There is an urgent medical need for an efficacious Salmonella vaccine with broad coverage of invasive serovars. One important bottleneck in the development of such a vaccine is the identification of suitable protective antigens. In this study, we identified broadly conserved S. Typhi antigen candidates that prolonged survival after S. Typhimurium challenge infection in the mouse typhoid fever model. The protectivity of some of these candidates should be confirmed with larger experimental groups to select the best antigen candidates for vaccine development in future studies.
Two siderophore receptors (IroN, CirA) enabled the longest survival () consistent with previous studies that revealed siderophore receptors including IroN as promising vaccine antigens in other models 
. Interestingly, siderophore receptors are induced by iron starvation and/or activation of the PhoPQ two component sensory system 
. IroN and CirA induction could thus contribute to increased protective efficacy of membrane preparations from iron-starved Salmonella
, or live attenuated Salmonella phoQ24
with constitutive hyperactivation of the PhoP response regulator 
On the other hand, all identified antigens still provided at most partial protection against challenge infection with virulent Salmonella suggesting a need for additional antigens. Unfortunately, protective Salmonella antigens might be rather rare as even among the 37 tested in vivo expressed antigens that were all highly immunogenic during infection, only a small minority enabled prolonged survival. OmpC, OmpD, and OmpF were previously proposed as potential protective antigens based on data obtained for enriched Salmonella membrane preparations. However, all three antigens failed to protect in our model. This could reflect higher stringency of our model (challenge infection with virulent Salmonella vs. highly attenuated mutant Salmonella), denatured three-dimensional structures of our recombinant antigen preparations vs. native antigens, and/or presence of undetected minor protective antigens (such as IroN and CirA) besides OmpC, OmpD, and OmpF in the previously used outer membrane antigen preparations.
Additional protective Salmonella
antigens could be identified by comprehensive immunization/challenge experiments, but this would require extensive animal experimentation. Antigen priorization using relevant antigen properties could help to narrow down the number of antigen candidates to more practical numbers. Unfortunately, some previously proposed antigen properties seemed to have limited relevance for protectivity in our model. This included Salmonella
in vivo expression levels, sequence-based antigenicity predictions, and immunodominance in convalescent individuals. Poor correlation of antigen immunodominance with protective efficacy has also been observed in tuberculosis 
. On the other hand, immune recognition in convalescent individuals can still provide valuable information about antigen expression during at least some stage of infection that might be difficult to obtain otherwise 
. Such data thus could greatly help to prioritize antigen candidates 
In contrast to antigen abundance and immunodominance, surface-association appeared to be an essential prerequisite. Surprisingly, some surface-associated proteins that enabled prolonged survival also included lipoproteins which were likely to reside in the inner leaflet of the outer membrane facing the internal periplasmic space with no exposure to the outside. It is possible that some lipoproteins might flip across the outer membrane as observed for other Gram-negative bacteria 
. Moreover, some lipoprotein fraction is constantly released to the outside through outer membrane vesicle shedding 
Several mechanisms could contribute to the striking superiority of surface-associated antigens. Antibody responses are important for full protection against virulent Salmonella
, and protective antibody responses must be directed against surface antigens 
. On the other hand, CD4 T cells are even more important for immunity to Salmonella
at least in the mouse typhoid fever model 
, and it is unclear why CD4 T cells should respond to surface-associated antigens in a fundamentally different way compared to the much larger number of internal antigens.
In fact, early cell culture experiments suggested no impact of Salmonella
antigen localization on CD4 T cell recognition of infected cells 
. However, in this study a large amount of antigen was already present in the inoculum, and rapid killing of the majority of phagocytosed Salmonella
would have released this antigen from all Salmonella
compartments. Several subsequent in vivo studies suggested that surface-associated model antigens might have intrinsically higher immunogenicity compared to internal model antigens 
. However, the various model antigen targeting constructs could have differed in antigen in vivo expression levels, antigen stability, and epitope processing. Fusion partners could also have direct immunomodulatory effects. We therefore re-visited this issue and tried to control some of these factors. Our results clearly supported the previous finding of superior immunogenicity of highly expressed surface-associated model antigens in Salmonella
In surprising contrast to these data from model antigens, however, humoral and cellular immune responses in Salmonella
-infected convalescent mice did not show any bias for surface-associated autologous Salmonella
antigens in this as well as in a recent large-scale study 
. Broad recognition of antigens from all pathogens compartments has also been observed in Salmonella
Typhi-infected or Chlamydia
-infected human patients 
. Model antigens and autologous antigens were also discordant with respect to the impact of antigen abundance. Specifically, our data for ovalbumin model antigens in this and a previous study 
, as well as similar findings for Mycobacterium bovis
BCG overexpressing Ag85b 
, suggested that high in vivo expression levels enhance antigen immunogenicity. However, for autologous Salmonella
antigens in vivo expression levels did not correlate with protectivity. Striking discrepancies between results for model antigens vs. autologous antigens have also been observed in other pathogens 
Some of the discrepancies could reflect technical issues. In particular, strong expression of foreign surface model antigens might induce subtle alteration in Salmonella in vivo properties such as increasing outer membrane vesicle shedding or alterations in protein secretion that could affect antigen presentation and immune recognition. Furthermore, model antigens might not be representative of autologous antigens that may have been shaped by host/pathogen co-evolution selecting for weak immunogenicity. Regardless of the actual causes of these discrepancies, our data indicated that in contrast to evidence from model antigens, protective Salmonella surface-associated antigens were not more immunogenic compared to internal antigens.
As an alternative explanation, surface-associated antigens might become more rapidly available for immune recognition compared to internal antigens that are only released after some pathogen damage. This could be relevant since early immune responses might facilitate infection control 
. In the mouse typhoid fever model, however, a detectable fraction of Salmonella
is rapidly killed early during infection as observed in this and previous studies 
similar to events during Mycobacterium
. Consistent with these observations, CD4 T cell induction kinetics in the ovalbumin model system were similar for Salmonella
strains with internal or surface-associated OVA-expression.
Instead, we propose an alternative explanation based on the observation that many live Salmonella resided alone, or together with other live Salmonella, in infected host cells with no dead Salmonella releasing their internal antigens. As a consequence, Salmonella internal antigens remained inaccessible for antigen processing and presentation in these cells. In contrast, surface-exposed Salmonella antigens, or antigens released by outer membrane vesicle shedding, could be accessible for processing and presentation to cognate CD4 T cells for initiation of protective anti-Salmonella effector mechanisms (). In comparison, CD4 T cells recognizing internal Salmonella antigens would have limited impact on infection control because they miss many cells containing live Salmonella and instead direct their responses to host cells containing already dead Salmonella. According to this model, surface-associated antigens thus differ fundamentally from internal antigens because they are uniquely accessible in host cells containing only live Salmonella.
Schematic model for cellular immunity to Salmonella.
Surface-associated/secreted antigens have been shown to be crucial for CD8 T cell-dependent immunity to Listeria
. Our data suggested that such antigens might also be crucial for CD4 T cell mediated immunity to Salmonella
and potentially other intracellular pathogens. Interestingly, some internal antigens have been shown to confer partial protection in infectious diseases caused by intracellular pathogens such as Leishmania
. In these infections live and dead pathogens often co-occur in the same host microenvironments 
suggesting that both internal and surface-associated antigens might be available for T cell recognition and initiation of antimicrobial immune effector mechanisms targeting both live and already dead pathogens 
. We speculate that full protection might still require immune detection of all live pathogens including those that reside in microenvironments with yet no accessible internal antigens from dead pathogens. Further studies are required to test this hypothesis.
This study suggested novel Salmonella antigens that conferred partial protection against virulent Salmonella in a stringent typhoid fever model. High sequence conservation among relevant Salmonella serovars and cross-protection of serovar Typhi antigens against serovar Typhimurium challenge infection, suggested that some of these antigens might help to pave the way for a broadly protective vaccine against systemic Salmonella infection. In addition, our findings suggested that surface-associated antigens might represent particular promising antigens for both humoral and cellular immunity to Salmonella, since recognition of surface antigens uniquely enables detection and destruction of live Salmonella in relevant host microenvironments. This crucial importance of antigen localization could facilitate discovery of additional protective antigens for Salmonella and potentially other intracellular pathogens.