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

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.

Fig. 1.

Mammalian expression plasmids for canine and mouse IL-18. (A) Schematic representation of IL-18 constructs. From top to bottom: full-length sequence of canine IL-18, mature canine IL-18 fused to the canine IL-12 signal sequence, mature canine IL-18 fused to the human IL-2 signal sequence and mature mouse IL-18 fused to the human IL-2 signal sequence were inserted into the pCAGGS and pFuse–hIgG2–Fc2 vectors. (B) The expression of IL-18 by transfected 293T cells was analyzed with immunoblotting using anti-GAPDH, anti-cIL-18 and anti-mIL-18 antibodies. 1. Mock; 2. pCAG–cIL18; 3. pCAG–cIL12ss–cIL18; 4. pFuse–hIL2ss–cIL18; 5. pFuse–hIL2ss–mIL18.

Fig. 2.

Induction of IFN-γ by incubating canine or mouse immune cells with the supernatant from transfected cells. (A) The supernatant from transfected 293T cells or mock-transfected 293T cells was cocultured with canine PBMCs. After 48 hr, the supernatant was examined for IFN-γ production with an ELISA. Three independent experiments were performed (n=3). (B) The supernatant from transfected 293T cells or mock-transfected 293T cells was cocultured with mouse spleen cells in a polyvinylidene difluoride (PVDF)-backed microplate coated with monoclonal antibody specific for mouse IFN-γ in a humidified 37°C incubator for 48 hr. The supernatant was examined for IFN-γ production by counting the individual blue–black spots under a stereomicroscope. Three independent experiments were performed (n=3).

Fig. 3.

Generation and in vitro characterization of rCDVs expressing mature canine or mouse IL-18 fused to the human IL-2 signal sequence. (A) Schematic model of the rCDV genome with the FseI site introduced between the N and P genes and hIL2ss–IL18 inserted at the FseI site. (B) The resulting viruses were harvested and identified by RT-PCR using primers complementary to the CDV-N gene and the canine IL-18 or mouse IL-18 gene. (C) Recombinant viruses were identified by immunoblotting analysis. Cell lysates were examined with anti-CDVN, anti-GAPDH, anti-cIL-18 or anti-mIL-18 antibody. 1: B95a; 2: parental CDV (Yanaka strain); 3: rCDV–cIL18; 4: rCDV–hIL2ss–cIL18; 5: rCDV–hIL2ss–mIL18.

Fig. 4.

Kinetics of recombinant viruses in B95a cells. The infected cells and supernatants were harvested separately every 24 hr after infection for 7 days. The titers of the viruses released into the supernatant (A) and the cell-associated viruses (B) were determined with a TCID₅₀ assay.

Fig. 5.

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