Limits...
The p40 subunit of interleukin (IL)-12 promotes stabilization and export of the p35 subunit: implications for improved IL-12 cytokine production.

Jalah R, Rosati M, Ganneru B, Pilkington GR, Valentin A, Kulkarni V, Bergamaschi C, Chowdhury B, Zhang GM, Beach RK, Alicea C, Broderick KE, Sardesai NY, Pavlakis GN, Felber BK - J. Biol. Chem. (2013)

Bottom Line: We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit.Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ~1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences.Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702-1201, USA.

ABSTRACT
IL-12 is a 70-kDa heterodimeric cytokine composed of the p35 and p40 subunits. To maximize cytokine production from plasmid DNA, molecular steps controlling IL-12p70 biosynthesis at the posttranscriptional and posttranslational levels were investigated. We show that the combination of RNA/codon-optimized gene sequences and fine-tuning of the relative expression levels of the two subunits within a cell resulted in increased production of the IL-12p70 heterodimer. We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit. This posttranslational regulation mediated by the p40 subunit is conserved in mammals. Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ~1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences. Such optimized DNA plasmids also produced significantly higher levels of systemic bioactive IL-12 upon in vivo DNA delivery in mice compared with plasmids expressing the wild type sequences. A single therapeutic injection of an optimized murine IL-12 DNA plasmid showed significantly more potent control of tumor development in the B16 melanoma cancer model in mice. Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.

Show MeSH

Related in: MedlinePlus

Optimized dual promoter expression vectors for improved IL-12p70 production.A, expression of the human IL-12p70 from different dual promoter plasmids. IL-12 production was measured by human IL-12p70-specific ELISA (top) and by a Western immunoblot assay (bottom). The plasmids contain the following: wild type human IL-12 coding sequences expressed from WLV-009M (plasmid WLV-103M; lane 1) and RNA-optimized coding sequences expressed from vectors WLV-009M (plasmid AG5 (lane 2); plasmid AG169 (lane 3) and pDP (plasmid AG183 (lane 4); plasmid AG181 (lane 5); plasmid AG259 (lane 6)). The two configurations of dual promoter (DP) human IL-12 plasmids expressing the p35 and p40 coding sequences from the huCMV or the siCMV promoters are shown on the right. The GFP values (mean ± S.E. (error bars)) for lanes 1–6 were 17.3 ± 5.7, 22.1 ± 2.3, 20.0 ± 1.6, 25.4 ± 3.3, 27.3 ± 4.0, and 25.6 ± 1.2 arbitrary units, respectively. B, expression of the rhesus macaque IL-12p70 from different dual promoter plasmids. The IL-12 production was measured by a macaque IL-12p70-specific ELISA (top) and by a Western immunoblot assay (bottom) from transfected cells. The plasmids contain the following: wild type macaque coding sequences expressed from the dual promoter plasmid WLV-009M (plasmid WLV-104M; lane 1) and RNA-optimized coding sequences expressed from vectors WLV-009M (plasmid AG3; lane 2) and pDP (plasmid AG159 (lane 3) and plasmid AG157 (lane 4), respectively). The GFP values (mean ± S.E.) for lanes 1–4 were 19.7 ± 1.0, 20.5 ± 1.3, 11.9 ± 1.6, and 16.2 ± 0.9 arbitrary units, respectively. C, expression of an optimized murine IL-12p70 plasmid. The IL-12 production was measured using a murine IL-12p70-specific ELISA (top) and by the Western immunoblot assay (bottom). The plasmids contain the following: wild type murine coding sequences expressed from the dual promoter plasmid WLV-009M (plasmid WLV-105M; lane 1) and RNA-optimized coding sequences expressed from vector pDP (plasmid AG250; lane 3). The GFP values (mean ± S.E.) for lanes 1 and 2 were 8.4 ± 0.8 and 11.6 ± 0.6 arbitrary units, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3585113&req=5

Figure 4: Optimized dual promoter expression vectors for improved IL-12p70 production.A, expression of the human IL-12p70 from different dual promoter plasmids. IL-12 production was measured by human IL-12p70-specific ELISA (top) and by a Western immunoblot assay (bottom). The plasmids contain the following: wild type human IL-12 coding sequences expressed from WLV-009M (plasmid WLV-103M; lane 1) and RNA-optimized coding sequences expressed from vectors WLV-009M (plasmid AG5 (lane 2); plasmid AG169 (lane 3) and pDP (plasmid AG183 (lane 4); plasmid AG181 (lane 5); plasmid AG259 (lane 6)). The two configurations of dual promoter (DP) human IL-12 plasmids expressing the p35 and p40 coding sequences from the huCMV or the siCMV promoters are shown on the right. The GFP values (mean ± S.E. (error bars)) for lanes 1–6 were 17.3 ± 5.7, 22.1 ± 2.3, 20.0 ± 1.6, 25.4 ± 3.3, 27.3 ± 4.0, and 25.6 ± 1.2 arbitrary units, respectively. B, expression of the rhesus macaque IL-12p70 from different dual promoter plasmids. The IL-12 production was measured by a macaque IL-12p70-specific ELISA (top) and by a Western immunoblot assay (bottom) from transfected cells. The plasmids contain the following: wild type macaque coding sequences expressed from the dual promoter plasmid WLV-009M (plasmid WLV-104M; lane 1) and RNA-optimized coding sequences expressed from vectors WLV-009M (plasmid AG3; lane 2) and pDP (plasmid AG159 (lane 3) and plasmid AG157 (lane 4), respectively). The GFP values (mean ± S.E.) for lanes 1–4 were 19.7 ± 1.0, 20.5 ± 1.3, 11.9 ± 1.6, and 16.2 ± 0.9 arbitrary units, respectively. C, expression of an optimized murine IL-12p70 plasmid. The IL-12 production was measured using a murine IL-12p70-specific ELISA (top) and by the Western immunoblot assay (bottom). The plasmids contain the following: wild type murine coding sequences expressed from the dual promoter plasmid WLV-009M (plasmid WLV-105M; lane 1) and RNA-optimized coding sequences expressed from vector pDP (plasmid AG250; lane 3). The GFP values (mean ± S.E.) for lanes 1 and 2 were 8.4 ± 0.8 and 11.6 ± 0.6 arbitrary units, respectively.

Mentions: IL-12 DNA-based in vivo applications require coordinated optimal expression of both IL-12 subunits within a cell, which can be better achieved with a single plasmid producing both p35 and p40 subunits. Two dual promoter vectors with slightly different plasmid backbones (vector pWLV-009M (31) and pDP (43)) were compared for the production of IL-12p70. Both vectors contain the strong huCMV and the weaker siCMV promoters, controlling the expression of the IL-12 subunits in counterclockwise orientation. The human, rhesus macaque, and murine IL-12 subunits were cloned into these vectors, taking into consideration the contribution of the RNA-optimized gene sequences and the importance of the relative ratio (see Fig. 1) of the subunits for optimal IL-12p70 production. Expression of the plasmids was compared upon transient transfection in HEK293 cells and analyzed by Western immunoblot and ELISA assays (Fig. 4).


The p40 subunit of interleukin (IL)-12 promotes stabilization and export of the p35 subunit: implications for improved IL-12 cytokine production.

Jalah R, Rosati M, Ganneru B, Pilkington GR, Valentin A, Kulkarni V, Bergamaschi C, Chowdhury B, Zhang GM, Beach RK, Alicea C, Broderick KE, Sardesai NY, Pavlakis GN, Felber BK - J. Biol. Chem. (2013)

Optimized dual promoter expression vectors for improved IL-12p70 production.A, expression of the human IL-12p70 from different dual promoter plasmids. IL-12 production was measured by human IL-12p70-specific ELISA (top) and by a Western immunoblot assay (bottom). The plasmids contain the following: wild type human IL-12 coding sequences expressed from WLV-009M (plasmid WLV-103M; lane 1) and RNA-optimized coding sequences expressed from vectors WLV-009M (plasmid AG5 (lane 2); plasmid AG169 (lane 3) and pDP (plasmid AG183 (lane 4); plasmid AG181 (lane 5); plasmid AG259 (lane 6)). The two configurations of dual promoter (DP) human IL-12 plasmids expressing the p35 and p40 coding sequences from the huCMV or the siCMV promoters are shown on the right. The GFP values (mean ± S.E. (error bars)) for lanes 1–6 were 17.3 ± 5.7, 22.1 ± 2.3, 20.0 ± 1.6, 25.4 ± 3.3, 27.3 ± 4.0, and 25.6 ± 1.2 arbitrary units, respectively. B, expression of the rhesus macaque IL-12p70 from different dual promoter plasmids. The IL-12 production was measured by a macaque IL-12p70-specific ELISA (top) and by a Western immunoblot assay (bottom) from transfected cells. The plasmids contain the following: wild type macaque coding sequences expressed from the dual promoter plasmid WLV-009M (plasmid WLV-104M; lane 1) and RNA-optimized coding sequences expressed from vectors WLV-009M (plasmid AG3; lane 2) and pDP (plasmid AG159 (lane 3) and plasmid AG157 (lane 4), respectively). The GFP values (mean ± S.E.) for lanes 1–4 were 19.7 ± 1.0, 20.5 ± 1.3, 11.9 ± 1.6, and 16.2 ± 0.9 arbitrary units, respectively. C, expression of an optimized murine IL-12p70 plasmid. The IL-12 production was measured using a murine IL-12p70-specific ELISA (top) and by the Western immunoblot assay (bottom). The plasmids contain the following: wild type murine coding sequences expressed from the dual promoter plasmid WLV-009M (plasmid WLV-105M; lane 1) and RNA-optimized coding sequences expressed from vector pDP (plasmid AG250; lane 3). The GFP values (mean ± S.E.) for lanes 1 and 2 were 8.4 ± 0.8 and 11.6 ± 0.6 arbitrary units, respectively.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3585113&req=5

Figure 4: Optimized dual promoter expression vectors for improved IL-12p70 production.A, expression of the human IL-12p70 from different dual promoter plasmids. IL-12 production was measured by human IL-12p70-specific ELISA (top) and by a Western immunoblot assay (bottom). The plasmids contain the following: wild type human IL-12 coding sequences expressed from WLV-009M (plasmid WLV-103M; lane 1) and RNA-optimized coding sequences expressed from vectors WLV-009M (plasmid AG5 (lane 2); plasmid AG169 (lane 3) and pDP (plasmid AG183 (lane 4); plasmid AG181 (lane 5); plasmid AG259 (lane 6)). The two configurations of dual promoter (DP) human IL-12 plasmids expressing the p35 and p40 coding sequences from the huCMV or the siCMV promoters are shown on the right. The GFP values (mean ± S.E. (error bars)) for lanes 1–6 were 17.3 ± 5.7, 22.1 ± 2.3, 20.0 ± 1.6, 25.4 ± 3.3, 27.3 ± 4.0, and 25.6 ± 1.2 arbitrary units, respectively. B, expression of the rhesus macaque IL-12p70 from different dual promoter plasmids. The IL-12 production was measured by a macaque IL-12p70-specific ELISA (top) and by a Western immunoblot assay (bottom) from transfected cells. The plasmids contain the following: wild type macaque coding sequences expressed from the dual promoter plasmid WLV-009M (plasmid WLV-104M; lane 1) and RNA-optimized coding sequences expressed from vectors WLV-009M (plasmid AG3; lane 2) and pDP (plasmid AG159 (lane 3) and plasmid AG157 (lane 4), respectively). The GFP values (mean ± S.E.) for lanes 1–4 were 19.7 ± 1.0, 20.5 ± 1.3, 11.9 ± 1.6, and 16.2 ± 0.9 arbitrary units, respectively. C, expression of an optimized murine IL-12p70 plasmid. The IL-12 production was measured using a murine IL-12p70-specific ELISA (top) and by the Western immunoblot assay (bottom). The plasmids contain the following: wild type murine coding sequences expressed from the dual promoter plasmid WLV-009M (plasmid WLV-105M; lane 1) and RNA-optimized coding sequences expressed from vector pDP (plasmid AG250; lane 3). The GFP values (mean ± S.E.) for lanes 1 and 2 were 8.4 ± 0.8 and 11.6 ± 0.6 arbitrary units, respectively.
Mentions: IL-12 DNA-based in vivo applications require coordinated optimal expression of both IL-12 subunits within a cell, which can be better achieved with a single plasmid producing both p35 and p40 subunits. Two dual promoter vectors with slightly different plasmid backbones (vector pWLV-009M (31) and pDP (43)) were compared for the production of IL-12p70. Both vectors contain the strong huCMV and the weaker siCMV promoters, controlling the expression of the IL-12 subunits in counterclockwise orientation. The human, rhesus macaque, and murine IL-12 subunits were cloned into these vectors, taking into consideration the contribution of the RNA-optimized gene sequences and the importance of the relative ratio (see Fig. 1) of the subunits for optimal IL-12p70 production. Expression of the plasmids was compared upon transient transfection in HEK293 cells and analyzed by Western immunoblot and ELISA assays (Fig. 4).

Bottom Line: We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit.Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ~1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences.Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.

View Article: PubMed Central - PubMed

Affiliation: Human Retrovirus Pathogenesis Section, Vaccine Branch, Center for Cancer Research, Frederick National Laboratory for Cancer Research, Frederick, Maryland 21702-1201, USA.

ABSTRACT
IL-12 is a 70-kDa heterodimeric cytokine composed of the p35 and p40 subunits. To maximize cytokine production from plasmid DNA, molecular steps controlling IL-12p70 biosynthesis at the posttranscriptional and posttranslational levels were investigated. We show that the combination of RNA/codon-optimized gene sequences and fine-tuning of the relative expression levels of the two subunits within a cell resulted in increased production of the IL-12p70 heterodimer. We found that the p40 subunit plays a critical role in enhancing the stability, intracellular trafficking, and export of the p35 subunit. This posttranslational regulation mediated by the p40 subunit is conserved in mammals. Based on these findings, dual gene expression vectors were generated, producing an optimal ratio of the two subunits, resulting in a ~1 log increase in human, rhesus, and murine IL-12p70 production compared with vectors expressing the wild type sequences. Such optimized DNA plasmids also produced significantly higher levels of systemic bioactive IL-12 upon in vivo DNA delivery in mice compared with plasmids expressing the wild type sequences. A single therapeutic injection of an optimized murine IL-12 DNA plasmid showed significantly more potent control of tumor development in the B16 melanoma cancer model in mice. Therefore, the improved IL-12p70 DNA vectors have promising potential for in vivo use as molecular vaccine adjuvants and in cancer immunotherapy.

Show MeSH
Related in: MedlinePlus