SBIR-STTR Award

Clostridium difficile is responsible for more than 29,000 deaths in the US each year and the infection represents an urgent threat to public health worldwide. Of most concern is that the incidence of C. difficile infection (CDI) and disease severity is rapidly increasing in recent years due to the emergence of hypervirulent and antibiotic-resistant strains. CDI is mainly caused by two major toxins TcdA and TcdB, and toxin- neutralizing antibodies (antitoxins) are responsible for effective immunity. Therefore, it is important to measure host antitoxin responses since such information will be crucial for assisting physicians to predict CDI progression and recurrence, as well as to guide the use of antibody-based therapeutics. However, current assays to measure patient antitoxin responses are based on serum inhibition of toxins' biological activities on cultured cells; such assays are time consuming, laborious, and difficult to standardize. We aim to develop a serological ELISA for predicting protective immunity against primary and recurrent CDI in patients. This will help clinical management of CDI and also may be used as a companion diagnostic for passive and active immunotherapies. We have identified a broadly neutralizing antibody (bNAb) N3 and established a N3-based competition ELISA in that CDI patient sera were assayed for inhibiting the binding of biotinylated N3 to TcdB. We found that those sera with high neutralizing anti-TcdB titers are also significantly more potent in inhibiting N3 binding to TcdB than sera with low neutralizing titers. In this project, we will identify additional bNAbs from a panel of anti-TcdB neutralizing antibodies we have developed and evaluate the bNAbs-based ELISA in 240 banked serum samples from CDI patients. Our ultimate goal is to develop a simple serological ELISA screen for antitoxin protective immunity, and thus help the clinical management of CDI and assist the future use of passive and active immunotherapies against this debilitating disease.

Public Health Relevance Statement:
Narrative: Clostridium difficile is responsible for more than 29,000 deaths in the US each year and host protective immunity against the infection is mediated via antitoxin antibodies. The goal of this project is to establish a simple diagnostic for antitoxin responses in patients.

Project Terms:
Animals; Antibiotics; Miscellaneous Antibiotic; Antibiotic Drugs; Antibiotic Agents; Antibodies; Epitopes; Binding Determinants; Antigenic Determinants; Antitoxins; B-Lymphocytes; Bursa-Equivalent Lymphocyte; Bursa-Dependent Lymphocytes; B-cell; B-Cells; B cells; B cell; B blood cells; Biological Assay; Biologic Assays; Bioassay; Assay; Cells; Cell Body; Cultured Cells; Clone Cells; Convalescence; Cessation of life; Death; Disease; Disorder; Enzyme-Linked Immunosorbent Assay; ELISA; Future; Goals; Human; Modern Man; Immunity; Incidence; Infection; Patients; Physicians; Public Health; Recurrence; Recurrent; Rest; Study of serum; Standardization; Testing; Time; Toxin; Vancomycin; Clostridium difficile; C.difficile; C. difficile; C. diff; C difficile; C diff; Active Immunotherapy; Measures; Antibiotic Resistance; antibiotic resistant; antibiotic drug resistance; Resistant to antibiotics; Resistance to antibiotics; Mediating; base; Clinical; Phase; Biological; Serologic; Serological; Blood Serum; Serum; a-Toxin; alpha Toxin; antibody-based treatment; antibody-based therapeutics; antibody treatment; antibody based therapies; Antibody Therapy; Diagnostic; neutralizing antibody; Severity of illness; disease severity; Passive Immunotherapy; passive immunotherapeutics; passive immune therapy; Disease Outcome; Sampling; response; Binding; Molecular Interaction; Multi-Institutional Clinical Trial; multi site clinical trial; multi site clinical study; multi center clinical trial; multi center clinical study; Multi-site clinical trial; Multi-site clinical study; Multi-center clinical trial; Multi-center clinical study; Clinical Management; Small Business Innovation Research; SBIR; Small Business Innovation Research Grant; standard treatment; standard care; resistance strain; resistant strain; intestinal tract microflora; intestinal microflora; intestinal microbiota; intestinal microbes; intestinal flora; gut microflora; gut microbiotic; gut microbial consortia; gut microbial composition; gut microbial community; gut microbe community; gut flora; gut community; gut commensal; gastrointestinal microbial flora; enteric microbiota; enteric microbial community; Gastrointestinal microbiota; GI microbiota; gut microbiota; phase II study; phase 2 study; companion diagnostics

This SBIR Phase II project will support the development of an in vitro diagnostic blood test to determine whether a person infected with Clostridioides difficile has mounted enough neutralizing antitoxin antibodies so that they are unlikely to suffer recurrence. Antibiotic-resistant C. difficile is responsible for more than 29,000 deaths in the US each year and the infection is an urgent threat to public health worldwide. The current standard treatment with antibiotics disrupts gut microbiota and induces high rates of recurrence, which is the most significant issue in clinical management of the disease. C. difficile infection (CDI) is mainly caused by two major exotoxins, and toxin-neutralizing antibodies are responsible for effective immunity. However, current assays to measure patient antitoxin responses are based on cell-culture neutralizing bioassay that is time consuming, laborious, and difficult to standardize, thus such bioassay is limited in research laboratories. In our SBIR Phase I (R43AI136176) study, we successfully established a simple and rapid serological ELISA, designated as BB-ELISA, and demonstrated that the BB-ELISA was able to measure antitoxin neutralizing activities in CDI blood samples. Based on these promising results, we propose to standardize this novel BB- ELISA through an experience CRO Corgenix and to collaborate with Merck to evaluate the assay for predicting CDI disease recurrence and outcomes. The completion of this Phase II SBIR will not only validate this novel ELISA for measuring neutralizing antibody responses in CDI patients and predicting disease outcomes, but also pave the way toward future commercialization of this novel ELISA assay.

Public Health Relevance Statement:
Narrative This SBIR Phase II project will support the development of a novel diagnostic blood test to determine whether a person infected with Clostridioides difficile has not mounted enough protective immune responses so that they are more likely to suffer recurrent disease.

Project Terms:
Age; ages; Animals; Antibiotics; Antibiotic Agents; Antibiotic Drugs; Miscellaneous Antibiotic; Antibodies; Epitopes; Antigenic Determinants; Binding Determinants; antitoxin; anti-toxin; Bacillus megaterium; B megaterium; B. megaterium; Bacillus megatherium; Biological Assay; Assay; Bioassay; Biologic Assays; Cell Culture Techniques; cell culture; Cell Line; CellLine; Strains Cell Lines; cultured cell line; Cells; Cell Body; Affinity Chromatography; affinity purification; Cessation of life; Death; Diarrhea; Disease; Disorder; Enzyme-Linked Immunosorbent Assay; ELISA; Exotoxins; Future; Goals; Blood Tests; Hematologic Tests; Hematological Tests; Hematology Testing; Recording of previous events; History; Human; Modern Man; Immunoglobulin G; 7S Gamma Globulin; IgG; Immunity; Infection; Laboratory Research; Persons; Patients; Physicians; Placebos; Sham Treatment; sham therapy; Public Health; Publications; Scientific Publication; Quality Control; Reagent; Recurrence; Recurrent; Rest; Risk Factors; Serology test; serology assay; Standardization; Testing; Time; Toxin; Vancomycin; Clostridium difficile; C diff; C difficile; C. diff; C. difficile; Clostridioides difficile; Active Immunotherapy; Measures; Chinese Hamster Ovary Cell; CHO Cells; Antibiotic Resistance; Resistance to antibiotics; Resistant to antibiotics; antibiotic drug resistance; antibiotic resistant; base; Blood specimen; Blood Sample; Procedures; Clinical; Phase; Serology; Serum; Blood Serum; Recurrent disease; Relapsed Disease; Antibody Therapy; antibody based therapies; antibody treatment; antibody-based therapeutics; antibody-based treatment; Phase III Clinical Trials; Phase 3 Clinical Trials; phase III protocol; Immunological response; host response; immune system response; immunoresponse; Immune response; Life; Disorder Management; Disease Management; Protocol; Protocols documentation; neutralizing antibody; disease severity; Severity of illness; experience; cohort; immunological status; passive immune therapy; passive immunotherapeutics; Passive Immunotherapy; Disease Outcome; novel; Sampling; response; assay development; Molecular Interaction; Binding; Pseudomembranous Colitis; Symptoms; Data; Recombinants; Reproducibility; Research Contracts; Clinical Management; Small Business Innovation Research Grant; SBIR; Small Business Innovation Research; Development; developmental; Sepsis; blood infection; bloodstream infection; Outcome; Consumption; novel diagnostics; new diagnostics; next generation diagnostics; antibody engineering; commercialization; standard care; standard treatment; gut microbiota; GI microbiota; Gastrointestinal microbiota; enteric microbial community; enteric microbiota; gastrointestinal microbial flora; gut commensal; gut community; gut flora; gut microbe community; gut microbial community; gut microbial composition; gut microbial consortia; gut microbiotic; gut microflora; intestinal flora; intestinal microbes; intestinal microbiota; intestinal microflora; intestinal tract microflora; phase 1 study; Phase I Study; phase 2 study; phase II study; operation; diagnostic assay; Antibody Response; companion diagnostics; predicting response; prediction of response; predictive response; predictor of response; response prediction; predictive test; predictive assay; secondary endpoint; secondary end point; primary endpoint; primary end point; recurrent infection; infection recurrence; in-vitro diagnostics