Fusogenic oncolytic vaccinia virus enhances systemic antitumor immune response by modulating the tumor microenvironment

Abstract

Oncolytic viruses induce antitumor immunity following direct viral oncolysis. However, their therapeutic effects are limited in distant untreated tumors because their antitumor function depends on indirect antitumor immunity. Here, we generated a novel fusogenic oncolytic vaccinia virus (FUVAC) and compared its antitumor activity with that of its parental non-fusogenic virus. Compared with the parent, FUVAC exerted the cytopathic effect and induced immunogenic cell death in human and murine cancer cells more efficiently. In a bilateral tumor-bearing syngeneic mouse model, FUVAC administration significantly inhibited tumor growth in both treated and untreated tumors. However, its antitumor effects were completely suppressed by CD8⁺ T cell depletion. Notably, FUVAC reduced the number of tumor-associated immune-suppressive cells in treated tumors, but not in untreated tumors. Mice treated with FUVAC before an immune checkpoint inhibitor (ICI) treatment achieved complete response (CR) in both treated and untreated tumors, whereas ICI alone did not show antitumor activity. Mice achieving CR rejected rechallenge with the same tumor cells, suggesting establishment of a long-term tumor-specific immune memory. Thus, FUVAC improves the tumor immune microenvironment and enhances systemic antitumor immunity, suggesting that, alone and in combination with ICI, it is a novel immune modulator for overcoming oncolytic virus-resistant tumors.

Keywords: antitumor immunity; cell membrane fusion; immune checkpoint blockade; oncolytic function; vaccinia virus.

Graphical abstract

Figure 1

Viral replication and oncolytic effect in MDRVV and FUVAC (A) Schematic representation of MDRVV and FUVAC. (B) A549 cells were infected with MDRVV- or FUVAC-LG/DsRed at an MOI of 0.01, 0.1, or 1, and the images were taken 3 days after infection. Scale bar, 500 μm. (C) Viability of the A549 cells 72 h after virus infection as described in (B) was examined with CellTiter 96 Aqueous Nonradioactive Cell Proliferation Assay (Promega). Data are represented as the percent survival of mock-infected cells. (D) A549 cells infected with MDRVV- or FUVAC-LG/DsRed at an MOI of 0.1 were harvested after infection for 24, 48, or 72 h, and the extracted intracellular viruses were titrated in RK13 cells. Data in (C) and (D) are presented as the means ± SEM (n = 3). ∗∗∗p < 0.001 (two-tailed unpaired t test).

Figure 2

Cytolytic fusion of FUVAC against several cancer cell lines (A) Several cancer cell lines were infected with MDRVV- or FUVAC-LG/DsRed at each MOI: Panc1, CaCO₂, MDA-MB-231, A549, and A431, 0.1; SKOV3 and PC3, 1; and B16-F10, CT26, and TC1, 5. Cells were photographed 3 days after virus infection, except for TC1 cells that were photographed after 2 days. Scale bar, 500 μm. (B) Viability of tumor cell lines described in (A). Data are presented as the percent survival of mock-infected cells and represented as the means ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (two-tailed unpaired t test).

Figure 3

Three types of cell death efficiently induced by FUVAC (A) A549 cells were infected with MDRVV- or FUVAC-Luc/LacZ at an MOI of 1, and the apoptotic or necrotic cells were detected by staining with annexin V or ethidium homodimer, respectively, 30 h after infection. HMGB1 release was detected using ELISA of the culture supernatant of the infected cells 60 h after infection. (B) CT26 cells were infected with MDRVV or FUVAC at an MOI of 10, and apoptosis or necrosis was detected as described in (A) 22 h after infection. Meanwhile, the culture supernatant of CT26 cells infected with MDRVV or FUVAC at an MOI of 5 for 60 h was used for HMGB1 detection as described in (A). Data in (A) and (B) are presented as the means ± SEM (n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (two-tailed unpaired t test).

Figure 4

In vivo viral replication and oncolytic effect of FUVAC (A) Schedule of MDRVV or FUVAC treatment against the bilateral CT26 tumor model. CT26 cells were bilaterally transplanted on BALB/c mice through subcutaneous injection. When the average tumor volume reached 60 mm³, 5 × 10⁷ PFUs of MDRVV or FUVAC-Luc/LacZ were intratumorally injected three times every 2 days. (B) In vivo bioluminescence imaging of the viral replication. Viral Fluc luminescence was detected by VivoGlo Luciferin (3 mg/mouse; Promega) on day 3. (C) Quantification of the viral luminescence on days 1, 3, 5, and 7 after the first virus injection. Fluc signals were separately quantified in the injection and non-injection sites. Mean luminescence (ph/s) ± SEM are shown. ∗p < 0.05 (two-way ANOVA). (D) Tumor growth was separately monitored in the injection and non-injection sites. Results are representative of two independent experiments comprising 6–8 mice/group (n = 12, 14, or 15 for PBS, MDRVV, or FUVAC, respectively). Mean tumor volume (mm³) ± SEM are shown. ∗∗p < 0.01, ∗∗∗p < 0.001 (two-way ANOVA).

Figure 5

Fusogenic findings in in vivo tumor tissues (A) Schedule of the tumor tissue collection for IHC. Mice bearing bilateral CT26 tumors were treated with 5 × 10⁷ PFUs of MDRVV- or FUVAC-LG/DsRed on days 0, 2, and 4, then tumors were collected on day 5. (B) Bioluminescence imaging before tumor collection on day 5. (C) Whole pictures of virus-treated or -untreated tumors. Upper images show the hematoxylin and eosin staining, and lower images show the anti-GFP antibody staining. Scale bar, 1,000 μm. (D) High-power images of the area marked in (C). Arrows show the fusogenic phenotype. Representative images are shown in (C) and (D). Pictures from the other two mice are shown in Figure S3. Scale bar, 50 μm.

Figure 6

Changing tumor immune microenvironment by FUVAC infection (A) Schedule of the immune microenvironment characterization after MDRVV or FUVAC treatment. Bilateral CT26-bearing mice were treated with MDRVV or FUVAC using the same method as Figure 4. 5 days after the first virus injection (1 day after the third injection), the virus-treated or -untreated tumors were recovered and processed for flow cytometry analysis. (B) The ratio of CD45⁺ total immune cells and total tumor cells, and the ratio of CD3⁺ T lymphocyte in CD45⁺ cells. (C) The ratios of CD3⁺/CD8⁺ and CD3⁺/CD4⁺ cells in CD45⁺ T lymphocyte, and the ratio of Treg in CD3⁺ T cell as determined by the expression levels of CD4, CD25, and FoxP3. (D) The ratio of TAM (CD11b⁺ F4/80⁺), M-MDSC (CD11b⁺ Ly6C⁺), or G-MDSC (CD11b⁺ Ly6C^– Ly6G⁺) in CD45⁺ cells. Data in (B)–(D) are presented as the means ± SEM (n = 7). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (two-tailed unpaired t test).

Figure 7

Effect of immune depletion against FUVAC treatment (A) Schedule of the CD4 or CD8 depletion. CT26-bearing mice were administrated with MDRVV or FUVAC on days 0, 2, and 4, and 200 μg of Isotype ctrl, anti-CD4, or anti-CD8 antibody was injected on days 3, 5, and 7. (B and C) Tumor growth curves of mice after the treatment of each inhibitor with MDRVV (B) or FUVAC (C). Mean tumor volume (mm³) ± SEM are shown (n = 6–7). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001 (two-way ANOVA).

Figure 8

Effect of PD-1 blockade under FUVAC treatment (A) Schedule of the combination of virus with PD-1 blockade. (B and C) Tumor growths in mice treated with PBS, MDRVV, or FUVAC + mock (B) or + anti-PD-1 antibodies (C). Data are presented as the mean tumor volume (mm³) ± SEM (n = 5–6). ∗∗∗p < 0.001 (two-way ANOVA). (D) Survival curves of mice associated with (B) and (C) generated using Kaplan-Meier analysis. FUVAC alone prolonged the survival of mice compared with the PBS treatment (∗p = 0.0112). FUVAC + anti-PD-1 prolonged the survival longer than anti-PD-1 antibody alone or MDRVV + anti-PD-1 (∗∗∗p = 0.0007 or ∗∗p = 0.0066, respectively, using log-rank test). (E) Tumor growths in CR mice and control mice after tumor rechallenge (n = 3).