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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

Class I- and Class II-dependent T cell responses to OVA. IFNγ ELISPOT analysis of splenic T cell responses to (a) Class-I and (b) Class-II MHC binding OVA peptides after vaccination. (White bars) g.g. (Dark Grey Bars) i.m. (Grey bars) i.na. immunized mice. Striped bars indicate the use of pFliC(-gly). Results are representative of two independent experiments (n = 7–8 mice/group). Data is expressed as the calculated geometric mean of the Ag-stimulated cells minus unstimulated cells. The error bars representSEM calculated from the mean SFC/106 splenocytes. Statistical analyses were conducted using a two-tailed Student t test. * Differences of the response relative to pOVA immunizations without pFliC(-gly) defined as p ≤ 0.05 were considered significant using an two-tailed unpaired student t test.
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vaccines-01-00415-f004: Class I- and Class II-dependent T cell responses to OVA. IFNγ ELISPOT analysis of splenic T cell responses to (a) Class-I and (b) Class-II MHC binding OVA peptides after vaccination. (White bars) g.g. (Dark Grey Bars) i.m. (Grey bars) i.na. immunized mice. Striped bars indicate the use of pFliC(-gly). Results are representative of two independent experiments (n = 7–8 mice/group). Data is expressed as the calculated geometric mean of the Ag-stimulated cells minus unstimulated cells. The error bars representSEM calculated from the mean SFC/106 splenocytes. Statistical analyses were conducted using a two-tailed Student t test. * Differences of the response relative to pOVA immunizations without pFliC(-gly) defined as p ≤ 0.05 were considered significant using an two-tailed unpaired student t test.

Mentions: MHC class I-dependent responses were analyzed by stimulation of splenocytes from immunized mice with peptide representing the immunodominant OVA H2-Kb restricted epitope. We observed significant increases in the numbers of antigen-specific IFNγ-producing cells in mice receiving either g.g. or i.na. immunization with pOVA and pFliC(-gly) when compared to mice receiving pOVA together with empty vector (Figure 4a). We also observed a reproducible trend of pFliC(-gly) to promote antigen-specific increases in IFNγ-producing cells when mice were vaccinated i.m. (Figure 4a). These Class I cellular responses were dependent on the dose of pFliC(-gly) delivered as mice given 0.1 or 0.2 μg of pFliC(-gly) by g.g or mice given 2 or 5 μg of pFliC(-gly) i.m. did not exhibit detectable Class I-dependent responses (data not shown; Table 1, Groups 2, 3 and 6, 7 respectively). When Class II-dependent cellular immune responses were studied by stimulating splenocytes with the immunodominant I-Ab binding OVA peptide we observed significant increases in the numbers of antigen-specific IFNγ-producing cells in mice receiving pOVA intranasally together with the highest amounts of pFliC(-gly), but not with pOVA and empty vector (Figure 4b). We did not observe any OVA-specific class II-restricted responses after i.m or g.g. immunization (Figure 4b).


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)

Class I- and Class II-dependent T cell responses to OVA. IFNγ ELISPOT analysis of splenic T cell responses to (a) Class-I and (b) Class-II MHC binding OVA peptides after vaccination. (White bars) g.g. (Dark Grey Bars) i.m. (Grey bars) i.na. immunized mice. Striped bars indicate the use of pFliC(-gly). Results are representative of two independent experiments (n = 7–8 mice/group). Data is expressed as the calculated geometric mean of the Ag-stimulated cells minus unstimulated cells. The error bars representSEM calculated from the mean SFC/106 splenocytes. Statistical analyses were conducted using a two-tailed Student t test. * Differences of the response relative to pOVA immunizations without pFliC(-gly) defined as p ≤ 0.05 were considered significant using an two-tailed unpaired student t test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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vaccines-01-00415-f004: Class I- and Class II-dependent T cell responses to OVA. IFNγ ELISPOT analysis of splenic T cell responses to (a) Class-I and (b) Class-II MHC binding OVA peptides after vaccination. (White bars) g.g. (Dark Grey Bars) i.m. (Grey bars) i.na. immunized mice. Striped bars indicate the use of pFliC(-gly). Results are representative of two independent experiments (n = 7–8 mice/group). Data is expressed as the calculated geometric mean of the Ag-stimulated cells minus unstimulated cells. The error bars representSEM calculated from the mean SFC/106 splenocytes. Statistical analyses were conducted using a two-tailed Student t test. * Differences of the response relative to pOVA immunizations without pFliC(-gly) defined as p ≤ 0.05 were considered significant using an two-tailed unpaired student t test.
Mentions: MHC class I-dependent responses were analyzed by stimulation of splenocytes from immunized mice with peptide representing the immunodominant OVA H2-Kb restricted epitope. We observed significant increases in the numbers of antigen-specific IFNγ-producing cells in mice receiving either g.g. or i.na. immunization with pOVA and pFliC(-gly) when compared to mice receiving pOVA together with empty vector (Figure 4a). We also observed a reproducible trend of pFliC(-gly) to promote antigen-specific increases in IFNγ-producing cells when mice were vaccinated i.m. (Figure 4a). These Class I cellular responses were dependent on the dose of pFliC(-gly) delivered as mice given 0.1 or 0.2 μg of pFliC(-gly) by g.g or mice given 2 or 5 μg of pFliC(-gly) i.m. did not exhibit detectable Class I-dependent responses (data not shown; Table 1, Groups 2, 3 and 6, 7 respectively). When Class II-dependent cellular immune responses were studied by stimulating splenocytes with the immunodominant I-Ab binding OVA peptide we observed significant increases in the numbers of antigen-specific IFNγ-producing cells in mice receiving pOVA intranasally together with the highest amounts of pFliC(-gly), but not with pOVA and empty vector (Figure 4b). We did not observe any OVA-specific class II-restricted responses after i.m or g.g. immunization (Figure 4b).

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