Ubiquitin variants potently inhibit SARS-CoV-2 PLpro and viral replication via a novel site distal to the protease active site.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has made it clear that combating coronavirus outbreaks benefits from a combination of vaccines and therapeutics. A promising drug target common to all coronaviruses-including SARS-CoV, MERS-CoV, and SARS-CoV-2-is the papain-like protease (PLpro). PLpro cleaves part of the viral replicase polyproteins into non-structural protein subunits, which are essential to the viral replication cycle. Additionally, PLpro can cleave both ubiquitin and the ubiquitin-like protein ISG15 from host cell substrates as a mechanism to evade innate immune responses during infection. These roles make PLpro an attractive antiviral drug target. Here we demonstrate that ubiquitin variants (UbVs) can be selected from a phage-displayed library and used to specifically and potently block SARS-CoV-2 PLpro activity. A crystal structure of SARS-CoV-2 PLpro in complex with a representative UbV reveals a dimeric UbV bound to PLpro at a site distal to the catalytic site. Yet, the UbV inhibits the essential cleavage activities of the protease in vitro and in cells, and it reduces viral replication in cell culture by almost five orders of magnitude.

Evaluation of Virtual Classes Teaching among first-year MBBS students during COVID-19 era in GMC Jammu

Background: The Covid-19 era has caused the shifting of medical teaching into an Online podium. This has provided us with an excellent opportunity to recognize MBBS-students’ preferences of these Online classes which otherwise did not form a significant part of the traditional medical teaching. We have undertaken online classes for theory & practical for the first time in our medical college at GMC Jammu. So the current study was conducted to assess & analyze the preference of first-year MBBS students between online classes & traditional classroom teaching. Objectives: To evaluate the predilection/inclination of first-year MBBS students between online classes and traditional classroom teaching. Materials & Methods: An institution-based descriptive cross-sectional study was performed on 180 MBBS students of 2020-21 batch at the Department of Physiology in GMC Jammu, after receiving Institutional ethics committee (IEC) clearance who have attended online classes for a minimum of 1 hour per day or 6 hours per week for 1-month duration using zoom or other video-based learning platforms. A pre-structured questionnaire-based feedback study was conducted after obtaining written consent from the first-year MBBS students. We designed a total of 24 questions related to their theory & practical classes, for assessing their preference for Online classes in comparison to traditional classroom teaching with multiple-choice options. Results: In our study, we found that overall, 61.11% of MBBS students prefer traditional classroom teaching over Online classes and 60% of students prefer traditional teaching methods over online learning for practical classes. However, 50% of students reported that accessibility to stable internet was causing difficulty in following Online classes. Moreover, 53.89% of students reported that they get distracted at home during Online classes. Further, 66.11% of students mentioned a lack of personal interaction & lonely experience on the Online platform. Conclusion: In our study, we found that medical students prefer the traditional teaching method to the Online platform for learning as well as examination. For better understanding and learning, the first phase of MBBS students prefers that traditional classroom teaching should be followed by Online classes that can be easily accessed by students at their convenient time. In this study, the majority of medical students reported limited access to stable internet, distractions & disturbances in the domestic environment, & lack of personal interaction as the barriers to Online learning.

Monoclonal antibodies binding data for SARS-CoV-2 proteins.

SARS-CoV-2 pandemic opens up the curiosity of understanding the coronavirus. This demand for the development of the regent, which can be used for academic and therapeutic applications. The present data provide the biochemical characterization of synthetically developed monoclonal antibodies for the SARS-CoV-2 proteins. The antibodies from phage-displayed antibody libraries were selected with the SARS-CoV-2 proteins immobilized in microwell plates. The clones which bind to the antigen in Fab-phage ELISA were selected, and a two-point competitive phage ELISA was performed. Antibodies binding kinetic of IgGs for SARS-CoV2 proteins further carried with B.L.I. Systematic analysis of binding with different control proteins and purified SARS-CoV-2 ensured the robustness of the antibodies.

Peptide-Antibody Fusions Engineered by Phage Display Exhibit an Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants.

The spread of COVID-19 has been exacerbated by the emergence of variants of concern (VoC). Many VoC contain mutations in the spike protein (S-protein) and are implicated in infection and response to therapeutics. Bivalent neutralizing antibodies (nAbs) targeting the S-protein receptor-binding domain (RBD) are promising therapeutics for COVID-19, but they are limited by low potency and vulnerability to RBD mutations in VoC. To address these issues, we used naïve phage-displayed peptide libraries to isolate and optimize 16-residue peptides that bind to the RBD or the N-terminal domain (NTD) of the S-protein. We fused these peptides to the N-terminus of a moderate-affinity nAb to generate tetravalent peptide-IgG fusions, and we showed that both classes of peptides were able to improve affinities for the S-protein trimer by >100-fold (apparent KD < 1 pM). Critically, cell-based infection assays with a panel of six SARS-CoV-2 variants demonstrated that an RBD-binding peptide was able to enhance the neutralization potency of a high-affinity nAb >100-fold. Moreover, this peptide-IgG was able to neutralize variants that were resistant to the same nAb in the bivalent IgG format, including the dominant B.1.1.529 (Omicron) variant that is resistant to most clinically approved therapeutic nAbs. To show that this approach is general, we fused the same peptide to a clinically approved nAb drug and showed that it enabled the neutralization of a resistant variant. Taken together, these results establish minimal peptide fusions as a modular means to greatly enhance affinities, potencies, and breadth of coverage of nAbs as therapeutics for SARS-CoV-2.

Ultrapotent and broad neutralization of SARS-CoV-2 variants by modular, tetravalent, bi-paratopic antibodies.

Neutralizing antibodies (nAbs) that target the SARS-CoV-2 spike protein have received emergency use approval for treatment of COVID-19. However, with the emergence of variants of concern, there is a need for new treatment options. We report a format that enables modular assembly of bi-paratopic tetravalent nAbs with antigen-binding sites from two distinct nAbs. The tetravalent nAb purifies in high yield and exhibits biophysical characteristics that are comparable to those of clinically used therapeutic antibodies. The tetravalent nAb binds to the spike protein trimer at least 100-fold more tightly than bivalent IgGs (apparent KD < 1 pM) and neutralizes a broad array of SARS-CoV-2 pseudoviruses, chimeric viruses, and authentic viral variants with high potency. Together, these results establish the tetravalent diabody-Fc-Fab as a robust, modular platform for rapid production of drug-grade nAbs with potencies and breadth of coverage that greatly exceed those of conventional bivalent IgGs.

Development of Monoclonal Antibodies to Detect for SARS-CoV-2 Proteins.

The COVID-19 pandemic caused by SARS-CoV-2 infection has impacted the world economy and healthcare infrastructure. Key reagents with high specificity to SARS-CoV-2 proteins are currently lacking, which limits our ability to understand the pathophysiology of SARS-CoV-2 infections. To address this need, we initiated a series of studies to generate and develop highly specific antibodies against proteins from SARS-CoV-2 using an antibody engineering platform. These efforts resulted in 18 monoclonal antibodies against nine SARS-CoV-2 proteins. Here we report the characterization of several antibodies, including those that recognize Nsp1, Nsp8, Nsp12, and Orf3b viral proteins. Our validation studies included evaluation for use of antibodies in ELISA, western blots, and immunofluorescence assays (IFA). We expect that availability of these antibodies will enhance our ability to further characterize host-viral interactions, including specific roles played by viral proteins during infection, to acquire a better understanding of the pathophysiology of SARS-CoV-2 infections.

Two Different Therapeutic Approaches for SARS-CoV-2 in hiPSCs-Derived Lung Organoids.

The global health emergency for SARS-CoV-2 (COVID-19) created an urgent need to develop new treatments and therapeutic drugs. In this study, we tested, for the first time on human cells, a new tetravalent neutralizing antibody (15033-7) targeting Spike protein and a synthetic peptide homologous to dipeptidyl peptidase-4 (DPP4) receptor on host cells. Both could represent powerful immunotherapeutic candidates for COVID-19 treatment. The infection begins in the proximal airways, namely the alveolar type 2 (AT2) cells of the distal lung, which express both ACE2 and DPP4 receptors. Thus, to evaluate the efficacy of both approaches, we developed three-dimensional (3D) complex lung organoid structures (hLORGs) derived from human-induced pluripotent stem cells (iPSCs) and resembling the in vivo organ. Afterward, hLORGs were infected by different SARS-CoV-2 S pseudovirus variants and treated by the Ab15033-7 or DPP4 peptide. Using both approaches, we observed a significant reduction of viral entry and a modulation of the expression of genes implicated in innate immunity and inflammatory response. These data demonstrate the efficacy of such approaches in strongly reducing the infection efficiency in vitro and, importantly, provide proof-of-principle evidence that hiPSC-derived hLORGs represent an ideal in vitro system for testing both therapeutic and preventive modalities against COVID-19.

Ultrapotent and Broad Neutralization of SARS-CoV-2 Variants by Modular, Tetravalent, Bi-paratopic Antibodies (preprint)

Neutralizing antibodies (nAbs) that target the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein (S-protein) are promising therapeutics for COVID-19. However, natural bivalent nAbs suffer from limited potency and are vulnerable to SARS-CoV-2 variants with mutated RBDs. We report a novel format that enables modular assembly of bi-paratopic, tetravalent nAbs with antigen-binding sites from two distinct nAbs. The diabody-Fc-Fab format consists of a central Fc with a bivalent diabody fused to its N-terminus and two Fabs fused to its C-terminus. The diabody and Fab modules do not interfere with each other, and thus, any diabody can be combined with any Fab in a facile manner. We engineered a diabody-Fc-Fab that contained the paratopes of two distinct nAbs derived from a phage-displayed library of synthetic Abs. The tetravalent nAb was purified in high yields with methods used to produce conventional IgGs, and it exhibited favorable biophysical characteristics comparable to those of approved therapeutic antibodies. The tetravalent nAb bound to the S-protein trimer at least 100-fold more tightly than the bivalent IgGs (apparent KD <1 pM). Most importantly, the tetravalent nAb exhibited extremely high potencies in neutralization assays across a panel of pseudoviruses representing seven natural SARS-CoV-2 variants (IC50 <5 ng/mL), including several that resisted IgGs and are known to evade approved IgG drugs. Taken together, our results showed that the tetravalent diabody-Fc-Fab is a robust, modular platform for rapid production of drug-grade nAbs with potencies and breadth of coverage that far exceed those of conventional bivalent IgGs.

Monitoring of epidemic outbreaks using social media data

The COVID-19 has become the most dangerous disease for the 21st Century. The infectious disease had still gone through outbreaks despite modern medical treatments. The most recent example being the COVID-19 which has infected over 108 million people over the world and resulted in the death of over 2.3 million people as of 13 February 2021. During the ongoing pandemic of COVID-19 people are making use of social media to express their concerns as well as events related to the pandemic in their personal life. There are also a lot of agencies/organizations that are using social media platforms to convey status regarding the pandemic. We have used this overwhelming amount of data that is available on social media, particularly Twitter, to find out the trend of the COVID-19 pandemic so that we can prove the correlation between volumes of tweets tweeted related to the pandemic and daily confirmed cases which will indeed help in getting early warning regarding immediate future cases so that government and medical agencies can take appropriate measures to handle the upcoming situation. We have used natural language processing techniques and classification algorithms to classify the tweets related to the current pandemic and find the trend of the pandemic. We have used sentiment analysis techniques to find out how the current situation of the epidemic is i.e. is it getting worse or is it getting better? We have also created a live epidemic monitoring system to monitor the live tweets on a map-based UI which will show real-time tweets related to any epidemic/pandemic outbreak on a map in real-time. © 2021 IEEE.

Peptide Platform as a Powerful Tool in the Fight against COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in a global pandemic causing over 195 million infections and more than 4 million fatalities as of July 2021.To date, it has been demonstrated that a number of mutations in the spike glycoprotein (S protein) of SARS-CoV-2 variants of concern abrogate or reduce the neutralization potency of several therapeutic antibodies and vaccine-elicited antibodies. Therefore, the development of additional vaccine platforms with improved supply and logistic profile remains a pressing need. In this work, we have validated the applicability of a peptide-based strategy focused on a preventive as well as a therapeutic purpose. On the basis of the involvement of the dipeptidyl peptidase 4 (DPP4), in addition to the angiotensin converting enzyme 2 (ACE2) receptor in the mechanism of virus entry, we analyzed peptides bearing DPP4 sequences by protein-protein docking and assessed their ability to block pseudovirus infection in vitro. In parallel, we have selected and synthetized peptide sequences located within the highly conserved receptor-binding domain (RBD) of the S protein, and we found that RBD-based vaccines could better promote elicitation of high titers of neutralizing antibodies specific against the regions of interest, as confirmed by immunoinformatic methodologies and in vivo studies. These findings unveil a key antigenic site targeted by broadly neutralizing antibodies and pave the way to the design of pan-coronavirus vaccines.

Tetravalent SARS-CoV-2 Neutralizing Antibodies Show Enhanced Potency and Resistance to Escape Mutations.

Neutralizing antibodies (nAbs) hold promise as therapeutics against COVID-19. Here, we describe protein engineering and modular design principles that have led to the development of synthetic bivalent and tetravalent nAbs against SARS-CoV-2. The best nAb targets the host receptor binding site of the viral S-protein and tetravalent versions block entry with a potency exceeding bivalent nAbs by an order of magnitude. Structural studies show that both the bivalent and tetravalent nAbs can make multivalent interactions with a single S-protein trimer, consistent with the avidity and potency of these molecules. Significantly, we show that the tetravalent nAbs show increased tolerance to potential virus escape mutants and an emerging variant of concern. Bivalent and tetravalent nAbs can be produced at large-scale and are as stable and specific as approved antibody drugs. Our results provide a general framework for enhancing antiviral therapies against COVID-19 and related viral threats, and our strategy can be applied to virtually any antibody drug.

Inhibition of HECT E3 ligases as potential therapy for COVID-19.

SARS-CoV-2 is responsible for the ongoing world-wide pandemic which has already taken more than two million lives. Effective treatments are urgently needed. The enzymatic activity of the HECT-E3 ligase family members has been implicated in the cell egression phase of deadly RNA viruses such as Ebola through direct interaction of its VP40 Protein. Here we report that HECT-E3 ligase family members such as NEDD4 and WWP1 interact with and ubiquitylate the SARS-CoV-2 Spike protein. Furthermore, we find that HECT family members are overexpressed in primary samples derived from COVID-19 infected patients and COVID-19 mouse models. Importantly, rare germline activating variants in the NEDD4 and WWP1 genes are associated with severe COVID-19 cases. Critically, I3C, a natural NEDD4 and WWP1 inhibitor from Brassicaceae, displays potent antiviral effects and inhibits viral egression. In conclusion, we identify the HECT family members of E3 ligases as likely novel biomarkers for COVID-19, as well as new potential targets of therapeutic strategy easily testable in clinical trials in view of the established well-tolerated nature of the Brassicaceae natural compounds.

Comparison of effectiveness of chlorine dioxide mouthwash and chlorhexidine gluconate mouthwash in reduction of oral viral load in patients with covid-19

Background: Air-borne droplets constitute the main route of transmission of COVID-19. Considering the exponential increase in number of cases, it has become the need of the hour to develop additional measures to limit the spread of infection. Materials and Methods: 40 patients were provided with Chlorhexidine gluconate (0.2%) mouthwash and Chlorine dioxide (0.1%) mouthwash to rinse and gargle thrice a day for one week. The qualitative COVID antigen test confirmed by Qualitative PCR on an oropharyngeal swab collected from the patients was compared for both the groups at baseline and post-intervention levels. Results: There was an improvement in symptoms such as cough, sore throat and bad breath in both the groups. The number of cases demonstrating reduction in intensity of symptoms as well as testing qualitatively negative for COVID-19 antigen were found to be greater in the group that was provided with Chlorine dioxide mouthwash. Conclusion: Regular use of Chlorine dioxide could effectively reduce the symptoms and oral viral load, thereby subsequently reducing the symptoms and risk of transmission of COVID-19. Use of Chlorine dioxide mouthwash may be recommended as a part of health policies and preprocedural protocols.

Synthetic Antibodies neutralize SARS-CoV-2 infection of mammalian cells (preprint)

Coronaviruses (CoV) are a large family of enveloped, RNA viruses that circulate in mammals and birds. Three highly pathogenic strains have caused zoonotic infections in humans that result in severe respiratory syndromes including the Middle East Respiratory Syndrome CoV (MERS), Severe Acute Respiratory Syndrome CoV (SARS), and the ongoing Coronavirus Disease 2019 (COVID-19) pandemic. Here, we describe a panel of synthetic monoclonal antibodies, built on a human IgG framework, that bind to the spike protein of SARS-CoV-2 (the causative agent of COVID-19), compete for ACE2 binding, and potently inhibit SARS-CoV-2. All antibodies that exhibited neutralization potencies at sub-nanomolar concentrations against SARS-CoV-2/USA/WA1 in Vero E6 cells, also bound to the receptor binding domain (RBD), suggesting competition for the host receptor ACE2. These antibodies represent strong immunotherapeutic candidates for treatment of COVID-19.