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. 2010 Feb;17(2):158-70.
doi: 10.1038/gt.2009.161. Epub 2009 Dec 17.

Single-cycle viral gene expression, rather than progressive replication and oncolysis, is required for VSV therapy of B16 melanoma

F Galivo  1 , R M Diaz, P Wongthida, J Thompson, T Kottke, G Barber, A Melcher, R Vile
Affiliations

Single-cycle viral gene expression, rather than progressive replication and oncolysis, is required for VSV therapy of B16 melanoma

F Galivo et al. Gene Ther. 2010 Feb.

Abstract

A fully intact immune system would be expected to hinder the efficacy of oncolytic virotherapy by inhibiting viral replication. Simultaneously, however, it may also enhance antitumor therapy through initiation of proinflammatory, antiviral cytokine responses at the tumor site. The aim of this study was to investigate the role of a fully intact immune system on the antitumor efficacy of an oncolytic virus. In this respect, injection of oncolytic vesicular stomatitis virus (VSV) into subcutaneous B16ova melanomas in C57Bl/6 mice leads to tumor regression, but it is not associated with viral replicative burst in the tumor. In contrast, intratumoral delivery of VSV induces an acute proinflammatory reaction, which quickly resolves concomitantly with virus clearance. Consistent with the hypothesis that therapy may not be dependent on the ability of VSV to undergo progressive rounds of replication, a single-cycle VSV is equally effective as a fully replication-competent VSV, whereas inactivated viruses do not generate therapy. Even though therapy is dependent on host CD8+ and natural killer cells, these effects are not associated with interferon-gamma-dependent responses against either the virus or tumor. There is, however, a strong correlation between viral gene expression, induction of proinflammatory reaction in the tumor and in vivo therapy. Overall, our results suggest that acute innate antiviral immune response, which rapidly clears VSV from B16ova tumors, is associated with the therapy observed in this model. Therefore, the antiviral immune response to an oncolytic virus mediates an intricate balance between safety, restriction of oncolysis and, potentially, significant immune-mediated antitumor therapy.

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Figures

Figure 1
Figure 1. Intratumoral VSV induced an acute proinflammatory reaction at the tumor site
A. (Left panel) B16ova cells were infected with VSV-GFP and the percentage of infected cells (GFP+ cells) were counted using flow cytometry at various time points. (Right panel) Overnight monolayer cultures of B16ova cells were infected with either VSV-XN2 or VSV-GFP (MOI=1.0). The number of infectious progeny viruses were determined from the culture supernatants harvested daily for 3 days using standard plaque assay in BHK cells. Values are averages of triplicate wells (+ SEM) and representative of two independent experiments. B. Effect of VSV dose-escalation on the survival of C57Bl/6 mice (n=8 per group) bearing subcutaneous B16ova tumors treated with three intratumoral injections of replication-competent VSV. *p<0.05, **p<0.01, ***p<0.001. C. Seven-day old subcutaneous B16ova tumors in C57Bl/6 mice (n=3 mice per group per time point) were infected with a single intratumoral dose of either VSV-GFP or HI-VSV (both using 5×108 pfu), harvested right after injection and on indicated days postinfection. The number of infectious virus was assayed using standard plaque assay. HI-VSV consistently gave no detectable titers (indicated by arrows). Values are averages of three tumors (+ SEM). D. Established B16ova tumors in immunocompetent mice (n=3 mice per group) were injected intratumorally with one dose of VSV (5×108 pfu), the injected tumors and corresponding draining lymph nodes were harvested at indicated times, the total RNA was extracted and used in a ribonuclease protection assay (RPA). The symbol (٭) corresponds to upregulated cytokine mRNA, while each lane corresponds to a sample from one mouse. E. Kaplan-Meier survival graph comparing the therapeutic efficacy of six intratumoral VSV in tumor-bearing C57Bl/6 mice (B6) or IFN-γ knockout mice (IFN-γko). Total of 8 mice per treatment group. *p<0.05, **p<0.01, ***p<0.001.
Figure 1
Figure 1. Intratumoral VSV induced an acute proinflammatory reaction at the tumor site
A. (Left panel) B16ova cells were infected with VSV-GFP and the percentage of infected cells (GFP+ cells) were counted using flow cytometry at various time points. (Right panel) Overnight monolayer cultures of B16ova cells were infected with either VSV-XN2 or VSV-GFP (MOI=1.0). The number of infectious progeny viruses were determined from the culture supernatants harvested daily for 3 days using standard plaque assay in BHK cells. Values are averages of triplicate wells (+ SEM) and representative of two independent experiments. B. Effect of VSV dose-escalation on the survival of C57Bl/6 mice (n=8 per group) bearing subcutaneous B16ova tumors treated with three intratumoral injections of replication-competent VSV. *p<0.05, **p<0.01, ***p<0.001. C. Seven-day old subcutaneous B16ova tumors in C57Bl/6 mice (n=3 mice per group per time point) were infected with a single intratumoral dose of either VSV-GFP or HI-VSV (both using 5×108 pfu), harvested right after injection and on indicated days postinfection. The number of infectious virus was assayed using standard plaque assay. HI-VSV consistently gave no detectable titers (indicated by arrows). Values are averages of three tumors (+ SEM). D. Established B16ova tumors in immunocompetent mice (n=3 mice per group) were injected intratumorally with one dose of VSV (5×108 pfu), the injected tumors and corresponding draining lymph nodes were harvested at indicated times, the total RNA was extracted and used in a ribonuclease protection assay (RPA). The symbol (٭) corresponds to upregulated cytokine mRNA, while each lane corresponds to a sample from one mouse. E. Kaplan-Meier survival graph comparing the therapeutic efficacy of six intratumoral VSV in tumor-bearing C57Bl/6 mice (B6) or IFN-γ knockout mice (IFN-γko). Total of 8 mice per treatment group. *p<0.05, **p<0.01, ***p<0.001.
Figure 2
Figure 2. Characteristics of replication-defective recombinant VSV in B16ova melanoma cells in vitro
A. cDNA representing the viral genomes of recombinant VSVs flanked by T7 RNA polymerase leader, T7 terminator, and hepatitis virus delta ribozyme (RBZ). VSV-XN2-ΔG and VSV-CD40L-ΔG were generated by removal of the G segment from VSV-XN2 and VSV-CD40L, respectively. B. 2×106 B16ova cells were infected with VSVs (MOI=0.01) for 24 hours and viral titers were measured in the supernatants. Values are averages of duplicate samples ± SEM. C. Using 96-well plates, 5×103 B16ova melanoma cells were infected with VSVs at an MOI of 1.0. The number of viable cells was measured using MTT assay at the indicated time points postinfection (hpi). Values are averages of triplicate samples (+ SEM) and representative of 2 independent experiments. D/E. B16ova cells were infected with replication competent (VSV-XN2 or VSV-CD40L) or single cycle (VSV-XN2-ΔG or VSVCD40L-ΔG) viruses in vitro at either low (replication-competent, MOI=0.001) or high (single-cycle, MOI>100) viral concentrations. 12 hours postinfection, cells were analyzed for expression of either viral VSV-G protein (D) or the CD40L transgene (E). F. Fresh BHK cells were exposed to undiluted supernatants harvested 48 hours postinfection from BHK cells infected with either replication competent VSV-XN2 (MOI=0.01), or single-cycle VSV-XN2-ΔG (MOI>100). Virus was allowed to expand in these cultures for 48hrs. Supernatants were harvested again and used to infect fresh cultures of BHK cells. 24hrs later, cells were analyzed for expression of the VSV-G protein by flow cytometry.
Figure 2
Figure 2. Characteristics of replication-defective recombinant VSV in B16ova melanoma cells in vitro
A. cDNA representing the viral genomes of recombinant VSVs flanked by T7 RNA polymerase leader, T7 terminator, and hepatitis virus delta ribozyme (RBZ). VSV-XN2-ΔG and VSV-CD40L-ΔG were generated by removal of the G segment from VSV-XN2 and VSV-CD40L, respectively. B. 2×106 B16ova cells were infected with VSVs (MOI=0.01) for 24 hours and viral titers were measured in the supernatants. Values are averages of duplicate samples ± SEM. C. Using 96-well plates, 5×103 B16ova melanoma cells were infected with VSVs at an MOI of 1.0. The number of viable cells was measured using MTT assay at the indicated time points postinfection (hpi). Values are averages of triplicate samples (+ SEM) and representative of 2 independent experiments. D/E. B16ova cells were infected with replication competent (VSV-XN2 or VSV-CD40L) or single cycle (VSV-XN2-ΔG or VSVCD40L-ΔG) viruses in vitro at either low (replication-competent, MOI=0.001) or high (single-cycle, MOI>100) viral concentrations. 12 hours postinfection, cells were analyzed for expression of either viral VSV-G protein (D) or the CD40L transgene (E). F. Fresh BHK cells were exposed to undiluted supernatants harvested 48 hours postinfection from BHK cells infected with either replication competent VSV-XN2 (MOI=0.01), or single-cycle VSV-XN2-ΔG (MOI>100). Virus was allowed to expand in these cultures for 48hrs. Supernatants were harvested again and used to infect fresh cultures of BHK cells. 24hrs later, cells were analyzed for expression of the VSV-G protein by flow cytometry.
Figure 3
Figure 3. Live replication-defective recombinant VSVs delayed the growth of established subcutaneous B16ova tumors in immunocompetent mice
A. VSV-injected B16ova tumors (n=3 per treatment group) were harvested four days after injection, dissociated to obtain single cell suspensions, and assayed for CD40L expression using flow cytometry. B. Seven-day old subcutaneous B16ova tumors were injected intratumorally six times with 5×107 pfu of either VSV-CD40L or VSV-CD40L-ΔG. Tumor growth and overall survival were monitored (n=8 per treatment group). C. Kaplan-Meier survival plot of subcutaneous B16ova tumor-bearing C57Bl/6 mice treated with six intratumoral injections (5×107 pfu/dose) of VSV-XN2-ΔG or VSV-CD40L-ΔG. *p<0.05, **p<0.01, ***p<0.001.
Figure 4
Figure 4. Effects of infecting B16ova with single-cycle recombinant VSVs on the adaptive and innate immune responses
A. IFN-γ ELISPOT assay of splenocytes harvested seven days after the third intratumoral virus injection. Two replicates of 1×105 splenocytes were plated in 96-well ELISPOT plates and cultured for 48 hours in the presence of the indicated peptides. B. Inguinal draining lymph nodes from mice treated with a single injection of intratumoral VSVs were harvested 4 days postinfection and assessed the frequency of CD45+ populations via flow cytometry. Three inguinal lymph nodes were pooled into a single sample per treatment group. Flow cytometric analysis of CD45+ populations were done in quadruplicates. C. From the same groups of mice in (a), blood was collected and the average serum antibody titer against VSV was determined (n=3 per group). *p<0.05, **p<0.01, ***p<0.001. D. RT-PCR was performed on total RNA from B16ova cells in vitro following 8 hours of infection with the following viruses using an MOI=1.0.
Figure 5
Figure 5. The effects of physical and chemical inactivation of replication-competent VSV on the efficacy of VSV virotherapy
A. Overnight cultures of 2×106 B16ova cells were infected with VSVs (MOI=0.01) for 24 hours. Top panels show dot plots of B16ova cells depicting surface expression of VSV-G. Representative photographs showing cytopathic effects (CPE) (bottom panels) after 24 hours. HI: Heat-inactivated VSV; UVI: ultraviolet-inactivated VSV; FF: Formalin-fixed VSV. B. Using 96-well plates, 5×103 B16ova melanoma cells were infected with either live or inactivated VSVs at an MOI of 1.0. The number of viable cells was measured using MTT assay at the indicated time points postinfection. Values are averages of triplicate samples (± SEM) and representative of 2 independent experiments. C. Kaplan-Meier survival plot of subcutaneous B16ova tumor-bearing C57Bl/6 mice treated with six intratumoral injections of either live VSV or inactivated forms of VSV (5×108 pfu/injection). *p<0.05, **p<0.01, ***p<0.001.
Figure 6
Figure 6. Immune responses after intratumoral injection of live and inactivated forms of VSV and therapeutic efficacy of intratumoral TLR agonist in B16ova tumors
A. Subcutaneous B16ova tumors were infected with 3 daily injections of either live or inactivated VSV (5×107 pfu/injection). Eight days after the last virus, spleens were harvested, dissociated and incubated with one of the four antigens indicated. Total IFN-γ spots (1×105 splenocytes/48h) were measured using ELISPOT (n=3 mice/group). B. At the time of sacrifice (day 17 after tumor challenge), blood was also extracted and serum neutralizing antibody titer was determined. *p<0.05, **p<0.01, ***p<0.001. C. RT-PCR for type I interferon-responsive genes was performed on total RNA from subcutaneous B16ova tumors—harvested 8 hours postinfection—given a single injection of 5×108 pfu either live or inactivated VSV. D. Kaplan-Meier survival plot of B16ova tumor-bearing C57Bl/6 mice (n=8 per group) treated with three (3) intratumoral injections of either VSV-GFP (5×108 pfu/dose) or 200 μg of lipopolysaccharide (LPS). *p<0.05, **p<0.01, ***p<0.001.
Figure 6
Figure 6. Immune responses after intratumoral injection of live and inactivated forms of VSV and therapeutic efficacy of intratumoral TLR agonist in B16ova tumors
A. Subcutaneous B16ova tumors were infected with 3 daily injections of either live or inactivated VSV (5×107 pfu/injection). Eight days after the last virus, spleens were harvested, dissociated and incubated with one of the four antigens indicated. Total IFN-γ spots (1×105 splenocytes/48h) were measured using ELISPOT (n=3 mice/group). B. At the time of sacrifice (day 17 after tumor challenge), blood was also extracted and serum neutralizing antibody titer was determined. *p<0.05, **p<0.01, ***p<0.001. C. RT-PCR for type I interferon-responsive genes was performed on total RNA from subcutaneous B16ova tumors—harvested 8 hours postinfection—given a single injection of 5×108 pfu either live or inactivated VSV. D. Kaplan-Meier survival plot of B16ova tumor-bearing C57Bl/6 mice (n=8 per group) treated with three (3) intratumoral injections of either VSV-GFP (5×108 pfu/dose) or 200 μg of lipopolysaccharide (LPS). *p<0.05, **p<0.01, ***p<0.001.
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