Remdesivir (RDV) is a nucleotide analog prodrug with demonstrated clinical benefit in patients with coronavirus disease 2019 (COVID-19). In October 2020, the U.S. FDA approved intravenous 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 nonhospitalized 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 intravenous administration of RDV at 10 mg/kg, an about 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 after inhaled RDV exposure. Inhalation RDV dosing resulted in lower systemic exposures to RDV and its metabolites as compared with intravenous 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 about 8 hours after infection. Moreover, the efficacy of inhaled RDV was similar to that of intravenous 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.

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.

The significant impact of the human virome on human physiology is beginning to emerge thanks to modern sequencing methods and bioinformatic tools ¹ . Anelloviruses, the principal constituent of the commensal human virome, are universally acquired in infancy and found throughout the body ^2,3,4 . Since the discovery of the original torque teno virus in 1997 ⁵ , three genera of the Anelloviridae family, each extremely diverse genetically, have been found in humans. These viruses elicit weak immune responses that permit multiple strains to co-exist and persist for years in a typical individual ⁶ . However, because they do not cause disease ⁷ and due to the lack of an in vitro culture system, anelloviruses remain poorly understood ^8,9 . Basic features of the virus, such as the identity of its structural protein, have been unclear until now. Here, we describe the first structure of an anellovirus particle, which includes a jelly roll domain that forms a 60-mer icosahedral particle core from which spike domains extend to form a salient part of the particle surface. The spike domains come together around the 5-fold symmetry axes to form crown-like features. Relatively conserved patches of amino acids are near the base of the spike domain while a hypervariable region is at the apex. We propose that this structure renders the particle less susceptible to antibody neutralization by hiding vulnerable conserved epitopes while exposing highly diverse epitopes as immunological decoys, thereby contributing to the immune evasion properties of anelloviruses. This would contrast with viruses such as beak and feather disease virus, canine parvovirus or adeno-associated virus which lack such pronounced surface features. These results shed light on the structure of anelloviruses and provide a framework to understand their interactions with the immune system.

RNA viruses with pandemic potential pose a significant global health risk. The adenosine nucleoside analog GS-441524 is metabolized to its active GS-443902 triphosphate metabolite to inhibit a broad spectrum of RNA viruses. Intravenous (IV) remdesivir (RDV) and oral obeldesivir (ODV) are phosphoramidate and isobutyryl-ester prodrugs of GS-441524, respectively. Following administration, both RDV and ODV show rapid and broad tissue distribution, form the same GS-443902 metabolite in target tissues, and demonstrate promising in vivo efficacy across several RNA virus infection models. In an African green monkey SARS-CoV-2 infection model, respective RDV and ODV treatments yielded similar antiviral efficacy. Here, we compare the in vitro and in vivo pharmacokinetics (PK) and metabolism of RDV and ODV to highlight both similarities and differences in their absorption, metabolism, distribution, and excretion profiles. The distinct route of administration and metabolic fate of each prodrug produced in vivo PK and metabolism profiles with differential GS-441524 to tissue GS-443902 relationships, thereby supporting alternate methods for predicting human efficacious doses. Overall, a metabolism-directed prodrug design enabled optimized delivery of the identical active GS-443902 metabolite through different routes of administration, supporting broader applications of the same nucleoside analog across an expanded spectrum of potential antiviral indications.

Flaviviruses are arthropod-borne RNA viruses associated with significant human diseases globally. There are no effective direct-acting antivirals approved to treat these viral infections. Given its critical role in viral replication, the RNA-dependent RNA polymerase (RdRp) is a logical target for antiviral drug development. Remdesivir (formerly GS-5734), a 1′-cyano modified C-adenosine monophosphate prodrug, was the first US Food and Drug Administration (FDA) approved antiviral for coronavirus disease 2019 (COVID-19) and was also shown to inhibit flavivirus replication. GS-7682, a 4′-cyano modified C-adenosine prodrug, exhibits a broad-spectrum antiviral activity. Here, we determined the anti-flavivirus potency of both remdesivir and GS-7682 and characterized their active triphosphate forms, GS-443902 and GS-646939, respectively, against a panel of purified flavivirus RdRps. These include dengue, Japanese encephalitis, West Nile, yellow fever, and Zika. Enzyme kinetics demonstrate efficient RNA incorporation of GS-443902 and GS-646939. GS-646939 acts as an immediate chain terminator. Conversely, GS-443902 acts through a template-dependent inhibition mechanism by impeding the incorporation of the complementary UTP. Both mechanisms correlate with anti-flavivirus activity, although remdesivir is generally superior. The data demonstrate that immediate chain termination is not necessarily a preferred mechanism of action of nucleotide analogs. Template-dependent inhibition should also be considered, especially for viruses lacking intrinsic proofreading activities.

Respiratory syncytial virus (RSV) is a significant cause of morbidity and mortality in high-risk populations. Although prophylactic options are available, there are no effective oral therapeutics for RSV infection. Obeldesivir (ODV) is an orally bioavailable prodrug of the nucleoside analog GS-441524, which is converted intracellularly to its active nucleoside triphosphate and inhibits the RSV RNA polymerase. Here we report the potent antiviral activity of ODV against geographically and temporally diverse RSV A and B clinical isolates (EC ₅₀ : 0.20-0.66 μM). Resistance selection studies with ODV and GS-441524 against RSV identified a single amino acid substitution, I777L, in the L polymerase with reduced susceptibility (3.3-3.8-fold) to ODV and GS-441524, indicating a high barrier for resistance development. In an African green monkey RSV infection model, once-daily oral ODV doses of 30 or 90 mg/kg initiated ∼24 hours post-infection significantly reduced log ₁₀ viral RNA copies/mL×day area under the curve by 69-92% in the upper and lower respiratory tracts. Together, these preclinical data support the clinical evaluation of ODV for the treatment of RSV infection.