This proposal is in response to RFA-AI-09-016 requesting applications to develop vaccines for selected pathogens including Clostridium difficile, the cause of pseudomembranous colitis, which accounts for a quarter of all cases of antibiotic-associated diarrhea. With the recent emergence of hypervirulent strains, the incidence of C. difficile infection (CDI) has increased significantly in both North America and Europe, causing lengthy hospitalization, substantial morbidity and mortality. CDI is thought to be mainly mediated by exotoxins TcdA and TcdB, which glucosylate low molecular mass GTPases of the Rho family, leading to massive fluid secretion, acute inflammation, and necrosis of the colonic mucosa. Protection against CDI was shown to be mediated through systemic and mucosal antibodies against the 2 key toxins, although other virulence attributes are known to exist which may also contribute to the manifestation of CDI. The goal of this proposal is to design a vaccine that targets both TcdA and TcdB, with a view to elicit strong systemic and mucosal immunity to prevent CDI, reduce the severity, or eliminate an ongoing chronic disease. We propose to exploit the recently expressed atoxic C. difficile holotoxin proteins in an endotoxin-free Bacillus megaterium system. Two immunogens will be evaluated: a mixture of atoxic full-length C. difficile toxin A and B generated by point mutations (designated as aTxAB), and a chimera protein containing full-length TcdB but with its receptor binding domain replaced with that of TcdA (designated as cTxAB). cTxAB has a small deletion (97 amino acids) in transmembrane domain rendering it non-toxic. Preliminary studies showed that atoxic TcdB vaccination induced antibody responses against a wide-spectrum of epitopes and potent protective immunity superior to toxoid;cTxAB immunization induced antibody and protective responses against both TcdA and TcdB. In this project, we will first evaluate the ability of these atoxic recombinant proteins to induce protective antibody responses following parenteral immunization followed by challenge with wild type toxins (Aim 1). This will be followed by evaluating several regimens of mucosal immunizations (oral, intranasal and sublingual) designed to induce protection against systemic and mucosal challenges with wild type toxins (Aim 2). We will test the protective efficacy of the various immunization regimens developed in Aims 1 and 2 in the recently described mouse acute infection model (Aim 3a), and the most efficient immunization method resulting from the mouse infection studies will undergo preclinical evaluation in the chronic piglet model of CDI developed in this laboratory (Aim 3b). Because at this early stage the nature of the candidate vaccine is unknown, nor is the adjuvant required, we are not in a position to form a suitable partnership. Abstract Narrative: Clostridium difficile-associated diarrhea and enteric inflammatory diseases are caused primarily by two secretory toxins. This project will use recombinant atoxic holotoxins produced in this lab as a basis for a vaccine to elicit strong systemic and mucosal immunity to prevent C. difficile infection, reduce the severity, or eliminate an ongoing chronic disease.

Public Health Relevance

Abstract Narrative: Clostridium difficile-associated diarrhea and enteric inflammatory diseases are caused primarily by two secretory toxins. This project will use recombinant atoxic holotoxins produced in this lab as a basis for a vaccine to elicit strong systemic and mucosal immunity to prevent C. difficile infection, reduce the severity, or eliminate an ongoing chronic disease.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI088748-04
Application #
8242822
Study Section
Special Emphasis Panel (ZAI1-TS-M (J1))
Program Officer
Ranallo, Ryan
Project Start
2010-04-15
Project End
2015-03-31
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
4
Fiscal Year
2012
Total Cost
$639,528
Indirect Cost
$149,825
Name
University of Maryland Baltimore
Department
Microbiology/Immun/Virology
Type
Schools of Dentistry
DUNS #
188435911
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Zhang, Yongrong; Feng, Hanping (2016) Pathogenic effects of glucosyltransferase from Clostridium difficile toxins. Pathog Dis 74:ftw024
Maldarelli, Grace A; Matz, Hanover; Gao, Si et al. (2016) Pilin Vaccination Stimulates Weak Antibody Responses and Provides No Protection in a C57Bl/6 Murine Model of Acute Clostridium difficile Infection. J Vaccines Vaccin 7:
Schmidt, Diane J; Beamer, Gillian; Tremblay, Jacqueline M et al. (2016) A Tetraspecific VHH-Based Neutralizing Antibody Modifies Disease Outcome in Three Animal Models of Clostridium difficile Infection. Clin Vaccine Immunol 23:774-84
Hamza, Therwa; Zhang, Zhifen; Melnyk, Roman A et al. (2016) Defective mutations within the translocation domain of Clostridium difficile toxin B impair disease pathogenesis. Pathog Dis 74:ftv098
Huang, Tuxiong; Perez-Cordon, Gregorio; Shi, Lianfa et al. (2015) Clostridium difficile toxin B intoxicated mouse colonic epithelial CT26 cells stimulate the activation of dendritic cells. Pathog Dis 73:
Li, Shan; Shi, Lianfa; Yang, Zhiyong et al. (2015) Critical roles of Clostridium difficile toxin B enzymatic activities in pathogenesis. Infect Immun 83:502-13
Zhang, Yongrong; Hamza, Therwa; Gao, Si et al. (2015) Masking autoprocessing of Clostridium difficile toxin A by the C-terminus combined repetitive oligo peptides. Biochem Biophys Res Commun 459:259-63
Yu, Hua; Chen, Kevin; Wu, Jianguo et al. (2015) Identification of toxemia in patients with Clostridium difficile infection. PLoS One 10:e0124235
Wang, Yuan-Kai; Yan, Ya-Xian; Kim, Hyeun Bum et al. (2015) A chimeric protein comprising the glucosyltransferase and cysteine proteinase domains of toxin B and the receptor binding domain of toxin A induces protective immunity against Clostridium difficile infection in mice and hamsters. Hum Vaccin Immunother 11:2215-22
Yuan, Pengfei; Zhang, Hongmin; Cai, Changzu et al. (2015) Chondroitin sulfate proteoglycan 4 functions as the cellular receptor for Clostridium difficile toxin B. Cell Res 25:157-68

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