Inhibitors of the RBD-ACE-2 Found among a Wide Range of Dyes by the Immunoassay Method

Angiotensin-converting enzyme 2 (ACE2), the functional receptor of SARS-CoV-2, plays a crucial role in the pathogenesis of COVID-19. ACE2 targeting holds the promise for preventing and inhibiting SARS-CoV-2 infection. In this work, we describe the development and use of a test system based on competitive ELISA for the primary screening of potential antiviral compounds. We studied the activity of the library of dyes of different groups. Several dyes (ortho-cresolphthalein, eosin (free acid), eosin (Na salt)) that inhibited the interaction of ACE2 with the spike proteins of SARS-CoV-2 have been identified among the candidates. A potential antiviral drug, methylene blue, did not show activity in our study. We believe that our results can help in the further search for inhibitors of interaction between the coronavirus spike protein and ACE2 receptor.

Antiviral Immunoglobulins of Chicken Egg Yolk for Potential Prevention of SARS-CoV-2 Infection.

Background: Some viruses cause outbreaks, which require immediate attention. Neutralizing antibodies could be developed for viral outbreak management. However, the development of monoclonal antibodies is often long, laborious, and unprofitable. Here, we report the development of chicken polyclonal neutralizing antibodies against SARS-CoV-2 infection. Methods: Layers were immunized twice with 14-day intervals using the purified receptor-binding domain (RBD) of the S protein of SARS-CoV-2/Wuhan or SARS-CoV-2/Omicron. Eggs were harvested 14 days after the second immunization. Polyclonal IgY antibodies were extracted. Binding of anti-RBD IgYs was analyzed by immunoblot and indirect ELISA. Furthermore, the neutralization capacity of anti-RBD IgYs was measured in Vero-E6 cells infected with SARS-CoV-2-mCherry/Wuhan and SARS-CoV-2/Omicron using fluorescence and/or cell viability assays. In addition, the effect of IgYs on the expression of SARS-CoV-2 and host cytokine genes in the lungs of Syrian Golden hamsters was examined using qRT-PCR. Results: Anti-RBD IgYs efficiently bound viral RBDs in situ, neutralized the virus variants in vitro, and lowered viral RNA amplification, with minimal alteration of virus-mediated immune gene expression in vivo. Conclusions: Altogether, our results indicate that chicken polyclonal IgYs can be attractive targets for further pre-clinical and clinical development for the rapid management of outbreaks of emerging and re-emerging viruses.

MultiOMICs landscape of SARS-CoV-2-induced host responses in human lung epithelial cells (preprint)

Despite the availability of vaccines and approved therapeutics, the COVID-19 pandemic continues to rise owing to the emergence of newer variants. Several multi-omics studies have made available extensive evidence on host-pathogen interactions and potential therapeutic targets. Nonetheless, an increased understanding of host signaling networks regulated by post-translational modifications and their ensuing effect on the biochemical and cellular dynamics is critical to expanding the current knowledge on the host response to SARS-CoV-2 infections. Here, employing unbiased global transcriptomics, proteomics, acetylomics, phosphoproteomics, and exometabolome analysis of a lung-derived human cell line, we show that SARS-CoV-2 Norway/Trondheim-S15 strain induces time-dependent alterations in the induction of type I IFN response, activation of DNA damage response, dysregulated Hippo signaling, among others. We provide evidence for the interplay of phosphorylation and acetylation dynamics on host proteins and its effect on the altered release of metabolites, especially organic acids and ketone bodies. Together, our findings serve as a resource of potential targets that can aid in designing novel host-directed therapeutic strategies.

Analyzing Successful Aging and Longevity: Risk Factors and Health Promoters in 2020 Older Adults.

Geriatric syndromes (GSs) and aging-associated diseases (AADs) are common side effects of aging. They are affecting the lives of millions of older adults and placing immense pressure on healthcare systems and economies worldwide. It is imperative to study the factors causing these conditions and develop a holistic framework for their management. The so-called long-lived individuals-people over the age of 90 who managed to retain much of their health and functionality-could be holding the key to understanding these factors and their health implications. We analyzed the health status and lifestyle of the long-lived individuals and identified risk factors for GSs. Family history greatly contributes to the health and prevention of cognitive decline in older adults. Lifestyle and certain socioeconomic factors such as education, the age of starting to work and retiring, job type and income level, physical activity, and hobby were also associated with certain GSs. Moreover, the levels of total protein, albumin, alpha-1 globulins, high-density lipoprotein, free triiodothyronine, and 25-hydroxyvitamin D were direct indicators of the current health status. The proposed mathematical model allows the prediction of successful aging based on family history, social and economic factors, and life-long physical activity (f1 score = 0.72, AUC = 0.68, precision = 0.83 and recall = 0.64).

A live measles-vectored COVID-19 vaccine induces strong immunity and protection from SARS-CoV-2 challenge in mice and hamsters

Several COVID-19 vaccines have now been deployed to tackle the SARSCoV- 2 pandemic, most of them based on messenger RNA or adenovirus vectors. The duration of protection afforded by these vaccines is unknown, as well as their capacity to protect from emerging new variants. To provide sufficient coverage for the world population, additional strategies need to be tested. The live pediatric measles vaccine (MeV) is an attractive approach, given its extensive safety and efficacy history, along with its established large-scale manufacturing capacity. We develop an MeVbased SARS-CoV-2 vaccine expressing the prefusion-stabilized, membrane-anchored full-length S antigen, which proves to be efficient at eliciting strong Th1-dominant T-cell responses and high neutralizing antibody titers. In both mouse and golden Syrian hamster models, these responses protect the animals from intranasal infectious challenge. Additionally, the elicited antibodies efficiently neutralize in vitro the three currently circulating variants of SARS-CoV-2.

CT follow-up of COVID-19 pneumonia in patients requiring hospitalization in the acute phase of the disease.

OBJECTIVE: There are studies discussing the lung CT abnormalities persisting 3-4 months after COVID-19 pneumonia. Very few studies have evaluated the radiological changes in longer perspective, especially in patients with a severe form of the disease. The aim of our study was to show and qualify the persistent CT changes and to evaluate the dynamics of their evolution in convalescent patients after discharge from hospital post moderate and severe COVID-19 pneumonia. PATIENTS AND METHODS: We retrospectively analyzed the lung involvement at acute phase and follow-up time of 6 months in 6 patients with COVID-19 pneumonia using high resolution computer tomography. RESULTS: Radiological changes in the course of SARS-CoV-2 infection persisted in all patients 6 months after the first CT examination. CONCLUSIONS: Our study confirms that the period of 3 months is too short for the follow-up CT examination in patients recovered after severe or moderate COVID-19 pneumonia.

Mono- and combinational drug therapies for global viral pandemic preparedness

Summary Broadly effective antiviral therapies must be developed to be ready for clinical trials, which should begin soon after the emergence of new life-threatening viruses. Here, we pave the way towards this goal by reviewing conserved druggable virus-host interactions, mechanisms of action, immunomodulatory properties of available broad-spectrum antivirals (BSAs), routes of BSA delivery, and interactions of BSAs with other antivirals. Based on the review, we concluded that the range of indications of BSAs can be expanded, and new pan- and cross-viral mono- and combinational therapies can be developed. We have also developed a new scoring algorithm that can help identify the most promising few of the thousands of potential BSAs and BSA-containing drug cocktails (BCCs) to prioritize their development during the critical period between the identification of a new virus and the development of virus-specific vaccines, drugs, and therapeutic antibodies. Graphical Pharmaceutical preparation;Pharmaceutical science;Pharmacology;Chemistry

Broad-Spectrum Antivirals and Antiviral Drug Combinations.

Viral diseases consistently pose a substantial economic and public health burden worldwide [...].

Fast and Accurate Surrogate Virus Neutralization Test Based on Antibody-Mediated Blocking of the Interaction of ACE2 and SARS-CoV-2 Spike Protein RBD.

The humoral response to the SARS-CoV-2 S protein determines the development of protective immunity against this infection. The standard neutralizing antibodies detection method is a live virus neutralization test. It can be replaced with an ELISA-based surrogate virus neutralization test (sVNT), measuring the ability of serum antibodies to inhibit complex formation between the receptor-binding domain (RBD) of the S protein and the cellular ACE2 receptor. There are conflicting research data on the sVNT methodology and the reliability of its results. We show that the performance of sVNT dramatically improves when the intact RBD from the Wuhan-Hu-1 virus variant is used as the plate coating reagent, and the HRP-conjugated soluble ACE2 is used as the detection reagent. This design omits the pre-incubation step in separate tubes or separate microplate and allows the simple quantification of the results using the linear regression, utilizing only 3-4 test sample dilutions. When this sVNT was performed for 73 convalescent plasma samples, its results showed a very strong correlation with VNT (Spearman's Rho 0.83). For the RBD, bearing three amino acid substitutions and corresponding to the SARS-CoV-2 beta variant, the inhibitory strength was diminished for 18 out of 20 randomly chosen serum samples, and the magnitude of this decrease was not similar to the change in overall anti-RBD IgG level. The sVNT assay design with the ACE2-HRP is preferable over the assay with the RBD-HRP reagent and is suitable for mass screening of neutralizing antibodies titers.

Antigenic properties of the SARS-CoV-2 nucleoprotein are altered by the RNA admixture.

Determining the presence of antibodies to the SARS-CoV-2 antigens is the best way to identify infected people, regardless of the development of symptoms of COVID-19. The nucleoprotein (NP) of the SARS-CoV-2 is an immunodominant antigen of the virus; anti-NP antibodies are detected in persons previously infected with the virus with the highest titers. Many test systems for detecting antibodies to SARS-CoV-2 contain NP or its fragments as antigen. The sensitivity and specificity of such test systems differ significantly, which can be explained by variations in the antigenic properties of NP caused by differences in the methods of its cultivation, isolation and purification. We investigated this effect for the Escherichia coli-derived SARS-CoV-2 NP, obtained from the cytoplasm in the soluble form. We hypothesized that co-purified nucleic acids that form a strong complex with NP might negatively affect NP's antigenic properties. Therefore, we have established the NP purification method, which completely eliminates the RNA in the NP preparation. Two stages of RNA removal were used: treatment of the crude lysate of E. coli with RNase A and subsequent selective RNA elution with 2 M NaCl solution. The resulting NP without RNA has a significantly better signal-to-noise ratio when used as an ELISA antigen and tested with a control panel of serum samples with antibodies to SARS-CoV-2; therefore, it is preferable for in vitro diagnostic use. The same increase of the signal-to-noise ratio was detected for the free N-terminal domain of the NP. Complete removal of RNA complexed with NP during purification will significantly improve its antigenic properties, and the absence of RNA in NP preparations should be controlled during the production of this antigen.

Apport de la microscopie 2-photon (M2P) dans l’exploration des mécanismes immunitaires après infection par le virus de la grippe d’un modèle murin

Introduction Même si depuis 18 mois, le SARS-Cov2 a fait disparaître toute trace des virus respiratoires hivernaux historiques, les virus de la grippe, responsables de plusieurs pandémies durant le 20e siècle et d’une mortalité non négligeable, restent un sujet d’étude d’actualité. De nombreux travaux ont étudié la réaction immune innée post infection grippale mais sous un angle quantitatif en terme cellulaire et cytokinique. Le développement de technologie de microscopie moderne permettant d’étudier un organe entier en profondeur en conservant une excellente résolution mérite d’être évaluée. Peu de données sont disponibles sur l’intérêt de la M2P dans l’exploration du poumon et aucune sur son intérêt dans l’exploration des mécanismes immunitaires post infection grippale. Le but de ce travail est donc d’étudier l’intérêt de la M2P dans l’exploration des dommages structurales pulmonaires et du recrutement cellulaire après infection par le virus de la grippe d’un modèle murin. Méthodes Nous avons infecté des souris C57bl/6J génétiquement modifiées, dont les cellules CD11C+ expriment une protéine fluorescente jaune, par voie inhalée avec Influenzavirus (adapté à la souris) auquel la protéine NS1 a été couplée à une protéine fluorescente rouge. En plus de cette fluorescente endogène, nous avons utilisé un Ac anti F4/80 couplé à une protéine fluorescente bleue pour visualiser les macrophages. Nous avons ensuite étudié les poumons à jour 1, 2, 3, 4, 5 post infection en cytométrie en flux pour quantification cellulaire, dosage cytokinique et exploration en M2P. Nous avons développé un protocole d’imagerie M2P Intravital ex vivo pour évaluer le trafficking cellulaire. Résultats M2P a permis de mettre en évidence des dommages structuraux pulmonaires, de localiser les sites infectés, d’étudier les interactions cellulaires et d’étudier l’évolution dans le temps post infection. Ces résultats enrichissent et complètent les données quantitatives déjà publiées (cytométrie) et permettent d’étudier dans le temps la cinétique et la localisation des lésions et du recrutement cellulaire. De plus, le protocole d’imagerie Intravitale ex vivo a été mis au point. Conclusion En complément des analyses usuelles (cytométrie en flux, dosage cytokines…), M2P est donc une technologie pertinente et prometteuse dans les études précliniques en infectiologie respiratoire nous a permis de décrire une base physiopathologique de référence pour de futures études notamment d’impact des traitements anti-viraux ou anti-inflammatoires.

Synergistic Interferon-Alpha-Based Combinations for Treatment of SARS-CoV-2 and Other Viral Infections.

BACKGROUND: There is an urgent need for new antivirals with powerful therapeutic potential and tolerable side effects. METHODS: Here, we tested the antiviral properties of interferons (IFNs), alone and with other drugs in vitro. RESULTS: While IFNs alone were insufficient to completely abolish replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), IFNα, in combination with remdesivir, EIDD-2801, camostat, cycloheximide, or convalescent serum, proved to be more effective. Transcriptome and metabolomic analyses revealed that the IFNα-remdesivir combination suppressed SARS-CoV-2-mediated changes in Calu-3 cells and lung organoids, although it altered the homeostasis of uninfected cells and organoids. We also demonstrated that IFNα combinations with sofosbuvir, telaprevir, NITD008, ribavirin, pimodivir, or lamivudine were effective against HCV, HEV, FLuAV, or HIV at lower concentrations, compared to monotherapies. CONCLUSIONS: Altogether, our results indicated that IFNα can be combined with drugs that affect viral RNA transcription, protein synthesis, and processing to make synergistic combinations that can be attractive targets for further pre-clinical and clinical development against emerging and re-emerging viral infections.

Lung cavitation as a consequence of coronavirus-19 pneumonia.

OBJECTIVE: There are reports confirming that the development of pulmonary cavities is an atypical CT finding in patients after COVID-19 pneumonia. Before the SARS-Cov-2 pandemic, we knew that the most common causes of pulmonary cavities were mycobacterial, fungal or parasitic infections. Rapidly increasing incidence of pneumonia in the course of COVID-19, and thus, tomographic examinations of the lungs proved that one of the rare complications of this disease may also be cavity development. The aim of the study was to assess the incidence of pulmonary cavities in patients after SARS-CoV-2 pneumonia. We also aimed to analyze the changes accompanying the pulmonary cavities in our patients. PATIENTS AND METHODS: We performed a retrospective analysis of 206 lung CT scans of patients with SARS-CoV-2 infection. In 28 of them, prior radiological examination revealed the presence of pulmonary lesions - these patients were disqualified for the study. RESULTS: Out of 178 enrolled patients, 6 developed pulmonary cavities (3.37% of all cases). The most frequent changes coexisting with cavitary lesions in our material were: ground glass opacities, reticular pattern, bronchiolectasis and subpleural bands. CONCLUSIONS: Our study confirms the similar incidence of pulmonary cavities after COVID-19 than previously reported. It also incites the clinicians to pay attention to the possibility of the occurrence of this complication.

Nafamostat-Interferon-α Combination Suppresses SARS-CoV-2 Infection In Vitro and In Vivo by Cooperatively Targeting Host TMPRSS2.

SARS-CoV-2 and its vaccine/immune-escaping variants continue to pose a serious threat to public health due to a paucity of effective, rapidly deployable, and widely available treatments. Here, we address these challenges by combining Pegasys (IFNα) and nafamostat to effectively suppress SARS-CoV-2 infection in cell culture and hamsters. Our results indicate that Serpin E1 is an important mediator of the antiviral activity of IFNα and that both Serpin E1 and nafamostat can target the same cellular factor TMPRSS2, which plays a critical role in viral replication. The low doses of the drugs in combination may have several clinical advantages, including fewer adverse events and improved patient outcome. Thus, our study may provide a proactive solution for the ongoing pandemic and potential future coronavirus outbreaks, which is still urgently required in many parts of the world.

Nafamostat-interferon-alpha combination suppresses SARS-CoV-2 infection in vitro and in vivo (preprint)

SARS-CoV-2 and its vaccine/immune-escaping variants continue to pose a serious threat to public health due to a paucity of effective, rapidly deployable, and widely available treatments. Here we address these challenges by combining Pegasys (IFNa) and nafamostat to effectively suppress SARS-CoV-2 infection in cell culture and hamsters. Our results indicate that Serpin E1 is an important mediator of the antiviral activity of IFNa and that both Serpin E1 and camostat can target the same cellular factor TMPRSS2, which plays a critical role in viral replication. The low doses of the drugs in combination may have several clinical advantages, including fewer adverse events and improved patient outcome. Thus, our study may provide a proactive solution for the ongoing pandemic and potential future coronavirus outbreaks, which is still urgently required in many parts of the world.

Epitope-resolved profiling of the SARS-CoV-2 antibody response identifies cross-reactivity with endemic human coronaviruses.

The SARS-CoV-2 proteome shares regions of conservation with endemic human coronaviruses (CoVs), but it remains unknown to what extent these may be cross-recognized by the antibody response. Here, we study cross-reactivity using a highly multiplexed peptide assay (PepSeq) to generate an epitope-resolved view of IgG reactivity across all human CoVs in both COVID-19 convalescent and negative donors. PepSeq resolves epitopes across the SARS-CoV-2 Spike and Nucleocapsid proteins that are commonly targeted in convalescent donors, including several sites also recognized in some uninfected controls. By comparing patterns of homologous reactivity between CoVs and using targeted antibody-depletion experiments, we demonstrate that SARS-CoV-2 elicits antibodies that cross-recognize pandemic and endemic CoV antigens at two Spike S2 subunit epitopes. We further show that these cross-reactive antibodies preferentially bind endemic homologs. Our findings highlight sites at which the SARS-CoV-2 response appears to be shaped by previous CoV exposures and which have the potential to raise broadly neutralizing responses.

Why does SARS-CoV-2 survive longer on plastic than on paper?

The Covid-19 coronavirus, SARS-CoV-2, is inactivated much faster on paper (3 h) than on plastic (7 d). By classifying materials according to virus stability on their surface, the following list is obtained (from long to short stability): polypropylene (mask), plastic, glass, stainless steel, pig skin, cardboard, banknote, cotton, wood, paper, tissue, copper. These observations and other studies suggest that SARS-CoV-2 may be inactivated by dryness on water absorbent porous materials but sheltered by long-persisting micro-droplets of water on waterproof surfaces. If such physical phenomenons were confirmed by direct evidence, the persistence of the virus on any surface could be predicted, and new porous objects could be designed to eliminate the virus faster.

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

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.