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

Figures

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  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.

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  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).…

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  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…

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  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…

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  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.