新型冠状病毒刺突蛋白的变异及其对中和活性的影响

2019年底发生的COVID-19疫情迅速传播至世界各地, 使全球公共卫生体系面临严峻考验。随着疫情的持续, 新型冠状病毒(severe acute respiratory syndrane coronavirus 2, SARS-CoV-2)变异株正在不断涌现。特别是病毒刺突蛋白的突变, 可以引起病毒感染性和抗原性改变, 从而导致病毒传染性增加, 以及现有疫苗保护效力的下降, 由此引起了流行毒株的替换。这也是目前疫情未得到有效控制的原因之一。目前主要的流行变异株都出现了一定程度的特性改变, 部分变异株与原始株相比对中和单抗、免疫血清及恢复期血清的中和敏感性出现了一定程度的下降。变异株的产生既与病毒本身特点有关, 也与传播宿主改变、免疫低下人群的慢性感染有关。应密切关注和监测不断出现的变异株, 并对其功能特性进行系统研究, 为疫苗研发和免疫策略的制定提供参考。     

新型冠状病毒疫苗及抗体药物研发进展EI北大核心CSCD

新型冠状病毒肺炎(COVID-19)疫情在全球的肆虐,不仅对公众的健康造成了重大威胁,也对全球卫生医疗系统和社会经济发展带来了巨大冲击。因此,加快疫苗和抗体药物的研发,为人们提供有效的保护和治疗措施,成为广大科学研究人员和医疗机构的首要任务。目前全球已有多款新冠疫苗和抗体药物进入临床研究或获批上市,本文对多款利用基因工程技术研发的基于S蛋白的新冠疫苗和抗体(包括亚单位疫苗、病毒载体疫苗、DNA疫苗、mRNA疫苗以及一些中和抗体药物)进行了简要阐述,同时也对疫苗和抗体药物的未来发展进行了探讨。     

SARS冠状病毒刺突蛋白的研究进展

SARS冠状病毒S蛋白是病毒颗粒最大的结构蛋白和最重要的表面抗原。该蛋白在介导病毒颗粒与宿主细胞受体结合、融合的过程中起着关键作用。它是病毒中和性抗体的靶点,在疫苗研究和抗病毒治疗研究中均占有重要地位。本文就SARS流行以来有关S蛋白的研究作一综述。     

预测的SARS冠状病毒刺突蛋白S2亚基B细胞表位肽在大肠杆菌中的表达及其抗原性的鉴定

目的研究预测的SARS冠状病毒刺突蛋白S2亚基B细胞表位肽在大肠杆菌中的表达及其模拟S2蛋白的抗原性。方法用DNAStar软件对S2亚基序列进行分析,预测B细胞表位所在肽段。通过4轮PCR反应人工搭建预测表位cDNA序列并克隆到含有伴侣10基因的pET28a( )载体中,构成重组载体pET28-chap10-S2epi。重组蛋白Chap10-S2epi在E.coliBL21(DE3)中表达并以SDS-PAGE和Westernblot进行鉴定。用纯化的chap10-S2epi免疫家兔制备抗血清,并通过ELISA判断Chap10-S2epi的抗原性。结果成功构建并在大肠杆菌中表达了Chap10-S2pei融合蛋白。Chap10-S2epi免疫的抗血清能识别真核细胞表达的SARS冠状病毒全长S2刺突蛋白。结论预测的SARS冠状病毒S2刺突蛋白B细胞表位肽能够诱导家兔产生针对S2蛋白的抗体,为研制抗SARS病毒基因工程疫苗奠定了基础。     

新型冠状病毒疫苗研发进展与分析

比较目前在全球范围内有代表性的新型冠状病毒候选疫苗,为新型冠状病毒疫苗研发提供更新的更全面的参考。本综述总结了新型冠状病毒疫苗的病毒学与免疫学基础,对部分基于不同技术的、有代表性的以及已经进入2a/3期临床试验的候选疫苗进行了详细的比较和分析。全球进入3期临床试验的多种新型冠状病毒疫苗免疫效果令人鼓舞,但疫苗免疫原性与...     

严重急性呼吸综合征冠状病毒2(SARS-CoV-2)刺突蛋白的B细胞及T细胞抗原表位的生物信息学分析

目的 使用生物信息学方法预测严重急性呼吸综合征冠状病毒2(SARS-CoV-2)刺突蛋白(S蛋白)的结构特点,并预测可能的B细胞和T细胞表位。方法 从NCBI数据库中获取SARS-CoV-2的S蛋白氨基酸序列,通过蛋白质基本性质分析工具ProtParam分析S蛋白的理化性质;利用综合性序列工具软件Lasergene和蛋白质二级结构分析软件SOMPA分析S蛋白二级结构;蛋白质同源物/类似物Y识别引擎Phyre2和分子图像观察软件Rasmol构建并分析S蛋白三级结构;B细胞表位预测工具ABCpred、 BepiPred和BcePred预测S蛋白的B细胞抗原表位;利用免疫表位数据库IEDB预测S蛋白的T细胞抗原表位。结果S蛋白由1273个氨基酸组成,理论等电点为6.24,原子组成为C₆₃₃₆H₉₇₇₀N₁₆₅₆O₁₈₉₄S₅₄,属于稳定亲水性蛋白。Lasergene软件的Gramier-Robson方法预测显示：α螺旋占23.5%、 β折叠占53.7%、转角区域占14.9%、无规则卷...     

新型冠状病毒疫苗不同接种剂次免疫效果研究

目的 了解郑州地区新型冠状病毒疫苗接种情况及接种后体内产生抗体情况,为新冠病毒的防控提供依据。方法收集1 437例病例的血清和个案资料信息,采用磁微粒化学发光法检测病例血清的新冠病毒IgG抗体水平,对病例的血清检测结果和个案资料信息进行统计分析。结果 0～3岁年龄组的307例全部未接种疫苗,4～6岁的学龄前儿童接种率为63.95%,7～59岁的接种率89.53%,60岁及以上老年人的接种率为70.00%。接种3剂次新冠病毒疫苗病例的IgG阳性率最高,为90.00%,60岁及以上的IgG阳性率明显低于18～59岁。男/女性别间接种新冠疫苗后产生IgG的总体阳性率无明显差别。4～17岁未成年组最后一针接种剂次有20.93%是在2022年内接种的,18岁及以上的成年组有58.70%为2022年内接种的。结论 郑州市4～6岁学龄前儿童和60岁以上老人的新冠疫苗接种率较低,多数4～17岁未成年人新冠疫苗最后剂次的接种时间为2022年以前,新冠抗体IgG阳性率较低。     

SARS冠状病毒棘突蛋白的S1蛋白诱导小鼠产生中和抗体的研究

目的 研究SARS冠状病毒棘突蛋白受体结合部位S1的免疫原性，为SARS的实验诊断和新型疫苗的研究提供依据。方法 用克隆有哺乳动物细胞密码子优化的SARS-CoV S1基因的质粒pcDNA3．1／S1或P-S1Ig转染293T细胞，用细胞的上清液纯化S1蛋白。以pcDNA3．1／S1质粒对BALB／c小鼠进行2次基因免疫，以纯化的S1蛋白进行加强免疫。用ELISA法检测小鼠抗SARS-CoV的特异性IgG抗体，并在Vero E6细胞上做体外中和实验，检测中和抗体。结果 S1蛋白诱导小鼠产生抗SARS-CoV的特异性抗体；1：1499．68稀释的S1蛋白免疫的小鼠血清可保护50％的细胞对1000TCID50的病毒攻击，而阴性对照血清不能保护细胞对病毒的感染。结论 SAPS冠状病毒棘突蛋白受体结合部位S1能有效诱导机体产生具有高效保护作用的中和抗体免疫反应，可望发展成为理想的SARS棘突蛋白亚单位疫苗。     

新型冠状病毒疫苗的研发进展及分析

随着新型冠状(简称:新冠)病毒疫情在全球愈演愈烈,越来越多的人们将疫情遏制的希望寄托于新冠病毒疫苗的研发。目前全球已经有多个研究团队,采用了不同的疫苗开发技术路线开展新冠病毒疫苗的研发。本文对目前不同路线新冠疫苗的开发与研究现状进行了综述和分析,同时也探讨了这些不同疫苗今后发展的可能性。     

Systemic and mucosal immunity in mice elicited by a single immunization with human adenovirus type 5 or 41 vector‐based vaccines carrying the spike protein of Middle East respiratory syndrome coronavirus

An ideal vaccine against mucosal pathogens such as Middle East respiratory syndrome coronavirus (MERS‐CoV) should confer sustained, protective immunity at both systemic and mucosal levels. Here, we evaluated the in vivo systemic and mucosal antigen‐specific immune responses induced by a single intramuscular or intragastric administration of recombinant adenoviral type 5 (Ad5) or type 41 (Ad41) ‐based vaccines expressing the MERS‐CoV spike (S) protein. Intragastric administration of either Ad5‐S or Ad41‐S induced antigen‐specific IgG and neutralizing antibody in serum; however, antigen‐specific T‐cell responses were not detected. In contrast, after a single intramuscular dose of Ad5‐S or Ad41‐S, functional antigen‐specific T‐cell responses were elicited in the spleen and pulmonary lymphocytes of the mice, which persisted for several months. Both rAd‐based vaccines administered intramuscularly induced systemic humoral immune responses (neutralizing IgG antibodies). Our results show that a single dose of Ad5‐S‐ or Ad41‐S‐based vaccines represents an appealing strategy for the control of MERS‐CoV infection and transmission.     

Emergence of novel coronavirus and progress toward treatment and vaccine

In late December 2019, a group of patients was observed with pneumonia‐like symptoms that were linked with a wet market in Wuhan, China. The patients were found to have a novel coronavirus genetically related to a bat coronavirus that was termed SARS‐CoV‐2. The virus gradually spread worldwide and was declared a pandemic by WHO. Scientists have started trials on potential preventive and treatment options. Currently, there is no specific approved treatment for SARS‐CoV‐2, and various clinical trials are underway to explore better treatments. Some previously approved antiviral and other drugs have shown some in vitro activity. Here we summarize the fight against this novel coronavirus with particular focus on the different treatment options and clinical trials exploring treatment as well as work done toward development of vaccines.     

SARS冠状病毒核衣壳蛋白的重组表达及其单克隆抗体的制备

目的: 表达SARS冠状病毒核衣壳蛋白(N蛋白),并以表达产物为免疫原制备特异性单克隆抗体 (mAb)。方法: 采用RT- PCR方法, 从灭活的病毒抗原标本中扩增编码N蛋白的基因。测序确认后, 再亚克隆至原核表达载体中。从SDS -PAGE凝胶中回收原核表达产物后免疫BALB/c小鼠, 经融合、筛选制备特异性mAb。结果: 从标本中扩增出 1 269bp的DNA, 测序结果证实为N蛋白基因。将该基因克隆至原核表达载体中后, 在大肠杆菌中获得了较好的表达。表达产物经SDS- PAGE后, 在Mr约为 43 000处可见 1条明显的诱导表达带。Westernblot的结果表明, 该电泳带与SARS患者的恢复期血清呈特异的免疫反应。从SDS- PAGE凝胶中回收表达产物并免疫BALB/c小鼠, 制备出 3株抗N蛋白的mAb。这些mAb与SDS- PAGE凝胶上Mr约为 43 000的蛋白带也呈现很强的免疫反应。结论: 所获的重组N蛋白及特异性mAb, 将为进一步建立SARS病毒感染早期诊断的方法奠定了基础。     

SARS-CoV S蛋白表位的表达及鉴定

目的:制备SARS冠状病毒S蛋白串联表位重组蛋白,为SARS的防治提供新型抗原蛋白。方法:应用抗原表位分析软件分析S蛋白的表位。选取其中16个表位,设计并合成表位串联重组蛋白编码基因Z,构建其原核细胞表达重组体,在大肠杆菌BL21(DE3)表达该重组表位蛋白Z,应用Ni离子亲合层析法纯化重组蛋白Z做抗原,采用皮下注射法免疫新西兰白兔。斑点杂交法测定抗Z-血清中S蛋白的抗体,ELISA法检测Z蛋白的抗原性。结果:构建了S蛋白重组表位蛋白Z的原核表达体,在BL21菌中表达了Z蛋白,Z蛋白免疫新西兰白兔后获得了抗Z血清。斑点杂交分析显示,抗Z血清识别哺乳动物细胞中表达的S蛋白。ELISA检测结果显示,抗Z血清识别SARS冠状病毒抗原。结论:建立了制备SARS冠状病毒S蛋白重组表位蛋白的方法,为防治SARS提供了新型抗原蛋白Z。     

Antibody isotype epitope mapping of SARS-CoV-2 Spike RBD protein: Targets for COVID-19 symptomatology and disease control

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) still poses a challenge for biomedicine and public health. To advance the development of effective diagnostic, prognostic, and preventive interventions, our study focused on high-throughput antibody binding epitope mapping of the SARS-CoV-2 spike RBD protein by IgA, IgM and IgG antibodies in saliva and sera of different cohorts from healthy uninfected individuals to SARS-CoV-2-infected unvaccinated and vaccinated asymptomatic, recovered, nonsevere, and severe patients. Identified candidate diagnostic (455-LFRKSNLKPFERD-467), prognostic (395-VYADSFVIRGDEV-407-C-KLH, 332-ITNLCPFGEV-342-C-KLH, 352-AWNRKRI-358-C-KLH, 524-VCGPKKSTNLVKN-536-KLH), and protective (MKLLE-487-NCYFPLQSYGFQPTNGVG-504-GGGGS-446-GGNYNYLYRLFRKSNLKPFERD-467) epitopes were validated with sera from prevaccine and postvaccine cohorts. The results identified neutralizing epitopes and support that antibody recognition of linear B-cell epitopes in RBD protein is associated with antibody isotype and disease symptomatology. The findings in asymptomatic individuals suggest a role for anti-RBD antibodies in the protective response against SARS-CoV-2. The possibility of translating results into diagnostic interventions for the early diagnosis of asymptomatic individuals and prognosis of disease severity provides new tools for COVID-19 surveillance and evaluation of risks in hospitalized patients. These results, together with other approaches, may contribute to the development of new vaccines for the control of COVID-19 and other coronavirus-related diseases using a quantum vaccinomics approach through the combination of protective epitopes.     

2019新型冠状病毒肺炎治疗研究现状

2019新型冠状病毒(2019 novel coronavirus,2019-nCoV)是继严重急性呼吸综合征冠状病毒(severe acute respiratory syndrome-coronavirus,SARS-CoV)和中东呼吸综合征冠状病毒(middle east respiratory syndrome-coronavirus,MERS-CoV)后出现的一种能引起人严重呼吸道疾病的新型冠状病毒。目前尚无特异性的靶向2019-nCoV的抗病毒治疗药物,面对新型冠状病毒肺炎日益严重的疫情和迫切需要解决的药物治疗策略,本文对新型冠状病毒肺炎抗病毒治疗研究现状与进展进行综述。     

新型冠状病毒肺炎的临床特征

本文总结了新型冠状病毒的特点,新型冠状病毒肺炎的发病特点、临床症状、实验室检查等临床特征,以及目前诊断标准包括临床症状、实验室检查、CT等,以提高公众对新型冠状病毒肺炎的认识和防控,同时为诊断研究提供基础。     

Spike gene deletion quasispecies in serum of patient with acute MERS‐CoV infection

The spike glycoprotein of the Middle East respiratory coronavirus (MERS‐CoV) facilitates receptor binding and cell entry. During investigation of a multi‐facility outbreak of MERS‐CoV in Taif, Saudi Arabia, we identified a mixed population of wild‐type and variant sequences with a large 530 nucleotide deletion in the spike gene from the serum of one patient. The out of frame deletion predicted loss of most of the S2 subunit of the spike protein leaving the S1 subunit with an intact receptor binding domain. This finding documents human infection with a novel genetic variant of MERS‐CoV present as a quasispecies. J. Med. Virol. 89:542–545, 2017 . © 2016 Wiley Periodicals, Inc.

     

Amino acid substitutions within the heptad repeat domain 1 of murine coronavirus spike protein restrict viral antigen spread in the central nervous system

Targeted recombination was carried out to select mouse hepatitis viruses (MHVs) in a defined genetic background, containing an MHV-JHM spike gene encoding either three heptad repeat 1 (HR1) substitutions (Q1067H, Q1094H, and L1114R) or L1114R alone. The recombinant virus, which expresses spike with the three substitutions, was nonfusogenic at neutral pH. Its replication was significantly inhibited by lysosomotropic agents, and it was highly neuroattenuated in vivo. In contrast, the recombinant expressing spike with L1114R alone mediated cell-to-cell fusion at neutral pH and replicated efficiently despite the presence of lysosomotropic agents; however, it still caused only subclinical morbidity and no mortality in animals. Thus, both recombinant viruses were highly attenuated and expressed viral antigen which was restricted to the olfactory bulbs and was markedly absent from other regions of the brains at 5 days postinfection. These data demonstrate that amino acid substitutions, in particular L1114R, within HR1 of the JHM spike reduced the ability of MHV to spread in the central nervous system. Furthermore, the requirements for low pH for fusion and viral entry are not prerequisites for the highly attenuated phenotype.     

Bovine coronavirus hemagglutinin protein

Treatment of purified bovine coronavirus (Mebus strain) with pronase destroyed the integrity of virion surface glycoproteins gp140, gp120, gp100, reduced the amount of gp26 and destroyed the hemagglutinating activity of the virus. Bromelain, on the other hand, destroyed the integrity of gp120, gp100 and gp26 but failed to remove gp140 and failed to destroy viral hemagglutinating activity. These experiments suggest that gp140 is the virion hemagglutinin. Immunoblotting studies using monospecific antiserum demonstrate that gp140 is a disulfide-linked dimeric structure reducible to monomers of 65 kDa.