Antiviral Ranpirnase TMR-001 Inhibits Rabies Virus Release and Cell-to-Cell Infection In Vitro

Abstract

Currently, no rabies virus-specific antiviral drugs are available. Ranpirnase has strong antitumor and antiviral properties associated with its ribonuclease activity. TMR-001, a proprietary bulk drug substance solution of ranpirnase, was evaluated against rabies virus in three cell types: mouse neuroblastoma, BSR (baby hamster kidney cells), and bat primary fibroblast cells. When TMR-001 was added to cell monolayers 24 h preinfection, rabies virus release was inhibited for all cell types at three time points postinfection. TMR-001 treatment simultaneous with infection and 24 h postinfection effectively inhibited rabies virus release in the supernatant and cell-to-cell spread with 50% inhibitory concentrations of 0.2-2 nM and 20-600 nM, respectively. TMR-001 was administered at 0.1 mg/kg via intraperitoneal, intramuscular, or intravenous routes to Syrian hamsters beginning 24 h before a lethal rabies virus challenge and continuing once per day for up to 10 days. TMR-001 at this dose, formulation, and route of delivery did not prevent rabies virus transit from the periphery to the central nervous system in this model (n = 32). Further aspects of local controlled delivery of other active formulations or dose concentrations of TMR-001 or ribonuclease analogues should be investigated for this class of drugs as a rabies antiviral therapeutic.

Keywords: antiviral; hamster; lyssavirus; onconase; rabies virus; ranpirnase TMR-001.

Figure 1

Time of treatment method. For cells treated 24 h preinfection, BSR cells (a clone of baby hamster kidney cells) were seeded in each well of an 8-well slide on day 1 and incubated overnight. On day 2, cells were treated with TMR-001 and incubated overnight. On day 3, treated cells were infected with rabies virus (RABV) strain Evelyn–Rokitnicki–Abelseth (ERA) and incubated for 48 h. On day 5, supernatant was collected, and cells were fixed and stained. For cells treated with TMR-001 simultaneously to infection, BSR cells were seeded in each well of an 8-well slide on day 1 and incubated overnight. On day 2, cells were treated with TMR-001, infected with RABV strain ERA, and incubated for 48 h. On day 4, supernatant was collected, and cells were fixed and stained. For cells treated 24 h postinfection, BSR cells were seeded in each well of an 8-well slide on day 1 and incubated overnight. On day 2, cells were infected with RABV strain ERA and incubated overnight. On day 3, infected cells were treated with TMR-001 and incubated overnight. On day 4, supernatant was collected, and cells were fixed and stained.

Figure 2

Inhibition of rabies virus release by TMR-001. TMR-001 was added 24 h preinfection at the given concentrations to a clone of baby hamster kidney cells (BSR), mouse neuroblastoma cells (MNA) and bat primary fibroblast cells (E03E). Culture supernatant was sampled at 24, 48, and 72 h postinfection. The 50% inhibitory concentration (IC50), 50% cytotoxicity concentration (CC50) and the 50% selective index (SI50) were calculated for each condition. The virus concentration in the supernatant was measured in fluorescent foci units per mL (ffu/mL) using direct fluorescent antibody (DFA) staining of MNA cells 24 h postinfection. The average and standard deviation were calculated from the log transformed data of six statistical replicates from at least two biological replicates and plotted on a linear axis. Comparison (2-way ANOVA, Dunnett’s adjusted p-values) of each concentration to the 0 µM TMR-001 (PBS) control are shown as <0.05 (*), <0.01 (**), <0.001 (***), <0.0001 (****). Note that no virus was detected in any of the replicates for BSR cells treated with 3 µM TMR-001 at 72 h postinfection, MNA cells treated with 10 µM TMR-001 at 72 h postinfection, as well as E03E cells treated with 10 µM TMR-001 at 24 h and 48 h postinfection.

Figure 3

Inhibition of rabies virus release and cell-to-cell infection regardless of TMR-001 treatment time. TMR-001 was added at the given concentrations to a clone of baby hamster kidney cells (BSR) at 24 h preinfection (blue circle), simultaneously to infection (red square), or 24 h postinfection (green triangle). The average and standard deviation were calculated from four statistical replicates from at least two biological replicates, plotted on a log₁₀ scale, and 50% inhibitory concentrations were calculated using a three-parameter fit, nonlinear regression. (a) Culture supernatant was sampled at 48 h postinfection, and virus concentration was measured in fluorescent foci units per mL (ffu/mL) using DFA staining of mouse neuroblastoma cells 24 h postinfection. Note that the titer for the first dilution (10^−1.54 mM ranpirnase) is below the axis limit for all three treatments and for the second dilution (10^−2.54 mM ranpirnase) is below the axis limit for 24 h preinfection and simultaneous treatments. The 50% inhibitory concentrations were 2 nM for 24 h preinfection, 0.4 nM for simultaneous, and 0.2 nM for 24 h postinfection. (b) The cell monolayer was fixed at 48 h postinfection, and relative cell-to-cell infection was measured by counting clusters of fluorescent foci using DFA staining and comparing counts to virus-only controls. The 50% inhibitory concentrations were 600 nM for 24 h preinfection, 100 nM for simultaneous, and 20 nM for 24 h postinfection. The amount of cell-to-cell infection was not significantly different between the different treatment time points (two-way ANOVA Tukey’s adjusted p-values > 0.05).

Figure 4

TMR-001 treatment does not prevent rabies in Syrian hamsters. Starting 24 h preinfection, 0.1 mg/kg TMR-001 was administered intraperitoneally (IP, green), intramuscularly (IM, red), intravenously (IV, blue), or no treatment (purple), once per day for 10 days. Group size was 12 hamsters, except the IV administration group, which contained 8 hamsters. All animals were infected with 10^3.5 mouse intracranial LD₅₀ of canine rabies virus IM in the hind leg. Days 7 to 21 postinfection, animals were observed twice daily and euthanized at the first clinical signs of rabies. The experiment was terminated 14 days postinfection due to a lack of significance between the groups (log-rank test p = 0.66). Percent survival over time is shown on a linear scale.