抗H5亚型禽流感病毒单链抗体在毕赤酵母中的分泌型表达和生物活性分析

在本实验室研制出的多株针对H5N1亚型禽流感血凝素单抗中,13D4单抗对所有H5亚型病毒均有血凝抑制和中和活性,具有特异性高、反应性强和识别广的特点,且在小鼠实验中显示了对各种代表株禽流感的感染和发病均具有良好的治疗效果。在此研究基础上,本实验通过基因工程构建含有13D4单链抗体(scFv)基因的毕赤酵母表达载体,实现目的蛋白的分泌性表达和纯化。经过竞争法和血凝抑制检测其活性,表明获得的单链抗体具有与原始鼠源抗体相近的反应活性和相同的识别表位。H5N1广谱中和单抗13D4的单链抗体的成功构建,为进一步研制针对H5N1禽流感病毒的治疗性抗体奠定了基础。     

Pichia pastoris酵母中表达人源中和性抗甲型肝炎病毒scFv-Fc融合抗体的研究

为了探讨人源抗甲型肝炎（甲肝）病毒scFv—Fc融合抗体在酵母中的表达特性,将获得的人源抗甲肝病毒中和性单链可变区抗体（scFv抗体）基因克隆入含信号肽及人IgG1Fc抗体基因的酵母细胞表达载体中,获得了一株中和性人源抗甲肝病毒pPiscFv—FcHA16融合抗体的分泌表达,并对表达产物进行了纯化。同时对表达产物的生物学特性进行了一系列鉴定。表达的pPiscFv—FcHA16融合抗体为具有不同糖基化形式的同源二聚体,与相应的CHO细胞表达的IgG抗体相比,pPiscFv—FcHA16融合抗体仍保持很好的抗原结合活性,以及与中和性鼠抗甲肝病毒单克隆抗体的竞争抑制能力。同时也保持了对甲肝病毒的体外中和活性。这些结果表明,在酵母中表达的单链可变区（scFv）与IgG1Fc区的融合抗体具有很好的生物学活性,有希望用做体外诊断,用纯化相应的抗原,或者可能用于体内预防与治疗。     

抗H3N2亚型犬流感病毒重组犬源化抗体的制备及特性分析

由于异源抗体在宠物临床治疗中易引起宿主免疫排斥反应,因此制备基于本动物源的基因工程抗体显示了更大的发展优势和前景。为制备H3N2亚型犬流感病毒(Canine influenza virus,CIV)的重组犬源化抗体scFv-Fc,采集CIV感染犬外周血淋巴细胞提取总RNA,利用PCR技术扩增IgG重链(VH)、轻链(VL)和Fc片段。利用重叠延伸PCR将VH和VL通过弹性肽链linker连接成单链抗体(scFv)片段,并转化大肠杆菌,挑取阳性菌测序。经分析筛选后,将目标scFv与Fc片段链接,构建scFv-Fc重组犬源抗体片段。经诱导表达,获得分子量约为50 kD的scFv和约为89 kD的重组抗体scFv-Fc。抗体活性测定结果表明,scFv能与CIV特异性结合,1 mg/mL scFv的ELISA效价为1∶2~(10),血凝抑制(HI)效价为1∶2~7,中和抗体效价为1∶20。1 mg/mL scFv-Fc的HI效价为1∶2~6,ELISA效价1∶2~5,中和抗体效价为1∶20。本研究成功制备了针对H3N2亚型犬流感病毒的重组犬源scFv-Fc,为该病毒感染的治疗提供了物质基础。     

人源中和性抗高致病性禽流感病毒H5N1基因工程全抗体的研制

运用噬菌体表面呈现技术,从禽流感病人恢复期血中获得淋巴细胞,通过基因工程手段,构建了人源抗H5NI禽流感病毒基因工程抗体文库.用纯化的人源H5N1禽流感病毒颗粒(A/Anhui/1/2005)及重组血凝素蛋白HA(A/Viet Nam/1203/2004)对Fab噬菌体抗体库进行富集筛选,成功地获得了抗禽流感病毒H5N1血凝素蛋白HA的人源单抗Fab段基因,并在大肠杆菌中获得有效表达.通过序列测定确定抗体轻重链型别,然后将阳性克隆的轻链和重链Fd段基因分别克隆入全抗体表达载体pAC-L-Fc后转染昆虫Sf9细胞,利用杆状病毒/昆虫细胞系统实现全抗体的分泌型表达.用ELISA、IFA和流式细胞术对所获人源单抗的功能特性进行鉴定.结果表明,我们获得了2株特异性针对H5N1禽流感病毒血凝素蛋白HA而与甲1型和甲3型人流感病毒无交叉反应的人源单抗(AVFlulgG01、AVFlulgG03).微量中和试验结果表明,除A/Guangdong/1/2006外,AVFlu-IgG01能够广泛地中和HA基因进化上属于Clade 2的中国南方、北方及中部地区的H5N1禽流感病毒分离株,同时还对属于Clade Ⅰ的越南H5N1分离株A/Viet Nam/1203/2004具有中和活性;AVFluIgG03虽然不能中和A/Viet Nam/1203/2004,但是对属于Clade 2的所有中国H5N1分离株均具有中和作用.人源中和性抗禽流感病毒H5N1基因工程全抗体的获得不仅为高致病性禽流感病毒H5N1的预防和治疗带来了希望,同时也为其疫苗研制提供了新的思路.     

幽门螺旋杆菌单链抗体的筛选及鉴定

为了获得幽门螺旋杆菌特异性单链抗体scFv,通过噬菌体展示技术,首次直接用幽门螺旋杆菌细胞Hp对噬菌体单链抗体文库Tomlinson进行单链抗体的筛选,经5轮筛选后,通过ELISA方法检测,从随机挑选的96个克隆中获得了8株阳性克隆。再分别将阳性克隆与10种常见菌进行ELISA的交叉反应,最终得到1株特异性表达抗Hp的scFv的噬菌体JH1。随后又进一步对JH1所表达的scFv基因进行PCR扩增,分别得到scFv的VH片段、VL片段,全长基因分别为527bp、368bp和935bp,这些包含着部分载体序列的DNA片段与理论值相符。通过对scFv全长基因进行测序,在NCBI中进行基因序列比对,与已报道的一种植物RNA病毒的复制酶单链抗体基因序列有96％同源性。     

鼠源性人乳腺癌单链抗体库的构建与乳腺癌相关抗体的筛选

目的:获得乳腺癌的噬菌体呈现型单链抗体(scFv)库,筛选与乳腺癌细胞特异结合的抗体,为乳腺癌的诊断和治疗奠定基础。方法:用乳腺癌细胞系MCF-7、T47D、MDA-MB-435免疫BALB/c小鼠,取脾脏提取总RNA,用RT-PCR分别扩增抗体重、轻链可变区(VH和VL)基因,经Linker连接形成scFv基因片段。将scFv基因片段与噬菌粒载体pCANTAB5E的连接产物转化大肠杆菌TG1。用辅助噬菌体M13KO7进行超感染,获得重组噬菌体抗体。选用乳腺癌细胞系MCF-7和人正常肝细胞系HL02做正负差异的筛选细胞,通过5轮筛选,随机挑取克隆,经phage-ELISA筛选特异性结合MCF-7细胞的scFv。结果:构建了1个库容为1.3×106的单链抗体库。筛选到2株与MCF-7细胞有较高结合活性的噬菌体-单链抗体scFv-873和scFv-874。数据库搜索表明这2株单链抗体基因是与以往抗体序列不同的新基因。用Westernblot检测了这2株单链抗体在琥珀密码子非抑制型菌株TOP10中的表达情况。结论:筛选到2个与乳腺癌细胞结合特异性较好的单链抗体,为乳腺癌的诊断和治疗研究奠定了基础。     

一株抗H5亚型禽流感病毒血凝素蛋白单克隆抗体的广谱中和活性

以H5N1禽流感病毒株Ck/HK/Yu22/02作为抗原,应用常规杂交瘤技术和血凝抑制实验筛选出抗H5亚型禽流感病毒血凝素蛋白的单抗8H5,单抗8H5经免疫荧光鉴定具有很好的H5特异性.选择33株2002～2006年不同地域,不同宿主中分离的不同遗传变异亚系的H5N1病毒代表株,对单抗8H5分别进行血凝抑制实验及中和试验分析,结果显示单抗8H5对所有H5亚型病毒均有较强反应,而对非H5亚型标准病毒株均不反应,说明8H5是一株广谱性抗H5特异性中和单抗,并提示单抗8H5的HA识别表位可能是一个相当保守的中和表位.并且单抗8H5双抗夹心系统的初步评价显示了其在诊断应用上的前景.     

抗H5N1病毒嵌合IgA抗体基因的构建及其在CHO细胞中的表达

为了表达具有中和活性的抗禽流感H5N1病毒人-鼠嵌合IgA抗体,采用RT-PCR法克隆具有中和活性的抗禽流感H5N1-HA鼠源单克隆抗体的轻重链可变区基因及相应的信号肽编码序列,分别与人免疫球蛋白IgA2重链恒定区、Kappa恒定区基因拼接,构建表达质粒pEF-IGHA9和pEF-IGK9,共转染二氢叶酸还原酶缺陷型CHO(CHO-dhfr-)细胞,用ELISA检测培养上清中嵌合IgA抗体的表达,对纯化的嵌合抗体进行SDS-PAGE、Western blotting印迹分析。结果成功地在CHO细胞中表达了抗禽流感H5N1病毒人-鼠嵌合IgA抗体,为制备抗H5N1重组分泌型IgA预防性抗体制剂奠定了良好的基础。     

高亲和力抗乙型肝炎病毒PreS1的人源单链抗体的获得:天然及免疫抗体库的对比研究

以健康人的外周血淋巴细胞为来源,以偶联BSA的乙型肝炎病毒PreS1肽体外免疫.分别从免疫和未经免疫的淋巴细胞提取RNA,扩增抗体基因,构建大容量天然单链抗体(scFv)噬菌体展示文库和体外免疫scFv抗体库.以PreS1肽进行3轮淘选后,抗原抗体反应结果显示,从免疫库中获得了亲和力10-7～10-8 M的抗乙型肝炎病毒PreS1的单链抗体,高于天然库的结果(10-6～10-7 M).测序结果表明两株抗体均为人抗体.为基因工程抗体用于临床治疗乙型肝炎奠定基础.同时证明淋巴细胞体外免疫方法构建的免疫抗体库优于大容量天然抗体库.     

高亲和力抗乙型肝炎病毒PreS1的人源单链抗体的获得:天然及免疫抗体库的对比研究( 英文)

以健康人的外周血淋巴细胞为来源 ,以偶联BSA的乙型肝炎病毒PreS1肽体外免疫 .分别从免疫和未经免疫的淋巴细胞提取RNA ,扩增抗体基因 ,构建大容量天然单链抗体 (scFv)噬菌体展示文库和体外免疫scFv抗体库 .以PreS1肽进行 3轮淘选后 ,抗原抗体反应结果显示 ,从免疫库中获得了亲和力 10 -7～ 10 -8M的抗乙型肝炎病毒PreS1的单链抗体 ,高于天然库的结果 (10 -6～ 10 -7M ) .测序结果表明两株抗体均为人抗体 .为基因工程抗体用于临床治疗乙型肝炎奠定基础 .同时证明淋巴细胞体外免疫方法构建的免疫抗体库优于大容量天然抗体库 .     

Influenza Neuraminidase Subtype N1: Immunobiological Properties and Functional Assays for Specific Antibody Response

Influenza neuraminidase (NA) proteins expressed in TK⁻ cells infected with recombinant vaccinia virus carrying NA gene of highly pathogenic avian influenza H5N1 virus or 2009 pandemic H1N1 (H1N1pdm) virus were characterized for their biological properties, i.e., cell localization, molecular weight (MW), glycosylation and sialidase activity.Immune sera collected from BALB/c mice immunized with these recombinant viruses were assayed for binding and functional activities of anti-NA antibodies. Recombinant NA proteins were found localized in cytoplasm and cytoplasmic membrane of the infected cells. H1N1pdm NA protein had MW at about 75 kDa while it was 55 kDa for H5N1 NA protein. Hyperglycosylation was more pronounced in H1N1pdm NA compared to H5N1 NA according to N-glycosidase F treatment. Three dimensional structures also predicted that H1N1 NA globular head contained 4 and that of H5N1 contained 2 potential glycosylation sites. H5N1 NA protein had higher sialidase activity than H1N1pdm NA protein as measured by both MUNANA-based assay and fetuin-based enzyme-linked lectin assay (ELLA). Plaque reduction assay demonstrated that anti-NA antibody could reduce number of plaques and plaque size through inhibiting virus release, not virus entry. Assay for neuraminidase-inhibition (NI) antibody by ELLA showed specific and cross reactivity between H5N1 NA and H1N1pdm NA protein derived from reverse genetic viruses or wild type viruses. In contrast, replication-inhibition assay in MDCK cells showed that anti-H1N1 NA antibody moderately inhibited viruses with homologous NA gene only, while anti-H5N1 NA antibody modestly inhibited the replication of viruses containing homologous NA gene and NA gene derived from H1N1pdm virus. Anti-H1N1 NA antibody showed higher titers of inhibiting virus replication than anti-H5N1 NA antibody, which are consistent with the results on reduction in plaque numbers and sizes as well as in inhibiting NA enzymatic activity. No assay showed cross reactivity with reassorted PR8 (H1N1) virus and H3N2 wild type viruses.     

高致病性禽流感病毒血凝素蛋白广谱中和表位模拟肽的筛选与鉴定

以H5N1型禽流感病毒HA蛋白广谱中和单抗8H5为基础,利用噬菌体展示肽库技术及类病毒颗粒融合表达技术研究HA模拟表位。ELISA检测结果显示:筛选获得模拟HA表位的模拟肽123,进行类病毒颗粒融合蛋白表达后,仍具有与8H5单抗特异结合的能力。免疫荧光检测结果说明,类病毒颗粒免疫小鼠后产生了能与HA交叉反应的抗体。禽流感病毒HA模拟表位的研究与性质的分析及类病毒颗粒融合蛋白的表达与活性分析、免疫原性分析,都为研制禽流感通用表位疫苗奠定了基础。     

Fab MAbs specific to HA of influenza virus with H5N1 neutralizing activity selected from immunized chicken phage library

Hemagglutinin protein (HA) was considered to be the primary target for monoclonal antibody production. This protein not only plays an important role in viral infections, but can also be used to differentiate H5N1 virus from other influenza A viruses. Hence, for diagnostic and therapeutic applications, it is important to develop anti-HA monoclonal antibody (MAb) with high sensitivity, specificity, stability, and productivity. Nine unique Fab MAbs were generated from chimeric chicken/human Fab phage display library constructed from cDNA derived from chickens immunized with recombinant hemagglutinin protein constructed from H5N1 avian influenza virus (A/Vietnam/1203/04). The obtained Fab MAbs showed several characteristics for further optimization and development—three clones were highly specific to only H5N1 virus. This finding can be applied to the development of H5N1 diagnostic testing. Another clone showed neutralization activity that inhibited H5N1 influenza virus infection in Madin-Darby canine kidney (MDCK) cells. In addition, one clone showed strong reactivity with several of the influenza A virus subtypes tested. The conversion of this clone to whole IgG is a promising study for a cross-neutralization activity test.     

Preexisting influenza-specific CD4+ T cells correlate with disease protection against influenza challenge in humans

Protective immunity against influenza virus infection is mediated by neutralizing antibodies, but the precise role of T cells in human influenza immunity is uncertain. We conducted influenza infection studies in healthy volunteers with no detectable antibodies to the challenge viruses H3N2 or H1N1. We mapped T cell responses to influenza before and during infection. We found a large increase in influenza-specific T cell responses by day 7, when virus was completely cleared from nasal samples and serum antibodies were still undetectable. Preexisting CD4+, but not CD8+, T cells responding to influenza internal proteins were associated with lower virus shedding and less severe illness. These CD4+ cells also responded to pandemic H1N1 (A/CA/07/2009) peptides and showed evidence of cytotoxic activity. These cells are an important statistical correlate of homotypic and heterotypic response and may limit severity of influenza infection by new strains in the absence of specific antibody responses. Our results provide information that may aid the design of future vaccines against emerging influenza strains.     

A human antibody recognizing a conserved epitope of H5 hemagglutinin broadly neutralizes highly pathogenic avian influenza H5N1 viruses

Influenza A virus infection is a persistent threat to public health worldwide due to its ability to evade immune surveillance through rapid genetic drift and shift. Current vaccines against influenza A virus provide immunity to viral isolates that are similar to vaccine strains. High-affinity neutralizing antibodies against conserved epitopes could provide immunity to diverse influenza virus strains and protection against future pandemic viruses. In this study, by using a highly sensitive H5N1 pseudotype-based neutralization assay to screen human monoclonal antibodies produced by memory B cells from an H5N1-infected individual and molecular cloning techniques, we developed three fully human monoclonal antibodies. Among them, antibody 65C6 exhibited potent neutralization activity against all H5 clades and subclades except for subclade 7.2 and prophylactic and therapeutic efficacy against highly pathogenic avian influenza H5N1 viruses in mice. Studies on hemagglutinin (HA)-antibody complexes by electron microscopy and epitope mapping indicate that antibody 65C6 binds to a conformational epitope comprising amino acid residues at positions 118, 121, 161, 164, and 167 (according to mature H5 numbering) on the tip of the membrane-distal globular domain of HA. Thus, we conclude that antibody 65C6 recognizes a neutralization epitope in the globular head of HA that is conserved among almost all divergent H5N1 influenza stains.     

一种新型H5N1禽流感病毒血凝素抗原快速检测试剂的建立

利用5株广谱特异性抗H5亚型血凝素单克隆抗体和酶联免疫渗滤技术成功地建立了一种适于现场检测H5亚型禽流感病毒血凝素蛋白的抗原快速检测试剂H5-HA(Ag)Dot-ELISA。该试剂对41株代表当前亚洲地区流行的各种遗传变异亚系H5N1禽流感病毒检测均为阳性,对多数毒株的分析灵敏度优于0.1个血凝滴度(HA titer),其中部分优于0.01个血凝滴度;比较该试剂与早期开发的同类ELISA试剂,发现前者对后者未能检出的H5N1新变异株检测均为阳性;利用该试剂和商品化Directigen Flu A(BD)试剂检测两株H5N1病毒株,提示前者灵敏度高于后者;该试剂对一株H5N1病毒的检测灵敏度与标准RT-PCR相当;该试剂对24株非H5亚型病毒检测均为阴性,显示出良好特异性。以上结果提示,此研究建立的H5N1病毒抗原快速检测试剂在H5禽流感现场检测上具有较好的应用前景。     

Altered specificity of single-chain antibody fragments bound to pandemic H1N1-2009 influenza virus after conversion of the phage-bound to the soluble form

ABSTRACT: BACKGROUND: In 2009, a novel influenza A/H1N1 virus (H1N1pdm) quickly spread worldwide and co-circulated with then-existing seasonal H1N1 virus (sH1N1). Distinguishing between these 2 viruses was necessary to better characterize the epidemiological properties of the emergent virus, including transmission patterns, pathogenesis, and anti-influenza drug resistance. This situation prompted us to develop a point-of-care virus differentiation system before entering the 2009--2010 influenza season. Aiming to establish H1N1pdm-specific detection tools rapidly, we employed phage display libraries to select H1N1pdm-specific single-chain variable fragments (scFvs). FINDINGS: Human single-fold scFv libraries (Tomlinson I + J) underwent selection for the ability to bind H1N1pdm virus particles. Three rounds of panning brought 1152 phage-bound scFvs, of which 58 clones reacted with H1N1pdm specifically or preferentially over sH1N1 in an enzyme-linked immunosorbent assay (ELISA). After conversion of the scFvs to soluble form, 7 clones demonstrating high/stable expression were finally obtained. However, all the soluble scFvs except No. 29 were found to have lost their specificity/preference for H1N1pdm in ELISA. The specificity/preference of No. 29 was also confirmed by immunofluorescence assay and immunoprecipitation, and the viral nucleoprotein was identified by ELISA as its target protein. The change in specificity associated with scFv conversion from phage-bound to soluble form could be due to loss of phage scaffold pIII protein, which likely provides structural support for the scFv antigen-binding site. It is also possible that the similar antigenic properties of H1N1pdm and sH1N1 led to the observed alterations in scFv specificity. DISCUSSION: Using a phage display library, we obtained 7 soluble scFv clones reactive against H1N1pdm; however, only 1 showed specificity/preference toward H1N1pdm. Our results confirmed that using phage display libraries was highly advantageous for the rapid development of molecules to detect target antigens. However, our results also indicated that this strategy might not have been effective for selecting H1N1pdm-specific antibodies during the 2009 pandemic, where the co-circulating sH1N1 virus shared similar antigenic properties. This suggests that it might be advisable to use a synthetic scFv phage display library by strategically considering the characteristics of target antigens and the potential situations.     

Discovery of novel benzoquinazolinones and thiazoloimidazoles, inhibitors of influenza H5N1 and H1N1 viruses, from a cell-based high-throughput screen

A highly reproducible and robust cell-based high-throughput screening (HTS) assay was adapted for screening of small molecules for antiviral activity against influenza virus strain A/Vietnam/1203/2004 (H5N1). The NIH Molecular Libraries Small Molecule Repository (MLSMR) Molecular Libraries Screening Centers Network (MLSCN) 100,000-compound library was screened at 50 μM. The "hit" rate (>25% inhibition of the viral cytopathic effect) from the single-dose screen was 0.32%. The hits were evaluated for their antiviral activity, cell toxicity, and selectivity in dose-response experiments. The screen yielded 5 active compounds (SI value >3). One compound showed an SI(50) value of greater than 3, 3 compounds had SI values ranging from greater than 14 to 34, and the most active compound displayed an SI value of 94. The active compounds represent 2 different classes of molecules, benzoquinazolinones and thiazoloimidazoles, which have not been previously identified as having antiviral/anti-influenza activity. These molecules were also effective against influenza A/California/04/2009 virus (H1N1) and other H1N1 and H5N1 virus strains in vitro but not H3N2 strains. Real-time qRT-PCR results reveal that these chemotypes significantly reduced M1 RNA levels as compared to the no-drug influenza-infected Madin Darby canine kidney cells.