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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.We also observed enhancement of cross-clade reactive IgA as well as a broadening of B cell epitope reactivity.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.

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, serum antibody responses to gp160, and virus neutralization titers. (a) Immunization and sample isolation timeline. Priming (ImmunogenP, plasmids) and boostings (ImmunogenB, rec proteins) are indicated in days while time after the final boost are indicated in weeks. Immunization details are listed in Table 2; (b) Serum IgG titer against rgp160 at 4 weeks post immunization in all seven study groups; (c) Serum titer anti-rgp160IgG1 isotype kinetics in the four first study groups in Table 2; (d) Serum end-point titer anti-rgp160 IgG2a isotype kinetics in the four first study groups. The concentration of rgp160-specific Abs are expressed as the end-point titers 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; (e) Serum neutralization of HIV shown as IC50 in serum samples of the four first study groups in Table 2. The TCID50 (the reciprocal of the virus dilution where 50% of the cultures were infected) of IIIB (LAI) or 6794 was incubated with sample mouse serum (dilutions: 20, 60, 180, 540, 1 620). 5 × 104 cells well were then added, incubated, washed, and incubated for 7 days. Culture supernatants were tested for virus production by HIV-1 p24 capture ELISA. The lowest serum concentration giving a 50% reduction (IC50) of ELISA absorbance value compared with the mean of the negative controls are presented [19]. Statistical analyses were conducted using a two-tailed unpaired Student t test. * Differences of the responses between compared groups defined as p ≤ 0.05 were considered significant. n.s. = non-significant. Comparisons between groups with the HIV-1 antigens were performed by using the non-parametric Mann-Whitney U test with Bonferroni correction, p < 0.05 was considered significant.
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vaccines-01-00415-f005: Vaccination schedule, serum antibody responses to gp160, and virus neutralization titers. (a) Immunization and sample isolation timeline. Priming (ImmunogenP, plasmids) and boostings (ImmunogenB, rec proteins) are indicated in days while time after the final boost are indicated in weeks. Immunization details are listed in Table 2; (b) Serum IgG titer against rgp160 at 4 weeks post immunization in all seven study groups; (c) Serum titer anti-rgp160IgG1 isotype kinetics in the four first study groups in Table 2; (d) Serum end-point titer anti-rgp160 IgG2a isotype kinetics in the four first study groups. The concentration of rgp160-specific Abs are expressed as the end-point titers 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; (e) Serum neutralization of HIV shown as IC50 in serum samples of the four first study groups in Table 2. The TCID50 (the reciprocal of the virus dilution where 50% of the cultures were infected) of IIIB (LAI) or 6794 was incubated with sample mouse serum (dilutions: 20, 60, 180, 540, 1 620). 5 × 104 cells well were then added, incubated, washed, and incubated for 7 days. Culture supernatants were tested for virus production by HIV-1 p24 capture ELISA. The lowest serum concentration giving a 50% reduction (IC50) of ELISA absorbance value compared with the mean of the negative controls are presented [19]. Statistical analyses were conducted using a two-tailed unpaired Student t test. * Differences of the responses between compared groups defined as p ≤ 0.05 were considered significant. n.s. = non-significant. Comparisons between groups with the HIV-1 antigens were performed by using the non-parametric Mann-Whitney U test with Bonferroni correction, p < 0.05 was considered significant.

Mentions: Experiments with pOVA and pFliC(-gly) indicated that delivery of plasmids using N3 and the intranasal route was able to promote cellular immune responses as well as humoral mucosal immune responses. To compare the effectiveness of secreted flagellin to promote immune responses to a clinical antigen using a heterologus prime/boost regime, priming intranasal DNA vaccinations were carried out using plasmid pgp160Lfai/pRev [16] with delivery lipid N3 alone or together with pFliC(-gly). Boostings were performed using recombinant gp160 proteins with a protein-delivery lipid L3B alone or together with N3 mixed with pFliC(-gly) (Table 2). In these experiments mice were given doses of antigen and adjuvant believed to maximize detectable responses. Mice were primed, boosted, and analyzed according to the indicated timeline (Figure 5a). Four weeks post-final boost serum total-IgG titers anti-rgp160 indicated that addition of N3 to pgp160 was able to strongly promote anti-gp160 antibody responses (Figure 5b). Similar to responses seen using OVA, addition of N3/pFliC(-gly) to the immunization regime enhanced antigen-specific antibody titers further (Figure 5b). The adjuvant effect was dependent on N3. Likely due to it’s ability to encapsulate plasmid DNA and protect it from the degradative environment of the mucosal compartment. These higher titers of antigen-specific IgG in the sera and the presence of antigen-specific mucosal IgA indicate that the mucosal adjuvant effects of pFliC(-gly) are not limited to experimental antigens.


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, serum antibody responses to gp160, and virus neutralization titers. (a) Immunization and sample isolation timeline. Priming (ImmunogenP, plasmids) and boostings (ImmunogenB, rec proteins) are indicated in days while time after the final boost are indicated in weeks. Immunization details are listed in Table 2; (b) Serum IgG titer against rgp160 at 4 weeks post immunization in all seven study groups; (c) Serum titer anti-rgp160IgG1 isotype kinetics in the four first study groups in Table 2; (d) Serum end-point titer anti-rgp160 IgG2a isotype kinetics in the four first study groups. The concentration of rgp160-specific Abs are expressed as the end-point titers 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; (e) Serum neutralization of HIV shown as IC50 in serum samples of the four first study groups in Table 2. The TCID50 (the reciprocal of the virus dilution where 50% of the cultures were infected) of IIIB (LAI) or 6794 was incubated with sample mouse serum (dilutions: 20, 60, 180, 540, 1 620). 5 × 104 cells well were then added, incubated, washed, and incubated for 7 days. Culture supernatants were tested for virus production by HIV-1 p24 capture ELISA. The lowest serum concentration giving a 50% reduction (IC50) of ELISA absorbance value compared with the mean of the negative controls are presented [19]. Statistical analyses were conducted using a two-tailed unpaired Student t test. * Differences of the responses between compared groups defined as p ≤ 0.05 were considered significant. n.s. = non-significant. Comparisons between groups with the HIV-1 antigens were performed by using the non-parametric Mann-Whitney U test with Bonferroni correction, p < 0.05 was considered significant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4494208&req=5

vaccines-01-00415-f005: Vaccination schedule, serum antibody responses to gp160, and virus neutralization titers. (a) Immunization and sample isolation timeline. Priming (ImmunogenP, plasmids) and boostings (ImmunogenB, rec proteins) are indicated in days while time after the final boost are indicated in weeks. Immunization details are listed in Table 2; (b) Serum IgG titer against rgp160 at 4 weeks post immunization in all seven study groups; (c) Serum titer anti-rgp160IgG1 isotype kinetics in the four first study groups in Table 2; (d) Serum end-point titer anti-rgp160 IgG2a isotype kinetics in the four first study groups. The concentration of rgp160-specific Abs are expressed as the end-point titers 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; (e) Serum neutralization of HIV shown as IC50 in serum samples of the four first study groups in Table 2. The TCID50 (the reciprocal of the virus dilution where 50% of the cultures were infected) of IIIB (LAI) or 6794 was incubated with sample mouse serum (dilutions: 20, 60, 180, 540, 1 620). 5 × 104 cells well were then added, incubated, washed, and incubated for 7 days. Culture supernatants were tested for virus production by HIV-1 p24 capture ELISA. The lowest serum concentration giving a 50% reduction (IC50) of ELISA absorbance value compared with the mean of the negative controls are presented [19]. Statistical analyses were conducted using a two-tailed unpaired Student t test. * Differences of the responses between compared groups defined as p ≤ 0.05 were considered significant. n.s. = non-significant. Comparisons between groups with the HIV-1 antigens were performed by using the non-parametric Mann-Whitney U test with Bonferroni correction, p < 0.05 was considered significant.
Mentions: Experiments with pOVA and pFliC(-gly) indicated that delivery of plasmids using N3 and the intranasal route was able to promote cellular immune responses as well as humoral mucosal immune responses. To compare the effectiveness of secreted flagellin to promote immune responses to a clinical antigen using a heterologus prime/boost regime, priming intranasal DNA vaccinations were carried out using plasmid pgp160Lfai/pRev [16] with delivery lipid N3 alone or together with pFliC(-gly). Boostings were performed using recombinant gp160 proteins with a protein-delivery lipid L3B alone or together with N3 mixed with pFliC(-gly) (Table 2). In these experiments mice were given doses of antigen and adjuvant believed to maximize detectable responses. Mice were primed, boosted, and analyzed according to the indicated timeline (Figure 5a). Four weeks post-final boost serum total-IgG titers anti-rgp160 indicated that addition of N3 to pgp160 was able to strongly promote anti-gp160 antibody responses (Figure 5b). Similar to responses seen using OVA, addition of N3/pFliC(-gly) to the immunization regime enhanced antigen-specific antibody titers further (Figure 5b). The adjuvant effect was dependent on N3. Likely due to it’s ability to encapsulate plasmid DNA and protect it from the degradative environment of the mucosal compartment. These higher titers of antigen-specific IgG in the sera and the presence of antigen-specific mucosal IgA indicate that the mucosal adjuvant effects of pFliC(-gly) are not limited to experimental antigens.

Bottom Line: Eliciting effective immune responses using non-living/replicating DNA vaccines is a significant challenge.We also observed enhancement of cross-clade reactive IgA as well as a broadening of B cell epitope reactivity.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.

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