Synergistic Interferon Alpha-based Drug Combinations Inhibit SARS-CoV-2 and other viral Infections in Vitro

Abstract There is an urgent need for new antivirals with powerful therapeutic potential and tolerable side effects. In the present study, we found that recombinant human interferon-alpha (IFNa) triggered cell intrinsic and extrinsic antiviral responses and reduced replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human lung epithelial Calu-3 cells. However, IFNa alone was insufficient to completely abolish SARS-CoV-2 replication. Combinations of IFNa with camostat, remdesivir, EIDD-2801, cycloheximide or convalescent serum showed strong synergy and effectively inhibited SARS-CoV-2 infection. Additionally, we demonstrated synergistic antiviral activity of IFNa2a with pimodivir against influenza A virus (FluAV) infection in human lung epithelial A549 cells, as well as of IFNa2a with lamivudine against human immunodeficiency virus 1 (HIV-1) infection in human TZM-bl cells. Our results indicate that IFNa2a-based combinational therapies help to reduce drug dose and improve efficacy in comparison with monotherapies, making them attractive targets for further pre-clinical and clinical development.

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Synergistic interferon alpha-based drug combinations inhibit SARS-CoV-2 and other viral infections in vitro

10.1101/2021.01.05.425331 ◽

2021 ◽

Author(s):

Aleksandr Ianevski ◽

Rouan Yao ◽

Eva Zusinaite ◽

Laura Sandra Lello ◽

Sainan Wang ◽

...

Keyword(s):

Interferon Alpha ◽

Influenza A ◽

Viral Infections ◽

Human Lung ◽

A549 Cells ◽

Drug Dose ◽

Human Interferon ◽

Emerging Viruses ◽

Lung Epithelial

AbstractAntiviral drugs are powerful tools to combat emerging viral diseases, one of the leading causes of morbidity and mortality in the world. However, most existing antivirals have failed to cure COVID-19. Accordingly, there is an urgent need for new therapeutics with powerful antiviral and tolerable side effects. Here, we observed that recombinant human interferon-alpha (IFNa) triggered cell intrinsic and extrinsic antiviral responses and reduced replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in human lung epithelial Calu-3 cells. However, IFNa alone was insufficient to completely abolish SARS-CoV-2 replication. The combinations of IFNa with camostat, remdesivir, EIDD-2801, cycloheximide or convalescent serum showed strong synergy and, therefore, effectively inhibited SARS-CoV-2 infection. Additionally, we demonstrated synergistic antiviral activity of IFNa2a with pimodivir against influenza A virus (FluAV) infection in human lung epithelial A549 cells, as well as IFNa2a with lamivudine against human immunodeficiency virus 1 (HIV-1) infection in human TZM-bl cells. Our results indicate that IFNa2a-based combinational therapies help to reduce drug dose and improve efficacy in comparison with monotherapies, making them attractive targets for further pre-clinical and clinical development. Additionally, they have powerful treatment potential, and can be leveraged for use in the inhibition of not only emerging or re-emerging viruses, but also immune-evading or drug-resistant viral variants, and viral co-infections.

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Identification of novel antiviral drug combinations in vitro and tracking their development

10.1101/2020.09.17.299933 ◽

2020 ◽

Author(s):

Aleksandr Ianevski ◽

Rouan Yao ◽

Svetlana Biza ◽

Eva Zusinaite ◽

Andres Männik ◽

...

Keyword(s):

Viral Infections ◽

Human Lung ◽

Antiviral Drug ◽

A549 Cells ◽

Drug Combinations ◽

Investigational Drug ◽

Synergistic Activity ◽

Lung Epithelial ◽

Reduced Toxicity

AbstractCombination therapies have become a standard for the treatment for HIV and HCV infections. They are advantageous over monotherapies due to better efficacy and reduced toxicity, as well as the ability to prevent the development of resistant viral strains and to treat viral co-infections. Here, we identify several new synergistic combinations against emerging and re-emerging viral infections in vitro. We observed synergistic activity of nelfinavir with investigational drug EIDD-2801 and convalescent serum against SARS-CoV-2 infection in human lung epithelial Calu-3 cells. We also demonstrated synergistic activity of vemurafenib combination with emetine, homoharringtonine, gemcitabine, or obatoclax against echovirus 1 infection in human lung epithelial A549 cells. We also found that combinations of sofosbuvir with brequinar and niclosamide were synergistic against HCV infection in hepatocyte derived Huh-7.5 cells, whereas combinations of monensin with lamivudine and tenofovir were synergistic against HIV-1 infection in human cervical TZM-bl cells. Finally, we present an online resource that summarizes novel and known antiviral drug combinations and their developmental status. Overall, the development of combinational therapies could have a global impact improving the preparedness and protection of the general population from emerging and re-emerging viral threats.

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Lysosome-associated membrane glycoprotein 3 is involved in influenza A virus replication in human lung epithelial (A549) cells

Virology Journal ◽

10.1186/1743-422x-8-384 ◽

2011 ◽

Vol 8 (1) ◽

pp. 384 ◽

Cited By ~ 19

Author(s):

Zhuo Zhou ◽

Qinghua Xue ◽

Yuli Wan ◽

Yaowu Yang ◽

Jianwei Wang ◽

...

Keyword(s):

Influenza A Virus ◽

Virus Replication ◽

Influenza A ◽

Human Lung ◽

A549 Cells ◽

Membrane Glycoprotein ◽

Lung Epithelial

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Cell culture model system utilizing engineered A549 cells to express high levels of ACE2 and TMPRSS2 for investigating SARS-CoV-2 infection and antivirals

10.1101/2021.12.31.474593 ◽

2022 ◽

Author(s):

Ching-Wen Chang ◽

Krishna Mohan Parsi ◽

Mohan Somasundaran ◽

Emma Vanderleeden ◽

John Cruz ◽

...

Keyword(s):

Epithelial Cell ◽

Virus Infection ◽

Viral Entry ◽

Human Lung ◽

A549 Cells ◽

Model System ◽

Lung Epithelial Cell ◽

Clinical Feature ◽

Lung Epithelial

Novel pathogenic severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to pose an imminent global threat since its initial outbreak in December 2019. A simple in vitro model system using cell lines highly susceptible to SARS-CoV-2 infection are critical to facilitate the study of the virus cycle and to discover effective antivirals against the virus. Human lung alveolar A549 cells are regarded as a useful and valuable model for respiratory virus infection. However, SARS-CoV-2 uses the ACE2 as receptor for viral entry and the TMPRSS2 to prime the Spike protein, both of which are negligibly expressed in A549 cells. Here, we report the generation of a robust human lung epithelial cell-based model by transducing ACE2 and TMPRSS2 into A549 cells and show that the ACE2 enriched A549ACE2/TMPRSS2 cells (ACE2plus) and its single-cell-derived subclone (ACE2plusC3) are highly susceptible to SARS-CoV-2 infection. These engineered ACE2plus showed higher ACE2 and TMPRSS2 mRNA expression levels than currently used Calu3 and commercial A549ACE2/TMPRSS2 cells. ACE2 and TMPRSS2 proteins were also highly and ubiquitously expressed in ACE2plusC3 cells. Additionally, antiviral drugs like Camostat mesylate, EIDD-1931, and Remdesivir strongly inhibited SARS-CoV-2 replication. Notably, multinucleated syncytia, a clinical feature commonly observed in severe COVID-19 patients was induced in ACE2plusC3 cells either by virus infection or by overexpressing the Spike proteins of different variants of SARS-CoV-2. Syncytial process was effectively blocked by the furin protease inhibitor, Decanoyl-RVKR-CMK. Taken together, we have developed a robust human A549 lung epithelial cell-based model that can be applied to probe SARS-CoV-2 replication and to facilitate the discovery of SARS-CoV-2 inhibitors.

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Identification and Tracking of Antiviral Drug Combinations

Viruses ◽

10.3390/v12101178 ◽

2020 ◽

Vol 12 (10) ◽

pp. 1178 ◽

Cited By ~ 4

Author(s):

Aleksandr Ianevski ◽

Rouan Yao ◽

Svetlana Biza ◽

Eva Zusinaite ◽

Andres Mannik ◽

...

Keyword(s):

Hepatitis C Virus ◽

Hepatitis C ◽

Human Lung ◽

Antiviral Drug ◽

A549 Cells ◽

Drug Combinations ◽

Synergistic Activity ◽

Lung Epithelial ◽

Hiv 1

Combination therapies have become a standard for the treatment for HIV and hepatitis C virus (HCV) infections. They are advantageous over monotherapies due to better efficacy, reduced toxicity, as well as the ability to prevent the development of resistant viral strains and to treat viral co-infections. Here, we identify new synergistic combinations against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), echovirus 1 (EV1), hepatitis C virus (HCV) and human immunodeficiency virus 1 (HIV-1) in vitro. We observed synergistic activity of nelfinavir with convalescent serum and with purified neutralizing antibody 23G7 against SARS-CoV-2 in human lung epithelial Calu-3 cells. We also demonstrated synergistic activity of nelfinavir with EIDD-2801 or remdesivir in Calu-3 cells. In addition, we showed synergistic activity of vemurafenib with emetine, homoharringtonine, anisomycin, or cycloheximide against EV1 infection in human lung epithelial A549 cells. We also found that combinations of sofosbuvir with brequinar or niclosamide are synergistic against HCV infection in hepatocyte-derived Huh-7.5 cells, and that combinations of monensin with lamivudine or tenofovir are synergistic against HIV-1 infection in human cervical TZM-bl cells. These results indicate that synergy is achieved when a virus-directed antiviral is combined with another virus- or host-directed agent. Finally, we present an online resource that summarizes novel and known antiviral drug combinations and their developmental status.

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In vitro reassortment and adaptation of influenza A viruses circulating in swine

Access Microbiology ◽

10.1099/acmi.ac2020.po0828 ◽

2020 ◽

Vol 2 (7A) ◽

Author(s):

VerÓnica Ferrando ◽

Yvonne Börgeling ◽

Alexander Mellmann ◽

Shrey Gandhi ◽

Linda Brunotte ◽

...

Keyword(s):

Lung Tissue ◽

Influenza A ◽

Human Lung ◽

Cell Clone ◽

Ex Vivo ◽

Influenza Pandemic ◽

A549 Cells ◽

Human Lung Tissue ◽

Zoonotic Risk

Since the last influenza pandemic in 2009, H1N1pdm has been introduced into the swine population in Europe where, in combination with swine influenza A virus (IAV) lineages, it started to generate a variety of reassortant viruses of unknown zoonotic risk for humans. To study these reassortment events, we isolated a wild swine lung cell clone (C22) susceptible to IAV infection. We established conditions for co-infection and passaging of H1N1pdm and swine avian-like H1N1. After 7 passages, we plaque-purified C22-adapted strains, characterized their genome composition by next-generation sequencing and analysed replication abilities in swine and human lung cell lines as well as in human lung tissue ex vivo. Among C22-adapted viruses isolated from co-infection, we revealed reassortants carrying PB1/PA/NA or only PB1/PA from H1N1pdm. We also detected exclusively swine H1N1-derived strains. All isolates carried distinct mutations. As expected, adapted viruses reached higher titers compared to both parental strains in swine lung cells. Furthermore, all C22-adapted viruses were able to replicate in human lung A549 cells without any prior adaptation to the human host. Strikingly, all reassortants were able to infect and efficiently replicate in human lung tissue ex vivo, indicating that these viruses might pose a zoonotic risk. To summarize, we successfully established an in vitro swine-like model to study reassortment and adaptation of IAVs currently circulating in swine. Our results indicate that our model might be a useful tool to prospectively evaluate the compatibility of different IAV strains to generate reassortants, which might represent a threat to the human population.

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Comparative study of the effect of a herbal preparation (Imupret®) dissolved in different solvents on LPS-primed type II-like human lung epithelial A549 cells

Planta Medica ◽

10.1055/s-0028-1084594 ◽

2008 ◽

Vol 74 (09) ◽

Author(s):

K Hostanska ◽

J Melzer ◽

A Amon ◽

R Saller

Keyword(s):

Comparative Study ◽

Human Lung ◽

A549 Cells ◽

Type Ii ◽

Herbal Preparation ◽

Lung Epithelial

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Copper(II) and Zinc(II) Complexes with the Clinically Used Fluconazole: Comparison of Antifungal Activity and Therapeutic Potential

Pharmaceuticals ◽

10.3390/ph14010024 ◽

2020 ◽

Vol 14 (1) ◽

pp. 24

Author(s):

Nevena Lj. Stevanović ◽

Ivana Aleksic ◽

Jakob Kljun ◽

Sanja Skaro Bogojevic ◽

Aleksandar Veselinovic ◽

...

Keyword(s):

Antifungal Activity ◽

Biofilm Formation ◽

Candida Parapsilosis ◽

Therapeutic Potential ◽

A549 Cells ◽

Candida Krusei ◽

Strong Inhibition ◽

Subinhibitory Concentrations ◽

Pyocyanin Production

Copper(II) and zinc(II) complexes with clinically used antifungal drug fluconazole (fcz), {[CuCl2(fcz)2].5H2O}n, 1, and {[ZnCl2(fcz)2]·2C2H5OH}n, 2, were prepared and characterized by spectroscopic and crystallographic methods. The polymeric structure of the complexes comprises four fluconazole molecules monodentately coordinated via the triazole nitrogen and two chlorido ligands. With respect to fluconazole, complex 2 showed significantly higher antifungal activity against Candida krusei and Candida parapsilosis. All tested compounds reduced the total amount of ergosterol at subinhibitory concentrations, indicating that the mode of activity of fluconazole was retained within the complexes, which was corroborated via molecular docking with cytochrome P450 sterol 14α-demethylase (CYP51) as a target. Electrostatic, steric and internal energy interactions between the complexes and enzyme showed that 2 has higher binding potency to this target. Both complexes showed strong inhibition of C. albicans filamentation and biofilm formation at subinhibitory concentrations, with 2 being able to reduce the adherence of C. albicans to A549 cells in vitro. Complex 2 was able to reduce pyocyanin production in Pseudomonas aeruginosa between 10% and 25% and to inhibit its biofilm formation by 20% in comparison to the untreated control. These results suggest that complex 2 may be further examined in the mixed Candida-P. aeruginosa infections.

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Comparative Toxic Effects of Manufactured Nanoparticles and Atmospheric Particulate Matter in Human Lung Epithelial Cells

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph18010022 ◽

2020 ◽

Vol 18 (1) ◽

pp. 22

Author(s):

Yun Wu ◽

Mei Wang ◽

Shaojuan Luo ◽

Yunfeng Gu ◽

Dongyang Nie ◽

...

Keyword(s):

Particulate Matter ◽

Human Lung ◽

A549 Cells ◽

Lung Epithelial Cells ◽

Atmospheric Particulate Matter ◽

Toxic Effects ◽

Ros Generation ◽

Atmospheric Particulate ◽

Significant Difference ◽

Lung Epithelial

Although nanoparticles (NPs) have been used as simplified atmospheric particulate matter (PM) models, little experimental evidence is available to support such simulations. In this study, we comparatively assessed the toxic effects of PM and typical NPs (four carbonaceous NPs with different morphologies, metal NPs of Fe, Al, and Ti, as well as SiO2 NPs) on human lung epithelial A549 cells. The EC50 value of PM evaluated by cell viability assay was 148.7 μg/mL, closest to that of SiO2 NPs, between the values of carbonaceous NPs and metal NPs. All particles caused varying degrees of reactive oxygen species (ROS) generation and adenosine triphosphate (ATP) suppression. TiO2 NPs showed similar performance with PM in inducing ROS production (p < 0.05). Small variations between two carbonaceous NPs (graphene oxides and graphenes) and PM were also observed at 50 μg/mL. Similarly, there was no significant difference in ATP inhibition between carbonaceous NPs and PM, while markedly different effects were caused by SiO2 NP and TiO2 NP exposure. Our results indicated that carbonaceous NPs could be served as potential surrogates for urban PM. The identification of PM model may help us further explore the specific roles and mechanisms of various components in PM.

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