Kinetics of interferon-λ and receptor expression in response to in vitro respiratory viral infection.

The major protective immune response against viruses is the production of type I and III interferons (IFNs). IFNs induce the expression of hundreds of IFN-stimulated genes (ISGs) that block viral replication and further viral spread. In this report, we analyzed the expression of IFNs and some ISGs (MxA, PKR, OAS-1, IFIT-1, RIG-1, MDA5, SOCS-1) in alveolar epithelial cells (A549) in response to infection with influenza A viruses (A/California/07/09 (H1N1pdm); A/Texas/50/12 (H3N2)); influenza B virus (B/Phuket/3073/13); adenovirus type 5 and 6; or respiratory syncytial virus (strain A2). Influenza B virus had the ability to most rapidly induce IFNs and ISGs as well as to stimulate excessive IFN-α, IFN-ß and IFN-λ secretion. It seems curious that IAV H1N1pdm did not induce IFN-λ secretion, but enhanced type I IFN and interleukin (IL)-6 production. We emphasized the importance of the negative regulation of virus-triggered signaling and cellular IFN response. We showed a decrease in IFNLR1 mRNA in the case of IBV infection. The attenuation of SOCS-1 expression in IAV H1N1pdm can be considered as the inability of the system to restore the immune status. Presumably, the lack of negative feedback loop regulation of proinflammatory immune response may be a factor contributing to the particular pathogenicity of several strains of influenza. Keywords: lambda interferons; MxA; influenza; respiratory syncytial virus; A549 cells.

Exosomes Released by Influenza-Virus-Infected Cells Carry Factors Capable of Suppressing Immune Defense Genes in Naïve Cells.

Background: Exosomes are involved in intercellular communication and can transfer regulatory molecules between cells. Consequently, they can participate in host immune response regulation. For the influenza A virus (IAV), there is very limited information on changes in exosome composition during cell infection shedding light on the potential role of these extracellular membrane vesicles. Thus, the aim of our work was to study changes in exosomal composition following IAV infection of cells, as well as to evaluate their effect on uninfected cells. Methods: To characterize changes in the composition of cellular miRNAs and mRNAs of exosomes during IAV infection of A549 cells, NGS was used, as well as PCR to identify viral genes. Naïve A549 cells were stimulated with infected-cell-secreted exosomes for studying their activity. Changes in the expression of genes associated with the cell's immune response were shown using PCR. The effect of exosomes on IAV replication was shown in MDCK cells using In-Cell ELISA and PCR of the supernatants. Results: A change in the miRNA composition (miR-21-3p, miR-26a-5p, miR-23a-5p, miR-548c-5p) and mRNA composition (RPL13A, MKNK2, TRIB3) of exosomes under the influence of the IAV was shown. Many RNAs were involved in the regulation of the immune response of the cell, mainly by suppressing it. After exosome stimulation of naïve cells, a significant decrease in the expression of genes involved in the immune response was shown (RIG1, IFIT1, MDA5, COX2, NFκB, AnxA1, PKR, IL6, IL18). When infecting MDCK cells, a significant decrease in nucleoprotein levels was observed in the presence of exosomes secreted by mock-infected cells. Viral levels in supernatants also decreased. Conclusions: Exosomes secreted by IAV-infected cells could reduce the immune response of neighboring intact cells, leading to more effective IAV replication. This may be associated both with regulatory functions of cellular miRNAs and mRNAs carried by exosomes, or with the presence of viral mRNAs encoding proteins with an immunosuppressive function.

Simultaneous Detection of RIG-1, MDA5, and IFIT-1 Expression Is a Convenient Tool for Evaluation of the Interferon-Mediated Response.

In this study, we developed a novel, multiplex qPCR assay for simultaneous detection of RIG-1, MDA5, and IFIT-1 at the mRNA level. The assay was validated in A549 cells transfected with in vitro transcribed RNAs. Both exogenous RNA-GFP and self-amplifying (saRNA-GFP) induced significant expression of RIG-1, MDA5, IFIT-1, as well as type I and III interferons. In contrast, native RNA from intact A549 cells did not upregulate expression of these genes. Next, we evaluated RIG-1, MDA5, and IFIT-1 mRNA levels in the white blood cells of patients with influenza A virus (H3N2) or SARS-CoV-2. In acute phase (about 4 days after disease onset) both viruses induced these genes expression. Clinical observations of SARS-CoV-2 typically describe a two-step disease progression, starting with a mild-to-moderate presentation followed by a secondary respiratory worsening 9 to 12 days after the first onset of symptoms. It revealed that the expression of RIG-1, MDA5, and MxA was not increased after 2 and 3 weeks from the onset the disease, while for IFIT-1 it was observed the second peak at 21 day post infection. It is well known that RIG-1, MDA5, and IFIT-1 expression is induced by the action of interferons. Due to the ability of SOCS-1 to inhibit interferon-dependent signaling, and the distinct antagonism of SARS-CoV-2 in relation to interferon-stimulated genes expression, we assessed SOCS-1 mRNA levels in white blood cells. SARS-CoV-2 patients had increased SOCS-1 expression, while the influenza-infected group did not differ from heathy donors. Moreover, SOCS-1 mRNA expression remained stably elevated during the course of the disease. It can be assumed that augmented SOCS-1 expression is one of multiple mechanisms that allow SARS-CoV-2 to escape from the interferon-mediated immune response. Our results implicate SOCS-1 involvement in the pathogenesis of SARS-CoV-2.

Influence of Lipid Composition of Cationic Liposomes 2X3-DOPE on mRNA Delivery into Eukaryotic Cells.

The design of cationic liposomes for efficient mRNA delivery can significantly improve mRNA-based therapies. Lipoplexes based on polycationic lipid 1,26-bis(cholest-5-en-3ß-yloxycarbonylamino)-7,11,16,20-tetraazahexacosane tetrahydrochloride (2X3) and helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) were formulated in different molar ratios (1:1, 1:2, 1:3) to efficiently deliver model mRNAs to BHK-21 and A549. The objective of this study was to examine the effect of 2X3-DOPE composition as well as lipid-to-mRNA ratio (amino-to-phosphate group ratio, N/P) on mRNA transfection. We found that lipoplex-mediated transfection efficiency depends on both liposome composition and the N/P ratio. Lipoplexes with an N/P ratio of 10/1 showed nanometric hydrodynamic size, positive ζ potential, maximum loading, and transfection efficiency. Liposomes 2X3-DOPE (1:3) provided the superior delivery of both mRNA coding firefly luciferase and mRNA-eGFP into BHK-21 cells and A549 cells, compared with commercial Lipofectamine MessengerMax.

Peripheral Blood Mononuclear Cell Cytokine mRNA Profiles in Acute Respiratory Infection Patients.

Introduction: Respiratory infections, collectively, are one of the World's most common and serious illness groups. As recent observations have shown, the most severe courses of acute respiratory infection, often leading to death, are due to uncontrolled cytokine production (hypercytokinemia). Methods: The study involved 364 patients with respiratory illness being treated in clinics in St. Petersburg (Russia) in 2018-2019 and 30 healthy controls. Cytokine analysis was carried out in the acute phase of illness (2-3 days from onset of initial symptoms) and in the stage of recovery (days 9-10). The research presented is devoted to the assessment of mRNA expression of specific cytokines (interleukin [IL]-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-18, tumor necrosis factor-α [TNF-α], and interferon-λ) and MxA in whole blood leukocytes, by means of real-time polymerase chain reaction. Results: In 70% of patients, bacterial or viral pathogens were identified, with influenza viral infections (types A and B) prevailing. Significant increases in the expression of IL-18, TNF, and IL-10 were observed, relative to controls, only with influenza viral infections. We have shown a difference in IL-6 mRNA expression in patients with bacterial or viral pathogens. No statistically significant difference was found in white blood cells IL-4 expression levels between patients and healthy controls. Conclusion: Investigation of the nuances of systemic cytokine production, in response to specific viral and bacterial pathogens, makes it possible to assess the risks of developing hypercytokinemia during respiratory infection with agents circulating in the human population and to predict the pathogenicity and virulence of circulating threats.

Clean and folded: Production of active, high quality recombinant human interferon-λ1.

Type III interferons exhibit antiviral activity against influenza viruses, coronaviruses, rotaviruses, and others. In addition, this type of interferon theoretically has therapeutic advantages, in comparison with type I interferons, due to its ability to activate a narrower group of genes in a relatively small group of target cells. Hence, it can elicit more targeted antiviral or immunomodulatory responses. Obtaining biologically-active interferon lambda (hIFN-λ1) is fraught with difficulties at the stage of expression in soluble form or, in the case of expression in the form of inclusion bodies, at the stage of refolding. In this work, hIFN-λ1 was expressed in the form of inclusion bodies, and a simple, effective refolding method was developed. Efficient and scalable methods for chromatographic purification of recombinant hIFN-λ1 were also developed. High-yield, high-purity product was obtained through optimization of several processes including: recombinant protein expression; metal affinity chromatography; cation exchange chromatography; and an intermediate protein refolding stage. The obtained protein was shown to feature expected specific biological activity in line with published effects: induction of MxA gene expression in A549 cells and antiviral activity against influenza A virus.

IFN-λ1 Displays Various Levels of Antiviral Activity In Vitro in a Select Panel of RNA Viruses.

Type III interferons (lambda IFNs) are a quite new, small family of three closely related cytokines with interferon-like activity. Attention to IFN-λ is mainly focused on direct antiviral activity in which, as with IFN-α, viral genome replication is inhibited without the participation of immune system cells. The heterodimeric receptor for lambda interferons is exposed mainly on epithelial cells, which limits its possible action on other cells, thus reducing the likelihood of developing undesirable side effects compared to type I IFN. In this study, we examined the antiviral potential of exogenous human IFN-λ1 in cellular models of viral infection. To study the protective effects of IFN-λ1, three administration schemes were used: 'preventive' (pretreatment); 'preventive/therapeutic' (pre/post); and 'therapeutic' (post). Three IFN-λ1 concentrations (from 10 to 500 ng/mL) were used. We have shown that human IFN-λ1 restricts SARS-CoV-2 replication in Vero cells with all three treatment schemes. In addition, we have shown a decrease in the viral loads of CHIKV and IVA with the 'preventive' and 'preventive/therapeutic' regimes. No significant antiviral effect of IFN-λ1 against AdV was detected. Our study highlights the potential for using IFN-λ as a broad-spectrum therapeutic agent against respiratory RNA viruses.

Rosin Derivatives as a Platform for the Antiviral Drug Design.

The increased complexity due to the emergence and rapid spread of new viral infections prompts researchers to search for potential antiviral and protective agents for mucous membranes among various natural objects, for example, plant raw materials, their individual components, as well as the products of their chemical modification. Due to their structure, resin acids are valuable raw materials of natural origin to synthesize various bioactive substances. Therefore, the purpose of this study was to confirm the possibility of using resin acid derivatives for the drug design. As a result, we studied the cytotoxicity and biological activity of resin acid derivatives. It was shown that a slight decrease in the viral load in the supernatants was observed upon stimulation of cells (II) compared with the control. When using PASS-online modeling (Prediction of Activity Spectra for Substances), the prediction of the biological activity spectrum showed that compound (I) is capable of exhibiting antiviral activity against the influenza virus. The use of the SWISS-ADME webserver to reveal the drug-like properties of compounds did not directly indicate the presence of antiviral activity. These results indicate the potential of resin acid derivatives as a starting point for extensive research in the study of biological activity.

Antibody microarray immunoassay for screening and differential diagnosis of upper respiratory tract viral pathogens.

Upper respiratory tract infections are the world's most common infectious disease. The etiologic agents behind upper respiratory tract infections (URTIs) are, in fact, a diverse set of pathogens such as influenza, parainfluenza, adenovirus, rhinovirus, and others. More than 200 pathogens are known to be involved. Differential diagnosis of viral infections is sometimes complicated by their diversity or similarity of clinical presentation. This work is devoted to the development of a method which enables simultaneous detection of six common viral URTI pathogens: IAV; IBV; RSV; hAdV; hPIV2; and hPIV3. Antibody microarray technology is utilized to accomplish the analysis. In preparation for protein microchip creation, we produced, characterized, and selected approximately 50 monoclonal antibodies; for each of the aforementioned pathogens, an optimal monoclonal antibody pair was selected. A protein microchip was created, and its core working conditions were optimized. With a balance between convenience and maximal assay sensitivity in mind, a one-step analysis approach was developed for accomplishing the ELISA-like "sandwich" interaction on the manufactured microchip (antibody microarray). Reference viral strains were used to establish the lower limits of detection (LoD) for the assay. For IAV, the LoD was 0.25 ng/ml total viral protein. For other viruses, the LoD ranged from 1 to 2 ng/ml total protein. These sensitivity limits are slightly better than those of standard ELISA, but inferior to those of PCR. Overall, we believe that the developed microchip is a good alternative to existing methods, allowing relatively quick (overnight), inexpensive, simultaneous screening of several pathogens. The design of the antibody microarray is conducive to further development, and the panel of analyzed pathogens can be expanded to include approximately 50 members.

Meglumine acridone acetate, the ionic salt of CMA and N-methylglucamine, induces apoptosis in human PBMCs via the mitochondrial pathway.

Meglumine acridone acetate (MA) is used in Russia for the treatment of influenza and other acute respiratory viral infections. It was assumed, until recently, that its antiviral effect was associated with its potential ability to induce type I interferon. Advanced studies, however, have shown the failure of 10-carboxymethyl-9-acridanone (CMA) to activate human STING. As such, MA's antiviral properties are still undergoing clarification. To gain insight into MA's mechanisms of action, we carried out RNA-sequencing analysis of global transcriptomes in MA-treated (MA+) human peripheral blood mononuclear cells (PBMCs). In response to treatment, approximately 1,223 genes were found to be differentially expressed, among which 464 and 759 were identified as either up- or down-regulated, respectively. To clarify the cellular and molecular processes taking place in MA+ cells, we performed a functional analysis of those genes. We have shown that evident MA subcellular localizations are: at the nuclear envelope; inside the nucleus; and diffusely in perinuclear cytoplasm. Postulating that MA may be a nuclear receptor agonist, we carried out docking simulations with PPARα and RORα ligand binding domains including prediction and molecular dynamics-based analysis of potential MA binding poses. Finally, we confirmed that MA treatment enhanced nuclear apoptosis in human PBMCs. The research presented here, in our view, indicates that: (i) MA activity is mediated by nuclear receptors; (ii) MA is a possible PPARα and/or RORα agonist; (iii) MA has an immunosuppressive effect; and (iv) MA induces apoptosis through the mitochondrial signaling pathway.

Development of a multiplex quantitative PCR assay for the analysis of human cytokine gene expression in influenza A virus-infected cells.

Cytokines are global mediators of cellular communications that are involved in broad array of biological processes, including the immunological and inflammatory mechanisms of virus-host interactions. Measuring the gene expression of simultaneously expressed cytokines is necessary for understanding the pathogenesis of many viral infections, including influenza. We developed a multiplex quantitative real-time PCR (qPCR) method for the detection of the following human cytokines: IL-1B, IL-2, IL-4, IL-6, IL-10, IL-12B, IL-18, IFN-γ and TNF. The assay consisted of three sets of multiple qPCRs; in each qPCR, three target cytokines and reference GAPDH genes were amplified. The assay provided a precise and sensitive quantification of cytokine gene expression with a 20fmol limit of detection and a 1.5% coefficient of variation. This method was successfully applied to cytokine profiling in epithelial A549 cells that were infected with A/California/07/09 (H1N1pdm2009) virus.