Oral dosing of the nucleoside analog obeldesivir is efficacious against RSV infection in African green monkeys

Abstract

Respiratory syncytial virus (RSV) is a significant cause of morbidity and mortality in high-risk populations. Although prophylactic options are available, there are no effective oral therapeutics for RSV infection. Obeldesivir (ODV) is an orally bioavailable prodrug of the nucleoside analog GS-441524, which is converted intracellularly to its active nucleoside triphosphate and inhibits the RSV RNA polymerase. Here we report the potent antiviral activity of ODV against geographically and temporally diverse RSV A and B clinical isolates (EC₅₀: 0.20-0.66 μM). Resistance selection studies with ODV and GS-441524 against RSV identify a single amino acid substitution, I777L, in the L polymerase with reduced susceptibility (3.3-3.8-fold) to ODV and GS-441524, indicating a high barrier for resistance development. In an African green monkey RSV infection model, once-daily oral ODV doses of 30 or 90 mg/kg initiated ~24 hours post-infection significantly reduces log₁₀ viral RNA copies/mL × day area under the curve by 69-92% in the upper and lower respiratory tracts. Together, these preclinical data support the clinical evaluation of ODV for the treatment of RSV infection.

Fig. 1. In vitro antiviral activity of ODV and its parent nucleoside against RSV laboratory strains and clinical isolates.

A Structure of ODV and its systemic metabolite GS-441524 (parent nucleoside). B Representative images for ODV dose response against laboratory strains RSV A2 and RSV B1 in HEp-2 cells. RSV F protein detection in green and nuclei detection in blue. C Representative dose-response curves presented as mean percent inhibition ± standard deviation for each concentration of ODV (blue) and GS-441524 (red) against RSV A2 and B1 from a single biological replicate with (n = 2) technical replicates. D Distribution of ODV (blue) and GS-441524 (red) half-maximal effective concentrations (EC₅₀s) of RSV A clinical isolates (n = 17) and RSV B clinical isolates (n = 15). Black horizontal solid lines represent the mean RSV clinical isolate EC₅₀ ± standard deviation. The mean RSV A2 EC₅₀ for ODV (red) and GS-441524 (blue) are represented by the dashed colored lines. Source data for these plots are provided in the source data file. ODV obeldesivir, RSV respiratory syncytial virus, F fusion glycoprotein.

Fig. 2. In vitro resistance selection of the RSV A2 laboratory strain and characterization of selected substitutions.

A Progression of continuous passage of RSV A2 for 91 days under escalating doses of ODV, GS-441524, or presatovir. Substitutions in the L polymerase (ODV and GS-441524 selections) or the F protein (presatovir selection) were determined by next-generation sequencing (NGS) analysis. Substitutions at >15% prevalence and in ≥2 successive passages, which were not identified in the DMSO controls, are indicated by dashed arrows (first detection) and solid arrows (detection at final passage). B, C Representative dose-response curves presented as mean percent inhibition ± standard deviation for each concentration of ODV and GS-441524 against I777L (B) or L1543W (C) recombinant RSV (rRSV) from a single biological replicate with (n = 2) technical replicates. Wildtype rRSV A2 (black) served as a reference control for rRSV with an L substitution. D ODV- or GS-441524-selected pools and cloned rRSV A2 bearing the I777L or L1453W substitutions in the L protein were tested against a panel of RSV inhibitors. Fold changes in EC₅₀ values from the wildtype RSV A2 control are shown (n = 2–4). The source data for these plots and tables is provided in the source data file. ODV obeldesivir, RDV remdesivir, RSV Respiratory syncytial virus, rRSV recombinant respiratory syncytial virus, F Fusion glycoprotein, N nucleoprotein, NNI non-nucleoside inhibitor.

Fig. 3. In vitro resistance selection of RSV A and B clinical isolates and characterization of the selected C319R substitution.

Progression of continuous passage of (A) RSV A clinical isolate HRSV/A/Texas.USA/79254/2004 or (B) RSV B clinical isolate HRSV/B/Texas.USA/79362/2005 for 91 days under escalating doses of ODV, GS-441524, or presatovir. Substitutions in the L polymerase (ODV and GS-441524 selections) or the F protein (presatovir selection) were determined following NGS analysis. Substitutions at >15% prevalence in ≥2 successive passages and not identified in DMSO controls are indicated by dashed arrows (first detection) and solid arrows (detection at final passage). C Representative dose-response curves presented as mean percent inhibition ± standard deviation for each concentration of ODV and GS-441524 against recombinant RSV (rRSV) A2 bearing C319R in the L polymerase. Reference wildtype rRSV A2 (black) was tested concurrently with C319R rRSV. D Cloned rRSV A2 bearing the C319R was tested against a panel of RSV inhibitors. Fold changes in EC₅₀ values from the wildtype rRSV A2 control are shown (n = 2); NNI (non-nucleoside inhibitor). The source data for these plots and tables is provided in the Source Data file. ODV obeldesivir, RDV remdesivir, rRSV recombinant respiratory syncytial virus, F Fusion glycoprotein, N nucleoprotein, NNI non-nucleoside inhibitor.

Fig. 4. Efficacy of oral ODV in RSV-infected African green monkeys.

A Schematic of study design for AGM study. A was created in BioRender at https://BioRender.com/v42e783. Animals (n = 5 per group) were infected with ~2.6 × 10⁶ TCID₅₀ RSV A2 by intranasal/intratracheal (IN/IT) instillation. Starting 20–28 hours post-infection, the animals were dosed orally (PO) with vehicle, 30 mg/kg ODV, or 90 mg/kg ODV once daily (QD) through day 6 post-infection (total of 6 doses). Labelled days represent timepoints at which throat swabs and bronchoalveolar lavage fluid (BALF) were collected for analysis. RSV RNA from throat swabs (B) or BALF samples (C) as determined by RT-qPCR for vehicle control (black dashed line), 30 mg/kg ODV (light blue line), and 90 mg/kg ODV (dark blue line). Samples with undetectable RNA levels were assigned a value of one-quarter the lower limit of quantification (LLOQ), and samples with detectable RNA levels below the LLOQ were assigned values of one-half the LLOQ before log₁₀ transformation for analysis. The LLOQs are 3.9 and 2.9 log₁₀ RNA copies/mL for the throat swab and BALF, respectively. The plots display the mean and standard deviation of the log₁₀-transformed values. Statistical significance relative to the vehicle control was calculated using a two-sided mixed-effects model with a Dunnett-Hsu correction for repeated measures. Only statistically significant (p < 0.05) differences are shown, exact p values can be found in Supplementary Table 7. ***p < 0.001. **** p < 0.0001. Source data for these plots are provided as a Source Data file. ODV obeldesivir, RSV Respiratory syncytial virus, TCID₅₀ half-maximal tissue culture infectious dose.

Fig. 5. Structural analysis of I777L within the RSV polymerase active site.

A Model of RSV L RNA-dependent RNA polymerase (RdRp) in its elongating state, with pre-incorporated GS-443902 NTP. The RdRp domain is shown in blue, the capping domain in green, the connector domain in orange, and the methyltransferase and carboxy-terminal domain (CTD) in red. The template RNA strand is in orange, and the nascent RNA strand is in yellow. The phosphoprotein (P) is in grey. B Detail of the RSV L RdRp active site with pre-incorporated GS-443902 NTP (purple). I777 is located on motif B and is in direct contact with several hydrophobic residues on motif F, including V614.