In the pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, epithelial populations in the distal lung expressing Angiotensin-converting enzyme 2 (ACE2) are infrequent, and therefore, the model of viral expansion and immune cell engagement remains incompletely understood. Using human lungs to investigate early host-viral pathogenesis, we found that SARS-CoV-2 had a rapid and specific tropism for myeloid populations. Human alveolar macrophages (AMs) reliably expressed ACE2 allowing both spike-ACE2–dependent viral entry and infection. In contrast to Influenza A virus, SARS-CoV-2 infection of AMs was productive, amplifying viral titers. While AMs generated new viruses, the interferon responses to SARS-CoV-2 were muted, hiding the viral dissemination from specific antiviral immune responses. The reliable and veiled viral depot in myeloid cells in the very early phases of SARS-CoV-2 infection of human lungs enables viral expansion in the distal lung and potentially licenses subsequent immune pathologies.

As new SARS-CoV-2 variants continue to emerge, it is important to evaluate the potency of antiviral drugs to support their continued use. Remdesivir (RDV; VEKLURY^®) an approved antiviral treatment for COVID-19, and obeldesivir (ODV) are inhibitors of the SARS-CoV-2 RNA-dependent RNA polymerase Nsp12. Here we show these two compounds retain antiviral activity against the Omicron variants BA.2.86, BF.7, BQ.1, CH.1.1, EG.1.2, EG.5.1, EG.5.1.4, FL.22, HK.3, HV.1, JN.1, JN.1.7, JN.1.18, KP.2, KP.3, LB.1, XBB.1.5, XBB.1.5.72, XBB.1.16, XBB.2.3.2, XBC.1.6, and XBF when compared with reference strains. Genomic analysis identified 29 Nsp12 polymorphisms in these and previous Omicron variants. Phenotypic analysis of these polymorphisms confirmed no impact on the antiviral activity of RDV or ODV and suggests Omicron variants containing these Nsp12 polymorphisms remain susceptible to both compounds. These data support the continued use of RDV in the context of circulating SARS-CoV-2 variants and the development of ODV as an antiviral therapeutic.

Remdesivir (RDV) is a broad-spectrum nucleotide analog prodrug approved for the treatment of COVID-19 in hospitalized and non-hospitalized patients with clinical benefit demonstrated in multiple Phase 3 trials. Here we present SARS-CoV-2 resistance analyses from the Phase 3 SIMPLE clinical studies evaluating RDV in hospitalized participants with severe or moderate COVID-19 disease. The severe and moderate studies enrolled participants with radiologic evidence of pneumonia and a room-air oxygen saturation of ≤94% or >94%, respectively. Virology sample collection was optional in the study protocols. Sequencing and related viral load data were obtained retrospectively from participants at a subset of study sites with local sequencing capabilities (10 of 183 sites) at timepoints with detectable viral load. Among participants with both baseline and post-baseline sequencing data treated with RDV, emergent Nsp12 substitutions were observed in 4 of 19 (21%) participants in the severe study and none of the 2 participants in the moderate study. The following 5 substitutions emerged: T76I, A526V, A554V, E665K, and C697F. The substitutions T76I, A526V, A554V, and C697F had an EC₅₀ fold change of ≤1.5 relative to the wildtype reference using a SARS-CoV-2 subgenomic replicon system, indicating no significant change in the susceptibility to RDV. The phenotyping of E665K could not be determined due to a lack of replication. These data reveal no evidence of relevant resistance emergence and further confirm the established efficacy profile of RDV with a high resistance barrier in COVID-19 patients.

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 [EC ₅₀ ]); 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 EC ₅₀ . 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 TRIALS: This study is registered with ClinicalTrials.gov as NCT04501952 .

Background: Obeldesivir is an oral nucleoside analog prodrug inhibitor of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Methods: Nonhospitalized adults with risk factors for developing severe coronavirus disease 2019 (COVID-19) were enrolled ≤5 days from COVID-19 symptom onset and randomized 1:1 to receive obeldesivir 350 mg or placebo twice daily for 5 days. The primary end point was COVID-19–related hospitalization or all-cause death by day 29. Other end points included time to symptom alleviation by day 15, change in SARS-CoV-2 viral RNA copy number and infectious viral titer, and incidence of adverse events and laboratory abnormalities. Results: Four hundred and sixty five participants were randomized and received ≥1 dose of study drug. Baseline characteristics were generally balanced between groups. Overall, 58% had received ≥1 COVID-19 vaccination, and 92% were seropositive for SARS-CoV-2 antibodies. COVID-19–related hospitalization or all-cause death by day 29 was reported in 0 of 211 (0%) participants with obeldesivir and 1 of 207 (0.5%) participants with placebo (log-rank P = .32). Time to COVID-19 symptom alleviation was numerically shorter with obeldesivir versus placebo. Obeldesivir reduced viral RNA copy number at day 5 and infectious titer at days 3 and 5 versus placebo. The safety profile was generally comparable across arms. Conclusions: Although underpowered in the context of a changing COVID-19 landscape, obeldesivir in nonhospitalized adults with targeted risk factors did not improve COVID-19–related hospitalization or all-cause death. Obeldesivir reduced viral RNA copy number and infectious titer, demonstrating its ability to inhibit SARS-CoV-2 replication, and resulted in numerically faster symptom alleviation. Clinical Trials Registration: NCT05603143; EudraCT 2022-002741-18.

With the ongoing emergence of SARS-CoV-2 variants, continued surveillance of antiviral susceptibility remains critical for detecting resistance that could compromise treatment efficacy. This study evaluated the activity of 2 SARS-CoV-2 RNA-dependent RNA polymerase (Nsp12) inhibitors against emerging Omicron variants: remdesivir (RDV), an approved antiviral for the treatment of COVID-19, and obeldesivir (ODV), an oral prodrug that shares the same parent nucleoside as RDV. Both RDV and ODV were shown to retain antiviral activity against the Omicron subvariants BA.2.86.1, JN.1.7, KP.2, KP.3.1.1, KP.3.3, LP.8.1, NB.1.8.1, XBB.2, XEC, and XFG compared with wild-type reference strains. Only 1 new lineage-defining Nsp12 substitution, D284Y (detected in NB.1.8.1), was observed. Phenotypic analysis demonstrated that a replicon containing this substitution remained susceptible to both RDV and ODV. These findings are consistent with previous studies showing that RDV and ODV retain potent activity against previously identified Omicron variants, support the continued clinical use of RDV against circulating SARS-CoV-2 variants, and reinforce the potential of ODV as an oral antiviral therapeutic.