Animals that became moribund were euthanized. CDI Challenge C57BL/6 mice were orally administered 105 spores from the UK1 (BI/NAP1/027) strain after receiving antibiotic treatment, as previously described [12]. a rationale for the Lofendazam development of multivalent VHHs that target both toxins and are broadly neutralizing for treating severe CDI. Keywords: is the most common cause of nosocomial antibiotic-associated diarrhea and is the etiologic agent of pseudomembranous colitis [1]. infection (CDI) is primarily caused by 2 large exotoxins, TcdA and TcdB. It is estimated that >500 000 cases of CDI occur annually in the United States, with the yearly mortality rate ranging Lofendazam from 3% to 17%, depending on the strains. The incidence of CDI-associated mortality among patients is increasing rapidly because of the emergence of hypervirulent and antibiotic-resistant strains [2], and systemic complications are the major cause of death in Rosetta-gami 2 (DE3)pLacI cells (Novagen). Generation of the VHH Heterotetramer AH3/E3/E3/AA6ABA (ABA) VHHs having the most potent neutralizing activity and the highest binding affinity to distinct, nonoverlapping epitopes targeting each toxins were chosen for inclusion within a multimeric, multivalent antibody. For TcdA, VHHs AH3 and AA6 were selected for their potent neutralizing activity. For TcdB, 2 copies of the E3 VHH were selected, because E3 is a potent TcdB-neutralizing VHH targeting the well-conserved glucosyltransferase domain with particularly high affinity. To generate ABA, the coding sequences of individual VHHs were amplified and fused under the cytomegalovirus promoter of a pSEC91 plasmid. DNA encoding a flexible linker sequence ([G3S]4) was installed between each of the 4 VHH-coding sequences. Both an immunoglobulin -chain leader (for protein secretion) and a His(6)-tag (for purification) were added to the N-terminus of the tetramer. The insert was sequenced to ensure that the proper sequence was obtained, and the final construct was transfected into HEK293 cells. ABA purified from cell culture supernatants of ABA-secreting stable 293 clones displayed a single dominant band during sodium dodecyl sulfateCpolyacrylamide gel electrophoresis (SDS-PAGE) after GelBlue (Pierce) staining. The purified ABA showed no toxicity to mice after intravenously injection of doses up to 10 mg/kg. ELISAs Microplates were coated with 0.5 g/mL recombinant TcdA or 0.5 g/mL TcdB [22] overnight at 4C and incubated with 50 L bacterial supernatants or purified VHHs. After washes, horseradish peroxidase (HRP)Cconjugated anti-E-tag antibody was added to plates, followed by analysis by a standard ELISA. For competition ELISA, serial dilutions of VHHs were mixed with serial dilutions Mouse monoclonal to WDR5 of ABA before adding to plates coated with TcdA or TcdB. After incubation and washes, the binding of monomer VHHs was measured by adding a biotinylated anti-thioredoxin VHH generated by us, followed by HRP-conjugated streptavidin. To determine whether ABA is capable of binding the 2 2 toxins simultaneously, plates were coated with TcdA or TcdB before adding serial dilutions of ABA. After washes, serial dilutions of TcdB or TcdA, respectively, were added to the wells. After extensive washing, mouse monoclonal antibodies against TcdB or TcdA (List Biological Laboratories, Campbell, CA), respectively, were added to the wells before the addition of HRP-conjugated antimouse antibodies for detection. In Vitro Neutralizing Assays Mouse colonic epithelial CT26 cells and African green monkey kidney Vero cells (ATCC, Manassas, VA) were cultured in Dulbecco’s modified Eagle’s medium (Invitrogen, Carlsbad, CA) with 10% fetal bovine serum, 1 Lofendazam mM sodium pyruvate, 2 mM L-glutamine, 100 U/mL penicillin G, and 40 g/mL streptomycin sulfate. Subconfluent CT26 or Vero cells (2.0 104 cells/well) were seeded in 96-well plates for 24 hours before the addition of toxin and VHH agents. Serially diluted VHHs and toxins were premixed using toxin at a concentration of 0. 2 ng/mL for TcdB or 10 ng/mL for TcdA and then added to each well. In some experiments, 10-L bacterial supernatants from 11 strains were mixed with ABA (10 g/mL) before addition to the Vero cell monolayer. This panel of strains was kindly provided by Dr Trevor Lawley and represent an assortment of genetically and geographically diverse clinical isolates [27, 28], Bacterial supernatant added without ABA acted as a control. After incubation for 24 hours, cells were observed under a phase-contrast microscope, and the percentage of cells that were rounded was assessed. Systemic Challenge Six-week-old female CD1 mice (Charles River Labs) were maintained in a pathogen-free animal biosafety level 2 facility. All mice used in the experiments were housed in groups of 5 per cage under the same conditions. Food, water, bedding, and cages were autoclaved. Mice (5 per group) were administered VHH monomers or ABA by intraperitoneal injection 1 hour before intraperitoneal challenge of a mixture of TcdA and TcdB (25 ng/mouse of.
Categories