Monoclonal antibody to dengue capsid protein: Its application in dengue studies

Dengue fever (DF) and dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are considered the most important arthropod-borne viral diseases in terms of morbidity and mortality. The emergency and severity of dengue (Den) infections increase the necessity of an early, quick and effective dengue laboratory diagnostic. Viral isolation is considered a gold standard for diagnosis of dengue infection using monoclonal antibodies (mAbs) as a tool for determining serotype specificity. Alternatives have been used to improve sensitivity and time to dengue diagnosis. Based on the early expression of dengue C protein in the life cycle, we focused our study on the application of an anti-dengue 2 virus capsid protein mAb in dengue diagnosis. The kinetic expression of dengue-2 capsid in mosquito cells and its immuno-localization in experimentally infected suckling albin Swiss (OF-1) mice brain tissues was established. The results demonstrate the possible utility of this mAb in early dengue diagnosis versus traditional isolation. In addition, a preliminary study of an enzyme immunoassay method using 8H8 mAb for specific detection of dengue C protein antigen was performed, making possible recombinant C protein quantification. The results suggest that detection of dengue capsid protein could be useful in the diagnosis of early dengue infection.Key words: monoclonal antibodies, capsid protein, dengue virus, diagnosis, immunoassays     

登革病毒衣壳蛋白在大肠杆菌中的表达及其自组装活性的研究

采用高保真RT-PCR自登革2型病毒43株基因组RNA中扩增全长C基因及缺失羧基端Cv片段,分别构建可表达C及Cv的重组质粒pLEX—C和pLEX—Cv,转化E．coliGI724后用色氨酸诱导表达。经SDS—PAGE分析,表达的C及Cv蛋白相对分子质量分别约为12000和10000,分别约占菌体蛋白总量的19％和13％。Western印迹检测表明重组表达的C蛋白均可被特异识别登革病毒衣壳蛋白的单克隆抗体特异识别。表达的蛋白经过硫酸铵沉淀和蔗糖密度梯度离心后,通过琼脂糖凝胶电泳和负染电镜均未能检测到衣壳样颗粒的存在,说明登革病毒衣壳蛋白可能不具体外自组装活性。     

Searching for drug leads targeted to the hydrophobic cleft of dengue virus capsid protein

We synthesised and screened 18 aromatic derivatives of guanylhydrazones and oximes aromatic for their capacity to bind to dengue virus capsid protein (DENVC). The intended therapeutic target was the hydrophobic cleft of DENVC, which is a region responsible for its anchoring in lipid droplets in the infected cells. The inhibition of this process completely suppresses virus infectivity. Using NMR, we describe five compounds able to bind to the α1-α2 interface in the hydrophobic cleft. Saturation transfer difference experiments showed that the aromatic protons of the ligands are important for the interaction with DENVC. Fluorescence binding isotherms indicated that the selected compounds bind at micromolar affinities, possibly leading to binding-induced conformational changes. NMR-derived docking calculations of ligands showed that they position similarly in the hydrophobic cleft. Cytotoxicity experiments and calculations of in silico drug properties suggest that these compounds may be promising candidates in the search for antivirals targeting DENVC.     

High affinity human antibody fragments to dengue virus non-structural protein 3

Background

The enzyme activities catalysed by flavivirus non-structural protein 3 (NS3) are essential for virus replication. They are distributed between the N-terminal protease domain in the first one-third and the C-terminal ATPase/helicase and nucleoside 5′ triphosphatase domain which forms the remainder of the 618-aa long protein.

Methodology/Principal Findings

In this study, dengue full-length NS3 protein with residues 49 to 66 of NS2B covalently attached via a flexible linker, was used as bait in biopanning with a naïve human Fab phage-display library. Using a range of truncated constructs spanning the NS2B cofactor region and the full-length NS3, 10 unique Fab were identified and characterized. Of these, monoclonal Fab 3F8 was shown to bind α3″ (residues 526 through 531) within subdomain III of the helicase domain. The antibody inhibits the ATPase and helicase activites of NS3 in biochemical assays and reduces DENV replication in HEK293 cells that were previously transfected with Fab 3F8 compared with mock transfected cells.

Conclusions/Significance

Antibodies such as 3F8 are valuable tools for studying the molecular mechanisms of flaviviral replication and for the monospecific detection of replicating dengue virus in vivo.     

The disordered N-terminal region of dengue virus capsid protein contains a lipid-droplet-binding motif

Dengue is the major arthropod-borne human viral disease, for which no vaccine or specific treatment is available. We used NMR, zeta potential measurements and atomic force microscopy to study the structural features of the interaction between dengue virus C (capsid) protein and LDs (lipid droplets), organelles crucial for infectious particle formation. C protein-binding sites to LD were mapped, revealing a new function for a conserved segment in the N-terminal disordered region and indicating that conformational selection is involved in recognition. The results suggest that the positively charged N-terminal region of C protein prompts the interaction with negatively charged LDs, after which a conformational rearrangement enables the access of the central hydrophobic patch to the LD surface. Taken together, the results allowed the design of a peptide with inhibitory activity of C protein-LD binding, paving the way for new drug development approaches against dengue.     

鼠源抗HFRSV衣壳蛋白McAbF3株单链抗体基因克隆构建及表达

为了获得原抗ＨＦＲＳＶ衣壳蛋白ＭｃＡｂＦ３＾１株轻链可变区基因,由连接肽体外连接获得单链抗体基因,在大肠杆菌中表达,从鼠源抗ＨＦＲＳＶ衣壳蛋白ＭｃＡｂＦ３株细胞中分离总ＲＮＡ,以ｏｌｉｇｏ（ｄＴ）１８为引物逆转录成ｃＤＮＡ,通过ＰＣＲ扩增出抗体的轻链（ＶＬ）和重链可变区（Ｖ１１）基因,由连接本外连接获得单链抗体（ＳｅＦｖ）基因。将此单链抗体（ＳｅＦｖ）基因插入原核表达载体ＰＥＴ２８ａ,经大肠杆菌（     

Monoclonal antibodies to reovirus reveal structure/function relationships between capsid proteins and genetics of susceptibility to antibody action.

Thirteen newly isolated monoclonal antibodies (MAbs) were used to study relationships between reovirus outer capsid proteins sigma 3, mu 1c, and lambda 2 (core spike) and the cell attachment protein sigma 1. We focused on sigma 1-associated properties of serotype specificity and hemagglutination (HA). Competition between MAbs revealed two surface epitopes on mu 1c that were highly conserved between reovirus serotype 1 Lang (T1L) and serotype 3 Dearing (T3D). There were several differences between T1L and T3D sigma 3 epitope maps. Studies using T1L x T3D reassortants showed that primary sequence differences between T1L and T3D sigma 3 proteins accounted for differences in sigma 3 epitope maps. Four of 12 non-sigma 1 MAbs showed a serotype-associated pattern of binding to 25 reovirus field isolates. Thus, for reovirus field isolates, different sigma 1 proteins are associated with preferred epitopes on other outer capsid proteins. Further evidence for a close structural and functional interrelationship between sigma 3/mu 1c and sigma 1 included (i) inhibition by sigma 3 and mu 1c MAbs of sigma 1-mediated HA, (ii) enhancement of sigma 1-mediated HA by proteolytic cleavage of sigma 3 and mu 1c, and (iii) genetic studies demonstrating that sigma 1 controlled the capacity of sigma 3 MAbs to inhibit HA. These data suggest that (i) epitopes on sigma 3 and mu 1c lie in close proximity to sigma 1 and that MAbs to these epitopes can modulate sigma 1-mediated functions, (ii) these spatial relationships have functional significance, since removal of sigma 3 and/or cleavage of mu 1c to delta can enhance sigma 1 function, (iii) in nature, the sigma 1 protein places selective constraints on the epitope structure of the other capsid proteins, and (iv) viral susceptibility to antibody action can be determined by genes other than that encoding an antibody's epitope.     

Development of cell lines stably expressing staphylococcal nuclease fused to dengue 2 virus capsid protein for CTVI

Dengue virus (DV) is one of the most significanthuman viral pathogens transmitted by arthropod vectorsand now present in over 100 countries. Half of the world’spopulation live in areas at risk of dengue virus infection[1]. The infection of DV causes a spectrum of diseasesranging from a debilitating, self-limited illness (denguefever), and life-threaten syndromes (dengue haemorrhagicfever/dengue shock syndrome). Annually, the fourserotypes of DV collectively cause 50–100 million casesof inf…     

Monoclonal antibody to GABA binding protein, a possible GABAB receptor.

A monoclonal antibody has been raised against a partially purified preparation for the GABAB receptor. The antibody recognized a protein of about 80 kDa in bovine brain synaptic membrane. Immunoabsorbent agarose beads conjugated with the antibody were able to remove, without visible changes in electrophoresed profiles of total proteins, over 90% of the baclofen suppressive GABA binding activity (designated herein, GABAB receptor binding activity) in the solubilized synaptic membrane fraction. Moreover, the addition of GB-1 antibody directly inhibited the GABA binding activity in the crude synaptic membrane fraction. These results indicate that the monoclonal antibody obtained here recognizes the GABA binding protein, or more specifically a GABAB receptor.     

Immune and antibody responses to an isolated capsid protein of foot-and-mouth disease virus.

The purified capsid proteins VP1, VP2, and VP3 of foot-and-mouth disease virus type A12 strain 119 emulsified with incomplete Freund's adjuvant were studied in swine and guinea pigs. Swine inoculated on days 0, 28, and 60 with 100-mug doses of VP3 were protected by day 82 against exposure to infected swine. Serums from animals inoculated with VP3 contained viral precipitating and neutralizing antibodies, but such serums recognized fewer viral antigenic determinants than did antiviral serums. Capsid proteins VP1 and VP2 did not produce detectable antiviral antibody in guinea pigs, and antiviral antibody responses in swine to a mixture of VP1, VP2, and VP3 were lower than the responses to VP3 alone. However, when swine were inoculated with VP1, VP2, and VP3 separately at different body sites, no interference with the response to VP3 was observed. Vaccine containing VP3 isolated from acetylethylenimine-treated virus appeared less protective for swine than vaccine containing VP3 from nontreated virus. Trypsinized virus, which contains the cleaved peptides VP3a and VP3b rather than intact VP3, produced approximately the same levels of antiviral antibody responses in guinea pigs as did virus. Conversely, an isolated mixture of VP3a and VP3b did not produce detectable antiviral antibody responses in guinea pigs. The VP3a-VP3b mixture did, however, sensitize guinea pigs to elicit such responses following reinoculation with a marginally effective dose of trypsinized virus.     

Monoclonal antibody, a novel probe for protein folding

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Monoclonal antibody to human lung angiotensin-converting enzyme

The hybridoma producing monoclonal antibody (IgG1) to human angiotensin-converting enzyme (ACE) has been prepared by fusion of murine myeloma P3O1 with spleen cells of BALB/c mice immunized with a purified human lung ACE preparation. A high specificity of monoclonal antibody (MAb) binding to immobilized ACE has been demonstrated by enzyme-linked immunosorbent assay and that of soluble ACE by an immunoadsorption test. The latter technique permits the use of impure ACE preparations for the screening procedure. This MAb did not affect ACE activity. We believe this antibody will be useful not only for immunoassay and immunopurification of ACE, but also as a tool for the investigation of the tissue distribution of the enzyme as well as for the study of the structure and mechanism of action of ACE.     

Monoclonal antibody to human plasma gelsolin.

Human plasma gelsolin was purified by column chromatography. The method yielded a protein of high purity and activity. Using this protein, we produced monoclonal antibody (Mab H6B11) against human plasma gelsolin by somatic cell fusion. This monoclonal antibody reacted in a dose-dependent manner with gelsolin derived from human plasma and platelets and neutralized depolymerizing activity to F-actin. It differed from the commercially available substance (Mab G4896; Sigma) in that the time required for the reaction between the antigen and antibody in the enzyme-linked immunosorbent assay could be shortened by one-third. The antibody was judged to be useful in assays for elucidating the physiological role of plasma gelsolin.     

Monoclonal antibody to a protein of the nucleus and mitotic spindle of mammalian cells

We report here the isolation of a monoclonal antibody, J17, that reacts with a conserved vertebrate protein antigen that is present in the spindle apparatus during mitosis but found within the nucleus during interphase. Immunofluorescence microscopy demonstrates that the J17 antigen is found in numerous punctate regions that are distinct from nucleoli. Furthermore, this antigen is not directly associated with kinetochores, the nuclear envelope, or with metaphase chromosomes. — Antibody J17 immunoprecipitates a single polypeptide of very high molecular weight (over 250000) from K562 human erythroleukemia cells pulse-labeled with ¹⁴C-leucine. This polypeptide is converted quantitatively to a stable 220-kilodalton product within one cellular generation. We discuss the possible relevance of this processing event for transport into the nucleus. The J17 antigen is synthesized throughout the cell cycle in Chinese hamster ovary cells.     

Monoclonal antibody analysis of human pancreatic juice components related to the pancreatic calculi protein

A CaCO3-crystal growth inhibitor has recently been isolated from the calculi of patients affected by pancreatic lithiasis. It is a phosphoglycoprotein, with a molecular weight of 14,000, whose probable physiological role is the stabilization of exocrine pancreatic secretion which is supersaturated with respect to CaCO3. In order to isolate this inhibitor from human pancreatic juice, monoclonal antibodies to the protein were prepared and an immunoadsorbent column was developed. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of proteins fixed by the immunoadsorbent reveals the form having a molecular weight of 14,000, in addition to other protein bands which have higher molecular weights (16,000, 16,800, 18,000, and 18,800). All of these different proteins are also recognized by a monospecific polyclonal antibody to the 14,000 molecular weight form. Using the same monospecific polyclonal antibody only one messenger RNA coding for this inhibitor has been demonstrated. Thus, this heterogeneity might be explained by post-translational modifications.     

Monoclonal antibody recognizes a conformational epitope in a random coil protein

The antigenic determinants for two monoclonal antibodies directed against horse apo-cytochrome c, a protein of disordered structure, as judged by spectroscopic and hydrodynamic criteria, have been studied by a combination of methods: antigen competition in solution by radio immunoassay and enzyme-linked immunoassay, and differential acetylation of free and antibody-bound antigen. In the latter method the accessibility of lysine residues of the antigen in the antigen-antibody complex is compared to the accessibility in the free antigen. The two antibodies against the heme-free protein do not recognize intact native cytochrome c, but they crossreact with the heme-containing peptides 1-38 and 1-65 of cytochrome c. The antigenic determinant recognized by monoclonal antibody SJL 2-4 is conformational and discontiguous, it is composed of residues close to the N-terminus and around position 25. The other monoclonal antibody, Cyt-1-59, seems to recognize a contiguous epitope close to the N-terminus. The present results show that even a seemingly disordered protein which is conventionally classified as a random coil may feature subtle spatial regularities. The presence of ordered conformational elements in apocytochrome c may be important for the enzyme-catalyzed covalent attachment of the heme and the import of cytochrome c into mitochondria. A discontiguous determinant for SJL 2-4 is particularly interesting because this antibody inhibits the proliferation of a T-cell clone specific for apo-cytochrome c [Corradin & Engers (1984) Nature (Lond.) 308, 547-548].     

Monoclonal antibody to glutamate dehydrogenase isolation and characterization

This paper describes the isolation and characterization of a monoclonal antibody to bovine liver glutamate dehydrogenase (GDH). Monoclonal antibody is mouse immunoglobulin subclass IgG2a and reacts strongly with the antigen in an enzyme-linked immunosorbent assay (ELISA). Its specificity was determined by an antigen binding assay and by Western blotting. Potential uses and possible applications are discussed.