3D-printed airway model as a tool for studying SARS-CoV-2 infection and antiviral therapeutics

The outbreak of the SARS-CoV-2 has caused the infection of numerous people, resulting in the majority of them suffering from respiratory disease. There is a need for an in vitro lung model in which antiviral drugs can be tested reliably and quickly against the novel coronavirus. A physiologically relevant respiratory model provides a drug screening platform to study SARS-CoV-2 infection. We recapitulated the multi-layered human airway structure consisting of pulmonary endothelium, extracellular matrix, and airway epithelium through automated inkjet and microextrusion bioprinting. The 3D microarchitecture exhibits cell-cell junction and mucus secretion which are the major respiratory barrier to viral infection and also expressed ACE2 and TMPRSS2 which are known to be involved in SARS-CoV-2 cell entry. We investigated the response following infection with SARS-CoV-2 in the 3D airway model. The infection induced cytopathic effect and barrier destruction in the model over time. Virus replication was effectively inhibited when an infected 3D airway model was treated with remdesivir and molnupiravir, approved for the treatment of COVID-19. Then the EC50 was determined for each drug in the model. The 3D-printed airway model can be used as a tool for studying viral infection and validating the efficacy of therapeutics against other respiratory infection viruses as well as SARS-CoV-2 .

Multifaceted roles of plant derived small molecule inhibitors on replication cycle of SARS-CoV-2.

INTRODUCTION: Coronavirus disease 2019 (COVID-19) is an illness caused by the new coronavirus severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2). It has affected public health and the economy globally. Currently approved vaccines and other drug candidates could be associated with several drawbacks which urges developing alternative therapeutic approaches. AIM: To provide a comprehensive review of anti-SARS-CoV-2 activities of plants and their bioactive compounds. METHODS: Information was gathered from diverse bibliographic platforms such as PubMed, Google Scholar, and ClinicalTrials.gov registry. RESULTS: The present review highlights the potential roles of crude extracts of plants as well as plant-derived small molecules in inhibiting SARS-CoV-2 infection by targeting viral or host factors essential for viral entry, polyprotein processing, replication, assembly and release. Their anti-inflammatory and antioxidant properties as well as plant-based therapies that are under development in the clinical trial phases-1 to 3 are also covered. CONCLUSION: This knowledge could further help understanding SARS-CoV-2 infection and anti-viral mechanisms of plant-based therapeutics.

Single-Domain Antibodies as Therapeutics for Respiratory RNA Virus Infections.

Over the years, infectious diseases with high morbidity and mortality disrupted human healthcare systems and devastated economies globally. Respiratory viruses, especially emerging or re-emerging RNA viruses, including influenza and human coronavirus, are the main pathogens of acute respiratory diseases that cause epidemics or even global pandemics. Importantly, due to the rapid mutation of viruses, there are few effective drugs and vaccines for the treatment and prevention of these RNA virus infections. Of note, a class of antibodies derived from camelid and shark, named nanobody or single-domain antibody (sdAb), was characterized by smaller size, lower production costs, more accessible binding epitopes, and inhalable properties, which have advantages in the treatment of respiratory diseases compared to conventional antibodies. Currently, a number of sdAbs have been developed against various respiratory RNA viruses and demonstrated potent therapeutic efficacy in mouse models. Here, we review the current status of the development of antiviral sdAb and discuss their potential as therapeutics for respiratory RNA viral diseases.

Advances in Nanomaterial-Based Platforms to Combat COVID-19: Diagnostics, Preventions, Therapeutics, and Vaccine Developments.

The COVID-19 pandemic caused by the SARS-CoV-2, a ribonucleic acid (RNA) virus that emerged less than two years ago but has caused nearly 6.1 million deaths to date. Recently developed variants of the SARS-CoV-2 virus have been shown to be more potent and expanded at a faster rate. Until now, there is no specific and effective treatment for SARS-CoV-2 in terms of reliable and sustainable recovery. Precaution, prevention, and vaccinations are the only ways to keep the pandemic situation under control. Medical and scientific professionals are now focusing on the repurposing of previous technology and trying to develop more fruitful methodologies to detect the presence of viruses, treat the patients, precautionary items, and vaccine developments. Nanomedicine or nanobased platforms can play a crucial role in these fronts. Researchers are working on many effective approaches by nanosized particles to combat SARS-CoV-2. The role of a nanobased platform to combat SARS-CoV-2 is extremely diverse (i.e., mark to personal protective suit, rapid diagnostic tool to targeted treatment, and vaccine developments). Although there are many theoretical possibilities of a nanobased platform to combat SARS-CoV-2, until now there is an inadequate number of research targeting SARS-CoV-2 to explore such scenarios. This unique mini-review aims to compile and elaborate on the recent advances of nanobased approaches from prevention, diagnostics, treatment to vaccine developments against SARS-CoV-2, and associated challenges.

Mechanistic Models of COVID-19: Insights into Disease Progression, Vaccines, and Therapeutics.

The COVID-19 pandemic has severely impacted health systems and economies worldwide. Significant global efforts are therefore ongoing to improve vaccine efficacies, optimize vaccine deployment, and develop new antiviral therapies to combat the pandemic. Mechanistic viral dynamics and quantitative systems pharmacology models of SARS-CoV-2 infection, vaccines, immunomodulatory agents, and antiviral therapeutics have played a key role in advancing our understanding of SARS-CoV-2 pathogenesis and transmission, the interplay between innate and adaptive immunity to influence the outcomes of infection, effectiveness of treatments, mechanisms and performance of COVID-19 vaccines, and the impact of emerging SARS-CoV-2 variants. Here, we review some of the critical insights provided by these models and discuss the challenges ahead.

Antiviral Efficacies of FDA-Approved Drugs against SARS-CoV-2 Infection in Ferrets.

Due to the urgent need of a therapeutic treatment for coronavirus (CoV) disease 2019 (COVID-19) patients, a number of FDA-approved/repurposed drugs have been suggested as antiviral candidates at clinics, without sufficient information. Furthermore, there have been extensive debates over antiviral candidates for their effectiveness and safety against severe acute respiratory syndrome CoV 2 (SARS-CoV-2), suggesting that rapid preclinical animal studies are required to identify potential antiviral candidates for human trials. To this end, the antiviral efficacies of lopinavir-ritonavir, hydroxychloroquine sulfate, and emtricitabine-tenofovir for SARS-CoV-2 infection were assessed in the ferret infection model. While the lopinavir-ritonavir-, hydroxychloroquine sulfate-, or emtricitabine-tenofovir-treated group exhibited lower overall clinical scores than the phosphate-buffered saline (PBS)-treated control group, the virus titers in nasal washes, stool specimens, and respiratory tissues were similar between all three antiviral-candidate-treated groups and the PBS-treated control group. Only the emtricitabine-tenofovir-treated group showed lower virus titers in nasal washes at 8 days postinfection (dpi) than the PBS-treated control group. To further explore the effect of immune suppression on viral infection and clinical outcome, ferrets were treated with azathioprine, an immunosuppressive drug. Compared to the PBS-treated control group, azathioprine-immunosuppressed ferrets exhibited a longer period of clinical illness, higher virus titers in nasal turbinate, delayed virus clearance, and significantly lower serum neutralization (SN) antibody titers. Taken together, all antiviral drugs tested marginally reduced the overall clinical scores of infected ferrets but did not significantly affect in vivo virus titers. Despite the potential discrepancy of drug efficacies between animals and humans, these preclinical ferret data should be highly informative to future therapeutic treatment of COVID-19 patients.IMPORTANCE The SARS-CoV-2 pandemic continues to spread worldwide, with rapidly increasing numbers of mortalities, placing increasing strain on health care systems. Despite serious public health concerns, no effective vaccines or therapeutics have been approved by regulatory agencies. In this study, we tested the FDA-approved drugs lopinavir-ritonavir, hydroxychloroquine sulfate, and emtricitabine-tenofovir against SARS-CoV-2 infection in a highly susceptible ferret infection model. While most of the drug treatments marginally reduced clinical symptoms, they did not reduce virus titers, with the exception of emtricitabine-tenofovir treatment, which led to diminished virus titers in nasal washes at 8 dpi. Further, the azathioprine-treated immunosuppressed ferrets showed delayed virus clearance and low SN titers, resulting in a prolonged infection. As several FDA-approved or repurposed drugs are being tested as antiviral candidates at clinics without sufficient information, rapid preclinical animal studies should proceed to identify therapeutic drug candidates with strong antiviral potential and high safety prior to a human efficacy trial.

Recent advances on therapeutic potentials of gold and silver nanobiomaterials for human viral diseases.

Viral diseases are prominent among the widely spread infections threatening human well-being. Real-life clinical successes of the few available therapeutics are challenged by pathogenic resistance and suboptimal delivery to target sites. Nanotechnology has aided the design of functionalised and non-functionalised Au and Ag nanobiomaterials through physical, chemical and biological (green synthesis) methods with improved antiviral efficacy and delivery. In this review, innovative designs as well as interesting antiviral activities of the nanotechnology-inclined biomaterials of Au and Ag, reported in the last 5 years were critically overviewed against several viral diseases affecting man. These include influenza, respiratory syncytial, adenovirus, severe acute respiratory syndromes (SARS), rotavirus, norovirus, measles, chikungunya, HIV, herpes simplex virus, dengue, polio, enterovirus and rift valley fever virus. Notably identified among the nanotechnologically designed promising antiviral agents include AuNP-M2e peptide vaccine, AgNP of cinnamon bark extract and AgNP of oseltamivir for influenza, PVP coated AgNP for RSV, PVP-AgNPs for SARS-CoV-2, AuNRs of a peptide pregnancy-induce d hypertension and AuNP nanocarriers of antigen for MERS-CoV and SARS-CoV respectively. Others are AgNPs of collagen and Bacillus subtilis for rotavirus, AgNPs labelled Ag30-SiO 2 for murine norovirus in water, AuNPs of Allium sativum and AgNPs of ribavirin for measles, AgNPs of Citrus limetta and Andrographis Paniculata for Chikungunya, AuNPs of efavirenz and stavudine, and AgNPs-curcumin for HIV, NPAuG3-S8 for HSV, AgNPs of Moringa oleifera and Bruguiera cylindrica for dengue while AgNPs of polyethyleneimine and siRNA analogues displayed potency against enterovirus. The highlighted candidates are recommended for further translational studies towards antiviral therapeutic designs.

Highly Specific Sigma Receptor Ligands Exhibit Anti-Viral Properties in SARS-CoV-2 Infected Cells.

(1) Background: There is a strong need for prevention and treatment strategies for COVID-19 that are not impacted by SARS-CoV-2 mutations emerging in variants of concern. After virus infection, host ER resident sigma receptors form direct interactions with non-structural SARS-CoV-2 proteins present in the replication complex. (2) Methods: In this work, highly specific sigma receptor ligands were investigated for their ability to inhibit both SARS-CoV-2 genome replication and virus induced cellular toxicity. This study found antiviral activity associated with agonism of the sigma-1 receptor (e.g., SA4503), ligation of the sigma-2 receptor (e.g., CM398), and a combination of the two pathways (e.g., AZ66). (3) Results: Intermolecular contacts between these ligands and sigma receptors were identified by structural modeling. (4) Conclusions: Sigma receptor ligands and drugs with off-target sigma receptor binding characteristics were effective at inhibiting SARS-CoV-2 infection in primate and human cells, representing a potential therapeutic avenue for COVID-19 prevention and treatment.

Exploiting Ubiquitin Ligases for Induced Target Degradation as an Antiviral Strategy.

Posttranslational modifications of targeted substrates alter their cellular fate. Ubiquitin is a highly conserved and ubiquitous covalent modifier protein that tags substrates with a single molecule or with a polyubiquitin chain. Monoubiquitination affects trafficking and signaling patterns of modified proteins. In contrast, polyubiquitination, particularly K48-linked polyubiquitination, targets the protein for degradation by the Ubiquitin-Proteasome System (UPS) resulting in a committed fate through irreversible inactivation of substrate. Given the diversity of cellular functions impacted by ubiquitination, it is no surprise that the wily pathogenic viruses have co-opted the UPS in myriad ways to ensure their survival. In this review, I describe viral exploitation of nondegradative ubiquitin signaling pathways to effect entry, replication, and egress. Additionally, viruses also harness the UPS to degrade antiviral cellular host factors. Finally, I describe how we can exploit the same proteolytic machinery to enable PROTACs (Proteolysis-Targeting Chimeras) to degrade essential viral proteins. Successful implementation of this modality will add to the arsenal of emerging antiviral therapies.

Report of the National Institutes of Health SARS-CoV-2 Antiviral Therapeutics Summit.

The NIH Virtual SARS-CoV-2 Antiviral Summit, held on 6 November 2020, was organized to provide an overview on the status and challenges in developing antiviral therapeutics for coronavirus disease 2019 (COVID-19), including combinations of antivirals. Scientific experts from the public and private sectors convened virtually during a live videocast to discuss severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) targets for drug discovery as well as the preclinical tools needed to develop and evaluate effective small-molecule antivirals. The goals of the Summit were to review the current state of the science, identify unmet research needs, share insights and lessons learned from treating other infectious diseases, identify opportunities for public-private partnerships, and assist the research community in designing and developing antiviral therapeutics. This report includes an overview of therapeutic approaches, individual panel summaries, and a summary of the discussions and perspectives on the challenges ahead for antiviral development.

Graphene Oxide Nanosheets Interact and Interfere with SARS-CoV-2 Surface Proteins and Cell Receptors to Inhibit Infectivity.

Nanotechnology can offer a number of options against coronavirus disease 2019 (COVID-19) acting both extracellularly and intracellularly to the host cells. Here, the aim is to explore graphene oxide (GO), the most studied 2D nanomaterial in biomedical applications, as a nanoscale platform for interaction with SARS-CoV-2. Molecular docking analyses of GO sheets on interaction with three different structures: SARS-CoV-2 viral spike (open state - 6VYB or closed state - 6VXX), ACE2 (1R42), and the ACE2-bound spike complex (6M0J) are performed. GO shows high affinity for the surface of all three structures (6M0J, 6VYB and 6VXX). When binding affinities and involved bonding types are compared, GO interacts more strongly with the spike or ACE2, compared to 6M0J. Infection experiments using infectious viral particles from four different clades as classified by Global Initiative on Sharing all Influenza Data (GISAID), are performed for validation purposes. Thin, biological-grade GO nanoscale (few hundred nanometers in lateral dimension) sheets are able to significantly reduce copies for three different viral clades. This data has demonstrated that GO sheets have the capacity to interact with SARS-CoV-2 surface components and disrupt infectivity even in the presence of any mutations on the viral spike. GO nanosheets are proposed to be further explored as a nanoscale platform for development of antiviral strategies against COVID-19.

Nanobody cocktails potently neutralize SARS-CoV-2 D614G N501Y variant and protect mice.

Neutralizing antibodies are important for immunity against SARS-CoV-2 and as therapeutics for the prevention and treatment of COVID-19. Here, we identified high-affinity nanobodies from alpacas immunized with coronavirus spike and receptor-binding domains (RBD) that disrupted RBD engagement with the human receptor angiotensin-converting enzyme 2 (ACE2) and potently neutralized SARS-CoV-2. Epitope mapping, X-ray crystallography, and cryo-electron microscopy revealed two distinct antigenic sites and showed two neutralizing nanobodies from different epitope classes bound simultaneously to the spike trimer. Nanobody-Fc fusions of the four most potent nanobodies blocked ACE2 engagement with RBD variants present in human populations and potently neutralized both wild-type SARS-CoV-2 and the N501Y D614G variant at concentrations as low as 0.1 nM. Prophylactic administration of either single nanobody-Fc or as mixtures reduced viral loads by up to 104-fold in mice infected with the N501Y D614G SARS-CoV-2 virus. These results suggest a role for nanobody-Fc fusions as prophylactic agents against SARS-CoV-2.

Potential "biopeptidal" therapeutics for severe respiratory syndrome coronaviruses: a review of antiviral peptides, viral mechanisms, and prospective needs.

Although great advances have been made on large-scale manufacturing of vaccines and antiviral-based drugs, viruses persist as the major cause of human diseases nowadays. The recent pandemic of coronavirus disease-2019 (COVID-19) mounts a lot of stress on the healthcare sector and the scientific society to search continuously for novel components with antiviral possibility. Herein, we narrated the different tactics of using biopeptides as antiviral molecules that could be used as an interesting alternative to treat COVID-19 patients. The number of peptides with antiviral effects is still low, but such peptides already displayed huge potentials to become pharmaceutically obtainable as antiviral medications. Studies showed that animal venoms, mammals, plant, and artificial sources are the main sources of antiviral peptides, when bioinformatics tools are used. This review spotlights bioactive peptides with antiviral activities against human viruses, especially the coronaviruses such as severe acute respiratory syndrome (SARS) virus, Middle East respiratory syndrome (MERS) virus, and severe acute respiratory syndrome coronavirus 2 (SARS-COV-2 or SARS-nCOV19). We also showed the data about well-recognized peptides that are still under investigations, while presenting the most potent ones that may become medications for clinical use.

Engineered Ultra-High Affinity Synthetic Antibodies for SARS-CoV-2 Neutralization and Detection.

The Covid-19 pandemic is a centenarial global catastrophe. Similar events are likely to be recurring with more frequency in the future. The inability to control the virus' impact is caused by many factors, but the lack of a technology infrastructure to detect and impede the virus at an early stage are principal shortcomings. Using phage display mutagenesis, we have generated a cohort of high performance antibody fragments (Fabs) that can be used in a sensitive point of care (POC) assay and are potent inhibitors (IC50-0.5 nM) to viral entry into cells. The POC assay is based on a split-enzyme (ß-lactamase) complementation strategy that detects virus particles at low nM levels. We have shown that this assay is equally effective for detecting other viruses like Ebola and Zika. Importantly, its components can be freeze dried and stored, but becomes fully active when rehydrated.

Advanced Nanomaterials for Preparedness Against (Re-)Emerging Viral Diseases.

While the coronavirus disease (COVID-19) accounts for the current global pandemic, the emergence of other unknown pathogens, named "Disease X," remains a serious concern in the future. Emerging or re-emerging pathogens continue to pose significant challenges to global public health. In response, the scientific community has been urged to create advanced platform technologies to meet the ever-increasing needs presented by these devastating diseases with pandemic potential. This review aims to bring new insights to allow for the application of advanced nanomaterials in future diagnostics, vaccines, and antiviral therapies, thereby addressing the challenges associated with the current preparedness strategies in clinical settings against viruses. The application of nanomaterials has advanced medicine and provided cutting-edge solutions for unmet needs. Herein, an overview of the currently available nanotechnologies is presented, highlighting the significant features that enable them to control infectious diseases, and identifying the challenges that remain to be addressed for the commercial production of nano-based products is presented. Finally, to conclude, the development of a nanomaterial-based system using a "One Health" approach is suggested. This strategy would require a transdisciplinary collaboration and communication between all stakeholders throughout the entire process spanning across research and development, as well as the preclinical, clinical, and manufacturing phases.

Engineered ACE2 receptor traps potently neutralize SARS-CoV-2.

An essential mechanism for severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection begins with the viral spike protein binding to the human receptor protein angiotensin-converting enzyme II (ACE2). Here, we describe a stepwise engineering approach to generate a set of affinity optimized, enzymatically inactivated ACE2 variants that potently block SARS-CoV-2 infection of cells. These optimized receptor traps tightly bind the receptor binding domain (RBD) of the viral spike protein and prevent entry into host cells. We first computationally designed the ACE2-RBD interface using a two-stage flexible protein backbone design process that improved affinity for the RBD by up to 12-fold. These designed receptor variants were affinity matured an additional 14-fold by random mutagenesis and selection using yeast surface display. The highest-affinity variant contained seven amino acid changes and bound to the RBD 170-fold more tightly than wild-type ACE2. With the addition of the natural ACE2 collectrin domain and fusion to a human immunoglobulin crystallizable fragment (Fc) domain for increased stabilization and avidity, the most optimal ACE2 receptor traps neutralized SARS-CoV-2-pseudotyped lentivirus and authentic SARS-CoV-2 virus with half-maximal inhibitory concentrations (IC50s) in the 10- to 100-ng/mL range. Engineered ACE2 receptor traps offer a promising route to fighting infections by SARS-CoV-2 and other ACE2-using coronaviruses, with the key advantage that viral resistance would also likely impair viral entry. Moreover, such traps can be predesigned for viruses with known entry receptors for faster therapeutic response without the need for neutralizing antibodies isolated from convalescent patients.

Broad Anti-coronavirus Activity of Food and Drug Administration-Approved Drugs against SARS-CoV-2 In Vitro and SARS-CoV In Vivo.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged in China at the end of 2019 and has rapidly caused a pandemic, with over 20 million recorded COVID-19 cases in August 2020 (https://covid19.who.int/). There are no FDA-approved antivirals or vaccines for any coronavirus, including SARS-CoV-2. Current treatments for COVID-19 are limited to supportive therapies and off-label use of FDA-approved drugs. Rapid development and human testing of potential antivirals is urgently needed. Numerous drugs are already approved for human use, and subsequently, there is a good understanding of their safety profiles and potential side effects, making them easier to fast-track to clinical studies in COVID-19 patients. Here, we present data on the antiviral activity of 20 FDA-approved drugs against SARS-CoV-2 that also inhibit SARS-CoV and Middle East respiratory syndrome coronavirus (MERS-CoV). We found that 17 of these inhibit SARS-CoV-2 at non-cytotoxic concentrations. We directly followed up seven of these to demonstrate that all are capable of inhibiting infectious SARS-CoV-2 production. Moreover, we evaluated two of these, chloroquine and chlorpromazine, in vivo using a mouse-adapted SARS-CoV model and found that both drugs protect mice from clinical disease.IMPORTANCE There are no FDA-approved antivirals for any coronavirus, including SARS-CoV-2. Numerous drugs are already approved for human use that may have antiviral activity and therefore could potentially be rapidly repurposed as antivirals. Here, we present data assessing the antiviral activity of 20 FDA-approved drugs against SARS-CoV-2 that also inhibit SARS-CoV and MERS-CoV in vitro We found that 17 of these inhibit SARS-CoV-2, suggesting that they may have pan-anti-coronaviral activity. We directly followed up seven of these and found that they all inhibit infectious-SARS-CoV-2 production. Moreover, we evaluated chloroquine and chlorpromazine in vivo using mouse-adapted SARS-CoV. We found that neither drug inhibited viral replication in the lungs, but both protected against clinical disease.

Development of cell-based pseudovirus entry assay to identify potential viral entry inhibitors and neutralizing antibodies against SARS-CoV-2.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative virus of the coronavirus disease 2019 (COVID-19) pandemic. To establish a safe and convenient assay system for studying entry inhibitors and neutralizing antibodies against SARS-CoV-2, we constructed a codon-optimized, full-length C-terminal mutant spike (S) gene of SARS-CoV-2. We generated a luciferase (Luc)-expressing pseudovirus containing the wild-type or mutant S protein of SARS-CoV-2 in the envelope-defective HIV-1 backbone. The key parameters for this pseudovirus-based assay, including the S mutants and virus incubation time, were optimized. This pseudovirus contains a Luc reporter gene that enabled us to easily quantify virus entry into angiotensin-converting enzyme 2 (ACE2)-expressing 293T cells. Cathepsin (Cat)B/L inhibitor E-64d could significantly block SARS-CoV-2 pseudovirus infection in 293T-ACE2 cells. Furthermore, the SARS-CoV-2 spike pseudotyped virus could be neutralized by sera from convalescent COVID-19 patients or recombinant ACE2 with the fused Fc region of human IgG1. Thus, we developed a pseudovirus-based assay for SARS-CoV-2, which will be valuable for evaluating viral entry inhibitors and neutralizing antibodies against this highly pathogenic virus.