Investigating the effect of reduced temperatures on the efficacy of rhabdovirus-based viral vector platforms

Abstract

Rhabdoviral vectors can induce lysis of cancer cells. While studied almost exclusively at 37 °C, viruses are subject to a range of temperatures in vivo, including temperatures ≤31 °C. Despite potential implications, the effect of temperatures <37 °C on the performance of rhabdoviral vectors is unknown. We investigated the effect of low anatomical temperatures on two rhabdoviruses, vesicular stomatitis virus (VSV) and Maraba virus (MG1). Using a metabolic resazurin assay, VSV- and MG1-mediated oncolysis was characterized in a panel of cell lines at 28, 31, 34 and 37 °C. The oncolytic ability of both viruses was hindered at 31 and 28 °C. Cold adaptation of both viruses was attempted as a mitigation strategy. Viruses were serially passaged at decreasing temperatures in an attempt to induce mutations. Unfortunately, the cold-adaptation strategies failed to potentiate the oncolytic activity of the viruses at temperatures <37 °C. Interestingly, we discovered that viral replication was unaffected at low temperatures despite the abrogation of oncolytic activity. In contrast, the proliferation of cancer cells was reduced at low temperatures. Equivalent oncolytic effects could be achieved if cells at low temperatures were treated with viruses for longer times. This suggests that rhabdovirus-mediated oncolysis could be compromised at low temperatures in vivo where therapeutic windows are limited.

Keywords: oncolytic; rhabdovirus; temperature; viral vector.

Fig. 1.. Illustrations of the vesicular stomatitis virus (VSV) and Maraba virus (MG1) vectors. (a) The VSV vector had a transgene for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein with the last 19 aa removed (VSV-SΔ19). The SΔ19 transgene was inserted in VSV between the VSV M and G viral genes. (b) The MG1 vector had an enhanced green fluorescent protein reporter transgene (MG1-eGFP). The eGFP transgene was inserted between the G and L viral genes.

Fig. 2.. Schematic of the proposed cold-adaptation strategy. Regardless of strategy, the passaging process for cold adaptation was the same. A dish of confluent Vero cells was infected at an m.o.i. of 0.1 and incubated at the desired temperature, depending on the cold-adaptation method used, until >80 % cytopathic effect (CPE) was observed via microscopy. Subsequently, the cells and supernatant were collected and centrifuged. The clarified supernatant was aliquoted and frozen. These aliquots were used to perform plaque assays to determine viral titres (p.f.u. ml^–1). This process was repeated with each passage.

Fig. 3.. The effect of decreased temperature on the oncolytic ability of Maraba virus (MG1-eGFP) and vesicular stomatitis virus (VSV-SΔ19). Graphs showing the results of 48 h metabolic resazurin assays for MG1-eGFP in (a) Vero, (b) B16-F10 and (c) HeLa cells, and VSV-SΔ19 in (d) Vero, (e) B16-F10 and (f) HeLa cells at 28, 31, 34 and 37 °C. Statistical analysis was performed using a two-way ANOVA with Dunnett’s multiple comparisons. Means and standard errors are shown (n=3; ****P<0.0001, ***P<0.001, *P<0.05; all data sets are compared to the 37 °C curves).

Fig. 4.. Comparing the oncolytic activities of Maraba virus (MG1-eGFP) and vesicular stomatitis virus (VSV-SΔ19) for which cold adaptation was attempted. Graphs show the results of 48 h metabolic resazurin assays at 28, 31, 34 and 37 °C for the parental viruses and VSV-SΔ19 that underwent the ‘immediate drop’ cold adaptation method in (a) Vero cells, (b) B16-F10 cells and (c) HeLa cells, MG1-eGFP that underwent the ‘immediate drop’ cold adaptation method in (d) Vero cells, (e) B16-F10 cells and (f) HeLa cells, and VSV-SΔ19 that underwent the ‘slow progression’ cold adaptation method in (g) Vero cells, (h) B16-F10 cells and (i) HeLa cells (n=3, ****P<0.0001, ***P<0.001, *P<0.05; all data sets are compared to the 37 °C curves).

Fig. 5.. The effect of temperatures <37 °C on the replicative ability of Maraba virus (MG1-eGFP) and vesicular stomatitis virus (VSV-SΔ19). Graphs showing the results of 60 h single-step in vitro growth curves for MG1-eGFP in (a) Vero, (b) B16-F10 and (c) HeLa cells, and VSV-SΔ19 in (d) Vero, (e) B16-F10 and (f) HeLa cells at 28, 30, 34 and 37 °C. Viruses were titrated using the TCID₅₀ assay and results were expressed in plaque-forming units per millilitre (p.f.u. ml^–1). Statistical analysis was performed using a two-way ANOVA with Dunnett’s multiple comparisons. Means and standard errors are shown. There were no significant differences between any of the curves (n=3 for Vero and B16-F10 cells, n=2 for HeLa cells).

Fig. 6.. The effect of temperatures <37 °C on the replicative ability of vesicular stomatitis virus (VSV-eGFP). Graphs showing the results of virus yield assays conducted in Vero cells at (a) 24 and (b) 48 h and B16-F10 cells at (c) 24 and (d) 48 h. Cells were infected at an m.o.i. of 0.01 and incubated at 28, 31, 34 and 37 °C. Viral supernatants were collected at 24 and 48 h time points and titrated using the standard plaque assay and are in plaque forming units per millilitre (p.f.u. ml^–1). Statistical analysis was performed using a one-way ANOVA with Tukey’s multiple comparisons. For both time points, none of the viral titres at temperatures <37 °C differed from the respective titre at 37 °C. Means and standard errors are shown (n=3 per treatment).

Fig. 7.. The effect of decreased temperature on the virus-mediated cell killing ability of vesicular stomatitis virus (VSV-eGFP). Photos taken with a fluorescence microscope. Representative brightfield images (photos on the left), and images of Hoechst staining (middle left) and green fluorescence (middle right) are shown alongside an overlay of all three (photos on the right). Photos are of Vero cell cultures in six-well plates that were treated with VSV-eGFP at an m.o.i. of 0.01 at 28, 31, 34 and 37 °C for 24 and 48 h.

Fig. 8.. The effect of temperatures <37 °C on cell proliferation. Vero and B16-F10 cells were stained with the fluorescent proliferation dye CFSE and then incubated at 28, 31, 34 and 37 °C. Fluorescence was analysed via flow cytometry at 0, 24, 48 and 72 h. Representative histograms show the fluorescence of CFSE (x-axes) of (a) Vero and (b) B16-F10 cells at 24, 48 and 72 h. Geometric means of CFSE fluorescence intensity were used to calculate the number of (c) Vero and (d) B16-F10 replication cycles relative to the 0 h time point for each temperature at 24, 48 and 72 h. Statistical analysis was performed using a two-way ANOVA with Tukey’s multiple comparisons. Means and standard errors are shown (n=3 per treatment; ****P<0.0001, ***P<0.001, **P<0.01, *P<0.05).

Fig. 9.. The effect of temperatures <37 °C on vesicular stomatitis virus (VSV-eGFP)-mediated cell killing at 48 and 72 h. Graphs showing the results of 48 and 72 h metabolic resazurin assays for VSV-eGFP in (a and b) Vero cells and (c and d) B16-F10 cells at 28, 31, 34 and 37 °C. Statistical analysis was performed using a two-way ANOVA with Tukey’s multiple comparisons. Means and standard errors are shown.

Fig. 10.. Oncolytic activity of parental VSV-S∆19 and MG1-eGFP in the 48 and 72 h resazurin assays. Graphs showing the results of the 48 and 72 h metabolic resazurin assays for VSV-S∆19 and MG1-eGFP in (a) Vero, (b) B16-F10 and (c) HeLa cells. Each graph shows the comparison of the 48 h 37 °C, 72 h 31 °C and 72 h 28 °C conditions. Statistical analysis was performed using a two-way ANOVA with Tukey’s multiple comparisons. Means and standard errors are shown.