狂犬病动物抗体水平检测和监测

检测人和动物体内狂犬病抗体的主要目的有三个方面,第一,对暴露前预防免疫的人或动物进行抗体监测,确定人的预防免疫效果和动物的免疫水平及免疫覆盖率;第二,对暴露后免疫治疗的人体内的中和抗体进行检测,以确定暴露后治疗效果;第三,对狂犬病疑似患者或患畜,通过测定患病早期和     

庆阳市狂犬病暴露人群的流行病学分析

目的了解狂犬病暴露人群的暴露部位、伤害程度、受伤时间等分布规律及暴露后的处置情况,为制定狂犬病防制对策提供依据。方法调查甘肃省庆阳市2013年有狂犬病发病的两个县的狂犬病暴露人群基本情况,采用描述流行病学方法进行统计学分析。结果 1 226例暴露者中9岁及以下(25.86%)和40岁~(22.92%)年龄组人群暴露率最高;暴露部位多为四肢、占84.41%,头面部最少、占2.70%;时间分布以5~10月为主、占71.04%,7月、8月达到高峰。结论狂犬病的暴露率高,群众的预防知识缺乏、防控意识淡薄,暴露后未及时进行规范的伤口处置、未及时接种狂犬病疫苗和被动免疫制剂是导致狂犬病发病的主要原因。     

冀、京、吉犬群犬腺病毒中和抗体调查

目的为表达狂犬病病毒糖蛋白的犬2型腺病毒（CAV-2）重组狂犬病口服疫苗的投放工作提供流行病学依据。方法通过病毒中和试验对随机采自河北省,北京市,吉林省等地的387份犬血清进行犬腺病毒中和抗体检测,以确定犬群腺病毒感染率。结果河北、吉林农村流浪犬或家养犬腺病毒中和抗体阳性率较低,阳性率为16％（30／192）,而北京市犬腺病毒中和抗体阳性率高达69％（135／195）,说明城乡犬只犬腺病毒中和抗体存在明显差异。结论农村流浪犬或家养犬腺病毒中和抗体阳性率较低,适用于CAV-2及其重组疫苗首次免疫、尤其是口服免疫。     

间接荧光抗体试验用于基本消灭丝虫病地区人群抗体水平的监测

本文报告了用马来丝虫成虫冰冻切片抗原作 IFAT 检测班氏丝虫微丝蚴血症者抗体阳性率为88.89％;非流行区健康居民抗体阳性率为3.92％。基本消灭丝虫病后8年的地区原微丝蚴血症者和阴性居民抗体阳性率分别为13.54％和10.84％;基本消灭后15年原微丝蚴血症者和阴性居民抗体阳性率分别为6.45％和5.16％;基本消灭后24年原微丝蚴血症者和阴性居民抗体阳性率分别为3.67％和3.96％。基本消灭丝虫病后15年的地区人群抗体阳性率已降到非流行区健康人群水平(X~2=0.48 P>0.05)。因此认为 IFAT 可作为我省丝虫病防治后期和基本消灭丝虫病后流行病学监测的主要方法之一。此外,观察到微丝蚴血症者血清抗体阳性率和阳性 GMRT 与微丝蚴密度无相关性。     

5119例狂犬病疫苗全程免疫接种者血清抗体水平检测结果分析

目的了解狂犬病疫苗全程免疫后抗体产生情况及其影响因素,为狂犬病疫苗预防接种工作提供依据。方法采用ELISA法对5119例于我院全程接种狂犬病疫苗者进行抗狂犬病病毒抗体检测并分析结果。结果5119份血清标本中抗体阳性5035份,总阳性率98．36％。其中男性及女性阳性率分别为97．91％、98．75％;P〈0．05。0—19岁、20～59岁及〉60岁者抗体阳性率分别为99．3％、98、4％、96．3％,两两比较,P均〈0．05;即随着年龄增大,接种疫苗后抗体阳性率呈下降趋势。结论性别对狂犬疫苗接种后抗体的产生有影响;随着年龄的增长,注射疫苗后抗体阳性率逐渐下降。对接种后抗体阴性者应再次进行全程免疫。     

2463例狂犬病疫苗免疫后抗体水平及其影响因素分析

狂犬病是一种人兽共患的自然疫源性疾病 ,目前尚无治疗药物 ,死亡率几乎为 10 0 %。随着人民生活水平的提高 ,宠物热日益升温 ,而“健康犬”的带毒率为 3 92 %～17 9% 〔1,2〕,患狂犬病的可能性亦随之增加。而预防发病的关键措施是开展有效的狂犬疫苗预防接种 ,快速、敏感的检测免疫者抗体情况也是非常必要的。现将 2 4 6 3例狂犬疫苗免疫后抗体结果分析如下。1　对象与方法1 1　对象　被犬等动物咬伤或抓伤者 ,常规 5针肌注狂犬疫苗 ,最后一针免疫后 15～ 2 0d ,采静脉血检测抗体 ,对阴性者再 0 ,7d加强免疫两针 ,15～ 2 0d后复检抗体。1 2…     

基本消灭疟疾地区人群抗体水平监测分析

为全面了解常州市现阶段疟疾传播的性质和流行态势,及早发现疟疾复燃的潜在危险因素,为调整疟疾防治措施提供科学依据,采用疟疾间接荧光抗体(IFA)检测不同人群的疟疾抗体水平,结果分析如下.     

猫用高效狂犬病灭活疫苗研究

目的研制水包油新型佐剂狂犬病疫苗开展猫的免疫试验。方法利用BHK-21细胞培养狂犬病病毒鼬獾源性JX04-45株,病毒滴度达到10^7.5 TCID₅₀/mL以上的病毒培养物用于灭活和疫苗制备,佐剂为新型水包油型;通过本实验室建立的荧光抗体病毒中和试验(FAVN)测定疫苗效力和猫免疫后的中和抗体,并进行了攻毒试验,评价本疫苗免疫效果。结果一个剂量的疫苗免疫小鼠后FAVN方法测定的疫苗效力≥3.0 IU/剂量;水包油佐剂型狂犬病灭活疫苗免疫猫后14 d时无不良反应,FAVN测得其诱导的中和抗体水平平均达16.04±4.29 IU/mL,28 d平均达37.13±6.12 IU/mL,56 d为21.01±7.61 IU/mL。180 d为6.71±3.44 IU/mL,360 d为2.66±2.01 IU/mL。至15个月时,咬肌攻毒10⁶MIC LD₅₀狂犬病病毒BD06株,观察90 d试验组无一死亡,对照组全部死亡。结论本疫苗诱导长达1年的免疫保护,可扩大安全评价和临床试验,以验证其实用价值。     

心脏病患者血清Coxsackie B组病毒中和抗体的调查

44例不明原因早搏患者的Coxsackie B组病毒(CBV)中和抗体效价明显高于献血员、风心病、冠心病及平均年龄基本配对的先心病组。效价≥1:320者亦明显高于其他4组,结果提示不明原因早搏与CBV感染可能有一定关系。     

上海地区2009年人群甲型流行性感冒病毒抗体水平检测分析

目的 了解上海地区人群季节性H1、H3亚型流感病毒抗体水平及职业人群中H5、H9亚型禽流感抗体的检出情况.方法 应用血凝抑制试验对上海地区2009年与禽类密切接触的职业人群356人以及一般人群332人各年龄组进行H1、H3、H5、H9亚型流感病毒抗体的血清学监测.结果 A/Brisbane/59/2007(H1N1)抗体阳性分布,一般人群有275人,占82.8%,职业人群有263人,占73.9%;A/Brisbane/10/2007(H3N2)抗体阳性,一般人群有168人,占50.6%,职业人群有195人,占54.8%;人群中A/Brisbane/59/2007(H1N1)抗体阳性率明显高于H3,与2008年上海地区流感病原学监测情况相吻合.职业人群与一般人群H5亚型抗体阳性率分别为4.2%(15/356)和0.3%(1/332);H9亚型抗体阳性率分别为34.6%(123/356)和2.4%(8/332).不同年龄组中,6个月～5岁年龄组和≥60岁的老年组A/Brisbane/59/2007(H1N1)、A/Brisbane/10/2007(H3N2)抗体阳性率较低.结论 上海地区人群对H1、H3季节性流感有较强的免疫保护,儿童和老年人群H1、H3季节性流感抗体水平较低.职业人群禽流感病毒H5、H9抗体有明显升高趋势,要加强对职业人群流感样患者病原学和血清流行病学监测.     

狂犬病毒新型快速荧光斑点抑制实验方法的建立

目的建立一种新型的快速荧光斑点抑制实验用于狂犬病毒抗体的检测。方法本研究以携带绿色荧光蛋白基因的嵌合狂犬病毒HEP-GFP株为基础毒株,建立了一种新型快速荧光斑点抑制实验(RFFIT-GFP);并对多份人、犬、猫的血清进行了检测,还将该检测结果与传统的RFFIT,ELISA相比较。结果与结论RFFIT-GFP和RFFIT,ELISA测定的结果基本相一致,特异性都比较高,而且RFFIT-GFP是一种比它们更为简便、经济的检测方法。     

1例特殊的疑似狂犬病死亡病例的实验室检测和确诊

目的采用实验室检测方法确诊一例特殊的疑似狂犬病病例。方法利用荧光抗体(FA)试验、双抗体夹心ELISA和逆转录-聚合酶链式反应(RT-PCR)方法对一例死亡的疑似狂犬病人的脑组织和此脑组织经乳鼠脑内接种传代后的鼠脑组织分别进行检测和分析。结果死亡病人脑组织的直接FA试验和双抗体夹心ELISA检测为阴性,但RT-PCR检测结果为阳性。将该病人脑组织经乳鼠传代后对鼠脑组织用上述三种方法检测的结果均为阳性。对该街毒株的RT-PCR产物中的核蛋白基因序列进行测定,用Mega3.1软件进行分子进化关系分析,证实所分离的街毒株为基因Ⅰ型狂犬病病毒,与以往在该地区所分离的街毒株有较近的遗传关系。结论该病例可确诊为狂犬病死亡病例,其主要和决定性的死因为狂犬病,但并发的肺部感染可能加速了死亡的进程。WHO认为FA技术是狂犬病诊断的金标准,但本病例说明,在某些复杂情况下,只有将多种实验室检测方法联合运用才能确诊狂犬病,而且病毒分离和RT-PCR可能比FA试验更敏感。     

抗狂犬病病毒N蛋白单克隆抗体的制备与鉴定

目的制备抗狂犬病病毒N蛋白单克隆抗体,为建立快速准确的狂犬病病毒抗原检测方法奠定基础。方法将狂犬病病毒Flury LEP株N基因克隆至pGEX-6P-1表达载体中,将纯化后的pGEX-6P-1-RV-N蛋白免疫BALB/c小鼠3次。取小鼠脾脏细胞和SP2/0细胞融合后,用pET-32a-RV-N重组蛋白作为筛选抗原,经3次亚克隆筛选后得到1D9、2B6、3C5、6B5及5F2 5株单克隆抗体细胞株。亚类鉴定结果表明,其中1D9、2B6、6B5 3株亚类为IgG1,3C5、5F2的亚类为IgM。间接ELISA方法检测2B6单抗细胞腹水效价可达1∶106。经Protein G亲和层析柱纯化和脱盐后可得到浓度为2.5mg/mL的高纯度的单克隆抗体。其免疫荧光试验结果表明5株单克隆抗体均能特异地与狂犬病病毒结合。结论制备的单抗具有良好的特异性和敏感性,为后期诊断方法的建立奠定了基础。     

一种不依赖于活病毒的中东呼吸综合征冠状病毒受体结合区特异性中和抗体检测方法的建立

目的 拟建立一种不依赖于活病毒和高等级生物安全条件,快速简便检测中东呼吸综合征冠状病毒(MERS-CoV)受体结合区(RBD)特异性的中和抗体。方法 利用哺乳动物表达系统表达重组rRBD-Fc蛋白;应用流式细胞术检测rRBD-Fc蛋白与Huh-7细胞结合的最佳浓度,建立通过流式细胞术检测rRBD-Fc与Huh-7结合活性的方法,并利用无关蛋白验证结合的特异性;在此基础上,利用RBD特异性中和抗体、RBD特异性非中和抗体、MERS-CoV RBD免疫小鼠血清和无关抗体建立能够检测中和抗体阻断RBD与Huh-7结合作用的RBD特异性中和抗体检测技术方法,并与实毒中和试验进行比较,评价方法的一致性;应用建立的中和抗体检测方法进行血清学检测。结果成功表达了rRBD-Fc蛋白,rRBD-Fc蛋白能够与Huh-7细胞特异性结合,并确定了rRBD-Fc与Huh-7细胞结合的最佳剂量;在此基础上建立了通过流式细胞术检测抗体阻断RBD与Huh-7结合作用的RBD特异性中和抗体检测技术方法,结果表明,检测方法能够区分具有中和活性的RBD特异性中和抗体、rRBD-Fc免疫小鼠血清和其他抗体,具有特异性,且能够反映中和活性的强弱。该方法在检测12株中和抗体时,与传统的实毒中和试验检测结果具有很好的一致性,能够有效应用于血清学检测。结论 成功建立了一种不依赖活病毒和高等级生物安全条件,即可快速检测MERS-CoV RBD特异性中和抗体的方法,为针对MERS-CoV疫苗设计和抗体效果快速评价、血清学调查和免疫保护机制研究提供了一种更加简便、安全的技术手段。     

Evaluation of Rapid Neutralizing Antibody Detection Test against Rabies Virus in Human Sera

Rapid and easy determination of protective neutralization antibody (NAb) against rabies in the field is very important for an early and effective response to rabies in both animal and human health sectors. The rapid neutralizing antibody detection test (RAPINA), first developed in 2009 and then improved in 2012, is a quick test allowing detection of 0.5 IU/ml antibodies in human and animal sera or plasma. This study aimed to assess the RAPINA test by comparison with rapid focus fluorescence inhibition test (RFFIT), using 214 sera of vaccinated and unvaccinated professional dog butchers, laboratory workers and rabies patients in Vietnam. The sensitivity, specificity, false negative rate, false positive rate and concordance of the RAPINA test as compared to RFFIT were 100%, 98.34%, 0%, 1.66% and 98.6%, respectively. The positive predictive value and negative predictive value were 91.7% and 100%, respectively when RAPINA test was used. With its remarkable sensitivity, specificity and easy implementation, RAPINA test can be used for rapid determination of NAb in the field.     

Discovery of Marburg virus neutralizing antibodies from virus-naïve human antibody repertoires using large-scale structural predictions

Marburg virus (MARV) disease is lethal, with fatality rates up to 90%. Neutralizing antibodies (Abs) are promising drug candidates to prevent or treat the disease. Current efforts are focused in part on vaccine development to induce such MARV-neutralizing Abs. We analyzed the antibody repertoire from healthy unexposed and previously MARV-infected individuals to assess if naïve repertoires contain suitable precursor antibodies that could become neutralizing with a limited set of somatic mutations. We computationally searched the human Ab variable gene repertoire for predicted structural homologs of the neutralizing Ab MR78 that is specific to the receptor binding site (RBS) of MARV glycoprotein (GP). Eight Ab heavy-chain complementarity determining region 3 (HCDR3) loops from MARV-naïve individuals and one from a previously MARV-infected individual were selected for testing as HCDR3 loop chimeras on the MR78 Ab framework. Three of these chimerized antibodies bound to MARV GP. We then tested a full-length native Ab heavy chain encoding the same 17-residue-long HCDR3 loop that bound to the MARV GP the best among the chimeric Abs tested. Despite only 57% amino acid sequence identity, the Ab from a MARV-naïve donor recognized MARV GP and possessed neutralizing activity against the virus. Crystallization of both chimeric and full-length native heavy chain-containing Abs provided structural insights into the mechanism of binding for these types of Abs. Our work suggests that the MARV GP RBS is a promising candidate for epitope-focused vaccine design to induce neutralizing Abs against MARV.

With the advent of high-throughput immune repertoire sequencing, the number of available human antibody (Ab) sequences is exploding rapidly from thousands to billions. These datasets provide a resource for understanding the natural process of Ab maturation through somatic mutations, quick identification of novel functional Abs, and engineering of improved Abs. Ultimately, such Ab repertoires may provide or add templates for developing epitope-focused (1) and germline-targeting (2, 3) vaccines. While Ab function sometimes can be predicted from sequence homology, often Abs of very different sequence have the same function by virtue of adopting a similar structure. However, despite substantial progress in computational methods of modeling Abs (4, 5) and Ab–antigen interactions (6–10), and the use of high-performance computing, it is still beyond the available computational resources to predict and study the structure of billions of Abs one by one. Therefore, in order to take advantage of the rapidly increasing Ab sequence databases, it is essential to find more effective ways of relating Ab sequence to function.The recently proposed position-specific structure scoring matrix (P3SM) approach is a new computational method specifically designed for rapid screening of large Ab sequence libraries (11, 12). This approach aims to predict whether a given Ab sequence can adopt the desired 3D conformation and thus correctly place critical-for-activity functional groups. This prediction is based on modeling of a small subset of structures using Rosetta (13) followed by evaluation of the compatibility of each of 20 amino acids in each of the analyzed positions with the desired structure and function. The resulting P3SM then can be used for rapid screening of the remaining Ab sequences. The best-scoring candidate sequences are fed back into Rosetta for a detailed energetic analysis and prioritization for experimental validation.The human monoclonal Ab MR78 was isolated previously from a B cell in the peripheral blood of an otherwise healthy individual with a prior history of naturally acquired Marburg virus (MARV) infection (14). A crystal structure of MARV glycoprotein (GP) in complex with MR78 (Protein Data Bank [PDB] ID 5UQY) revealed that the Ab heavy-chain complementarity determining region 3 (HCDR3) contacts the receptor binding domain (RBD) on MARV GP that interacts with the natural receptor on human cells (Niemann–Pick disease, type C1 [NPC1] protein) (15). Here, we applied the P3SM approach to search for structurally homologous Abs to MR78 based on this Ab–antigen cocrystal structure.     

抗TNFR1中和抗体对急性肝衰竭小鼠肝细胞的保护作用

目的探讨抗TNFR1中和抗体对急性肝衰竭小鼠肝细胞的作用。方法联合应用D-氨基半乳糖和肿瘤坏死因子-α造模，酶联免疫吸附试验测定血清ALT、AST浓度。HE染色检查肝脏组织病理变化、脱氧核糖核苷酸转移酶介导的缺口末端标记（TUNEL）技术检测肝细胞的凋亡程度。结果模型组各时间点（4、8、12h）均可见明显的肝细胞凋亡，治疗组各时间点亦可见少量凋亡细胞，但与模型组比较各时间点均明显减少（P〈0．01），其血清ALT、AST浓度较模型组也有下降。结论抗TNFR1中和抗体对TNF—α能诱导肝细胞损伤具有保护作用肝。     

Structures of complexes formed by H5 influenza hemagglutinin with a potent broadly neutralizing human monoclonal antibody

H5N1 avian influenza viruses remain a threat to public health mainly because they can cause severe infections in humans. These viruses are widespread in birds, and they vary in antigenicity forming three major clades and numerous antigenic variants. The most important features of the human monoclonal antibody FLD194 studied here are its broad specificity for all major clades of H5 influenza HAs, its high affinity, and its ability to block virus infection, in vitro and in vivo. As a consequence, this antibody may be suitable for anti-H5 therapy and as a component of stockpiles, together with other antiviral agents, for health authorities to use if an appropriate vaccine was not available. Our mutation and structural analyses indicate that the antibody recognizes a relatively conserved site near the membrane distal tip of HA, near to, but distinct from, the receptor-binding site. Our analyses also suggest that the mechanism of infectivity neutralization involves prevention of receptor recognition as a result of steric hindrance by the Fc part of the antibody. Structural analyses by EM indicate that three Fab fragments are bound to each HA trimer. The structure revealed by X-ray crystallography is of an HA monomer bound by one Fab. The monomer has some similarities to HA in the fusion pH conformation, and the monomer’s formation, which results from the presence of isopropanol in the crystallization solvent, contributes to considerations of the process of change in conformation required for membrane fusion.The initial steps in influenza virus infection involve sialic acid receptor binding and membrane fusion, both of which are functions of the hemagglutinin (HA) virus membrane glycoprotein. Anti-HA antibodies that block these functions neutralize virus infectivity. Such antibodies are induced by infection and by vaccination, and the immune pressure that they impose on subsequently infecting viruses is responsible for the antigenic drift for which influenza viruses are notorious. Zoonotic infections, which can lead to new pandemics, occur periodically, and H5N1, H7N9, and H10N8 avian viruses are recent examples of this sort. The threat that zoonotic infections present is based, in part, on the lack of immunity in the human population to the novel HAs that they contain. In attempts to substitute for this deficiency, human immune sera have been used successfully to treat severe infections (1), and monoclonal antibodies have been prepared from mice and from humans for potential use in immunotherapy.Analyses of antibodies produced by cloned immune cells derived from infected patients have revealed that antibodies are induced that are either subtype- or group-specific and others that cross-react with HAs of both groups (2). To date, cross-reactive antibodies have been shown to recognize both membrane-distal and membrane-proximal regions of HA (3). Subtype-specific antibodies, on the other hand, bind to the membrane-distal region, covering the receptor-binding site and, in some cases, inserting into it (4, 5).In the studies reported here, a human monoclonal antibody is described that recognizes the HAs of viruses of all three clades of the H5 subtype that have caused human infection and is shown to be effective in protecting mice from lethal challenge. EM and X-ray crystallography studies of HA-Fab complexes indicate that the antibody binds to a site containing residue 122, located on the membrane-distal surface of the HA trimer. We describe the antibody-binding site in detail to show that binding occurs at a distance from the receptor-binding site. Infectivity neutralization and receptor-binding experiments, together with these observations, lead to the conclusion that the antibody neutralizes viruses by blocking receptor binding in a way that is dependent on the Fc region of the bound antibody. We compare the site with similar sites reported by others (6–9) for antibodies that have not as yet given crystalline HA-Fab complexes.Under the conditions that we obtain crystals of the HA-Fab complex, the HA dissociates and reveals the structure of a monomeric HA. We consider the structure of the monomer in relation to the structure that HA has been shown to assume after exposure to the pH of membrane fusion.     

宠物犬自然感染乙型脑炎病毒中和抗体动态变化的研究

目的 以宠物犬为研究对象,监测在自然感染条件下其体内乙型脑炎病毒(JEV)中和抗体随流行季节的变化规律, 进而评估JEV感染人的风险性。方法 按月采集不同年龄的宠物犬血液,分离血清,用微量细胞中和试验检测血清JEV中和抗体。结果 共得到宠物犬血清253份,中和抗体阳性率达到28.1%。不同季节间犬JEV中和抗体阳性率差异显著,JEV流行季节(6-9月)之前(3-5月)和之后(10-12月)的3个月中和抗体阳性率分别为16.3%和43.4%,差异有统计学意义(χ²=14.432, P<0.01)。小于1岁与大于1岁的宠物犬中和抗体阳性率分别为24.1%和39.0%,二者差异显著(χ²=5.633, P<0.05)。而犬的性别间中和抗体阳性率显著无统计学意义(χ²=1.860, P>0.05)。结论 在JE流行季节宠物犬受到JEV自然感染的机率较高,可作为哨兵动物来评估JEV感染人的风险。