Outer membrane particles as Shigella vaccine candidate: generation, proteomic and immunoproteomic analysis

Shigellae are Gram-negative enteric bacteria representing the major cause of dysenteric diseases in infants and young children in developing countries. The genus Shigella comprises 50 different serotypes characterized by the carbohydrate composition of the O-antigen of the lipopolysaccharide. In natural infection the immune response to the O-antigen has been shown to be protective but serotype-specific, and no vaccine against Shigella is currently available. Novartis Vaccines Institute for Global Health (NVGH) aims to develop a broadly protective protein-based vaccine using outer membrane particles. These particles, called Generalized Modules for Membrane Antigens (GMMA), also known as outer membrane vesicles (OMV), are naturally shed from the surface of Gram-negative bacteria and expose the host to proteins of the outer membrane in their native orientation. They are different from detergent-extracted OMV which are depleted of lipooligosaccharides and lipoproteins. As Shigella naturally produce low amounts of GMMA, the tolR gene was deleted to induce GMMA overproduction. To avoid immuno-dominant immune responses to the O-antigen, the genes of the O-antigen biosynthesis gene cluster were deleted. In addition, the msbB gene was deleted to reduce the endotoxicity of the lipopolysaccharide (LPS). GMMA were generated under iron-restricted conditions to induce the expression of highly conserved iron siderophore receptors that have been shown to be protective antigens against Salmonella infection. The proteomics analysis of S. sonnei 53G and S. flexneri 2a 2457T GMMA confirmed that GMMA are highly enriched in outer membrane and periplasmic proteins. Approximately 65% and 71% of the total predicted outer membrane proteins of S. flexneri and S. sonnei, respectively, and 50% of the total predicted periplasmic proteins were found to be present in GMMA. In contrast, only 4% of the total predicted cytoplasmic proteins were indentified in GMMA. We identified 50 outer membrane proteins and 2 predicted extracellular proteins that were conserved in S. sonnei and S. flexneri GMMA. Among these proteins we identified 3 proteins encoded on the virulence plasmid (MxiD, MxiM and IcsA/VirG), and other 3 proteins with a virulence-associated function (SepA, SigA and TieB) which are specific for Shigella and pathogenic E. coli. The proteomic results also confirmed the overexpression of 8 iron-regulated proteins in GMMA of which 5 are conserved in S. sonnei and S. flexneri. Mice were immunized using purified GMMA and immune sera were analyzed by ELISA and bi-dimensional Western blots. All GMMA elicited similarly high levels of total GMMA-specific IgG. By 2 dimensional Western blots (2-D WB) analysis mainly periplasmic proteins and only 3 outer membrane proteins were identified, suggesting that a different approach was needed to identify outer membrane proteins in their native conformation. In this work we present a novel approach to identify immunogenic surface proteins based on immunoprecipitation studies with live bacteria using sera raised against GMMA. We identified 30 immunogenic surface proteins and among those we found OmpA, OmpX and YaeT confirming our previous 2-D WB results. In addition, we identified proteins that were not found by 2-D WB but are known to be immunogenic in Shigella, e.g. TolC, supporting the power of this approach. Furthermore, OmpC, FepA, FhuA, and CirA were identified using this approach suggesting that the native conformation of the epitopes is important for antibody binding and that these epitopes are lost by 2-D WB analysis. A quantitative proteomic analysis of GMMA proteomes, based on stable isotope dimethyl labeling followed by GeLC-MS/MS, revealed that the majority of the 30 immunogenic proteins are the most abundant proteins in GMMA. Interestingly, YiaD, YtfM and one of the iron-regulated proteins, FepA, raised a strong antibody response but are of low abundance in GMMA. The majority of the immunogenic proteins identified in S. sonnei are also present and highly conserved in S. flexneri GMMA. Thus, it is likely that these proteins might raise cross-reactive immune responses. The present work shows for the first time an extensive qualitative and quantitative proteomic analysis of Shigella GMMA. In addition, the analysis of immunogenic surface proteins under native conditions identified proteins that are highly conserved among Shigella serotypes. This work provides the basis for further characterization of GMMA as a protein-based vaccine against Shigella