Search

The ongoing evolution of SARS-CoV-2 to evade vaccines and therapeutics underlines the need for novel therapies with high genetic barriers to resistance. The small molecule PAV-104, identified through a cell-free protein synthesis and assembly screen, was recently shown to target host protein assembly machinery in a manner specific to viral assembly. Here, we investigated the capacity of PAV-104 to inhibit SARS-CoV-2 replication in human airway epithelial cells (AECs). Our data demonstrate that PAV-104 inhibited > 99% of infection with diverse SARS-CoV-2 variants in primary and immortalized human AECs. PAV-104 suppressed SARS-CoV-2 production without affecting viral entry or protein synthesis. PAV-104 interacted with SARS-CoV-2 nucleocapsid (N) and interfered with its oligomerization, blocking particle assembly. Transcriptomic analysis revealed that PAV-104 reversed SARS-CoV-2 induction of the Type-I interferon response and the ‘maturation of nucleoprotein’ signaling pathway known to support coronavirus replication. Our findings suggest that PAV-104 is a promising therapeutic candidate for COVID-19.

BackgroundLung transplant recipients undergo bronchoalveolar lavage (BAL) to detect antecedents of chronic lung allograft dysfunction (CLAD), but routine assessment of BAL cytology is controversial. We hypothesized that inflammation on BAL cytology would predict CLAD-free survival.MethodsIn a single-center retrospective cohort, associations between cytology results and clinical characteristics were compared using generalized-estimating equation-adjusted regression. The association between BAL inflammation and CLAD or death risk was assessed using time-dependent Cox models.ResultsIn 3365 cytology reports from 451 subjects, inflammation was the most common finding (6.2%, 210 cases), followed by fungal forms (5.3%, 178 cases, including 24 cases of suspectedAspergillus). Inflammation on BAL cytology was more common in procedures for symptoms (8.5%) versus surveillance (3.2%, p&nbsp

Advanced donor age is a risk factor for poor survival following lung transplantation. However, recent work identifying epigenetic determinants of aging has shown that biologic age may not always reflect chronologic age and that stressors can accelerate biologic aging. We hypothesized that lung allografts that experienced primary graft dysfunction (PGD), characterized by poor oxygenation in the first three post-transplant days, would have increased biologic age. We cultured airway epithelial cells isolated by transbronchial brush at 1-year bronchoscopies from 13 subjects with severe PGD and 15 controls matched on age and transplant indication. We measured epigenetic age using the Horvath epigenetic clock. Linear models were used to determine the association of airway epigenetic age with chronologic ages and PGD status, adjusted for recipient PGD risk factors. Survival models assessed the association with chronic lung allograft dysfunction (CLAD) or death. Distributions of promoter methylation within pathways were compared between groups. DNA methyltransferase (DNMT) activity was quantified in airway epithelial cells under hypoxic or normoxic conditions. Airway epigenetic age appeared younger but was strongly associated with the age of the allograft (slope 0.38 per year, 95% CI 0.27–0.48). There was no correlation between epigenetic age and recipient age (P = 0.96). Epigenetic age was 6.5 years greater (95% CI 1.7–11.2) in subjects who had experienced PGD, and this effect remained significant after adjusting for donor and recipient characteristics (P = 0.03). Epigenetic age was not associated with CLAD-free survival risk (P = 0.11). Analysis of differential methylation of promoters of key biologic pathways revealed hypomethylation in regions related to hypoxia, inflammation, and metabolism-associated pathways. Accordingly, airway epithelial cells cultured in hypoxic conditions showed suppressed DNMT activity. While airway methylation age was primarily determined by donor chronologic age, early injury in the form of PGD was associated with increased allograft epigenetic age. These data show how PGD might suppress key promoter methylation resulting in long-term impacts on the allograft.

Author(s): Hoover, Jonathan; Mintz, Michelle A; Deiter, Fred; Aminian, Emily; Chen, Joy; Hays, Steven R; Singer, Jonathan P; Calabrese, Daniel R; Kukreja, Jasleen; Greenland, John R | Abstract: BackgroundAirway infections are difficult to distinguish from acute rejection in lung transplant recipients. Traditional culture techniques take time that may delay treatment. We hypothesized that a rapid multiplex molecular assay could improve time to diagnosis and appropriate clinical decision making.MethodsIn a prospective observational study of recipients undergoing bronchoscopy, we assessed the BioFire® FilmArray® Pneumonia Panel (BFPP) in parallel to standard of care (SOC) diagnostics. Research clinicians performed shadow (research only) clinical decision making in real time. Time to report and interpretation were reported as median and interquartile ranges and compared by Wilcoxon signed-ranked test. Agreement was defined based on detection of any species targeted in the molecular assay.ResultsFor the 150 enrolled subjects, BFPP results were available 3.8nhours (IQR 2.8-5.1) following bronchoscopy, compared to 13nhours for viral SOC (IQR 10-34, Pnln.001) results and 48nhours for bacterial SOC (IQR 46-70, Pnln.001) results. Positive BFPP results were interpreted in 9nhours (IQR 5-20) following bronchoscopy, compared to 74nhours for SOC (IQR 37-110, Pnln.001). Assays agreed for 138 (92%) of the 150 subjects. Of 22 BFPP diagnoses, five (23%) resulted in a shadow antibiotic recommendation. Notable BFPP deficiencies included fungal species and H parainfluenzae, accounting for 15 (27%) and 13 (23%) of the 56 actionable SOC results, respectively.ConclusionsThis molecular diagnostic including bacterial targets has the potential to shorten time to diagnosis and augment current clinical decision making but cannot replace SOC culture methods.

Antiviral compounds displaying several remarkable features have been identified by a uniquely enabling drug screen and advanced through validation in two animal models, and in human primary bronchial epithelial cells grown to an air-liquid interface (ALI) and infected with SARS-CoV-2 (Brazil). Activity is observed in the nanomolar range in mammalian cells in vitro against the six viral families causing most human respiratory viral disease, irrespective of strain, including SARS-CoV-2 delta variant. A substantial barrier to development of viral resistance is demonstrated for influenza (FLUV). The drug target is an allosteric site on a novel host multi-protein complex (MPC) formed transiently, in an energy-dependent fashion, and composed of proteins implicated in viral lifecycles and innate immunity. The composition of this host MPC is modified in viral family-specific ways by FLUV and CoV, and substantially restored to the uninfected state with drug treatment. SQSTM1/p62, a key regulator of the autophagy pathway of innate immunity is i) found in the target MPC from uninfected cells, ii) lost upon infection, and iii) restored by drug treatment of infected cells, as assessed by drug resin affinity chromatography. A small subset of 14-3-3 is identified as the host protein to which the drug is bound. Advanced compounds with good oral bioavailability, half-life, lung exposure, and safety are approaching criteria for a Target Product Profile. We propose these novel drug targets to comprise a previously unappreciated molecular basis for homeostasis that is modified by viruses to facilitate their propagation and is restored by treatment with the therapeutic compounds presented. This discovery has transformative implications for treating respiratory viral-related disease, applicable to everything from COVID-19, seasonal influenza, common ‘winter viruses’ (respiratory syncytial virus, parainfluenza virus, rhinovirus, etc.), emerging respiratory viruses, and prevention of virus-induced asthma/COPD exacerbations. Treating respiratory viral disease with these host-targeted pan-respiratory viral family active compounds early, upon onset of symptoms of viral upper respiratory infection, irrespective of cause, should protect against progression to lower respiratory tract or systemic infection, the hallmarks of serious illness.

Chronic lung allograft dysfunction (CLAD) is the major barrier to long-term survival following lung transplantation, and new mechanistic biomarkers are needed. Lymphocytic bronchitis (LB) precedes CLAD and has a defined molecular signature. We hypothesized that this LB molecular signature would be associated with CLAD in small airway brushings independent of infection. We quantified RNA expression from small airway brushings and transbronchial biopsies, using RNAseq and digital RNA counting, respectively, for 22 CLAD cases and 27 matched controls. LB metagene scores were compared across CLAD strata by Wilcoxon rank sum test. We performed unbiased host transcriptome pathway and microbial metagenome analysis in airway brushes and compared machine-learning classifiers between the two tissue types. This LB metagene score was increased in CLAD airway brushes (P = 0.002) and improved prediction of graft failure (P = 0.02). Gene expression classifiers based on airway brushes outperformed those using transbronchial biopsies. While infection was associated with decreased microbial alpha-diversity (P ≤0.04), neither infection nor alpha-diversity was associated with LB gene expression. In summary, CLAD was associated with small airway gene expression changes not apparent in transbronchial biopsies in this cohort. Molecular analysis of airway brushings for diagnosing CLAD merits further examination in multicenter cohorts.

Purpose Final culture results are often unavailable at the time of donor organ offers, despite their importance in the evaluation of donor organ quality. We hypothesized that a rapid multiplex molecular assay could improve time to results and facilitate treatment initiation in the donors prior to organ recovery. Methods The “Rapid Detection of Airway Pathogens for Lung Transplantation” trial (NCT03933878) is a prospective observational clinical trial investigating the efficiency and accuracy of the BioFire Pneumonia Panel in the context of donor bronchoscopy. This assay tests the presence and/or quantity for 26 common airway pathogens in 2 hours. Bronchoalveolar lavage was collected from brain dead donors at the time of lung procurement. Molecular assay results were compared to standard of care cultures. Time to result between molecular assay and standard of care was compared by Wilcoxon signed-rank test. Agreement between assays was assessed by Cohen's kappa. Results A total of 35 donor samples were included. From the time of sample collection to time to result, the median time was 7.01 hours [6.59–9.18] versus 57.7 hours [44.6–72.6] (p Conclusion Molecular detection of lower respiratory pathogens significantly reduces the time to result while maintaining agreement with standard of care clinical cultures in the context of candidate lung allografts. Such data could inform quality assessment and facilitate timely donor management prior to organ recovery.

Purpose Rejection and infection can be antecedents of chronic lung allograft dysfunction (CLAD), the major limitation to long term survival for lung transplant recipients. BAL cytology is routinely performed but recently some have advocated abandoning this practice because of the low diagnostic yield. We hypothesized that inflammation observed on BAL cytology would predict CLAD risk. Methods We categorized diagnostic findings on BAL cytology between 2012 and 2019. Bronchoscopy indication, infection treatment, BAL and biopsy results, and CLAD-free survival were abstracted from medical records. Cytology associations with clinical outcomes were compared using generalized-estimating equation-adjusted logistic regression. The association between BAL inflammation and CLAD or death were determined using time-dependent Cox proportional hazards models adjusted for age, gender, diagnosis, lung allocation score, and transplant type. Results We evaluated 3,578 cytology reports from 491 subjects. Inflammation was the most common finding (6.4%), followed by fungi (5.2%). There were 5 cases of malignancy and 2 cases of CMV. Inflammation on BAL cytology was more common in procedures performed for symptoms, associated with antimicrobial initiation (a proxy for clinically significant infection), associated with acute rejection, and linked to increased BAL neutrophil and lymphocyte concentrations (Figure 1A). Inflammation on BAL cytology was present for 32% of subjects on at least one sample, more frequent around the time of CLAD onset, and associated a 2.6-fold hazard ratio (CI 1.2-5.3) for CLAD or death (Figure 1B). However, this association was not significant after adjusting for BAL cell counts and acute rejection. Conclusion Inflammation on BAL cytology specimens is clinically significant, suggesting acute rejection or infection and increased risk of CLAD or death. However, other indicators of allograft inflammation can substitute for some of the information provided by BAL cytology.