt, Germany) and transformed into E. coli BL21 (DE3). The sequence of the recombinant plasmid was confirmed by a commercial sequencing company (Genomics, Taiwan). Expression of recombinant 6xHis-tagged TcdB protein was induced by adding 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) when the cells reached an O.D.₆₀₀ of 0.6, and further incubated at 37°C for 4 h. Cells were centrifuged at 8000 rpm for 30 min at 4°C, resuspended in phosphate buffer saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 2 mM KH2PO4, pH 6.5), and disrupted by sonication on ice. Next, the supernatants were loaded into an Ni-NTA column (GE Healthcare Life Sciences, Pittsburg, PA, United States) and contaminant proteins were eliminated through a washing procedure by using 50 mM imidazole in wash buffer (20 mM NaH2PO4, 500 mM NaCl, 50 mM imidazole, pH 6.5). Proteins were eluted with 500 mM imidazole in wash buffer. After SDS-PAGE analysis, proteins were concentrated by Amicon^® Ultra 30-kDa cut-off unit (Merck Darmstadt, Germany). The 6xHis-tag of rTcdB was removed by the Thrombin CleanCleave^TM kit (GE Healthcare Life Sciences, Pittsburg, PA, United States) according to the manufacturer’s instructions. Tag-less recombinant proteins were verified by Western blots. All recombinant proteins were subsequently stored at 4°C for future use.

The antigen loaded chitosan/γ-PGA NPs were prepared by flush mixing of an aqueous γ-PGA (1 ml, unfractionated γ-PGA) into an aqueous chitosan (pH 6.0, 5 ml) at various weight ratios under magnetic stirring at room temperature (Lin and Chen, 2017). The obtained nanoparticle solution was then dialyzed (MWCO: 10000, Spectrum Labs, Rancho Dominguez, CA, United States) against deionized water for 3 days. The stock solutions of chitosan and γ–PGA were prepared by mixing γ-PGA with chitosan solution. After vacuum drying, nanoparticles were homogenized with phosphate-buffered saline (PBS, pH 7.0). The particle size, polydispersity index (PDI) and zeta potential of the prepared nanoparticles were measured using a Zetasizer (3000HS, Malvern Instruments, Malvern, United Kingdom). For the preparation and characterization of polymer-based nanoparticle encapsulated rTcdB, rTcdB protein was premixed with aqueous γ-PGA and added into aqueous chitosan under magnetic stirring in 10 mM phosphate buffer (pH 6.0) at room temperature. The samples were concentrated to 1/10 of volume and stored at 4°C. The particle size and zeta potential of the prepared nanoparticles were measured using a quasi-elastic light scattering (QELS) analyzer (3000HS, Malvern Instruments, Malvern, United Kingdom).

Specific-pathogen-free 6-weeks old male C57BL/6 mice were housed in the Laboratory Animal Center of National Cheng Kung University. All mice were maintained and handled according to the guidelines of the Institutional Animal Care and Use Committee (IACUC) of National Cheng Kung University (NCKU). All animal studies were performed following a protocol approved by the IACUC of NCKU (approval NCKU-IACUC-102-149). Mice were vaccinated intraperitoneally every other week for a total of three injections. For optimization of nanoparticle sizes, eight groups of three mice each were injected intraperitoneally with the following inoculant: (1) sterile PBS control; (2) NPs only (200 nm); (3) purified rTcdB; (4) NP₂₀₀; (5) NP₃₅₀; (6) NP₅₀₀; (7) NP₇₅₀; (8) 100 μg of rTcdB in PBS mixed with aluminum hydroxide [Al(OH)3; 1:1 by volume] (Thermo). For C. difficile challenge experiments, mice in groups of 5 were vaccinated with the following: (1) sterile PBS control; (2) empty NPs only; and (3) NP_750. A total of 20 μg of rTcdB were administered to each mouse per injection. All injections were performed intraperitoneally. Serum samples were collected from each animal via submandibular collection 1 week after each vaccination and stored at -80°C prior to use.

TcdB-specific IgA and IgA were determined by enzyme-linked immunosorbent assay (ELISA). Purified rTcdB proteins were coated onto ELISA plates (Nunc, Roskilde, Denmark) using coating buffer (20 mM NaCO3, 35 mM NaHCO3, pH 9.6) at 4°C overnight. The wells were then blocked with 10% skim milk in PBS (pH 7.4) at room temperature. To detect the antigen-specific antibody, mouse serum samples were diluted in PBS and incubated for 1 h at 37°C. Plates were washed 3 times with 0.05% Tween 20 in PBS (PBS/T) and then incubated with HRP-conjugated anti-mouse IgG or IgA for 1 h at 37°C. Colors were developed by tetramethylbenzidine substrate (TMB) and the reaction stopped by adding stop solution (2M H₂SO₄). Absorbance was measured at 450 nm using iMark^TM Microplate Reader (Bio-Rad, Hercules, CA, United States).

Vero cells were cultured in Dulbecco’s modified Eagles medium (DMEM) containing Penicillin-Streptomycin and 10% fetal bovine serum (FBS) in a humidified incubator containing 5% CO₂ at 37°C. Cells were detached using 1000 U/ml trypsin and 0.5 mM EDTA and counted by LUNA-FL^TM Dual Fluorescence Cell Counter (Logos Biosystems, Anyang, South Korea). Then cells were seeded into a 96 well tissue culture test plate (SPL life sciences, Pocheon, South Korea) at a density of 5 × 10⁴ cells per well and incubated at 37°C containing 5% CO₂ overnight.

Toxin neutralizing properties of antiserum were determined using Vero cells and C. difficile purified toxin B (List Biological Labs, Campbell, CA, United States). Serum samples obtained from all immunized mice were serially diluted in DMEM and incubated for 1 h at 37°C with toxin B (final concentration 0.5 ng/ml). The toxin-serum mixtures were then added to 96 well plates containing Vero cells and the plates were incubated at 37°C containing 5% CO2 for 18 h. After incubation, the culture supernatant was collected and incubated with the substrate mixture from the Cytotoxicity detection kit (Roche, Basel, Switzerland) for 30 min in dark. The lactate dehydrogenase (LDH) activity was then determined at 492 nm using iMark^TM Microplate Reader (Bio-Rad, Hercules, CA, United States), and cytotoxicity was calculated from the following equation. Statistical analyses were performed using GraphPad Prism version 6.0.

Background control = medium only + reagent only

Low control = spontaneous LDH release

High control = maximum LDH release

After pre-vaccination, mice were fed drinking water containing an antibiotic mixture, which included 0.4 mg/ml vancomycin, 0.215 mg/ml metronidazole, 0.4 mg/ml kanamycin, 0.035 mg/mL gentamycin, and 850 U/ml colistin, for a total of 5 days before challenge. All antibiotics were purchased from Sigma-Aldrich. Vancomycin and metronidazole were omitted to avoid disrupting C. difficile colonization on the day before challenge. 1 × 10⁶ CFU of C. difficile R20291 spores were administered orogastrically and 4 mg/kg of clindamycin was injected intraperitoneally. 2 days post infection, all animals were sacrificed. Serum and organs were extracted for downstream analysis. Serum samples were stored at -80°C prior to use. For survival rate analysis, following oral challenge, mice were monitored daily for a total of 5 days for diarrhea and other signs of disease, and moribund animals were euthanized.

The concentrations of gastrointestinal lavage (GAL) cytokines and chemokines were measured by DuoSet^® ELISA Development system (R&D Systems, Minneapolis, MN, United States) according to the manufacturer’s instruction. Absorbance was measured at 450 nm using iMark^TM Microplate Reader (Bio-Rad, Hercules, CA, United States). Samples were measured in triplicate and statistical analyses were performed using GraphPad Prism version 6.0.

Fecal samples (premixed in PBS) were collected from animals and immediately subjected to heat treatment at 65°C for 20 min and then serially diluted onto BHI agar containing 0.1% taurocholate. Plates were incubated anaerobically at 37°C for 48 h and colonies were counted for CFU determination.

The colon samples were extracted with RNeasy^® Plus Mini kit (QIAGEN, Venlo, Netherlands). RNA yield and quality were checked by NanoDrop Spectrophotometer (Thermo Scientific, Waltham, MA, United States). Reverse transcription was performed with SuperScript^TM II Reverse Transcriptase (Invitrogen, Waltham, MA, United States). The expression level of proinflammatory cytokines and chemokines were measured by quantitative reverse transcription-polymerase chain reaction using RealQ Plus 2X Master Mix Green (Ampliqon, Denmark) with β-actin as the reference gene in each reaction (Table 1). The data were analyzed by using the ΔΔCt method and expressed as the fold change in transcript level under the test condition compared to the average for the indicated control and then normalized to the reference gene β-actin. Statistical analyses were done by using GraphPad Prism 6.0.

All data were expressed as the mean ± standard deviations and statistical comparisons among the groups were analyzed by Student’s t-test. Multiple intergroup comparisons were assessed by one-way ANOVA, followed by post hoc Tukey’s test with GraphPad Prism version 6.0. Statistical significance was set at P < 0.05.

As the immunogenicity of TcdB has been well studied, in order to evaluate the potential of using nanoparticles as C. difficile vaccine adjuvants, our vaccine design started with the expression of recombinant C. difficile toxin B RBD. The RBD of TcdB is non-toxic, have been used in other vaccine studies in the past, and is relatively easy to express and purify when compared to the full length toxin (Wang et al., 2012; Baliban et al., 2014; Spencer et al., 2014; Huang et al., 2015). The fragment comprised only the C-terminal domain region (rTcdB, amino acids 1852–2363) to avoid cytotoxicity (Figure 1A). PCR products were cloned into pET-21b and then transformed into E. coli. rTcdB was expressed in E. coli and purified from soluble extracts by Ni2+-NTA affinity chromatography (Figure 1B). Proteins were further purified to homogeneity by size-exclusion chromatography. The identity of the recombinant proteins were confirmed by Western blot analysis using Anti-His and Anti-TcdB antibody (Figure 1C). The overall purification yield from E. coli extracts was 4.5–5.0 mg/L of E. coli culture. His-tag of purified rTcdB was removed by thrombin cleavage.

Nanoparticles were produced using an electro-kinetic approach involving the ionic attraction of chitosan (containing positively charged –NH3 group) and γ-PGA (containing negatively charged –C00- group) which are both FDA-approved biodegradable polymers. Specific procedures for the encapsulation of rTcdB are described in the Section “Materials and Methods.” As the size of nanoparticles might influence the immunogenicity of the vaccine, we generated four different rTcdB-encapsulated nanoparticles (NP-rTcdB) with the designation NP₂₀₀, NP₃₅₀, NP₅₀₀, and NP₇₅₀ with each type of particles having a mean particle diameter of 200, 350, 500, and 750 nm, respectively. All NPs, regardless of size, contained same amount of antigen.

To evaluate the immunogenicity of rTcdB-encapsulated nanoparticles, mice were vaccinated three times intraperitoneally with prepared nanoparticle vaccines. Sera were collected from mice before primary immunization and 1 week after each booster. No significant rTcdB specific IgGs were detected in mice injected with empty nanoparticles as expected, while IgGs were not detected in mice injected with purified recombinant rTcdB until 1 week after the third immunization (Figure 2). In contrast, significant IgGs were detected as earlier as 1 week after the second immunization for all NP-rTcdB immunized mice compared to the control groups (Figure 2). Immunization with Alum-mixed rTcdB induced detectable IgG responses albeit at a significantly lower level compared to NP-rTcdB injected groups. In terms of differences in nanoparticle sizes, while mice vaccinated with NP₂₀₀ appeared to have higher IgGs after the first injection, NP₅₀₀ and NP₇₅₀ elicited a higher level of IgG with subsequent boosters. The highest IgG level detected for all sizes of nanoparticles were after the third immunization.

On the other hand, rTcdB-specific IgAs were observed only after the third immunization (Figure 3). All four sizes of nanoparticle vaccines induced a detectable level of IgAs with a general trend toward larger NPs inducing higher antibody responses. Only baseline titers were observed in empty NPs immunized control, purified rTcdB immunized control, and the PBS control groups. Alum-mixed rTcdB induced little to no IgAs. Taken together, these results showed that vaccination with NP-rTcdB was much more effective in inducing antigen-specific IgG and IgA than the traditional aluminum hydroxide adjuvant.

To determine the functional capacity of anti-rTcdB antibodies induced by our nanoparticle vaccine to neutralize native toxin B, neutralizing efficacy was assessed against purified toxin B (Figure 4). After incubation of Vero cells with serial dilutions of serum, the cells were assayed for the release of lactate dehydrogenase (LDH). Antibodies raised against nanoparticle vaccines were found to have neutralizing activity in a dose-dependent manner and no significant differences were observed between all four NP sizes (Figure 4). Control animals that received adjuvant alone or rTcdB alone did not produce sufficient antibodies to neutralize the cytotoxic effect of toxin B (data not shown).

To further evaluate the potency of various sizes of NP-rTcdB as vaccine candidates, serum antibody responses of immunized mice were measured for up to 6 months after the last immunization. As shown in Figures 5A,B, both IgG and IgA levels detected in all NP-rTcdB vaccinated mice peaked after the third immunization and were still detectable 6 months after the last immunization (Figure 5). Antibody titer for NP₇₅₀ vaccinated mice at 6-month after immunization were either not significantly different (IgGs) or higher (IgAs) than those that were immunized with alum mixed rTcdB (P < 0.05). In terms of nanoparticle size, IgG titer of NP₇₅₀ immunized mice at 6-month post-immunization were significantly higher than mice immunized with smaller size nanoparticle vaccines (P < 0.05).

In order to determine the protective efficacy of immunization with nanoparticle vaccines upon challenge with C. difficile spores. The high-toxin producing C. difficile strain R20291 was used for infection. We immunized C57BL/6 mice by i.p. injection with NP₇₅₀ three times. Control groups include mice immunized with PBS alone, and empty nanoparticle alone. After the third immunization, the normal gut microbiota of immunized mice was perturbed with antibiotic cocktails dissolved in the drinking water for 5 days followed by a single i.p. injection of clindamycin prior to challenge with purified R20291 C. difficile spores (5 × 10⁵ spores). Infected mice in the control group receiving either PBS or empty nanoparticle (NP) showed signs of CDI, including loss of body and cecal weight and a decrease in colon length (Figures 6A–C). Gross view of colon and cecum indicated acute stage of colitis (Figure 6D). In contrast, NP₇₅₀ immunized animal displayed no body weight loss and cecum weight and colon length were similar to that of the healthy control (Vehicle). Gross view of colon and cecum from NP₇₅₀ vaccinated mice also were similar in appearance to the healthy control with visibly formed fecal samples being retained in the colon (Figure 6D). Serum samples obtained before and post-infection were obtained and evaluated by ELISA to assess the development of specific antibody response. Serum from NP₇₅₀ immunized group induced significant serum IgG and IgA responses post infection, while only baseline titers were detected from the PBS and NP control group (Figures 7A,B). In addition to systemic antibody response, the GAL were collected from each mouse to test for intestinal antigen-specific antibody responses. As shown in Figure 7C, significantly higher IgG responses were detected from GAL samples of NP₇₅₀ immunized mice compared to control groups. As expected, no detectable IgAs were observed in samples from NP100 group since the vaccine was delivered intraperitoneally. To assess whether immunization with NP-rTcdBs would also influence C. difficile replication, fecal samples were collected from animals 2 days post infection. As shown in Supplementary Figure 1, no significant differences were observed between all groups.

Consistent with the results observed above, the expression of proinflammatory cytokines interleukin 6 (IL-6), interleukin 1β (IL-1β), tumor necrosis factor α (TNF-α), interferon γ (IFN-γ), macrophage inflammatory protein 2 (MIP-2), monocyte chemoattractant protein 1 (MCP-1), and interleukin 17A (IL-17A), was significantly increased in colons of the control group mice vaccinated with PBS or empty nanoparticle compared to healthy control (Figure 8A). In contrast, colons from the NP₇₅₀ immunized group contained significantly lower level of inflammatory cytokines and chemokines level with the exception of IL-17A (Figure 8A). In addition, proinflammatory cytokines and chemokines within GAL were measured by ELISA (Figure 8B). The level of IL-6, IL-1β, TNF-α, and MCP-1 was increased in GAL of mice immunized with PBS or empty nanoparticles compared to the healthy control. However, mice immunized with NP₇₅₀ displayed significant decrease level of cytokines and chemokines tested (Figure 8B).

Finally, the survival rate of i.p. immunized animal post infection was recorded for up to 6 days post-infection (Figure 9). Eighty of mice in control groups vaccinated with PBS or empty nanoparticle were susceptible to C. difficile infection and died on day 3 post infection. In contrast, all mice vaccinated with NP₇₅₀ were completely protected against the lethal C. difficile spore challenge (P < 0.001). In short, these results suggest that immunization with nanoparticle vaccines can protect mice from severe CDI.