靶向SARS-CoV spike中和抗体的检测

目的 建立靶向SARS-CoV spike中和抗体的检测模型.方法 构建优化的全基因SARS-CoV spike质粒免疫小鼠产生S1190抗血清,与合成的结合受体ACE2的S318-510蛋白进行作用,通过荧光显微镜观察对SARS假病毒感染抑制作用的变化以确证中和抗体的高效性.结果 SARS-CoV spike质粒诱导产生的S蛋白全长抗血清不但可以有效中和SARS假病毒进入HEK293E/ACE2-Myc细胞,S318-510蛋白的预处理可以降低抗血清对SARS假病毒的进入抑制效应.结论 受体结合部(RBD)的S318-510蛋白靶向SARS-CoV spike中和抗体的检测模型得到有效的验证.可以作为直观迅速判断中和抗体是否高效的模型推广至不具备P3级别的实验室.     

SARS患者特异性SARS-CoV抗体变化的观察

目的　研究SARS患者特异性SARS CoV抗体IgG、IgM动态变化规律 ,探讨机体感染SARS CoV后免疫应答的可能模式及流行病学意义 ,为科学的防治SARS提供理论依据。方法　ELISA测定北京地区最早感染SARS CoV的 3条传播链的SARS患者 4 7例 ,12 4份血清中SARS CoV特异性抗体IgG、IgM。其中 ,山西链 9人 ,13份血清 ;香港链 31人 ,84份血清 ;北京链 7人 ,2 7份血清。结果　 3条传播链SARS患者IgM阳性率 73% ,IgG阳性率 86 %。IgM最早于发病第 6天出现 ,阳性率高峰在病程 2～ 3周 ;持续时间短 ,1月后 6 0 %患者IgM逐渐阴转。IgG最早也可在发病第 6天出现 ,阳性率高峰在病程 3～ 4周 ,3月后IgG抗体阳性率减少。机体感染SARS CoV后 ,抗体出现有 3种模式。结论　机体感染SARS CoV后 ,近 90 %患者可出现特异性IgG ,病程 3～ 4周阳性率最高 ;特异性IgM抗体在病程2～ 3周阳性率最高 ,但多数只持续 2 0～ 30d。特异性IgG抗体表现 3种免疫应答模式。     

SARS-CoV蚀斑形成技术的建立

自2002年11月我国出现第一例传染性非典型肺炎(SARS)病例以来，至2003年8月已造成5000多例临床病例，其中死亡300多例，对我国国民经济和社会稳定造成了巨大影响。现在已确定了SARS-冠状病毒(SARS-CoV)为其病原体，也成功分离到多株SARS-CoV，并能在Veto细胞和2BS等细胞系中培养增殖，这为我们研发疫苗和研究病原学等方面提供了基础和基本条件。由于蚀斑试验和蚀斑减少中和试验用于测定病毒滴度和中和抗体效价的检测较其他方法准确，且特异性高，因此，用蚀斑方法可以准确测定病毒滴度、SARS患者恢复期抗体水平、疫苗免疫后的抗体产生水平以及评价疫苗的质量和有效性、抗病毒药物的筛选等。     

抗SARS-CoV免疫球蛋白F(ab'''')2治疗性抗体对SARS-CoVBJ-01株的中和作用

目的观察马抗SARS-CoV免疫球蛋白F（ab’）2治疗性抗体对SARS-CoVBJ-01毒株的中和作用。方法采用细胞病变（cytopathiceffect，CPE）、四甲基偶氮唑盐（nlethylthiazolyl tetrazolium assay，MTT）测定马抗SARS-CoV免疫球蛋白F（ab’）2对SARS-CoVBJ-01株的中和作用。结果三批马抗SARS-CoV免疫球蛋白F（ab’）2对SARS-CoVBJ-01株有很好的中和作用，CPE和MTT法测得中和效价分别为1：6400、1：6400、1：12800。结论研制的马抗SARS-CoV免疫球蛋白F（ab’）2对SARS-CoV BJ-0l1株有很好的中和作用，并且两种方法结果一致，为SARS的治疗提供了可靠的依据。     

HIV-1自然感染中的中和抗体反应

中和抗体(Nab)可以防止I型人类免疫缺陷病毒(HIV-1)侵入靶细胞.HIV-1感染数周后即可诱导产生Nab,这些早期抗体只能特异性地中和自体病毒但不能中和异源性病毒.在一些慢性感染者体内则可以检测到可同时中和同源性和异源性病毒的广谱中和抗体(BNab).BNab的靶点通常位于包膜蛋白的保守区域.HIV-1 BNab的产生还受到病毒变异及结构遮盖等因素的限制,同时Nab的中和广度与病毒载量具有相关性.     

抗SARS-CoV血清抗体体外抗SARS病毒实验研究

目的研究三个批号的抗SARS-Cov血清抗体体外对SARS-CoV的抑制作用。方法采用MTT法,观察药物对SARS-Cov的抑制作用。结果MTT法测得三个批号的抗SARS-CoV血清抗体的最大无毒浓度均小于10μg/mL。不同时间点的治疗指数（TI）：2h组〉6h组〉12h组〉24h组;不同批号的治疗指数（TI）200307批〉200306批〉200308批。结论在本实验条件下抗SARS-CoV血清抗体体外有一定的抗SARS-CoV作用,为其治疗SARS-CoV感染提供了实验依据。     

抗SARS-CoV免疫球蛋白F(ab′)_2治疗性抗体对SARS-CoVBJ-01株的中和作用

目的观察马抗SARS-CoV免疫球蛋白F(ab′)2治疗性抗体对SARS-CoVBJ-01毒株的中和作用。方法采用细胞病变法(cytopathiceffect,CPE)、四甲基偶氮唑盐(methylthiazolyltetrazoliumassay,MTT)测定马抗SARS-CoV免疫球蛋白F(ab′)2对SARS-CoVBJ-01株的中和作用。结果三批马抗SARS-CoV免疫球蛋白F(ab′)2对SARS-CoVBJ-01株有很好的中和作用,CPE和MTT法测得中和效价分别为1∶6400、1∶6400、1∶12800。结论研制的马抗SARS-CoV免疫球蛋白F(ab′)2对SARS-CoVBJ-01株有很好的中和作用,并且两种方法结果一致,为SARS的治疗提供了可靠的依据。     

针对SARS⁃CoV2不同突变株广谱特异性中和抗体的筛选与鉴定

目的 筛选对严重急性呼吸综合征冠状病毒2(SARS?CoV2)不同突变株具有广谱特异性的、高亲和性中和抗体,并进行功能验证.方法 用脂质体包被的SARS?CoV2刺突蛋白(S蛋白)mRNA疫苗对Balb/c小鼠进行2次免疫,测定小鼠血清中特异性抗体含量,选取抗体含量较高的小鼠进行冲击免疫;取冲击免疫后小鼠脾细胞与骨髓瘤...     

基于量子点免疫层析技术的新型冠状病毒中和抗体检测方法建立

目的:建立简便、快速、低成本的2019新型冠状病毒(2019 novel coronavirus,2019-nCoV)中和抗体检测方法。方法:基于量子点荧光免疫层析原理,建立新冠RBD IgG检测方法,并用新型冠状病毒肺炎(coronavirus disease 2019,COVID-19)患者恢复期血清( ...     

抗SARS-CoV病毒N蛋白的单链抗体(scFv)筛选

目的筛选出抗SARS-CoV病毒N蛋白的单链抗体。方法利用原核表达所获得的SARS病毒N蛋白,筛选人源单链抗体噬菌体展示库,经特异性的检测,以期得到抗SARS-CoV病毒N蛋白的特异单链抗体。结果获得了8个抗SARS病毒N蛋白的候选克隆。经测序,获得了编码抗体可变区的基因序列,并进行了原核表达。结论筛选得到的抗SARS-CoV病毒N蛋白单链抗体具有高度的特异性,可以用作临床实验或研究SARS病毒过程中快速检测SARSN蛋白或SARS病毒粒子的候选抗体。     

Inhibition of severe acute respiratory syndrome-associated coronavirus infection by equine neutralizing antibody in golden Syrian hamsters

Equine anti-severe acute respiratory syndrome-associated coronavirus F(ab')(2) has been verified to protect mice from infection with severe acute respiratory syndrome-associated coronavirus (SARS-CoV). However, before potential clinical application, the antibody needs to be tested in as many animal models as possible to ensure its safety and efficiency. In this study, after verification by various methods that the golden Syrian hamster constitutes a model susceptible to SARS-CoV infection, we confirmed that the antibody could protect animals completely from SARS-CoV infection in the preventive setting. More importantly, the antibody could reduce viral titers or copies by approximately 10(3)- to 10(4)-fold in animal lung after virus exposure, compared with negative control. These data provide further evidence to warrant clinical studies of this antibody in the treatment and prevention of SARS.     

Protection from infection with severe acute respiratory syndrome coronavirus in a Chinese hamster model by equine neutralizing F(ab')2

To warrant potential clinical testing, the equine anti-severe acute respiratory syndrome coronavirus (SARS-CoV) F(ab')(2) requires evaluation in as many animal models as possible. In this study, we established a new animal model, the Chinese hamster, susceptible to SARS-CoV infection. SARS-CoV could propagate effectively and sustain high levels for 1 wk in animal lungs. All animals were protected from SARS-CoV infection in preventive settings. Further, when used therapeutically this antibody led to an approximately 4-log(10) decrease in viral burden in infected animal lungs. The pathological changes in lungs correlated closely with the dose of antibody administered. The excellent preventive and therapeutic roles of equine anti-SARS-CoV F(ab')(2) in several animal models, including the novel Chinese hamster model described in this study, have provided exciting data concerning its potential clinical study.     

一种新型冠状病毒假病毒的制备及初步应用

目的 制备一种新型冠状病毒(SARS-CoV-2)假病毒,并将其应用于抗体中和能力检测和抗体广谱性评估.方法 整合2种近期出现的SARS-CoV-2变异病毒株(20A.EU1和B1.1.7)刺突(spike,S)蛋白的突变序列、以及对胞浆区肽段19个氨基酸进行局部缺失突变,构建突变型S蛋白表达质粒,转染293T细胞进行...     

Use of antibody avidity assays for diagnosis of severe acute respiratory syndrome coronavirus infection

An indirect immunofluorescent assay (Euroimmun AG, Luebeck, Germany) was used to investigate the avidity of immunoglobulin G (IgG), IgM, IgA, and total Ig (IgGAM) antibody responses to severe acute respiratory syndrome coronavirus (SARS CoV) infections. Serial serum samples from eight patients collected during the first, third, and ninth months after the onset of infection were evaluated. It was found that low-avidity IgG antibodies were detected in 15/15 (100%), 1/5 (20%), and 0/8 (0%) serum samples collected during the first, third, and ninth months after the onset of symptoms, respectively. Low-avidity antibodies of IgA and IgM subclasses were detected in 14/14 (100%) and 3/14 (21%) serum samples, respectively, collected in the first month after the onset of infection. However, IgA antibodies remained low in avidity in a proportion of patients even during late convalescence. As a consequence, IgG antibody avidity assays gave better discrimination between acute-phase and late-convalescent-phase serum samples than IgM, IgA, or IgGAM assays. In two of these patients, sequential serum samples were also tested for IgG avidity against human CoV strains OC43 and 229E in parallel. While SARS CoV infections induced an anamnestic IgG antibody response to the 229E and OC43 viruses, these cross-reactive antibodies remained of high avidity from early (the first month) postinfection. The results showed that assays to detect low-avidity antibody may be useful for discriminating early from late antibody responses and also for distinguishing anamnestic cross-reactive antibody responses from primary specific responses. This may be useful in some clinical situations.     

Recombinant adeno-associated virus expressing the receptor-binding domain of severe acute respiratory syndrome coronavirus S protein elicits neutralizing antibodies: Implication for developing SARS vaccines

Development of an effective vaccine for severe acute respiratory syndrome (SARS) remains to be a priority to prevent possible re-emergence of SARS coronavirus (SARS-CoV). We previously demonstrated that the receptor-binding domain (RBD) of SARS-CoV S protein is a major target of neutralizing antibodies. This suggests that the RBD may serve as an ideal vaccine candidate. Recombinant adeno-associated virus (rAAV) has been proven to be an effective system for gene delivery and vaccine development. In this study, a novel vaccine against SARS-CoV was developed based on the rAAV delivery system. The gene encoding RBD was cloned into a pAAV-IRES-hrGFP plasmid. The immunogenicity induced by the resulting recombinant RBD-rAAV was evaluated in BALB/c mice. The results demonstrated that (1) a single dose of RBD-rAAV vaccination could induce sufficient neutralizing antibody against SARS-CoV infection; (2) two more repeated doses of the vaccination boosted the neutralizing antibody to about 5 times of the level achieved by a single dose of the immunization and (3) the level of the antibody continued to increase for the entire duration of the experiment of 5.5 months. These results suggested that RBD-rAAV is a promising SARS candidate vaccine.     

Elucidating the molecular physiopathology of acute respiratory distress syndrome in severe acute respiratory syndrome patients

Acute respiratory distress syndrome (ARDS) is a severe form of acute lung injury. It is a response to various diseases of variable etiology, including SARS-CoV infection. To date, a comprehensive study of the genomic physiopathology of ARDS (and SARS) is lacking, primarily due to the difficulty of finding suitable materials to study the disease process at a tissue level (instead of blood, sputa or swaps). Hereby we attempt to provide such study by analyzing autopsy lung samples from patient who died of SARS and showed different degrees of severity of the pulmonary involvement. We performed real-time quantitative PCR analysis of 107 genes with functional roles in inflammation, coagulation, fibrosis and apoptosis; some key genes were confirmed at a protein expression level by immunohistochemistry and correlated to the degree of morphological severity present in the individual samples analyzed. Significant expression levels were identified for ANPEP (a receptor for CoV), as well as inhibition of the STAT1 pathway, IFNs production and CXCL10 (a T-cell recruiter). Other genes unassociated to date with ARDS/SARS include C1Qb, C5R1, CASP3, CASP9, CD14, CD68, FGF7, HLA-DRA, IGF1, IRF3, MALAT-1, MSR1, NFIL3, SLPI, USP33, CLC, GBP1 and TAC1. As a result, we proposed to therapeutically target some of these genes with compounds such as ANPEP inhibitors, SLPI and dexamethasone. Ultimately, this study may serve as a model for future, tissue-based analyses of fibroinflammatory conditions affecting the lung.     

Epitope mapping and biological function analysis of antibodies produced by immunization of mice with an inactivated Chinese isolate of severe acute respiratory syndrome-associated coronavirus (SARS-CoV)

Inactivated severe acute respiratory syndrome-associated coronavirus (SARS-CoV) has been tested as a candidate vaccine against the re-emergence of SARS. In order to understand the efficacy and safety of this approach, it is important to know the antibody specificities generated with inactivated SARS-CoV. In the current study, a panel of twelve monoclonal antibodies (mAbs) was established by immunizing Balb/c mice with the inactivated BJ01 strain of SARS-CoV isolated from the lung tissue of a SARS-infected Chinese patient. These mAbs could recognize SARS-CoV-infected cells by immunofluorescence analysis (IFA). Seven of them were mapped to the specific segments of recombinant spike (S) protein: six on S1 subunit (aa 12-798) and one on S2 subunit (aa 797-1192). High neutralizing titers against SARS-CoV were detected with two mAbs (1A5 and 2C5) targeting at a subdomain of S protein (aa 310-535), consistent with the previous report that this segment of S protein contains the major neutralizing domain. Some of these S-specific mAbs were able to recognize cleaved products of S protein in SARS-CoV-infected Vero E6 cells. None of the remaining five mAbs could recognize either of the recombinant S, N, M, or E antigens by ELISA. This study demonstrated that the inactivated SARS-CoV was able to preserve the immunogenicity of S protein including its major neutralizing domain. The relative ease with which these mAbs were generated against SARS-CoV virions further supports that subunit vaccination with S constructs may also be able to protect animals and perhaps humans. It is somewhat unexpected that no N-specific mAbs were identified albeit anti-N IgG was easily identified in SARS-CoV-infected patients. The availability of this panel of mAbs also provided potentially useful agents with applications in therapy, diagnosis, and basic research of SARS-CoV.     

Pathogenicity of severe acute respiratory coronavirus deletion mutants in hACE-2 transgenic mice

Recombinant severe acute respiratory virus (SARS-CoV) variants lacking the group specific genes 6, 7a, 7b, 8a, 8b and 9b (rSARS-CoV-Delta[6-9b]), the structural gene E (rSARS-CoV-DeltaE), and a combination of both sets of genes (rSARS-CoV-Delta[E,6-9b]) have been generated. All these viruses were rescued in monkey (Vero E6) cells and were also infectious for human (Huh-7, Huh7.5.1 and CaCo-2) cell lines and for transgenic (Tg) mice expressing the SARS-CoV receptor human angiotensin converting enzyme-2 (hACE-2), indicating that none of these proteins is essential for the viral cycle. Furthermore, in Vero E6 cells, all the viruses showed the formation of particles with the same morphology as the wt virus, indicating that these proteins do not have a high impact in the final morphology of the virions. Nevertheless, in the absence of E protein, release of virus particles efficacy was reduced. Viruses lacking E protein grew about 100-fold lower than the wt virus in lungs of Tg infected mice but did not grow in the brains of the same animals, in contrast to the rSARS-CoV-Delta[6-9b] virus, which grew almost as well as the wt in both tissues. Viruses lacking E protein were highly attenuated in the highly sensitive hACE-2 Tg mice, in contrast to the minimal rSARS-CoV-Delta[6-9b] and wt viruses. These data indicate that E gene might be a virulence factor influencing replication level, tissue tropism and pathogenicity of SARS-CoV, suggesting that DeltaE attenuated viruses are promising vaccine candidates.