No Remdesivir Resistance Observed in the Phase 3 Severe and Moderate COVID-19 SIMPLE Trials.

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.

Oral administration of obeldesivir protects nonhuman primates against Sudan ebolavirus.

Obeldesivir (ODV, GS-5245) is an orally administered prodrug of the parent nucleoside of remdesivir (RDV) and is presently in phase 3 trials for COVID-19 treatment. In this work, we show that ODV and its circulating parent nucleoside metabolite, GS-441524, have similar in vitro antiviral activity against filoviruses, including Marburg virus, Ebola virus, and Sudan virus (SUDV). We also report that once-daily oral ODV treatment of cynomolgus monkeys for 10 days beginning 24 hours after SUDV exposure confers 100% protection against lethal infection. Transcriptomics data show that ODV treatment delayed the onset of inflammation and correlated with antigen presentation and lymphocyte activation. Our results offer promise for the further development of ODV to control outbreaks of filovirus disease more rapidly.

Characterization of the Cynomolgus Macaque Model of Marburg Virus Disease and Assessment of Timing for Therapeutic Treatment Testing.

Marburg virus (MARV) causes severe disease and high mortality in humans. The objective of this study was to characterize disease manifestations and pathogenesis in cynomolgus macaques exposed to MARV. The results of this natural history study may be used to identify features of MARV disease useful in defining the ideal treatment initiation time for subsequent evaluations of investigational therapeutics using this model. Twelve cynomolgus macaques were exposed to a target dose of 1000 plaque-forming units MARV by the intramuscular route, and six control animals were mock-exposed. The primary endpoint of this study was survival to Day 28 post-inoculation (PI). Anesthesia events were minimized with the use of central venous catheters for periodic blood collection, and temperature and activity were continuously monitored by telemetry. All mock-exposed animals remained healthy for the duration of the study. All 12 MARV-exposed animals (100%) became infected, developed illness, and succumbed on Days 8-10 PI. On Day 4 PI, 11 of the 12 MARV-exposed animals had statistically significant temperature elevations over baseline. Clinically observable signs of MARV disease first appeared on Day 5 PI, when 6 of the 12 animals exhibited reduced responsiveness. Ultimately, systemic inflammation, coagulopathy, and direct cytopathic effects of MARV all contributed to multiorgan dysfunction, organ failure, and death or euthanasia of all MARV-exposed animals. Manifestations of MARV disease, including fever, systemic viremia, lymphocytolysis, coagulopathy, and hepatocellular damage, could be used as triggers for initiation of treatment in future therapeutic efficacy studies.

Viral Resistance Analyses From the Remdesivir Phase 3 Adaptive COVID-19 Treatment Trial-1 (ACTT-1).

BACKGROUND: Remdesivir is approved for treatment of coronavirus disease 2019 (COVID-19) in nonhospitalized and hospitalized adult and pediatric patients. Here we present severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) resistance analyses from the phase 3 ACTT-1 randomized placebo-controlled trial conducted in adult participants hospitalized with COVID-19. METHODS: Swab samples were collected at baseline and longitudinally through day 29. SARS-CoV-2 genomes were sequenced using next-generation sequencing. Phenotypic analysis was conducted directly on participant virus isolates and/or using SARS-CoV-2 subgenomic replicons expressing mutations identified in the Nsp12 target gene. RESULTS: Among participants with both baseline and postbaseline sequencing data, emergent Nsp12 substitutions were observed in 12 of 31 (38.7%) and 12 of 30 (40.0%) participants in the remdesivir and placebo arms, respectively. No emergent Nsp12 substitutions in the remdesivir arm were observed in more than 1 participant. Phenotyping showed low to no change in susceptibility to remdesivir relative to wild-type Nsp12 reference for the substitutions tested: A16V (0.8-fold change in EC50), P323L + V792I (2.2-fold), C799F (2.5-fold), K59N (1.0-fold), and K59N + V792I (3.4-fold). CONCLUSIONS: The similar rate of emerging Nsp12 substitutions in the remdesivir and placebo arms and the minimal change in remdesivir susceptibility among tested substitutions support a high barrier to remdesivir resistance development in COVID-19 patients. Clinical Trials Registration. NCT04280705.

Late remdesivir treatment initiation partially protects African green monkeys from lethal Nipah virus infection.

Remdesivir is a nucleotide prodrug with preclinical efficacy against lethal Nipah virus infection in African green monkeys when administered 1 day post inoculation (dpi) (Lo et al., 2019). Here, we determined whether remdesivir treatment was still effective when treatment administration initiation was delayed until 3 dpi. Three groups of six African green monkeys were inoculated with a lethal dose of Nipah virus, genotype Bangladesh. On 3 dpi, one group received a loading dose of 10 mg/kg remdesivir followed by daily dosing with 5 mg/kg for 11 days, one group received 10 mg/kg on 12 consecutive days, and the remaining group received an equivalent volume of vehicle solution. Remdesivir treatment initiation on 3 dpi provided partial protection from severe Nipah virus disease that was dose dependent, with 67% of animals in the high dose group surviving the challenge. However, remdesivir treatment did not prevent clinical disease, and surviving animals showed histologic lesions in the brain. Thus, early administration seems critical for effective remdesivir treatment during Nipah virus infection.

Effect of remdesivir post-exposure prophylaxis and treatment on pathogenesis of measles in rhesus macaques.

Measles is a systemic disease initiated in the respiratory tract with widespread measles virus (MeV) infection of lymphoid tissue. Mortality can be substantial, but no licensed antiviral therapy is available. We evaluated both post-exposure prophylaxis and treatment with remdesivir, a broad-spectrum antiviral, using a well-characterized rhesus macaque model of measles. Animals were treated with intravenous remdesivir for 12 days beginning either 3 days after intratracheal infection (post-exposure prophylaxis, PEP) or 11 days after infection at the onset of disease (late treatment, LT). As PEP, remdesivir lowered levels of viral RNA in peripheral blood mononuclear cells, but RNA rebounded at the end of the treatment period and infectious virus was continuously recoverable. MeV RNA was cleared more rapidly from lymphoid tissue, was variably detected in the respiratory tract, and not detected in urine. PEP did not improve clinical disease nor lymphopenia and reduced the antibody response to infection. In contrast, LT had little effect on levels of viral RNA or the antibody response but also did not decrease clinical disease. Therefore, remdesivir transiently suppressed expression of viral RNA and limited dissemination when provided as PEP, but virus was not cleared and resumed replication without improvement in the clinical disease parameters evaluated.

Expanded profiling of Remdesivir as a broad-spectrum antiviral and low potential for interaction with other medications in vitro.

Remdesivir (GS-5734; VEKLURY) is a single diastereomer monophosphoramidate prodrug of an adenosine analog (GS-441524). Remdesivir is taken up by target cells and metabolized in multiple steps to form the active nucleoside triphosphate (GS-443902), which acts as a potent inhibitor of viral RNA-dependent RNA polymerases. Remdesivir and GS-441524 have antiviral activity against multiple RNA viruses. Here, we expand the evaluation of remdesivir's antiviral activity to members of the families Flaviviridae, Picornaviridae, Filoviridae, Orthomyxoviridae, and Hepadnaviridae. Using cell-based assays, we show that remdesivir can inhibit infection of flaviviruses (such as dengue 1-4, West Nile, yellow fever, Zika viruses), picornaviruses (such as enterovirus and rhinovirus), and filoviruses (such as various Ebola, Marburg, and Sudan virus isolates, including novel geographic isolates), but is ineffective or is significantly less effective against orthomyxoviruses (influenza A and B viruses), or hepadnaviruses B, D, and E. In addition, remdesivir shows no antagonistic effect when combined with favipiravir, another broadly acting antiviral nucleoside analog, and has minimal interaction with a panel of concomitant medications. Our data further support remdesivir as a broad-spectrum antiviral agent that has the potential to address multiple unmet medical needs, including those related to antiviral pandemic preparedness.

In Vitro Selection of Remdesivir-Resistant SARS-CoV-2 Demonstrates High Barrier to Resistance.

In vitro selection of remdesivir-resistant severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) revealed the emergence of a V166L substitution, located outside of the polymerase active site of the Nsp12 protein, after 9 passages of a single lineage. V166L remained the only Nsp12 substitution after 17 passages (10 µM remdesivir), conferring a 2.3-fold increase in 50% effective concentration (EC50). When V166L was introduced into a recombinant SARS-CoV-2 virus, a 1.5-fold increase in EC50 was observed, indicating a high in vitro barrier to remdesivir resistance.

Repurposing an In Vitro Measles Virus Dissemination Assay for Screening of Antiviral Compounds.

Measles virus (MV) is a highly contagious respiratory virus responsible for outbreaks associated with significant morbidity and mortality among children and young adults. Although safe and effective measles vaccines are available, the COVID-19 pandemic has resulted in vaccination coverage gaps that may lead to the resurgence of measles when restrictions are lifted. This puts individuals who cannot be vaccinated, such as young infants and immunocompromised individuals, at risk. Therapeutic interventions are complicated by the long incubation time of measles, resulting in a narrow treatment window. At present, the only available WHO-advised option is treatment with intravenous immunoglobulins, although this is not approved as standard of care. Antivirals against measles may contribute to intervention strategies to limit the impact of future outbreaks. Here, we review previously described antivirals and antiviral assays, evaluate the antiviral efficacy of a number of compounds to inhibit MV dissemination in vitro, and discuss potential application in specific target populations. We conclude that broadly reactive antivirals could strengthen existing intervention strategies to limit the impact of measles outbreaks.

Remdesivir efficacy against yellow fever in a hamster model.

Yellow fever virus (YFV) continues to cause periodic outbreaks of severe disease throughout tropical regions of South America and Africa despite the availability of an effective vaccine. Despite efforts to control this virus for the last century, no antivirals have been approved for the treatment of YFV. The purpose of this study was to evaluate the broadly active antiviral compound remdesivir (RDV) in a hamster model of disease. Yellow fever (YF) disease in hamsters was prevented when treatment with RDV was initiated just prior to virus challenge, which was confirmed in a second study. Disease parameters including viremia, serum ALT and weight loss were significantly improved with RDV treatment in a dose-dependent manner. RDV was also effective when treatment was initiated as late as 4 days post-virus infection (dpi). These results demonstrate therapeutic efficacy of RDV in the treatment of YF in a relevant animal model of disease.

Remdesivir and GS-441524 Retain Antiviral Activity against Delta, Omicron, and Other Emergent SARS-CoV-2 Variants.

Genetic variation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in the emergence and rapid spread of multiple variants throughout the pandemic, of which Omicron is currently the predominant variant circulating worldwide. SARS-CoV-2 variants of concern/variants of interest (VOC/VOI) have evidence of increased viral transmission, disease severity, or decreased effectiveness of vaccines and neutralizing antibodies. Remdesivir (RDV [VEKLURY]) is a nucleoside analog prodrug and the first FDA-approved antiviral treatment of COVID-19. Here, we present a comprehensive antiviral activity assessment of RDV and its parent nucleoside, GS-441524, against 10 current and former SARS-CoV-2 VOC/VOI clinical isolates by nucleoprotein enzyme-linked immunosorbent assay (ELISA) and plaque reduction assay. Delta and Omicron variants remained susceptible to RDV and GS-441524, with 50% effective concentration (EC50) values 0.30- to 0.62-fold of those observed against the ancestral WA1 isolate. All other tested variants exhibited EC50 values ranging from 0.13- to 2.3-fold of the observed EC50 values against WA1. Analysis of nearly 6 million publicly available variant isolate sequences confirmed that Nsp12, the RNA-dependent RNA polymerase (RdRp) target of RDV and GS-441524, is highly conserved across variants, with only 2 prevalent changes (P323L and G671S). Using recombinant viruses, both RDV and GS-441524 retained potency against all viruses containing frequent variant substitutions or their combination. Taken together, these results highlight the conserved nature of SARS-CoV-2 Nsp12 and provide evidence of sustained SARS-CoV-2 antiviral activity of RDV and GS-441524 across the tested variants. The observed pan-variant activity of RDV supports its continued use for the treatment of COVID-19 regardless of the SARS-CoV-2 variant.

Intravenous delivery of GS-441524 is efficacious in the African green monkey model of SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the COVID-19 pandemic, has infected over 260 million people over the past 2 years. Remdesivir (RDV, VEKLURY®) is currently the only antiviral therapy fully approved by the FDA for the treatment of COVID-19. The parent nucleoside of RDV, GS-441524, exhibits antiviral activity against numerous respiratory viruses including SARS-CoV-2, although at reduced in vitro potency compared to RDV in most assays. Here we find in both human alveolar and bronchial primary cells, GS-441524 is metabolized to the pharmacologically active GS-441524 triphosphate (TP) less efficiently than RDV, which correlates with a lower in vitro SARS-CoV-2 antiviral activity. In vivo, African green monkeys (AGM) orally dosed with GS-441524 yielded low plasma levels due to limited oral bioavailability of <10%. When GS-441524 was delivered via intravenous (IV) administration, although plasma concentrations of GS-441524 were significantly higher, lung TP levels were lower than observed from IV RDV. To determine the required systemic exposure of GS-441524 associated with in vivo antiviral efficacy, SARS-CoV-2 infected AGMs were treated with a once-daily IV dose of either 7.5 or 20 mg/kg GS-441524 or IV RDV for 5 days and compared to vehicle control. Despite the reduced lung TP formation compared to IV dosing of RDV, daily treatment with IV GS-441524 resulted in dose-dependent efficacy, with the 20 mg/kg GS-441524 treatment resulting in significant reductions of SARS-CoV-2 replication in the lower respiratory tract of infected animals. These findings demonstrate the in vivo SARS-CoV-2 antiviral efficacy of GS-441524 and support evaluation of its orally bioavailable prodrugs as potential therapies for COVID-19.

Mutations in the SARS-CoV-2 RNA-dependent RNA polymerase confer resistance to remdesivir by distinct mechanisms.

The nucleoside analog remdesivir (RDV) is a Food and Drug Administration-approved antiviral for treatment of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections. Thus, it is critical to understand factors that promote or prevent RDV resistance. We passaged SARS-CoV-2 in the presence of increasing concentrations of GS-441524, the parent nucleoside of RDV. After 13 passages, we isolated three viral lineages with phenotypic resistance as defined by increases in half-maximal effective concentration from 2.7- to 10.4-fold. Sequence analysis identified nonsynonymous mutations in nonstructural protein 12 RNA-dependent RNA polymerase (nsp12-RdRp): V166A, N198S, S759A, V792I, and C799F/R. Two lineages encoded the S759A substitution at the RdRp Ser759-Asp-Asp active motif. In one lineage, the V792I substitution emerged first and then combined with S759A. Introduction of S759A and V792I substitutions at homologous nsp12 positions in murine hepatitis virus demonstrated transferability across betacoronaviruses; introduction of these substitutions resulted in up to 38-fold RDV resistance and a replication defect. Biochemical analysis of SARS-CoV-2 RdRp encoding S759A demonstrated a roughly 10-fold decreased preference for RDV-triphosphate (RDV-TP) as a substrate, whereas nsp12-V792I diminished the uridine triphosphate concentration needed to overcome template-dependent inhibition associated with RDV. The in vitro-selected substitutions identified in this study were rare or not detected in the greater than 6 million publicly available nsp12-RdRp consensus sequences in the absence of RDV selection. The results define genetic and biochemical pathways to RDV resistance and emphasize the need for additional studies to define the potential for emergence of these or other RDV resistance mutations in clinical settings.

Combination therapy with remdesivir and monoclonal antibodies protects nonhuman primates against advanced Sudan virus disease.

A major challenge in managing acute viral infections is ameliorating disease when treatment is delayed. Previously, we reported the success of a 2-pronged mAb and antiviral remdesivir therapeutic approach to treat advanced illness in rhesus monkeys infected with Marburg virus (MARV). Here, we explored the benefit of a similar combination therapy for Sudan ebolavirus (Sudan virus; SUDV) infection. Importantly, no licensed anti-SUDV therapeutics currently exist, and infection of rhesus macaques with SUDV results in a rapid disease course similar to MARV with a mean time to death of 8.3 days. When initiation of therapy with either remdesivir or a pan-ebolavirus mAb cocktail (MBP431) was delayed until 6 days after inoculation, only 20% of macaques survived. In contrast, when remdesivir and MBP431 treatment were combined beginning 6 days after inoculation, significant protection (80%) was achieved. Our results suggest that combination therapy may be a viable treatment for patients with advanced filovirus disease that warrants further clinical testing in future outbreaks.

Subcutaneous remdesivir administration prevents interstitial pneumonia in rhesus macaques inoculated with SARS-CoV-2.

The utility of remdesivir treatment in COVID-19 patients is currently limited by the necessity to administer this antiviral intravenously, which has generally limited its use to hospitalized patients. Here, we tested a novel, subcutaneous formulation of remdesivir in the rhesus macaque model of SARS-CoV-2 infection that was previously used to establish the efficacy of remdesivir against this virus in vivo. Compared to vehicle-treated animals, macaques treated with subcutaneous remdesivir from 12 h through 6 days post inoculation showed reduced signs of respiratory disease, a reduction of virus replication in the lower respiratory tract, and an absence of interstitial pneumonia. Thus, early subcutaneous administration of remdesivir can protect from lower respiratory tract disease caused by SARS-CoV-2.

Inhaled remdesivir reduces viral burden in a nonhuman primate model of SARS-CoV-2 infection.

Remdesivir (RDV) is a nucleotide analog prodrug with demonstrated clinical benefit in patients with coronavirus disease 2019 (COVID-19). In October 2020, the US FDA approved intravenous (IV) RDV as the first treatment for hospitalized COVID-19 patients. Furthermore, RDV has been approved or authorized for emergency use in more than 50 countries. To make RDV more convenient for non-hospitalized patients earlier in disease, alternative routes of administration are being evaluated. Here, we investigated the pharmacokinetics and efficacy of RDV administered by head dome inhalation in African green monkeys (AGM). Relative to an IV administration of RDV at 10 mg/kg, an approximately 20-fold lower dose administered by inhalation produced comparable concentrations of the pharmacologically active triphosphate in lower respiratory tract tissues. Distribution of the active triphosphate into the upper respiratory tract was also observed following inhaled RDV exposure. Inhalation RDV dosing resulted in lower systemic exposures to RDV and its metabolites as compared with IV RDV dosing. An efficacy study with repeated dosing of inhaled RDV in an AGM model of SARS-CoV-2 infection demonstrated reductions in viral replication in bronchoalveolar lavage fluid and respiratory tract tissues compared with placebo. Efficacy was observed with inhaled RDV administered once daily at a pulmonary deposited dose of 0.35 mg/kg beginning approximately 8 hours post-infection. Moreover, the efficacy of inhaled RDV was similar to that of IV RDV administered once at 10 mg/kg followed by 5 mg/kg daily in the same study. Together, these findings support further clinical development of inhalation RDV.

Efficient incorporation and template-dependent polymerase inhibition are major determinants for the broad-spectrum antiviral activity of remdesivir.

Remdesivir (RDV) is a direct-acting antiviral agent that is approved in several countries for the treatment of coronavirus disease 2019 caused by the severe acute respiratory syndrome coronavirus 2. RDV exhibits broad-spectrum antiviral activity against positive-sense RNA viruses, for example, severe acute respiratory syndrome coronavirus and hepatitis C virus, and nonsegmented negative-sense RNA viruses, for example, Nipah virus, whereas segmented negative-sense RNA viruses such as influenza virus or Crimean-Congo hemorrhagic fever virus are not sensitive to the drug. The reasons for this apparent efficacy pattern are unknown. Here, we expressed and purified representative RNA-dependent RNA polymerases and studied three biochemical parameters that have been associated with the inhibitory effects of RDV-triphosphate (TP): (i) selective incorporation of the nucleotide substrate RDV-TP, (ii) the effect of the incorporated RDV-monophosphate (MP) on primer extension, and (iii) the effect of RDV-MP in the template during incorporation of the complementary UTP. We found a strong correlation between antiviral effects and efficient incorporation of RDV-TP. Inhibition in primer extension reactions was heterogeneous and usually inefficient at higher NTP concentrations. In contrast, template-dependent inhibition of UTP incorporation opposite the embedded RDV-MP was seen with all polymerases. Molecular modeling suggests a steric conflict between the 1'-cyano group of the inhibitor and residues of the structurally conserved RNA-dependent RNA polymerase motif F. We conclude that future efforts in the development of nucleotide analogs with a broader spectrum of antiviral activities should focus on improving rates of incorporation while capitalizing on the inhibitory effects of a bulky 1'-modification.

Remdesivir is efficacious in rhesus monkeys exposed to aerosolized Ebola virus.

Efficacious therapeutics for Ebola virus disease are in great demand. Ebola virus infections mediated by mucosal exposure, and aerosolization in particular, present a novel challenge due to nontypical massive early infection of respiratory lymphoid tissues. We performed a randomized and blinded study to compare outcomes from vehicle-treated and remdesivir-treated rhesus monkeys in a lethal model of infection resulting from aerosolized Ebola virus exposure. Remdesivir treatment initiated 4 days after exposure was associated with a significant survival benefit, significant reduction in serum viral titer, and improvements in clinical pathology biomarker levels and lung histology compared to vehicle treatment. These observations indicate that remdesivir may have value in countering aerosol-induced Ebola virus disease.

Prevention and therapy of SARS-CoV-2 and the B.1.351 variant in mice.

Improving clinical care for individuals infected with SARS-CoV-2 variants is a global health priority. Small-molecule antivirals like remdesivir (RDV) and biologics such as human monoclonal antibodies (mAbs) have demonstrated therapeutic efficacy against SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19). It is not known whether combination RDV/mAb will improve outcomes over single-agent therapies or whether antibody therapies will remain efficacious against variants. Here, we show that a combination of two mAbs in clinical trials, C144 and C135, have potent antiviral effects against even when initiated 48 h after infection and have therapeutic efficacy in vivo against the B.1.351 variant of concern (VOC). Combining RDV and antibodies provided a modest improvement in outcomes compared with single agents. These data support the continued use of RDV to treat SARS-CoV-2 infections and the continued clinical development of the C144 and C135 antibody combination to treat patients infected with SARS-CoV-2 variants.