Journal: Communications medicine

Article Title: Fusion peptide is superior to co-expressing subunits for arming oncolytic herpes virus with interleukin 12.

doi: 10.1038/s43856-023-00270-4

Figure Lengend Snippet: Fig. 1 The structures of T-mfIL12 and T-mIL12-IRES. The boxes (top line) represent inverted repeat sequences flanking the long (UL) and short (US) unique sequences of HSV-1 DNA. Oncolytic HSV-1 uses G47Δ as the backbone and contains 1.0 kb deletions in both copies of the γ34.5 gene, a 0.3 kb deletion in the α47 gene, and a 0.9 kb deletion in the ICP6 gene. The lacZ gene and the cytomegalovirus (CMV) promoter-driven interleukin-12 (IL-12) gene are oriented in opposite directions, and inserted into the deleted ICP6 locus. T-mfIL12 contains a cassette carrying the p35 and p40 genes of murine IL-12 linked by two bovine elastin motifs. Murine IL-12 is expressed as a single fusion peptide and folds to become active. T-mIL12- IRES contains a cassette in which the p40 and p35 genes of murine IL-12 are separated by an internal ribosome entry site (IRES) sequence from the encephalomyocarditis virus (EMCV). The p40 and p35 subunits are efficiently co-expressed to form active murine IL-12. T-01 contains a cassette with no transgene.

Article Snippet: Supernatants were collected, and the concentration of IL-12 was measured using mouse IL12 p70 immunoassay (M1270, R&D Systems Inc., IL) with a detection limit of 7.8 pg/ ml.

Techniques: Sequencing, Virus

Journal: Communications medicine

Article Title: Fusion peptide is superior to co-expressing subunits for arming oncolytic herpes virus with interleukin 12.

doi: 10.1038/s43856-023-00270-4

Figure Lengend Snippet: Fig. 2 In vitro replication capabilities and murine IL-12 expressions of T-mfIL12 and T-mIL12-IRES. a In vitro replication assay. Vero cells were infected with G47Δ, T-01, T-mfIL12 candidate (4 clones) or T-mIL12-IRES candidate (4 clones) at a multiplicity of infection (MOI) of 0.01, the progeny virus was recovered 48 h after infection, and the number determined by plaque assay. T-mfIL12 and T-mfIL12-IRES showed no significant difference in replication capability (p = 0.736, t-test). b In vitro murine IL-12 expression. Vero cells were infected with G47Δ, T-01, T-mfIL12 candidate (4 clones) or T-mIL12-IRES candidate (4 clones) at an MOI of 1, and the amount of murine interleukin-12 (IL-12) (p70) secreted was determined by enzyme-linked immune-sorbent assay (ELISA). T-mfIL12 expressed a significantly higher amount of p70 IL-12 than T-mIL12-IRES (p < 0.001, t-test). c The time course of viral yields. Vero cells were infected with G47Δ, T-01, T-mfIL12 or T-mIL12-IRES in duplicate at an MOI of 0.01, the progeny virus was recovered 0 h, 6 h, 24 h and 48 h after infection, and titrated by plaque assay. The time course for the viral yields of T-mfIL12 was comparable to that of T-mIL12-IRES. d In vitro murine IL-12 expression in murine tumor cell lines. Neuro2a, Pr14-2 or TRAMP-C2 cells were infected with T-mfIL12 or T-mIL12-IRES at an MOI of 1, and the amount of murine IL-12 (p70) secreted was determined by ELISA. T-mfIL12 expressed a higher amount of p70 IL-12 than T-mIL12-IRES in all three cell lines. All assays were performed in duplicate. ***, p < 0.001; NS, not significant.

Article Snippet: Supernatants were collected, and the concentration of IL-12 was measured using mouse IL12 p70 immunoassay (M1270, R&D Systems Inc., IL) with a detection limit of 7.8 pg/ ml.

Techniques: In Vitro, Infection, Clone Assay, Virus, Plaque Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Communications medicine

Article Title: Fusion peptide is superior to co-expressing subunits for arming oncolytic herpes virus with interleukin 12.

doi: 10.1038/s43856-023-00270-4

Figure Lengend Snippet: Fig. 5 Immune responses by T-mfIL12 and T-mIL12-IRES. a Immunohistochemistry. Bilateral subcutaneous Neuro2a tumors were generated in A/J mice, left tumors only were inoculated with T-mfIL12, T-mIL12-IRES, T-01 (2 × 105 pfu) or mock on days 0 and 3, and the tumors were harvested on day 6 (n = 3 per group). An increased infiltration of CD4+ and CD8+ lymphocytes were observed in the tumor for all three viruses, both in the treated and the untreated side, most prominently with T-mfIL12 (Fig. 5a). HSV-1 positive cells were observed in the tumor with all viruses in the treated side, but not in the untreated side. HE, hematoxylin and eosin. Scale bars, 100 μm. b In vivo levels of interleukin-12 (IL-12) and Interferon γ (IFNγ). In the unilateral subcutaneous Neuro2a model, established tumors were inoculated with T-01, T-mfIL12, T-mIL12-IRES (2 × 106 pfu) or mock, sera and tumor samples were collected on days 1, 3 and 6 (n = 3 per group), and the levels of mouse IL-12 and IFNγ were measured by ELISA. The intratumoral IL-12 levels for T-mfIL12 were significantly higher than those for T-mIL12-IRES at all time points (p = 0.018, p = 0.016 and p = 0.046 for days 1, 3 and 6, respectively). The levels of IL-12 detected from the serum were remarkably lower than those in the tumor. Correlating with the intratumoral IL-12, the serum IL-12 level for T-mfIL12 was higher than that for T-mIL12-IRES on day 1 (p = 0.047). The IFNγ levels of T-mfIL12 were significantly higher than those of T-mIL12-IRES both in the tumor and serum on day 1 (p = 0.014 and p = 0.027, tumor and serum, respectively). c Immune responses specific to Neuro2a cells. In the unilateral subcutaneous Neuro2a model, established tumors were inoculated with T-01, T-mfIL12, T-mIL12-IRES (5 × 104 pfu) or mock on days 0 and 3, and the spleen was harvested on day 6. By ELISpot assay, splenocytes from T-mfIL12-treated mice showed a significantly higher number of IFNγ release stimulated by Neuro2a cells than those from T-01- and T-mIL12-IRES- treated ones (p = 0.005 and p = 0.004 vs T-01 and T-mIL12-IRES, respectively). No significant difference in number of IL-4 releasing splenocytes was observed among the three virus-treated groups. The IFNγ or IL-4 releases specific to Neuro2a cells were calculated by subtracting the numbers of SaI/N responding spots from those of Neuro2a responding spots. For b and c, Graphs show the means. Dots represent individual data. Bars, SD. *, p < 0.05; **, p < 0.01; NS, not significant; one-way ANOVA with Tukey’s multiple comparisons.

Article Snippet: Supernatants were collected, and the concentration of IL-12 was measured using mouse IL12 p70 immunoassay (M1270, R&D Systems Inc., IL) with a detection limit of 7.8 pg/ ml.

Techniques: Immunohistochemistry, Generated, In Vivo, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Virus

Journal: Communications medicine

Article Title: Fusion peptide is superior to co-expressing subunits for arming oncolytic herpes virus with interleukin 12.

doi: 10.1038/s43856-023-00270-4

Figure Lengend Snippet: Fig. 6 Comparison of in vivo efficacy of T-mfIL12 and direct intratumoral injection with recombinant interleukin-12 (rIL-12). In the bilateral subcutaneous Neuro2a model, rIL-12, T-01 (5 × 104 pfu) without or with rIL-12, T-mfIL12 (5 × 104 pfu) or mock was inoculated into the left tumors only on days 0 and 4 (n = 10 per group). Three different doses were used for rIL-12; 500 ng (a), 50 ng (b) and 1 ng (c). The dose 50 ng represents the intratumoal IL-12 level treated with T-mfIL12 at 2 × 106 pfu and 1 ng represents that at 5 × 104 pfu, the T-mfIL12 dose used in these experiments. a When the dose of 500 ng was used for rIL-12, rIL-12, T-01, T-01+rIL-12 and T-mfIL12 were all significantly more efficacious than mock in the treated side (p < 0.001 vs mock for all). In the untreated side, rIL-12 alone showed no significant antitumor effect compared with mock, whereas T-01+rIL-12 and T-mfIL12 showed a significantly higher efficacy than mock (p = 0.039 and p < 0.001 vs mock, respectively). Further, in the untreated side, T-mfIL12, but not T-01+rIL-12, was significantly more efficacious than rIL-12 alone (p = 0.003). b Results similar to a were obtained when the dose of 50 ng was used for rIL-12. c When the dose of 1 ng was used for rIL-12, rIL-12 alone showed no significant efficacy in both treated and untreated sides, and T-mfIL12, but not T-01+rIL-12, was significantly more efficacious than rIL-12 alone in the treated side. Tumor volume = length × width × height × 0.52. Results represent the mean. Bars, standard error of the mean (SEM). *, p < 0.05; **, p < 0.01; ***, p < 0.001; ns, not significant; Two-way ANOVA with Bonferroni’s multiple comparisons test.

Article Snippet: Supernatants were collected, and the concentration of IL-12 was measured using mouse IL12 p70 immunoassay (M1270, R&D Systems Inc., IL) with a detection limit of 7.8 pg/ ml.

Techniques: Comparison, In Vivo, Injection, Recombinant

Journal: International Journal of Nanomedicine

Article Title: IL-12-Overexpressed Nanoparticles Suppress the Proliferation of Melanoma Through Inducing ICD and Activating DC, CD8+ T, and CD4+ T Cells

doi: 10.2147/ijn.s442446

Figure Lengend Snippet: Figure 2 CPP transferred IL-12 into B16F10 cells and CPP-IL-12 induced immunogenic cell death. (A) The structure of IL-12 vector. (B) Western blot detection of IL-12 expression. **P < 0.01, Student’s t-test. (C) ELISA. CPP-IL-12 increased IL-12 levels in the supernatant of B16-F10 cells, and 2.0 W/cm2 of laser further increased IL-12 levels. **P < 0.01, ANOVA. (D) Live/dead staining assay. Scale bars=100 μm. (E) The number of dead cells. CPP-IL-12 increased and 2.0 W/cm2 of laser further increased the number of dead cells. **P < 0.01, ANOVA. (F) Cell apoptosis analysis. CPP-IL-12 increased and laser treatment further increased the number of apoptotic cells. (G) Immunofluorescence. Green color, CALR expression. Scale bars=2 μm. (H, I) ELISA analysis of extracellular and intracellular HMGB1 levels, respectively. *P < 0.05, **P < 0.01, ANOVA. (J) ATP levels. The ATP levels decreased in CPP-IL-12-treated cells, and laser treatment further reduced ATP content. **P < 0.01, ANOVA. (K) CPP increased CD80+/CD86+ expression of DC.

Article Snippet: IL-12 and HMGB1 levels were estimated using ELISA kits (CSB-E04600m and CSB-E08225M, CUSABIO, Wuhan, China) in accordance with the manufacturer′s instructions.

Techniques: Plasmid Preparation, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Staining, Immunofluorescence