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Construction of an expression system for bioactive IL-18 and generation of recombinant canine distemper virus expressing IL-18.

Liu Y, Sato H, Hamana M, Moonan NA, Yoneda M, Xia X, Kai C - J. Vet. Med. Sci. (2014)

Bottom Line: However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1.The expressed proIL-18 proteins were processed to their mature forms in the cells.These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Animal Research Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

ABSTRACT
Interleukin 18 (IL-18) plays an important role in the T-helper-cell type 1 immune response against intracellular parasites, bacteria and viral infections. It has been widely used as an adjuvant for vaccines and as an anticancer agent. However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1. In this study, we constructed mammalian expression vectors carrying cDNA encoding mature canine IL-18 (cIL-18) or mouse IL-18 (mIL-18) fused to the human IL-2 (hIL-2) signal sequence. The expressed proIL-18 proteins were processed to their mature forms in the cells. The supernatants of cells transfected with these plasmids induced high interferon-γ production in canine peripheral blood mononuclear cells or mouse splenocytes, respectively, indicating the secretion of bioactive IL-18. Using reverse genetics, we also generated a recombinant canine distemper virus that expresses cIL-18 or mIL-18 fused to the hIL-2 signal sequence. As expected, both recombinant viruses produced mature IL-18 in the infected cells, which secreted bioactive IL-18. These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18. These recombinant viruses can also potentially be used as immunoadjuvants and agents for anticancer therapies in vivo.

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Related in: MedlinePlus

Induction of IFN-γ production in canine or mouse immune cells after coculture withsupernatant harvested from different recombinant-virus-infected B95a cells. (A) B95acells were infected with parental CDV (Yanaka strain), rCDV–cIL18 orrCDV–hIL2ss–cIL18 for 48 hr. The supernatants were then harvestedand cocultured with canine PBMCs for 48 hr. The supernatants were examined for IFN-γproduction with an ELISA. Three independent experiments were performed (n=3). (B) B95acells were infected with parental CDV (Yanaka strain) orrCDV–hIL2ss–mIL18 for 48 hr. The supernatants from the infected B95acells or mock-infected B95a cells were then cocultured with mouse spleen cells in aPVDF-backed microplate coated with monoclonal antibody specific for mouse IFN-γ in ahumidified 37°C incubator for 48 hr. The supernatants were then examined for IFN-γproduction by counting the individual blue–black spots under a stereomicroscope. Threeindependent experiments were performed (n=3).
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fig_005: Induction of IFN-γ production in canine or mouse immune cells after coculture withsupernatant harvested from different recombinant-virus-infected B95a cells. (A) B95acells were infected with parental CDV (Yanaka strain), rCDV–cIL18 orrCDV–hIL2ss–cIL18 for 48 hr. The supernatants were then harvestedand cocultured with canine PBMCs for 48 hr. The supernatants were examined for IFN-γproduction with an ELISA. Three independent experiments were performed (n=3). (B) B95acells were infected with parental CDV (Yanaka strain) orrCDV–hIL2ss–mIL18 for 48 hr. The supernatants from the infected B95acells or mock-infected B95a cells were then cocultured with mouse spleen cells in aPVDF-backed microplate coated with monoclonal antibody specific for mouse IFN-γ in ahumidified 37°C incubator for 48 hr. The supernatants were then examined for IFN-γproduction by counting the individual blue–black spots under a stereomicroscope. Threeindependent experiments were performed (n=3).

Mentions: Bioactivity of IL-18 produced by rCDV: To investigatewhether the mature IL-18 protein expressed by rCDV was bioactive, weharvested the supernatants of the virus-infected cells and cocultured them with caninePBMCs. The supernatant contained infectious CDV released from the cells, but the parentalCDV induced little IFN-γ production in the PBMCs (Fig.5AFig. 5.


Construction of an expression system for bioactive IL-18 and generation of recombinant canine distemper virus expressing IL-18.

Liu Y, Sato H, Hamana M, Moonan NA, Yoneda M, Xia X, Kai C - J. Vet. Med. Sci. (2014)

Induction of IFN-γ production in canine or mouse immune cells after coculture withsupernatant harvested from different recombinant-virus-infected B95a cells. (A) B95acells were infected with parental CDV (Yanaka strain), rCDV–cIL18 orrCDV–hIL2ss–cIL18 for 48 hr. The supernatants were then harvestedand cocultured with canine PBMCs for 48 hr. The supernatants were examined for IFN-γproduction with an ELISA. Three independent experiments were performed (n=3). (B) B95acells were infected with parental CDV (Yanaka strain) orrCDV–hIL2ss–mIL18 for 48 hr. The supernatants from the infected B95acells or mock-infected B95a cells were then cocultured with mouse spleen cells in aPVDF-backed microplate coated with monoclonal antibody specific for mouse IFN-γ in ahumidified 37°C incubator for 48 hr. The supernatants were then examined for IFN-γproduction by counting the individual blue–black spots under a stereomicroscope. Threeindependent experiments were performed (n=3).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4197152&req=5

fig_005: Induction of IFN-γ production in canine or mouse immune cells after coculture withsupernatant harvested from different recombinant-virus-infected B95a cells. (A) B95acells were infected with parental CDV (Yanaka strain), rCDV–cIL18 orrCDV–hIL2ss–cIL18 for 48 hr. The supernatants were then harvestedand cocultured with canine PBMCs for 48 hr. The supernatants were examined for IFN-γproduction with an ELISA. Three independent experiments were performed (n=3). (B) B95acells were infected with parental CDV (Yanaka strain) orrCDV–hIL2ss–mIL18 for 48 hr. The supernatants from the infected B95acells or mock-infected B95a cells were then cocultured with mouse spleen cells in aPVDF-backed microplate coated with monoclonal antibody specific for mouse IFN-γ in ahumidified 37°C incubator for 48 hr. The supernatants were then examined for IFN-γproduction by counting the individual blue–black spots under a stereomicroscope. Threeindependent experiments were performed (n=3).
Mentions: Bioactivity of IL-18 produced by rCDV: To investigatewhether the mature IL-18 protein expressed by rCDV was bioactive, weharvested the supernatants of the virus-infected cells and cocultured them with caninePBMCs. The supernatant contained infectious CDV released from the cells, but the parentalCDV induced little IFN-γ production in the PBMCs (Fig.5AFig. 5.

Bottom Line: However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1.The expressed proIL-18 proteins were processed to their mature forms in the cells.These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18.

View Article: PubMed Central - PubMed

Affiliation: Laboratory Animal Research Center, Institute of Medical Science, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan.

ABSTRACT
Interleukin 18 (IL-18) plays an important role in the T-helper-cell type 1 immune response against intracellular parasites, bacteria and viral infections. It has been widely used as an adjuvant for vaccines and as an anticancer agent. However, IL-18 protein lacks a typical signal sequence and requires cleavage into its mature active form by caspase 1. In this study, we constructed mammalian expression vectors carrying cDNA encoding mature canine IL-18 (cIL-18) or mouse IL-18 (mIL-18) fused to the human IL-2 (hIL-2) signal sequence. The expressed proIL-18 proteins were processed to their mature forms in the cells. The supernatants of cells transfected with these plasmids induced high interferon-γ production in canine peripheral blood mononuclear cells or mouse splenocytes, respectively, indicating the secretion of bioactive IL-18. Using reverse genetics, we also generated a recombinant canine distemper virus that expresses cIL-18 or mIL-18 fused to the hIL-2 signal sequence. As expected, both recombinant viruses produced mature IL-18 in the infected cells, which secreted bioactive IL-18. These results indicate that the signal sequence from hIL-2 is suitable for the secretion of mature IL-18. These recombinant viruses can also potentially be used as immunoadjuvants and agents for anticancer therapies in vivo.

Show MeSH
Related in: MedlinePlus