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

Galivo F, Diaz RM, Wongthida P, Thompson J, Kottke T, Barber G, Melcher A, Vile R - Gene Ther. (2009)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55905, USA.

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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The effects of physical and chemical inactivation of replication-competent VSV on the efficacy of VSV virotherapyA. 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.
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Figure 5: The effects of physical and chemical inactivation of replication-competent VSV on the efficacy of VSV virotherapyA. 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.

Mentions: Next, we investigated whether viral antigen load alone is sufficient to trigger the antiviral innate response associated with antitumor therapy, or whether viral gene expression is required. To this end, we generated inactivated viral preparations which would contain similar antigen loads but are incapable of even a single cycle of infection and viral gene expression. Viral inactivation by heat (HI), exposure to ultraviolet (UVI), or by formalin-fixation (FF) was confirmed by the inability of B16ova cells exposed to different inactivated viral preparations in vitro to express viral glycoprotein G (Fig. 5A, top panels), to exhibit CPE (Fig. 5A, bottom panels) or to be killed in vitro (Fig. 5B). Intratumoral injection of any of the three inactivated viral preparations was, however, ineffective in generating antitumor therapy compared to the replication competent viruses VSV or VSV-CD40L (Fig. 5C). These results indicate that live, viable virus which can express its genome is required for antitumor effects of VSV.


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

Galivo F, Diaz RM, Wongthida P, Thompson J, Kottke T, Barber G, Melcher A, Vile R - Gene Ther. (2009)

The effects of physical and chemical inactivation of replication-competent VSV on the efficacy of VSV virotherapyA. 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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3934361&req=5

Figure 5: The effects of physical and chemical inactivation of replication-competent VSV on the efficacy of VSV virotherapyA. 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.
Mentions: Next, we investigated whether viral antigen load alone is sufficient to trigger the antiviral innate response associated with antitumor therapy, or whether viral gene expression is required. To this end, we generated inactivated viral preparations which would contain similar antigen loads but are incapable of even a single cycle of infection and viral gene expression. Viral inactivation by heat (HI), exposure to ultraviolet (UVI), or by formalin-fixation (FF) was confirmed by the inability of B16ova cells exposed to different inactivated viral preparations in vitro to express viral glycoprotein G (Fig. 5A, top panels), to exhibit CPE (Fig. 5A, bottom panels) or to be killed in vitro (Fig. 5B). Intratumoral injection of any of the three inactivated viral preparations was, however, ineffective in generating antitumor therapy compared to the replication competent viruses VSV or VSV-CD40L (Fig. 5C). These results indicate that live, viable virus which can express its genome is required for antitumor effects of VSV.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Medicine, Mayo Clinic, Rochester, MN 55905, USA.

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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Related in: MedlinePlus