EDP-938 Has a High Barrier to Resistance in Healthy Adults Experimentally Infected with Respiratory Syncytial Virus

Figures

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  Figure 1.. Variants detected in the human challenge trial. A, Diagram detailing selection of participants for sequencing analysis. B, Theoretical viral load patterns for each group. These data are not from any participants but an example of viral load patterns that could be observed in each group. C and D, Viral shedding patterns for (C) LLOQ and (D) N:L139I for each participant from the human challenge trial with treatment-emergent mutations. Shaded triangles indicate EDP-938 dosing. Participant 20027 was assigned to a once daily treatment group. As such, this participant received a placebo for 1 of the 2 daily doses (open triangles). Light dashed line indicates the LLOQ (2.8 log10 copies/mL). Bold dashed line indicates the LLOD (1.4 log10 copies/mL). No mapped reads indicate no sequencing reads were able to be aligned to the RSV M37b reference genome. Abbreviations: LD, loading dose; LLOD, lower limit of detection; LLOQ, lower limit of quantification; N, nucleoprotein; RSV, respiratory syncytial virus; WT, wild type.

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  Figure 2.. N:L139I mediates resistance to EDP-938. HEp-2 cells were infected with the indicated recombinant strain of RSV-A expressing an mKATE reporter at a multiplicity of infection of 0.1. At the time of infection, cells were treated with the indicated concentration of the listed compound or DMSO vehicle control: (A) EDP-938, (B) EDP-323, (C) AK0529, and (D) RV521. Five days postinfection, the mKATE fluorescence intensity was measured. Percent viral inhibition at each concentration was calculated. Data are expressed as the mean percent viral inhibition at each concentration ± standard deviation from at least 3 independent biological replicates. Dotted lines indicate 50% and 90% viral inhibition. Abbreviations: DMSO, dimethyl sulfoxide; N, nucleoprotein; RSV, respiratory syncytial virus; WT, wild type.

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  Figure 3.. N:L139I has in vitro fitness defects compared to WT. A and C, HEp-2 cells were infected with the indicated recombinant strain of RSV-A expressing an mKATE reporter at a multiplicity of infection of 0.1. At each day postinfection, mKATE florescence intensity was measured (A) or cells and supernatant were harvested and subject to TCID50 analysis (C). Data are expressed as the mean mKATE florescence intensity (A) or TCID50/mL (C) at each day postinfection ± standard deviation from at least 3 independent biological replicates. LoD was 3.16E + 0 TCID50/mL. Values at or below the LoD are set at 2.0E + 0 TCID50/mL. B and D, For each replicate, the AUC was determined using a linear-linear plot. The AUC was normalized to WT and expressed as the percent AUC relative to WT. Data are expressed as the mean ± standard deviation AUC. *P < .05 by 1-way repeated measured ANOVA followed by Dunnett multiple comparison test. Abbreviations: AUC, area under the curve; LOD, limit of detection; N, nucleoprotein; ns, not significant; RSV, respiratory syncytial virus; TCID50, 50% tissue culture infectious dose; WT, wild type.

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  Figure 4.. Mapping of observed mutations on the nucleoprotein (N) crystal structure. N crystal structure [9] (Protein Date Bank file No. 4UC6) of amino acids 32–251 containing all observed mutations in this region. Observed mutated residues are shown as spheres.