ABSTRACT
Background: SARS-CoV-2 evolution has contributed to successive waves of infections and severely compromised the efficacy of available SARS-CoV-2 monoclonal antibodies. Decaying vaccine-induced immunity, vaccine hesitancy, and limited vaccine protection in older and immunocompromised populations further compromises vaccine efficacy at the population level. Early antiviral treatments, including intravenous remdesivir (RDV), reduce hospitalization and severe disease due to COVID-19. An orally bioavailable RDV analog could facilitate earlier widespread administration to non-hospitalized COVID-19 patients. Method(s): We synthesized monoalkyl glyceryl ether phosphodiesters of GS-441524 (RVn), lysophospholipid analogs which allow for oral bioavailability and stability in plasma. We evaluated the in vivo efficacy of our lead compound, 1-O-octadecyl-2-O-benzyl-sn-glyceryl-3-phospho-RVn (V2043), in an oral treatment model of murine SARS-CoV-2 infection. We then synthesized numerous phospholipid analogs of RVn and determined which modifications enhanced in vitro antiviral activity and selectivity. The most effective compounds against SARS-CoV-2 were then evaluated for antiviral activity against other RNA viruses. Result(s): Oral treatment of SARS-CoV-2 infected BALB/c mice with V2043 (60 mg/kg once daily for 5 days, starting 12 hrs after infection) reduced lung viral load by more than 100-fold versus vehicle at day 2 and to below the LOD at day 5. V2043 inhibited previous and contemporary SARS-CoV-2 Variants of concern to a similar degree, as measured by the half maximal effective concentration (EC50) in a human lung epithelial cell line (Calu-3). Evaluation of multiple RVn analogs with hydrophobic esters at the sn-2 of glycerol revealed that in vitro antiviral activity was improved by the introduction of a 3-fluoro-4-methoxysubstituted benzyl or a 3-or 4-cyano-substituted benzyl. These compounds showed a 2-to 6-fold improvement in antiviral activity compared to analogs having an unsubstituted benzyl, such as V2043, and were more active than RDV. These compounds also showed enhanced antiviral activity against multiple contemporary and emerging RNA viruses. Conclusion(s): Collectively, our data support the development of RVn phospholipid prodrugs as oral antiviral agents for prevention and treatment of SARS-CoV-2 infections and as preparation for future outbreaks of pandemic RNA viruses.
ABSTRACT
The current, rapidly diversifying pandemic has accelerated the need for efficient and effective identification of potential drug candidates for COVID-19. Knowledge on host-immune response to SARS-CoV-2 infection, however, remains limited with few drugs approved to date. Viable strategies and tools are rapidly arising to address this, especially with repurposing of existing drugs offering significant promise. Here we introduce a systems biology tool, the PHENotype SIMulator, which -by leveraging available transcriptomic and proteomic databases-allows modeling of SARS-CoV-2 infection in host cells in silico to i) determine with high sensitivity and specificity (both>96%) the viral effects on cellular host-immune response, resulting in specific cellular SARS-CoV-2 signatures and ii) utilize these cell-specific signatures to identify promising repurposable therapeutics. Powered by this tool, coupled with domain expertise, we identify several potential COVID-19 drugs including methylprednisolone and metformin, and further discern key cellular SARS-CoV-2-affected pathways as potential druggable targets in COVID-19 pathogenesis.
ABSTRACT
Purpose/Objectives: Acute and chronic respiratory diseases constitute a substantial socioeconomic burden on a global scale, as made abundantly clear in the last two years with the rampant coronavirus disease 2019 (COVID-19) pandemic. Alas, the development of new therapies for pathological respiratory conditions has been hindered by the inadequacy of current preclinical models, which often fail to provide reliable predictions on drug safety and efficacy in humans. In particular, considerable anatomical and physiological differences between the respiratory systems of commonly used animal models and humans are one of the main issues leading to high drug attrition and clinical failure rates. Accordingly, the generation of physiologically relevant preclinical lung models for early drug development and pharmaceutical research is urgently needed. In this work, poly(ϵ-caprolactone) (PCL) and gelatin were used as raw materials to produce electrospun scaffolds for in vitro lung tissue engineering, in order to generate human biomimetic platforms for preclinical drug safety and efficacy testing. Methodology: PCL and gelatin were mixed at varying volume ratios: 1:0 (PP), 6:1 (PPG61), 4:1 (PPG41), and 2:1 (PPG21), so as to determine the optimal gelatin concentration for cell adhesion and growth. Poly(vinylpyrrolidone) (PVP) was added to every polymer mixture to facilitate the electrospinning process, and electrospun fibrous matrices were fabricated using a needleless electrospinning technique. Scaffold morphology, chemical composition, and wettability were characterized with scanning electron microscopy (SEM), Fourier transform infrared (FTIR) spectroscopy, and water contact angle analysis, respectively. Biocompatibility testing was performed using human bronchial (16HBE) and alveolar (A549) epithelial cell lines, consisting of cell metabolic activity, proliferation, and adhesion evaluation over two weeks of in vitro culture. Results: All polymer blends resulted in the formation of electrospun scaffolds with a nanofibrous structure. The addition of gelatin in PPG61 scaffolds improved fiber morphology compared to PP formulations, but increasing proportions of this polymer in PPG41 and PPG21 mats caused a larger number of defects, such as beading and branching. FTIR analysis confirmed the presence of PCL and PVP in PP scaffolds, as well as the addition of gelatin in all PPG blends. Moreover, as expected, all scaffolds were hydrophilic, with water contact angles below 90°, being suitable for protein adsorption and cell adhesion. Regarding 16HBE and A549 cell viability, surprisingly, no major differences were found between the different formulations over the two-week culture period, showing that all polymer blends were equally capable of promoting cell adhesion and growth. While PP scaffolds significantly outperformed PPG electrospun mats in early timepoints, no such differences were identified at the end of the experimental period. Conclusion/Significance: These results suggested that PCL, PVP, and/or gelatin blend electrospun scaffolds are conducive to lung epithelial cell adhesion and proliferation. Nevertheless, further studies investigating epithelial cell differentiation and function should be conducted to fully assess the suitability of these biomaterials as platforms for in vitro lung tissue engineering.
ABSTRACT
Introduction: The rapid emergence of the SARS-CoV-2 Omicron variant that evades many monoclonal antibody therapies illustrates the need for anti-viral treatments with low susceptibility to evolutionary escape. The small molecule PAV-104, identified through a moderate-throughput screen involving cell-free protein synthesis, was recently shown to target a subset of host protein assembly machinery in a manner specific to viral assembly. This compound has minimal host toxicity, including once daily oral dosing in rats that achieves >200-fold of the 90% effective concentration (EC90) in blood. The chemotype shows broad activity against respiratory viral pathogens, including Orthomyxoviridae, Paramyxoviridae, Adenoviridae, Herpesviridae, and Picornaviridae, with low suceptability to evolutionary escape. We hypothesized that PAV-104 would be active against SARSCoV- 2 variants in human airway epithelial cells. Methods: Airway epithelial cells were differentiated from lung transplant tissue at air-liquid interface (ALI) for four weeks prior to challenge with Alpha (Pango lineage designation B.1.1.7), Beta (B.1.351), Gamma (P.1), and Delta (B.1.617.2) SARS-CoV-2 variants. Viral replication was determined by quantitative PCR measurement of the SARS-CoV-2 nucleocapsid (N) gene. Dose-dependent virus inhibition and cytotoxicity of PAV-104 in the Calu-3 airway epithelial cell line was determined by PCR and MTT assay. Student's t-tests were used to evaluate statistical significance. Results: Alpha, Beta, Gamma, and Delta variants of SARS-CoV-2 showed comparable infectivity in human primary airway epithelial cells at ALI (N=3 donors), 47- to 550-fold higher than the parent (USA-WA1/2020) strain. PAV-104 reached 50% cytotoxicity in Calu-3 cells at 240 nM (Fig. 1A). Dose-response studies in Calu-3 cells demonstrated PAV-104 has a 6 nM 50% inhibitory concentration (IC50) for blocking replication of SARS-CoV-2 (USA-WA1/2020) (Fig.1B). In primary cells at ALI from 3 donors tested, there was >99% inhibition of infection by SARS-CoV-2 Gamma variant (N=3, MOI 0.1, P <0.01) with 100 nM PAV-104 (Fig. 1C). Addition of 100 nM PAV-104 2-hours post-infection, but not pre-infection, resulted in >99% suppression of viral replication, indicating a post-entry drug mechanism. PAV-104 bound a small subset of the known allosteric modulator 14-3-3, itself implicated in the interactome of SARS-CoV-2. Conclusion: PAV-104 is a host-targeted, orally bioavailable, pan-viral small molecule inhibitor with promising activity against SARS-CoV-2 variants in human primary airway epithelial cells. (Figure Presented).
ABSTRACT
Introduction: Management of acute respiratory distress including COVID-19 pneumonia involves O2 supplementation, which is lifesaving, but causes severe hyperoxic acute lung injury (HALI). AT2 cells are the most affected cell type in hyperoxia (HO). NADPH oxidase (NOX) is a major source of reactive oxygen species (ROS) in HO. NOX4, the only functionally active NOX present in mitochondria, and primarily produces H2O2 as well as mtROS has been shown to be involved in several human pathologies. Not much is known about NOX4-induced mitochondrial injury in HALI. The current study aims to determine the role of AT2 epithelial cell NOX4 in HALI and the impact of HO on the modulation of mtROS and mitochondrial dynamics in HALI. Methods: Nox4-/-Spc-Cre animals were generated using tamoxifen induction and the knockdown was validated. The Nox4- /-Spc-Cre knockout (KO) and wild type (WT) mice were exposed to room air (NO) or 95% O2 (HO) for 66h to study the structural and functional changes in the lung. Transmission Electron Microscopy (TEM) was used to study the HO-induced changes in mitochondria. Isolated primary AT2 and/ mouse lung epithelial (MLE) cell line was investigated for mtROS, mt dynamics and apoptosis. Mitochondrial injury was assessed in Nox4 WT and Nox4 silenced cells. Results: C57BL/6J WT animals subjected to HO for 66h showed increased expression of NOX4, determining the role of NOX4 in HALI. The H&E staining demonstrated significant HALI in Nox4 WT animals exposed to HO compared to Nox4 KO as determined by increased infiltration of neutrophils, alveolar wall thickening and presence of proteinaceous debris in the alveolar space. Further, increased BAL cell count and protein levels, increased AT2 cell death and elevation of the proinflammatory cytokine IL- 6 and the chemokine KC was seen in WT animals compared to Nox4 KO. Analysis of lung tissues by TEM showed mitochondrial swelling, cristae damage and mitophagy in AT2 cells due to HO. Changes in mt injury markers were also observed. HO-induced NOX4 increase in primary AT2/ MLE-12 cells resulted in increased mtROS production and apoptosis, which was reduced with Nox4 siRNA silencing. Conclusion: This study suggests that the HO induced NOX4 expression in mouse lung, and deletion of Nox4 gene in AT2 cells reduced mtROS production and apoptosis and protected the lungs from severe hyperoxic lung injury. These results suggest NOX4 as a potential target for the treatment of HALI.
ABSTRACT
Background: Interaction between HIV and SARS-CoV-2 infection has not yet been fully characterized. To this purpose, an in-vitro HIV/SARS-CoV-2 coinfection assay was set up. Furthermore, the results obtained in the in-vitro model were verified in a cohort of HIV/SARS-CoV-2 coinfected young individuals. Methods: We designed an in-vitro SARS-CoV-2/HIV coinfection. We challenged PBMCs derived from 10 healthy volunteers with 1 ng/1×106 cells of HIV-1BaL and subsequently co-cultured them with a human lung epithelial cell line (CaLu3) infected with SARS-CoV-2 at 0.015 MOI. At 96 hours post HIV-1 infection, both PBMCs and CaLu3 cells were harvested for mRNA expression and proteomic analysis. Furthermore, we enrolled 85 ART-treated HIV-vertically transmitted patients (mean age 22.4 years) followed at the Unit of Pediatric Infectious Diseases, Sacco Hospital in Milan, Italy. Real-time PCR was performed to detect SARS-CoV-2 and plasma samples were tested for anti-SARS-CoV-2-specific IgG (Euroimmun Kit). The subjects who contracted SARS-CoV-2 infection (H+/S+) were compared to the HIV-positive, SARS-CoV-2 negative ones (H+/S-) and to a cohort of SARS-CoV-2 positive, HIV-negative age-matched patients (H-/S+, mean age 22.8 years). We evaluated mRNA expression of factors involved in the anti-viral immune response on PBMCs upon stimulation with SARS-CoV-2 antigens (Quantigene Plex assay) and secreted cytokines/chemokines on plasma (Multiplex Cytokine Array). Results: We observed a significant reduction of SARS-CoV-2 replication on CaLu3 cells when exposed to HIV-pre-infected PBMCs in-vitro. IL-10 expression and production were significantly higher in the coinfected condition, in both CaLu3 cells and PBMCs. The upregulation of IL-10 was associated to higher expression levels of STAT3. In the HIV-vertically transmitted cohort, 4 out of 85 subjects contracted SARS-CoV-2 infection (H+/S+). All H+/S+ patients were asymptomatic. Similarly to the data obtained in-vitro, a significant increase in both expression and production of IL-10 emerged in comparison to H+/S-and H-/S+. Conclusion: In-vitro, a dampening in SARS-CoV-2 replication, along with a higher IL-10 mRNA expression and production, have been observed in the HIV/SARS-CoV-2 coinfected condition. Presumably, IL-10 exerted its activity through the STAT3 pathway. These results were confirmed in HIV/SARS-CoV-2 coinfected subjects in which an upregulation of IL-10 was observed. Our data might be useful defining HIV/SARS-CoV-2 coinfected young individuals pathogenesis.
ABSTRACT
Background: Galectin-9 (Gal-9) is a β-galactoside-binding lectin involved in immune regulation and viral immunopathogenesis. Multiple recent reports demonstrate that plasma levels of Gal-9 are elevated in the setting of severe COVID-19 disease. However, a causal role of Gal-9 in SARS-CoV-2 pathology remains to be elucidated. Here, we determined the impact of Gal-9 on SARS-CoV-2 replication and pro-inflammatory signaling in immortalized and primary human airway epithelial cells (AECs). Methods: Dose-dependent cytotoxicity of recombinant human Gal-9 in the Calu-3 AEC line was determined by MTT assay. Calu-3 cells were infected with SARS-CoV-2 isolate USA-WA1/2020 (MOI=0.01). Primary AECs were isolated from healthy donor lung transplant tissue, cultured at air liquid interface (ALI), and infected with SARS-CoV-2 lineage P.1 (MOI=0.1). SARS-CoV-2 replication was assessed by RT-PCR quantitation of the nucleocapsid (N) gene, immunofluorescence assay (IFA) of N protein, and titration of supernatant (TCID50). Viral entry was measured using luciferase activity of VSV-SARS-CoV-2 S-ΔG-Luciferase reporter pseudovirus. ACE2 and TMPRSS2 cell-surface expression were measured by flow cytometry. Pro-inflammatory factors (IL-6, IL-8, and TNFα) were detected by RT-PCR. Total RNA-seq was used to evaluate Gal-9 effects on the host transcriptome. Groups were compared by Student's t-test, and differential expression analyses were performed using DESeq2. Results: Gal-9 reached 50% cytotoxicity in Calu-3 cells at 597 nM. Gal-9 significantly increased SARS-CoV-2 expression (8.1 to 25.5 fold;p<0.0001) and infectious virus release (1.9 to 17.8 fold;p<0.038) in a dose-dependent manner in Calu-3 cells. Pseudovirus entry into Calu-3 cells was enhanced by Gal-9 (2.4 to 5.6 fold;p<0.0016), and the enhanced entry was inhibited by anti-ACE2 antibody (p<0.0027). Cell surface ACE2 and TMPRSS2 expression were unaffected by Gal-9. Gal-9 treatment accelerated virus-induced expression of IL-6, IL-8, and TNFα (p<0.018) in Calu-3 cells. Gal-9 increased SARS-CoV-2 production (p=0.03) and pro-inflammatory factor expression (p<0.05) in primary AECs (N=5 donors). RNA-seq data revealed that Gal-9 significantly induced IL-17, EIF2, IL-8 and IL-6 signaling pathways in the setting of SARS-CoV-2 infection. Conclusion: Gal-9 facilitates SARS-CoV-2 entry, replication, and virus-induced pro-inflammatory signaling in AECs ex vivo. Our data suggest that pharmacologic manipulation of Gal-9 should be explored as a SARS-CoV-2 therapeutic strategy.
ABSTRACT
COVID-19 presents a unique threat to patients with lung cancer, with mortality rates as high as ∼32%. Given the convergence of these two deadly diseases, the lung cancer research and advocacy communities rapidly mobilized in early 2020 to create the COVID Lung Cancer Consortium (CLCC), a global assembly of leading experts in thoracic oncology, immunology, virology, vaccines and patient advocacy. With ongoing robust exchange of data and shared learning and rational planning for clinical and laboratory investigations, the CLCC is bringing its collective expertise to bear on beginning to address the question of why patients with lung cancer are at such elevated risk of worse outcomes from COVID-19. These efforts led to a recently funded U54 CA260560 grant as part of the National Cancer Institute's SeroNet initiative to study the magnitude, quality and duration of antibody responses to SARS-CoV-2 infection in patients with lung cancer compared to healthy controls. In the first project, our Mt. Sinai U54 Serological Center of Excellence will follow a prospective lung cancer cohort (750 patients) and a matched non-lung cancer control group (750 individuals) to determine if there are differences in antibody responses related to age, gender, tobacco history, and race/ethnicity. Given that effective SARS-CoV-2 vaccines are now being deployed, the study will also analyze antibody responses to vaccination across these two patient cohorts. The second project will examine biological determinants correlating with susceptibility to infection, including analysis of both ACE2 and TMPRSS2 levels, and antibody-mediated neutralization in pre-clinical models of established lung cancer and normal lung epithelial cell lines. In order to capture a diverse and inclusive patient population, this effort will be supported by GO2 Foundation for Lung Cancer through its national network of Centers of Excellence. This rapid global mobilization of the lung cancer community through the CLCC and the resulting Serological Center of Excellence is positioned to answer fundamental questions regarding the susceptibility of patients with lung cancer to SARS-CoV-2 infection and severe COVID-19 disease and provide information to allow assessment of the value of vaccination and the utility of specifically designed vaccine programs for this high-risk patient population.
ABSTRACT
Coronavirus disease 2019, caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), leads to a series of clinical symptoms of respiratory and pulmonary inflammatory reactions via unknown pathologic mechanisms related to the viral infection process in tracheal or bronchial epithelial cells. Investigation of this viral infection in the human bronchial epithelial cell line (16HBE) suggests that SARS-CoV-2 can enter these cells through interaction between its membrane-localized S protein with the angiotensin-converting enzyme 2 molecule on the host cell membrane. Further observation indicates distinct viral replication with a dynamic and moderate increase, whereby viral replication does not lead to a specific cytopathic effect but maintains a continuous release of progeny virions from infected cells. Although messenger RNA expression of various innate immune signaling molecules is altered in the cells, transcription of interferons-α (IFN-α), IFN-ß, and IFN-γ is unchanged. Furthermore, expression of some interleukins (IL) related to inflammatory reactions, such as IL-6, IL-2, and IL-8, is maintained at low levels, whereas that of ILs involved in immune regulation is upregulated. Interestingly, IL-22, an IL that functions mainly in tissue repair, shows very high expression. Collectively, these data suggest a distinct infection process for this virus in respiratory epithelial cells, which may be linked to its clinicopathological mechanism.