Generation and characterisation of an outer membrane particle-based vaccine against non - typhoidal salmonella

Non-typhoidal Salmonella enterica (NTS) serovars are a major cause of bacteraemia in Sub-Saharan Africa, affecting mainly children and immunocompromised adults, leading to case-fatality rates as high as 24%. Despite the high burden of disease and increasing emergence of multidrug resistant strains, there are currently no vaccines available against NTS disease. Vaccination is the most cost-effective way of disease prevention, so a broad-spectrum, effective and affordable vaccine against NTS would be highly desirable. NTS vaccine research efforts have focused both on the development of live attenuated strains and subunit vaccines. Whereas live attenuated strains might pose an important safety problem in populations with a high number of immunocompromised adults as found in an African setting, O antigen (Oag)-based vaccines are serovar specific and might elicit an immune response that generates inhibitory antibodies against the LPS and subsequently impair Salmonella killing in humans. Protein-based vaccines might be the most suitable approach in NTS vaccine development. Several protein antigens have shown protection against Salmonella infection in mouse models and it has been shown that antibodies against the outer membrane proteins effect Salmonella killing in human serum. In this study, we investigated the use of Generalised Modules for Membrane Antigens, or GMMA, as a broad-spectrum protein-based vaccine against NTS. GMMA are outer membrane particles naturally released by Salmonella during growth and contain high amounts of periplasmic and outer membrane proteins. GMMA are usually known in literature as outer membrane vesicles (OMV), but are distinct from detergent-extracted OMV in their generation, content, and immunogenicity. GMMA are released in small amounts during Salmonella growth, so we genetically engineered tolR deletion mutants of two NTS serovars, S. Typhimurium strain SL1344 and S. Enteritidis strain P12109, for increased GMMA release. These GMMA were optimised for protein antigen delivery by deleting the Oag, through disruption of the wbaP gene, thus overcoming the serospecificity of the Oag and unmasking protein antigens on the GMMA surface. A further genetic deletion in the msbB gene was done to overcome the GMMA reactogenicity associated with the lipid A portion of LPS. Overexpression of the highly conserved Salmonella iron-regulated outer membrane proteins (IROMPs) on the GMMA was achieved by growing the GMMA-overproducing S. Typhimurium and S. Enteritidis mutants under iron-limiting conditions. The proteomic content of S. Typhimurium and S. Enteritidis GMMA was characterised and, as expected, found to contain a high number of outer membrane and periplasmic proteins. Salmonella GMMA were also tested for their ability to elicit an immune response in mice and were determined to be highly immunogenic by both subcutaneous and intranasal immunisation routes, generating high levels of GMMA-specific IgG. Immunoblots using GMMA immune sera showed that IgG antibodies were raised against many S. Typhimurium GMMA proteins and that these were cross-reactive in S. Enteritidis. In vitro, however, the presence of an Oag layer in live Salmonella was shown to impair the binding of protein antibodies to the bacterial surface, an effect that was abrogated in Salmonella strains lacking Oag. In a murine challenge model of acute Salmonella infection, S. Typhimurium and S. Enteritidis GMMA lacking Oag impaired S. Typhimurium infection in the spleen, indicating that a protein-based response was able to impair homologous and heterologous challenge, respectively. S. Typhimurium GMMA with Oag provided the same protective effect as the positive control, the live attenuated strain S. Typhimurium SL3261. This is the first time that heterologous protection against Salmonella infection has been demonstrated for GMMA, indicating that they have the potential to be developed as a broad-spectrum protein-based vaccine against NTS.