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)
Type
Research Project (R01)
Project #
5R01AI088748-06
Application #
8648989
Study Section
Special Emphasis Panel (ZAI1)
Program Officer
Ranallo, Ryan
Project Start
Project End
Budget Start
Budget End
Support Year
6
Fiscal Year
2014
Total Cost
Indirect Cost
Name
University of Maryland Baltimore
Department
Microbiology/Immun/Virology
Type
Schools of Dentistry/Oral Hygn
DUNS #
City
Baltimore
State
MD
Country
United States
Zip Code
21201
Kim, Hyeun Bum; Zhang, Quanshun; Sun, Xingmin et al. (2014) Beneficial effect of oral tigecycline treatment on Clostridium difficile infection in gnotobiotic piglets. Antimicrob Agents Chemother 58:7560-4
Yang, Zhiyong; Schmidt, Diane; Liu, Weilong et al. (2014) A novel multivalent, single-domain antibody targeting TcdA and TcdB prevents fulminant Clostridium difficile infection in mice. J Infect Dis 210:964-72
Huang, Tuxiong; Li, Shan; Li, Guangchao et al. (2014) Utility of Clostridium difficile toxin B for inducing anti-tumor immunity. PLoS One 9:e110826
Perez-Cordon, Gregorio; Yang, Guilin; Zhou, Boping et al. (2013) Interaction of Cryptosporidium parvum with mouse dendritic cells leads to their activation and parasite transportation to mesenteric lymph nodes. Pathog Dis :
Steele, Jennifer; Mukherjee, Jean; Parry, Nicola et al. (2013) Antibody against TcdB, but not TcdA, prevents development of gastrointestinal and systemic Clostridium difficile disease. J Infect Dis 207:323-30
Zhang, Quanshun; Widmer, Giovanni; Tzipori, Saul (2013) A pig model of the human gastrointestinal tract. Gut Microbes 4:193-200
Zhang, Yongrong; Shi, Lianfa; Li, Shan et al. (2013) A segment of 97 amino acids within the translocation domain of Clostridium difficile toxin B is essential for toxicity. PLoS One 8:e58634
Li, Shan; Shi, Lianfa; Yang, Zhiyong et al. (2013) Cytotoxicity of Clostridium difficile toxin B does not require cysteine protease-mediated autocleavage and release of the glucosyltransferase domain into the host cell cytosol. Pathog Dis 67:11-8
Steele, Jennifer; Zhang, Quanshun; Beamer, Gillian et al. (2013) MBX-500 is effective for treatment of Clostridium difficile infection in gnotobiotic piglets. Antimicrob Agents Chemother 57:4039-41
Steele, Jennifer; Chen, Kevin; Sun, Xingmin et al. (2012) Systemic dissemination of Clostridium difficile toxins A and B is associated with severe, fatal disease in animal models. J Infect Dis 205:384-91

Showing the most recent 10 out of 13 publications