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.

Establishment of monoclonal antibodies broadly neutralize infection of hepatitis B virus.

Antibodies against hepatitis B virus S protein can protect against hepatitis B virus (HBV) infection. Therefore, hepatitis B immunoglobulin (HBIG), which contains HBsAb, is used clinically as a therapy for HBV infection. In this study, a series of monoclonal antibodies that recognize multiple HBV genotypes was obtained. All the antibodies recognized conformational epitopes of S protein, but not linear epitopes. Several antibodies neutralized HBV infection and exhibited strong affinities and neutralizing activities. Antigenic epitope analysis demonstrated that they recognized residue Ile152 of S protein, which is localized outside the "a" determinant. Ile152 is highly conserved, and a mutation in this residue resulted in reduced expression of large hepatitis B surface proteins (L protein), suggesting that the amino acid at this position is involved in the expression of L protein. In addition, the antibodies neutralized the infection of hepatitis D virus possessing a Gly145 mutation to Arg in S protein, which is a well-known escape mutation against HBIG treatment. Using mouse monoclonal antibodies, a humanized antibody possessing affinities and neutralizing activities similar to those of the original mouse antibody was successfully established. The antibodies generated in this study may have the potential for use in alternative antibody therapies for HBV infection.

Apical cell membranes are shed into urine from injured podocytes: a novel phenomenon of podocyte injury.

Previously it was shown that urine from patients with nephritis contains podocytes and their fragments (podocalyxin [PCX]-positive granular structures [PPGS]), reflecting the degree of podocyte injury. The present study was designed to trace PPGS to their origin. Urine samples and renal biopsy specimens from 53 children with nephrotic syndrome and nephritis were examined immunohistochemically. Immunofluorescence studies of kidney sections using an anti-PCX antibody demonstrated that PPGS originated from the glomerulus and flowed into the tubular lumen. Electron microscopic examination revealed that PPGS originated from microvillous or vesicle-like structures on injured podocytes in the glomerulus. For examining the origin of the PPGS, apical, slit-diaphragmatic, and basal portions of the podocytes were specifically stained, revealing that PPGS are composed primarily of apical podocyte membranes. Several newly developed antibodies that are reactive with various segments of the PCX molecule were used to analyze more detailed membrane structures, and it was found that PPGS contained intact PCX molecules, indicating that cell membrane structures are excreted in urine. The quantification of PCX content and podocyte numbers revealed that urinary sediment PCX (u-sed-PCX) content per urinary podocyte was much higher than PCX content per podocyte from isolated glomeruli of normal controls, suggesting that u-sed-PCX are derived from sources other than just the cell debris of detached podocytes. Analysis of the correlation between u-sed-PCX and renal histology revealed that the presence of PPGS reflects acute glomerular injury. In conclusion, podocyte apical cell membranes are shed into the urine from injured podocytes, indicating a previously unrecognized manifestation of podocyte injury.