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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

Mucosal antibody responses to OVA. (a) Fecal anti-OVA IgG and (b) IgA responses; (c) Vaginal anti-OVA IgA responses. (White bars) g.g. (Dark Grey Bars) i.m. (Grey bars) i.na. immunized mice. Striped bars indicate the use of pFliC(-gly); (d) Lung anti-OVA IgA responses shown are from mice only vaccinated i.na. and immunizations given are shown below the axis. Results are representative of two independent experiments (n = 7–8 mice/group). The concentration of OVA-specific Abs in samples are expressed as OD equal to, or higher than, the mean OD of the values of samples from unimmunized mice. The error bars represent SEM calculated from the mean OD. * 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-f003: Mucosal antibody responses to OVA. (a) Fecal anti-OVA IgG and (b) IgA responses; (c) Vaginal anti-OVA IgA responses. (White bars) g.g. (Dark Grey Bars) i.m. (Grey bars) i.na. immunized mice. Striped bars indicate the use of pFliC(-gly); (d) Lung anti-OVA IgA responses shown are from mice only vaccinated i.na. and immunizations given are shown below the axis. Results are representative of two independent experiments (n = 7–8 mice/group). The concentration of OVA-specific Abs in samples are expressed as OD equal to, or higher than, the mean OD of the values of samples from unimmunized mice. The error bars represent SEM calculated from the mean OD. * 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 assess whether DNA vaccination of mice with pOVA and empty vector or pFliC(-gly) by various routes could elicit antibody responses in mucosal compartments, we collected extracts from fecal pellets, vaginal washes, and extracts from lung homogenates. We observed no significant differences in total amounts of total IgG or IgA immunoglobulins isolated from g.g., i.m. or i.na.-immunized animals (data not shown). Fecal extract samples were assessed for the relative amount of anti-OVA total IgG and IgA. We were able to detect significant increases in fecal anti-OVA IgG and IgA in the groups of mice vaccinated intranasally with pOVA together with the highest amounts of pFliC(-gly), but not when the same plasmids were delivered by g.g. or i.m. (Figure 3a–b). Similarly, only animals receiving the highest doses of pFliC(-gly) and pOVA intranasally developed measurable anti-OVA IgA in the vaginal washes (Figure 3c) and lungs (Figure 3d).


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)

Mucosal antibody responses to OVA. (a) Fecal anti-OVA IgG and (b) IgA responses; (c) Vaginal anti-OVA IgA responses. (White bars) g.g. (Dark Grey Bars) i.m. (Grey bars) i.na. immunized mice. Striped bars indicate the use of pFliC(-gly); (d) Lung anti-OVA IgA responses shown are from mice only vaccinated i.na. and immunizations given are shown below the axis. Results are representative of two independent experiments (n = 7–8 mice/group). The concentration of OVA-specific Abs in samples are expressed as OD equal to, or higher than, the mean OD of the values of samples from unimmunized mice. The error bars represent SEM calculated from the mean OD. * 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
Show All Figures
getmorefigures.php?uid=PMC4494208&req=5

vaccines-01-00415-f003: Mucosal antibody responses to OVA. (a) Fecal anti-OVA IgG and (b) IgA responses; (c) Vaginal anti-OVA IgA responses. (White bars) g.g. (Dark Grey Bars) i.m. (Grey bars) i.na. immunized mice. Striped bars indicate the use of pFliC(-gly); (d) Lung anti-OVA IgA responses shown are from mice only vaccinated i.na. and immunizations given are shown below the axis. Results are representative of two independent experiments (n = 7–8 mice/group). The concentration of OVA-specific Abs in samples are expressed as OD equal to, or higher than, the mean OD of the values of samples from unimmunized mice. The error bars represent SEM calculated from the mean OD. * 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 assess whether DNA vaccination of mice with pOVA and empty vector or pFliC(-gly) by various routes could elicit antibody responses in mucosal compartments, we collected extracts from fecal pellets, vaginal washes, and extracts from lung homogenates. We observed no significant differences in total amounts of total IgG or IgA immunoglobulins isolated from g.g., i.m. or i.na.-immunized animals (data not shown). Fecal extract samples were assessed for the relative amount of anti-OVA total IgG and IgA. We were able to detect significant increases in fecal anti-OVA IgG and IgA in the groups of mice vaccinated intranasally with pOVA together with the highest amounts of pFliC(-gly), but not when the same plasmids were delivered by g.g. or i.m. (Figure 3a–b). Similarly, only animals receiving the highest doses of pFliC(-gly) and pOVA intranasally developed measurable anti-OVA IgA in the vaginal washes (Figure 3c) and lungs (Figure 3d).

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