Outer membrane vesicles derived from Gram-negative enteric pathogens

The release of outer membrane vesicles (OMVs) is a common feature of Gram-negative bacteria. OMVs are non- living facsimiles of the donor cells and interesting candidates for vaccine development, since they carry multiple native bacterial surface antigens. Recently, we have demonstrated that OMVs derived from the human pathogen V. cholerae are potent and stable cholera vaccine candidates. In the first activity of this proposal we want to improve and further characterize the cholera vaccine based on OMVs. This includes the reduction of the endotoxicity by genetic modification of the lipid A, which will broaden the spectrum of applicable administration routes, including the widely used subcutaneous and intramuscular injections. Identification of the protective antigen(s) will not only reveal the important immunogenic components of the OMVs, but might also unravel a common principle of protection by cholera vaccines through inhibition of motility. Immunization studies including enterotoxic Escherichia coli (ETEC) antigens will demonstrate, whether this protection mechanism can also be applied for other pathogens. In addition, we aim to create a combined vaccine against V. cholerae and ETEC, which are two important bacterial pathogens causing diarrhea. All aims of this first activity can be achieved independently, but may synergistically contribute to an improved OMV vaccine candidate. In the second activity of this proposal, we will investigate the OMV biogenesis using V. cholerae as a model organism. Several models for vesiculation have been proposed, but there is little evidence supporting these mechanisms and our knowledge of their biological roles and the formation process is very limited. We already obtained preliminary data, demonstrating that the cargo of V. cholerae OMVs is altered by changing environmental stimuli (e.g.: during induction of virulence genes) or by mutations affecting LPS biosynthesis. We aim to learn more on the V. cholerae OMV cargo and use a genetic approach via transposon- mutagenesis to identify pathways involved in OMV biogenesis. The collaborating laboratories of G. Daum and M. Feldman will provide their expertise on lipid, protein and LPS analysis. In summary, results obtained within this proposed work should improve our understanding of the protective immune response induced upon immunization with OMVs, broaden the spectrum for the use of OMVs as vaccine candidates and identify pathways involved in the OMV secretion and packaging of the OMV content.

The release of outer membrane vesicles (OMVs) is a common feature of Gram- negative bacteria. OMVs are non-living facsimiles of the donor cells and interesting candidates for vaccine development, since they carry multiple native bacterial surface antigens. Prior to the project, we could demonstrate that OMVs derived from the human pathogen V. cholerae are potent and stable cholera vaccine candidates. In the first activity of the project we improved and further characterized the cholera vaccine based on OMVs. This included the reduction of the endotoxicity by genetic modification of the lipid A, which extended the spectrum of applicable administration routes, including the widely used subcutaneous and intramuscular injections. Furthermore we charactreized the O antigen as the protective antigen, which additionally revealed a common principle of protection mechanism by cholera vaccines through inhibition of motility. Immunization studies including enterotoxic Escherichia coli (ETEC) antigens extended our studies to other enteric pathogens. We successfully described a combined OMV-based vaccine against V. cholerae and ETEC, which are two important bacterial pathogens causing diarrhea. In the second activity of this project, we investigated the OMV biogenesis of Gram negative bacteria. Via genetic approaches we identified a retrograde phospholipid trafficking system, which affects vesiculation. Repression or inactivation of the phospholipid trafficking system resulted in accumualtion of phospholipids in the outer mebrane promoting the release of vesicles. This mechanism is regulated by iron availability in all Gram-negative bacteria tested. Our findings indicate that iron limitation leads to a ferric uptake regulator (Fur)- dependent downregulation of the phospholipid trafficking system, which ultimately results in increased OMV production. In summary, results obtained within this project improved our understanding of the protective immune response induced upon immunization with OMVs, extended the spectrum of OMVs as vaccine candidates and identified a novel and conserved mechanism for OMV release in Gram negative bacteria.