Neutralization of Clostridium difficile Toxin B Mediated by Engineered Lactobacilli That Produce Single-Domain Antibodies

Abstract

cClostridium difficile is the primary cause of nosocomial antibiotic-associated diarrhea in the Western world. The major virulence factors of C. difficile are two exotoxins, toxin A (TcdA) and toxin B (TcdB), which cause extensive colonic inflammation and epithelial damage manifested by episodes of diarrhea. In this study, we explored the basis for an oral antitoxin strategy based on engineered Lactobacillus strains expressing TcdB-neutralizing antibody fragments in the gastrointestinal tract. Variable domain of heavy chain-only (VHH) antibodies were raised in llamas by immunization with the complete TcdB toxin. Four unique VHH fragments neutralizing TcdB in vitro were isolated. When these VHH fragments were expressed in either secreted or cell wall-anchored form in Lactobacillus paracasei BL23, they were able to neutralize the cytotoxic effect of the toxin in an in vitro cell-based assay. Prophylactic treatment with a combination of two strains of engineered L. paracasei BL23 expressing two neutralizing anti-TcdB VHH fragments (VHH-B2 and VHH-G3) delayed killing in a hamster protection model where the animals were challenged with spores of a TcdA ؊ TcdB ؉ strain of C. difficile (P < 0.05). Half of the hamsters in the treated group survived until the termination of the experiment at day 5 and showed either no damage or limited inflammation of the colonic mucosa despite having been colonized with C. difficile for up to 4 days. The protective effect in the hamster model suggests that the strategy could be explored as a supplement to existing therapies for patients.

Effect of Bifidobacterium upon Clostridium difficile Growth and Toxicity When Co-cultured in Different Prebiotic Substrates

“…There is a great scientific interest on the development of interventions for preventing or treating CDI, including vaccines (Senoh et al, 2015 ), antimicrobials (Gebhart et al, 2015 ; Vickers et al, 2015 ), anti-toxin antibodies (Yang et al, 2015 ), or genetically engineered bacteria producing them (Andersen et al, 2015 ), among others. Fecal transplants have demonstrated a high efficacy to treat recurrent CDI (Lee et al, 2016 ), underlining the importance of the gut microbiota in this disease.…”

“…Similarly, the ability to produce antimicrobials inhibiting the growth of C. difficile in vitro has been repeatedly reported (Lee et al, 2013 ; Schoster et al, 2013 ). However, other potential targets of probiotics and prebiotics on CDI, such as their impact on toxin production by the pathogen, and/or toxin activity, have been explored to a lesser extent and have not attracted attention until recently (Kondepudi et al, 2014 ; Yun et al, 2014 ; Andersen et al, 2015 ). Ambalam et al ( 2015 ) recently reported the ability of cell-free supernatants from some Lactobacillus strains, and a probiotic mix, to inhibit the growth of C. difficile strains in variable way depending on the carbon source used.…”

Expression of Human Immunodeficiency Virus Type 1 Neutralizing Antibody Fragments Using Human VaginalLactobacillus

“… 40 , 41 Most recently, Andersen et al described the use of single dAbs to neutralize Clostridium difficile toxin B. 42 …”

An update on antibody-based immunotherapies for <em>Clostridium difficile</em> infection

“…A mixture of B2, G3, and D8 dosed orally was not protective, possibly due to its degradation and/or transitory nature in the GI tract. Interestingly, Andersen et al 81 also immunized llamas with whole TcdA, but failed to isolate any neutralizing antibodies, which is exactly the opposite of Hussack et al 76 who isolated TcdA neutralizers but not TcdB neutralizers.…”

“…More recently, Andersen et al 81 reported the isolation of four llama V H Hs against the TcdB RBD after immunization with whole TcdB toxin. The four V H Hs (B2, E2, G3, and D8) bind to three unique TcdB RBD epitopes (B2, E2/G3, and D8).…”