Protein expression and purification
Point mutations were made in WT VPI10463 TcdA and WT TcdB2 as described previously18,19,20. Primer information can be found in Supplementary Table 2. TcdA, TcdB and CspC were recombinantly expressed and purified as previously described18,41. Plasmid information for all antigens is provided in Supplementary Table 2. Plasmids encoding NTAs were codon-optimized versions of the candidate NTAs from the C. difficile R20291 background which were synthesized at Genscript into a pET47b(+) vector and included a C-terminal 6×-histidine tag for protein purification. C40 peptidase 2, FlgG, FlgE, FlgK, CspC and polysaccharide deacetylase were transformed into E. coli BL21 (DE3) STARs (Supplementary Table 2). To express each NTA, 12 l of lysogeny broth medium supplemented with 50 mg l−1 kanamycin were inoculated with an overnight culture to an optical density at 600 nm (OD 600 ) of 0.1. Cells were grown at 37 °C and 220 rpm. Expression was induced with 1 mM Isopropyl-β-D-1-thiogalactopyranoside (IPTG) once cells reached an OD 600 of 0.4–0.6. After 4 h, cells were centrifuged and the pellets were resuspended in 20 mM Tris (pH 8.0), 500 mM NaCl, 2% lysis mix (phenylmethylsulfonyl fluoride (0.1 mM), leupeptin (2 mg ml−1), pepstatin (2 mg ml−1), 2% DNase (2 mg ml−1) and 2% lysozyme (10 mg ml−1)). Bacterial suspensions were lysed three times using an EmulsiFlex C3 microfluidizer (Avestin) at 15,000 lb in−2. Lysates were centrifuged at 40,000g for 45 min at 4 °C. NTAs were initially isolated from supernatant using a Ni2+-affinity column (HisTrap FastFlow Crude; GE Healthcare). NTA eluents were further purified using an S-200 size-exchange column (GE Healthcare) in 20 mM HEPES (pH 6.9) with 50 mM NaCl on the ÄKTA Pure fast protein liquid chromatography system (Cytiva). All of the samples were treated using an endotoxin removal kit (Thermo Fisher Scientific) and sterile filtered through a 0.22-µm filter before being aliquoted for immunization studies and stored at −80 °C.
The ternary complex of FlgGEK was produced by co-purifying FlgG, FlgE and FlgK. In brief, supernatants of the three proteins were mixed in a 1:1:1 ratio after lysis and centrifugation, before purification by Ni2+-affinity and S-200 size-exchange chromatography and subsequent endotoxin removal, filtration and freezing, as described above.
dmLT was provided by PATH (Acknowledgements and Data Availability) in 1× PBS supplemented with 0.05% Tween-20 (0.6 mg ml−1).
Animals and study design
Male and female C57BL/6J mice (Jackson Laboratories, 000664) were used in all studies. Mice were assimilated to the new facility 1 week before immunization. Mice were maintained at Vanderbilt University Medical Center under 12 h–12 h light–dark cycles under an ambient temperature of 23 °C (±3 °C) and 50% humidity (±20%), with ad libitum access to chow pellets and water. These studies were approved by the Institutional Animal Care and Use Committee at Vanderbilt University Medical Center and were performed using protocol M2200087-00. All animals were randomly assigned to experimental groups. Researchers were not blinded to groups throughout the animal experiments to properly monitor individual weight loss and morbidity during C. difficile infection according to institutional euthanasia guidelines.
For NTA-cocktail and individual NTA studies, 6-week-old male and female mice were immunized three times over the course of 28 days, with 14 days spanning between injections. Intraperitoneally injected mice received 5 µg of dmLT adjuvant with 5 µg of FlgGEK, C40 peptidase 2, CspC and/or polysaccharide deacetylase in sterile PBS in a total volume of 100 µl per injection. r.i.-treated mice received 25 µg of dmLT adjuvant with 5 µg of FlgGEK, C40 peptidase 2, CspC and/or polysaccharide deacetylase suspended in 200 µl of PBS. Mice were rectally instilled after faecal collection to empty the colon. r.i. occurred under anaesthesia using a sterilized metal ball-end gavage needle that was inserted into the rectum. The vaccine formula was pulsed into the colon, and the rectum was manually squeezed shut for 15 s after administration to prevent leakage, as described previously42. All vaccinations were administered within 2 h of antigen thaw. Faecal and serum samples were obtained before each vaccination and challenge. Mice were challenged 14 days after the final boost, as previously described43,44. Antibiotic treatment was administered by providing 0.5 mg ml−1 cefoperazone in the drinking water ad libitum for 5 days, followed by a 2-day recovery period where normal water was provided before CDI through oral gavage. Two different C. difficile strains were used where indicated: WT R20291 and R20291 ∆A∆B45, both administered at a dose of 1 × 103 spores per mouse. Mice were monitored daily for survival and weight loss. Animal cages were kept the same (left unchanged) for the entirety of the infection. Mice were humanely euthanized when weight loss exceeded 20% of their original body weight. Faecal samples were obtained daily during challenge for CFU enumeration.
For studies to analyse the toxicity of various TcdA and TcdB point mutants, 6-week-old mice were intraperitoneally injected as described above with 5 µg of dmLT and either 1 or 5 µg of the following toxins/toxin combinations: TcdA GTX ; TcdB2 GTX,L1106K ; TcdA GTX + TcdB2 GTX,L1106K ; TcdB2 GTX,L1106K,D1812G ; or TcdB2 GTX,L1106K + TcdB2 GTX,L1106K,D1812G . For combination vaccines with two antigens, 1 or 5 µg of each antigen was injected for a combined total antigenic amount of 2 or 10 µg. The mice were monitored for signs of morbidity and mortality for 7 days after injection.
For studies to optimize the amount of dmLT to include in vaccination, 6-week-old mice were either i.p. injected or rectally instilled with varying amounts of dmLT. Mice were intraperitoneally injected twice over 2 weeks with 0, 0.5, 1, 2.5 or 5 µg of dmLT alongside 5 µg of TcdA GTX or rectally instilled twice over 2 weeks with 0, 10, 15, 20 and 25 µg dmLT with 5 µg of TcdA GTX . Sera and faeces were collected at days 0 (first dose), 14 (second dose), 28, 58 and 88 for enzyme-linked immunosorbent assays (ELISA) analysis of vaccine-induced humoral immune responses.
For studies comparing vaccination of dmLT adjuvant alone to toxin mutants with dmLT and toxin mutants with the NTA cocktail and dmLT, 2 cohorts of 6- and 12-week-old mice were immunized twice with vaccinations spaced 14 days apart. Intraperitoneally injected mice received 1 µg dmLT; 1 µg dmLT with 5 µg each of TcdA GTX , TcdB2 GTX,L1106K , TcdB2 GTX,L1106K,D1812G ; or 1 µg dmLT alongside 5 µg each of TcdA GTX , TcdB2 GTX,L1106K , TcdB2 GTX,L1106K,D1812G , CspC, C40 peptidase 2 and FlgGEK. r.i.-treated mice received: 15 µg dmLT; 15 µg dmLT with 5 µg each of TcdA GTX , TcdB2 GTX,L1106K and TcdB2 GTX,L1106K,D1812G ; or 15 µg dmLT alongside 5 µg each of TcdA GTX , TcdB2 GTX,L1106K , TcdB2 GTX,L1106K,D1812G , CspC, C40 peptidase 2 and FlgGEK. Serum and faecal samples were collected as described above. Mice were challenged as stated above with WT C. difficile R20291. Mice were either euthanized for histopathological analysis and flow cytometry analysis on day 3 after infection or were monitored for 10 (6-week-old mice) or 15 (12-week-old mice) days after infection as described above.
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