4’-Fluorouridine mitigates lethal infection with pandemic human and highly pathogenic avian influenza viruses

Abstract

Influenza outbreaks are associated with substantial morbidity, mortality and economic burden. Next generation antivirals are needed to treat seasonal infections and prepare against zoonotic spillover of avian influenza viruses with pandemic potential. Having previously identified oral efficacy of the nucleoside analog 4-Fluorouridine (4-FlU, EIDD-2749) against SARS-CoV-2 and respiratory syncytial virus, we explored activity of the compound against seasonal and highly pathogenic influenza (HPAI) viruses in cell culture, human airway epithelium organoids, and/or two animal models, ferrets and mice, that assess IAV transmission and lethal viral pneumonia, respectively. 4-FlU inhibited a panel of relevant influenza A and B viruses with nanomolar potency in organoids. In vitro polymerase assays revealed immediate chain termination of IAV polymerase after 4-FlU incorporation, in contrast to delayed chain termination of SARS-CoV-2 and RSV polymerase. Once-daily oral treatment of ferrets with 2 mg/kg 4-FlU initiated 12 hours after infection rapidly stopped virus shedding and prevented direct-contact transmission to untreated sentinels. Treatment of mice infected with a lethal inoculum of pandemic A/CA/07/2009 (H1N1) (Ca09) with 2 mg/kg 4-FlU alleviated pneumonia. Three doses mediated complete survival when treatment was initiated up to 60 hours after infection, indicating an unusually broad window for effective intervention. Therapeutic oral 4-FlU ensured survival of animals infected with HPAI A/VN/12/2003 (H5N1) and of immunocompromised mice infected with pandemic Ca09. Recoverees were fully protected against homologous reinfection. This study defines the mechanistic foundation for high sensitivity of influenza viruses to 4-FlU and supports 4-FlU as developmental candidate for the treatment of seasonal and pandemic influenza.

Influenza A virus resistance to 4’-fluorouridine coincides with viral attenuationin vitroandin vivo

“…Dose-response assessment of 4'-FlU against recCA09 returned 50 and 90% inhibitory concentrations (EC50 and EC90, respectively) of 0.14 and 0.24 µM (S1 Fig), which was consistent with our previous observations [29].…”

“…None of the combinations of mutations required for moderate resistance to 4'-FlU existed in complete IAV genome sequences available in the NCBI Virus database (S4 Fig) . Although some mutations were present individually, all remained below 1% relative frequency except PB1 M339I and PB2 E191K in complete and partial sequences (S6 Table ), confirming our previous observation that circulating human and avian IAVs are efficiently inhibited by the compound [29].…”

“…At every passage, progeny virus titers were determined, viral RNA extracted, and fresh cells infected at a controlled multiplicity of infection (MOI) of 0.01 TCID50 units/cell. We rebuilt all mutations in polymerase subunits in a recCA09-maxGFP background, which expresses a maxGFP from the HA segment [29,30] Lineage #4 co-replicated with the genetic parent without substantial change in relative allele frequencies, and one substitution each of lineages #5 and #6, V285I in PB1 and Y488C in PB2, respectively, disappeared rapidly, whereas others co-existed with the parent allele.…”

“…We localized all resistance mutations in structural models of the influenza virus polymerase complex, built on the coordinates released for influenza virus C (ICV) [33], the 1918 H1N1 IAV [34], and a bat IAV [35] Overall, altered geometry of the polymerase active site, achieved through combinations of mutations in distinct sets, emerged as the common theme across all resistance lineages (S7 Table ). These changes may either increase selectivity of the polymerase complex for substrate triphosphates or, more likely based on in vitro RdRP assay results of immediate chain termination [29], increase spatial flexibility at the active site to better accommodate secondary structure changes introduced by incorporation of the 4'-FlU analog into the nascent RNA strand [27,29]. Structural limits for flexibility appear to be tight, however, since different artificially engineered combinations of independently emerged signature resistance mutations did not result in bioactive polymerase complexes that could support productive virus replication (S8 Table ).…”

“…We have recently identified 4'-FlU, an orally efficacious nucleoside analog with broad spectrum activity against multiple positive and negative strand RNA viruses including betacoronaviruses [27,28], RSV and paramyxoviruses [27], and both seasonal and highly pathogenic avian influenza viruses [29]. Inducing immediate chain termination of the IAV polymerase, 4'-FlU displayed an unusually wide therapeutic time window in the lethal pdm09 H1N1 mouse model, mediating complete survival of animals when treatment was initiated up to 60 hours after infection [29].…”