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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 of IL-12p70 plasmids in mice. Expression of the human IL-12p70 DNA was tested in BALB/c mice upon hydrodynamic DNA delivery. The animals (n = 10–11/group) were injected with 100 ng of a plasmid expressing the IL-12 from wild type (WLV-103M; lanes 2 and 5) or from optimized coding sequences (plasmid AG181; lanes 3 and 6). The sham control group (n = 3) was injected with 100 ng of the empty vector pCMVkan (lanes 1 and 4). The mice were bled at day 1 (lanes 1–3) and sacrificed at day 3 (lanes 4–6) postinjection. Plasma samples were analyzed by a human IL-12p70-specific ELISA, and the values of individual mice and median are shown. p values using the Mann-Whitney t test are shown.
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Figure 5: In vivo expression of IL-12p70 plasmids in mice. Expression of the human IL-12p70 DNA was tested in BALB/c mice upon hydrodynamic DNA delivery. The animals (n = 10–11/group) were injected with 100 ng of a plasmid expressing the IL-12 from wild type (WLV-103M; lanes 2 and 5) or from optimized coding sequences (plasmid AG181; lanes 3 and 6). The sham control group (n = 3) was injected with 100 ng of the empty vector pCMVkan (lanes 1 and 4). The mice were bled at day 1 (lanes 1–3) and sacrificed at day 3 (lanes 4–6) postinjection. Plasma samples were analyzed by a human IL-12p70-specific ELISA, and the values of individual mice and median are shown. p values using the Mann-Whitney t test are shown.

Mentions: We further evaluated the in vivo production of human IL-12p70 from different plasmids upon hydrodynamic DNA delivery into BALB/c mice (Fig. 5). The animals received 100 ng of plasmids expressing the IL-12p70 from wild type sequences (Plasmid WLV-103M, wt) and from RNA-optimized coding sequences (Plasmid AG181, opt) or sham DNA, respectively. At days 1 and 3 postinjection, the levels of the IL-12p70 heterodimer were measured in plasma using the human IL-12p70-specific ELISA (Fig. 4). Sham DNA-injected mice (lanes 1 and 4) did not show any detectable human IL-12 (threshold of the assay 0.015 ng/ml). At day 1, the mice receiving the wild type plasmid had a median IL-12p70 plasma level of 37 ng/ml (lane 2), whereas the mice receiving the optimized plasmid (lane 3) had a median of 489 ng/ml. These data show a significant ∼13-fold higher IL-12p70 production from the optimized human IL-12 plasmid and are in agreement with the observed difference between the plasmids upon transient transfection (Fig. 4A). This difference also remained significant at day 3 (lanes 5 and 6), with 1.8 and 107 ng/ml IL-12p70 for the mice that received the wild type and the optimized plasmids, respectively. Interestingly, we also noted a significantly slower apparent decay (p = 0.0004) of IL-12 in the plasma levels produced from optimized plasmid (∼4-fold decline, p = 0.0015; lanes 3 and 6) compared with the wild type plasmid (∼20-fold decline, p = 0.0004; lanes 2 and 5), suggesting that the optimized RNA is more stable in vivo, resulting in longer IL-12p70 expression. Because the human IL-12 is not active in mice, bioactivity could be tested in this experiment.


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 of IL-12p70 plasmids in mice. Expression of the human IL-12p70 DNA was tested in BALB/c mice upon hydrodynamic DNA delivery. The animals (n = 10–11/group) were injected with 100 ng of a plasmid expressing the IL-12 from wild type (WLV-103M; lanes 2 and 5) or from optimized coding sequences (plasmid AG181; lanes 3 and 6). The sham control group (n = 3) was injected with 100 ng of the empty vector pCMVkan (lanes 1 and 4). The mice were bled at day 1 (lanes 1–3) and sacrificed at day 3 (lanes 4–6) postinjection. Plasma samples were analyzed by a human IL-12p70-specific ELISA, and the values of individual mice and median are shown. p values using the Mann-Whitney t test are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: In vivo expression of IL-12p70 plasmids in mice. Expression of the human IL-12p70 DNA was tested in BALB/c mice upon hydrodynamic DNA delivery. The animals (n = 10–11/group) were injected with 100 ng of a plasmid expressing the IL-12 from wild type (WLV-103M; lanes 2 and 5) or from optimized coding sequences (plasmid AG181; lanes 3 and 6). The sham control group (n = 3) was injected with 100 ng of the empty vector pCMVkan (lanes 1 and 4). The mice were bled at day 1 (lanes 1–3) and sacrificed at day 3 (lanes 4–6) postinjection. Plasma samples were analyzed by a human IL-12p70-specific ELISA, and the values of individual mice and median are shown. p values using the Mann-Whitney t test are shown.
Mentions: We further evaluated the in vivo production of human IL-12p70 from different plasmids upon hydrodynamic DNA delivery into BALB/c mice (Fig. 5). The animals received 100 ng of plasmids expressing the IL-12p70 from wild type sequences (Plasmid WLV-103M, wt) and from RNA-optimized coding sequences (Plasmid AG181, opt) or sham DNA, respectively. At days 1 and 3 postinjection, the levels of the IL-12p70 heterodimer were measured in plasma using the human IL-12p70-specific ELISA (Fig. 4). Sham DNA-injected mice (lanes 1 and 4) did not show any detectable human IL-12 (threshold of the assay 0.015 ng/ml). At day 1, the mice receiving the wild type plasmid had a median IL-12p70 plasma level of 37 ng/ml (lane 2), whereas the mice receiving the optimized plasmid (lane 3) had a median of 489 ng/ml. These data show a significant ∼13-fold higher IL-12p70 production from the optimized human IL-12 plasmid and are in agreement with the observed difference between the plasmids upon transient transfection (Fig. 4A). This difference also remained significant at day 3 (lanes 5 and 6), with 1.8 and 107 ng/ml IL-12p70 for the mice that received the wild type and the optimized plasmids, respectively. Interestingly, we also noted a significantly slower apparent decay (p = 0.0004) of IL-12 in the plasma levels produced from optimized plasmid (∼4-fold decline, p = 0.0015; lanes 3 and 6) compared with the wild type plasmid (∼20-fold decline, p = 0.0004; lanes 2 and 5), suggesting that the optimized RNA is more stable in vivo, resulting in longer IL-12p70 expression. Because the human IL-12 is not active in mice, bioactivity could be tested in this experiment.

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