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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

B cell epitope mapping to C2-C5 region of gp160 after immunization with gp160 with and without adjuvant. ELISA was performed using group-pooled serum (equal volumes) from immunization group 2 (n = 35) or 4 (n = 35) against individual peptides. Priming (ImmunogenP, plasmids) and boosting (ImmunogenB, rec proteins) groups are shown in the key. Immunization details are listed in Table 2. The concentration of gp160-peptide 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. 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.
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vaccines-01-00415-f007: B cell epitope mapping to C2-C5 region of gp160 after immunization with gp160 with and without adjuvant. ELISA was performed using group-pooled serum (equal volumes) from immunization group 2 (n = 35) or 4 (n = 35) against individual peptides. Priming (ImmunogenP, plasmids) and boosting (ImmunogenB, rec proteins) groups are shown in the key. Immunization details are listed in Table 2. The concentration of gp160-peptide 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. 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.

Mentions: To determine the breadth of antibody responses against hypervariable regions of gp160 within antigen-specific IgG we performed B cell epitope mapping of group-pooled serum against individual 20-mers of gp160 from AA249–499 containing the V3 variable loop region. We observed clear populations of IgG anti-gp160 peptide immune responses in the sera of mice immunized with pgp160 with N3 followed by boosting with rgp160 protein with L3B (Figure 7a). However, the addition of pFliC(-gly) to immunizations expanded the number of detectable populations by five (Figure 7a). Analysis of amino acid identities and similarities between FliC and clade B LAI gp160 within the region encoded by the peptides were performed using NCBI BLASTP analysis (v2.2.26+) with default settings. Two regions were identified containing identity and similarity. Of these two regions only one (containing 36% identities and 55% similarity) overlapped with a region of increased reactivity (peptides AA 439 and 444) and was excluded. There were no regions of alignment identified within the 5 annotated populations that exhibited equal or greater identity and similarity than the 22AA region.


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)

B cell epitope mapping to C2-C5 region of gp160 after immunization with gp160 with and without adjuvant. ELISA was performed using group-pooled serum (equal volumes) from immunization group 2 (n = 35) or 4 (n = 35) against individual peptides. Priming (ImmunogenP, plasmids) and boosting (ImmunogenB, rec proteins) groups are shown in the key. Immunization details are listed in Table 2. The concentration of gp160-peptide 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. 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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494208&req=5

vaccines-01-00415-f007: B cell epitope mapping to C2-C5 region of gp160 after immunization with gp160 with and without adjuvant. ELISA was performed using group-pooled serum (equal volumes) from immunization group 2 (n = 35) or 4 (n = 35) against individual peptides. Priming (ImmunogenP, plasmids) and boosting (ImmunogenB, rec proteins) groups are shown in the key. Immunization details are listed in Table 2. The concentration of gp160-peptide 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. 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.
Mentions: To determine the breadth of antibody responses against hypervariable regions of gp160 within antigen-specific IgG we performed B cell epitope mapping of group-pooled serum against individual 20-mers of gp160 from AA249–499 containing the V3 variable loop region. We observed clear populations of IgG anti-gp160 peptide immune responses in the sera of mice immunized with pgp160 with N3 followed by boosting with rgp160 protein with L3B (Figure 7a). However, the addition of pFliC(-gly) to immunizations expanded the number of detectable populations by five (Figure 7a). Analysis of amino acid identities and similarities between FliC and clade B LAI gp160 within the region encoded by the peptides were performed using NCBI BLASTP analysis (v2.2.26+) with default settings. Two regions were identified containing identity and similarity. Of these two regions only one (containing 36% identities and 55% similarity) overlapped with a region of increased reactivity (peptides AA 439 and 444) and was excluded. There were no regions of alignment identified within the 5 annotated populations that exhibited equal or greater identity and similarity than the 22AA region.

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