Enhanced immunogenicity of a Clostridioides difficile TcdB vaccine adjuvanted with a synthetic dual-TLR ligand adjuvant

Antigen and adjuvant

Inactivated toxin B (toxoided TcdB) was acquired from TechLab, Inc. (Blacksburg, VA). Alum (Catalog # AL007) was supplied by AAHI for the immunization studies. GLA 3M-052 LS has been described previously and was also supplied by AAHI⁹.

Animals and immunizations

Animal procedures were conducted in accordance with the guidelines approved by the Institutional Animal Care and Use Committee at the University of Virginia (IACUC). Eight-week-old C57BL/6J mice were procured from room EMO3 of the Jackson Laboratory and distributed randomly across the groups. All the treatments (immunizations, infection) were also randomly allocated to ensure equal representation across the groups. Toxoided TcdB (20 μg) was mixed with either Alum (100 µg) or Liposome adjuvant (10 µg GLA, 4 µg 3M-O52) formulations just before immunizations. Volume was adjusted to 100 μL for intramuscular and 50 μL for intranasal immunization using saline (n = 8 per group). Intramuscular injections targeted the thigh region. Intranasal immunizations were performed with 25 μL of inactivated TcdB-adjuvant mix administered per nostril after the animals were anesthetized using an intraperitoneal Ketamine/ Xylazine (60–80/5–10 mg/kg) injection. Animals were observed until fully awake after intranasal immunizations and all efforts were made to minimize animal suffering. A two-week interval was maintained between successive immunizations across all regimens, totaling three immunizations. Immunogenicity was evaluated four weeks after the final immunization. Animals were euthanized using an overdose of anesthesia (twice the anesthetic dose) via intraperitoneal route followed by cervical dislocation.

Fecal toxin detection

Fecal toxin A/B in the cecal contents was detected using the C. DIFFICILE TOX A/B II ELISA kit (TechLab Inc., catalog #T5015). C. difficile glutamate dehydrogenase was measured using the C. DIFF CHEK – 60 kit (TechLab Inc., catalog #TL5025). A standard curve was used to represent bacterial load as arbitrary units.

ELISA

Plasma, cecal contents, and stool samples were stored at −80 °C until they were analyzed for TcdB-specific IgG and IgA antibody titers using an enzyme-linked immunosorbent assay (ELISA). 96-well ELISA half-area plates (Corning Costar, Cat# 3690) were coated with 2 µg/well of toxin B (Techlab) in 50 mM bicarbonate buffer (pH 9.3, Sigma-C3041) overnight at 4 °C. After coating, the plates were washed three times with 1x PBS-Tween 20 (PBST; Thermo Scientific, Cat# 28352) using a BioTek plate washer and then blocked with commercial ELISA blocking buffer (Thermo Scientific, Cat# N502) for 1 h at room temperature. Subsequently, the plates were air-dried and stored until further use.

Prior to mucosal antibody analysis, all stool and cecal content samples were processed as follows: to each milligram of stool or cecal content, 10 µL of PBS containing protease inhibitors (Thermo Scientific, Cat# A32953) was added. The samples were homogenized using a bead beater (Qiagen, Germantown, MD) at 250 rev/min for 10 minutes and then centrifuged at 14,000 × g at 4 °C for 10 min. The resulting supernatants were carefully transferred into 1.5-mL microcentrifuge tubes (Axygen), and phenylmethylsulfonyl fluoride was added to a final concentration of 1 mM before storage at −80 °C.

Thawed plasma samples were used directly without additional processing. Serial dilutions of plasma were prepared with ELISA blocking buffer and added to the toxin B-coated wells of the ELISA plates, followed by incubation for 1 hour at 37 °C. After washing with PBST, horseradish peroxidase (HRP)-conjugated antibodies (Goat Anti-Mouse IgG1 at 1:5000, Goat Anti-Mouse IgG2c at 1:1000, Goat Anti-Mouse IgG at 1:5000, and Goat Anti-Mouse IgA at 1:1000, sourced from SouthernBiotech and catalog #1040-05) were added and incubated for 1 h at 37 °C. The color was developed using 1-Step™ Ultra TMB-ELISA Substrate Solution (Thermo Scientific 34028), and the reaction was stopped with 1:100 diluted sulfuric acid (Sigma-Aldrich). Optical densities (ODs) were measured at 450 nm using an ELISA reader (Synergy, BioTek). Endpoint titers were determined with OD cutoffs of 0.5 for plasma IgG and 0.1 for mucosal IgA.

IgG avidity assay

The avidity index (AI) was measured as previously described²⁵. The avidity of antibodies specific to toxin B was assessed using a urea disruption ELISA. To summarize, ELISA plates were coated with 2 µg/mL of toxin B. Plasma samples were diluted to achieve a value of 1.0 at OD_450nm ensuring that the antibody concentrations were within a linear range. Samples were plated in triplicate on two sets of ELISA plates: one set washed with standard PBST and the other with a dissociation buffer (PBS containing 6 M urea). Both sets were then incubated with an HRP-conjugated secondary antibody, developed, and read. The avidity percentage was determined using the following formula: Avidity Index (AI) = [(average absorbance of urea-treated sample/average absorbance of PBST-treated matched sample) × 100].

In-vitro neutralization assay

The neutralization of TcdB by plasma antibodies was assessed using Chinese hamster ovary (CHO) cell lines. CHO cells were seeded at a density of 3 × 10⁴ cells per well in a 96-well microtiter plate and incubated for 24 h at 37 °C with 5% CO2 in Ham’s F-12K medium (Thermo Fisher #21127022) supplemented with 10% heat-inactivated fetal bovine serum (HI FBS) and 1% penicillin-streptomycin, to reach 70–80% confluence. Toxin B was mixed with the plasma from immunized mice at various concentrations in PBS and incubated for 60 minutes at 37 °C. The CHO cells were washed with PBS, resuspended in 75 µL of media, overlaid with 25 µL of the plasma-toxin mix, and incubated at 37 °C for 24 hours in 5% CO2. The cytotoxic effect of non-neutralized toxins was evaluated microscopically, indicated by the presence of cells exhibiting partial to complete rounding. Complete neutralization of the toxin was characterized by the presence of visually undamaged cells. Toxin neutralization by sera from immunized mice was tested using twofold dilutions (ranging from 1/10 to 1/80) incubated with 2 ng/mL of toxin B.

ELISpot assays

B cell ELISpot assays were performed to quantify the memory B cell (B_MEM) pool, which is essential for generating a secondary immune response upon antigen re-exposure. Ninety-six-well multi-screen filter plates (Millipore, Cat# MSIPS4W10) were pre-treated with 35% ethanol and subsequently rinsed with sterile PBS. The wells were then coated with 2 µg/mL of TcdB to evaluate vaccine-specific responses. As controls, additional plates were also coated with 15 µg/mL of anti-mouse IgG or anti-mouse IgA capture antibodies to measure total IgG and IgA-secreting cells. Bone marrow cells were harvested from the femurs of immunized mice and cultured in RPMI with 10% FBS, and activated with the B-Poly-S Polyclonal B Cell Activator (1 mg/ml R848, 1μg/ml IL-2, Mabtech Catalog # 3661-1) for 48 hours before spot development. Approximately 500,000 activated cells were seeded per well in 200 µL of medium and incubated at 37 °C in a CO2 incubator for 48 h. An AEC substrate solution was added for up to 15 minutes to visualize the spots, and the reaction was terminated by rinsing under running distilled water. The plates were dried in the dark for a minimum of 2 days, and the spots were counted using an ELISpot reader. The reported values were averaged from two dilutions. Reference plasma from toxoid B-immunized mice served as a positive control, while plasma from unimmunized mice served as a negative control.

Bacterial strains and culture

Mice were infected with 10⁵ CFU/mL of Clostridioides difficile VPI 10643 strain spores. C. difficile strains were cultured on Brain Heart Infusion (BHI) agar from glycerol stocks and incubated overnight at 37 °C under anaerobic conditions. Columbia, clospore, and BHI broths were pre-reduced for a minimum of 24 h. VPI spore stocks were prepared according to a previously described protocol²⁶. Briefly, a single colony was inoculated into 15 mL of Columbia broth and incubated overnight at 37 °C. Subsequently, 5 mL of this culture was anaerobically transferred to 45 mL of Clospore broth and incubated for 7 days at 37 °C. The resulting culture was washed at least five times with cold, sterile water and resuspended in 1 mL of sterile water. Spores were stored in 1.5 mL twist-cap tubes at 4 °C (Corning #4309309). Each mouse received 100 μL of the inoculum via oral gavage. The actual inoculum concentration was verified by plating on BHI agar supplemented with 0.032 mg/mL cefoxitin, 1 mg/mL D-cycloserine, and 1% sodium taurocholate (Sigma), followed by anaerobic incubation at 37 °C overnight.

Infection model

Mice were infected with C. difficile as previously described²⁷. Briefly, mice (n = 17 per group) received an antibiotic cocktail in their drinking water consisting of 215 mg/L metronidazole (Hospira), 35 mg/L colistin (Sigma), 45 mg/L vancomycin (Mylan), and 35 mg/L gentamicin (Sigma) for 3 days, followed by regular drinking water. One day prior to infection, clindamycin (Hospira) was administered intraperitoneally at a dose of 0.016 mg/g. Post-infection, mice were monitored twice daily to assess clinical scoring parameters and weight loss. The scoring criteria included weight loss, coat condition, eye condition, activity level, diarrhea, and posture, as described²⁷. Mice were humanely euthanized using an overdose of anesthesia followed by cervical dislocation as described if they reached a clinical score of 14 or experienced more than 25% weight loss. The investigators were not blinded during the studies.

FITC-dextran gut permeability assay

After fasting for four hours, mice were gavaged with fluorescein isothiocyanate (FITC)-dextran solution (Sigma-Aldrich, # 46944-500MG-F) at a dose of 44 mg per 100 g of body weight. Four hours post-gavage, the mice were euthanized, and plasma samples were collected. The FITC-dextran present in the plasma was measured using a spectrophotometer at wavelengths of 485/530 nm.

RNA isolation, sequencing, and analysis

RNAlater-stabilized cecal tissue samples were excised and subsequently immersed in 1 mL of TRIzol per tissue sample. Homogenization of the tissues was carried out using buffer RLT with 1% β-mercaptoethanol (Qiagen). The aqueous phase was isolated and purified utilizing the RNeasy Plus mini kit (Qiagen) following the manufacturer’s instructions. RNA sequencing was performed by Novogene, employing polyA enrichment and sequencing on an Illumina NovaSeq X Plus platform with paired-end 150 bp reads. Quality assessment of unprocessed FASTQ files was conducted using FastQC (v0.12.1)²⁸ and MultiQC (v1.14)²⁹. The reads were pseudo-aligned to the murine genome (v109 from EnsemblDB) using Kallisto (v0.44.0)³⁰. Count tables were imported into R with TxImport (v1.30.0)³¹, and the DESeq2 package (v1.42.0)³² was utilized to filter low-count genes, normalize the data, estimate dispersions, and fit counts using a negative binomial model. Differential gene expression analysis was conducted, ranking genes according to Wald’s statistic from most upregulated to most downregulated post-FMT. This ranked list was used for Gene Set Enrichment Analysis (GSEA) with the fgsea package (v1.20.0)³³ using Gene Ontology databases³⁴.

Statistical analyses

Statistical analyses were performed using Graph Pad Prism software and Microsoft Excel (www.biostathandbook.com/welchanova.xls). Statistically significant differences were determined by one-way ANOVA with appropriate correction for multiple comparisons. No animals were excluded during the analysis.