Volume 12, Issue 5, Pages 789-802 (November 2005) HSV oncolytic therapy upregulates interferon-inducible chemokines and recruits immune effector cells in ovarian cancer  Fabian Benencia, Maria C. Courrèges, José R. Conejo-García, Alisha Mohamed-Hadley, Lin Zhang, Ronald J. Buckanovich, Richard Carroll, Nigel Fraser, George Coukos  Molecular Therapy  Volume 12, Issue 5, Pages 789-802 (November 2005) DOI: 10.1016/j.ymthe.2005.03.026 Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 1 Chemokine and cytokine induction by HSV-1716 in murine monocytes and DCs. (A) Flow cytometry analysis of surface antigens on in vitro generated murine monocytes. (B) Real-time quantitative RT-PCR analysis showed higher IFN-γ, IP-10, and MIG mRNA expression by murine monocytes 72 h after infection (1 m.o.i.) with live HSV-1716 compared to UV-inactivated virus (mock). (C) High levels of IFN-γ, MIG, and IP-10 proteins were detected by ELISA in supernatants of murine monocyte cultures 72 h after infection with live HSV-1716. (D) Flow cytometry analysis of surface antigens on in vitro generated murine DCs. (E) Time course upregulation of MIG and IP-10 mRNAs in response to HSV-1716 infection. (MIG: 12 h P < 0.01, 24 h P < 0.05, 48 and 72 h, ns. IP-10: 12 h P < 0.01, 24 h P < 0.01, 48 h P < 0.01, 72 h P < 0.05. ANOVA, Tukey–Kramer multicomparison posttest.) (F) Real-time quantitative RT-PCR analysis showed higher IFN-γ mRNA expression by murine DCs 72 h after infection (1 m.o.i.) with live HSV-1716 compared to UV-inactivated virus (mock). (G) High levels of IFN-γ, MIG. and IP-10 proteins were detected by ELISA in supernatants of murine DC cultures 72 h after infection with live HSV-1716 UV-inactivated virus (mock). Molecular Therapy 2005 12, 789-802DOI: (10.1016/j.ymthe.2005.03.026) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 2 ELISA of (A, C) IP-10 and (B, D) MIG in supernatants of monocyte (A, B) or DC (C, D) cultures 72 h after HSV-1716 infection (1 m.o.i.). Immediately after infection, 104 U/ml anti-mouse IFN-α or IFN-β or both was added to cell cultures where indicated. Control cultures were infected with UV-inactivated HSV-1716 (mock). (A) HSV-1716 vs mock, P < 0.001; HSV-1716 vs anti-IFN-α, P < 0.01; HSV-1716 vs anti-IFN-β, P < 0.001; HSV-1716 vs anti-IFN-α/β, P < 0.001. (B) HSV-1716 vs mock, P < 0.001; HSV-1716 vs anti-IFN-α, P < 0.001; HSV-1716 vs anti-IFN-β, P < 0.001; HSV-1716 vs anti-IFN-α/β, P < 0.001. (C) HSV-1716 vs mock, P < 0.001; HSV-1716 vs anti-IFN-α, P < 0.05; HSV-1716 vs anti-IFN-β, P < 0.001; HSV-1716 vs anti-IFN-α/β, P < 0.001. (D) HSV-1716 vs mock, P < 0.001; HSV-1716 vs anti-IFN-α, ns; HSV-1716 vs anti-IFN-β, ns; HSV-1716 vs anti-IFN-α/β, ns. ANOVA, Tukey–Kramer multicomparison posttest. Molecular Therapy 2005 12, 789-802DOI: (10.1016/j.ymthe.2005.03.026) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 3 HSV-1716 oncolytic effects in vitro and in vivo. (A) ID8-VEGF cells were susceptible to infection by HSV-1716 as assessed by viral growth curve. (B) HSV-1716 killed ID8-VEGF. Cells were exposed to HSV-1716 at 0.01, 0.1, and 1 m.o.i. Cell survival was assessed by the MTS colorimetric assay. *P < 0.05, **P < 0.01. (C) Survival curves of animals treated ip with 5 × 106 PFU of live or UV-inactivated HSV-1716 (mock) given 6 h after ip tumor implantation (arrow). Data represent means ± SD, n = 5, Mann–Whitney test. The experiment was repeated two times with similar results. (D). Survival curves of animals treated ip with two doses of live or UV-inactivated HSV-1716 (5 × 106 PFU) given when the animals exhibited visible ascites (arrows). Data represent means ± SD, n = 5, Mann–Whitney test. (E, F) Tumor growth upon repeated intratumoral injection of live or UV-inactivated HSV-1716 (5 × 106 PFU, arrows). Data represent means ± SD, n = 5, Mann–Whitney test. *P < 0.05. The experiment was repeated three times with similar results. (F) Left, tumors treated with HSV-1716; right, tumors treated with UV-inactivated virus. Molecular Therapy 2005 12, 789-802DOI: (10.1016/j.ymthe.2005.03.026) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 4 Chemokine and cytokine induction by therapeutic HSV-1716 treatment in a syngeneic model of ovarian carcinoma. (A) ELISA of ascites showed an increase in the levels of IFN-γ (n = 6), MIG (n = 10), and IP-10 (n = 10) together with a decrease in IL-4 protein (n = 6) 5 days after inoculation of live HSV-1716 (5 × 106 PFU ip) compared to UV-inactivated virus (mock) in the intraperitoneal model of murine ovarian carcinoma. (B) Real-time quantitative RT-PCR analysis of IFN-γ, IL-4, IP-10, and MIG mRNA expression in solid tumors (n = 5) after intratumoral treatment with HSV-1716 (5 × 106 PFU) or UV-inactivated virus (mock). Molecular Therapy 2005 12, 789-802DOI: (10.1016/j.ymthe.2005.03.026) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 5 HSV-1716 promotes tumor infiltration of CD3+ and NK1.1+ cells. (A) Flow cytometry analysis shows an increase in the proportion of peritoneal CD45+ cells in ascites of animals treated with HSV-1716 (5 × 106 PFU) (left) compared to UV-inactivated virus (mock, right) 5 days after virus inoculation. (B) Higher frequency of CD3+ and NK1.1+ cells is seen in ascites of HSV-1716-treated animals compared to controls treated with UV-inactivated virus (mock). (C) An increase in tumor-infiltrating CD8+ leukocytes was observed in mouse ovarian tumors treated with HSV-1716 compared to controls as determined by immunohistochemical analysis. Nuclei were counterstained with hematoxylin (original magnification ×100). (D) Higher frequency of CD8+ infiltrating cells was observed in HSV-1716-treated tumors compared to mock-treated tumors. (E) Immunofluorescent staining demonstrates an increase in NK1.1+ tumor-infiltrating leukocytes in HSV-1716-treated tumors. Original magnification ×200. Nuclei were counterstained with DAPI. (F) Higher frequency of NK1.1+ infiltrating cells was observed in tumors of HSV-1716-treated animals compared to mock-treated tumors. Molecular Therapy 2005 12, 789-802DOI: (10.1016/j.ymthe.2005.03.026) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 6 Tumor-infiltrating CD11c+ cells were observed in (A) ascites or (B) solid ovarian carcinomas (original magnification ×200) as determined by flow cytometry or immunohistochemical analysis, respectively. Similarly, CD14+ cells were also present in (F) ascites and (G) solid tumor samples (original magnification ×200) as determined by flow cytometry and immunofluorescence analysis, respectively. Flow cytometry analysis showed expression of HSV glycoprotein D in (C) CD11c+ and (H) CD14+ cells from ascites 24 h after ip treatment with 5 × 106 PFU of live HSV-1716 (solid line) compared to mock (UV-inactivated virus; gray area). Dashed line, isotype control. (D) CD11c+ and (I) CD14+ cells immunopurified from ascites of animals treated with live HSV-1716 showed higher expression of MIG and IP-10 mRNA as determined by real-time quantitative RT-PCR. (E) CD11c+ and (J) CD14+ cells immunopurified from ascites of HSV-1716-treated animals showed higher expression of IFN-γ mRNA as determined by real-time quantitative RT-PCR. ND, not detected. Molecular Therapy 2005 12, 789-802DOI: (10.1016/j.ymthe.2005.03.026) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 7 Chemoattraction of CD8+ T and NK cells by HSV-1716. (A) Chemotaxis of splenic CD8+ T and NK cells was higher toward ascites obtained from HSV-1716-treated mice compared to mock-infected controls. Some experiments were performed in the presence of neutralizing anti-mouse IP-10 (anti-IP-10) or MIG (anti-MIG) antibodies. Left: CD8+ cells. HSV-1716 vs mock (P < 0.05), HSV-1716 vs HSV-1716 + anti-IP-10 (P < 0.05), HSV-1716 vs HSV-1716 + anti-MIG (P < 0.05). Right: NK cells. HSV-1716 vs mock (P < 0.01), HSV-1716 vs HSV-1716 + anti-IP-10 (P < 0.05), HSV-1716 vs HSV-1716 + anti-MIG (P < 0.05). (B) Tumor-infiltrating CD8+ T and NK cells obtained from ascites of mice treated with HSV-1716 ip showed higher chemotactic response toward IP-10 and MIG compared to similar cells extracted from ascites of mock-treated animals (left, CD8+ cells; right, NK cells). (C) Flow cytometry analysis showed increased expression of CXCR3 in tumor-infiltrating CD8+ T and NK1.1+ cells obtained from HSV-1716-treated animals (gray area) compared to controls (solid line). Dashed line, isotype control. Molecular Therapy 2005 12, 789-802DOI: (10.1016/j.ymthe.2005.03.026) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions

Fig. 8 Human ovarian carcinomas are infiltrated with CD11c+CD83+, CD14+HLA DR+, and CD68+HLA DR+ cells. (A) Immunohistochemical analysis of solid human ovarian carcinomas revealed the presence of tumor-infiltrating leukocytes showing dendritic markers CD1a, CD83, and CD11c and monocyte markers CD14 and CD68 (original magnification ×200). (B) Similarly, CD11c+CD83+, CD14+HLA DR+, and CD68+HLA DR+ cells were detected in samples of human ascites as determined by flow cytometry analysis. (C) HSV-1716 induced production of IFN-γ protein by human peripheral blood mononuclear cell-derived DC cultures as determined by ELISA of supernatants 72 h after infection (1 m.o.i.). (D) Real-time quantitative RT-PCR analysis of monocyte-derived human DCs showed an upregulation of IP-10 and MIG mRNA expression 72 h after infection with HSV-1716 (1 m.o.i.). Molecular Therapy 2005 12, 789-802DOI: (10.1016/j.ymthe.2005.03.026) Copyright © 2005 The American Society of Gene Therapy Terms and Conditions