Assessment of Helicobacter pylori antioxidant enzymes as therapeutic targets

Abstract

© 2012 Dr. Andrew William StentHelicobacter pylori is a significant gastric pathogen in humans, with gastritis and gastric neoplasia frequently resulting from chronic colonisation. The organism is reliant on its antioxidant enzyme system for survival, and this thesis proposes that components of this system – namely, the antioxidant enzymes superoxide dismutase (HpSOD), catalase (HpKatA), and thiolperoxidase (HpTpx) - present a viable therapeutic target in H. pylori infection. Using a pET Escherichia coli expression system, recombinant forms of the three enzymes from H. pylori were created and then trialled as vaccine antigens administered with an appropriate adjuvant via systemic and mucosal routes. Moreover, in order to assess for synergistic action, the enzymes were assessed both individually and in combination. These trials demonstrated that superoxide dismutase and thiolperoxidase are protective antigens, but no additional protection was observed with the combination vaccine. The essential requirement for the antioxidant system in H. pylori further led to speculation that inhibition of enzyme function may be an alternative mode of therapy. Monoclonal antibodies directed against the three enzymes were generated via hybridomas derived from hyperimmunised mice. Antibodies were characterised by class and isotype, binding properties and ability to inhibit enzyme function, in order to determine the most promising candidates for therapeutic trials. After demonstrating the presence of systemic antibodies at the gastrointestinal mucosal surface in a mouse trial, the candidate antibodies were administered via intraperitoneal injection to mice infected with H. pylori. Although the antibody therapy did not impair colonisation, there was a significant increase in a number of pro-inflammatory cytokines within gastric tissue following administration of the anti-HpSOD antibody. Investigating further, it was found that incubation of macrophages in the presence of recombinant HpSOD attenuated the cytokine response to lipopolysaccharide. These findings suggested that HpSOD may possess previously undescribed immunosuppressive properties, potentially facilitating long-term colonization. Possible pathway mechanisms are discussed, as well as supportive findings from work on other chronic pathogens