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Co-Administration of Molecular Adjuvants Expressing NF-Kappa B Subunit p65/RelA or Type-1 Transactivator T-bet Enhance Antigen Specific DNA Vaccine-Induced Immunity.

Shedlock DJ, Tingey C, Mahadevan L, Hutnick N, Reuschel EL, Kudchodkar S, Flingai S, Yan J, Kim JJ, Ugen KE, Weiner DB, Muthumani K - Vaccines (Basel) (2014)

Bottom Line: Specifically the co-delivery of (a) RelA, a subunit of the NF-κB transcription complex or (b) T-bet, a Th1-specific T box transcription factor, along with a prototypical DNA vaccine expressing HIV-1 proteins was evaluated.As such, this study demonstrated that co-delivery of either adjuvant resulted in enhanced T and B cell responses, specifically characterized by increased T cell numbers, IFN-γ production, as well as enhanced antibody responses.This study demonstrates the use of cellular transcription factors as adjuvants for enhancing DNA vaccine-induced immunity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology & Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. shedlock@mail.med.upenn.edu.

ABSTRACT
DNA vaccine-induced immunity can be enhanced by the co-delivery of synthetic gene-encoding molecular adjuvants. Many of these adjuvants have included cytokines, chemokines or co-stimulatory molecules that have been demonstrated to enhance vaccine-induced immunity by increasing the magnitude or type of immune responses and/or protective efficacy. In this way, through the use of adjuvants, immune responses can be highly customizable and functionally tailored for optimal efficacy against pathogen specific (i.e., infectious agent) or non-pathogen (i.e., cancer) antigens. In the novel study presented here, we examined the use of cellular transcription factors as molecular adjuvants. Specifically the co-delivery of (a) RelA, a subunit of the NF-κB transcription complex or (b) T-bet, a Th1-specific T box transcription factor, along with a prototypical DNA vaccine expressing HIV-1 proteins was evaluated. As well, all of the vaccines and adjuvants were administered to mice using in vivo electroporation (EP), a technology demonstrated to dramatically increase plasmid DNA transfection and subsequent transgene expression with concomitant enhancement of vaccine induced immune responses. As such, this study demonstrated that co-delivery of either adjuvant resulted in enhanced T and B cell responses, specifically characterized by increased T cell numbers, IFN-γ production, as well as enhanced antibody responses. This study demonstrates the use of cellular transcription factors as adjuvants for enhancing DNA vaccine-induced immunity.

No MeSH data available.


Related in: MedlinePlus

Molecular adjuvant construction and expression. (A) Mouse RelA or T-bet primary sequences were genetically optimized, synthesized, and then subcloned into modified pVax1 expression vectors. Optimization entailed inclusion of a IgE leader peptide (IgE), preceded by a Kozak sequence, fused at the N-terminus. The figure indicates the restrictions enzymes used for subcloning, the translation initiation site (forward arrow), IgE leader peptide (IgE; hatched bar), protein length (aa), and transgenes (black with white lettering); (B) Protein expression from the nuclear extract was analyzed by Western immunoblotting following transfection of HEK 293T cells with pRelA, pTbet, or empty vector control (pVax1). The relative size (kDa) of the proteins are determined by detection analysis using protein-specific Abs as indicated; (C) Over expression of RelA potently induces κB dependent transcription. HeLa cells were transiently transfected with a NF-κB-dependent luciferase reporter gene together with expression vectors encoding RelA/p65. The cotransfected cells were subsequently grown for 48 h, and the luciferase activity was determined as described in the Materials and Methods; (D). Overexpression of T-bet stimulates production of IFN-γ: Naive CD4 T cells were transfected with either pT-bet or pVax1 and stimulated with anti-CD3 plus anti-CD28 followed the measurement of IFN-γ production by enzyme-linked immunosorbent assay (ELISA) as described Materials and Methods. IFN-γ levels are expressed as μg/mL
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vaccines-02-00196-f001: Molecular adjuvant construction and expression. (A) Mouse RelA or T-bet primary sequences were genetically optimized, synthesized, and then subcloned into modified pVax1 expression vectors. Optimization entailed inclusion of a IgE leader peptide (IgE), preceded by a Kozak sequence, fused at the N-terminus. The figure indicates the restrictions enzymes used for subcloning, the translation initiation site (forward arrow), IgE leader peptide (IgE; hatched bar), protein length (aa), and transgenes (black with white lettering); (B) Protein expression from the nuclear extract was analyzed by Western immunoblotting following transfection of HEK 293T cells with pRelA, pTbet, or empty vector control (pVax1). The relative size (kDa) of the proteins are determined by detection analysis using protein-specific Abs as indicated; (C) Over expression of RelA potently induces κB dependent transcription. HeLa cells were transiently transfected with a NF-κB-dependent luciferase reporter gene together with expression vectors encoding RelA/p65. The cotransfected cells were subsequently grown for 48 h, and the luciferase activity was determined as described in the Materials and Methods; (D). Overexpression of T-bet stimulates production of IFN-γ: Naive CD4 T cells were transfected with either pT-bet or pVax1 and stimulated with anti-CD3 plus anti-CD28 followed the measurement of IFN-γ production by enzyme-linked immunosorbent assay (ELISA) as described Materials and Methods. IFN-γ levels are expressed as μg/mL

Mentions: The pRelA and pTbet plasmids encode the full-length mouse NF-kappa B subunit p65/RelA and Type-1 transactivator T-bet, respectively. Each was genetically optimized, synthesized, and subcloned into modified pVax1 mammalian expression vectors (Figure 1A). To test for expression of these plasmids, HEK 293T cells were transfected with each and protein production was assessed by standard Western immunoblotting. An approximately 65 kDa protein corresponding to RelA was detected, using a specific Ab, in cell lysates harvested both 24 h and 48 h post-transfection (Figure 1B). Likewise, T-bet was detected as an approximately 56 kDa protein using an anti-T-bet Ab. Binding was specific for their respective proteins since neither bound to lysates from cells transfected with empty vector control plasmid pVax1. These data demonstrate that each of the molecular adjuvants expresses their respective encoded proteins upon in vitro transfection of HEK 293T cells. Further, IκB-dependent transcription was accessed in the HeLa cells luciferase expressing cell system (Figure 1C) to confirm the activation of RelA (p65). An increase in RelA expression as measured by relative luciferase activity was observed in a dose dependent manner. That is, increasing the plasmid from 3 μg to 5 μg or 10 μg resulted in an increase in the relative luciferase activity approximately 1.5 or 2.5 fold. T-bet expression correlates with IFN-γ expression in T cell and NK cells [60] and therefore in this assay IFN-γ serves as surrogate for the functional expression of T-bet (Figure 1D).


Co-Administration of Molecular Adjuvants Expressing NF-Kappa B Subunit p65/RelA or Type-1 Transactivator T-bet Enhance Antigen Specific DNA Vaccine-Induced Immunity.

Shedlock DJ, Tingey C, Mahadevan L, Hutnick N, Reuschel EL, Kudchodkar S, Flingai S, Yan J, Kim JJ, Ugen KE, Weiner DB, Muthumani K - Vaccines (Basel) (2014)

Molecular adjuvant construction and expression. (A) Mouse RelA or T-bet primary sequences were genetically optimized, synthesized, and then subcloned into modified pVax1 expression vectors. Optimization entailed inclusion of a IgE leader peptide (IgE), preceded by a Kozak sequence, fused at the N-terminus. The figure indicates the restrictions enzymes used for subcloning, the translation initiation site (forward arrow), IgE leader peptide (IgE; hatched bar), protein length (aa), and transgenes (black with white lettering); (B) Protein expression from the nuclear extract was analyzed by Western immunoblotting following transfection of HEK 293T cells with pRelA, pTbet, or empty vector control (pVax1). The relative size (kDa) of the proteins are determined by detection analysis using protein-specific Abs as indicated; (C) Over expression of RelA potently induces κB dependent transcription. HeLa cells were transiently transfected with a NF-κB-dependent luciferase reporter gene together with expression vectors encoding RelA/p65. The cotransfected cells were subsequently grown for 48 h, and the luciferase activity was determined as described in the Materials and Methods; (D). Overexpression of T-bet stimulates production of IFN-γ: Naive CD4 T cells were transfected with either pT-bet or pVax1 and stimulated with anti-CD3 plus anti-CD28 followed the measurement of IFN-γ production by enzyme-linked immunosorbent assay (ELISA) as described Materials and Methods. IFN-γ levels are expressed as μg/mL
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4494262&req=5

vaccines-02-00196-f001: Molecular adjuvant construction and expression. (A) Mouse RelA or T-bet primary sequences were genetically optimized, synthesized, and then subcloned into modified pVax1 expression vectors. Optimization entailed inclusion of a IgE leader peptide (IgE), preceded by a Kozak sequence, fused at the N-terminus. The figure indicates the restrictions enzymes used for subcloning, the translation initiation site (forward arrow), IgE leader peptide (IgE; hatched bar), protein length (aa), and transgenes (black with white lettering); (B) Protein expression from the nuclear extract was analyzed by Western immunoblotting following transfection of HEK 293T cells with pRelA, pTbet, or empty vector control (pVax1). The relative size (kDa) of the proteins are determined by detection analysis using protein-specific Abs as indicated; (C) Over expression of RelA potently induces κB dependent transcription. HeLa cells were transiently transfected with a NF-κB-dependent luciferase reporter gene together with expression vectors encoding RelA/p65. The cotransfected cells were subsequently grown for 48 h, and the luciferase activity was determined as described in the Materials and Methods; (D). Overexpression of T-bet stimulates production of IFN-γ: Naive CD4 T cells were transfected with either pT-bet or pVax1 and stimulated with anti-CD3 plus anti-CD28 followed the measurement of IFN-γ production by enzyme-linked immunosorbent assay (ELISA) as described Materials and Methods. IFN-γ levels are expressed as μg/mL
Mentions: The pRelA and pTbet plasmids encode the full-length mouse NF-kappa B subunit p65/RelA and Type-1 transactivator T-bet, respectively. Each was genetically optimized, synthesized, and subcloned into modified pVax1 mammalian expression vectors (Figure 1A). To test for expression of these plasmids, HEK 293T cells were transfected with each and protein production was assessed by standard Western immunoblotting. An approximately 65 kDa protein corresponding to RelA was detected, using a specific Ab, in cell lysates harvested both 24 h and 48 h post-transfection (Figure 1B). Likewise, T-bet was detected as an approximately 56 kDa protein using an anti-T-bet Ab. Binding was specific for their respective proteins since neither bound to lysates from cells transfected with empty vector control plasmid pVax1. These data demonstrate that each of the molecular adjuvants expresses their respective encoded proteins upon in vitro transfection of HEK 293T cells. Further, IκB-dependent transcription was accessed in the HeLa cells luciferase expressing cell system (Figure 1C) to confirm the activation of RelA (p65). An increase in RelA expression as measured by relative luciferase activity was observed in a dose dependent manner. That is, increasing the plasmid from 3 μg to 5 μg or 10 μg resulted in an increase in the relative luciferase activity approximately 1.5 or 2.5 fold. T-bet expression correlates with IFN-γ expression in T cell and NK cells [60] and therefore in this assay IFN-γ serves as surrogate for the functional expression of T-bet (Figure 1D).

Bottom Line: Specifically the co-delivery of (a) RelA, a subunit of the NF-κB transcription complex or (b) T-bet, a Th1-specific T box transcription factor, along with a prototypical DNA vaccine expressing HIV-1 proteins was evaluated.As such, this study demonstrated that co-delivery of either adjuvant resulted in enhanced T and B cell responses, specifically characterized by increased T cell numbers, IFN-γ production, as well as enhanced antibody responses.This study demonstrates the use of cellular transcription factors as adjuvants for enhancing DNA vaccine-induced immunity.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology & Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA. shedlock@mail.med.upenn.edu.

ABSTRACT
DNA vaccine-induced immunity can be enhanced by the co-delivery of synthetic gene-encoding molecular adjuvants. Many of these adjuvants have included cytokines, chemokines or co-stimulatory molecules that have been demonstrated to enhance vaccine-induced immunity by increasing the magnitude or type of immune responses and/or protective efficacy. In this way, through the use of adjuvants, immune responses can be highly customizable and functionally tailored for optimal efficacy against pathogen specific (i.e., infectious agent) or non-pathogen (i.e., cancer) antigens. In the novel study presented here, we examined the use of cellular transcription factors as molecular adjuvants. Specifically the co-delivery of (a) RelA, a subunit of the NF-κB transcription complex or (b) T-bet, a Th1-specific T box transcription factor, along with a prototypical DNA vaccine expressing HIV-1 proteins was evaluated. As well, all of the vaccines and adjuvants were administered to mice using in vivo electroporation (EP), a technology demonstrated to dramatically increase plasmid DNA transfection and subsequent transgene expression with concomitant enhancement of vaccine induced immune responses. As such, this study demonstrated that co-delivery of either adjuvant resulted in enhanced T and B cell responses, specifically characterized by increased T cell numbers, IFN-γ production, as well as enhanced antibody responses. This study demonstrates the use of cellular transcription factors as adjuvants for enhancing DNA vaccine-induced immunity.

No MeSH data available.


Related in: MedlinePlus