Biology subjects

Sobolik, E. B.

Publications and source records attributed to Sobolik, E. B..

6 recordsLinked to original sources

bioRxiv2025

Impact of Mutations Affecting 4'-Fluorouridine Susceptibility on Fitness and Treatment Outcomes for Venezuelan Equine Encephalitis Virus

Venezuelan equine encephalitis virus (VEEV) is a prototypical encephalitic alphavirus. Members of the Alphavirus genus are found across the globe, transmitted by arthropod vectors, and cause significant disease burdens in humans and animals. There are currently no FDA-approved antivirals against any member of the Alphavirus genus. While a vaccine exists against chikungunya virus (CHIKV), a member of the arthitogenic alphaviruses, FDA-approved vaccines are not available for other members of this genus, particularly the encephalitic alphaviruses such as VEEV, Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV). 4-Fluorouridine (4-FlU, EIDD-2749) was recently identified as a broad-spectrum antiviral against multiple RNA viruses, including alphaviruses. 4-FlU can potently inhibit VEEV-TC83 replication, with submicromolar potency in cell culture. However, the emergence of antiviral resistance represents a hurdle for antiviral drug development and the implementation of effective treatment strategies. Here, we have identified novel mutations in the VEEV nsP4 RNA-dependent RNA polymerase that reduce susceptibility to 4-FlU, including P187A, Q191L, L289F, and T296I. We rebuilt each mutation in recombinant VEEV-TC83 and characterized the effects of these mutations on fitness and pathogenicity. In addition, we assessed the impact of mutations reducing sensitivity to 4-FlU in a mouse model. Although mutations against 4-FlU arise quickly in vitro, treatment can still alleviate severe disease and lethal encephalitis. Together, these data highlight the promising therapeutic potential of 4-FlU for the treatment of alphavirus encephalitis. ImportanceVEEV is one of several mosquito-spread viruses that can cause serious brain infections in people and animals. Unlike CHIKV, against which an approved vaccine exists, there are no countermeasures to prevent or treat VEEV infections or block its close relatives, EEEV and WEEV. 4-FlU inhibits many different RNA viruses, including VEEV, and is currently being developed to treat multiple viral infections. A major problem with small-molecule antivirals is the appearance of virus populations that are less susceptible to treatment. In this study, we identified mutations in VEEVs RNA-dependent RNA polymerase (nsP4) that confer reduced susceptibility to 4-FlU. We then engineered these mutations into full-length infectious clones and assessed if viruses encoding for these mutations were still pathogenic. In addition, we treated infected mice with 4-FlU and measured how well the compound inhibited virus replication and prevented severe disease, even when mice were infected with viruses harboring mutations that reduced susceptibility to 4-FlU. Although VEEV can develop moderate resistance to 4-FlU in vitro, administration of 4-FlU still reduced severe disease and prevented lethality in the animals infected with viruses that possess mutations that decrease susceptibility to 4-FlU. These results suggest that 4-FlU has strong potential as a future treatment for alphavirus infections like VEEV encephalitis.

microbiology↗

bioRxiv2025

DEVELOPMENT OF A POTENT MONOCLONAL ANTIBODY FOR TREATMENT OF HUMAN METAPNEUMOVIRUS INFECTIONS

Human metapneumovirus (HMPV) is a major cause of respiratory infections, particularly among vulnerable populations, yet effective therapeutics remain unavailable. Monoclonal antibodies (mAbs) offer a promising approach for both treatment and prevention. Here, we describe the discovery and characterization of 4F11, a highly potent and broadly neutralizing mAb with demonstrated in vitro and in vivo efficacy against HMPV. Using cryo-electron microscopy, we defined a unique mechanism of binding HMPV employed by 4F11, which distinguishes it from previously characterized RSV and HMPV mAbs. 4F11 targets an epitope located at the apex of the prefusion F protein (site O) with a 1:1 stoichiometry, distinct from the 3:1 stoichiometry observed with other HMPV site O antibodies. Unlike other site O antibodies, which penetrate the glycan shield between Asn57 and Asn172, 4F11 binds vertically and directly interacts with the Asn172 glycan, representing a unique glycan-dependent mode of recognition. In vitro, 4F11 displayed high potency and broad neutralization across diverse HMPV strains. It also showed a low propensity for resistance development, with only a single escape mutation (K179E) identified, a mutation not found in any published HMPV sequence to date. Viruses rescued with the K179E escape mutation had significantly decreased fitness in vitro compared to wild-type virus. In a hamster challenge model, 4F11 significantly reduced viral loads in both the lungs and nasal turbinates. These findings highlight 4F11 as a promising candidate for therapeutic development, particularly for immunocompromised individuals and other high-risk groups.

immunology↗

bioRxiv2025

A luminescent attenuated SARS-CoV-2 for the identification and validation of drug-resistant mutants

The emergence of SARS-CoV-2 variants has necessitated continuous updating of vaccines. In contrast, antivirals remained effective as they target conserved viral proteins that are essential for the viral life cycle. However, several mutations in SARS-CoV-2 that may affect the efficacy of United States (US) Food and Drug Administration (FDA)-approved antivirals have been recently identified. Detecting drug-resistant SARS-CoV-2 mutants and investigating their escape mechanism(s) are critical to guide the selection of effective antiviral therapies. In this study, we constructed an attenuated recombinant (r)SARS-CoV-2 lacking the open reading frame (ORF) proteins 3a and 7b but expressing nanoluciferase (Nluc), rSARS-CoV-2 {Delta}3a7b-Nluc, to facilitate tracking viral infection. Using this virus, we selected drug-resistant mutants to the main viral protease (Mpro) inhibitor nirmatrelvir. After passaging {Delta}3a7b-Nluc 10 times in the presence of increasing concentrations of nirmatrelvir, a virus population with enhanced resistance was selected. We identified two non-synonymous mutations (L50F and R188G) in Mpro, encoded by the non-structural protein 5 (NSP5) gene. Using reverse genetics, we generated rSARS-CoV-2 {Delta}3a7b-Nluc containing the identified L50F and R188G mutations, individually or in combination, and assessed their contribution to nirmatrelvir resistance. Our results indicate that both mutations are involved in escaping from nirmatrelvir. Altogether, our results demonstrate the feasibility of using rSARS-CoV-2 {Delta}3a7b-Nluc variant to identify and validate mutations that confer resistance to FDA-approved antiviral drugs without the concern of conducting gain of function (GoF) experiments with wild-type (WT) forms of SARS-CoV-2. IMPORTANCESmall-molecule antiviral drugs have been used for the treatment of SARS-CoV-2 infections. However, drug-resistant SARS-CoV-2 mutants to currently US FDA-approved Mpro targeting antivirals have been identified. Information on SARS-CoV-2 escape mutants and mutations affecting the antiviral activity of licensed antivirals remain limited. In this study, we developed a nanoluciferase (Nluc)-expressing attenuated recombinant (r)SARS-CoV-2 lacking the ORF 3a and 7b proteins ({Delta}3a7b-Nluc) to identify nirmatrelvir resistant mutants without the biosafety concerns associated with gain-of-function (GoF) research using wild-type (WT) SARS-CoV-2. Using {Delta}3a7b-Nluc, we have selected variants with reduced sensitivity to nirmatrelvir that were validated by the generation of rSARS-CoV-2 {Delta}3a7b-Nluc containing the candidate L50F and R188G mutations in Mpro. These results demonstrate the feasibility of using rSARS-CoV-2 {Delta}3a7b-Nluc to safely identify and validate drug-resistant mutants overcoming concerns originating from adaptation studies using WT SARS-CoV-2.

microbiology↗

bioRxiv2024

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. ImportanceFour 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.

microbiology↗

bioRxiv2023

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. Author SummaryReduced 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.

microbiology↗

bioRxiv2023

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

After years of the COVID-19 pandemic, over 40 million children worldwide are at risk of measles due to delayed vaccination1 and temporary SARS-CoV-2 viral dominance2. Acute measles has a case-fatality rate of [~]1%, but most morbidity and mortality arise post-measles due to destruction of pre-existing immune memory by lymphotropic measles virus (MeV)3,4, a paramyxovirus of the Morbillivirus genus. MeV-induced immune amnesia is not mitigated by post-exposure vaccination and the impact of unrelated respiratory virus disease history on measles severity has not been defined. We used a lethal canine distemper virus (CDV)-ferret model as surrogate for human morbillivirus disease5 and employed the orally efficacious broad-spectrum paramyxovirus polymerase inhibitor GHP-883096 to establish measles treatment paradigms. Applying a receptor tropism-intact recombinant CDV with low lethality, we provide in vivo confirmation of the morbillivirus immune amnesia hypothesis and reveal an 8-day advantage of antiviral treatment versus therapeutic vaccination in preserving immune memory. Infection of ferrets with non-lethal influenza A virus (IAV) A/CA/07/2009 (H1N1) or respiratory syncytial virus (RSV) four weeks prior to CDV caused exacerbated CDV disease that rapidly advanced to fatal hemorrhagic pneumonia associated with lung onslaught by commensal bacteria. RNAseq of BAL samples and lung tissue identified CDV-induced expression of trefoil factor (TFF) peptides, which was absent in animals pre-infected with IAV, thus highlighting that immune priming by unrelated respiratory viruses influences morbillivirus infection outcome. Non-invasive pulmonary ferret MRI revealed that severe outcomes of consecutive IAV/CDV infections were prevented by oral GHP-88309 treatment even when initiated after peak clinical signs of CDV. These findings validate the morbillivirus immune amnesia hypothesis, define treatment paradigms for measles, identify prior disease history as risk factor for exacerbated morbillivirus disease, and demonstrate that treating morbillivirus infection with direct-acting oral antivirals provides therapeutic benefit regardless of whether the time window to mitigate primary clinical signs of infection has closed.

microbiology↗