Plasmid DNA as a platform for the development of prophylactic and therapeutic vaccines against Clostridium difficile in aging models of disease

Arjun Ramamurthi

doi:10.17918/etd-7521

Clostridium difficile associated disease (CDAD) is a gastrointestinal disease whose symptoms range from mild diarrhea to pseudomembranous colitis and toxic megacolon. Statistically, it is among the most frequent hospital acquired infections in the United States, and is the leading cause of antibiotic-associated diarrhea in the developed world. Affecting roughly 500,000 people in the U. S in the year 2011 alone, it was also identified as a top drug resistance concern in 2013. A significant fiscal burden, acute nosocomial infections alone cost $4.8 billion. Mortality in the elderly due to CDAD has skyrocketed in recent years, second only to septicemia and pneumonia, with the bulk of these cases being individuals >85 years of age. Advanced age, immunosuppression and antibiotic usage are the chief risk factors predisposing patients to CDAD. CDAD is mediated by two large exotoxins, tcdA and tcdB. These toxins bind to receptors on the surface of epithelial cells, and glycosylate Rho and Rac GTPases, causing actin cytoskeletal rearrangements and cell death. Protection from CDAD is mediated by antitoxin IgG antibodies, and there is a strong correlation between antitoxin IgG titers and disease outcomes. Immunosenescence, the age associated weakening of the immune system, leads to a deficiency in these antibodies, causing the heightened susceptibility of aging individuals. There is a paucity of efficacious, protective vaccines against CDAD for the elderly, which is highly relevant given the high risk of recurrent disease in this demographic. While numerous platforms have been studied, very few data describe the potential of prophylactic and therapeutic DNA vaccination against CDAD in an aging mouse model of disease. Here, we test the immunogenicity of a DNA vaccine encoding the receptor binding domain (RBD) of the two exotoxins in an aging murine model. We hypothesized that aging mice that receive the DNA vaccine delivered by in vivo electroporation would produce a lower, but functional, antitoxin antibody response than young mice, and this response protects mice from a lethal infection. In this thesis, I tested highly optimized plasmids encoding the RBD from TcdA and TcdB and immunized aged C57BL/6 mice intramuscularly followed by in vivo electroporation. In the young adult mice, vaccination induced significant levels of anti-RBD antibodies within the serum that could neutralize toxins in an in vitro cytotoxicity assay. Aged animals had lower endpoint and anti-toxin neutralization titers compared to young mice post vaccination, but still elicited functional antibody. Both young and aged immunized mice were protected following orogastric challenge with strains of 105 cfu/ml C. difficile spores that were homologous to our vaccine antigens (VPI 10463; n=6/6). In addition, bacterial burden in immunized mice post challenge was analyzed, and revealed that aging mice had a higher bacteria load in their intestines when compared to young adults, but lower levels when compared to mice that received an empty plasmid immunization. These data demonstrate the protective immunogenicity and efficacy of a TcdA/B RBD-based DNA vaccine in young adult and aged mouse models of acute toxin-associated disease. We also tested an immediate immune therapy platforms that could deliver anti-toxin antibodies during severe acute infection for protection against CDAD in this thesis. We posit that a new platform of DNA-encoded human monoclonal antibodies may be a viable option for an immediate treatment of CDAD upon diagnosis. We tested two DNA-encoded monoclonal antibodies (DMAbs) against the RBD of the toxins of C. difficile and evaluated their expression and functionality in vitro and in vivo. We show that the DMAbs are expressed by transfected cells, and specifically bind toxoids of the toxins. Additionally, we tested the temporal stability of the antibodies in vivo following a single vaccination in NOD/SCID Gamma mice and detected toxin specific antibody through day 21 post vaccination. Future studies will determine the ex vivo toxin neutralization by these antibodies, and the DMAbs will be tested for protection against lethal spore challenge in aged mice. In conclusion, the novel findings of this work are 1) the DNA vaccine encoding the RBD of C. difficile toxins A and B elicits a humoral response in aged mice, 2) antibodies elicited by this vaccine are functional, 3) Two immunizations protects young and aged mice from a lethal spore challenge, 4) infected old mice have a higher bacterial load than younger mice, but are still protected from morbidity and mortality, 5) DNA-encoded monoclonal antibodies are produced in vitro and are stable for at least 21 days in nude mice after a single immunization, and 6) these monoclonal antibodies are capable of binding toxoid antigen. These findings highlight the potential for in vivo electroporation delivered toxin-based DNA as a vaccine platform for prevention of CDAD, due to the ability to generate a strong, persistent humoral response that is protective in an aging mouse model of antibiotic associated diarrhea. In addition, testing of DNA-encoded anti-toxin human monoclonal antibodies may be a viable option for an immediate treatment of CDAD upon diagnosis in high risk individuals, such as the elderly.

Plasmid DNA as a platform for the development of prophylactic and therapeutic vaccines against Clostridium difficile in aging models of disease

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Plasmid DNA as a platform for the development of prophylactic and therapeutic vaccines against Clostridium difficile in aging models of disease

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