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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 gp160. (a) Nasal IgA anti-rgp160; (b) Nasal IgA anti-gp160 cross-reactivity against clade A, B, and C envelope antigens. Priming (ImmunogenP, plasmids) and boosting (ImmunogenB, rec proteins) groups are shown in the key. Immunization details are listed in Table 2. ELISA was performed using individual serum from the indicated immunization groups. 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-f006: Mucosal antibody responses to gp160. (a) Nasal IgA anti-rgp160; (b) Nasal IgA anti-gp160 cross-reactivity against clade A, B, and C envelope antigens. Priming (ImmunogenP, plasmids) and boosting (ImmunogenB, rec proteins) groups are shown in the key. Immunization details are listed in Table 2. ELISA was performed using individual serum from the indicated immunization groups. 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: Although we observe enhanced titers of anti-viral spike antigen antibodies in the serum of animals immunized with antigen and pFliC(-gly) adjuvant the antibodies in mucosal secretions may be more likely to potentially neutralize viral particles. Studies of vaginal IgA anti-gp160 responses had assay backgrounds that precluded the determination of antigen-specific titers (data not shown). As a surrogate location representative of antigen-specific IgA responses we studied the titers of IgA anti-gp160 harvested from nasal washes. We observed trends similar to those found in the serum. Addition of N3 to pgp160 vaccinations followed by L3B protein boostings lead to clear IgA anti-gp160 titers which could be further enhanced by the addition of pFliC(-gly) (Figure 6a). The ability of nasal IgA anti-gp160 to cross-react against homologous (clade B) as well as heterologus clades (A and C) of HIV-1 gp160 was also tested. We observed nasal wash reactivity to clades A and B (Figure 6b). As with serum IgG, N3 could promote detectable anti-gp160 antibodies while pFliC(-gly) could enhance responses even further (Figure 6b). These results indicate that higher titers of antigen-specific clade B160 IgA also correlate with higher titers of antibody able to cross react with HIV-1 clade A gp160. Increases in cross-clade reactivity may likely be a behavior central to the development of an effective vaccine.


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 gp160. (a) Nasal IgA anti-rgp160; (b) Nasal IgA anti-gp160 cross-reactivity against clade A, B, and C envelope antigens. Priming (ImmunogenP, plasmids) and boosting (ImmunogenB, rec proteins) groups are shown in the key. Immunization details are listed in Table 2. ELISA was performed using individual serum from the indicated immunization groups. 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-f006: Mucosal antibody responses to gp160. (a) Nasal IgA anti-rgp160; (b) Nasal IgA anti-gp160 cross-reactivity against clade A, B, and C envelope antigens. Priming (ImmunogenP, plasmids) and boosting (ImmunogenB, rec proteins) groups are shown in the key. Immunization details are listed in Table 2. ELISA was performed using individual serum from the indicated immunization groups. 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: Although we observe enhanced titers of anti-viral spike antigen antibodies in the serum of animals immunized with antigen and pFliC(-gly) adjuvant the antibodies in mucosal secretions may be more likely to potentially neutralize viral particles. Studies of vaginal IgA anti-gp160 responses had assay backgrounds that precluded the determination of antigen-specific titers (data not shown). As a surrogate location representative of antigen-specific IgA responses we studied the titers of IgA anti-gp160 harvested from nasal washes. We observed trends similar to those found in the serum. Addition of N3 to pgp160 vaccinations followed by L3B protein boostings lead to clear IgA anti-gp160 titers which could be further enhanced by the addition of pFliC(-gly) (Figure 6a). The ability of nasal IgA anti-gp160 to cross-react against homologous (clade B) as well as heterologus clades (A and C) of HIV-1 gp160 was also tested. We observed nasal wash reactivity to clades A and B (Figure 6b). As with serum IgG, N3 could promote detectable anti-gp160 antibodies while pFliC(-gly) could enhance responses even further (Figure 6b). These results indicate that higher titers of antigen-specific clade B160 IgA also correlate with higher titers of antibody able to cross react with HIV-1 clade A gp160. Increases in cross-clade reactivity may likely be a behavior central to the development of an effective vaccine.

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