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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

Vaccination schedule and serum antibody responses to OVA. (a) Immunization and sample isolation timeline; (b) Anti-OVA total IgG responses. Anti-OVA IgG1 (c), IgG2b (d), IgG2c (e) responses. (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). The concentration of OVA-specific Abs are expressed as the reciprocal of the last dilution of samples giving an OD equal to, or higher than, the mean + 3 SDs (the determined cutoff value for the assay) of the values of serum samples from unimmunized mice. Absorbance values equal to or above the cutoff value were considered positive. The error bars represent 95% confidence intervals calculated from the geometric mean titers. * Differences of the response relative to pOVA immunizations without pFliC(-gly) defined as p ≤ 0.05 were considered significant using a two-tailed unpaired Student t test.
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vaccines-01-00415-f002: Vaccination schedule and serum antibody responses to OVA. (a) Immunization and sample isolation timeline; (b) Anti-OVA total IgG responses. Anti-OVA IgG1 (c), IgG2b (d), IgG2c (e) responses. (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). The concentration of OVA-specific Abs are expressed as the reciprocal of the last dilution of samples giving an OD equal to, or higher than, the mean + 3 SDs (the determined cutoff value for the assay) of the values of serum samples from unimmunized mice. Absorbance values equal to or above the cutoff value were considered positive. The error bars represent 95% confidence intervals calculated from the geometric mean titers. * Differences of the response relative to pOVA immunizations without pFliC(-gly) defined as p ≤ 0.05 were considered significant using a two-tailed unpaired Student t test.

Mentions: To compare the effectiveness of secreted flagellin (pFliC(-gly)) as a DNA adjuvant by various routes, DNA vaccinations were carried out using plasmid pOVA together with empty vector (pcDNA3.1/Zeo(+)) or with vector expressing pFliC(-gly). Empty vector control was used to include possible adjuvant effects contributed by sensing of B-DNA by innate immune receptors [22,23] but to exclude adjuvant effects contributed by secreted flagellin (Table 1). Vaccinated mice were primed once, boosted once and then sampled 9 and 10 days later (Figure 2a). The amount of total DNA given to the mice varied and was dependent on the limitations of the delivery route (Table 1). Mice were vaccinated by three different routes representative of dermal (gene-gun, g.g.), systemic (intramuscular, i.m.), and mucosal tissues (intra-nasal, i.na.). A constant sub-optimal amount of pOVA was used with each route (0.5 μg/g.g., 10 μg/i.m., 5 μg/i.na.) to allow the study of the adjuvant effects of flagellin.


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)

Vaccination schedule and serum antibody responses to OVA. (a) Immunization and sample isolation timeline; (b) Anti-OVA total IgG responses. Anti-OVA IgG1 (c), IgG2b (d), IgG2c (e) responses. (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). The concentration of OVA-specific Abs are expressed as the reciprocal of the last dilution of samples giving an OD equal to, or higher than, the mean + 3 SDs (the determined cutoff value for the assay) of the values of serum samples from unimmunized mice. Absorbance values equal to or above the cutoff value were considered positive. The error bars represent 95% confidence intervals calculated from the geometric mean titers. * Differences of the response relative to pOVA immunizations without pFliC(-gly) defined as p ≤ 0.05 were considered significant using a two-tailed unpaired Student t test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4494208&req=5

vaccines-01-00415-f002: Vaccination schedule and serum antibody responses to OVA. (a) Immunization and sample isolation timeline; (b) Anti-OVA total IgG responses. Anti-OVA IgG1 (c), IgG2b (d), IgG2c (e) responses. (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). The concentration of OVA-specific Abs are expressed as the reciprocal of the last dilution of samples giving an OD equal to, or higher than, the mean + 3 SDs (the determined cutoff value for the assay) of the values of serum samples from unimmunized mice. Absorbance values equal to or above the cutoff value were considered positive. The error bars represent 95% confidence intervals calculated from the geometric mean titers. * Differences of the response relative to pOVA immunizations without pFliC(-gly) defined as p ≤ 0.05 were considered significant using a two-tailed unpaired Student t test.
Mentions: To compare the effectiveness of secreted flagellin (pFliC(-gly)) as a DNA adjuvant by various routes, DNA vaccinations were carried out using plasmid pOVA together with empty vector (pcDNA3.1/Zeo(+)) or with vector expressing pFliC(-gly). Empty vector control was used to include possible adjuvant effects contributed by sensing of B-DNA by innate immune receptors [22,23] but to exclude adjuvant effects contributed by secreted flagellin (Table 1). Vaccinated mice were primed once, boosted once and then sampled 9 and 10 days later (Figure 2a). The amount of total DNA given to the mice varied and was dependent on the limitations of the delivery route (Table 1). Mice were vaccinated by three different routes representative of dermal (gene-gun, g.g.), systemic (intramuscular, i.m.), and mucosal tissues (intra-nasal, i.na.). A constant sub-optimal amount of pOVA was used with each route (0.5 μg/g.g., 10 μg/i.m., 5 μg/i.na.) to allow the study of the adjuvant effects of flagellin.

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