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Screening and identification of HTNV_(pv)entry inhibitors with high-throughput pseudovirus-based chemiluminescence

Virologica Sinica 2022年第4期37卷 531-537页

作者： Xiaojing Wen Li Zhang Qiang Liu Xinyue Xiao weijin huang Youchun Wang Division of HIV/AIDS and Sex-transmitted Virus Vaccines Institute for Biological Product ControlNational Institutes for Food and Drug Control(NIFDC)and WHO Collaborating Center for Standardization and Evaluation of BiologicalsBeijing 102629China Department of Microbiological Laboratory Technology School of Public HealthShandong UniversityJinan 250012China Institute for Reference Standards and Standardization National Institutes for Food and Drug ControlBeijing 102629China

Hantaviruses,such as Hantaan virus(HTNV)and Seoul virus,are the causative agents of Hantavirus cardiopulmonary syndrome(HCPS)and hemorrhagic fever with renal syndrome(HFRS),and are important zoonotic *** has the highest incidence of HFRS,which is mainly caused by HTNV and Seoul *** approved antiviral drugs are available for these hantaviral ***,a chemiluminescence-based highthroughput-screening(HTS)assay was developed and used to screen HTNV pseudovirus(HTNVpv)inhibitors in a library of 1813 approved drugs and 556 small-molecule compounds from traditional Chinese medicine *** identified six compounds with in vitro anti-HTNVpvactivities in the low-micromolar range(EC50values of0.1–2.2μmol/L;selectivity index of 40–900).Among the six selected compounds,cepharanthine not only showed good anti-HTNVpvactivity in vitro but also inhibited HTNVpv-fluc infection in Balb/c mice 5 h after infection by94%(180 mg/kg/d,P<0.01),93%(90 mg/kg/d,P<0.01),or 92%(45 mg/kg/d,P<0.01),respectively,in a bioluminescent imaging mouse model.A time-of-addition analysis suggested that the antiviral mechanism of cepharanthine involves the membrane fusion and entry ***,we have established a HTS method for antiviral drugs screening,and shown that cepharanthine is a candidate for HCPS and HFRS *** findings may offer a starting point for the treatment of patients infected with hantaviruses.

关键词： Hantaan virus(HTNV) High-throughput screening(HTS) Inhibitor Drug repurposing

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Three amino acid residues in the envelope of human immunodeficiency virus type 1 CRF07_BC regulate viral neutralization susceptibility to the human monoclonal neutralizing antibody IgG1b12

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Virologica Sinica 2014年第5期29卷 299-307页

作者： Jianhui Nie Juan Zhao Qingqing Chen weijin huang Youchun Wang College of Life Science Jilin University Key Laboratory of the Ministry of Health for Research on Quality and Standardization of Biotech Products Division of HIV/AIDS and sex-transmitted virus vaccines National Institutes for Food and Drug Control (NIFDC)

The CD4 binding site(CD4bs) of envelope glycoprotein(Env) is an important conserved target for anti-human immunodeficiency virus type 1(HIV-1) neutralizing antibodies. Neutralizing monoclonal antibodies IgG1 b12(b12) could recognize conformational epitopes that overlap the CD4 bs of Env. Different virus strains, even derived from the same individual, showed distinct neutralization susceptibility to b12. We examined the key amino acid residues affecting b12 neutralization susceptibility using single genome amplification and pseudovirus neutralization assay. Eleven amino acid residues were identified that affect the sensitivity of Env to b12. Through site-directed mutagenesis, an amino acid substitution at position 182 in the V2 region of Env was confirmed to play a key role in regulating the b12 neutralization susceptibility. The introduction of V182 L to a resistant strain enhanced its sensitivity to b12 more than twofold. Correspondingly, the introduction of L182 V to a sensitive strain reduced its sensitivity to b12 more than tenfold. Amino acid substitution at positions 267 and 346 could both enhance the sensitivity to b12 more than twofold. However, no additive effect was observed when the three site mutageneses were introduced into the same strain, and the sensitivity was equivalent to the single V182 L mutation. CRF07_BC is a major circulating recombinant form of HIV-1 prevalent in China. Our data may provide important information for understanding the molecular mechanism regulating the neutralization susceptibility of CRF07_BC viruses to b12 and may be helpful for a vaccine design targeting the CD4 bs epitopes.

关键词： human immunodeficiency virus type 1 CRF07_BC envelope glycoprotein IgG1b12 neutralizing antibody single genome amplification

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Monitoring Neutralization Property Change of Evolving Hantaan and Seoul Viruses with a Novel Pseudovirus-Based Assay

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Virologica Sinica 2021年第1期36卷 104-112页

作者： Tingting Ning Ling Wang Shuo Liu Jian Ma Jianhui Nie weijin huang Xuguang Li Yuhua Li Youchun Wang Division of HIV/AIDS and Sexually Transmitted Virus Vaccines National Institutes for Food and Drug Control(NIFDC)Beijing 102629China Division of Arboviruses Vaccine National Institutes for Food and Drug Control(NIFDC)Beijing 102629China Centre for Biologics Evaluation Biologics and Genetic Therapies DirectorateHealth CanadaOttawaON K2J 4X9Canada

The Hantaan virus(HTNV)and Seoul virus(SEOV)mutants have accumulated over *** is important to determine whether their neutralizing epitopes have evolved,thereby making the current vaccine ***,it is impossible to determine by using traditional plaque reduction neutralization test(PRNT),because it requires large numbers of live mutant ***-based neutralization assays(PBNA)were developed by employing vesicular stomatitis virus(VSV)backbone incorporated with HTNV or SEOV glycoproteins(VSVDG*-HTNVG or VSVDG*-SEOVG).56 and 51 single amino acid substitutions of glycoprotein(GP)in HTNV and SEOV were selected and introduced into the reference *** the mutant pseudoviruses were generated and tested by *** PBNA results were highly correlated with PRNT ones with R2 being 0.91 for VSVDG*-HTNVG and 0.82 for VSVDG*-SEOVG.53 HTNV mutant pseudoviruses and 46 SEOV mutants were successfully ***,by using PBNA,we found that HTNV or SEOV immunized antisera could neutralize all the corresponding 53 HTNV mutants or the 46 SEOV mutants *** novel PBNA enables us to closely monitor the effectiveness of vaccines against large numbers of evolving HTNV and *** the current vaccine remains to be effective for the naturally occurring mutants.

关键词： Hemorrhagic fever with renal syndrome(HFRS) Hantaan virus(HTNV) Seoul virus(SEOV) Pseudovirus-based neutralization assay(PBNA) Amino acid substitution Vaccine

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A non-ACE2-blocking neutralizing antibody against Omicron-included SARS-CoV-2 variants

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Signal Transduction and Targeted Therapy 2022年第2期7卷 372-374页

作者： Xiaomin Duan Rui Shi Pulan Liu Qingrui huang Fengze Wang Xinyu Chen Hui Feng weijin huang Junyu Xiao Jinghua Yan CAS Key Laboratory of Pathogenic Microbiology and Immunology Institute of MicrobiologyChinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100049China State Key Laboratory of Protein and Plant Gene Research School of Life SciencesPeking-Tsinghua Center for Life SciencesBeijing Advanced Innovation Center for GenomicsPeking UniversityBeijing 100871China Shanghai Junshi Biosciences Co.Ltd Shanghai 200126China Division of HIV/AIDS and Sextransmitted Virus Vaccines Institute for Biological Product ControlNational Institutes for Food and Drug Control(NIFDC)Beijing 102629China

Dear Editor,The emergence of severe acute respiratory syndrome coronavirus-2(SARS-CoV-2)variants threatens efforts to contain the coronavirus disease 2019(COVID-19)***(B.1.1.529),the fifth novel SARS-CoV-2 variant of concern(VOC),harbors 15 mutations in the receptor-binding domain(RBD)of the spike(S)protein.1 These mutations include almost all the sites of existing VOCs(Alpha/B.1.1.7,Beta/B.1.351,Gamma/P.1,and Delta/B.1.617.2).2–4 Importantly,the key mutations directly interact with the ACE2 receptor and constitute the main target of neutralizing antibodies(NAbs),which is believed to alter the sensitivity to a large number of NAbs.

关键词： ACE2 acute neutral

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海藻玉壶汤治疗甲状腺结节的疗效与争议概述

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山东中医杂志 2024年第10期43卷 1164-1168页

作者：黄薇瑾曲笑啸何燕铭张丹杨宏杰上海中医药大学附属岳阳中西医结合医院内分泌科上海200437

甲状腺结节属中医学瘿病范畴,海藻玉壶汤是治疗瘿病的代表方之一。研究发现,海藻玉壶汤可以缩小甲状腺结节,改善患者临床症状。然而,由于碘盐普及后群众碘摄入量提高,甲状腺疾病谱发生了相应变化,临床对于是否能用以海藻玉壶汤为代表的... 详细信息

甲状腺结节属中医学瘿病范畴,海藻玉壶汤是治疗瘿病的代表方之一。研究发现,海藻玉壶汤可以缩小甲状腺结节,改善患者临床症状。然而,由于碘盐普及后群众碘摄入量提高,甲状腺疾病谱发生了相应变化,临床对于是否能用以海藻玉壶汤为代表的富碘复方治疗甲状腺结节颇有争议。本文对相关内容进行综述,为临床应用海藻玉壶汤治疗甲状腺结节提供参考。参考文献52篇。

关键词：甲状腺结节海藻玉壶汤碘自身免疫性甲状腺病甲状腺功能亢进

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Screening and Identification of Marburg Virus Entry Inhibitors Using Approved Drugs

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Virologica Sinica 2020年第2期35卷 235-239页

作者： Li Zhang Shan Lei Hui Xie Qianqian Li Shuo Liu Qiang Liu weijin huang Xinyue Xiao Youchun Wang Division of HIV/AIDS and Sexually-transmitted Virus Vaccines National Institutes for Food and Drug ControlBeijing 102629China Institute for Reference Standards and Standardization National Institutes for Food and Drug ControlBeijing 102629China

Dear Editor,Marburg virus(MARV)belongs to the Filoviridae family,along with the related Ebola virus(EBOV).Although MARV is less renowned than EBOV,it causes an equally devastating disease,with clinical symptoms similar to EBOV infection and a remarkably high mortality *** date,about 600 MARV cases have been reported globally,with an average mortality rate of around 80%(Brauburger et al.2012).Due to the severity of disease caused by MARV,as well as its threat to global public health,MARV is classified as a risk group 4 pathogen by WHO,necessitating containment measures equivalent to biosafety level 4(BSL-4).

关键词： mortality al. clinical

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Cellular tropism and antigenicity of mink-derived SARS-CoV-2 variants

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Signal Transduction and Targeted Therapy 2021年第6期6卷 1668-1670页

作者： Li Zhang Qianqian Li Jianhui Nie Ruxia Ding Haixin Wang Jiajing Wu Xuguang Li Xiaoming Yang weijin huang Youchun Wang Division of HIV/AIDS and Sex-transmitted Virus Vaccines Institute for Biological Product ControlNational Institutes for Food and Drug Control(NIFDC)and WHO Collaborating Center for Standardization and Evaluation of BiologicalsBeijingChina Centre for Biologies Evaluation Biologic and Radiopharmaceutical Drugs DirectorateHPFBHealth CanadaOttawaONCanada China National Biotec Group Company Limited BeijingChina

Dear Editor,The outbreak of SARS-CoV-2 in minks has been observed recently,raising serious concerns over cross-species transmission and the emergence of variants capable of rendering antibody therapy and vaccines less ***,the species tropism and antigenicity of the spike protein of ten variants were analyzed in pseudovirus-based assays involving 25 cell lines as well as 293T cells expressing ACE2 receptor from 14 species.

关键词： ACE2 mink variants

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基于稳定表达萤火虫荧光素酶Vero细胞系体外病毒中和活性检测方法的建立及应用

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中国生物制品学杂志 2024年第4期37卷 481-486,492页

作者：丁如霞李晓玉赵晨燕刘强姚佳维黄维金王佑春中国食品药品检定研究院北京102629 北京百奥赛图基因生物技术有限公司北京102629

目的建立一种基于稳定表达萤火虫荧光素酶(firefly luciferase,fluc)Vero细胞系的高通量检测方法,以评估样品的体外抗病毒中和活性。方法基于成簇规律间隔短回文重复序列(clustered regularly interspaced short palindromic repeats,CR... 详细信息

目的建立一种基于稳定表达萤火虫荧光素酶(firefly luciferase,fluc)Vero细胞系的高通量检测方法,以评估样品的体外抗病毒中和活性。方法基于成簇规律间隔短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)/Cas9靶向基因编辑技术,构建一种稳定表达fluc的Vero细胞系,在此基础上建立痘病毒和呼吸道合胞病毒(respiratory syncytial virus,RSV)感染细胞活性检测方法,利用该方法评估痘病毒免疫血清及RSV单抗的中和活性,并与噬斑抑制法及细胞活力检测(adenosine triphosphate,ATP)法检测结果进行比较。结果成功构建了Vero-fluc细胞系,其细胞量与表达的fluc相关性良好(R^(2)=0.9948)。利用Vero-fluc细胞作为感染细胞验证的痘病毒中和活性检测方法具有良好的稳定性、可靠性[信噪比(signal/background ratio,S/B)=25],可满足高通量的检测需求(Z’=0.9),相比传统噬斑抑制法一致性强(R2=0.76),免疫血清的ID50值较噬斑法平均高出2倍。建立的RSV中和活性检测方法,检测的抗RSV单抗中和活性与ATP活性试剂盒检测结果一致,Vero-fluc细胞法在1∶103~1∶105的抗体滴度水平上抑制率显著高于ATP法。结论建立的基于Vero-fluc细胞系的病毒中和活性检测方法与传统方法相比,具有良好的相关性,结果可靠,且具有更高的灵敏度及检测通量,是一种通用型体外抗病毒中和活性评价方法。

关键词：萤火虫荧光素酶 Vero细胞高通量检测抗病毒活性中和活性

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Durability of neutralizing antibodies and T-cell response post SARS-CoV-2 infection

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Frontiers of Medicine 2020年第6期14卷 746-751页

作者： Yun Tan Feng Liu Xiaoguang Xu Yun Ling weijin huang Zhaoqin Zhu Mingquan Guo Yixiao Lin Ziyu Fu Dongguo Liang Tengfei Zhang Jian Fan Miao Xu Hongzhou Lu Saijuan Chen Shanghai Institute of Hematology State Key Laboratory of Medical GenomicsNational Research Center for Translational Medicine at ShanghaiRuijin Hospital Affiliated to Shanghai Jiao Tong University School of MedicineShanghai 200025China Shanghai Public Health Clinical Center Shanghai 201508China National Institutes for Food and Drug Control(NIFDC) Beijing 102629China

The ongoing pandemic of coronavirus disease 19(COVID-19)is caused by a newly discoveredβcoronavirus named severe acute respiratory syndrome coronavirus 2(SARS-CoV-2).How long the adaptive immunity triggered by SARS-CoV-2 can last is of critical clinical relevance in assessing the probability of second infection and efficacy of *** we examined,using ELISA,the IgG antibodies in serum specimens collected from 17 COVID-19 patients at 6–7 months after diagnosis and the results were compared to those from cases investigated 2 weeks to 2 months *** samples were positive for IgGs against the S-and N-proteins of ***,14 samples available at 6–7 months post-infection all showed significant neutralizing activities in a pseudovirus assay,with no difference in blocking the cell-entry of the 614D and 614G variants of ***,in 10 blood samples from cases at 6–7 months post-infection used for memory T-cell tests,we found that interferonγ-producing CD4+and CD8+cells were increased upon SARS-CoV-2 antigen ***,these results indicate that durable anti-SARS-CoV-2 immunity is common in convalescent population,and vaccines developed from 614D variant may offer protection from the currently predominant 614D variant of SARS-CoV-2.

关键词： SARS-CoV-2 neutralizing antibodies T-cell response

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Monkeypox virus quadrivalent mRNA vaccine induces immune response and protects against vaccinia virus

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Signal Transduction and Targeted Therapy 2023年第5期8卷 2423-2433页

作者： Ye Sang Zhen Zhang Fan Liu Haitao Lu Changxiao Yu Huisheng Sun Jinrong Long Yiming Cao Jierui Mai Yiqi Miao Xin Wang Jiaxin Fang Youchun Wang weijin huang Jing Yang Shengqi Wang Bioinformatics center of AMMS Beijing100850P.R.China Division of HIV/AIDS and Sex-transmitted Virus Vaccines Institute for Biological Product ControlNational Institutes for Food and Drug Control(NIFDC)Beijing102629P.R.China Institute of Medical Biology Chinese Academy of Medical SciencesKunming650031P.R.China

Monkeypox has been declared a public health emergency by the World Health *** is an urgent need for efficient and safe vaccines against the monkeypox virus(MPXV)in response to the rapidly spreading monkeypox *** the age of COVID-19,mRNA vaccines have been highly successful and emerged as platforms enabling rapid development and large-scale ***,we develop two MPXV quadrivalent mRNA vaccines,named mRNA-A-LNP and mRNA-B-LNP,based on two intracellular mature virus specific proteins(A29L and M1R)and two extracellular enveloped virus specific proteins(A35R and B6R).By administering mRNA-A-LNP and mRNA-B-LNP intramuscularly twice,mice induce MPXV specific IgG antibodies and potent vaccinia virus(VACV)specific neutralizing ***,it elicits efficient MPXV specific Th-1 biased cellular immunity,as well as durable effector memory T and germinal center B cell responses in *** addition,two doses of mRNA-A-LNP and mRNA-B-LNP are protective against the VACV challenge in ***,the passive transfer of sera from mRNA-A-LNP and mRNA-B-LNP-immunized mice protects nude mice against the VACV ***,our results demonstrate that mRNA-A-LNP and mRNA-B-LNP appear to be safe and effective vaccine candidates against monkeypox epidemics,as well as against outbreaks caused by other orthopoxviruses,including the smallpox virus.

关键词： doses protective immunity

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