Fig 2: Combination therapy by oHSV-CD40L and PD-1 blockade. (A, B) Cell suspensions from treated Pan02_HVEM tumors were stained with indicated fluorescence-conjugated antibodies on day 20. The expression of MHC-I (A) and PD-L1 (B) on tumor cells was analyzed by flow cytometry and plotted in scatter graphs (n=5). Statistical analysis was performed by two-tailed Student’s t-test. (C) Scheme of combination therapy of oHSVs and PD-1 antibody. (D) After indicated treatments, tumor volumes were measured every third day and plotted as the vertical axis (n=6). (E) The survival of mice was subjected to Kaplan-Meier analysis and log-rank test (n=6). (F) Experimental layout of combination therapy of oHSVs and PD-1 antibody for the bilateral tumor mouse model. The growth of tumor in the treated flank (G) and the contralateral flank (H) was monitored and plotted with volumes in the vertical axis (n=4). Data were presented as mean±SD (A and B) or mean+SD (D, G, and H). *p<0.05; **p<0.01; ****p<0.0001. MHC, major histocompatibility complex; oHSV, oncolytic herpes simplex virus-1; PBS, phosphate buffered saline; PD-1, programmed cell death-1; PD-L1, programmed death-ligand 1.

Fig 3: The construction and cytolytic effects of oHSV-CD40L. (A) Schematic representations of oHSV and oHSV-CD40L genomes. For oHSV (upper row), both ICP34.5 loci were replaced with GFP-coding sequences, and the ICP47 gene region was deleted. oHSV-CD40L (lower row) was derived from oHSV by replacing the GFP sites with murine CD40L-encoding sequence. (B) Pan02_HVEM cells were mock-infected or infected with HSV-1 or oHSV-CD40L, and co-cultured with Jurkat/NF-κB-GFP-CD40 reporter cells as described in the ‘Materials and methods’ section. These cells were then subjected to flow cytometry analysis. The histograms were shown with fluorescence intensity of GFP as the horizontal axis. The reporter cells that were not co-cultured with Pan02_HVEM cells were designated as blank. The percentage of GFP-positive cells was labeled inside. (C) Pan02_HVEM cells were mock-infected or infected with wild-type HSV-1, oHSV, or oHSV-CD40L (MOI=5), respectively, and the cytopathic effects were monitored under microscopy at 24 hpi. Scale bar denotes 100 μm. (D) Pan02_HVEM cells were mock-infected or infected with wild-type HSV-1, oHSV, or oHSV-CD40L (MOI=1) for 24 hours, respectively. Cells were then stained with calreticulin (CRT) antibody for flow cytometry and the percentages of CRT+ cells were plotted in columns. The media were harvested, and the levels of released ATP were determined and plotted in E. (F) Total mRNA was isolated from cells infected as indicated, and IFN-β mRNA was quantitated by real-time quantitative PCR and plotted as a ratio relative to that of β-actin. Data were representative of three independent experiments. All values were presented as mean±SD. Statistics were performed using two-tailed Student’s t-test (D–F). *p<0.05; **p<0.01; ***p<0.001. hpi, hours post infection; IFN, interferon; MOI, multiplicity of infection; oHSV, oncolytic herpes simplex virus-1.

Fig 4: Antitumor effect of oHSV-CD40L. (A) Experimental layout of oHSVs therapy in Pan02_HVEM model. Mice were inoculated with Pan02_HVEM cells on day 0. The treatments were performed on days 8, 11, and 14 (arrows) with PBS, oHSV, or oHSV-CD40L, respectively. (B) Tumor volumes were measured every third day and plotted as the vertical axis (n=6). (C) The survival of Pan02_HVEM-bearing mice was plotted using Kaplan-Meier analysis and log-rank test (n=6). (D) On day 20, mice were executed and Pan02_HVEM tumors were isolated from individual mice and pictured with the treatments labeled on the left. The weight of each tumor sample was presented in E (n=5). (F) Experimental layout of oHSVs therapy in subcutaneous KPC model. The treatments were performed on day 4, 7, and 10 with PBS, oHSV, or oHSV-CD40L, respectively. (G) Tumor volumes were monitored and plotted as the vertical axis (n=6). (H) The survival of KPC-bearing mice was subjected to Kaplan-Meier analysis and log-rank test (n=6). (I) On day 16, KPC tumors were isolated from individual mice and pictured. The weight of each tumor sample was presented in J (n=6). Data were presented as mean+SD (B and G) or ±SD (E and J). Two-tailed Student’s t-test was performed in B (on day 20), E, G (on day 19), and J. *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. oHSV, oncolytic herpes simplex virus-1; PBS, phosphate buffered saline.

Fig 5: Repeated dosing therapy by oHSV-CD40L. (A) Repeated dosing schedule of oHSVs therapy. Mice were treated with oHSVs for 3 or 9 times in a row of every third day. Tumor volumes were monitored and plotted as the vertical axis in B, and the survival of tumor-bearing mice was monitored by Kaplan-Meier analysis in C. PBS, oHSV, or oHSV-CD40L×3 (n=5); oHSV or oHSV-CD40L×9 (n=6). (D) Scheme of rechallenged experiment. The oHSV-CD40L cured mice on the 80th day after the initial tumor graft or age-matched mice were inoculated with Pan02_HVEM cells subcutaneously (renamed as day 0). The survival of rechallenged mice (red) and age-matched mice (blue) was monitored by Kaplan-Meier analysis (n=5). Data were presented as mean+SD in B. Log-rank test on mice survival was performed in C and E. *p<0.05; **p<0.01; ***p<0.001. oHSV, oncolytic herpes simplex virus-1; PBS, phosphate buffered saline.