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Immunity to intracellular Salmonella depends on surface-associated antigens.

Barat S, Willer Y, Rizos K, Claudi B, Mazé A, Schemmer AK, Kirchhoff D, Schmidt A, Burton N, Bumann D - PLoS Pathog. (2012)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Focal Area Infection Biology, Biozentrum, University of Basel, Basel, Switzerland.

ABSTRACT
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.

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Related in: MedlinePlus

CD4 T cell responses to Salmonella expressing an ovalbumin model antigen in various compartments.A) Schematic overview of fusion proteins that target an immunodominant ovalbumin epitope (OVA) to various Salmonella cell compartments. B) Flow cytometric analysis of ovalbumin-specific CD4 T cell activation in a T cell receptor-transgenic adoptive transfer model. Mice were infected with control Salmonella expressing GFP (left) or Salmonella expressing LPP_OVA (right). Ovalbumin-specific transgenic CD4 T cells were detected with a clonotypic monoclonal antibody and analyzed for forward scatter and expression of the very early activation marker CD69. The dashed line was used to count CD4 T cell blasts. Similar observations were made for more than hundred mice in several independent experiments. C) Relationship between Salmonella Peyer's patches colonization and OVA-specific CD4 T cell induction in mice infected with Salmonella expressing high levels of LPP_OVA (filled circles) or low levels of GFP_OVA (open circles). Data represent means ± SEM's for groups of five to six animals from three independent experiments. CD4 T cell blasts correlated with Salmonella Peyer's patches colonization for both strains (Spearman test, P<0.05 in both cases). The slopes of the two curves differed (ANCOVA; P<0.05). D) OVA-specific CD4 T cell induction in mice infected with Salmonella expressing OVA at various levels (open circles, low abundance; filled circles, high abundance) in four different compartments. The dashed line represents CD4 T cell responses to saturating levels of cytosolic OVA. The star represents data for Salmonella expressing moderate levels of cytosolic OVA together with cholera toxin B and AIDA. Data represent means ± SEM's for groups of ten to twenty mice. Statistical significance of differences to Salmonella expressing saturating levels of cytosolic OVA were tested using Mann-Whitney U test (*, P<0.05; **, P<0.01).
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ppat-1002966-g006: CD4 T cell responses to Salmonella expressing an ovalbumin model antigen in various compartments.A) Schematic overview of fusion proteins that target an immunodominant ovalbumin epitope (OVA) to various Salmonella cell compartments. B) Flow cytometric analysis of ovalbumin-specific CD4 T cell activation in a T cell receptor-transgenic adoptive transfer model. Mice were infected with control Salmonella expressing GFP (left) or Salmonella expressing LPP_OVA (right). Ovalbumin-specific transgenic CD4 T cells were detected with a clonotypic monoclonal antibody and analyzed for forward scatter and expression of the very early activation marker CD69. The dashed line was used to count CD4 T cell blasts. Similar observations were made for more than hundred mice in several independent experiments. C) Relationship between Salmonella Peyer's patches colonization and OVA-specific CD4 T cell induction in mice infected with Salmonella expressing high levels of LPP_OVA (filled circles) or low levels of GFP_OVA (open circles). Data represent means ± SEM's for groups of five to six animals from three independent experiments. CD4 T cell blasts correlated with Salmonella Peyer's patches colonization for both strains (Spearman test, P<0.05 in both cases). The slopes of the two curves differed (ANCOVA; P<0.05). D) OVA-specific CD4 T cell induction in mice infected with Salmonella expressing OVA at various levels (open circles, low abundance; filled circles, high abundance) in four different compartments. The dashed line represents CD4 T cell responses to saturating levels of cytosolic OVA. The star represents data for Salmonella expressing moderate levels of cytosolic OVA together with cholera toxin B and AIDA. Data represent means ± SEM's for groups of ten to twenty mice. Statistical significance of differences to Salmonella expressing saturating levels of cytosolic OVA were tested using Mann-Whitney U test (*, P<0.05; **, P<0.01).

Mentions: To better understand these discrepancies between model antigens and autologous Salmonella antigens, we re-visited the impact of antigen localization using a well-characterized, sensitive model system in which a MHC II-restricted T cell epitope from ovalbumin comprising amino acids 319 to 343 (OVA) is recognized by adoptively transferred cognate T cell receptor transgenic CD4 T cells [51], [52]. We targeted the OVA epitope to different Salmonella compartments by fusing it to various proteins with known localization: GFP_OVA (cytosol [53]), OVA_MglB (periplasm [54]), Lpp_OVA (inner leaflet of the outer membrane [55]), and OVA_AIDA (outer leaflet of the outer membrane [56]) (Fig. 6A). To modulate expression levels, we used ribosome binding sites with differential translation initiation efficiency [12]. We expressed these fusion proteins from an in vivo inducible promoter [57] in an attenuated Salmonella enterica serovar Typhimurium aroA strain [58]. Antigen expression and localization was validated in in vitro cultures using cell fractionation followed by western blotting, trypsin treatment, and antibody binding (Fig. S2). Interestingly, small fractions of both outer membrane antigens LPP_OVA and partially processed OVA_AIDA were released to the extracellular surroundings when expressed at high levels (Fig. S2C) in agreement with previous findings for similar proteins [59]–[61].


Immunity to intracellular Salmonella depends on surface-associated antigens.

Barat S, Willer Y, Rizos K, Claudi B, Mazé A, Schemmer AK, Kirchhoff D, Schmidt A, Burton N, Bumann D - PLoS Pathog. (2012)

CD4 T cell responses to Salmonella expressing an ovalbumin model antigen in various compartments.A) Schematic overview of fusion proteins that target an immunodominant ovalbumin epitope (OVA) to various Salmonella cell compartments. B) Flow cytometric analysis of ovalbumin-specific CD4 T cell activation in a T cell receptor-transgenic adoptive transfer model. Mice were infected with control Salmonella expressing GFP (left) or Salmonella expressing LPP_OVA (right). Ovalbumin-specific transgenic CD4 T cells were detected with a clonotypic monoclonal antibody and analyzed for forward scatter and expression of the very early activation marker CD69. The dashed line was used to count CD4 T cell blasts. Similar observations were made for more than hundred mice in several independent experiments. C) Relationship between Salmonella Peyer's patches colonization and OVA-specific CD4 T cell induction in mice infected with Salmonella expressing high levels of LPP_OVA (filled circles) or low levels of GFP_OVA (open circles). Data represent means ± SEM's for groups of five to six animals from three independent experiments. CD4 T cell blasts correlated with Salmonella Peyer's patches colonization for both strains (Spearman test, P<0.05 in both cases). The slopes of the two curves differed (ANCOVA; P<0.05). D) OVA-specific CD4 T cell induction in mice infected with Salmonella expressing OVA at various levels (open circles, low abundance; filled circles, high abundance) in four different compartments. The dashed line represents CD4 T cell responses to saturating levels of cytosolic OVA. The star represents data for Salmonella expressing moderate levels of cytosolic OVA together with cholera toxin B and AIDA. Data represent means ± SEM's for groups of ten to twenty mice. Statistical significance of differences to Salmonella expressing saturating levels of cytosolic OVA were tested using Mann-Whitney U test (*, P<0.05; **, P<0.01).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475680&req=5

ppat-1002966-g006: CD4 T cell responses to Salmonella expressing an ovalbumin model antigen in various compartments.A) Schematic overview of fusion proteins that target an immunodominant ovalbumin epitope (OVA) to various Salmonella cell compartments. B) Flow cytometric analysis of ovalbumin-specific CD4 T cell activation in a T cell receptor-transgenic adoptive transfer model. Mice were infected with control Salmonella expressing GFP (left) or Salmonella expressing LPP_OVA (right). Ovalbumin-specific transgenic CD4 T cells were detected with a clonotypic monoclonal antibody and analyzed for forward scatter and expression of the very early activation marker CD69. The dashed line was used to count CD4 T cell blasts. Similar observations were made for more than hundred mice in several independent experiments. C) Relationship between Salmonella Peyer's patches colonization and OVA-specific CD4 T cell induction in mice infected with Salmonella expressing high levels of LPP_OVA (filled circles) or low levels of GFP_OVA (open circles). Data represent means ± SEM's for groups of five to six animals from three independent experiments. CD4 T cell blasts correlated with Salmonella Peyer's patches colonization for both strains (Spearman test, P<0.05 in both cases). The slopes of the two curves differed (ANCOVA; P<0.05). D) OVA-specific CD4 T cell induction in mice infected with Salmonella expressing OVA at various levels (open circles, low abundance; filled circles, high abundance) in four different compartments. The dashed line represents CD4 T cell responses to saturating levels of cytosolic OVA. The star represents data for Salmonella expressing moderate levels of cytosolic OVA together with cholera toxin B and AIDA. Data represent means ± SEM's for groups of ten to twenty mice. Statistical significance of differences to Salmonella expressing saturating levels of cytosolic OVA were tested using Mann-Whitney U test (*, P<0.05; **, P<0.01).
Mentions: To better understand these discrepancies between model antigens and autologous Salmonella antigens, we re-visited the impact of antigen localization using a well-characterized, sensitive model system in which a MHC II-restricted T cell epitope from ovalbumin comprising amino acids 319 to 343 (OVA) is recognized by adoptively transferred cognate T cell receptor transgenic CD4 T cells [51], [52]. We targeted the OVA epitope to different Salmonella compartments by fusing it to various proteins with known localization: GFP_OVA (cytosol [53]), OVA_MglB (periplasm [54]), Lpp_OVA (inner leaflet of the outer membrane [55]), and OVA_AIDA (outer leaflet of the outer membrane [56]) (Fig. 6A). To modulate expression levels, we used ribosome binding sites with differential translation initiation efficiency [12]. We expressed these fusion proteins from an in vivo inducible promoter [57] in an attenuated Salmonella enterica serovar Typhimurium aroA strain [58]. Antigen expression and localization was validated in in vitro cultures using cell fractionation followed by western blotting, trypsin treatment, and antibody binding (Fig. S2). Interestingly, small fractions of both outer membrane antigens LPP_OVA and partially processed OVA_AIDA were released to the extracellular surroundings when expressed at high levels (Fig. S2C) in agreement with previous findings for similar proteins [59]–[61].

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Focal Area Infection Biology, Biozentrum, University of Basel, Basel, Switzerland.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus