Pathogenesis And Pharmacological Mitigation Of Influenza Viruses, Respiratory Syncytial Virus And SARS-CoV-2

Innumerable future deaths and hospitalizations could be avoided through improved pandemic preparedness and the use of personalized medicine. Here, various animal models have been used to improve an understanding of the pathogenicity and therapeutic treatment potential of three viral pathogens that have a significant impact on public health: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus (RSV), and influenza virus. Roborovski dwarf hamsters were developed as a novel animal model to study SARS-CoV-2-induced acute lower respiratory tract injury. Dwarf hamsters recapitulate the pathogenicity of SARS-CoV-2 variants of concern (VOC) and demonstrate that the effect size of molnupiravir and paxlovid-like nirmatrelvir/ritonavir is VOC-dependent. Additionally, the ferret model of upper respiratory disease was used to study human-to-human-like transmission of SARS-CoV-2 and define a therapeutic window and human effect-size equivalent dose (HESED) for the approved drugs molnupiravir and paxlovid. Therapeutic administration of molnupiravir successfully prevented contact transmission, whereas paxlovid-like nirmatrelvir/ritonavir only partially reduced upper respiratory viral shedding. Moreover, the oral prodrug of remdesivir parent GS-441524 was shown to be efficacious against SARS-CoV-2 in ferrets. A broad-spectrum polymerase inhibitor, 4’-Fluorouridine (4’-FlU), was identified and characterized, revealing potent activity against influenza virus, RSV, and SARS-CoV-2. This pre-clinical candidate is orally available and was shown to block replication of RSV in mice and SARS-CoV-2 in ferrets. In the mouse and ferret model, 4’-FlU mitigated lethal infection with pandemic human and highly pathogenic avian influenza. 4’-FlU could overcome moderate resistance, making this candidate a promising first line defense broad-spectrum antiviral. In parallel, an allosteric orally efficacious AVG-inhibitor series, which targets a dynamic interface in the RSV polymerase, was further developed to proof-of-concept in vivo efficacy testing. To explore a relationship between the gut microbiota and the host response to respiratory viral infections, the impact of the intestinal microbiome on influenza virus infection outcome was investigated. Colonization with segmented filamentous bacteria (SFB) in the gut stably and broadly altered the phenotype of alveolar macrophages (AM) enabling these cells to better protect their hosts from lethal respiratory viral infections. Moreover, gut microbiota-derived metabolites were discovered which conferred protection against SARS-CoV-2

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

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 (RSV), we explored activity of the compound against seasonal and highly pathogenic influenza (HPAI) viruses in cell culture, human airway epithelium (HAE) models, 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 to sub-micromolar potency in HAE cells. 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 transmission to untreated sentinels. Treatment of mice infected with a lethal inoculum of pandemic A/CA/07/2009 (H1N1)pdm09 (pdmCa09) with 4'-FlU alleviated pneumonia. Three doses mediated complete survival when treatment was initiated up to 60 hours after infection, indicating a broad time 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 pdmCa09. Recoverees were 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

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

Abstract 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

Detailed histopathological scores of all animals examined in this study.

Individual clinical scores for alveolitis, bronchiolitis, perivascular cuffing (PVC), vasculitis, interstitial pneumonia (IP), and pleuritis. Columns show data medians, symbols represent individual animals; 1-way ANOVA with Tukey’s post hoc test; p values are specified; n values are specified in Fig 4F. (TIF)</p

Effect of 4’-FlU on the antiviral immune response and lung histopathology.

a)In vivo Bioplex and histopathology study schematic. b) Lung viral titers in animals treated as in (a). c-d) Changes in IL-6 (c) and TNF-α (d) levels present in BALF of animals treated as in (a), relative to levels at time of infection. Lines in (b) intersect, and symbols show, geometric means ± SD, lines in (c-d) intersect, and symbols show, data medians with 95% CI; 2-way ANOVA with Tukey’s post hoc test; P values are given. e) Representative photomicrographs of lung tissue extracted 5 days after infection of animals treated as in (a). Tissues of two individual animals per study arm are shown at 10× magnification; scale bar denotes 100 μm; Br, Bronchiole; Bl, Blood vessel. f) Histopathology scores of animals treated as in (a). Lungs were extracted 5 days after infection. Scores for each animal represents a mean of individual alveolitis, bronchiolitis, vasculitis, pleuritis, perivascular cuffing (PVC), and interstitial pneumonia (IP) scores. Columns represent data medians with 95% CI; symbols show mean scores for each individual animal; 1-way ANOVA with Dunnett’s post hoc test, P values and n values for each study arm are specified.</p

Clinical signs of mice infected and reinfected with pdmCa09.

a-d) Body weight measurements taken twice daily (a, c), and rectal body temperature determined once daily (b, d). Lines intersect, and symbols show, data means ± SD. Results are shown for animals after the original infection (a-b) and after homotypic reinfection (c-d). Dashed lines in (a,c) specify predefined endpoint. (TIF)</p

Clinical signs of mice involved in mitigation of histopathology study.

a-b) Body weight measurements taken twice daily (a), and rectal body temperature determined once daily (b). Lines intersect, and symbols show, data means ± SD. Dashed line in (a) specifies predefined endpoint. (TIF)</p

Urea-PAGE fractionation (uncropped) of RNA transcripts.

a) Template and primer used in the reaction. b-e) Independent repeats of primer extension assay by IAV polymerase. Dashed rectangle shows insert presented in Fig 1E. Replicates were included in quantitations presented in Fig 1E and 1F. (TIF)</p

All statistical analyses performed for this study.

All statistical analyses performed for this study.</p

Clinical signs in pdmCa09-infected immunocompromised mice treated with 4’-FlU.

a-c) Shown are body weight measurements taken twice daily (a, c), and rectal body temperature (b, d) determined once daily. Lines intersect, and symbols show, data means ± SD; n values are specified in Fig 5. Results are shown for RAG1 KO (a-b) and IFNAR1 KO (c-d) animals. Dashed lines in (a,c) specify predefined endpoint. (TIF)</p