Ensitrelvir treatment-emergent amino acid substitutions in SARS-CoV-2 3CLpro detected in the SCORPIO-SR phase 3 trial

The impact of treatment-emergent amino acid substitutions (TEAASs) in severe acute coronavirus 2 (SARS-CoV-2) 3C-like protease (3CL(pro)) on clinical and virologic outcomes was evaluated in patients with mild-to-moderate coronavirus disease 2019 (COVID-19) who received ensitrelvir 125 mg in the SCORPIO-SR trial. Individuals were randomised to ensitrelvir or matched placebo once daily for 5 days (first dose <72 h after disease onset). 3CL(pro)-TEAASs were identified by sequencing nsp5 encoding 3CL(pro) from pre- and post-treatment nasopharyngeal swabs. Time to resolution of a composite of five characteristic COVID-19 symptoms (TTR) was compared between patients with and without the most common 3CL(pro)-TEAASs in the ensitrelvir arm. The ensitrelvir and placebo intention-to-treat populations comprised 345 and 341 patients, respectively. 3CL(pro)-TEAASs were detected in 19/204 (9.3%) ensitrelvir-treated and 3/137 (2.2%) placebo-treated patients with paired sequence data. The most common 3CL(pro)-TEAASs in the ensitrelvir arm were M49L (n = 12), M49I (n = 3) and S144A (n = 2). In the placebo arm, all 3CL(pro)-TEAASs occurred in <= 1 patient. Median (95% confidence interval) TTR was comparable between patients with and without those TEAASs (158.8 h [112.1-281.9] vs 189.7 h [151.4-234.4]). Mean viral RNA levels declined more slowly in patients with M49L/I or S144A versus those without. Reductions in viral titre were unaffected by these TEAASs. The characteristics of recombinant SARS-CoV-2 with 3CL(pro) mutations were explored in vitro. Recombinant viruses with some 3CL(pro) mutations had reduced susceptibility to ensitrelvir in vitro, with limited effects on viral and competitive fitness. Continued surveillance is warranted to monitor the spread of viruses with 3CL(pro) mutations.