Immunity to intracellular Salmonella depends on surface-associated antigens.

Invasive Salmonella infection is an important health problem that is worsening because of rising antimicrobial resistance and changing Salmonella serovar spectrum. Novel vaccines with broad serovar coverage are needed, but suitable protective antigens remain largely unknown. Here, we tested 37 broadly conserved Salmonella antigens in a mouse typhoid fever model, and identified antigen candidates that conferred partial protection against lethal disease. Antigen properties such as high in vivo abundance or immunodominance in convalescent individuals were not required for protectivity, but all promising antigen candidates were associated with the Salmonella surface. Surprisingly, this was not due to superior immunogenicity of surface antigens compared to internal antigens as had been suggested by previous studies and novel findings for CD4 T cell responses to model antigens. Confocal microscopy of infected tissues revealed that many live Salmonella resided alone in infected host macrophages with no damaged Salmonella releasing internal antigens in their vicinity. In the absence of accessible internal antigens, detection of these infected cells might require CD4 T cell recognition of Salmonella surface-associated antigens that could be processed and presented even from intact Salmonella. In conclusion, our findings might pave the way for development of an efficacious Salmonella vaccine with broad serovar coverage, and suggest a similar crucial role of surface antigens for immunity to both extracellular and intracellular pathogens.

Antigen selection based on expression levels during infection facilitates vaccine development for an intracellular pathogen.

Vaccines effective against intracellular pathogens could save the lives of millions of people every year, but vaccine development has been hampered by the slow largely empirical search for protective antigens. In vivo highly expressed antigens might represent a small attractive antigen subset that could be rapidly evaluated, but experimental evidence supporting this rationale, as well as practical strategies for its application, is largely lacking because of technical difficulties. Here, we used Salmonella strains expressing differential amounts of a fluorescent model antigen during infection to show that, in a mouse typhoid fever model, CD4 T cells preferentially recognize abundant Salmonella antigens. To identify a large number of natural Salmonella antigens with high expression levels during infection, we used a quantitative in vivo screening strategy. Immunization studies with five particularly attractive candidates revealed two highly protective antigens that might permit the development of an improved typhoid fever vaccine. In conclusion, we have established a rationale and an experimental strategy that will substantially facilitate vaccine development for Salmonella and possibly other intracellular pathogens.

Autodisplay: efficacious surface exposure of antigenic UreA fragments from Helicobacter pylori in Salmonella vaccine strains.

Live attenuated Salmonella strains expressing antigens of pathogens are promising oral vaccine candidates. There is growing evidence that the topology of expression of the foreign antigens can have a dramatic impact on the immunogenicity. We examined the potential of the AIDA-I (Escherichia coli adhesin involved in diffuse adherence) autotransporter domain to display antigenic fragments of the urease A subunit of Helicobacter pylori for the induction of a protective immune response. In the murine H. pylori model, protection is mainly mediated by CD4(+) T cells, and we therefore used the AIDA-I expression system to successfully express both nearly full-length UreA and defined T-helper-cell epitopes on the surface of an attenuated Salmonella enterica serovar Typhimurium vaccine strain. Surface exposure of the large UreA fragment or of one UreA T-cell epitope mediated a significant reduction in the level of H. pylori in immunized mice after challenge infection, whereas conventional cytoplasmic expression of UreA in Salmonella had no effect. These results support the concept that surface display increases the immunogenicity of recombinant antigens expressed on oral live vaccine carriers and further demonstrate the feasibility of immunizing against H. pylori with Salmonella vaccine strains expressing CD4(+) T-cell epitopes.

Autodisplay: development of an efficacious system for surface display of antigenic determinants in Salmonella vaccine strains.

To optimize antigen delivery by Salmonella vaccine strains, a system for surface display of antigenic determinants was established by using the autotransporter secretion pathway of gram-negative bacteria. A modular system for surface display allowed effective targeting of heterologous antigens or fragments thereof to the bacterial surface by the autotransporter domain of AIDA-I, the Escherichia coli adhesin involved in diffuse adherence. A major histocompatibility complex class II-restricted epitope, comprising amino acids 74 to 86 of the Yersinia enterocolitica heat shock protein Hsp60 (Hsp60(74-86)), was fused to the AIDA-I autotransporter domain, and the resulting fusion protein was expressed at high levels on the cell surface of E. coli and Salmonella enterica serovar Typhimurium. Colonization studies in mice vaccinated with Salmonella strains expressing AIDA-I fusion proteins demonstrated high genetic stability of the generated vaccine strain in vivo. Furthermore, a pronounced T-cell response against Yersinia Hsp60(74-86) was induced in mice vaccinated with a Salmonella vaccine strain expressing the Hsp60(74-86)-AIDA-I fusion protein. This was shown by monitoring Yersinia Hsp60-stimulated IFN-gamma secretion and proliferation of splenic T cells isolated from vaccinated mice. These results demonstrate that the surface display of antigenic determinants by the autotransporter pathway deserves special attention regarding the application in live attenuated Salmonella vaccine strains.