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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.

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In vivo expression and bioactivity of murine IL-12 plasmids.A, production of bioactive murine IL-12p70 in vivo. Plasmids (5 μg/animal) expressing the murine IL-12p70 were injected intramuscularly, followed by in vivo electroporation into BALB/c mice (n = 5/group). The dual gene expression plasmids contain the following: wild type coding sequences (WLV-105M; lanes 2 and 5) or optimized coding sequences (plasmid AG250; lanes 3 and 6). The sham control group (n = 3) was injected with 100 μg of the empty vector (CMVkan; lanes 1 and 4). The mice were bled at day 3 and sacrificed at day 6 postinjection. Individual mouse plasma samples were analyzed for the levels of murine IL-12p70 (top) and murine IFN-γ (bottom) by the respective ELISA. The values from the individual mice and median values are shown. p values using Mann-Whitney two-tailed t test are shown. B, r and p values using Spearman nonparametric correlation between the murine IL-12p70 levels and the murine IFN-γ levels from the mice, depicted in Fig. 5A.
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Figure 6: In vivo expression and bioactivity of murine IL-12 plasmids.A, production of bioactive murine IL-12p70 in vivo. Plasmids (5 μg/animal) expressing the murine IL-12p70 were injected intramuscularly, followed by in vivo electroporation into BALB/c mice (n = 5/group). The dual gene expression plasmids contain the following: wild type coding sequences (WLV-105M; lanes 2 and 5) or optimized coding sequences (plasmid AG250; lanes 3 and 6). The sham control group (n = 3) was injected with 100 μg of the empty vector (CMVkan; lanes 1 and 4). The mice were bled at day 3 and sacrificed at day 6 postinjection. Individual mouse plasma samples were analyzed for the levels of murine IL-12p70 (top) and murine IFN-γ (bottom) by the respective ELISA. The values from the individual mice and median values are shown. p values using Mann-Whitney two-tailed t test are shown. B, r and p values using Spearman nonparametric correlation between the murine IL-12p70 levels and the murine IFN-γ levels from the mice, depicted in Fig. 5A.

Mentions: Next, we compared expression of murine IL-12p70 plasmids in BALB/c mice (Fig. 6). The plasmids consisting of the wild type (WLV-105M, wt) and the RNA-optimized (AG250, opt) murine IL-12p70, described in Fig. 2, or sham DNA were delivered by intramuscular injection followed by in vivo electroporation (5 μg of DNA/mouse; n = 5/group). Plasma samples were collected at days 3 and 6 post-DNA injection, and cytokine production was measured by a murine IL-12p70-specific ELISA (Fig. 6A). Sham DNA-injected mice (lanes 1 and 4) did not show detectable IL-12p70 levels (threshold of the assay, 0.015 ng/ml). The optimized plasmid produced significantly higher IL-12p70 levels with a median of 4 ng/ml plasma (lane 3) compared with 0.46 ng/ml (lane 2) from the wild type plasmid at day 3 postinjection. Thus, the production from the optimized plasmid in mice is ∼10-fold higher, and we found a similar difference upon transient transfection of these plasmids in human HEK293 cells. This ∼1 log difference in IL-12 levels also remained significant at day 6, with IL-12p70 levels of 0.09 ng/ml obtained from the wild type plasmid and 1.07 ng/ml from the optimized plasmid, respectively.


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)

In vivo expression and bioactivity of murine IL-12 plasmids.A, production of bioactive murine IL-12p70 in vivo. Plasmids (5 μg/animal) expressing the murine IL-12p70 were injected intramuscularly, followed by in vivo electroporation into BALB/c mice (n = 5/group). The dual gene expression plasmids contain the following: wild type coding sequences (WLV-105M; lanes 2 and 5) or optimized coding sequences (plasmid AG250; lanes 3 and 6). The sham control group (n = 3) was injected with 100 μg of the empty vector (CMVkan; lanes 1 and 4). The mice were bled at day 3 and sacrificed at day 6 postinjection. Individual mouse plasma samples were analyzed for the levels of murine IL-12p70 (top) and murine IFN-γ (bottom) by the respective ELISA. The values from the individual mice and median values are shown. p values using Mann-Whitney two-tailed t test are shown. B, r and p values using Spearman nonparametric correlation between the murine IL-12p70 levels and the murine IFN-γ levels from the mice, depicted in Fig. 5A.
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Figure 6: In vivo expression and bioactivity of murine IL-12 plasmids.A, production of bioactive murine IL-12p70 in vivo. Plasmids (5 μg/animal) expressing the murine IL-12p70 were injected intramuscularly, followed by in vivo electroporation into BALB/c mice (n = 5/group). The dual gene expression plasmids contain the following: wild type coding sequences (WLV-105M; lanes 2 and 5) or optimized coding sequences (plasmid AG250; lanes 3 and 6). The sham control group (n = 3) was injected with 100 μg of the empty vector (CMVkan; lanes 1 and 4). The mice were bled at day 3 and sacrificed at day 6 postinjection. Individual mouse plasma samples were analyzed for the levels of murine IL-12p70 (top) and murine IFN-γ (bottom) by the respective ELISA. The values from the individual mice and median values are shown. p values using Mann-Whitney two-tailed t test are shown. B, r and p values using Spearman nonparametric correlation between the murine IL-12p70 levels and the murine IFN-γ levels from the mice, depicted in Fig. 5A.
Mentions: Next, we compared expression of murine IL-12p70 plasmids in BALB/c mice (Fig. 6). The plasmids consisting of the wild type (WLV-105M, wt) and the RNA-optimized (AG250, opt) murine IL-12p70, described in Fig. 2, or sham DNA were delivered by intramuscular injection followed by in vivo electroporation (5 μg of DNA/mouse; n = 5/group). Plasma samples were collected at days 3 and 6 post-DNA injection, and cytokine production was measured by a murine IL-12p70-specific ELISA (Fig. 6A). Sham DNA-injected mice (lanes 1 and 4) did not show detectable IL-12p70 levels (threshold of the assay, 0.015 ng/ml). The optimized plasmid produced significantly higher IL-12p70 levels with a median of 4 ng/ml plasma (lane 3) compared with 0.46 ng/ml (lane 2) from the wild type plasmid at day 3 postinjection. Thus, the production from the optimized plasmid in mice is ∼10-fold higher, and we found a similar difference upon transient transfection of these plasmids in human HEK293 cells. This ∼1 log difference in IL-12 levels also remained significant at day 6, with IL-12p70 levels of 0.09 ng/ml obtained from the wild type plasmid and 1.07 ng/ml from the optimized plasmid, respectively.

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