Virus-Free and Live-Cell Visualizing SARS-CoV-2 Cell Entry for Studies of Neutralizing Antibodies and Compound Inhibitors

新型冠状病毒SARS-CoV-2在全球蔓延，给全球公共卫生带来严重威胁。快速研制疫苗、抗体和治疗药物成为科学界面临的重大挑战。由于SARS-CoV-2的高度传染性，采用病毒感染模型进行中和抗体及小分子抑制剂的药效评估需要在高等级生物安全实验室中进行，且常需要数天时间才能完成检测，限制了抗体和药物筛选的效率。发展快速、可视、不依赖于活病毒的新冠病毒入胞检测探针和细胞模型，对于加速新冠病毒抗体和药物的研究有重要意义。夏宁邵教授团队通过CHO真核表达系统高效表达制备出C端融合抗酸荧光蛋白Gamillus的重组新冠病毒spike蛋白STG。STG经SEC分子筛和冷冻电镜确认呈现与天然病毒刺突高度相似的三聚体结构，且与ACE2有很高的亲和力（18.2nM）。STG具备良好的细胞相容性和荧光性质，研究者进一步开发了可定量测定感染恢复期血清、疫苗免疫血清中和抗体（入胞阻断抗体）水平的CSBT检测方法。除了抗体检测评估方面的应用外，该研究发展的探针和模型还可用于筛选分析抑制新冠病毒入胞及胞内转运的小分子化合物。 我校博士后张雅丽，博士生王邵娟、巫洋涛，博士后侯汪衡、袁伦志和深圳市第三人民医院沈晨光博士为共同第一作者。厦门大学夏宁邵教授、袁权教授、程通教授为该论文共同通讯作者。The ongoing corona virus disease 2019 (COVID-19) pandemic, caused by SARS-CoV-2 infection, has resulted in hundreds of thousands of deaths. Cellular entry of SARS-CoV-2, which is mediated by the viral spike protein and ACE2 receptor, is an essential target for the development of vaccines, therapeutic antibodies, and drugs. Using a mammalian cell expression system,a genetically engineered sensor of fluorescent protein (Gamillus)-fused SARS-CoV-2 spike trimer (STG) to probe the viral entry process is developed.In ACE2-expressing cells, it is found that the STG probe has excellent performance in the live-cell visualization of receptor binding, cellular uptake, and intracellular trafficking of SARS-CoV-2 under virus-free conditions. The new system allows quantitative analyses of the inhibition potentials and detailed influence of COVID-19-convalescent human plasmas, neutralizing antibodies and compounds, providing a versatile tool for high-throughput screening and phenotypic characterization of SARS-CoV-2 entry inhibitors. This approach may also be adapted to develop a viral entry visualization system for other viruses.This study was supported by National Natural Science Foundation of China (81993149041 for N.X.; 81902057 for Y.Z.; 81871316 and U1905205 for Q.Y.), the National Science and Technology Major Project of Infectious Diseases (No. 2017ZX10304402‐002‐003 for T.C. and No. 2017ZX10202203‐009 for Q.Y.), the National Science and Technology Major Projects for Major New Drugs Innovation and Development (No. 2018ZX09711003‐005‐003 for T.C.), the Science and Technology Major Project of Fujian (2020YZ014001), the Science and Technology Major Project of Xiamen (3502Z2020YJ01), and the Guangdong Basic and Applied Basic Research Foundation (2020A1515010368 for C.S.). 该研究得到了国家自然科学基金、传染病防治国家科技重大专项、福建省应急科技攻关项目和厦门应急科技攻关项目的支持

Revealing the Origin of Heterogeneous Phase Transition and Deformation Behavior in Au-Ag-Cu-Based Multicomponent Alloys

Local chemical heterogeneity of highly-concentrated multicomponent alloys has drawn much attention as it can produce novel material behaviors and remarkable properties. In Au-Ag-Cu-based multicomponent alloys, phase separation and ordering have long been recognized to correlate with grain boundaries (GBs), but there is still a lack of atomic-scale understanding of the heterogeneous phase transition and how the microstructures respond to deformation. In this paper, a joint experimental and theoretical study was conducted on a medium-entropy polycrystalline model alloy, which is a representative Au-Ag-Cu-based multicomponent alloy with important applications in fields such as photocatalyst and micro-/nano-electromechanical systems. The GB regions are observed to preferentially nucleate two-phase lamellar structures, which are softer than grain interiors featuring short-range-order and modulated morphologies. First-principles calculations suggest the GB segregation of Ag and depletion of Cu are energetically favorable, consequently creating compositions that facilitate phase separation and impede ordering. Calculations of elasticity-based mechanical properties, stacking fault and surface energies reveal the GB lamellar structures are intrinsically soft with heterogeneous deformation capabilities. Furthermore, design strategies based on GB segregation engineering and tuning the dual-phase compositions are proposed to control heterogeneities. The results provide new insights into GB segregation, phase nucleation precursor and mechanical properties of noble-metal multicomponent alloys

Revealing the Origin of Heterogeneous Phase Transition and Deformation Behavior in Au-Ag-Cu-Based Multicomponent Alloys

Local chemical heterogeneity of highly-concentrated multicomponent alloys has drawn much attention as it can produce novel material behaviors and remarkable properties. In Au-Ag-Cu-based multicomponent alloys, phase separation and ordering have long been recognized to correlate with grain boundaries (GBs), but there is still a lack of atomic-scale understanding of the heterogeneous phase transition and how the microstructures respond to deformation. In this paper, a joint experimental and theoretical study was conducted on a medium-entropy polycrystalline model alloy, which is a representative Au-Ag-Cu-based multicomponent alloy with important applications in fields such as photocatalyst and micro-/nano-electromechanical systems. The GB regions are observed to preferentially nucleate two-phase lamellar structures, which are softer than grain interiors featuring short-range-order and modulated morphologies. First-principles calculations suggest the GB segregation of Ag and depletion of Cu are energetically favorable, consequently creating compositions that facilitate phase separation and impede ordering. Calculations of elasticity-based mechanical properties, stacking fault and surface energies reveal the GB lamellar structures are intrinsically soft with heterogeneous deformation capabilities. Furthermore, design strategies based on GB segregation engineering and tuning the dual-phase compositions are proposed to control heterogeneities. The results provide new insights into GB segregation, phase nucleation precursor and mechanical properties of noble-metal multicomponent alloys

Additional file 1 of A potent synthetic nanobody with broad-spectrum activity neutralizes SARS-CoV-2 virus and the Omicron variant BA.1 through a unique binding mode

Additional file 1: Table S1. Epitope binning of spike nanobodies. Table S2. Cryo-EM data collection, refinement and validation statistics. Table S3. Conservation of epitopic residues in RBD for C5G2 binding from natural occurring SARS-Cov-2 variants

supplementary_Figure_1 - Efficacy and Safety of Non-Vitamin K Antagonist Oral Anticoagulants in Asians With Nonvalvular Atrial Fibrillation: A Network Meta-Analysis

supplementary_Figure_1 for Efficacy and Safety of Non-Vitamin K Antagonist Oral Anticoagulants in Asians With Nonvalvular Atrial Fibrillation: A Network Meta-Analysis by Qinmei Xiong, Cen Wang, Hualong Liu, Zhaochong Tan, Chen Chen, Juxiang Li, Gregory Y. H. Lip and Kui Hong in Clinical and Applied Thrombosis/Hemostasi

DataSheet_1_Potential of conserved antigenic sites in development of universal SARS-like coronavirus vaccines.docx

Given pandemic risks of zoonotic SARS-CoV-2 variants and other SARS-like coronaviruses in the future, it is valuable to perform studies on conserved antigenic sites to design universal SARS-like coronavirus vaccines. By using antibodies obtained from convalescent COVID-19 patients, we succeeded in functional comparison of conserved antigenic sites at multiple aspects with each other, and even with SARS-CoV-2 unique antigenic sites, which promotes the cognition of process of humoral immune response to the conserved antigenic sites. The conserved antigenic sites between SARS-CoV-2 and SARS-CoV can effectively induce affinity maturation of cross-binding antibodies, finally resulting in broadly neutralizing antibodies against multiple variants of concern, which provides an important basis for universal vaccine design, however they are subdominant, putatively due to their lower accessibility relative to SARS-CoV-2 unique antigenic sites. Furthermore, we preliminarily design RBDs to improve the immunogenicity of these conserved antigenic sites. Our study focusing on conserved antigenic sites provides insights for promoting the development of universal SARS-like coronavirus vaccines, thereby enhancing our pandemic preparedness.</p