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DNA-Encoded Flagellin Activates Toll-Like Receptor 5 (TLR5), Nod-like Receptor Family CARD Domain-Containing Protein 4 (NRLC4), and Acts as an Epidermal, Systemic, and Mucosal-Adjuvant.

Nyström S, Bråve A, Falkeborn T, Devito C, Rissiek B, Johansson DX, Schröder U, Uematsu S, Akira S, Hinkula J, Applequist SE - Vaccines (Basel) (2013)

Bottom Line: Eliciting effective immune responses using non-living/replicating DNA vaccines is a significant challenge.By all three routes we observed increases in antigen-specific antibodies in serum as well as MHC Class I-dependent cellular immune responses when pFliC(-gly) adjuvant was added.We also observed enhancement of cross-clade reactive IgA as well as a broadening of B cell epitope reactivity.

View Article: PubMed Central - PubMed

Affiliation: Center for Infectious Medicine, F59, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm 141 86, Sweden.

ABSTRACT
Eliciting effective immune responses using non-living/replicating DNA vaccines is a significant challenge. We have previously shown that ballistic dermal plasmid DNA-encoded flagellin (FliC) promotes humoral as well as cellular immunity to co-delivered antigens. Here, we observe that a plasmid encoding secreted FliC (pFliC(-gly)) produces flagellin capable of activating two innate immune receptors known to detect flagellin; Toll-like Receptor 5 (TLR5) and Nod-like Receptor family CARD domain-containing protein 4 (NRLC4). To test the ability of pFliC(-gly) to act as an adjuvant we immunized mice with plasmid encoding secreted FliC (pFliC(-gly)) and plasmid encoding a model antigen (ovalbumin) by three different immunization routes representative of dermal, systemic, and mucosal tissues. By all three routes we observed increases in antigen-specific antibodies in serum as well as MHC Class I-dependent cellular immune responses when pFliC(-gly) adjuvant was added. Additionally, we were able to induce mucosal antibody responses and Class II-dependent cellular immune responses after mucosal vaccination with pFliC(-gly). Humoral immune responses elicited by heterologus prime-boost immunization with a plasmid encoding HIV-1 from gp160 followed by protein boosting could be enhanced by use of pFliC(-gly). We also observed enhancement of cross-clade reactive IgA as well as a broadening of B cell epitope reactivity. These observations indicate that plasmid-encoded secreted flagellin can activate multiple innate immune responses and function as an adjuvant to non-living/replicating DNA immunizations. Moreover, the capacity to elicit mucosal immune responses, in addition to dermal and systemic properties, demonstrates the potential of flagellin to be used with vaccines designed to be delivered by various routes.

No MeSH data available.


Related in: MedlinePlus

Flagellin (FliC)(-gly) variants for in vivo and in vitro use activate innate immune responses. (a) Depiction of pFliC(-gly) and variants relative to FliC polypeptide and domains produced by S. typhimurium. Grey domains D0/1 indicate conserved regions important for activating innate immune responses. L and HA indicates a leader and HA-epitope tag domains respectively. Names of the four FliC(-gly) constructs used in this study are indicated to the right of the drawings; (b) Western blot analysis of cellular lysates from 293T cells transfected with the indicated constructs. Apparent molecular weights were determined by comparison to the standard depicted to the left of the blot. Signals were not detected from cells transfected with empty vector (data not shown); (c) Release of TNFα from B6 alveolar macrophages but not Toll-like Receptor 5 (TLR5) −/− alveolar macrophages after stimulation with FliC(-gly) and FliC(-gly)Δ34. Supernatant from 293T cells transfected with pFliC(-gly) and pFliC(-gly)Δ34 vectors was incubated with cells for 4 h followed by analysis of secreted TNFα by ELISA. Data are mean ± SEM of triplicate samples representative of two independent experiments; (d) Activation of pyroptotic cell death by retroviral transduction of BcgR macrophages with constructs expressing FliC(-gly) but not FliC(-gly)Δ34 as determined by GFP expression. Upper panels represent representative data from BcgR cells transduced with FliC(-gly), FliC(-gly)Δ34 and controls (as indicated) when comparing GFP and forward-scatter (FS) parameters. Quantitative data of the percentage of GFP positive BcgR cells from each construct after transduction. Lower panel represent representative data from 293T cells transduced in identical fashion. Data are mean ± SEM of GFP positive cells observed during three independent transduction experiments. * Differences of the response relative to the FliC(-gly) construct without Δ34 defined as p ≤ 0.05 calculated using a two-tailed unpaired Student t test.
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vaccines-01-00415-f001: Flagellin (FliC)(-gly) variants for in vivo and in vitro use activate innate immune responses. (a) Depiction of pFliC(-gly) and variants relative to FliC polypeptide and domains produced by S. typhimurium. Grey domains D0/1 indicate conserved regions important for activating innate immune responses. L and HA indicates a leader and HA-epitope tag domains respectively. Names of the four FliC(-gly) constructs used in this study are indicated to the right of the drawings; (b) Western blot analysis of cellular lysates from 293T cells transfected with the indicated constructs. Apparent molecular weights were determined by comparison to the standard depicted to the left of the blot. Signals were not detected from cells transfected with empty vector (data not shown); (c) Release of TNFα from B6 alveolar macrophages but not Toll-like Receptor 5 (TLR5) −/− alveolar macrophages after stimulation with FliC(-gly) and FliC(-gly)Δ34. Supernatant from 293T cells transfected with pFliC(-gly) and pFliC(-gly)Δ34 vectors was incubated with cells for 4 h followed by analysis of secreted TNFα by ELISA. Data are mean ± SEM of triplicate samples representative of two independent experiments; (d) Activation of pyroptotic cell death by retroviral transduction of BcgR macrophages with constructs expressing FliC(-gly) but not FliC(-gly)Δ34 as determined by GFP expression. Upper panels represent representative data from BcgR cells transduced with FliC(-gly), FliC(-gly)Δ34 and controls (as indicated) when comparing GFP and forward-scatter (FS) parameters. Quantitative data of the percentage of GFP positive BcgR cells from each construct after transduction. Lower panel represent representative data from 293T cells transduced in identical fashion. Data are mean ± SEM of GFP positive cells observed during three independent transduction experiments. * Differences of the response relative to the FliC(-gly) construct without Δ34 defined as p ≤ 0.05 calculated using a two-tailed unpaired Student t test.

Mentions: A secreted variant of flagellin with reduced glycosylation (called pFliC(-gly)), based on the pFliC-Tm(-gly) plasmid [5], was constructed by removing the human transmembrane PDGFR domain from the ORF to eliminate potential immune responses to this region and to prepare a base vector for adjuvant use. Three additional variants of pFliC(-gly) were also constructed to test the ability of FliC(-gly) to activate the two known innate immune receptors capable of sensing flagellin TLR5 and NLRC4/Naip5. These four constructs are depicted in Figure 1a relative to the defined domains of Salmonella typhimurium FliC. To prepare pFliC(-gly) control variants capable of activating cytoplasmically expressed NLRC4/Naip5 we recloned the FliC(-gly) insert sans leader sequence (pcFliC(-gly)). We also prepared additional control versions of pFliC(-gly) and pcFliC(-gly) removing the COOH-34 amino-acids of FliC(-gly) shown to activate NLRC4/Naip5 [20]. These versions were designated pFliC(-gly)Δ34 and pcFliC(-gly)Δ34 respectively. All four vectors were capable of expressing proteins of predicted size with an apparent polypeptide of approximately 52 kDa for pFliC(-gly) and pcFliC(-gly) and approximately 48 kDa for pFliC(-gly)Δ34 and pcFliC(-gly)Δ34 (Figure 1b). To determine if secreted FliC(-gly) protein produced from pFliC vectors could activate TLR5 culture supernatants from pFliC(-gly), pFliC(-gly)Δ34 transfected 293 cells, or recombinant FliC protein were applied to alveolar macrophages from B6 or TLR5-deficient mice. Plasmid vectors produced full-length or Δ34 secreted FliC(-gly) able to activate B6 alveolar macrophages to produce TNFα but not macrophages from TLR5-deficient mice (Figure 1c). To determine if secreted FliC(-gly) has the potential to activate cytoplasmic NLRC4/Naip5 inflammasome responses we performed a retroviral lethality screen using the macrophage cell line BcgR which undergoes pyroptosis in the presence of the COOH-terminal tail of FliC [21]. This assay detects the ability of macrophages virally transduced with genes expressing GFP as well as various flagellin constructs to undergo pyroptotic cell death in response to whole flagellin dependent on the NLRC4/Naip5 35 amino-acid carboxy-terminal activating domain [20,21]. GFP positive BcgR cells are taken as evidence of a lack of NLRC4/Naip5 activation while GFP negative cells, relative to GFP positive identically transduced 293T control cells, are taken as evidence of NLRC4/Naip5 activation. FliC(-gly), cytoplasmic expressed FliC (cFLiC(-gly)), and a variant of each lacking the final 34 amino-acid COOH-tail (Δ34) (Figure 1a) were subcloned into the retroviral vector pMSCV-IRES-GFP which are designed to produce FliC(-gly) and variants as well as GFP upon transduction. Various FliC(-gly) constructs were Amphotropic packaged, and used to transduce BcgR or 293T cells. Using GFP as a reporter for FliC expression we observed that all versions of FliC(-gly) were expressed at nearly equal frequency in 293T cells indicating that all vectors were packaged with equal efficiency and could deliver GFP and FliC genes (Figure 1d). However, when identical vector preparations expressing FliC(-gly) were trandsuced into BcgR cells we observed GFP expression only with Δ34 versions (Figure 1d). These results demonstrate that secreted form of FliC(-gly) we use as an in vivo adjuvant has the ability to activate NLRC4/Naip5 pyroptotic cell death when expressed in a responsive cell type.


DNA-Encoded Flagellin Activates Toll-Like Receptor 5 (TLR5), Nod-like Receptor Family CARD Domain-Containing Protein 4 (NRLC4), and Acts as an Epidermal, Systemic, and Mucosal-Adjuvant.

Nyström S, Bråve A, Falkeborn T, Devito C, Rissiek B, Johansson DX, Schröder U, Uematsu S, Akira S, Hinkula J, Applequist SE - Vaccines (Basel) (2013)

Flagellin (FliC)(-gly) variants for in vivo and in vitro use activate innate immune responses. (a) Depiction of pFliC(-gly) and variants relative to FliC polypeptide and domains produced by S. typhimurium. Grey domains D0/1 indicate conserved regions important for activating innate immune responses. L and HA indicates a leader and HA-epitope tag domains respectively. Names of the four FliC(-gly) constructs used in this study are indicated to the right of the drawings; (b) Western blot analysis of cellular lysates from 293T cells transfected with the indicated constructs. Apparent molecular weights were determined by comparison to the standard depicted to the left of the blot. Signals were not detected from cells transfected with empty vector (data not shown); (c) Release of TNFα from B6 alveolar macrophages but not Toll-like Receptor 5 (TLR5) −/− alveolar macrophages after stimulation with FliC(-gly) and FliC(-gly)Δ34. Supernatant from 293T cells transfected with pFliC(-gly) and pFliC(-gly)Δ34 vectors was incubated with cells for 4 h followed by analysis of secreted TNFα by ELISA. Data are mean ± SEM of triplicate samples representative of two independent experiments; (d) Activation of pyroptotic cell death by retroviral transduction of BcgR macrophages with constructs expressing FliC(-gly) but not FliC(-gly)Δ34 as determined by GFP expression. Upper panels represent representative data from BcgR cells transduced with FliC(-gly), FliC(-gly)Δ34 and controls (as indicated) when comparing GFP and forward-scatter (FS) parameters. Quantitative data of the percentage of GFP positive BcgR cells from each construct after transduction. Lower panel represent representative data from 293T cells transduced in identical fashion. Data are mean ± SEM of GFP positive cells observed during three independent transduction experiments. * Differences of the response relative to the FliC(-gly) construct without Δ34 defined as p ≤ 0.05 calculated using a two-tailed unpaired Student t test.
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vaccines-01-00415-f001: Flagellin (FliC)(-gly) variants for in vivo and in vitro use activate innate immune responses. (a) Depiction of pFliC(-gly) and variants relative to FliC polypeptide and domains produced by S. typhimurium. Grey domains D0/1 indicate conserved regions important for activating innate immune responses. L and HA indicates a leader and HA-epitope tag domains respectively. Names of the four FliC(-gly) constructs used in this study are indicated to the right of the drawings; (b) Western blot analysis of cellular lysates from 293T cells transfected with the indicated constructs. Apparent molecular weights were determined by comparison to the standard depicted to the left of the blot. Signals were not detected from cells transfected with empty vector (data not shown); (c) Release of TNFα from B6 alveolar macrophages but not Toll-like Receptor 5 (TLR5) −/− alveolar macrophages after stimulation with FliC(-gly) and FliC(-gly)Δ34. Supernatant from 293T cells transfected with pFliC(-gly) and pFliC(-gly)Δ34 vectors was incubated with cells for 4 h followed by analysis of secreted TNFα by ELISA. Data are mean ± SEM of triplicate samples representative of two independent experiments; (d) Activation of pyroptotic cell death by retroviral transduction of BcgR macrophages with constructs expressing FliC(-gly) but not FliC(-gly)Δ34 as determined by GFP expression. Upper panels represent representative data from BcgR cells transduced with FliC(-gly), FliC(-gly)Δ34 and controls (as indicated) when comparing GFP and forward-scatter (FS) parameters. Quantitative data of the percentage of GFP positive BcgR cells from each construct after transduction. Lower panel represent representative data from 293T cells transduced in identical fashion. Data are mean ± SEM of GFP positive cells observed during three independent transduction experiments. * Differences of the response relative to the FliC(-gly) construct without Δ34 defined as p ≤ 0.05 calculated using a two-tailed unpaired Student t test.
Mentions: A secreted variant of flagellin with reduced glycosylation (called pFliC(-gly)), based on the pFliC-Tm(-gly) plasmid [5], was constructed by removing the human transmembrane PDGFR domain from the ORF to eliminate potential immune responses to this region and to prepare a base vector for adjuvant use. Three additional variants of pFliC(-gly) were also constructed to test the ability of FliC(-gly) to activate the two known innate immune receptors capable of sensing flagellin TLR5 and NLRC4/Naip5. These four constructs are depicted in Figure 1a relative to the defined domains of Salmonella typhimurium FliC. To prepare pFliC(-gly) control variants capable of activating cytoplasmically expressed NLRC4/Naip5 we recloned the FliC(-gly) insert sans leader sequence (pcFliC(-gly)). We also prepared additional control versions of pFliC(-gly) and pcFliC(-gly) removing the COOH-34 amino-acids of FliC(-gly) shown to activate NLRC4/Naip5 [20]. These versions were designated pFliC(-gly)Δ34 and pcFliC(-gly)Δ34 respectively. All four vectors were capable of expressing proteins of predicted size with an apparent polypeptide of approximately 52 kDa for pFliC(-gly) and pcFliC(-gly) and approximately 48 kDa for pFliC(-gly)Δ34 and pcFliC(-gly)Δ34 (Figure 1b). To determine if secreted FliC(-gly) protein produced from pFliC vectors could activate TLR5 culture supernatants from pFliC(-gly), pFliC(-gly)Δ34 transfected 293 cells, or recombinant FliC protein were applied to alveolar macrophages from B6 or TLR5-deficient mice. Plasmid vectors produced full-length or Δ34 secreted FliC(-gly) able to activate B6 alveolar macrophages to produce TNFα but not macrophages from TLR5-deficient mice (Figure 1c). To determine if secreted FliC(-gly) has the potential to activate cytoplasmic NLRC4/Naip5 inflammasome responses we performed a retroviral lethality screen using the macrophage cell line BcgR which undergoes pyroptosis in the presence of the COOH-terminal tail of FliC [21]. This assay detects the ability of macrophages virally transduced with genes expressing GFP as well as various flagellin constructs to undergo pyroptotic cell death in response to whole flagellin dependent on the NLRC4/Naip5 35 amino-acid carboxy-terminal activating domain [20,21]. GFP positive BcgR cells are taken as evidence of a lack of NLRC4/Naip5 activation while GFP negative cells, relative to GFP positive identically transduced 293T control cells, are taken as evidence of NLRC4/Naip5 activation. FliC(-gly), cytoplasmic expressed FliC (cFLiC(-gly)), and a variant of each lacking the final 34 amino-acid COOH-tail (Δ34) (Figure 1a) were subcloned into the retroviral vector pMSCV-IRES-GFP which are designed to produce FliC(-gly) and variants as well as GFP upon transduction. Various FliC(-gly) constructs were Amphotropic packaged, and used to transduce BcgR or 293T cells. Using GFP as a reporter for FliC expression we observed that all versions of FliC(-gly) were expressed at nearly equal frequency in 293T cells indicating that all vectors were packaged with equal efficiency and could deliver GFP and FliC genes (Figure 1d). However, when identical vector preparations expressing FliC(-gly) were trandsuced into BcgR cells we observed GFP expression only with Δ34 versions (Figure 1d). These results demonstrate that secreted form of FliC(-gly) we use as an in vivo adjuvant has the ability to activate NLRC4/Naip5 pyroptotic cell death when expressed in a responsive cell type.

Bottom Line: Eliciting effective immune responses using non-living/replicating DNA vaccines is a significant challenge.By all three routes we observed increases in antigen-specific antibodies in serum as well as MHC Class I-dependent cellular immune responses when pFliC(-gly) adjuvant was added.We also observed enhancement of cross-clade reactive IgA as well as a broadening of B cell epitope reactivity.

View Article: PubMed Central - PubMed

Affiliation: Center for Infectious Medicine, F59, Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm 141 86, Sweden.

ABSTRACT
Eliciting effective immune responses using non-living/replicating DNA vaccines is a significant challenge. We have previously shown that ballistic dermal plasmid DNA-encoded flagellin (FliC) promotes humoral as well as cellular immunity to co-delivered antigens. Here, we observe that a plasmid encoding secreted FliC (pFliC(-gly)) produces flagellin capable of activating two innate immune receptors known to detect flagellin; Toll-like Receptor 5 (TLR5) and Nod-like Receptor family CARD domain-containing protein 4 (NRLC4). To test the ability of pFliC(-gly) to act as an adjuvant we immunized mice with plasmid encoding secreted FliC (pFliC(-gly)) and plasmid encoding a model antigen (ovalbumin) by three different immunization routes representative of dermal, systemic, and mucosal tissues. By all three routes we observed increases in antigen-specific antibodies in serum as well as MHC Class I-dependent cellular immune responses when pFliC(-gly) adjuvant was added. Additionally, we were able to induce mucosal antibody responses and Class II-dependent cellular immune responses after mucosal vaccination with pFliC(-gly). Humoral immune responses elicited by heterologus prime-boost immunization with a plasmid encoding HIV-1 from gp160 followed by protein boosting could be enhanced by use of pFliC(-gly). We also observed enhancement of cross-clade reactive IgA as well as a broadening of B cell epitope reactivity. These observations indicate that plasmid-encoded secreted flagellin can activate multiple innate immune responses and function as an adjuvant to non-living/replicating DNA immunizations. Moreover, the capacity to elicit mucosal immune responses, in addition to dermal and systemic properties, demonstrates the potential of flagellin to be used with vaccines designed to be delivered by various routes.

No MeSH data available.


Related in: MedlinePlus