Efficacy of late-onset antiviral treatment in immune-compromised hosts with persistent SARS-CoV-2 infection.

The immunocompromised are at high risk of prolonged SARS-CoV-2 infection and progression to severe COVID-19. However, efficacy of late-onset direct-acting antiviral (DAA) therapy with therapeutics in clinical use and experimental drugs to mitigate persistent viral replication is unclear. In this study, we employed an immunocompromised mouse model, which supports prolonged replication of SARS-CoV-2 to explore late-onset treatment options. Tandem immuno-depletion of CD4 + and CD8 + T cells in C57BL/6 mice followed by infection with SARS-CoV-2 variant of concern (VOC) beta B.1.351 resulted in prolonged infection with virus replication for five weeks after inoculation. Early-onset treatment with nirmatrelvir/ritonavir (paxlovid) or molnupiravir was only moderately efficacious, whereas the experimental therapeutic 4'-fluorourdine (4'-FlU, EIDD-2749) significantly reduced virus load in upper and lower respiratory compartments four days post infection (dpi). All antivirals significantly lowered virus burden in a 7-day treatment regimen initiated 14 dpi, but paxlovid-treated animals experienced rebound virus replication in the upper respiratory tract seven days after treatment end. Viral RNA was detectable 28 dpi in paxlovid-treated animals, albeit not in the molnupiravir or 4'-FlU groups, when treatment was initiated 14 dpi and continued for 14 days. Low-level virus replication continued 35 dpi in animals receiving vehicle but had ceased in all treatment groups. These data indicate that late-onset DAA therapy significantly shortens the duration of persistent virus replication in an immunocompromised host, which may have implications for clinical use of antiviral therapeutics to alleviate the risk of progression to severe disease in highly vulnerable patients. Importance: Four years after the onset of the global COVID-19 pandemic, the immunocompromised are at greatest risk of developing life-threatening severe disease. However, specific treatment plans for this most vulnerable patient group have not yet been developed. Employing a CD4 + and CD8 + T cell-depleted immunocompromised mouse model of SARS-CoV-2 infection, we explored therapeutic options of persistent infections with standard-of-care paxlovid, molnupiravir, and the experimental therapeutic 4'-FlU. Late-onset treatment initiated 14 days after infection was efficacious, but only 4'-FlU was rapidly sterilizing. No treatment-experienced viral variants with reduced susceptibility to the drugs emerged, albeit virus replication rebounded in animals of the paxlovid group after treatment end. This study supports the use of direct-acting antivirals for late-onset management of persistent SARS-CoV-2 infection in immunocompromised hosts. However, treatment courses likely require to be extended for maximal therapeutic benefit, calling for appropriately powered clinical trials to meet the specific needs of this patient group.

Influenza A virus resistance to 4'-fluorouridine coincides with viral attenuation in vitro and in vivo.

Pre-existing or rapidly emerging resistance of influenza viruses to approved antivirals makes the development of novel therapeutics to mitigate seasonal influenza and improve preparedness against future influenza pandemics an urgent priority. We have recently identified the chain-terminating broad-spectrum nucleoside analog clinical candidate 4'-fluorouridine (4'-FlU) and demonstrated oral efficacy against seasonal, pandemic, and highly pathogenic avian influenza viruses in the mouse and ferret model. Here, we have resistance-profiled 4'-FlU against a pandemic A/CA/07/2009 (H1N1) (CA09). In vitro viral adaptation yielded six independently generated escape lineages with distinct mutations that mediated moderate resistance to 4'-FlU in the genetically controlled background of recombinant CA09 (recCA09). Mutations adhered to three distinct structural clusters that are all predicted to affect the geometry of the active site of the viral RNA-dependent RNA polymerase (RdRP) complex for phosphodiester bond formation. Escape could be achieved through an individual causal mutation, a combination of mutations acting additively, or mutations functioning synergistically. Fitness of all resistant variants was impaired in cell culture, and all were attenuated in the mouse model. Oral 4'-FlU administered at lowest-efficacious (2 mg/kg) or elevated (10 mg/kg) dose overcame moderate resistance when mice were inoculated with 10 LD50 units of parental or resistant recCA09, demonstrated by significantly reduced virus load and complete survival. In the ferret model, invasion of the lower respiratory tract by variants representing four adaptation lineages was impaired. Resistant variants were either transmission-incompetent, or spread to untreated sentinels was fully blocked by therapeutic treatment of source animals with 4'-FlU.

Therapeutic mitigation of measles-like immune amnesia and exacerbated disease after prior respiratory virus infections in ferrets.

Measles cases have surged pre-COVID-19 and the pandemic has aggravated the problem. Most measles-associated morbidity and mortality arises from destruction of pre-existing immune memory by measles virus (MeV), a paramyxovirus of the morbillivirus genus. Therapeutic measles vaccination lacks efficacy, but little is known about preserving immune memory through antivirals and the effect of respiratory disease history on measles severity. We use a canine distemper virus (CDV)-ferret model as surrogate for measles and employ an orally efficacious paramyxovirus polymerase inhibitor to address these questions. A receptor tropism-intact recombinant CDV with low lethality reveals an 8-day advantage of antiviral treatment versus therapeutic vaccination in maintaining immune memory. Infection of female ferrets with influenza A virus (IAV) A/CA/07/2009 (H1N1) or respiratory syncytial virus (RSV) four weeks pre-CDV causes fatal hemorrhagic pneumonia with lung onslaught by commensal bacteria. RNAseq identifies CDV-induced overexpression of trefoil factor (TFF) peptides in the respiratory tract, which is absent in animals pre-infected with IAV. Severe outcomes of consecutive IAV/CDV infections are mitigated by oral antivirals even when initiated late. These findings validate the morbillivirus immune amnesia hypothesis, define measles treatment paradigms, and identify priming of the TFF axis through prior respiratory infections as risk factor for exacerbated morbillivirus disease.

Influenza A virus resistance to 4'-fluorouridine coincides with viral attenuation in vitro and in vivo.

Pre-existing or rapidly emerging resistance of influenza viruses to approved antivirals makes the development of novel therapeutics to mitigate seasonal influenza and improve preparedness against future influenza pandemics an urgent priority. We have recently identified the chain-terminating broad-spectrum nucleoside analog clinical candidate 4'-fluorouridine (4'-FlU) and demonstrated oral efficacy against seasonal, pandemic, and highly pathogenic avian influenza viruses in the mouse and ferret model. Here, we have resistance-profiled 4'-FlU against a pandemic A/CA/07/2009 (H1N1) (CA09). In vitro viral adaptation yielded six independently generated escape lineages with distinct mutations that mediated moderate resistance to 4'-FlU in the genetically controlled background of recombinant CA09 (recCA09). Mutations adhered to three distinct structural clusters that are all predicted to affect the geometry of the active site of the viral RNA-dependent RNA polymerase (RdRP) complex for phosphodiester bond formation. Escape could be achieved through an individual causal mutation, a combination of mutations acting additively, or mutations functioning synergistically. Fitness of all resistant variants was impaired in cell culture, and all were attenuated in the mouse model. Oral 4'-FlU administered at lowest-efficacious (2 mg/kg) or elevated (10 mg/kg) dose overcame moderate resistance when mice were inoculated with 10 LD 50 units of parental or resistant recCA09, demonstrated by significantly reduced virus load and complete survival. In the ferret model, invasion of the lower respiratory tract by variants representing four adaptation lineages was impaired. Resistant variants were either transmission-incompetent, or spread to untreated sentinels was fully blocked by therapeutic treatment of source animals with 4'-FlU. Author Summary: Reduced sensitivity to FDA-approved influenza drugs is a major obstacle to effective antiviral therapy. We have previously demonstrated oral efficacy of a novel clinical candidate drug, 4'-FlU, against seasonal, pandemic, and highly pathogenic avian influenza viruses. In this study, we have determined possible routes of influenza virus escape from 4'-FlU and addressed whether resistance imposes a viral fitness penalty, affecting pathogenicity or ability to transmit. We identified three distinct clusters of mutations that lead to moderately reduced viral sensitivity to the drug. Testing of resistant variants against two chemically unrelated nucleoside analog inhibitors of influenza virus, conditionally approved favipiravir and the broad-spectrum SARS-CoV-2 drug molnupiravir, revealed cross-resistance of one cluster with favipiravir, whereas no viral escape from molnupiravir was noted. We found that the resistant variants are severely attenuated in mice, impaired in their ability to invade the lower respiratory tract and cause viral pneumonia in ferrets, and transmission-defective or compromised. We could fully mitigate lethal infection of mice with the resistant variants with standard or 5-fold elevated oral dose of 4'-FlU. These results demonstrate that partial viral escape from 4'-FlU is feasible in principle, but escape mutation clusters are unlikely to reach clinical significance or persist in circulating influenza virus strains.

Genomic Characterization of Respiratory Syncytial Virus during 2022-23 Outbreak, Washington, USA.

We sequenced 54 respiratory syncytial virus (RSV) genomes collected during 2021-22 and 2022-23 outbreaks in Washington, USA, to determine the origin of increased RSV cases. Detected RSV strains have been spreading for >10 years, suggesting a role for diminished population immunity from low RSV exposure during the COVID-19 pandemic.