Structural Basis and Mode of Action for Two Broadly Neutralizing Antibodies Against SARS-CoV-2 Emerging Variants of Concern

Emerging variants of concern for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can transmit more efficiently and partially evade protective immune responses, thus necessitating continued refinement of antibody therapies and immunogen design. Here we elucidate the structural basis and mode of action for two potent SARS-CoV-2 Spike (S) neutralizing monoclonal antibodies CV3-1 and CV3-25 that remained effective against emerging variants of concern in vitro and in vivo. CV3-1 bound to the (485-GFN-487) loop within the receptor-binding domain (RBD) in the "RBD-up" position and triggered potent shedding of the S1 subunit. In contrast, CV3-25 inhibited membrane fusion by binding to an epitope in the stem helix region of the S2 subunit that is highly conserved among {beta}-coronaviruses. Thus, vaccine immunogen designs that incorporate the conserved regions in RBD and stem helix region are candidates to elicit pan-coronavirus protective immune responses.

Analysis of glycosylation and disulfide bonding of wild-type SARS-CoV-2 spike glycoprotein

The SARS-CoV-2 coronavirus, the etiologic agent of COVID-19, uses its spike (S) glycoprotein anchored in the viral membrane to enter host cells. The S glycoprotein is the major target for neutralizing antibodies elicited by natural infection and by vaccines. Approximately 35% of the SARS-CoV-2 S glycoprotein consists of carbohydrate, which can influence virus infectivity and susceptibility to antibody inhibition. We found that virus-like particles produced by coexpression of SARS-CoV-2 S, M, E and N proteins contained spike glycoproteins that were extensively modified by complex carbohydrates. We used a fucose-selective lectin to enrich the Golgi-resident fraction of a wild-type SARS-CoV-2 S glycoprotein trimer, and determined its glycosylation and disulfide bond profile. Compared with soluble or solubilized S glycoproteins modified to prevent proteolytic cleavage and to retain a prefusion conformation, more of the wild-type S glycoprotein N-linked glycans are processed to complex forms. Even Asn 234, a significant percentage of which is decorated by high-mannose glycans on soluble and virion S trimers, is predominantly modified in the Golgi by processed glycans. Three incompletely occupied sites of O-linked glycosylation were detected. Viruses pseudotyped with natural variants of the serine/threonine residues implicated in O-linked glycosylation were generally infectious and exhibited sensitivity to neutralization by soluble ACE2 and convalescent antisera comparable to that of the wild-type virus. Unlike other natural cysteine variants, a Cys15Phe (C15F) mutant retained partial, but unstable, infectivity. These findings enhance our understanding of the Golgi processing of the native SARS-CoV-2 S glycoprotein carbohydrates and could assist the design of interventions.

Spike glycoprotein and host cell determinants of SARS-CoV-2 entry and cytopathic effects

SARS-CoV-2, a betacoronavirus, is the cause of the COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein trimer mediates virus entry into host cells and cytopathic effects. We studied the contribution of several S glycoprotein features to these functions, focusing on those that differ among related coronaviruses. Acquisition of the furin cleavage site by the SARS-CoV-2 S glycoprotein decreased virus stability and infectivity, but greatly enhanced the ability to form lethal syncytia. Notably, the D614G change found in globally predominant SARS-CoV-2 strains restored infectivity, modestly enhanced responsiveness to the ACE2 receptor and susceptibility to neutralizing sera, and tightened association of the S1 subunit with the trimer. Apparently, two unique features of the SARS-CoV-2 S glycoprotein, the furin cleavage site and D614G, have evolved to balance virus infectivity, stability, cytopathicity and antibody vulnerability. Although the endodomain (cytoplasmic tail) of the S2 subunit was not absolutely required for virus entry or syncytium formation, alteration of palmitoylated cysteine residues in the cytoplasmic tail decreased the efficiency of these processes. As proteolytic cleavage contributes to the activation of the SARS-CoV-2 S glycoprotein, we evaluated the ability of protease inhibitors to suppress S glycoprotein function. Matrix metalloprotease inhibitors suppressed S-mediated cell-cell fusion, but not virus entry. Synergy between inhibitors of matrix metalloproteases and TMPRSS2 suggests that both proteases can activate the S glycoprotein during the process of syncytium formation. These results provide insights into SARS-CoV-2 S glycoprotein-host cell interactions that likely contribute to the transmission and pathogenicity of this pandemic agent. IMPORTANCEThe development of an effective and durable SARS-CoV-2 vaccine is essential for combating the growing COVID-19 pandemic. The SARS-CoV-2 spike (S) glycoprotein is the main target of neutralizing antibodies elicited during virus infection or following vaccination. Knowledge of the spike glycoprotein evolution, function and interactions with host factors will help researchers to develop effective vaccine immunogens and treatments. Here we identify key features of the spike glycoprotein, including the furin cleavage site and the D614G natural mutation, that modulate viral cytopathic effects, infectivity and sensitivity to inhibition. We also identify two inhibitors of host metalloproteases that block S-mediated cell-cell fusion, which contributes to the destruction of the virus-infected cell.

3D printing pre-operation assisted with custom-made plate for internal fixation of femoral shaft fractures after poliomyelitis / 中国组织工程研究

BACKGROUND: Patients with poliomyelitis often have abnormalities in the femur and the conventional plant matching degree of fracture is low. There are few reports on individualized treatment for severe deformity of femoral shaft fracture in poliomyelitis. OBJECTIVE: To summarize the clinical features of femoral shaft fracture in poliomyelitis and discuss the operative outcome of 3D printing pre-operation assisted with custom-made plate. METHODS: From May 2015 to May 2018, data of 19 patients with poliomyelitis surgically treated with 3D printing pre-operation assisted with custom-made plate were retrospectively analyzed. There were 11 males and 8 females with an average age of 52.5 years (range, 38-62 years). There were 7 cases on the left side, and 12 cases on the right side. According to AO/OTA classification of femoral shaft fracture, there were type A1 in 6 cases, type A2 in 3 cases, type A3 in 1 case, type B1 in 4 cases, type B2 in 4 cases and type C1 in 1 case. All patients signed the informed consent. This study was approved by the Hospital Ethics Committee. The 3D printed 1:1 femur model and custom-made plate were designed. Operation was simulated in vitro in femoral model. The placement of the plate was identified. The direction and length of the screws were measured and recorded. The final internal fixation of fracture was performed according to the pre-operation results. Hip and knee functions in the affected limb were assessed based on Harris evaluation criterion and hospital for special surgery knee score evaluation criterion. RESULTS AND CONCLUSION: (1) All 19 patients were followed up for 12-18 months. Bone healing was obtained in all fractures, with a healing time of 5-12 months, averaging 6.6 months. (2) Harris score was (84.95±5.18) before injury, and (84.42±4.83) 1 year after operation. Hospital for special surgery knee score was (84.53±4.36) before injury, and (83.63±3.90) 1 year after operation. Statistical analysis showed no statistical difference between the two groups before and after the injury (P > 0.05). (3) All patients had no internal fixation loosening or fracture. One patient had delayed bone healing and healed after iliac bone grafting. One patient developed wound infection after surgery, and received adequate drainage. Wound was healed after intravenous application of sensitive antibiotics. (4) Patients with poliomyelitis often have abnormalities in the femur with varying degrees of osteoporosis. For femoral shaft fractures that cannot be fixed with conventional internal fixation, 3D printing pre-operation combined with custom-made steel plate provides a new choice for the treatment of femoral shaft fractures in poliomyelitis.

Effects of flapless versus flap implant surgery on tissues surrounding implants in esthetic zone / 中华医学美学美容杂志

Objective To evaluate the effects of flapless versus flap implant surgery on the tissues surrounding the implants in the esthetic zone.Methods A controlled study was conducted in 20 patients with a single implant in the anterior esthetic zone,as compared with the flapless and traditional flap implant placement.6 and 12 months after the implant ation,the vertical reduction of labial and lingual bone were assessed by cone beam computed tomography (CBCT);6 and 12 months after the crowns restoration,the peri-implant soft tissues were examined by probing depth (PD),modification sulcus bleeding index (mSBI),papillary index (PPI) and evaluated by pink esthetic score (PES) standard.Results The vertical reduction of the flapless labial and lingual bone was lower than that of the flap,in which the change of labial bone was statistical differences at 6 months after implant surgery between groups (P ＜ 0.05).The differences of PES between two groups were not statistically significant,although PES increased over time in both groups,which the flapless group had a significantly change in PES from crown placement to 12 months (P＜0.05).Conclusions A flapless implant surgery in sufficient bone support may provide a better short-term esthetic result,whereas the longterm effects need further studies.