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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

Kinetic analysis of T cell responses to immunizations with gp160 with and without adjuvant combinations. (a) Anti-mIFNγ ELISA was performed on cells restimulated with rgp160. Values shown were adjusted for baseline values seen using identical stimulations using splenocytes from naive mice. Priming (ImmunogenP, plasmids) and boosting (ImmunogenB, rec proteins) groups are shown in the key. Immunization details are listed in Table 2; (b) Anti-mIL-5 ELISA was performed on cells restimulated with rgp160. Values shown were adjusted for baseline values seen using identical stimulations using splenocytes from naïve mice; (c) Proliferative response to stimulation with rgp160 defined as stimulation index relative to identical stimulations using splenocytes from naïve mice. Statistical analyses were conducted using a two-tailed unpaired Student t test. * Differences of the responses between compared groups at week 4 after final boost 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-f008: Kinetic analysis of T cell responses to immunizations with gp160 with and without adjuvant combinations. (a) Anti-mIFNγ ELISA was performed on cells restimulated with rgp160. Values shown were adjusted for baseline values seen using identical stimulations using splenocytes from naive mice. Priming (ImmunogenP, plasmids) and boosting (ImmunogenB, rec proteins) groups are shown in the key. Immunization details are listed in Table 2; (b) Anti-mIL-5 ELISA was performed on cells restimulated with rgp160. Values shown were adjusted for baseline values seen using identical stimulations using splenocytes from naïve mice; (c) Proliferative response to stimulation with rgp160 defined as stimulation index relative to identical stimulations using splenocytes from naïve mice. Statistical analyses were conducted using a two-tailed unpaired Student t test. * Differences of the responses between compared groups at week 4 after final boost 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: To study T cell immune responses to DNA-prime/protein-boost i.na. immunizations we chose to assay standard cytokines associated with Th1-like (IFNγ) or Th2-like (IL-5) populations. Responses to gp160 were assayed following individual splenocytes harvesting at 4, 8, 12, 24, and 36 weeks after final boost and restimulation with rgp160. Observed response trends were similar to those seen when studying antibody responses. Addition of N3 to pgp160 vaccinations followed by L3B protein boostings lead to clear and significant IFNγ, IL-5, and proliferative responses over mice immunized without adjuvant (Figure 8a–c). The IFNγ and proliferative responses could be further enhanced by the addition of pFliC(-gly) but not IL-5 (Figure 8a–c) demonstrating the ability of pFliC(-gly) to act as an adjuvant but with a propensity to strengthen Th1-like responses.


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)

Kinetic analysis of T cell responses to immunizations with gp160 with and without adjuvant combinations. (a) Anti-mIFNγ ELISA was performed on cells restimulated with rgp160. Values shown were adjusted for baseline values seen using identical stimulations using splenocytes from naive mice. Priming (ImmunogenP, plasmids) and boosting (ImmunogenB, rec proteins) groups are shown in the key. Immunization details are listed in Table 2; (b) Anti-mIL-5 ELISA was performed on cells restimulated with rgp160. Values shown were adjusted for baseline values seen using identical stimulations using splenocytes from naïve mice; (c) Proliferative response to stimulation with rgp160 defined as stimulation index relative to identical stimulations using splenocytes from naïve mice. Statistical analyses were conducted using a two-tailed unpaired Student t test. * Differences of the responses between compared groups at week 4 after final boost 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

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

vaccines-01-00415-f008: Kinetic analysis of T cell responses to immunizations with gp160 with and without adjuvant combinations. (a) Anti-mIFNγ ELISA was performed on cells restimulated with rgp160. Values shown were adjusted for baseline values seen using identical stimulations using splenocytes from naive mice. Priming (ImmunogenP, plasmids) and boosting (ImmunogenB, rec proteins) groups are shown in the key. Immunization details are listed in Table 2; (b) Anti-mIL-5 ELISA was performed on cells restimulated with rgp160. Values shown were adjusted for baseline values seen using identical stimulations using splenocytes from naïve mice; (c) Proliferative response to stimulation with rgp160 defined as stimulation index relative to identical stimulations using splenocytes from naïve mice. Statistical analyses were conducted using a two-tailed unpaired Student t test. * Differences of the responses between compared groups at week 4 after final boost 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: To study T cell immune responses to DNA-prime/protein-boost i.na. immunizations we chose to assay standard cytokines associated with Th1-like (IFNγ) or Th2-like (IL-5) populations. Responses to gp160 were assayed following individual splenocytes harvesting at 4, 8, 12, 24, and 36 weeks after final boost and restimulation with rgp160. Observed response trends were similar to those seen when studying antibody responses. Addition of N3 to pgp160 vaccinations followed by L3B protein boostings lead to clear and significant IFNγ, IL-5, and proliferative responses over mice immunized without adjuvant (Figure 8a–c). The IFNγ and proliferative responses could be further enhanced by the addition of pFliC(-gly) but not IL-5 (Figure 8a–c) demonstrating the ability of pFliC(-gly) to act as an adjuvant but with a propensity to strengthen Th1-like responses.

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