SARS-CoV-2 resistance analyses from the Phase 3 PINETREE study of remdesivir treatment in nonhospitalized participants

Abstract

Remdesivir inhibits the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp; Nsp12). Here, we conducted viral resistance analyses from the Phase 3 PINETREE trial of remdesivir in nonhospitalized participants at risk of severe COVID-19. Nasopharyngeal swabs (collected at baseline [Day 1], Days 2, 3, 7, and 14) were eligible for analysis if their viral load was above the lower limit of quantification for the RT-qPCR assay (2228 copies/mL). The SARS-CoV-2 genome was sequenced for all remdesivir participants and 50% of placebo participants (baseline, Days 3, 7, and 14) and for participants who progressed to COVID-19-related hospitalization or all-cause death (all time points). Emergent substitutions in Nsp12 and other replication complex proteins were phenotyped using site-directed mutagenesis in a SARS-CoV-2 subgenomic replicon system. Overall, emergent Nsp12 substitutions were detected in 8/115 (7.0%) remdesivir participants and 7/129 (5.4%) placebo participants (1 substitution overlap between groups). Based on a structural analysis, none of the emergent Nsp12 substitutions were in direct contact with the incoming nucleoside triphosphate substrate, the RNA, or the RNA template 5' overhang. One substitution (A376V) showed reduced susceptibility to remdesivir (12.6-fold change in remdesivir half-maximal concentration [EC₅₀]); it also showed reduced fitness when introduced in the SARS-CoV-2 replicon and virus in vitro. Other substitutions had <1.1-fold change in remdesivir EC₅₀. None of the emergent substitutions in Nsp8, Nsp10, Nsp13, or Nsp14 (remdesivir, 10/115 [8.7%]; placebo, 10/129 [7.8%]) showed reduced remdesivir susceptibility. In conclusion, emergent substitutions in the SARS-CoV-2 RdRp complex with reduced remdesivir susceptibility were uncommon, indicating a high barrier to remdesivir resistance.CLINICAL TRIALSThis study is registered with ClinicalTrials.gov as NCT04501952.

Keywords: drug therapy.

Fig 1

PINETREE study design. AE, adverse event; IV, intravenous; QD, once daily; RDV, remdesivir.

Fig 2

Cryo-EM map of observed amino acid substitutions within the SARS-CoV-2 RNA-dependent polymerase complex in the remdesivir group. Cryo-EM, cryo-electron microscopy; RDV-TP, triphosphate form of remdesivir. On the left is the complex formed from Nsp12, Nsp7, two subunits of Nsp8, and two subunits of Nsp13, modeled on a composite of two cryo-EM structures (7UO4 and 7RDX) (38). A pre-incorporated RDV-TP (colored in cyan) is seen in the polymerase active site, as captured in the 7UO4 structure. On the right is the complex formed by Nsp14 and Nsp10, modeled from the cryo-EM structure 7N0B. Substitutions are widely dispersed across the two complexes. Of note, the Nsp13 A598V substitution is not shown. It is located in the C-terminal tail of the protein, which is not resolved in observed structures, implying significant disorder.

Fig 3

Viral load decrease and symptom alleviation in participants with Nsp12 substitution A376V. RDV, remdesivir; WT, wild-type. ^aSelf-reported symptom scale: (4) very much, (3) quite a bit, (2) somewhat, (1) a little bit, and (0) not at all.

Fig 4

Enzymatic activity of A376V- and A376V/P323L-mutant RdRps on full-length RNA synthesis relative to WT. ATP, adenosine triphosphate; CTP, cytidine triphosphate; GTP, guanosine triphosphate; UTP, uridine triphosphate; RdRp, RNA-dependent RNA polymerase; WT, wild-type. RNA template and primer sequences used are shown at the top. RNA products synthesized by the WT, A376V-, and A376V/P323L-mutant RdRps. The reaction mixture contained [α-³²P] CTP for detection, the indicated nucleotide-triphosphates for full-length RNA extension, 5 mM MgCl₂ cofactor, and increasing concentrations of RdRps. The sizes of extended RNA products in nucleotides are shown on the left. m denotes a radioactively labeled, 4-nucleotide primer used as a size marker. C (red) indicates the position at which [α-³²P] CTP is incorporated. *Indicates RNA products resulting from terminal transferase activity. Results are representative of a total of two replicates.