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Dissertation
Development of a DNA vaccine encoding the receptor-binding domain of Clostridium difficile toxin A and toxin B, and the role of immune suppression in increased susceptibility to Clostridium difficile infection
Doctor of Philosophy (Ph.D.), Drexel University
Jul 2014
DOI:
https://doi.org/10.17918/00000466
Abstract
Clostridium difficile infection (CDI) constitutes the most serious cause of antibiotic-associated diarrhea resulting in over 330,000 hospitalizations, 30,000 deaths and $8.2 billion in C. difficile-related hospital costs annually. C. difficile-associated disease (CDAD) is mediated by toxin A (TcdA) and toxin B (TcdB) which contain epitopes within the receptor-binding domain (RBD) that are important for toxin neutralization. We created DNA vaccine plasmids encoding TcdA- and TcdB-RBD and immunized mice and rhesus macaques intramuscularly followed by in vivo electroporation. Vaccination induced serum and stool anti-RBD antibodies that neutralized toxins in vitro. Mice actively immunized with the RBD plasmids or passively administered rhesus macaque hyperimmune sera were protected from challenge with purified toxin as well as strains of C. difficile that were homologous (VPI 10463) and heterologous (UK1) to our vaccine antigens. We next sought to model individuals at high risk for CDI and severe CDAD with the longterm goal of testing our DNA vaccine strategy in this susceptible cohort. Higher anti-toxin antibody responses are indicative of lesser CDAD severity and risk of recurrence. Advanced age and opioid usage are associated with increased risk of recurrent CDI, which may reflect an inability to mount protective antibody responses. Compared to controls, infected aged or morphine-treated mice were more susceptible to sub-lethal CDI, displaying higher mortality and advanced gut inflammation. After multiple reinfections, older animals displayed worse intestinal pathology and higher frequencies of mucosal CD4 + T cells expressing either PD-1 or a follicular phenotype. In summary, we are the first to 1) create DNA vaccine plasmids expressing N-linked glycosylation null RBDs for enhanced recognition of native bacterial toxin, (2) demonstrate the immunogenicity and efficacy of a TcdB-RBD-expressing plasmid, (3) test our RBD plasmids in mice and non-human primates utilizing CELLECTRA® 2000 in vivo electroporation, (4) show efficacy against challenge with both purified toxin and homologous and heterologous stains of C. difficile, (5) observe protection after only two immunizations, which is especially important given the acute onset of CDAD, and (6) establish animal models of CDI in the context of host immune suppression, which will provide an ideal means to study underlying host factors leading to increased susceptibility.
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Details
- Title
- Development of a DNA vaccine encoding the receptor-binding domain of Clostridium difficile toxin A and toxin B, and the role of immune suppression in increased susceptibility to Clostridium difficile infection
- Creators
- Scott M. Baliban
- Contributors
- Michele Kutzler (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xv, 170 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Microbiology and Immunology; College of Medicine; Drexel University
- Other Identifier
- 991014970227804721