Ennis, Matthew K.

Abstract The use of replication-competent viruses as oncolytic agents is rapidly expanding, with several oncolytic viruses approved for cancer therapy. As responses to therapy are highly variable, understanding the dynamics of therapy is critical for optimal application of virotherapy in practice. Although mathematical models have been developed to understand the dynamics of tumor virotherapy, a scarcity of in vivo data has made difficult parametrization of these models. To tackle this problem, we studied the in vitro and in vivo spread of two oncolytic measles viruses that induce expression of the sodium iodide symporter (NIS) in cells. NIS expression enabled infected cells to concentrate radioactive isotopes that could be reproducibly and quantitatively imaged using SPECT/CT. We observed a strong linear relationship in vitro between infectious virus particles, viral N and NIS gene expression, and radioactive isotope uptake. In vivo radioisotope uptake was highly correlated with viral N and NIS gene expression. Similar expression patterns between viral N and NIS gene expression in vitro and in vivo implied that the oncolytic virus behaved similarly in both scenarios. Significant titers of viable virus were consistently isolated from tumors explanted from mice that had been injected with oncolytic measle viruses. We observed a weaker but positive in vivo relationship between radioisotope uptake and the viable virus titer recovered from tumors; this was likely due to anisotropies in the viral distribution in vivo. These data suggest that methods that enable quantitation of in vivo anisotropies are required for continuing development of oncolytic virotherapy. Significance: These findings address a fundamental gap in our knowledge of oncolytic virotherapy by presenting technology that gives insight into the behavior of oncolytic viruses in vivo. Cancer Res; 78(20); 5992–6000. ©2018 AACR.

Abstract Oncolytic replication competent viruses are increasingly being tested in clinical trials. Their optimal use would be greatly facilitated by technologies that enable repeated and quantitative estimation of the location and population size of the virus as it propagates within the tumor. To address this, we have generated two replication competent measles viruses (MV) that express the thyroidal sodium iodide symporter (NIS) in injected cells. One virus, MV-NIS is in Phase 1 clinical trials and kills cells by formation of syncytia. The second virus, MV-I98A-NIS has a mutation in the MV hemagglutinin protein that prevents syncytium formation and kills cells by lysis. Cells infected with MV-I98A-NIS remain viable for longer compared to MV-NIS and are expected to express higher concentrations of NIS, facilitating in vivo imaging. BxPC-3 human pancreatic carcinoma cells were implanted in the flank of nude mice. Subsequently, the mice were injected with Opti-MEM (control), MV-NIS or MV-I98A-NIS (1x107 TCID50/ml) via the tail vein. On days 3, 5, 8, 10, 12, 15, 17 and 19 after virus injection, mice from the control, and virus injected groups were imaged with SPECT/CT (U-SPECT II) 1 hour after injection of 99mTc. Immediately afterwards, the mice were euthanized, the tumors excised, weighed and isotope activity measured. The tumors were divided into parts for histologic analysis and RNA extraction. NIS gene expression was determined by Q-RT-PCR and immunohistochemistry, while viral genome copy number was determined by Q-RT-PCR for the nucleoprotein (N). Image analysis using ROI was performed using AMIDE with background correction across all imaging planes. There was no correlation between tumor mass (volume) and isotope uptake (R2=0.0001 and 0.0051 for MV-NIS and MV-I98A-Nis respectively) suggesting that isotope uptake was strictly linked to virus infection. There was a strong, linear correlation between the levels of virus dependent NIS expression and isotope activity within the tumor (R2=0.79 and 0.93 for MV-NIS and MV-I98A-NIS respectively). Tumors infected with MV-I98A-NIS had a higher concentration of isotope compared with MV-NIS (p<0.004) and a higher correlation coefficient of NIS isotope uptake versus NIS expression (14.1 mCi/NIS copies/ng RNA versus 11.5, p=0.003), compatible with the observation that tumor cells infected with MV-I98A-NIS remain viable for longer compared to MV-NIS. Activity within tumors increased as a function of time, compatible with ongoing virus replication and spread within the tumors. Correlation of imaging studies with NIS expression and viral genome copy number is ongoing. Our preliminary studies suggest that in vivo imaging can be used to accurately determine the size of the oncolytic virus population within a tumor as a function of time. These observations will be used to understand the dynamics of virus spread within tumors and optimize therapy. Citation Format: Mi-Yeon Jung, Matthew K. Ennis, Chetan P. Offord, David Dingli. Quantitative in vivo imaging of oncolytic virus replication. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4947. doi:10.1158/1538-7445.AM2014-4947

ABSTRACT Measles virus (MV) entry requires at least 2 viral proteins, the hemagglutinin (H) and fusion (F) proteins. We describe the rescue and characterization of a measles virus with a specific mutation in the stalk region of H (I98A) that is able to bind normally to cells but infects at a lower rate than the wild type due to a reduction in fusion triggering. The mutant H protein binds to F more avidly than the parent H protein does, and the corresponding virus is more sensitive to inhibition by fusion-inhibitory peptide. We show that after binding of MV to its receptor, H-F dissociation is required for productive infection.