Fig 1.
Inhibition of RSV in vitro by EDP-938.
(A) Visualization of HEp-2 cell monolayers following RSV-A Long infection with or without treatment at indicated levels of EDP-938 with 5-day post infection (dpi) endpoint. (B) Percent cell viability measured by ATPlite and (C) percent viral RNA reduction measured by RT-qPCR in cells infected with RSV-A Long at MOI of 0.1. Data are mean ± standard error of the mean (SEM) from 89 and 77 individual experiments for EDP-938 and RSV-604, respectively, in the CPE assay and 8 separate assay runs for the RT-qPCR endpoint.
Table 1.
In vitro activity of EDP-938 against RSV laboratory strains.
Fig 2.
In vitro activity of EDP-938 against RSV clinical isolates.
(A) Average EC50 of EDP-938 against RSV-A and RSV-B USA derived clinical isolates. (B) EC50 and EC90 (half maximal and 90% inhibitory concentration) of EDP-938 against 10 RSV-A and RSV-B clinical isolates from the Netherlands. Bold bars indicate the mean ± SEM.
Table 2.
Characterization of EDP-938 resistant viruses.
Table 3.
Analysis of the impact of individual or combination of RSV N protein mutations on the antiviral activity of EDP-938 using the RSV reverse genetics system.
Fig 3.
Growth kinetics of EDP-938 resistance mutants vs. wild-type (WT) virus.
Viral Fitness analysis. (A) Cell viability compared to uninfected cells quantified by cellular ATP levels. (B) Plaque forming units (PFU) produced per well per day. Data are mean ± SEM, N = 3–7 for all runs.
Fig 4.
In vitro time-of-addition studies with EDP-938.
(A) Cell viability of RSV-A Long-infected HEp-2 cells with EDP-938 treatment given at indicated times relative to infection. Cells were infected at an MOI of 0.1 and harvested 5 days post infection. All data are mean ± SD, N = 3–6 per time point. (B) Single-round RSV infection comparing EDP-938 and fusion inhibitor GS-5806 treated to untreated (DMSO) samples. A cold-synchronized infection was performed with virus at an MOI of 3. Viral RNA was quantitated by RT-qPCR at endpoint 24 hpi. 2x and 20x EC50 values are 100 nM and 1,000 nM for EDP-938, 2 nM and 20 nM for GS-5806. All data are mean ± SD, N = 2 per time point.
Fig 5.
EDP-938 prevents primary transcription of RSV mRNAs.
Following a synchronized infection with RSV-A2 (MOI of 3), DMSO or 1000 nM EDP-938 was added at 0 hpi. Immunofluorescence analysis of inclusion bodies at 8 hpi in the presence of DMSO (A) or EDP-938 (B). N protein was detected with an N specific monoclonal antibody (red) and cell nuclei were stained with DAPI (blue). (C) RT-qPCR analysis of RSV N gene expression between 0 and 6 hpi. Samples were quantified using the ΔΔCt method relative to the mean expression of housekeeping gene GAPDH. The relative expression of positive sensitive RNA is shown. Data are representative of two independent experiments.
Fig 6.
In vivo efficacy of EDP-938 in RSV-infected African Green monkey (AGM) model.
(A) Viral load in bronchoalveolar lavage (BAL) in RSV infected AGMs. Copies of RSV RNA per mL of BAL at indicated days relative to infection challenge. EDP-938 or RSV-604 treatment period shown as shaded area. Bars represent SD. (B) Reduction in RSV RNA per mL in BAL on Day 5 or in nasopharyngeal (NP) on Day 7 post infection. LOD (limit of detection) was 100 copies/mL Bars represent SD.
Fig 7.
Locations of mutations mapped to RSV-A N protein.
Crystal structure of amino acids 32–251. RSV-A mutations shown in red, RSV-B mutations shown in blue, purple indicates mutation seen in both. PDB file 4UC6 used for this analysis.