The oral drug obeldesivir protects nonhuman primates against lethal Ebola virus infection

Abstract

Obeldesivir (ODV; GS-5245) is an orally administered ester prodrug of the parent nucleoside GS-441524 that has broad spectrum antiviral activity inhibiting viral RNA-dependent RNA polymerases. We recently showed that ODV completely protects cynomolgus macaques against lethal infection with Sudan virus when given 24 hours after parenteral exposure. Here, we report that once daily oral ODV treatment of cynomolgus and rhesus macaques for 10 days confers 80 and 100% protection, respectively, against lethal Ebola virus infection when treatment is initiated 24 hours after mucosal (conjunctival) exposure. ODV treatment delayed viral replication to abate excessive inflammation and promote adaptive immunity. For outbreak response, oral antivirals might present substantial advantages over now approved intravenous drugs, such as easy supply, storage, distribution, and administration. Furthermore, these results support the potential of ODV as an oral postexposure prophylaxis with broad spectrum activity across filoviruses.

Fig. 1.. Survival analysis, clinical scoring, and virus replication kinetics in cynomolgus macaques challenged with EBOV and treated with ODV.

(A) Kaplan-Meier survival curves for EBOV-challenged (Makona variant) cynomolgus macaques. The in-study control is plotted separately; however, for statistical comparison, the in-study control was grouped with the historical controls (HC) from a previous study (14). Differences in curves were tested by the Mantel-Cox log-rank test. (B) Clinical scores were assigned on the basis of daily cage-side observations of behavior and apparent physical health. The horizontal dashed line indicates the minimum clinical score by which euthanasia criteria were met. (C and D) Viral load was determined by RT-qPCR of RNA from whole blood (C) or plaque titration of plasma (D) collected at predetermined sampling points or at euthanasia. For both panels, individual data points represent the mean of two technical replicates. For HC, the geometric mean ± geometric SD for the cohort is plotted. To fit on a log scale axis, zero values [below limit of quantitation (LOQ)] are plotted as “1” (10⁰). (E) Viral load in selected tissues harvested at necropsy as determined by RT-qPCR detection of vRNA. The geometric mean titer ± SD is plotted for HC animals. For (C) to (E), dashed horizontal lines indicate the lower limit of quantitation (LLOQ) for the assay (1000 GEq/ml for RT-qPCR; 25 PFU/ml for plaque titration). To fit on a log scale axis, zero values (below LLOQ) are plotted.

Fig. 2.. Survival analysis, clinical scoring, and virus replication kinetics in rhesus macaques challenged with EBOV and treated with ODV.

(A) Kaplan-Meier survival curves for EBOV-challenged (Makona variant) rhesus macaques. The in-study control for the ODV treatment study is plotted separately; however, for statistical comparison, the in-study control was grouped with the untreated animals from the pilot study (PC). Differences in curves were tested by the Mantel-Cox log-rank test. (B) Clinical scores were assigned on the basis of daily cage-side observations of behavior and apparent physical health. The horizontal dashed line indicates the minimum clinical score by which euthanasia criteria were met. (C to F) Viral load was determined by RT-qPCR of RNA from whole blood [(C) and (E)] or plaque titration of plasma [(D) and (F)] collected at predetermined sampling points or at euthanasia. For (C) to (F), individual data points represent the mean of two technical replicates. Data plotted for PC represents the geometric mean ± geometric SD for the cohort. To fit on a log scale axis, zero values (below LOQ) are plotted as 1 (10⁰). (G and H) Viral load in selected tissues harvested at necropsy from untreated animals in the pilot study (G) and ODV-treated animals in the second study (H) as determined by RT-qPCR detection of vRNA. For (C) to (H), horizontal dashed lines indicate the LLOQ for the assay (1000 GEq/ml for RT-qPCR; 25 PFU/ml for plaque titration). To fit on a log scale axis, zero values (below LLOQ) are plotted as 1 (10⁰).

Fig. 3.. Anti-EBOV GP antibody titers in control and ODV-treated macaques.

Plasma samples from each study [(A and B) ODV cynomolgus macaque (N = 6); (C and D) pilot rhesus (N = 4); (E and F) ODV rhesus macaque (N = 7)] were tested for circulating anti-EBOV GP-specific IgG [(A), (C), and (E)] and IgM [(B), (D), and (F)] binding antibody responses by indirect ELISA. Line graphs (log₂ y axis) depicting the average reciprocal dilution titer for individual subjects at each time point (0, 1, 4, 7, 10, 14, 21, 28, terminal, or 35 DPI) are shown. To fit on a log scale axis, zero values are plotted as 1 (10⁰). The dotted line (1:100 dilution) represents the limit of detection for the assay.

Fig. 4.. Transcriptional profiling of ODV-treated versus control macaques exposed to EBOV.

RNA samples from all subjects [N = 6 cynomolgus macaques (N = 5 treated including 1 fatal subject and N = 1 in-study control subject); N = 11 rhesus macaques (N = 5 treated all survivors and N = 6 control subjects with two in-study controls and four pilot study controls] were included at the following time points: 0, 4, 7, and 10 DPI. Groups: Rhesus control (N = 6), rhesus ODV (N = 5), cyno control (N = 1), cyno fatal (N = 1), cyno survivor (N = 4). Heatmaps of the most up-regulated (A) and down-regulated (B) transcripts in whole blood from ODV-treated versus control macaques exposed to EBOV. Any differentially expressed genes (DEGs) with a Benjamini-Hochberg FDR corrected P value less than 0.05 were deemed significant; the dataset was filtered by the ODV rhesus group at 10 DPI in order of fold change. Red indicates increased expression; blue indicates decreased expression; white indicates no change in expression. (C) Ingenuity Pathway Analysis–based enrichment of transcripts in ODV-treated versus control macaques exposed to EBOV at 0, 4, 7, and 10 DPI. Up-regulated pathways were classified by a >1.5 z score, and down-regulated pathways were classified by a <−1.5 z score. Pathways are sorted by the topmost positive (top) or negative (bottom) z score for the ODV rhesus group at 10 DPI. (D and E) Immune cell transcriptional profiling of controls and ODV-treated rhesus (D) and cynomolgus (E) macaques exposed to EBOV. Higher cell type scores indicate a higher abundance of transcripts mapping to the specific cell subset. All samples (0, 4, 7, and 10 DPI) were combined to generate cell type quantities for each group. Cyno, cynomolgus macaque group; T_H1, T helper cell 1.

Fig. 5.. Plasma levels of cytokines/chemokines and coagulation markers in ODV-treated versus control macaques exposed to EBOV.

Expression of circulating proinflammatory mediators and thrombosis analytes in the plasma of EBOV-exposed cynomolgus (N = 12) and rhesus (N = 11) macaques at 0, 4, 7, 10, and 35 DPI or the terminal time point. To help identify trends, the in-study cynomolgus macaque control subject (CCy-1) was grouped with historical controls (N = 6; CCy-2, CCy-3, CCy-4, CCy-5, CCy-6, and CCy-7) from a previous study that were exposed by the same route at the same target dose (14). Depicted are fold change values for each analyte with respect to a prechallenge baseline (0 DPI) for each subject. Red indicates increased expression; blue indicates decreased expression; white indicates no change in expression. Individual data points represent the mean of two technical replicates. GM-CSF, granulocyte-macrophage colony-stimulating factor.