Epitope Mapping of a Bactericidal Monoclonal Antibody against the Factor H Binding Protein of Neisseria meningitidis

The factor H binding protein (fHbp) is a 27-kDa membrane-anchored lipoprotein of Neisseria meningitidis that allows the survival of the bacterium in human plasma; it is also a major component of a universal vaccine against meningococcus B.In this study, we used nuclear magnetic resonance spectroscopy, mutagenesis, and in silico modeling to map the epitope recognized by MAb502, a bactericidal monoclonal antibody elicited by fHbp. The data show that the antibody recognizes a conformational epitope within a well-defined area of the immunodominant C-terminal domain of the protein that is formed by two loops connecting different β-strands of a β-barrel and a short α-helix brought in spatial proximity by the protein folding. The identification of the protective epitopes of fHbp is an important factor for understanding the mechanism(s) of an effective immune response and provides valuable guidelines for designing variants of the protein able to induce broadly protective immunity.     

The Marburgvirus-Neutralizing Human Monoclonal Antibody MR191 Targets a Conserved Site to Block Virus Receptor Binding

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Monoclonal Antibody Developed against a Hemolysin of Bacillus thuringiensis

A total of five hybridoma cell lines that produced monoclonal antibodies (MAb) against a hemolysin (Bt-hemolysin) produced by Bacillus thuringiensis were established and characterized. All of these monoclonal antibodies reacted similarly not only to Bt-hemolysin but also to a hemolysin (Bc-hemolysin) produced by B. cereus, suggesting that the two hemolysins are immunologically indistinguishable. The MAb developed in this study was also successfully applied for rapid and simple purification of both Bt- and Bc-hemolysins by immunoaffinity column chromatography. The partial N-terminal amino acid sequence of the purified hemolysins was determined to be Ile-Glu-Gln-Thr.     

Production,Binding Characteristics and Protective Immunity of Monoclonal Antibody to Pneumococcal Type-9V Conjugate

A monoclonal antibody (MAb) to pneumococcal type-9V polysaccharide (PS) was produced using PS conjugated to inactivated pneumolysin as the immunogen. The MAb to 9V PS was of the IgG₁ subclass. The antigen-antibody reaction increased rapidly at low concentrations and reached a plateau at 10 μg PS/ml as measured by nephelometry of the group 9 PS against 9V MAb binding. In contrast, the binding of group 9 PS against rabbit 9V antiserum (AS) increased proportionally and continued to increase up to the highest concentration of PS tested (20 μg PS/ml). The 9V MAb reacted with all group 9 PSs (9A, 9L, 9N and 9V) by immunodiffusion. In the homologous 9V Ag-MAb reaction, there were marked differences in the inhibition of binding by the cross-reactive 9L PS (19.2% inhibition) and the 9N PS (0.2%). In contrast, inhibition of the homologous 9V Ag-rabbit AS binding by cross-reactive 9L and 9N PSs ranged from 57.8 to 62.7%. Removal of the O-acetyl group from 9V PS by alkali hydrolysis resulted in decreased binding with rabbit 9V AS. However, the binding reaction with 9V MAb was less affected by the loss of O-acetyl content. The 9V MAb was both opsonic and passively protected young mice against challenge with type-9V pneumococci.     

抗人线粒体核糖体小亚基蛋白17单克隆抗体的制备和鉴定

以纯化人线粒体核糖体小亚基蛋白17(MRPS17)免疫BALB/c小鼠,经细胞融合和ELISA法筛选成功获得1株抗MRPS17杂交瘤细胞。以所获特异性单抗作为一抗,使用Western印迹、免疫组化和免疫荧光等方法检测标本中MRPS17。结果显示:Western印迹检测人骨骼肌组织、黑素瘤组织和体外培养HeLa细胞提取蛋白质,在分子量约13kDa处有一特异性条带,与阳性对照纯化MRPS17相一致;免疫组化检测石蜡切片标本显示人骨骼肌细胞和恶性黑素瘤细胞胞浆中强阳性着色;细胞免疫荧光检测于培养的HeLa细胞,可见细胞核周围胞浆部位颗粒状绿色荧光,其分布与线粒体特异性荧光探针(MitoTrackerRedCM-H2XRos)的荧光分布一致。说明成功制备了具有高度特异性并可适用于多种检测方法的抗人MRPS17单抗,应用该单克隆抗体对人MRPS17进行了亚细胞水平定位,为线粒体生物学相关研究提供了新的研究工具。     

抗人肝素酶单克隆抗体的制备与鉴定

目的:肝素酶在白细胞游走和恶性肿瘤转移的过程中发挥重要作用,肝素酶抗体的制备对于自身免疫病和肿瘤的良恶性鉴别诊断具有重要意义。制备抗人肝素酶单克隆抗体,用于肝素酶的研究及临床恶性肿瘤的鉴别诊断。方法:通过杂交瘤技术将分泌抗人肝素酶单抗的小鼠B细胞与小鼠骨髓瘤细胞Sp2/0融合,获得稳定分泌抗人肝素酶单抗的杂交瘤细胞;用有限稀释法获得单克隆,以重组人肝素酶及含肝素酶的血小板裂解液对抗体进行Western印迹检测。结果:Western印迹结果显示制备的单抗与人肝素酶具有特异性免疫识别特性。结论:获得了能够特异性免疫识别人肝素酶的分泌性抗人肝素酶单克隆抗体。     

A Human Monoclonal Antibody against Hepatitis B Surface Antigen with Potent Neutralizing Activity

We describe the production and characterization of human monoclonal antibodies (mAb) specific for the major hepatitis B virus (HBV) S protein. The mAbs, two IgG1κ and one IgG1λ, were secreted by B-cell clones obtained from peripheral blood mononuclear cells (PBMC) of one person convalescent from acute hepatitis B and one vaccinated individual. The former recognized a denaturation-insensitive epitope within the p24 protein whereas the latter recognized a denaturation-sensitive, conformational epitope located within the HBsAg common “a” determinant. This mAb, denominated ADRI-2F3, displayed a very high protective titer of over 43,000 IU/mg mAb and showed an extremely potent neutralizing activity in the in vitro model of HBV infection using primary hepatocytes from Tupaia belangeri as target. Recombinant variable heavy and light domain sequences derived from mAb ADRI-2F3 were cloned into eukaryotic expression vectors and showed identical fine specificity and 1 log₁₀ higher titer than the original IgG1λ. It is envisaged that such mAb will be able to efficiently prevent HBV reinfection after liver transplantation for end-stage chronic HBV infection or infection after needle-stick exposure, providing an unlimited source of valuable protective anti-HBs antibody.     

Implications of a Vasodilatory Human Monoclonal Autoantibody in Postural Hypotension

Functional autoantibodies to the autonomic receptors are increasingly recognized in the pathophysiology of cardiovascular diseases. To date, no human activating monoclonal autoantibodies to these receptors have been available. In this study, we describe for the first time a β₂-adrenergic receptor (β₂AR)-activating monoclonal autoantibody (C5F2) produced from the lymphocytes of a patient with idiopathic postural hypotension. C5F2, an IgG3 isotype, recognizes an epitope in the N terminus of the second extracellular loop (ECL2) of β₂AR. Surface plasmon resonance analysis revealed high binding affinity for the β₂AR ECL2 peptide. Immunoblotting and immunofluorescence demonstrated specific binding to β₂AR in H9c2 cardiomyocytes, CHO cells expressing human β₂AR, and rat aorta. C5F2 stimulated cyclic AMP production in β₂AR-transfected CHO cells and induced potent dilation of isolated rat cremaster arterioles, both of which were specifically blocked by the β₂AR-selective antagonist ICI-118551 and by the β₂AR ECL2 peptide. This monoclonal antibody demonstrated sufficient activity to produce postural hypotension in its host. Its availability provides a unique opportunity to identify previously unrecognized causes and new pharmacological management of postural hypotension and other cardiovascular diseases.     

Reactivity of a Monoclonal Antibody Raised against Human Leukemic Cells to Embryonic and Adult Tissues of the Mouse and Teratocarcinomas

C-9-1, a monoclonal IgM antibody raised against human null cell acute lymphocytic leukemia cells reacted with restricted regions of embryonic and adult tissues of the mouse. The antigen positive sites in the embryos included embryonic ectoderm, visceral endoderm, trophoblastic cells invading the maternal decidua of 5∼7-day embryos, primordial germ cells of 10∼12-day embryos, epithelium of nasal chamber, the bronchus, Mullerian duct, epididymis and bladder of 12∼17-day embryos. In the adult mice, C-9-1 antigen was detected in renal tubules, a part of stomach, bladder, endometrium and epididymal sperm. Embryonal carcinoma cells, but not endodermal cells of teratocarcinoma expressed the antigen. Thus, C-9-1 antigen showed distribution similar to SSEA-1. However, C-9-1 antigen was not detected in preimplantation embryos, nor in oviduct, both of which are positive for SSEA-1.     

猪细小病毒单克隆抗体的制备和鉴定

目的制备猪细小病毒（PPV）杂交瘤细胞株,并对其分泌的PPV单克隆抗体进行鉴定。方法按常规方法制备并获得2株杂交瘤细胞。用染色体分析对杂交瘤细胞进行鉴定,用间接ELISA、免疫过氧化物酶单层试验（IPMA）和间接免疫荧光试验（IFA）对其分泌的单克隆抗体进行效价测定、亚型鉴定和特异性鉴定。结果得到2株分泌单克隆抗体的杂交瘤细胞株2H9、1F9,染色体数目介于90～110之间。细胞上清效价均达1∶1×104,腹水效价均达1∶1×107,其亚型分别为IgG1、IgM,均为kappa链。2H9、1F9单抗与猪繁殖与呼吸综合征病毒（PRRSV）、猪瘟病毒（CSFV）、猪伪狂犬病毒（PRV）、猪圆环病毒I型（PCV-1）、猪圆环病毒Ⅱ型（PCV-2）、乙脑病毒（JEV）等均无交叉反应。IPMA和IFA检测结果显示2H9、1F9单抗均能与接种于PK-15细胞的PPV发生特异性反应。结论成功制备了2株抗PPV杂交瘤细胞株,证实其产生的单克隆抗体具有良好的特异性和敏感性。     

Development of a Highly Protective Combination Monoclonal Antibody Therapy against Chikungunya Virus

Chikungunya virus (CHIKV) is a mosquito-transmitted alphavirus that causes global epidemics of a debilitating polyarthritis in humans. As there is a pressing need for the development of therapeutic agents, we screened 230 new mouse anti-CHIKV monoclonal antibodies (MAbs) for their ability to inhibit infection of all three CHIKV genotypes. Four of 36 neutralizing MAbs (CHK-102, CHK-152, CHK-166, and CHK-263) provided complete protection against lethality as prophylaxis in highly susceptible immunocompromised mice lacking the type I IFN receptor (Ifnar^−/−) and mapped to distinct epitopes on the E1 and E2 structural proteins. CHK-152, the most protective MAb, was humanized, shown to block viral fusion, and require Fc effector function for optimal activity in vivo. In post-exposure therapeutic trials, administration of a single dose of a combination of two neutralizing MAbs (CHK-102+CHK-152 or CHK-166+CHK-152) limited the development of resistance and protected immunocompromised mice against disease when given 24 to 36 hours before CHIKV-induced death. Selected pairs of highly neutralizing MAbs may be a promising treatment option for CHIKV in humans.     

化学突变具有底物结合部位的单克隆抗体制备含硒抗体酶

开发了一种制备抗体酶的新方法。用二硝基氯苯（ＤＮＣＢ）专一地与谷胱甘肽（ＧＳＨ）的巯基反应,合成出半抗原ＧＳＨ－Ｓ－ＤＮＰ。用戊二醛将半抗原偶联到牛血清白蛋白（ＢＳＡ）上,制成全抗原。再用标准的单抗制备法获得具有ＧＳＨ结合部位的单抗（４Ａ４ＩｇＧ）。用苯甲基磺酞氟（ＰＭＳＦ）和Ｈ２Ｓｅ相继处理该单抗,则将单拉结合部位上的丝氨酸（Ｓｅｒ）突变成硒代半胱氨酸（ＳｅＣｙｓ,因而在单抗结合部位上引入了谷胱甘肽过氧化物酶（ＧＰＸ）的催化基团。突变后的单抗具有ＧＰＸ活性,其活力已达到天然ＧＰＸ的数量级水平。动力学行为也与天然ＧＰＸ类似。这种新的含硒抗体酶有优于ＧＰＸ的一些特点。     

A Neutralizing Human Monoclonal Antibody Protects against Lethal Disease in a New Ferret Model of Acute Nipah Virus Infection

Nipah virus is a broadly tropic and highly pathogenic zoonotic paramyxovirus in the genus Henipavirus whose natural reservoirs are several species of Pteropus fruit bats. Nipah virus has repeatedly caused outbreaks over the past decade associated with a severe and often fatal disease in humans and animals. Here, a new ferret model of Nipah virus pathogenesis is described where both respiratory and neurological disease are present in infected animals. Severe disease occurs with viral doses as low as 500 TCID₅₀ within 6 to 10 days following infection. The underlying pathology seen in the ferret closely resembles that seen in Nipah virus infected humans, characterized as a widespread multisystemic vasculitis, with virus replicating in highly vascular tissues including lung, spleen and brain, with recoverable virus from a variety of tissues. Using this ferret model a cross-reactive neutralizing human monoclonal antibody, m102.4, targeting the henipavirus G glycoprotein was evaluated in vivo as a potential therapeutic agent. All ferrets that received m102.4 ten hours following a high dose oral-nasal Nipah virus challenge were protected from disease while all controls died. This study is the first successful post-exposure passive antibody therapy for Nipah virus using a human monoclonal antibody.     

Functional Relevance of Improbable Antibody Mutations for HIV Broadly Neutralizing Antibody Development

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人源单克隆抗人免疫缺陷病毒1型抗体Fab段基因的获得

应用噬苏体抗体库技术有效地筛选出了多株抗ＨＩＶ－１人源单克隆抗体。以逆转录聚合酶链反应（ＲＴ－ＰＣＲ）从ＨＩＶ－１感染者外周血淋巴细胞中扩增抗体轻重链可变区基因,插入载体ｐＣＯＭＢ３,建立噬菌体抗体库。分别以ＨＩＶ－１ｇｐ１２０和ｇｐ１６０为固相抗原,经过多轮筛选,从中获得了多株抗ＨＩＶ－１ｇｐ４１、ｇｐ１２０和ｇｐ１６０的单克隆抗体Ｆａｂ段基因。抗ＨＩＶ特异性噬菌体抗体随抗体库的筛选高度富集,抗     

Structural Insights into the Neutralization Mechanism of Monoclonal Antibody 6C2 against Ricin

Ricin belongs to the type II ribosome-inactivating proteins that depurinate the universally conserved α-sarcin loop of rRNA. The RNA N-glycosidase activity of ricin also largely depends on the ribosomal proteins that play an important role during the process of rRNA depurination. Therefore, the study of the interaction between ricin and the ribosomal elements will be better to understand the catalysis mechanism of ricin. The antibody 6C2 is a mouse monoclonal antibody exhibiting unusually potent neutralizing ability against ricin, but the neutralization mechanism remains unknown. Here, we report the 2.8 Å crystal structure of 6C2 Fab in complex with the A-chain of ricin (RTA), which reveals an extensive antigen-antibody interface that contains both hydrogen bonds and van der Waals contacts. The complementarity-determining region loops H1, H2, H3, and L3 form a pocket to accommodate the epitope on the RTA (residues Asp⁹⁶–Thr¹¹⁶). ELISA results show that Gln⁹⁸, Glu⁹⁹, Glu¹⁰², and Thr¹⁰⁵ (RTA) are the key residues that play an important role in recognizing 6C2. With the perturbation of the 6C2 Fab-RTA interface, 6C2 loses its neutralization ability, measured based on the inhibition of protein synthesis in a cell-free system. Finally, we propose that the neutralization mechanism of 6C2 against ricin is that the binding of 6C2 hinders the interaction between RTA and the ribosome and the surface plasmon resonance and pulldown results confirm our hypothesis. In short, our data explain the neutralization mechanism of mAb 6C2 against ricin and provide a structural basis for the development of improved antibody drugs with better specificity and higher affinity.