Characterization of a neutralizing antibody that recognizes a loop region adjacent to the receptor-binding interface of the SARS-CoV-2 spike receptor-binding domain.

Although the global crisis caused by the coronavirus disease 2019 (COVID-19) pandemic is over, the global epidemic of the disease continues. Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the cause of COVID-19, initiates infection via the binding of the receptor-binding domain (RBD) of its spike protein to the human angiotensin-converting enzyme II (ACE2) receptor, and this interaction has been the primary target for the development of COVID-19 therapeutics. Here, we identified neutralizing antibodies against SARS-CoV-2 by screening mouse monoclonal antibodies and characterized an antibody, CSW1-1805, that targets a narrow region at the RBD ridge of the spike protein. CSW1-1805 neutralized several variants in vitro and completely protected mice from SARS-CoV-2 infection. Cryo-EM and biochemical analyses revealed that this antibody recognizes the loop region adjacent to the ACE2-binding interface with the RBD in both a receptor-inaccessible "down" state and a receptor-accessible "up" state and could stabilize the RBD conformation in the up-state. CSW1-1805 also showed different binding orientations and complementarity determining region properties compared to other RBD ridge-targeting antibodies with similar binding epitopes. It is important to continuously characterize neutralizing antibodies to address new variants that continue to emerge. Our characterization of this antibody that recognizes the RBD ridge of the spike protein will aid in the development of future neutralizing antibodies.IMPORTANCESARS-CoV-2 cell entry is initiated by the interaction of the viral spike protein with the host cell receptor. Therefore, mechanistic findings regarding receptor recognition by the spike protein help uncover the molecular mechanism of SARS-CoV-2 infection and guide neutralizing antibody development. Here, we characterized a SARS-CoV-2 neutralizing antibody that recognizes an epitope, a loop region adjacent to the receptor-binding interface, that may be involved in the conformational transition of the receptor-binding domain (RBD) of the spike protein from a receptor-inaccessible "down" state into a receptor-accessible "up" state, and also stabilizes the RBD in the up-state. Our mechanistic findings provide new insights into SARS-CoV-2 receptor recognition and guidance for neutralizing antibody development.

Electrolyzed hypochlorous acid water exhibits potent disinfectant activity against various viruses through irreversible protein aggregation.

It is essential to employ efficient measures to prevent the transmission of pathogenic agents during a pandemic. One such method involves using hypochlorous acid (HClO) solution. The oxidative properties of HClO water (HAW) can contribute to its ability to eliminate viral particles. Here, we examined a highly purified slightly acidic hypochlorous acid water (Hp-SA-HAW) obtained from the reverse osmosis membrane treatment of an electrolytically-generated SA-HAW for its anti-viral activity and mode of action on viral proteins. Hp-SA-HAW exhibited broad-spectrum antiviral effects against various viruses, including adenovirus, hepatitis B virus, Japanese encephalitis virus (JEV), and rotavirus. Additionally, Hp-SA-HAW treatment dose-dependently resulted in irreversibly aggregated multimers of the JEV envelope and capsid proteins. However, Hp-SA-HAW treatment had no discernible effect on viral RNA, indicating that Hp-SA-HAW acts against amino acids rather than nucleic acids. Furthermore, Hp-SA-HAW substantially reduced the infectivity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), including the ancestral variant and other multiple variants. Hp-SA-HAW treatment induced the aggregation of the SARS-CoV-2 spike and nuclear proteins and disrupted the binding of the purified spike protein of SARS-CoV-2 to human ACE2. This study demonstrates that the broad-spectrum virucidal activity of highly purified HClO is attributed to viral protein aggregation of virion via protein oxidation.

Epoxidized graphene grid for highly efficient high-resolution cryoEM structural analysis.

Functionalization of graphene is one of the most important fundamental technologies in a wide variety of fields including industry and biochemistry. We have successfully achieved a novel oxidative modification of graphene using photoactivated ClO2· as a mild oxidant and confirmed the oxidized graphene grid is storable with its functionality for at least three months under N2 atmosphere. Subsequent chemical functionalization enabled us to develop an epoxidized graphene grid (EG-grid™), which effectively adsorbs protein particles for electron cryomicroscopy (cryoEM) image analysis. The EG-grid dramatically improved the particle density and orientation distribution. The density maps of GroEL and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were reconstructed at 1.99 and 2.16 Å resolution from only 504 and 241 micrographs, respectively. A sample solution of 0.1 mg ml-1 was sufficient to reconstruct a 3.10 Å resolution map of SARS-CoV-2 spike protein from 1163 micrographs. The map resolutions of ß-galactosidase and apoferritin easily reached 1.81 Å and 1.29 Å resolution, respectively, indicating its atomic-resolution imaging capability. Thus, the EG-grid will be an extremely powerful tool for highly efficient high-resolution cryoEM structural analysis of biological macromolecules.

Structural insights into the rational design of a nanobody that binds with high affinity to the SARS-CoV-2 spike variant.

The continuous emergence of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) variants associated with the adaptive evolution of the virus is prolonging the global coronavirus disease 2019 (COVID-19) pandemic. The modification of neutralizing antibodies based on structural information is expected to be a useful approach to rapidly combat emerging variants. A dimerized variable domain of heavy chain of heavy chain antibody (VHH) P17 that has highly potent neutralizing activity against SARS-CoV-2 has been reported but the mode of interaction with the epitope remains unclear. Here, we report the X-ray crystal structure of the complex of monomerized P17 bound to the SARS-CoV-2 receptor binding domain (RBD) and investigated the binding activity of P17 toward various variants of concern (VOCs) using kinetics measurements. The structure revealed details of the binding interface and showed that P17 had an appropriate linker length to have an avidity effect and recognize a wide range of RBD orientations. Furthermore, we identified mutations in known VOCs that decrease the binding affinity of P17 and proposed methods for the acquisition of affinity toward the Omicron RBD because Omicron is currently the most predominant VOC. This study provides information for the rational design of effective VHHs for emerging VOCs.

Establishment and clinical application of SARS-CoV-2 catch column.

BACKGROUND: A certain number of patients with coronavirus disease 2019 (COVID-19), particularly those who test positive for SARS-CoV-2 in the serum, are hospitalized. Further, some even die. We examined the effect of blood adsorption therapy using columns that can eliminate SARS-CoV-2 on the improvement of the prognosis of severe COVID-19 patients. METHODS: This study enrolled seven patients receiving mechanical ventilation. The patients received viral adsorption therapy using SARS-catch column for 3 days. The SARS-catch column was developed by immobilizing a specific peptide, designed based on the sequence of human angiotensin-converting enzyme 2 (hACE2), to an endotoxin adsorption column (PMX). In total, eight types of SARS-CoV-2-catch (SCC) candidate peptides were developed. Then, a clinical study on the effects of blood adsorption therapy using the SARS-catch column in patients with severe COVID-19 was performed, and the data in the present study were compared with historical data of severe COVID-19 patients. RESULTS: Among all SCC candidate peptides, SCC-4N had the best adsorption activity against SARS-CoV-2. The SARS-catch column using SCC-4N removed 65% more SARS-CoV-2 than PMX. Compared with historical data, the weaning time from mechanical ventilation was faster in the present study. In addition, the rate of negative blood viral load in the present study was higher than that in the historical data. CONCLUSION: The timely treatment with virus adsorption therapy may eliminate serum SARS-CoV-2 and improve the prognosis of patients with severe COVID-19. However, large-scale studies must be performed in the future to further assess the finding of this study (jRCTs052200134).

Human antibody recognition and neutralization mode on the NTD and RBD domains of SARS-CoV-2 spike protein.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19). Variants of concern (VOCs) such as Delta and Omicron have developed, which continue to spread the pandemic. It has been reported that these VOCs reduce vaccine efficacy and evade many neutralizing monoclonal antibodies (mAbs) that target the receptor binding domain (RBD) of the glycosylated spike (S) protein, which consists of the S1 and S2 subunits. Therefore, identification of optimal target regions is required to obtain neutralizing antibodies that can counter VOCs. Such regions have not been identified to date. We obtained 2 mAbs, NIBIC-71 and 7G7, using peripheral blood mononuclear cells derived from volunteers who recovered from COVID-19. Both mAbs had neutralizing activity against wild-type SARS-CoV-2 and Delta, but not Omicron. NIBIC-71 binds to the RBD, whereas 7G7 recognizes the N-terminal domain of the S1. In particular, 7G7 inhibited S1/S2 cleavage but not the interaction between the S protein and angiotensin-converting enzyme 2; it suppressed viral entry. Thus, the efficacy of a neutralizing mAb targeting inhibition of S1/2 cleavage was demonstrated. These results suggest that neutralizing mAbs targeting blockade of S1/S2 cleavage are likely to be cross-reactive against various VOCs.

A panel of nanobodies recognizing conserved hidden clefts of all SARS-CoV-2 spike variants including Omicron.

We are amid the historic coronavirus infectious disease 2019 (COVID-19) pandemic. Imbalances in the accessibility of vaccines, medicines, and diagnostics among countries, regions, and populations, and those in war crises, have been problematic. Nanobodies are small, stable, customizable, and inexpensive to produce. Herein, we present a panel of nanobodies that can detect the spike proteins of five SARS-CoV-2 variants of concern (VOCs) including Omicron. Here we show via ELISA, lateral flow, kinetic, flow cytometric, microscopy, and Western blotting assays that our nanobodies can quantify the spike variants. This panel of nanobodies broadly neutralizes viral infection caused by pseudotyped and authentic SARS-CoV-2 VOCs. Structural analyses show that the P86 clone targets epitopes that are conserved yet unclassified on the receptor-binding domain (RBD) and contacts the N-terminal domain (NTD). Human antibodies rarely access both regions; consequently, the clone buries hidden crevasses of SARS-CoV-2 spike proteins that go undetected by conventional antibodies.

Secretory glycoprotein NS1 plays a crucial role in the particle formation of flaviviruses.

Flaviviruses, which are globally distributed and cause a spectrum of potentially severe illnesses, pose a major threat to public health. Although Flaviviridae viruses, including flaviviruses, possess similar genome structures, only the flaviviruses encode the non-structural protein NS1, which resides in the endoplasmic reticulum (ER) and is secreted from cells after oligomerization. The ER-resident NS1 is known to be involved in viral genome replication, but the essential roles of secretory NS1 in the virus life cycle are not fully understood. Here we characterized the roles of secretory NS1 in the particle formation of flaviviruses. We first identified an amino acid residue essential for the NS1 secretion but not for viral genome replication by using protein-protein interaction network analyses and mutagenesis scanning. By using the recombinant flaviviruses carrying the identified NS1 mutation, we clarified that the mutant flaviviruses employed viral genome replication. We then constructed a recombinant NS1 with the identified mutation and demonstrated by physicochemical assays that the mutant NS1 was unable to form a proper oligomer or associate with liposomes. Finally, we showed that the functions of NS1 that were lost by the identified mutation could be compensated for by the in trans-expression of Erns of pestiviruses and host exchangeable apolipoproteins, which participate in the infectious particle formation of pestiviruses and hepaciviruses in the family Flaviviridae, respectively. Collectively, our study suggests that secretory NS1 plays a role in the particle formation of flaviviruses through its interaction with the lipid membrane.

Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). Although multiple mutations have been observed in SARS-CoV-2, functional analysis of each mutation of SARS-CoV-2 has been limited by the lack of convenient mutagenesis methods. In this study, we establish a PCR-based, bacterium-free method to generate SARS-CoV-2 infectious clones. Recombinant SARS-CoV-2 could be rescued at high titer with high accuracy after assembling 10 SARS-CoV-2 cDNA fragments by circular polymerase extension reaction (CPER) and transfection of the resulting circular genome into susceptible cells. The construction of infectious clones for reporter viruses and mutant viruses could be completed in two simple steps: introduction of reporter genes or mutations into the desirable DNA fragments (∼5,000 base pairs) by PCR and assembly of the DNA fragments by CPER. This reverse genetics system may potentially advance further understanding of SARS-CoV-2.

Synthesis of low-molecular weight fucoidan derivatives and their binding abilities to SARS-CoV-2 spike proteins.

Fucoidan derivatives 10-13, whose basic sugar chains are composed of repeating α(1,4)-linked l-fucopyranosyl residues with different sulfation patterns, were designed and systematically synthesized. A structure-activity relationship (SAR) study examined competitive inhibition by thirteen fucoidan derivatives against heparin binding to the SARS-CoV-2 spike (S) protein. The results showed for the first time that 10 exhibited the highest inhibitory activity of the fucoidan derivatives used. The inhibitory activity of 10 was much higher than that of fondaparinux, the reported ligand of SARS-CoV-2 S protein. Furthermore, 10 exhibited inhibitory activities against the binding of heparin with several mutant SARS-CoV-2 S proteins, but was found to not inhibit factor Xa (FXa) activity that could otherwise lead to undesirable anticoagulant activity.

Ponesimod suppresses hepatitis B virus infection by inhibiting endosome maturation.

The discovery of novel antivirals to treat hepatitis B virus (HBV) infection is urgently needed, as the currently available drugs mainly target viral proteins at replication step, whereas host factors also play significant roles in HBV infection. Although numerous studies have reported candidate drugs for HBV treatment, there remains a need to find a new drug that may target other steps of the HBV life cycle. In this study, by drug screening of a 533 G-protein-coupled receptors (GPCRs)-associated compound library, we identified ponesimod, a selective agonist of sphingosine-1-phosphate receptor 1 (S1P1), as a drug candidate for the suppression of HBV infection. However, the anti-HBV effect of ponesimod is independent of S1P1 and other sphingosine-1-phosphate receptors (S1PRs). Treatment with ponesimod at an early step of infection but not at a post-entry step significantly reduced the HBV relaxed circular DNA (rcDNA) level in a dose-dependent manner. Ponesimod treatment did not inhibit attachment, binding, or internalization of HBV particles via endocytosis through an interaction with sodium taurocholate cotransporting polypeptide (NTCP) or epidermal growth factor receptor (EGFR). Importantly, during the transportation of HBV particles to the nucleus, co-localization of HBV with early endosomes but not with late endosomes and lysosomes was induced by the treatment with ponesimod, suggesting that ponesimod interferes with the conversion of early endosomes to late endosomes without significant damage to cellular growth. Conclusion: Ponesimod is a promising anti-HBV drug targeting the endosome maturation of HBV. This finding can be applied to the development of novel antivirals that target the trafficking pathway of HBV particles.

Oxidative misfolding of Cu/Zn-superoxide dismutase triggered by non-canonical intramolecular disulfide formation.

Misfolded Cu/Zn-superoxide dismutase (SOD1) is a pathological species in a subset of amyotrophic lateral sclerosis (ALS). Oxidative stress is known to increase in affected spinal cords of ALS and is thus considered to cause damages on SOD1 leading to the misfolding and aggregation. Despite this, it still remains elusive what triggers misfolding of SOD1 under oxidizing environment. Here, we show that a thiol group of Cys111 in SOD1 is oxidized to a sulfenic acid with hydrogen peroxide and reveal that further dissociation of the bound metal ions from the oxidized SOD1 allows another free Cys residue (Cys6) to nucleophilically attack the sulfenylated Cys111. As a result, an intra-molecular disulfide bond forms between Cys6 and Cys111. Such an abnormal SOD1 with the non-canonical disulfide bond was conformationally extended with significant cytotoxicity as well as high propensity to aggregate. Taken together, we propose a new model of SOD1 misfolding under oxidizing environment, in which formation of the non-canonical intramolecular disulfide bond plays a pivotal role.

Immunochemical characterization on pathological oligomers of mutant Cu/Zn-superoxide dismutase in amyotrophic lateral sclerosis.

BACKGROUND: Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (SOD1-ALS) with accumulation of misfolded SOD1 proteins as intracellular inclusions in spinal motor neurons. Oligomerization of SOD1 via abnormal disulfide crosslinks has been proposed as one of the misfolding pathways occurring in mutant SOD1; however, the pathological relevance of such oligomerization in the SOD1-ALS cases still remains obscure. METHODS: We prepared antibodies exclusively recognizing the SOD1 oligomers cross-linked via disulfide bonds in vitro. By using those antibodies, immunohistochemical examination and ELISA were mainly performed on the tissue samples of transgenic mice expressing mutant SOD1 proteins and also of human SOD1-ALS cases. RESULTS: We showed the recognition specificity of our antibodies exclusively toward the disulfide-crosslinked SOD1 oligomers by ELISA using various forms of purified SOD1 proteins in conformationally distinct states in vitro. Furthermore, the epitope of those antibodies was buried and inaccessible in the natively folded structure of SOD1. The antibodies were then found to specifically detect the pathological SOD1 species in the spinal motor neurons of the SOD1-ALS patients as well as the transgenic model mice. CONCLUSIONS: Our findings here suggest that the SOD1 oligomerization through the disulfide-crosslinking associates with exposure of the SOD1 structural interior and is a pathological process occurring in the SOD1-ALS cases.

A misfolded dimer of Cu/Zn-superoxide dismutase leading to pathological oligomerization in amyotrophic lateral sclerosis.

Misfolding of mutant Cu/Zn-superoxide dismutase (SOD1) is a pathological hallmark in a familial form of amyotrophic lateral sclerosis. Pathogenic mutations have been proposed to monomerize SOD1 normally adopting a homodimeric configuration and then trigger abnormal oligomerization of SOD1 proteins. Despite this, a misfolded conformation of SOD1 leading to the oligomerization at physiological conditions still remains ambiguous. Here, we show that, around the body temperature (∼37°C), mutant SOD1 maintains a dimeric configuration but lacks most of its secondary structures. Also, such an abnormal SOD1 dimer with significant structural disorder was prone to irreversibly forming the oligomers crosslinked via disulfide bonds. The disulfide-crosslinked oligomers of SOD1 were detected in the spinal cords of the diseased mice expressing mutant SOD1. We hence propose an alternative pathway of mutant SOD1 misfolding that is responsible for oligomerization in the pathologies of the disease.

Screening of Drugs Inhibiting In vitro Oligomerization of Cu/Zn-Superoxide Dismutase with a Mutation Causing Amyotrophic Lateral Sclerosis.

Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) gene have been shown to cause a familial form of amyotrophic lateral sclerosis (SOD1-ALS). A major pathological hallmark of this disease is abnormal accumulation of mutant SOD1 oligomers in the affected spinal motor neurons. While no effective therapeutics for SOD1-ALS is currently available, SOD1 oligomerization will be a good target for developing cures of this disease. Recently, we have reproduced the formation of SOD1 oligomers abnormally cross-linked via disulfide bonds in a test tube. Using our in vitro model of SOD1 oligomerization, therefore, we screened 640 FDA-approved drugs for inhibiting the oligomerization of SOD1 proteins, and three effective classes of chemical compounds were identified. Those hit compounds will provide valuable information on the chemical structures for developing a novel drug candidate suppressing the abnormal oligomerization of mutant SOD1 and possibly curing the disease.

Conformational Disorder of the Most Immature Cu, Zn-Superoxide Dismutase Leading to Amyotrophic Lateral Sclerosis.

Misfolding of Cu,Zn-superoxide dismutase (SOD1) is a pathological change in the familial form of amyotrophic lateral sclerosis caused by mutations in the SOD1 gene. SOD1 is an enzyme that matures through the binding of copper and zinc ions and the formation of an intramolecular disulfide bond. Pathogenic mutations are proposed to retard the post-translational maturation, decrease the structural stability, and hence trigger the misfolding of SOD1 proteins. Despite this, a misfolded and potentially pathogenic conformation of immature SOD1 remains obscure. Here, we show significant and distinct conformational changes of apoSOD1 that occur only upon reduction of the intramolecular disulfide bond in solution. In particular, loop regions in SOD1 lose their restraint and become significantly disordered upon dissociation of metal ions and reduction of the disulfide bond. Such drastic changes in the solution structure of SOD1 may trigger misfolding and fibrillar aggregation observed as pathological changes in the familial form of amyotrophic lateral sclerosis.

A primary role for disulfide formation in the productive folding of prokaryotic Cu,Zn-superoxide dismutase.

Enzymatic activation of Cu,Zn-superoxide dismutase (SOD1) requires not only binding of a catalytic copper ion but also formation of an intramolecular disulfide bond. Indeed, the disulfide bond is completely conserved among all species possessing SOD1; however, it remains obscure how disulfide formation controls the enzymatic activity of SOD1. Here, we show that disulfide formation is a primary event in the folding process of prokaryotic SOD1 (SodC) localized to the periplasmic space. Escherichia coli SodC was found to attain ß-sheet structure upon formation of the disulfide bond, whereas disulfide-reduced SodC assumed little secondary structure even in the presence of copper and zinc ions. Moreover, reduction of the disulfide bond made SodC highly susceptible to proteolytic degradation. We thus propose that the thiol-disulfide status in SodC controls the intracellular stability of this antioxidant enzyme and that the oxidizing environment of the periplasm is required for the enzymatic activation of SodC.