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999 In Vitro and In Vivo Characterization of Neutralizing Monoclonal Antibodies Against Clostridium difficile Toxins a and B
999 In Vitro and In Vivo Characterization of Neutralizing Monoclonal Antibodies Against Clostridium difficile Toxins a and B Dominiek Staelens, Marlies Van de Wouwer, Els Brouwers, Silvia Caluwaerts, Borden Lacy, Pieter Rottiers, Peter Vanhoenacker, Nick Geukens, Paul J. Declerck, Severine Vermeire, Paul J. Rutgeerts, Gert A. Van Assche Introduction - Clostridium difficile associated disease (CDAD) is increasing in incidence and severity with significant morbidity and mortality. Antibiotic treatment is not invariably successful with a relapse rate around 30%. Since only toxin A and/or B (TcdA, TcdB) producing strains are proven to be pathogenic, we aim to develop a novel treatment modality based on targeted mucosal delivery of anti- Clostridium difficile toxin-antigen binding fragments. Methods - SJL/J mice were immunized with Clostridium difficile toxoid by a standard protocol for hybridoma generation. Monoclonal antibody (MA) production was evaluated on coated TcdA and TcdB. Antigen binding fragments (Fabs) were generated using a standard protocol based on papain treatment. Selected MAs were evaluated for their in vitro neutralization capacities using human fibroblasts (IMR-90) and TcdA or TcdB at 570 pM or 56 pM, respectively. Affinities were determined on a Biacore 3000. Relative epitopemapping was based on pair-wise combination of MAs in sandwich ELISAs. Western blot with different domains of TcdA and TcdB and selected MAs as primary antibody were used for initial epitope mapping. In vivo neutralization capacities of the MAs were evaluated by murine ileal loops in C57BL/6 mice (65 nM TcdA, 4 hours incubation, 2 cm loops either with or without 72 - 113 μM MA). Gene expression was analyzed by standard RNA extraction and RealTime-PCR. Results - Out of 99 monoclonal antibodies that were reactive against TcdA and/or TcdB, 4 neutralized TcdA in vitro (average EC50: 15 nM), 6 neutralized TcdB in vitro (average EC50: 3.02 nM) and 1 antibody neutralized both toxins (EC50-TcdA: 13 nM, EC50-TcdB: 140 nM) in vitro. Biacore affinity studies on 4 TcdA neutralizing MAs demonstrated high affinity for TcdA and moreover, very slow dissociation rates. Relative epitope mapping resulted in the selection of 6 neutralizing MAs representing 3 distinct epitopes at each toxin. Western blot experiments indicate that the three selected MAs against TcdA are binding at the receptor-binding domain, while the selected MAs against TcdB bind at the glucosyltransferase domain. In vivo neutralization activity was confirmed for three selected MAs by a decrease in mRNA levels of IL6, CXCL1 and TNF α after injection in murine ileal loops of TcdA and the MA, compared to injection with TcdA alone. Fabs derived from the best neutralizing MAs showed in vitro neutralization of TcdA or TcdB without significant loss in affinity. Discussion - This study identifies several monoclonal antibodies with high affinity and bioactivity in vitro as well as in vivo. We will further seek in vivo confirmation in the well-established Syrian gold hamster model of CDAD with local delivery of Fab fragments of the selected MAs using the ActoGeniX Lactococcus-based mucosal delivery system. 1000
998
Elucidating Intestinal Microbiota Components That Play a Protective or Deleterious Role During Clostridium difficile Infections Louis P. Schenck, Simon A. Hirota, Glen D. Armstrong, Justin A. MacDonald, Paul L. Beck
Long-Term Follow-up Study of Fecal Microbiota Transplantation (FMT) for Severe or Complicated Clostridium difficile Infection (CDI) Olga C. Aroniadis, Lawrence J. Brandt, Adam Greenberg, Thomas J. Borody, Colleen Kelly, Mark Mellow, Christina Surawicz, Leslie A. Cagle, Leila Neshatian
Aims: Clostridium difficile infections (CDI) are a world-wide epidemic and the major cause of hospital-acquired disease. The link between the gut microbiota and CDI has been apparent since the 1970's, being linked as the main cause of antibiotic-associated diarrhea. Recent case reports and small trials have generated excitement around the development of fecal transplants as a treatment, however determining the exact components of the microbiota needed for protection have remained elusive with the safety of these transplants in question. My aim is to assess the specific intestinal microbiota components involved in the prevention or exacerbation of CDI. Methods: C57/Bl6 mice from two different vendors (A or B) were exposed to antibiotics before gavage with C. difficile (Cdif) spores. Four days post gavage, mice were sacrificed and analyzed for histological and inflammatory changes. Mice from A and B were cohoused for 14 days, followed by exposure to antibiotics and Cdif spores. Microbiota diversity and composition were analyzed using terminal restriction fragment length polymorphism (TRFLP) and fluorescent in situ hybridization (FISH) analysis of fecal samples. Results: Mice from the two vendors were found to have markedly distinct microbiota profiles both before and after antibiotic exposure via TRFLP analysis. Mice from A developed severe CDI as evidenced by greater histological damage, increased MPO levels and increased inflammatory cytokines, including KC and IL-1beta. Mice from B were almost completely resistant to CDI with minimal histological and cytokine changes from baseline. Interestingly, FISH analysis revealed decreased overall microbial RNA levels in feces from vendor A mice compared to vendor B post antibiotic exposure, indicating decreased activity of the microbiota. Additionally, Lactobacilli levels were higher in the susceptible mice whereas Bacteroides-Prevotella were higher in the non-susceptible mice. Following cohousing, microbial analysis determined that the microbiota of mice from vendor A and B merged and interestingly their response to CDI merged, with mice from vendor A having less severe CDI and mice from vendor B having more severe CDI. Conclusions: Our data are the first to identify specific patterns in the intestinal microbiota that confer susceptibility to CDI. Furthermore, we have leads on the bacterial populations that may be critical for preventing or reducing the severity of CDI. This study may lead to development of clinical biomarkers to determine patient susceptibility to CDI and create more targeted bacteriotherapy for the treatment and prevention of CDI.
Introduction: CDI has increased to epidemic proportions over the past 15 yrs and the emergence of virulent strains has been associated with heightened morbidity and mortality. FMT is highly successful for the treatment of recurrent CDI, although its benefit in severe or complicated CDI has not been elucidated. The aim of this study was to investigate the efficacy of FMT in hospitalized patients with severe or complicated CDI. Methods: A multicenter long-term follow-up study was performed on the use of FMT in hospitalized pts with severe or complicated CDI. CDI was defined as severe in the presence of abdominal tenderness, albumin ,3.0 g/dL or WBC .15,000 cells/μL and as complicated if there was admission to the intensive care unit (as a consequence of CDI); hypotension with or without the use of vasopressors; change in mental status; WBC .35,000 cells/μL or ,2,000 cells/μL; serum lactate levels ≥ 2.2 mmol/L; end-organ failure; fever ≥ 38.5°C; ileus or significant abdominal tenderness. A 43-item questionnaire soliciting demographic and pre- and post-FMT data was completed for all 13 eligible pts identified. Study outcomes included primary and secondary cure rates and time to resolution of symptoms. Results: 77% of subjects were women and their average age was 70 yrs (range: 38-89 yrs). The follow-up period between FMT and data collection ranged from 1-42 months (mean: 15 mos). All pts were hospitalized at the time of FMT and diagnosed with severe (84%) or complicated CDI (92%). 12 patients (92%) experienced diarrhea. After FMT, diarrhea resolved in 9 pts (75%) within 1-7 days (mean: 4.5 days) and improved in the remaining 3 pts (25%). Of the 8 pts (62%) with abdominal pain, resolution was seen after FMT in 6 pts (75%) within 1-7 days (mean 3.3 days) and improvement was seen in 2 pts (25%). The primary cure rate was 84%. 2 pts were continued on short courses of vancomycin (4 days) and fidaxomicin (10 days) postFMT. 1 pt was asymptomatic for 1 month, then discharged to hospice and died of unknown cause 1 month later. 2 pts (15%) experienced early CDI recurrence ( ≤ 90 days) after FMT. In 1 of these 2 pts CDI recurrence was associated with antibiotic treatment of a urinary tract infection and the pt was treated with and maintained on oral vancomycin after repeat FMT failed. The other pt was unable to retain the initial FMT but was successfully treated with second FMT after premedication with loperamide. Thus, the secondary cure rate was 92%. Late CDI recurrence (≥ 90 days) after FMT was seen in 1 of 13 pts (8%) in association with antibiotic treatment of diverticulitis. This pt was successfully treated with a repeat FMT and has not experienced recurrent CDI. Conclusions: FMT is a successful treatment option in pts with severe or complicated CDI. In this small multi-center case series, primary and secondary cure rates were 84% and 92%, respectively.
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AGA Abstracts
AGA Abstracts
from each other. Comparison of fecal microbiota (weighted UniFrac) showed that healthy donor and post-FT patient samples were relatively similar to each other.In the first patient the bacterial flora was similar to the donor even 1 year after the FMT. Conclusion: These results suggest a case-specific disturbance (dysbiosis) of the fecal microbiota of patients with RCDI as compared to healthy donors. RCDI is not always associated with similar microbiota alterations in all the patients.Antibiotcs appear to alter the fecal microbiota from a healthy state to a state resembling untreated RCDI. FMT appears to change the fecal microbiome in patients with RCDI, with post-FMT samples resembling the donor microbiota. Moreover, changes in microbiota in patients with RCDI appear to remain stable over time in the absence of recurrent antibiotic therapy. Course of Recurrent Clostridium difficile Infection in study patients, attempted antibiotic treatment and patient response to Fecal Microbiota Therapy.
998
Elucidating Intestinal Microbiota Components That Play a Protective or Deleterious Role During Clostridium difficile Infections Louis P. Schenck, Simon A. Hirota, Glen D. Armstrong, Justin A. MacDonald, Paul L. Beck
Long-Term Follow-up Study of Fecal Microbiota Transplantation (FMT) for Severe or Complicated Clostridium difficile Infection (CDI) Olga C. Aroniadis, Lawrence J. Brandt, Adam Greenberg, Thomas J. Borody, Colleen Kelly, Mark Mellow, Christina Surawicz, Leslie A. Cagle, Leila Neshatian
Aims: Clostridium difficile infections (CDI) are a world-wide epidemic and the major cause of hospital-acquired disease. The link between the gut microbiota and CDI has been apparent since the 1970's, being linked as the main cause of antibiotic-associated diarrhea. Recent case reports and small trials have generated excitement around the development of fecal transplants as a treatment, however determining the exact components of the microbiota needed for protection have remained elusive with the safety of these transplants in question. My aim is to assess the specific intestinal microbiota components involved in the prevention or exacerbation of CDI. Methods: C57/Bl6 mice from two different vendors (A or B) were exposed to antibiotics before gavage with C. difficile (Cdif) spores. Four days post gavage, mice were sacrificed and analyzed for histological and inflammatory changes. Mice from A and B were cohoused for 14 days, followed by exposure to antibiotics and Cdif spores. Microbiota diversity and composition were analyzed using terminal restriction fragment length polymorphism (TRFLP) and fluorescent in situ hybridization (FISH) analysis of fecal samples. Results: Mice from the two vendors were found to have markedly distinct microbiota profiles both before and after antibiotic exposure via TRFLP analysis. Mice from A developed severe CDI as evidenced by greater histological damage, increased MPO levels and increased inflammatory cytokines, including KC and IL-1beta. Mice from B were almost completely resistant to CDI with minimal histological and cytokine changes from baseline. Interestingly, FISH analysis revealed decreased overall microbial RNA levels in feces from vendor A mice compared to vendor B post antibiotic exposure, indicating decreased activity of the microbiota. Additionally, Lactobacilli levels were higher in the susceptible mice whereas Bacteroides-Prevotella were higher in the non-susceptible mice. Following cohousing, microbial analysis determined that the microbiota of mice from vendor A and B merged and interestingly their response to CDI merged, with mice from vendor A having less severe CDI and mice from vendor B having more severe CDI. Conclusions: Our data are the first to identify specific patterns in the intestinal microbiota that confer susceptibility to CDI. Furthermore, we have leads on the bacterial populations that may be critical for preventing or reducing the severity of CDI. This study may lead to development of clinical biomarkers to determine patient susceptibility to CDI and create more targeted bacteriotherapy for the treatment and prevention of CDI.
Introduction: CDI has increased to epidemic proportions over the past 15 yrs and the emergence of virulent strains has been associated with heightened morbidity and mortality. FMT is highly successful for the treatment of recurrent CDI, although its benefit in severe or complicated CDI has not been elucidated. The aim of this study was to investigate the efficacy of FMT in hospitalized patients with severe or complicated CDI. Methods: A multicenter long-term follow-up study was performed on the use of FMT in hospitalized pts with severe or complicated CDI. CDI was defined as severe in the presence of abdominal tenderness, albumin ,3.0 g/dL or WBC .15,000 cells/μL and as complicated if there was admission to the intensive care unit (as a consequence of CDI); hypotension with or without the use of vasopressors; change in mental status; WBC .35,000 cells/μL or ,2,000 cells/μL; serum lactate levels ≥ 2.2 mmol/L; end-organ failure; fever ≥ 38.5°C; ileus or significant abdominal tenderness. A 43-item questionnaire soliciting demographic and pre- and post-FMT data was completed for all 13 eligible pts identified. Study outcomes included primary and secondary cure rates and time to resolution of symptoms. Results: 77% of subjects were women and their average age was 70 yrs (range: 38-89 yrs). The follow-up period between FMT and data collection ranged from 1-42 months (mean: 15 mos). All pts were hospitalized at the time of FMT and diagnosed with severe (84%) or complicated CDI (92%). 12 patients (92%) experienced diarrhea. After FMT, diarrhea resolved in 9 pts (75%) within 1-7 days (mean: 4.5 days) and improved in the remaining 3 pts (25%). Of the 8 pts (62%) with abdominal pain, resolution was seen after FMT in 6 pts (75%) within 1-7 days (mean 3.3 days) and improvement was seen in 2 pts (25%). The primary cure rate was 84%. 2 pts were continued on short courses of vancomycin (4 days) and fidaxomicin (10 days) postFMT. 1 pt was asymptomatic for 1 month, then discharged to hospice and died of unknown cause 1 month later. 2 pts (15%) experienced early CDI recurrence ( ≤ 90 days) after FMT. In 1 of these 2 pts CDI recurrence was associated with antibiotic treatment of a urinary tract infection and the pt was treated with and maintained on oral vancomycin after repeat FMT failed. The other pt was unable to retain the initial FMT but was successfully treated with second FMT after premedication with loperamide. Thus, the secondary cure rate was 92%. Late CDI recurrence (≥ 90 days) after FMT was seen in 1 of 13 pts (8%) in association with antibiotic treatment of diverticulitis. This pt was successfully treated with a repeat FMT and has not experienced recurrent CDI. Conclusions: FMT is a successful treatment option in pts with severe or complicated CDI. In this small multi-center case series, primary and secondary cure rates were 84% and 92%, respectively.
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AGA Abstracts
AGA Abstracts
from each other. Comparison of fecal microbiota (weighted UniFrac) showed that healthy donor and post-FT patient samples were relatively similar to each other.In the first patient the bacterial flora was similar to the donor even 1 year after the FMT. Conclusion: These results suggest a case-specific disturbance (dysbiosis) of the fecal microbiota of patients with RCDI as compared to healthy donors. RCDI is not always associated with similar microbiota alterations in all the patients.Antibiotcs appear to alter the fecal microbiota from a healthy state to a state resembling untreated RCDI. FMT appears to change the fecal microbiome in patients with RCDI, with post-FMT samples resembling the donor microbiota. Moreover, changes in microbiota in patients with RCDI appear to remain stable over time in the absence of recurrent antibiotic therapy. Course of Recurrent Clostridium difficile Infection in study patients, attempted antibiotic treatment and patient response to Fecal Microbiota Therapy.