Design of a chimeric ACE-2/Fc-silent fusion protein with ultrahigh affinity and neutralizing capacity for SARS-CoV-2 variants.

Background: As SARS-CoV-2 continues to mutate into Variants of Concern (VOC), there is growing and urgent need to develop effective antivirals to combat COVID-19. Monoclonal antibodies developed earlier are no longer capable of effectively neutralizing currently active VOCs. This report describes the design of variant-agnostic chimeric molecules consisting of an Angiotensin-Converting Enzyme 2 (ACE-2) domain mutated to retain ultrahigh affinity binding to a wide variety of SARS-CoV-2 variants, coupled to an Fc-silent immunoglobulin domain that eliminates antibody-dependent enhancement and extends biological half-life. Methods: Molecular modeling, Surrogate Viral Neutralization tests (sVNTs) and infection studies of human airway organoid cultures were performed with synthetic chimeras, SARS-CoV-2 spike protein mimics and SARS-CoV-2 Omicron variants B.1.1.214, BA.1, BA.2 and BA.5. Results: ACE-2 mutations L27, V34 and E90 resulted in ultrahigh affinity binding of the LVE-ACE-2 domain to the widest variety of VOCs, with KDs of 93 pM and 73 pM for binding to the Alpha B1.1.7 and Omicron B.1.1.529 variants, and notably, 78fM, 133fM and 1.81pM affinities to the Omicron BA.2, BA2.75 and BQ.1.1 subvariants, respectively. sVNT assays revealed titers of ≥4.9 ng/ml, for neutralization of recombinant viral proteins corresponding to the Alpha, Delta and Omicron variants. The values above were obtained with LVE-ACE-2/mAB chimeras containing the FcRn-binding Y-T-E sequence which extends biological half-life 3-4-fold. Conclusions: The ACE-2-mutant/Fc silent fusion proteins described have ultrahigh affinity to a wide variety of SARS-CoV-2 variants including Omicron. It is proposed that these chimeric ACE-2/mABs will constitute variant-agnostic and cost-effective prophylactics against SARS-CoV-2, particularly when administered nasally.

Non-uniform aspects of the SARS-CoV-2 intraspecies evolution reopen question of its origin.

Several hypotheses have been presented on the origin of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) from its identification as the agent causing the current coronavirus disease 19 (COVID-19) pandemic. So far, no solid evidence has been found to support any hypothesis on the origin of this virus, and the issue continue to resurface over and over again. Here we have unfolded a pattern of distribution of several mutations in the SARS-CoV-2 proteins in 24 geo-locations across different continents. The results showed an evenly uneven distribution of the unique protein variants, distinct mutations, unique frequency of common conserved residues, and mutational residues across these 24 geo-locations. Furthermore, ample mutations were identified in the evolutionarily conserved invariant regions in the SARS-CoV-2 proteins across almost all geo-locations studied. This pattern of mutations potentially breaches the law of evolutionary conserved functional units of the beta-coronavirus genus. These mutations may lead to several novel SARS-CoV-2 variants with a high degree of transmissibility and virulence. A thorough investigation on the origin and characteristics of SARS-CoV-2 needs to be conducted in the interest of science and for the preparation of meeting the challenges of potential future pandemics.

Dendritic cell-based vaccine: the state-of-the-art vaccine platform for COVID-19 management.

INTRODUCTION: A correlation between new coronaviruses and host immunity, as well as the role of defective immune function in host response, would be extremely helpful in understanding coronavirus disease (COVID-19) pathogenicity, and a coherent structure of treatments and vaccines. As existing vaccines may be inadequate for new viral variants emerging in various regions of the world, it is a vital requirement for fresh and effective therapeutic alternatives. AREA COVERED: Immunotherapy may give a viable protective option for COVID-19, a disease that is currently a big burden on global health and economic systems. Herein, we have outlined three dendritic cell (DC)-based vaccines for COVID-19 which are in human clinical trials and have shown encouraging outcomes. EXPERT OPINION: With existing knowledge of the virus, and the nature of DC, DC-based vaccines may be proven to be effective in inducing long-lasting protective immunity, especially T cell responses.

Would New SARS-CoV-2 Variants Change the War against COVID-19?

The scientific, private, and industrial sectors use a wide variety of technological platforms available to achieve protection against SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2), including vaccines. However, the virus evolves continually into new highly virulent variants, which might overcome the protection provided by vaccines and may re-expose the population to infections. Mass vaccinations should be continued in combination with more or less mandatory non-pharmaceutical interventions. Therefore, the key questions to be answered are: (i) How to identify the primary and secondary infections of SARS-CoV-2? (ii) Why are neutralizing antibodies not long-lasting in both cases of natural infections and post-vaccinations? (iii) Which are the factors responsible for this decay in neutralizing antibodies? (iv) What strategy could be adapted to develop long-term herd immunity? (v) Is the Spike protein the only vaccine target or is a vaccine cocktail better?

Interindividual immunogenic variants: Susceptibility to coronavirus, respiratory syncytial virus and influenza virus.

The coronavirus disease (Covid-19) pandemic is the most serious event of the year 2020, causing considerable global morbidity and mortality. The goal of this review is to provide a comprehensive summary of reported associations between inter-individual immunogenic variants and disease susceptibility or symptoms caused by the coronavirus strains severe acute respiratory syndrome-associated coronavirus, severe acute respiratory syndrome-associated coronavirus-2, and two of the main respiratory viruses, respiratory syncytial virus and influenza virus. The results suggest that the genetic background of the host could affect the levels of proinflammatory and anti-inflammatory cytokines and might modulate the progression of Covid-19 in affected patients. Notably, genetic variations in innate immune components such as toll-like receptors and mannose-binding lectin 2 play critical roles in the ability of the immune system to recognize coronavirus and initiate an early immune response to clear the virus and prevent the development of severe symptoms. This review provides promising clues related to the potential benefits of using immunotherapy and immune modulation for respiratory infectious disease treatment in a personalized manner.

Implications derived from S-protein variants of SARS-CoV-2 from six continents.

The spike (S) protein is a critical determinant of the infectivity and antigenicity of SARS-CoV-2. Several mutations in the S protein of SARS-CoV-2 have already been detected, and their effect in immune system evasion and enhanced transmission as a cause of increased morbidity and mortality are being investigated. From pathogenic and epidemiological perspectives, S proteins are of prime interest to researchers. This study focused on the unique variants of S proteins from six continents: Asia, Africa, Europe, Oceania, South America, and North America. In comparison to the other five continents, Africa had the highest percentage of unique S proteins (29.1%). The phylogenetic relationship implies that unique S proteins from North America are significantly different from those of the other five continents. They are most likely to spread to the other geographic locations through international travel or naturally by emerging mutations. It is suggested that restriction of international travel should be considered, and massive vaccination as an utmost measure to combat the spread of the COVID-19 pandemic. It is also further suggested that the efficacy of existing vaccines and future vaccine development must be reviewed with careful scrutiny, and if needed, further re-engineered based on requirements dictated by new emerging S protein variants.

Periodically aperiodic pattern of SARS-CoV-2 mutations underpins the uncertainty of its origin and evolution.

Various lineages of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) have contributed to prolongation of the Coronavirus Disease 2019 (COVID-19) pandemic. Several non-synonymous mutations in SARS-CoV-2 proteins have generated multiple SARS-CoV-2 variants. In our previous report, we have shown that an evenly uneven distribution of unique protein variants of SARS-CoV-2 is geo-location or demography-specific. However, the correlation between the demographic transmutability of the SARS-CoV-2 infection and mutations in various proteins remains unknown due to hidden symmetry/asymmetry in the occurrence of mutations. This study tracked how these mutations are emerging in SARS-CoV-2 proteins in six model countries and globally. In a geo-location, considering the mutations having a frequency of detection of at least 500 in each SARS-CoV-2 protein, we studied the country-wise percentage of invariant residues. Our data revealed that since October 2020, highly frequent mutations in SARS-CoV-2 have been observed mostly in the Open Reading Frame (ORF) 7b and ORF8, worldwide. No such highly frequent mutations in any of the SARS-CoV-2 proteins were found in the UK, India, and Brazil, which does not correlate with the degree of transmissibility of the virus in India and Brazil. However, we have found a signature that SARS-CoV-2 proteins were evolving at a higher rate, and considering global data, mutations are detected in the majority of the available amino acid locations. Fractal analysis of each protein's normalized factor time series showed a periodically aperiodic emergence of dominant variants for SARS-CoV-2 protein mutations across different countries. It was noticed that certain high-frequency variants have emerged in the last couple of months, and thus the emerging SARS-CoV-2 strains are expected to contain prevalent mutations in the ORF3a, membrane, and ORF8 proteins. In contrast to other beta-coronaviruses, SARS-CoV-2 variants have rapidly emerged based on demographically dependent mutations. Characterization of the periodically aperiodic nature of the demographic spread of SARS-CoV-2 variants in various countries can contribute to the identification of the origin of SARS-CoV-2.

Urgent Need for Field Surveys of Coronaviruses in Southeast Asia to Understand the SARS-CoV-2 Phylogeny and Risk Assessment for Future Outbreaks.

Phylogenetic analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is focused on a single isolate of bat coronaviruses (bat CoVs) which does not adequately represent genetically related coronaviruses (CoVs) [...].

SARS-CoV-2 research using human pluripotent stem cells and organoids.

Experimental cell models are indispensable for clarifying the pathophysiology of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, and for developing therapeutic agents. To recapitulate the symptoms and drug response of COVID-19 patients in vitro, SARS-CoV-2 studies using physiologically relevant human embryonic stem (ES)/induced pluripotent stem (iPS) cell-derived somatic cells and organoids are ongoing. These cells and organoids have been used to show that SARS-CoV-2 can infect and damage various organs including the lung, heart, brain, intestinal tract, kidney, and pancreas. They are also being used to develop COVID-19 therapeutic agents, including evaluation of their antiviral efficacy and safety. The relationship between COVID-19 aggravation and human genetic backgrounds has been investigated using genetically modified ES/iPS cells and patient-derived iPS cells. This review summarizes the latest results and issues of SARS-CoV-2 research using human ES/iPS cell-derived somatic cells and organoids.

COVID-19 Vaccines and Thrombosis-Roadblock or Dead-End Street?

Two adenovirus-based vaccines, ChAdOx1 nCoV-19 and Ad26.COV2.S, and two mRNA-based vaccines, BNT162b2 and mRNA.1273, have been approved by the European Medicines Agency (EMA), and are invaluable in preventing and reducing the incidence of coronavirus disease-2019 (COVID-19). Recent reports have pointed to thrombosis with associated thrombocytopenia as an adverse effect occurring at a low frequency in some individuals after vaccination. The causes of such events may be related to SARS-CoV-2 spike protein interactions with different C-type lectin receptors, heparan sulfate proteoglycans (HSPGs) and the CD147 receptor, or to different soluble splice variants of the spike protein, adenovirus vector interactions with the CD46 receptor or platelet factor 4 antibodies. Similar findings have been reported for several viral diseases after vaccine administration. In addition, immunological mechanisms elicited by viral vectors related to cellular delivery could play a relevant role in individuals with certain genetic backgrounds. Although rare, the potential COVID-19 vaccine-induced immune thrombotic thrombocytopenia (VITT) requires immediate validation, especially in risk groups, such as the elderly, chronic smokers, and individuals with pre-existing incidences of thrombocytopenia; and if necessary, a reformulation of existing vaccines.

Notable sequence homology of the ORF10 protein introspects the architecture of SARS-CoV-2.

The current Coronavirus Disease 19 (COVID-19) pandemic, caused by Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) shows similar pathology to MERS and SARS-CoV, with a current estimated fatality rate of 1.4%. Open reading frame 10 (ORF10) is a unique SARS-CoV-2 accessory protein, which contains eleven cytotoxic T lymphocyte (CTL) epitopes each of nine amino acids in length. Twenty-two unique SARS-CoV-2 ORF10 variants have been identified based on missense mutations found in sequence databases. Some of these mutations are predicted to decrease the stability of ORF10 in silico physicochemical and structural comparative analyses were carried out on SARS-CoV-2 and Pangolin-CoV ORF10 proteins, which share 97.37% amino acid (aa) homology. Though there is a high degree of ORF10 protein similarity of SARS-CoV-2 and Pangolin-CoV, there are differences of these two ORF10 proteins related to their sub-structure (loop/coil region), solubility, antigenicity and shift from strand to coil at aa position 26 (tyrosine). SARS-CoV-2 ORF10, which is apparently expressed in vivo since reactive T cell clones are found in convalescent patients should be monitored for changes which could correlate with the pathogenesis of COVID-19.

A unique view of SARS-CoV-2 through the lens of ORF8 protein.

Immune evasion is one of the unique characteristics of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) attributed to its ORF8 protein. This protein modulates the adaptive host immunity through down-regulation of MHC-1 (Major Histocompatibility Complex) molecules and innate immune responses by surpassing the host's interferon-mediated antiviral response. To understand the host's immune perspective in reference to the ORF8 protein, a comprehensive study of the ORF8 protein and mutations possessed by it have been performed. Chemical and structural properties of ORF8 proteins from different hosts, such as human, bat, and pangolin, suggest that the ORF8 of SARS-CoV-2 is much closer to ORF8 of Bat RaTG13-CoV than to that of Pangolin-CoV. Eighty-seven mutations across unique variants of ORF8 in SARS-CoV-2 can be grouped into four classes based on their predicted effects (Hussain et al., 2021) [1]. Based on the geo-locations and timescale of sample collection, a possible flow of mutations was built. Furthermore, conclusive flows of amalgamation of mutations were found upon sequence similarity analyses and consideration of the amino acid conservation phylogenies. Therefore, this study seeks to highlight the uniqueness of the rapidly evolving SARS-CoV-2 through the ORF8.

COVID-19-A Theory of Autoimmunity Against ACE-2 Explained.

The COVID-19 pandemic caused by the coronavirus SARS-COV-2 has cost many lives worldwide. In dealing with affected patients, the physician is faced with a very unusual pattern of organ damage that is not easily explained on the basis of prior knowledge of viral-induced pathogenesis. It is established that the main receptor for viral entry into tissues is the protein angiotensin-converting enzyme-2 ["ACE-2", (1)]. In a recent publication (2), a theory of autoimmunity against ACE-2, and/or against the ACE-2/SARS-COV-2 spike protein complex or degradation products thereof, was proposed as a possible explanation for the unusual pattern of organ damage seen in COVID-19. In the light of more recent information, this manuscript expands on the earlier proposed theory and offers additional, testable hypotheses that could explain both the pattern and timeline of organ dysfunction most often observed in COVID-19.

Carbon-Based Nanomaterials: Promising Antiviral Agents to Combat COVID-19 in the Microbial-Resistant Era.

Therapeutic options for the highly pathogenic human severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing the current pandemic coronavirus disease (COVID-19) are urgently needed. COVID-19 is associated with viral pneumonia and acute respiratory distress syndrome causing significant morbidity and mortality. The proposed treatments for COVID-19 have shown little or no effect in the clinic so far. Additionally, bacterial and fungal pathogens contribute to the SARS-CoV-2-mediated pneumonia disease complex. The antibiotic resistance in pneumonia treatment is increasing at an alarming rate. Therefore, carbon-based nanomaterials (CBNs), such as fullerene, carbon dots, graphene, and their derivatives constitute a promising alternative due to their wide-spectrum antimicrobial activity, biocompatibility, biodegradability, and capacity to induce tissue regeneration. Furthermore, the antimicrobial mode of action is mainly physical (e.g., membrane distortion), characterized by a low risk of antimicrobial resistance. In this Review, we evaluated the literature on the antiviral activity and broad-spectrum antimicrobial properties of CBNs. CBNs had antiviral activity against 13 enveloped positive-sense single-stranded RNA viruses, including SARS-CoV-2. CBNs with low or no toxicity to humans are promising therapeutics against the COVID-19 pneumonia complex with other viruses, bacteria, and fungi, including those that are multidrug-resistant.

Viewpoint: Origin of SARS-CoV-2.

The origin of the severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) virus causing the COVID-19 pandemic has not yet been fully determined. Despite the consensus about the SARS-CoV-2 origin from bat CoV RaTG13, discrepancy to host tropism to other human Coronaviruses exist. SARS-CoV-2 also possesses some differences in its S protein receptor-binding domain, glycan-binding N-terminal domain and the surface of the sialic acid-binding domain. Despite similarities based on cryo-EM and biochemical studies, the SARS-CoV-2 shows higher stability and binding affinity to the ACE2 receptor. The SARS-CoV-2 does not appear to present a mutational "hot spot" as only the D614G mutation has been identified from clinical isolates. As laboratory manipulation is highly unlikely for the origin of SARS-CoV-2, the current possibilities comprise either natural selection in animal host before zoonotic transfer or natural selection in humans following zoonotic transfer. In the former case, despite SARS-CoV-2 and bat RaTG13 showing 96% identity some pangolin Coronaviruses exhibit very high similarity to particularly the receptor-binding domain of SARS-CoV-2. In the latter case, it can be hypothesized that the SARS-CoV-2 genome has adapted during human-to-human transmission and based on available data, the isolated SARS-CoV-2 genomes derive from a common origin. Before the origin of SARS-CoV-2 can be confirmed additional research is required.

Possible Transmission Flow of SARS-CoV-2 Based on ACE2 Features.

Angiotensin-converting enzyme 2 (ACE2) is the cellular receptor for the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) that is engendering the severe coronavirus disease 2019 (COVID-19) pandemic. The spike (S) protein receptor-binding domain (RBD) of SARS-CoV-2 binds to the three sub-domains viz. amino acids (aa) 22-42, aa 79-84, and aa 330-393 of ACE2 on human cells to initiate entry. It was reported earlier that the receptor utilization capacity of ACE2 proteins from different species, such as cats, chimpanzees, dogs, and cattle, are different. A comprehensive analysis of ACE2 receptors of nineteen species was carried out in this study, and the findings propose a possible SARS-CoV-2 transmission flow across these nineteen species.

The structural basis of accelerated host cell entry by SARS-CoV-2†.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of the pandemic coronavirus disease 2019 (COVID-19) that exhibits an overwhelming contagious capacity over other human coronaviruses (HCoVs). This structural snapshot describes the structural bases underlying the pandemic capacity of SARS-CoV-2 and explains its fast motion over respiratory epithelia that allow its rapid cellular entry. Based on notable viral spike (S) protein features, we propose that the flat sialic acid-binding domain at the N-terminal domain (NTD) of the S1 subunit leads to more effective first contact and interaction with the sialic acid layer over the epithelium, and this, in turn, allows faster viral 'surfing' of the epithelium and receptor scanning by SARS-CoV-2. Angiotensin-converting enzyme 2 (ACE-2) protein on the epithelial surface is the primary entry receptor for SARS-CoV-2, and protein-protein interaction assays demonstrate high-affinity binding of the spike protein (S protein) to ACE-2. To date, no high-frequency mutations were detected at the C-terminal domain of the S1 subunit in the S protein, where the receptor-binding domain (RBD) is located. Tight binding to ACE-2 by a conserved viral RBD suggests the ACE2-RBD interaction is likely optimal. Moreover, the viral S subunit contains a cleavage site for furin and other proteases, which accelerates cell entry by SARS-CoV-2. The model proposed here describes a structural basis for the accelerated host cell entry by SARS-CoV-2 relative to other HCoVs and also discusses emerging hypotheses that are likely to contribute to the development of antiviral strategies to combat the pandemic capacity of SARS-CoV-2.