Sample records for aberrant o-glycopeptide epitopes

  1. Seromic profiling of colorectal cancer patients with novel glycopeptide microarray.

    PubMed

    Pedersen, Johannes W; Blixt, Ola; Bennett, Eric P; Tarp, Mads A; Dar, Imran; Mandel, Ulla; Poulsen, Steen S; Pedersen, Anders E; Rasmussen, Susanne; Jess, Per; Clausen, Henrik; Wandall, Hans H

    2011-04-15

    Cancer-associated autoantibodies hold promise as sensitive biomarkers for early detection of cancer. Aberrant post-translational variants of proteins are likely to induce autoantibodies, and changes in O-linked glycosylation represent one of the most important cancer-associated post-translational modifications (PTMs). Short aberrant O-glycans on proteins may introduce novel glycopeptide epitopes that can elicit autoantibodies because of lack of tolerance. Technical barriers, however, have hampered detection of such glycopeptide-specific autoantibodies. Here, we have constructed an expanded glycopeptide array displaying a comprehensive library of glycopeptides and glycoproteins derived from a panel of human mucins (MUC1, MUC2, MUC4, MUC5AC, MUC6 and MUC7) known to have altered glycosylation and expression in cancer. Seromic profiling of patients with colorectal cancer identified cancer-associated autoantibodies to a set of aberrant glycopeptides derived from MUC1 and MUC4. The cumulative sensitivity of the array analysis was 79% with a specificity of 92%. The most prevalent of the identified autoantibody targets were validated as authentic cancer immunogens by showing expression of the epitopes in cancer using novel monoclonal antibodies. Our study provides evidence for the value of glycopeptides and other PTM-peptide arrays in diagnostic measures. Copyright © 2011 UICC.

  2. Immunological Evaluation of Recent MUC1 Glycopeptide Cancer Vaccines

    PubMed Central

    Hossain, Md Kamal; Wall, Katherine A.

    2016-01-01

    Aberrantly glycosylated mucin 1 (MUC1) is a recognized tumor-specific antigen on epithelial cell tumors. A wide variety of MUC1 glycopeptide anti-cancer vaccines have been formulated by many research groups. Some researchers have used MUC1 alone as an immunogen whereas other groups used different antigenic carrier proteins such as bovine serum albumin or keyhole limpet hemocyanin for conjugation with MUC1 glycopeptide. A variety of adjuvants have been used with MUC1 glycopeptides to improve their immunogenicity. Fully synthetic multicomponent vaccines have been synthesized by incorporating different T helper cell epitopes and Toll-like receptor agonists. Some vaccine formulations utilized liposomes or nanoparticles as vaccine delivery systems. In this review, we discuss the immunological evaluation of different conjugate or synthetic MUC1 glycopeptide vaccines in different tumor or mouse models that have been published since 2012. PMID:27472370

  3. Antibody recognition of a unique tumor-specific glycopeptide antigen

    PubMed Central

    Brooks, Cory L.; Schietinger, Andrea; Borisova, Svetlana N.; Kufer, Peter; Okon, Mark; Hirama, Tomoko; MacKenzie, C. Roger; Wang, Lai-Xi; Schreiber, Hans; Evans, Stephen V.

    2010-01-01

    Aberrant glycosylation and the overexpression of certain carbohydrate moieties is a consistent feature of cancers, and tumor-associated oligosaccharides are actively investigated as targets for immunotherapy. One of the most common aberrations in glycosylation patterns is the presentation of a single O-linked N-acetylgalactosamine on a threonine or serine residue known as the “Tn antigen.” Whereas the ubiquitous nature of Tn antigens on cancers has made them a natural focus of vaccine research, such carbohydrate moieties are not always tumor-specific and have been observed on embryonic and nonmalignant adult tissue. Here we report the structural basis of binding of a complex of a monoclonal antibody (237mAb) with a truly tumor-specific glycopeptide containing the Tn antigen. In contrast to glycopeptide-specific antibodies in complex with simple peptides, 237mAb does not recognize a conformational epitope induced in the peptide by sugar substitution. Instead, 237mAb uses a pocket coded by germ-line genes to completely envelope the carbohydrate moiety itself while interacting with the peptide moiety in a shallow groove. Thus, 237mAb achieves its striking tumor specificity, with no observed physiological cross-reactivity to the unglycosylated peptide or the free glycan, by a combination of multiple weak but specific interactions to both the peptide and to the glycan portions of the antigen. PMID:20479270

  4. Analysis of O-Glycopeptides by Acetone Enrichment and Capillary Electrophoresis-Mass Spectrometry.

    PubMed

    Mancera-Arteu, Montserrat; Giménez, Estela; Benavente, Fernando; Barbosa, José; Sanz-Nebot, Victòria

    2017-11-03

    Acetone precipitation was evaluated as a rapid, simple, low-cost, and efficient method for the selective purification of O-glycopeptides from enzymatic digests of glycoproteins. Ovalbumin (OVA), human and bovine α 1 -acid glycoprotein (hAGP and bAGP), human apolipoprotein C-III (APO-C3), and recombinant human erythropoietin (rhEPO) were used to obtain enzymatic digests with a broad and varied set of peptides, N-glycopeptides, and O-glycopeptides. After digestion and before capillary electrophoresis mass spectrometry (CE-MS) analysis, the amount of ice-cold acetone added to the digests was optimized to maximize recoveries of O-glycopeptides. Furthermore, the different behavior of peptides, N- and O-glycopeptides was explained by studying with multivariate data analysis methods the influence of several physicochemical parameters and properties related to their composition and structure. Principal component analysis (PCA) and, afterward, partial least-squares discriminant analysis (PLS-DA) were used to identify the most significant variables and their importance to differentiate between peptides, N-glycopeptides and O-glycopeptides, or within these classes. This information was useful to understand precipitation of these compounds after addition of acetone and for the selection of the optimal conditions for purification of specific O-glycopeptide biomarkers. Special attention was paid to O 126 -glycopeptide glycoforms of rhEPO because of their applicability in biopharmaceutical quality control and doping analysis.

  5. Targeted, Site-specific quantitation of N- and O-glycopeptides using 18O-labeling and product ion based mass spectrometry.

    PubMed

    Srikanth, Jandhyam; Agalyadevi, Rathinasamy; Babu, Ponnusamy

    2017-02-01

    The site-specific quantitation of N- and O-glycosylation is vital to understanding the function(s) of different glycans expressed at a given site of a protein under physiological and disease conditions. Most commonly used precursor ion intensity based quantification method is less accurate and other labeled methods are expensive and require enrichment of glycopeptides. Here, we used glycopeptide product (y and Y0) ions and 18 O-labeling of C-terminal carboxyl group as a strategy to obtain quantitative information about fold-change and relative abundance of most of the glycoforms attached to the glycopeptides. As a proof of concept, the accuracy and robustness of this targeted, relative quantification LC-MS method was demonstrated using Rituximab. Furthermore, the N-glycopeptide quantification results were compared with a biosimilar of Rituximab and validated with quantitative data obtained from 2-AB-UHPLC-FL method. We further demonstrated the intensity fold-change and relative abundance of 46 unique N- and O-glycopeptides and aglycopeptides from innovator and biosimilar samples of Etanercept using both the normal-MS and product ion based quantitation. The results showed a very similar site-specific expression of N- and O-glycopeptides between the samples but with subtle differences. Interestingly, we have also been able to quantify macro-heterogeneity of all N- and O-glycopetides of Etanercept. In addition to applications in biotherapeutics, the developed method can also be used for site-specific quantitation of N- and O-glycopeptides and aglycopeptides of glycoproteins with known glycosylation pattern.

  6. Status Report on the High-Throughput Characterization of Complex Intact O-Glycopeptide Mixtures

    NASA Astrophysics Data System (ADS)

    Pap, Adam; Klement, Eva; Hunyadi-Gulyas, Eva; Darula, Zsuzsanna; Medzihradszky, Katalin F.

    2018-05-01

    A very complex mixture of intact, human N- and O-glycopeptides, enriched from the tryptic digest of urinary proteins of three healthy donors using a two-step lectin affinity enrichment, was analyzed by LC-MS/MS, leading to approximately 45,000 glycopeptide EThcD spectra. Two search engines, Byonic and Protein Prospector, were used for the interpretation of the data, and N- and O-linked glycopeptides were assigned from separate searches. The identification rate was very low in all searches, even when results were combined. Thus, we investigated the reasons why was it so, to help to improve the identification success rate. Focusing on O-linked glycopeptides, we noticed that in EThcD, larger glycan oxonium ions better survive the activation than those in HCD. These fragments, combined with reducing terminal Y ions, provide important information about the glycan(s) present, so we investigated whether filtering the peaklists for glycan oxonium ions indicating the presence of a tetra- or hexasaccharide structure would help to reveal all molecules containing such glycans. Our study showed that intact glycans frequently do not survive even mild supplemental activation, meaning one cannot rely on these oxonium ions exclusively. We found that ETD efficiency is still a limiting factor, and for highly glycosylated peptides, the only information revealed in EThcD was related to the glycan structures. The limited overlap of results delivered by the two search engines draws attention to the fact that automated data interpretation of O-linked glycopeptides is not even close to being solved. [Figure not available: see fulltext.

  7. Concurrent Automated Sequencing of the Glycan and Peptide Portions of O-Linked Glycopeptide Anions by Ultraviolet Photodissociation Mass Spectrometry

    PubMed Central

    Madsen, James A.; Ko, Byoung Joon; Xu, Hua; Iwashkiw, Jeremy A.; Robotham, Scott A.; Shaw, Jared B.; Feldman, Mario F.; Brodbelt, Jennifer S.

    2013-01-01

    O -glycopeptides are often acidic owing to the frequent occurrence of acidic saccharides in the glycan, rendering traditional proteomic workflows that rely on positive mode tandem mass spectrometry (MS/MS) less effective. In this report, we demonstrate the utility of negative mode ultraviolet photodissociation (UVPD) MS for the characterization of acidic O-linked glycopeptide anions. This method was evaluated for a series of singly- and multiply-deprotonated glycopeptides from the model glycoprotein kappa casein, resulting in production of both peptide and glycan product ions that afforded 100% sequence coverage of the peptide and glycan moieties from a single MS/MS event. The most abundant and frequent peptide sequence ions were a/x-type products, which, importantly, were found to retain the labile glycan modifications. The glycan-specific ions mainly arose from glycosidic bond cleavages (B, Y, C, and Z ions) in addition to some less common cross-ring cleavages. Based on the UVPD fragmentation patterns, an automated database searching strategy (based on the MassMatrix algorithm) was designed that is specific for the analysis of glycopeptide anions by UVPD. This algorithm was used to identify glycopeptides from mixtures of glycosylated and non-glycosylated peptides, sequence both glycan and peptide moieties simultaneously, and pinpoint the correct site(s) of glycosylation. This methodology was applied to uncover novel site-specificity of the O-linked glycosylated OmpA/MotB from the “superbug” A. baumannii to help aid in the elucidation of the functional role that protein glycosylation plays in pathogenesis. PMID:24006841

  8. Two New Tools for Glycopeptide Analysis Researchers: A Glycopeptide Decoy Generator and a Large Data Set of Assigned CID Spectra of Glycopeptides.

    PubMed

    Lakbub, Jude C; Su, Xiaomeng; Zhu, Zhikai; Patabandige, Milani W; Hua, David; Go, Eden P; Desaire, Heather

    2017-08-04

    The glycopeptide analysis field is tightly constrained by a lack of effective tools that translate mass spectrometry data into meaningful chemical information, and perhaps the most challenging aspect of building effective glycopeptide analysis software is designing an accurate scoring algorithm for MS/MS data. We provide the glycoproteomics community with two tools to address this challenge. The first tool, a curated set of 100 expert-assigned CID spectra of glycopeptides, contains a diverse set of spectra from a variety of glycan types; the second tool, Glycopeptide Decoy Generator, is a new software application that generates glycopeptide decoys de novo. We developed these tools so that emerging methods of assigning glycopeptides' CID spectra could be rigorously tested. Software developers or those interested in developing skills in expert (manual) analysis can use these tools to facilitate their work. We demonstrate the tools' utility in assessing the quality of one particular glycopeptide software package, GlycoPep Grader, which assigns glycopeptides to CID spectra. We first acquired the set of 100 expert assigned CID spectra; then, we used the Decoy Generator (described herein) to generate 20 decoys per target glycopeptide. The assigned spectra and decoys were used to test the accuracy of GlycoPep Grader's scoring algorithm; new strengths and weaknesses were identified in the algorithm using this approach. Both newly developed tools are freely available. The software can be downloaded at http://glycopro.chem.ku.edu/GPJ.jar.

  9. Synthetic Self-Adjuvanting Glycopeptide Cancer Vaccines

    NASA Astrophysics Data System (ADS)

    Payne, Richard; McDonald, David; Byrne, Scott

    2015-10-01

    Due to changes in glycosyltransferase expression during tumorigenesis, the glycoproteins of cancer cells often carry highly truncated carbohydrate chains compared to those on healthy cells. These glycans are known as tumor-associated carbohydrate antigens, and are prime targets for use in vaccines for the prevention and treatment of cancer. Herein, we review the state-of-the-art in targeting the immune system towards tumor-associated glycopeptide antigens via synthetic self adjuvanting vaccines, in which the antigenic and adjuvanting moieties of the vaccines are present in the same molecule. The majority of the self-adjuvanting glycopeptide cancer vaccines reported to date employ antigens from mucin 1, a protein which is highly over-expressed and aberrantly glycosylated in many forms of cancer. The adjuvants used in these vaccines predominantly include lipopeptide- or lipoamino acid-based TLR2 agonists, although studies investigating stimulation of TLR9 and TLR4 are also discussed. Most of these adjuvants are highly lipophilic, and, upon conjugation to antigenic peptides, provide amphiphilic vaccine molecules. The amphiphilic nature of these vaccine constructs can lead to the formation of higher-order structures by vaccines in solution, which are likely to be important for their efficacy in vivo.

  10. Separation and Identification of Isomeric Glycopeptides by High Field Asymmetric Waveform Ion Mobility Spectrometry

    PubMed Central

    2012-01-01

    The analysis of intact glycopeptides by mass spectrometry is challenging due to the numerous possibilities for isomerization, both within the attached glycan and the location of the modification on the peptide backbone. Here, we demonstrate that high field asymmetric wave ion mobility spectrometry (FAIMS), also known as differential ion mobility, is able to separate isomeric O-linked glycopeptides that have identical sequences but differing sites of glycosylation. Two glycopeptides from the glycoprotein mucin 5AC, GT(GalNAc)TPSPVPTTSTTSAP and GTTPSPVPTTST(GalNAc)TSAP (where GalNAc is O-linked N-acetylgalactosamine), were shown to coelute following reversed-phase liquid chromatography. However, FAIMS analysis of the glycopeptides revealed that the compensation voltage ranges in which the peptides were transmitted differed. Thus, it is possible at certain compensation voltages to completely separate the glycopeptides. Separation of the glycopeptides was confirmed by unique reporter ions produced by supplemental activation electron transfer dissociation mass spectrometry. These fragments also enable localization of the site of glycosylation. The results suggest that glycan position plays a key role in determining gas-phase glycopeptide structure and have implications for the application of FAIMS in glycoproteomics. PMID:22280549

  11. Automated glycopeptide analysis—review of current state and future directions

    PubMed Central

    Dallas, David C.; Martin, William F.; Hua, Serenus

    2013-01-01

    Glycosylation of proteins is involved in immune defense, cell–cell adhesion, cellular recognition and pathogen binding and is one of the most common and complex post-translational modifications. Science is still struggling to assign detailed mechanisms and functions to this form of conjugation. Even the structural analysis of glycoproteins—glycoproteomics—remains in its infancy due to the scarcity of high-throughput analytical platforms capable of determining glycopeptide composition and structure, especially platforms for complex biological mixtures. Glycopeptide composition and structure can be determined with high mass-accuracy mass spectrometry, particularly when combined with chromatographic separation, but the sheer volume of generated data necessitates computational software for interpretation. This review discusses the current state of glycopeptide assignment software—advances made to date and issues that remain to be addressed. The various software and algorithms developed so far provide important insights into glycoproteomics. However, there is currently no freely available software that can analyze spectral data in batch and unambiguously determine glycopeptide compositions for N- and O-linked glycopeptides from relevant biological sources such as human milk and serum. Few programs are capable of aiding in structural determination of the glycan component. To significantly advance the field of glycoproteomics, analytical software and algorithms are required that: (i) solve for both N- and O-linked glycopeptide compositions, structures and glycosites in biological mixtures; (ii) are high-throughput and process data in batches; (iii) can interpret mass spectral data from a variety of sources and (iv) are open source and freely available. PMID:22843980

  12. [Preparation of cysteine-click maltose modified silica as a hydrophilic interaction liquid chromatography material for the enrichment of glycopeptides].

    PubMed

    Sun, Xudong; Zhang, Lingyi; Zhang, Weibing

    2017-07-08

    Because of the low abundance of glycoprotein and glycopeptide in complex biological samples, it is urgent to develop an efficient method for glycopeptide enrichment in comprehensive and in-depth glycoproteomes research. Herein, a novel hydrophilic silica was developed through surface modification with cysteine-click maltose (Cys-Mal@SiO 2 ). The developed hydrophilic silica was packed into a solid phase extraction (SPE) column, and applied to the highly selective enrichment and identification of N -linked glycopeptides. The Cys-Mal@SiO 2 demonstrated better identification capability over Cys@SiO 2 , Mal@SiO 2 and commercial hydrophilic interaction liquid chromatography (HILIC) in glycopeptide enrichment due to the synergistic effect of the two kinds of hydrophilic molecules. In the selective enrichment of tryptic digest from human immunoglobulin G, glycopeptides with higher signal-to-noises were detected by Cys-Mal@SiO 2 . In addition, 1551 unique glycopeptides with 906 N -glycosylation sites from 466 different N -linked glycoproteins were identified from the proteins extracted from mouse liver after the enrichment with Cys-Mal@SiO 2 . In contrast, the numbers of identified glycopeptides, glycoproteins and N -glycosylation sites identified by Cys@SiO 2 were 211, 67, 127 respectively less than by Cys-Mal@SiO 2 , and the corresponding numbers were 289, 76, 193 by Mal@SiO 2 . These results showed that the developed Cys-Mal@SiO 2 is a promising affinity material for N -glycoproteomics research of real complex biological samples.

  13. Glycopeptide Analysis, Recent Developments and Applications*

    PubMed Central

    Desaire, Heather

    2013-01-01

    Glycopeptide-based analysis is used to inform researchers about the glycans on one or more proteins. The method's key attractive feature is its ability to link glycosylation information to exact locations (glycosylation sites) on proteins. Numerous applications for glycopeptide analysis are known, and several examples are described herein. The techniques used to characterize glycopeptides are still emerging, and recently, research focused on facilitating aspects of glycopeptide analysis has advanced significantly in the areas of sample preparation, MS fragmentation, and automation of data analysis. These recent developments, described herein, provide the foundation for the growth of glycopeptide analysis as a blossoming field. PMID:23389047

  14. Glycopeptide antibiotic biosynthesis.

    PubMed

    Yim, Grace; Thaker, Maulik N; Koteva, Kalinka; Wright, Gerard

    2014-01-01

    Glycopeptides such as vancomycin, teicoplanin and telavancin are essential for treating infections caused by Gram-positive bacteria. Unfortunately, the dwindled pipeline of new antibiotics into the market and the emergence of glycopeptide-resistant enterococci and other resistant bacteria are increasingly making effective antibiotic treatment difficult. We have now learned a great deal about how bacteria produce antibiotics. This information can be exploited to develop the next generation of antimicrobials. The biosynthesis of glycopeptides via nonribosomal peptide assembly and unusual amino acid synthesis, crosslinking and tailoring enzymes gives rise to intricate chemical structures that target the bacterial cell wall. This review seeks to describe recent advances in our understanding of both biosynthesis and resistance of these important antibiotics.

  15. Facile synthesis of thiol-polyethylene glycol functionalized magnetic titania nanomaterials for highly efficient enrichment of N-linked glycopeptides.

    PubMed

    Wang, Jiawen; Yao, Jizong; Sun, Nianrong; Deng, Chunhui

    2017-08-25

    As protein N-glycosylation involved in generation and development of various cancers and diseases, it is vital to capture glycopeptides from complex biological samples for biomarker discovery. In this work, by taking advantages of the interaction between titania and thiol groups, thiol-polyethylene glycol functionalized magnetic titania nanomaterials (denoted as Fe 3 O 4 @TiO 2 @PEG) were firstly fabricated as an excellent hydrophilic adsorbent of N-linked glycopeptides. On one hand, the special interaction of titanium-thiol makes the synthetic manipulation simple and provides a new idea for design and synthesis of novel nanomaterials; on the other hand, strong magnetic response could realize rapid separation and the outstanding hydrophilicity of polyethylene glycol makes Fe 3 O 4 @TiO 2 @PEG nanomaterials show superior performance for glycopeptides enrichment with ultralow limit of detection (0.1mol/μL) and high selectivity (1:100). As a result, 24 and 33 glycopeptides enriched from HRP and IgG digests were identified respectively by MALDI-TOF MS, and 300 glycopeptides corresponding to 106 glycoproteins were recognized from merely 2μL human serum, indicating a great potential of Fe 3 O 4 @TiO 2 @PEG nanomaterials for glycoproteomic research. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Facile synthesis of zwitterionic polymer-coated core-shell magnetic nanoparticles for highly specific capture of N-linked glycopeptides

    NASA Astrophysics Data System (ADS)

    Chen, Yajing; Xiong, Zhichao; Zhang, Lingyi; Zhao, Jiaying; Zhang, Quanqing; Peng, Li; Zhang, Weibing; Ye, Mingliang; Zou, Hanfa

    2015-02-01

    Highly selective and efficient capture of glycosylated proteins and peptides from complex biological samples is of profound significance for the discovery of disease biomarkers in biological systems. Recently, hydrophilic interaction liquid chromatography (HILIC)-based functional materials have been extensively utilized for glycopeptide enrichment. However, the low amount of immobilized hydrophilic groups on the affinity material has limited its specificity, detection sensitivity and binding capacity in the capture of glycopeptides. Herein, a novel affinity material was synthesized to improve the binding capacity and detection sensitivity for glycopeptides by coating a poly(2-(methacryloyloxy)ethyl)-dimethyl-(3-sulfopropyl) ammonium hydroxide (PMSA) shell onto Fe3O4@SiO2 nanoparticles, taking advantage of reflux-precipitation polymerization for the first time (denoted as Fe3O4@SiO2@PMSA). The thick polymer shell endows the nanoparticles with excellent hydrophilic property and several functional groups on the polymer chains. The resulting Fe3O4@SiO2@PMSA demonstrated an outstanding ability for glycopeptide enrichment with high selectivity, extremely high detection sensitivity (0.1 fmol), large binding capacity (100 mg g-1), high enrichment recovery (above 73.6%) and rapid magnetic separation. Furthermore, in the analysis of real complicated biological samples, 905 unique N-glycosylation sites from 458 N-glycosylated proteins were reliably identified in three replicate analyses of a 65 μg protein sample extracted from mouse liver, showing the great potential of Fe3O4@SiO2@PMSA in the detection and identification of low-abundance N-linked glycopeptides in biological samples.Highly selective and efficient capture of glycosylated proteins and peptides from complex biological samples is of profound significance for the discovery of disease biomarkers in biological systems. Recently, hydrophilic interaction liquid chromatography (HILIC)-based functional materials have

  17. Intact glycopeptide characterization using mass spectrometry.

    PubMed

    Cao, Li; Qu, Yi; Zhang, Zhaorui; Wang, Zhe; Prytkova, Iya; Wu, Si

    2016-05-01

    Glycosylation is one of the most prominent and extensively studied protein post-translational modifications. However, traditional proteomic studies at the peptide level (bottom-up) rarely characterize intact glycopeptides (glycosylated peptides without removing glycans), so no glycoprotein heterogeneity information is retained. Intact glycopeptide characterization, on the other hand, provides opportunities to simultaneously elucidate the glycan structure and the glycosylation site needed to reveal the actual biological function of protein glycosylation. Recently, significant improvements have been made in the characterization of intact glycopeptides, ranging from enrichment and separation, mass spectroscopy (MS) detection, to bioinformatics analysis. In this review, we recapitulated currently available intact glycopeptide characterization methods with respect to their advantages and limitations as well as their potential applications.

  18. Facile synthesis of zwitterionic polymer-coated core-shell magnetic nanoparticles for highly specific capture of N-linked glycopeptides.

    PubMed

    Chen, Yajing; Xiong, Zhichao; Zhang, Lingyi; Zhao, Jiaying; Zhang, Quanqing; Peng, Li; Zhang, Weibing; Ye, Mingliang; Zou, Hanfa

    2015-02-21

    Highly selective and efficient capture of glycosylated proteins and peptides from complex biological samples is of profound significance for the discovery of disease biomarkers in biological systems. Recently, hydrophilic interaction liquid chromatography (HILIC)-based functional materials have been extensively utilized for glycopeptide enrichment. However, the low amount of immobilized hydrophilic groups on the affinity material has limited its specificity, detection sensitivity and binding capacity in the capture of glycopeptides. Herein, a novel affinity material was synthesized to improve the binding capacity and detection sensitivity for glycopeptides by coating a poly(2-(methacryloyloxy)ethyl)-dimethyl-(3-sulfopropyl) ammonium hydroxide (PMSA) shell onto Fe3O4@SiO2 nanoparticles, taking advantage of reflux-precipitation polymerization for the first time (denoted as Fe3O4@SiO2@PMSA). The thick polymer shell endows the nanoparticles with excellent hydrophilic property and several functional groups on the polymer chains. The resulting Fe3O4@SiO2@PMSA demonstrated an outstanding ability for glycopeptide enrichment with high selectivity, extremely high detection sensitivity (0.1 fmol), large binding capacity (100 mg g(-1)), high enrichment recovery (above 73.6%) and rapid magnetic separation. Furthermore, in the analysis of real complicated biological samples, 905 unique N-glycosylation sites from 458 N-glycosylated proteins were reliably identified in three replicate analyses of a 65 μg protein sample extracted from mouse liver, showing the great potential of Fe3O4@SiO2@PMSA in the detection and identification of low-abundance N-linked glycopeptides in biological samples.

  19. Changes in the expression of N- and O-glycopeptides in patients with colorectal cancer and hepatocellular carcinoma quantified by full-MS scan FT-ICR and multiple reaction monitoring.

    PubMed

    Darebna, Petra; Novak, Petr; Kucera, Radek; Topolcan, Ondrej; Sanda, Miloslav; Goldman, Radoslav; Pompach, Petr

    2017-02-05

    Alternations in the glycosylation of proteins have been described in connection with several cancers, including hepatocellular carcinoma (HCC) and colorectal cancer. Analytical tools, which use combination of liquid chromatography and mass spectrometry, allow precise and sensitive description of these changes. In this study, we use MRM and FT-ICR operating in full-MS scan, to determine ratios of intensities of specific glycopeptides in HCC, colorectal cancer, and liver metastasis of colorectal cancer. Haptoglobin, hemopexin and complement factor H were detected after albumin depletion and the N-linked glycopeptides with fucosylated glycans were compared with their non-fucosylated forms. In addition, sialylated forms of an O-linked glycopeptide of hemopexin were quantified in the same samples. We observe significant increase in fucosylation of all three proteins and increase in bi-sialylated O-glycopeptide of hemopexin in HCC of hepatitis C viral (HCV) etiology by both LC-MS methods. The results of the MRM and full-MS scan FT-ICR analyses provide comparable quantitative readouts in spite of chromatographic, mass spectrometric and data analysis differences. Our results suggest that both workflows allow adequate relative quantification of glycopeptides and suggest that HCC of HCV etiology differs in glycosylation from colorectal cancer and liver metastasis of colorectal cancer. The article compares N- and O-glycosylation of several serum proteins in different diseases by a fast and easy sample preparation procedure in combination with high resolution Fourier transform ion cyclotron resonance mass spectrometry. The results show successful glycopeptides relative quantification in a complex peptide mixture by the high resolution instrument and the detection of glycan differences between the different types of cancer diseases. The presented method is comparable to conventional targeted MRM approach but allows additional curation of the data. Copyright © 2016 Elsevier B

  20. Changes in the expression of N- and O-glycopeptides in patients with colorectal cancer and hepatocellular carcinoma quantified by full-MS scan FT-ICR and multiple reaction monitoring

    PubMed Central

    Darebna, Petra; Novak, Petr; Kucera, Radek; Topolcan, Ondrej; Sanda, Miloslav; Goldman, Radoslav; Pompach, Petr

    2018-01-01

    Alternations in the glycosylation of proteins have been described in connection with several cancers, including hepatocellular carcinoma (HCC) and colorectal cancer. Analytical tools, which use combination of liquid chromatography and mass spectrometry, allow precise and sensitive description of these changes. In this study, we use MRM and FT-ICR operating in full-MS scan, to determine ratios of intensities of specific glycopeptides in HCC, colorectal cancer, and liver metastasis of colorectal cancer. Haptoglobin, hemopexin and complement factor H were detected after albumin depletion and the N-linked glycopeptides with fucosylated glycans were compared with their non-fucosylated forms. In addition, sialylated forms of an O-linked glycopeptide of hemopexin were quantified in the same samples. We observe significant increase in fucosylation of all three proteins and increase in bisialylated O-glycopeptide of hemopexin in HCC of hepatitis C viral (HCV) etiology by both LC-MS methods. The results of the MRM and full-MS scan FT-ICR analyses provide comparable quantitative readouts in spite of chromatographic, mass spectrometric and data analysis differences. Our results suggest that both workflows allow adequate relative quantification of glycopeptides and suggest that HCC of HCV etiology differs in glycosylation from colorectal cancer and liver metastasis of colorectal cancer. Significance The article compares N- and O-glycosylation of several serum proteins in different diseases by a fast and easy sample preparation procedure in combination with high resolution Fourier transform ion cyclotron resonance mass spectrometry. The results show successful glycopeptides relative quantification in a complex peptide mixture by the high resolution instrument and the detection of glycan differences between the different types of cancer diseases. The presented method is comparable to conventional targeted MRM approach but allows additional curation of the data. PMID:27646713

  1. Homoserine as an Aspartic Acid Precursor for Synthesis of Proteoglycan Glycopeptide Containing Aspartic Acid and a Sulfated Glycan Chain.

    PubMed

    Yang, Weizhun; Ramadan, Sherif; Yang, Bo; Yoshida, Keisuke; Huang, Xuefei

    2016-12-02

    Among many hurdles in synthesizing proteoglycan glycopeptides, one challenge is the incorporation of aspartic acid in the peptide backbone and acid sensitive O-sulfated glycan chains. To overcome this, a new strategy was developed utilizing homoserine as an aspartic acid precursor. The conversion of homoserine to aspartic acid in the glycopeptide was successfully accomplished by late stage oxidation using (2,2,6,6-tetramethyl-piperidin-1-yl)oxyl (TEMPO) and bis(acetoxy)iodobenzene (BAIB). This is the first time that a glycopeptide containing aspartic acid and an O-sulfated glycan was synthesized.

  2. N-Glycopeptide Profiling in Arabidopsis Inflorescence

    DOE PAGES

    Xu, Shou-Ling; Medzihradszky, Katalin F.; Wang, Zhi-Yong; ...

    2016-04-11

    This study presents the first large scale analysis of plant intact glycopeptides. Using wheat germ agglutinin lectin weak affinity chromatography to enrich modified peptides, followed by ETD fragmentation tandem mass spectrometry, glycan compositions on over 1100 glycopeptides from 270 proteins found in Arabidopsis inflorescence tissue were characterized. While some sites were only detected with a single glycan attached, others displayed up to 16 different glycoforms. Among the identified glycopeptides were four modified in non-consensus glycosylation motifs. Finally, while most of the modified proteins are secreted, membrane, ER or Golgi localized proteins, surprisingly N-linked sugars were detected on a protein predictedmore » to be cytosolic or nuclear.« less

  3. Click Synthesis of Hydrophilic Maltose-Functionalized Iron Oxide Magnetic Nanoparticles Based on Dopamine Anchors for Highly Selective Enrichment of Glycopeptides.

    PubMed

    Bi, Changfen; Zhao, Yingran; Shen, Lijin; Zhang, Kai; He, Xiwen; Chen, Langxing; Zhang, Yukui

    2015-11-11

    The development of methods to isolate and enrich low-abundance glycopeptides from biological samples is crucial to glycoproteomics. Herein, we present an easy and one-step surface modification strategy to prepare hydrophilic maltose functionalized Fe3O4 nanoparticles (NPs). First, based on the chelation of the catechol ligand with iron atoms, azido-terminated dopamine (DA) derivative was assembled on the surface of magnetic Fe3O4 nanoparticles by sonication. Second, the hydrophilic maltose-functionalized Fe3O4 (Fe3O4-DA-Maltose) NPs were obtained via copper(I)-catalyzed azide-alkyne cycloaddition (click chemistry). The morphology, structure, and composition of Fe3O4-DA-Maltose NPs were investigated by Fourier transform infrared spectroscopy (FT-IR), transmission electron microscopy (TEM), X-ray powder diffraction (XRD), X-ray photoelectron spectrometer (XPS), and vibrating sample magnetometer (VSM). Meanwhile, hydrophilicity of the obtained NPs was evaluated by water contact angle measurement. The hydrophilic Fe3O4-DA-Maltose NPs were applied in isolation and enrichment of glycopeptides from horseradish peroxidase (HRP), immunoglobulin (IgG) digests. The MALDI-TOF mass spectrometric analysis indicated that the novel NPs exhibited high detection sensitivity in enrichment from HRP digests at concentration as low as 0.05 ng μL(-1), a large binding capacity up to 43 mg g(-1), and good recovery for glycopeptides enrichment (85-110%). Moreover, the Fe3O4-DA-Maltose NPs were applied to enrich glycopeptides from human renal mesangial cells (HRMC) for identification of N-glycosylation sites. Finally, we identified 115 different N-linked glycopeptides, representing 93 gene products and 124 glycosylation sites in HRMC.

  4. Cysteine S-glycosylation, a new post-translational modification found in glycopeptide bacteriocins.

    PubMed

    Stepper, Judith; Shastri, Shilpa; Loo, Trevor S; Preston, Joanne C; Novak, Petr; Man, Petr; Moore, Christopher H; Havlíček, Vladimír; Patchett, Mark L; Norris, Gillian E

    2011-02-18

    O-Glycosylation is a ubiquitous eukaryotic post-translational modification, whereas early reports of S-linked glycopeptides have never been verified. Prokaryotes also glycosylate proteins, but there are no confirmed examples of sidechain glycosylation in ribosomal antimicrobial polypeptides collectively known as bacteriocins. Here we show that glycocin F, a bacteriocin secreted by Lactobacillus plantarum KW30, is modified by an N-acetylglucosamine β-O-linked to Ser18, and an N-acetylhexosamine S-linked to C-terminal Cys43. The O-linked N-acetylglucosamine is essential for bacteriostatic activity, and the C-terminus is required for full potency (IC(50) 2 nM). Genomic context analysis identified diverse putative glycopeptide bacteriocins in Firmicutes. One of these, the reputed lantibiotic sublancin, was shown to contain a hexose S-linked to Cys22. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  5. Study on behaviors and performances of universal N-glycopeptide enrichment methods.

    PubMed

    Xue, Yu; Xie, Juanjuan; Fang, Pan; Yao, Jun; Yan, Guoquan; Shen, Huali; Yang, Pengyuan

    2018-04-16

    Glycosylation is a crucial process in protein biosynthesis. However, the analysis of glycopeptides through MS remains challenging due to the microheterogeneity and macroheterogeneity of the glycoprotein. Selective enrichment of glycopeptides from complex samples prior to MS analysis is essential for successful glycoproteome research. In this work, we systematically investigated the behaviors and performances of boronic acid chemistry, ZIC-HILIC, and PGC of glycopeptide enrichment to promote understanding of these methods. We also optimized boronic acid chemistry and ZIC-HILIC enrichment methods and applied them to enrich glycopeptides from mouse liver. The intact N-glycopeptides were interpreted using the in-house analysis software pGlyco 2.0. We found that boronic acid chemistry in this study preferred to capture glycopeptides with high mannose glycans, ZIC-HILIC enriched most N-glycopeptides and did not show significant preference during enrichment and PGC was not suitable for separating glycopeptides with a long amino acid sequence. We performed a detailed study on the behaviors and performances of boronic acid chemistry, ZIC-HILIC, and PGC enrichment methods and provide a better understanding of enrichment methods for further glycoproteomics research.

  6. Highly Selective Enrichment of Glycopeptides Based on Zwitterionically Functionalized Soluble Nanopolymers

    NASA Astrophysics Data System (ADS)

    Cao, Weiqian; Huang, Jiangming; Jiang, Biyun; Gao, Xing; Yang, Pengyuan

    2016-07-01

    Efficient glycopeptides enrichment prior to mass spectrometry analysis is essential for glycoproteome study. ZIC-HILIC (zwitterionic hydrophilic interaction liquid chromatography) based glycopeptides enrichment approaches have been attracting more attention for several benefits like easy operating, high enrichment specificity and intact glycopeptide retained. In this study, Poly (amidoamine) dendrimer (PAMAM) was adopted for the synthesis of zwitterionically functionalized (ZICF) materials for glycopeptide enrichment. The multiple branched structure and good solubility of ZICF-PAMAM enables a sufficient interaction with glycopeptides. The ZICF-PAMAM combined with the FASP-mode enrichment strategy exhibits more superior performance compared with the existing methods. It has the minimum detectable concentration of femtomolar level and high recovery rate of over 90.01%, and can efficiently enrich glycopeptides from complex biological samples even for merely 0.1 μL human serum. The remarkable glycopeptides enrichment capacity of ZICF-PAMAM highlights the potential application in in-depth glycoproteome research, which may open up new opportunities for the development of glycoproteomics.

  7. Highly Selective Enrichment of Glycopeptides Based on Zwitterionically Functionalized Soluble Nanopolymers.

    PubMed

    Cao, Weiqian; Huang, Jiangming; Jiang, Biyun; Gao, Xing; Yang, Pengyuan

    2016-07-14

    Efficient glycopeptides enrichment prior to mass spectrometry analysis is essential for glycoproteome study. ZIC-HILIC (zwitterionic hydrophilic interaction liquid chromatography) based glycopeptides enrichment approaches have been attracting more attention for several benefits like easy operating, high enrichment specificity and intact glycopeptide retained. In this study, Poly (amidoamine) dendrimer (PAMAM) was adopted for the synthesis of zwitterionically functionalized (ZICF) materials for glycopeptide enrichment. The multiple branched structure and good solubility of ZICF-PAMAM enables a sufficient interaction with glycopeptides. The ZICF-PAMAM combined with the FASP-mode enrichment strategy exhibits more superior performance compared with the existing methods. It has the minimum detectable concentration of femtomolar level and high recovery rate of over 90.01%, and can efficiently enrich glycopeptides from complex biological samples even for merely 0.1 μL human serum. The remarkable glycopeptides enrichment capacity of ZICF-PAMAM highlights the potential application in in-depth glycoproteome research, which may open up new opportunities for the development of glycoproteomics.

  8. Ana o 1 and Ana o 2 cashew allergens share cross-reactive CD4+ T-cell epitopes with other tree nuts

    PubMed Central

    Archila, Luis Diego; Chow, I-Ting; McGinty, John W.; Renand, Amedee; Jeong, David; Robinson, David; Farrington, Mary L.; Kwok, William.W.

    2017-01-01

    Background Allergies to cashew are increasing in prevalence, with clinical symptoms ranging from oral pruritus to fatal anaphylactic reaction. Yet, cashew-specific T-cell epitopes and T-cell cross-reactivity amongst cashew and other tree nut allergens in humans remain uncharacterized. Objectives In this study, we characterized cashew specific T-cell responses in cashew allergic subjects and examined cross-reactivity of these cashew specific cells toward other tree nut allergens. Methods CD154 up-regulation assay was used to determine immunodominance hierarchy among cashew major allergens at the T cell level. The phenotype, magnitude and functionality of cashew-specific T-cells was determined by utilizing ex vivo staining with MHC class II tetramers. Dual tetramer staining and proliferation experiments were used to determine cross-reactivity to other tree nuts. Results CD4+ T-cell responses were directed towards cashew allergens Ana o 1 and Ana o 2. Multiple Ana o 1 and Ana o 2 T-cell epitopes were then identified. These epitopes elicited either TH2 or TH2/TH17 responses in allergic subjects, which were either cashew unique epitope or cross-reactive epitopes. For clones that recognized the cross-reactive epitope, T-cell clones responded robustly to cashew, hazelnut and/or pistachio but not to walnut. Conclusions Phylogenetically diverse tree nut allergens can activate cashew reactive T-cells and elicit a TH2 type response at an epitope specific level. Clinical relevance Lack of cross-reactivity between walnut and cashew suggest that cashew peptide immunotherapy approach may not be most effective for walnut. PMID:27129138

  9. Ana o 1 and Ana o 2 cashew allergens share cross-reactive CD4(+) T cell epitopes with other tree nuts.

    PubMed

    Archila, L D; Chow, I-T; McGinty, J W; Renand, A; Jeong, D; Robinson, D; Farrington, M L; Kwok, W W

    2016-06-01

    Allergies to cashew are increasing in prevalence, with clinical symptoms ranging from oral pruritus to fatal anaphylactic reaction. Yet, cashew-specific T cell epitopes and T cell cross-reactivity amongst cashew and other tree nut allergens in humans remain uncharacterized. In this study, we characterized cashew-specific T cell responses in cashew-allergic subjects and examined cross-reactivity of these cashew-specific cells towards other tree nut allergens. CD154 up-regulation assay was used to determine immunodominance hierarchy among cashew major allergens at the T cell level. The phenotype, magnitude and functionality of cashew-specific T cells were determined by utilizing ex vivo staining with MHC class II tetramers. Dual tetramer staining and proliferation experiments were used to determine cross-reactivity to other tree nuts. CD4(+) T cell responses were directed towards cashew allergens Ana o 1 and Ana o 2. Multiple Ana o 1 and Ana o 2 T cell epitopes were then identified. These epitopes elicited either TH 2 or TH 2/TH 17 responses in allergic subjects, which were either cashew unique epitope or cross-reactive epitopes. For clones that recognized the cross-reactive epitope, T cell clones responded robustly to cashew, hazelnut and/or pistachio but not to walnut. Phylogenetically diverse tree nut allergens can activate cashew-reactive T cells and elicit a TH 2-type response at an epitope-specific level. Lack of cross-reactivity between walnut and cashew suggests that cashew peptide immunotherapy approach may not be most effective for walnut. © 2016 John Wiley & Sons Ltd.

  10. Antibody induction directed against the tumor-associated MUC4 glycoprotein.

    PubMed

    Cai, Hui; Palitzsch, Björn; Hartmann, Sebastian; Stergiou, Natascha; Kunz, Horst; Schmitt, Edgar; Westerlind, Ulrika

    2015-04-13

    Mucin glycoproteins are important diagnostic and therapeutic targets for cancer treatment. Although several strategies have been developed to explore anti-tumor vaccines based on MUC1 glycopeptides, only few studies have focused on vaccines directed against the tumor-associated MUC4 glycoprotein. MUC4 is an important tumor marker overexpressed in lung cancer and uniquely expressed in pancreatic ductual adenocarcinoma. The aberrant glycosylation of MUC4 in tumor cells results in an exposure of its peptide backbone and the formation of tumor-associated glycopeptide antigens. Due to the low immunogenicity of these endogenous structures, their conjugation with immune stimulating peptide or protein carriers are required. In this study, MUC4 tandem-repeat glycopeptides were conjugated to the tetanus toxoid and used for vaccination of mice. Immunological evaluations showed that our MUC4-based vaccines induced very strong antigen-specific immune responses. In addition, antibody binding epitope analysis on glycopeptide microarrays, were demonstrating a clear glycosylation site dependence of the induced antibodies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. Automatic and rapid identification of glycopeptides by nano-UPLC-LTQ-FT-MS and proteomic search engine.

    PubMed

    Giménez, Estela; Gay, Marina; Vilaseca, Marta

    2017-01-30

    Here we demonstrate the potential of nano-UPLC-LTQ-FT-MS and the Byonic™ proteomic search engine for the separation, detection, and identification of N- and O-glycopeptide glycoforms in standard glycoproteins. The use of a BEH C18 nanoACQUITY column allowed the separation of the glycopeptides present in the glycoprotein digest and a baseline-resolution of the glycoforms of the same glycopeptide on the basis of the number of sialic acids. Moreover, we evaluated several acquisition strategies in order to improve the detection and characterization of glycopeptide glycoforms with the maximum number of identification percentages. The proposed strategy is simple to set up with the technology platforms commonly used in proteomic labs. The method allows the straightforward and rapid obtention of a general glycosylated map of a given protein, including glycosites and their corresponding glycosylated structures. The MS strategy selected in this work, based on a gas phase fractionation approach, led to 136 unique peptides from four standard proteins, which represented 78% of the total number of peptides identified. Moreover, the method does not require an extra glycopeptide enrichment step, thus preventing the bias that this step could cause towards certain glycopeptide species. Data are available via ProteomeXchange with identifier PXD003578. We propose a simple and high-throughput glycoproteomics-based methodology that allows the separation of glycopeptide glycoforms on the basis of the number of sialic acids, and their automatic and rapid identification without prior knowledge of protein glycosites or type and structure of the glycans. Copyright © 2016 Elsevier B.V. All rights reserved.

  12. Use of CID/ETD Mass Spectrometry to Analyze Glycopeptides

    PubMed Central

    Mechref, Yehia

    2013-01-01

    Collision-induced dissociation (CID) tandem mass spectrometry (MS) does not allow the characterization of glycopeptides because of the fragmentation of their glycan structures and limited fragmentation of peptide backbones. Electron-transfer dissociation (ETD) tandem MS, on the other hand, offers an alternative approach allowing the fragmentation of only peptide backbones of glycopeptides. Characterization of glycopeptides using both CID and ETD is summarized in this unit. While CID provide information related to the composition of glycan moiety attached to a peptide backbone, ETD permits de novo sequencing of peptides, since it prompts only peptide backbone fragmentation while keeping posttranslational modifications intact. Radical anions transfer of electrons to peptide backbone which induces cleavage of the N-Cα bond is observed in ETD. The glycan moiety is retained on the peptide backbone, largely unaffected by the ETD process. Accordingly, ETD allows not only the identification of the amino acid sequence of a glycopeptide, but also the unambiguous assignment of its glycosylation site. When data acquired from both fragmentation techniques are combined, it is possible to characterize comprehensively the entire glycopeptide. This is achieved using an instrument capable of alternating between CID and ETD experiments during an LC-MS/MS analysis. This unit discusses the different fragmentation of glycopeptides observed in CID and ETD. Tables of residue masses associated with oxonium ions observed in CID are provided to help in the interpretation of CID mass spectra. The utility of both CID and ETD for better characterization of glycopeptides are demonstrated for a model glycoprotein. PMID:22470127

  13. Parallel Comparison of N-Linked Glycopeptide Enrichment Techniques Reveals Extensive Glycoproteomic Analysis of Plasma Enabled by SAX-ERLIC.

    PubMed

    Totten, Sarah M; Feasley, Christa L; Bermudez, Abel; Pitteri, Sharon J

    2017-03-03

    Protein glycosylation is of increasing interest due to its important roles in protein function and aberrant expression with disease. Characterizing protein glycosylation remains analytically challenging due to its low abundance, ion suppression issues, and microheterogeneity at glycosylation sites, especially in complex samples such as human plasma. In this study, the utility of three common N-linked glycopeptide enrichment techniques is compared using human plasma. By analysis on an LTQ-Orbitrap Elite mass spectrometer, electrostatic repulsion hydrophilic interaction liquid chromatography using strong anion exchange solid-phase extraction (SAX-ERLIC) provided the most extensive N-linked glycopeptide enrichment when compared with multilectin affinity chromatography (M-LAC) and Sepharose-HILIC enrichments. SAX-ERLIC enrichment yielded 191 unique glycoforms across 72 glycosylation sites from 48 glycoproteins, which is more than double that detected using other enrichment techniques. The greatest glycoform diversity was observed in SAX-ERLIC enrichment, with no apparent bias toward specific glycan types. SAX-ERLIC enrichments were additionally analyzed by an Orbitrap Fusion Lumos mass spectrometer to maximize glycopeptide identifications for a more comprehensive assessment of protein glycosylation. In these experiments, 829 unique glycoforms were identified across 208 glycosylation sites from 95 plasma glycoproteins, a significant improvement from the initial method comparison and one of the most extensive site-specific glycosylation analysis in immunodepleted human plasma to date. Data are available via ProteomeXchange with identifier PXD005655.

  14. Characterization of Glycan Structures of Chondroitin Sulfate-Glycopeptides Facilitated by Sodium Ion-Pairing and Positive Mode LC-MS/MS

    NASA Astrophysics Data System (ADS)

    Nilsson, Jonas; Noborn, Fredrik; Gomez Toledo, Alejandro; Nasir, Waqas; Sihlbom, Carina; Larson, Göran

    2017-02-01

    Purification and liquid chromatography-tandem mass spectrometry (LC-MS/MS) characterization of glycopeptides, originating from protease digests of glycoproteins, enables site-specific analysis of protein N- and O-glycosylations. We have described a protocol to enrich, hydrolyze by chondroitinase ABC, and characterize chondroitin sulfate-containing glycopeptides (CS-glycopeptides) using positive mode LC-MS/MS. The CS-glycopeptides, originating from the Bikunin proteoglycan of human urine samples, had ΔHexAGalNAcGlcAGalGalXyl- O-Ser hexasaccharide structure and were further substituted with 0-3 sulfate and 0-1 phosphate groups. However, it was not possible to exactly pinpoint sulfate attachment residues, for protonated precursors, due to extensive fragmentation of sulfate groups using high-energy collision induced dissociation (HCD). To circumvent the well-recognized sulfate instability, we now introduced Na+ ions to form sodiated precursors, which protected sulfate groups from decomposition and facilitated the assignment of sulfate modifications. Sulfate groups were pinpointed to both Gal residues and to the GalNAc of the hexasaccharide structure. The intensities of protonated and sodiated saccharide oxonium ions were very prominent in the HCD-MS2 spectra, which provided complementary structural analysis of sulfate substituents of CS-glycopeptides. We have demonstrated a considerable heterogeneity of the bikunin CS linkage region. The realization of these structural variants should be beneficial in studies aimed at investigating the importance of the CS linkage region with regards to the biosynthesis of CS and potential interactions to CS binding proteins. Also, the combined use of protonated and sodiated precursors for positive mode HCD fragmentation analysis will likely become useful for additional classes of sulfated glycopeptides.

  15. Facile and easily popularized synthesis of L-cysteine-functionalized magnetic nanoparticles based on one-step functionalization for highly efficient enrichment of glycopeptides.

    PubMed

    Feng, Xiaoyan; Deng, Chunhui; Gao, Mingxia; Zhang, Xiangmin

    2018-01-01

    Protein glycosylation is one of the most important post-translational modifications. Also, efficient enrichment and separation of glycopeptides from complex samples are crucial for the thorough analysis of glycosylation. Developing novel hydrophilic materials with facile and easily popularized synthesis is an urgent need in large-scale glycoproteomics research. Herein, for the first time, a one-step functionalization strategy based on metal-organic coordination was proposed and Fe 3 O 4 nanoparticles were directly functionalized with zwitterionic hydrophilic L-cysteine (L-Cys), greatly simplifying the synthetic procedure. The easily synthesized Fe 3 O 4 /L-Cys possessed excellent hydrophilicity and brief composition, contributing to affinity for glycopeptides and reduction in nonspecific interaction. Thus, Fe 3 O 4 /L-Cys nanoparticles showed outstanding sensitivity (25 amol/μL), high selectivity (mixture of bovine serum albumin and horseradish peroxidase tryptic digests at a mass ratio of 100:1), good reusability (five repeated times), and stability (room temperature storage of 1 month). Encouragingly, in the glycosylation analysis of human serum, a total of 376 glycopeptides with 393 N-glycosylation sites corresponding to 118 glycoproteins were identified after enrichment with Fe 3 O 4 /L-Cys, which was superior to ever reported L-Cys modified magnetic materials. Furthermore, applying the one-step functionalization strategy, cysteamine and glutathione respectively direct-functionalized Fe 3 O 4 nanoparticles were successfully synthesized and also achieved efficient glycopeptide enrichment in human serum. The results indicated that we have presented an efficient and easily popularized strategy in glycoproteomics as well as in the synthesis of novel materials. Graphical abstract Fe 3 O 4 /L-Cys nanoparticles based on one-step functionalization for highly efficient enrichment of glycopeptides.

  16. Electron-Transfer/Higher-Energy Collision Dissociation (EThcD)-Enabled Intact Glycopeptide/Glycoproteome Characterization

    NASA Astrophysics Data System (ADS)

    Yu, Qing; Wang, Bowen; Chen, Zhengwei; Urabe, Go; Glover, Matthew S.; Shi, Xudong; Guo, Lian-Wang; Kent, K. Craig; Li, Lingjun

    2017-09-01

    Protein glycosylation, one of the most heterogeneous post-translational modifications, can play a major role in cellular signal transduction and disease progression. Traditional mass spectrometry (MS)-based large-scale glycoprotein sequencing studies heavily rely on identifying enzymatically released glycans and their original peptide backbone separately, as there is no efficient fragmentation method to produce unbiased glycan and peptide product ions simultaneously in a single spectrum, and that can be conveniently applied to high throughput glycoproteome characterization, especially for N-glycopeptides, which can have much more branched glycan side chains than relatively less complex O-linked glycans. In this study, a redefined electron-transfer/higher-energy collision dissociation (EThcD) fragmentation scheme is applied to incorporate both glycan and peptide fragments in one single spectrum, enabling complete information to be gathered and great microheterogeneity details to be revealed. Fetuin was first utilized to prove the applicability with 19 glycopeptides and corresponding five glycosylation sites identified. Subsequent experiments tested its utility for human plasma N-glycoproteins. Large-scale studies explored N-glycoproteomics in rat carotid arteries over the course of restenosis progression to investigate the potential role of glycosylation. The integrated fragmentation scheme provides a powerful tool for the analysis of intact N-glycopeptides and N-glycoproteomics. We also anticipate this approach can be readily applied to large-scale O-glycoproteome characterization. [Figure not available: see fulltext.

  17. Peptidoglycan Cross-Linking in Glycopeptide-Resistant Actinomycetales

    PubMed Central

    Hugonnet, Jean-Emmanuel; Haddache, Nabila; Veckerlé, Carole; Dubost, Lionel; Marie, Arul; Shikura, Noriyasu; Mainardi, Jean-Luc; Rice, Louis B.

    2014-01-01

    Synthesis of peptidoglycan precursors ending in d-lactate (d-Lac) is thought to be responsible for glycopeptide resistance in members of the order Actinomycetales that produce these drugs and in related soil bacteria. More recently, the peptidoglycan of several members of the order Actinomycetales was shown to be cross-linked by l,d-transpeptidases that use tetrapeptide acyl donors devoid of the target of glycopeptides. To evaluate the contribution of these resistance mechanisms, we have determined the peptidoglycan structure of Streptomyces coelicolor A(3)2, which harbors a vanHAX gene cluster for the production of precursors ending in d-Lac, and Nonomuraea sp. strain ATCC 39727, which is devoid of vanHAX and produces the glycopeptide A40296. Vancomycin retained residual activity against S. coelicolor A(3)2 despite efficient incorporation of d-Lac into cytoplasmic precursors. This was due to a d,d-transpeptidase-catalyzed reaction that generated a stem pentapeptide recognized by glycopeptides by the exchange of d-Lac for d-Ala and Gly. The contribution of l,d-transpeptidases to resistance was limited by the supply of tetrapeptide acyl donors, which are essential for the formation of peptidoglycan cross-links by these enzymes. In the absence of a cytoplasmic metallo-d,d-carboxypeptidase, the tetrapeptide substrate was generated by hydrolysis of the C-terminal d-Lac residue of the stem pentadepsipeptide in the periplasm in competition with the exchange reaction catalyzed by d,d-transpeptidases. In Nonomuraea sp. strain ATCC 39727, the contribution of l,d-transpeptidases to glycopeptide resistance was limited by the incomplete conversion of pentapeptides into tetrapeptides despite the production of a cytoplasmic metallo-d,d-carboxypeptidase. Since the level of drug production exceeds the level of resistance, we propose that l,d-transpeptidases merely act as a tolerance mechanism in this bacterium. PMID:24395229

  18. Peptidoglycan cross-linking in glycopeptide-resistant Actinomycetales.

    PubMed

    Hugonnet, Jean-Emmanuel; Haddache, Nabila; Veckerlé, Carole; Dubost, Lionel; Marie, Arul; Shikura, Noriyasu; Mainardi, Jean-Luc; Rice, Louis B; Arthur, Michel

    2014-01-01

    Synthesis of peptidoglycan precursors ending in D-lactate (D-Lac) is thought to be responsible for glycopeptide resistance in members of the order Actinomycetales that produce these drugs and in related soil bacteria. More recently, the peptidoglycan of several members of the order Actinomycetales was shown to be cross-linked by L,D-transpeptidases that use tetrapeptide acyl donors devoid of the target of glycopeptides. To evaluate the contribution of these resistance mechanisms, we have determined the peptidoglycan structure of Streptomyces coelicolor A(3)2, which harbors a vanHAX gene cluster for the production of precursors ending in D-Lac, and Nonomuraea sp. strain ATCC 39727, which is devoid of vanHAX and produces the glycopeptide A40296. Vancomycin retained residual activity against S. coelicolor A(3)2 despite efficient incorporation of D-Lac into cytoplasmic precursors. This was due to a D,D-transpeptidase-catalyzed reaction that generated a stem pentapeptide recognized by glycopeptides by the exchange of D-Lac for D-Ala and Gly. The contribution of L,D-transpeptidases to resistance was limited by the supply of tetrapeptide acyl donors, which are essential for the formation of peptidoglycan cross-links by these enzymes. In the absence of a cytoplasmic metallo-D,D-carboxypeptidase, the tetrapeptide substrate was generated by hydrolysis of the C-terminal D-Lac residue of the stem pentadepsipeptide in the periplasm in competition with the exchange reaction catalyzed by D,D-transpeptidases. In Nonomuraea sp. strain ATCC 39727, the contribution of L,D-transpeptidases to glycopeptide resistance was limited by the incomplete conversion of pentapeptides into tetrapeptides despite the production of a cytoplasmic metallo-D,D-carboxypeptidase. Since the level of drug production exceeds the level of resistance, we propose that L,D-transpeptidases merely act as a tolerance mechanism in this bacterium.

  19. Isolation and Structure Characterization of an Antioxidative Glycopeptide from Mycelial Culture Broth of a Medicinal Fungus

    PubMed Central

    Wu, Jian-Yong; Chen, Xia; Siu, Ka-Chai

    2014-01-01

    A novel glycopeptide (Cs-GP1) with an average molecular weight (Mw) of 6.0 kDa was isolated and purified by column chromatography from the lower Mw fraction of exopolysaccharide (EPS) produced by a medicinal fungus Cordyceps sinensis Cs-HK1. Its carbohydrate moiety was mainly composed of glucose and mannose at 3.2:1.0 mole ratio, indicating an O-linked glycopeptide. The peptide chain contained relatively high mole ratios of aspartic acid, glutamic acid and glycine (3.3–3.5 relative to arginine) but relatively low ratios of tyrosine and histidine. The peptide chain sequence analyzed after trypsin digestion by LC-MS was KNGIFQFGEDCAAGSISHELGGFREFREFLKQAGLE. Cs-GP1 exhibited remarkable antioxidant capacity with a Trolox equivalent antioxidant capacity of 1183.8 μmol/g and a ferric reducing ability of 611.1 μmol Fe(II)/g, and significant protective effect against H2O2-induced PC12 cell injury at a minimum dose of 10 μg/mL. This is the first report on the structure and bioactivity of an extracellular glycopeptide from the Cordyceps species. PMID:25268609

  20. Hexose rearrangements upon fragmentation of N-glycopeptides and reductively aminated N-glycans.

    PubMed

    Wuhrer, Manfred; Koeleman, Carolien A M; Deelder, André M

    2009-06-01

    Tandem mass spectrometry of glycans and glycoconjugates in protonated form is known to result in rearrangement reactions leading to internal residue loss. Here we studied the occurrence of hexose rearrangements in tandem mass spectrometry of N-glycopeptides and reductively aminated N-glycans by MALDI-TOF/TOF-MS/MS and ESI-ion trap-MS/MS. Fragmentation of proton adducts of oligomannosidic N-glycans of ribonuclease B that were labeled with 2-aminobenzamide and 2-aminobenzoic acid resulted in transfer of one to five hexose residues to the fluorescently tagged innermost N-acetylglucosamine. Glycopeptides from various biological sources with oligomannosidic glycans were likewise shown to undergo hexose rearrangement reactions, resulting in chitobiose cleavage products that have acquired one or two hexose moieties. Tryptic immunoglobulin G Fc-glycopeptides with biantennary N-glycans likewise showed hexose rearrangements resulting in hexose transfer to the peptide moiety retaining the innermost N-acetylglucosamine. Thus, as a general phenomenon, tandem mass spectrometry of reductively aminated glycans as well as glycopeptides may result in hexose rearrangements. This characteristic of glycopeptide MS/MS has to be considered when developing tools for de novo glycopeptide structural analysis.

  1. Antibodies against Escherichia coli O24 and O56 O-Specific Polysaccharides Recognize Epitopes in Human Glandular Epithelium and Nervous Tissue

    PubMed Central

    Korzeniowska-Kowal, Agnieszka; Kochman, Agata; Gamian, Elżbieta; Lis-Nawara, Anna; Lipiński, Tomasz; Seweryn, Ewa; Ziółkowski, Piotr; Gamian, Andrzej

    2015-01-01

    Lipopolysaccharide (LPS), the major component of the outer membrane of Gram-negative bacteria, contains the O-polysaccharide, which is important to classify bacteria into different O-serological types within species. The O-polysaccharides of serotypes O24 and O56 of E. coli contain sialic acid in their structures, already established in our previous studies. Here, we report the isolation of specific antibodies with affinity chromatography using immobilized lipopolysaccharides. Next, we evaluated the reactivity of anti-O24 and anti-O56 antibody on human tissues histologically. The study was conducted under the assumption that the sialic acid based molecular identity of bacterial and tissue structures provides not only an understanding of the mimicry-based bacterial pathogenicity. Cross-reacting antibodies could be used to recognize specific human tissues depending on their histogenesis and differentiation, which might be useful for diagnostic purposes. The results indicate that various human tissues are recognized by anti-O24 and anti-O56 antibodies. Interestingly, only a single specific reactivity could be found in the anti-O56 antibody preparation. Several tissues studied were not reactive with either antibody, thus proving that the presence of cross-reactive antigens was tissue specific. In general, O56 antibody performed better than O24 in staining epithelial and nervous tissues. Positive staining was observed for both normal (ganglia) and tumor tissue (ganglioneuroma). Epithelial tissue showed positive staining, but an epitope recognized by O56 antibody should be considered as a marker of glandular epithelium. The reason is that malignant glandular tumor and its metastasis are stained, and also epithelium of renal tubules and glandular structures of the thyroid gland are stained. Stratified epithelium such as that of skin is definitely not stained. Therefore, the most relevant observation is that the epitope recognized by anti-O56 antibodies is a new marker

  2. Identification of novel HLA-A(*)0201-restricted CTL epitopes from Pokemon.

    PubMed

    Yuan, Bangqing; Zhao, Lin; Xian, Ronghua; Zhao, Gang

    2012-01-01

    Pokemon is a member of the POK family of transcriptional repressors and aberrant overexpressed in various human cancers. Therefore, the related peptide epitopes derived from Pokemon is essential for the development of specific immunotherapy of malignant tumors. In this study, we predicted and identified HLA-A(*)0201-restricted cytotoxic T lymphocyte (CTL) epitopes derived from Pokemon with computer-based epitope prediction, peptide-binding assay and testing of the induced CTLs toward different kinds of carcinoma cells. The results demonstrated that effectors induced by peptides of Pokemon containing residues 32-40, 61-69, 87-95, and 319-327 could specifically secrete IFN-γ and lyse tumor cell lines of Pokemon-positive and HLA-A2-matched. The results suggest that Pokemon32, Pokemon61, Pokemon87, and Pokemon319 peptides are novel HLA-A(*)0201-restricted restricted CTL epitopes, and could be utilized in the cancer immunotherapy against a broad spectrum of tumors. Copyright © 2012 Elsevier Inc. All rights reserved.

  3. Antifreeze glycopeptide diastereomers.

    PubMed

    Nagel, Lilly; Budke, Carsten; Dreyer, Axel; Koop, Thomas; Sewald, Norbert

    2012-01-01

    Antifreeze glycopeptides (AFGPs) are a special class of biological antifreeze agents, which possess the property to inhibit ice growth in the body fluids of arctic and antarctic fish and, thus, enable life under these harsh conditions. AFGPs are composed of 4-55 tripeptide units -Ala-Ala-Thr- glycosylated at the threonine side chains. Despite the structural homology among all the fish species, divergence regarding the composition of the amino acids occurs in peptides from natural sources. Although AFGPs were discovered in the early 1960s, the adsorption mechanism of these macromolecules to the surface of the ice crystals has not yet been fully elucidated. Two AFGP diastereomers containing different amino acid configurations were synthesized to study the influence of amino acid stereochemistry on conformation and antifreeze activity. For this purpose, peptides containing monosaccharide-substituted allo-L- and D-threonine building blocks were assembled by solid-phase peptide synthesis (SPPS). The retro-inverso AFGP analogue contained all amino acids in D-configuration, while the allo-L-diastereomer was composed of L-amino acids, like native AFGPs, with replacement of L-threonine by its allo-L-diastereomer. Both glycopeptides were analyzed regarding their conformational properties, by circular dichroism (CD), and their ability to inhibit ice recrystallization in microphysical experiments.

  4. Antifreeze glycopeptide diastereomers

    PubMed Central

    Nagel, Lilly; Budke, Carsten; Dreyer, Axel; Koop, Thomas

    2012-01-01

    Summary Antifreeze glycopeptides (AFGPs) are a special class of biological antifreeze agents, which possess the property to inhibit ice growth in the body fluids of arctic and antarctic fish and, thus, enable life under these harsh conditions. AFGPs are composed of 4–55 tripeptide units -Ala-Ala-Thr- glycosylated at the threonine side chains. Despite the structural homology among all the fish species, divergence regarding the composition of the amino acids occurs in peptides from natural sources. Although AFGPs were discovered in the early 1960s, the adsorption mechanism of these macromolecules to the surface of the ice crystals has not yet been fully elucidated. Two AFGP diastereomers containing different amino acid configurations were synthesized to study the influence of amino acid stereochemistry on conformation and antifreeze activity. For this purpose, peptides containing monosaccharide-substituted allo-L- and D-threonine building blocks were assembled by solid-phase peptide synthesis (SPPS). The retro-inverso AFGP analogue contained all amino acids in D-configuration, while the allo-L-diastereomer was composed of L-amino acids, like native AFGPs, with replacement of L-threonine by its allo-L-diastereomer. Both glycopeptides were analyzed regarding their conformational properties, by circular dichroism (CD), and their ability to inhibit ice recrystallization in microphysical experiments. PMID:23209499

  5. Affinity entrapment of oligosaccharides and glycopeptides using free lectin solution.

    PubMed

    Yodoshi, Masahiro; Oyama, Takehiro; Masaki, Ken; Kakehi, Kazuaki; Hayakawa, Takao; Suzuki, Shigeo

    2011-01-01

    Two procedures were proposed for the specific recovery of fluorescent derivatives of glycoprotein-derived oligosaccharides and tryptic glycopeptides using certain plant lectins. The first was based on the salting out of oligosaccharide-lectin conjugates with ammonium sulfate. Oligosaccharides specifically bound to lectins were recovered free from lectins using ethanol precipitation after dissolution in water. This method enabled group separation of 2-aminopyridine-labeled oligosaccharides derived from ovalbumin to galacto-oligosaccharides and agalacto-oligosaccharides by Ricinus communis agglutinin, and to high mannose- and hybrid-type oligosaccharides by wheat-germ agglutinin. Fractional precipitation based on differences in affinity for concanavalin A was accomplished by adding an appropriate concentration of methyl α-mannoside as an inhibitor. In the second method, tryptic digests of glycoproteins were mixed with a lectin solution, and the glycopeptide-lectin conjugates were specifically trapped on a centrifugal ultrafiltration membrane with cut-off of 10 kD. Trapped glycopeptides, as retentates, were passed through membranes by resuspension in diluted acid. This method is particularly useful for the enrichment of glycopeptides in protease digestion mixtures for glycosylation analyses by liquid chromatography-mass spectrometry.

  6. Synthesis, Properties, and Mechanism of Action of New Generation of Polycyclic Glycopeptide Antibiotics.

    PubMed

    Olsufyeva, Eugenia N; Tevyashova, Anna N

    2017-01-01

    The increased resistance of glycopeptide based antibiotics has become a serious problem for the chemotherapy of infections triggered by resistant Gram-positive bacteria. This has motivated the urgent sincere efforts to develop potent glycopeptide-based antibiotics in both academy and industry research laboratories. Understanding of the mechanism of action of natural and modified glycopeptides is considered as the basis for the rational design of compounds with valuable properties to achieve the fundamental results. Several hydrophobic glycopeptide analogues active against resistant strains were developed during the last two decades. Three drugs, namely, oritavancin, telavancin and dalbavancin were approved by FDA in 2013-2014. It was found that hydrophobic derivatives act through different mechanisms without binding with the modified target of resistant bacteria. Types: Different types of chemical modifications led to several glycopeptide analogues active against Gram-negative bacteria as advocated by in vitro studies or demonstrating potent antiviral activity in the cell models. A new class of glycopeptide antibiotics with potent activity against sensitive and resistant bacterial strains has been recently reported with the aim to overcome the resistance, however, there are a lot of obscure problems in the complete understanding of their mechanisms of actions. In this review, we summarized the achievements of synthetic methods devoted to the construction of new polycyclic glycopeptide antibiotics and described the studies related to their mechanism of actions. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  7. Synthesis and study of antibacterial activities of antibacterial glycopeptide antibiotics conjugated with benzoxaboroles.

    PubMed

    Printsevskaya, S S; Reznikova, M I; Korolev, A M; Lapa, G B; Olsufyeva, E N; Preobrazhenskaya, M N; Plattner, J J; Zhang, Y K

    2013-04-01

    The ability of boron-containing compounds to undergo a number of novel binding interactions with drug target functional groups has recently been described. In an extension of this work, we have incorporated a boron-containing scaffold, the benzoxaborole, into several glycopeptides antibiotics. The aim of this work is to exploit the inherent reactivity of boron to gain additional interactions with the bacterial cell wall components to improve binding affinity and to thereby overcome resistance. Three antibacterial glycopeptides (vancomycin, eremomycin and teicoplanin aglycone) have been selected for the construction of a series of 12 new benzoxaborole-glycopeptide conjugates. The hybrid antibiotics, in which the benzoxaborole and glycopeptide moieties were separated by a linker, exhibited excellent antibacterial activity against Gram-positive bacteria, including those with intermediate susceptibility to glycopeptides. Some analogs also demonstrated activity against vancomycin-resistant enterococci. Conjugation of antibiotics with benzoxaborole derivatives provides antibiotics with new and useful properties. Teicoplanin aglycone-benzoxaborole derivatives overcome resistance of Gram-positive bacteria to vancomycin.

  8. Supercharging Reagent for Enhanced Liquid Chromatographic Separation and Charging of Sialylated and High-Molecular-Weight Glycopeptides for NanoHPLC-ESI-MS/MS Analysis.

    PubMed

    Lin, Chia-Wei; Haeuptle, Micha A; Aebi, Markus

    2016-09-06

    Recent developments in proteomic techniques have led to the development of mass spectrometry (MS)-based methods to characterize site-specific glycosylation of proteins. However, appropriate analytical tools to characterize acidic and high-molecular-weight (hMW) glycopeptides are still lacking. In this study, we demonstrate that the addition of supercharging reagent, m-nitrobenzyl alcohol (m-NBA), into mobile phases greatly facilitates the analysis of acidic and hMW glycopeptides. Using commercial glycoproteins, we demonstrated that in the presence of m-NBA the charge state of sialylated glycopeptides increased and the chromatographic separation of neutral and acidic glycopeptides revealed a remarkable improvement. Next, we applied this system to the characterization of a glycoconjugate vaccine candidate consisting of a genetically detoxified exotoxin A of Pseudomonas aeruginosa covalently linked to Shigella flexneri type 2a O-antigen (Sf2E) produced by engineered Escherichia coli. The addition of m-NBA, allowed us to identify peptides with glycan chains of unprecedented size, up to 20 repeat units (98 monosaccharides). Our results indicated that incorporation of m-NBA into reversed-phase liquid chromatography (LC) solvents improves sensitivity, charging, and chromatographic resolution for acidic and hMW glycopeptides.

  9. It is all about the solvent: on the importance of the mobile phase for ZIC-HILIC glycopeptide enrichment.

    PubMed

    Alagesan, Kathirvel; Khilji, Sana Khan; Kolarich, Daniel

    2017-01-01

    Glycopeptide enrichment is a crucial step in glycoproteomics for which hydrophilic interaction chromatography (HILIC) has extensively been applied due to its low bias towards different glycan types. A systematic evaluation of applicable HILIC mobile phases on glycopeptide enrichment efficiency and selectivity is, to date, however, still lacking. Here, we present a novel, simplified technique for HILIC enrichment termed "Drop-HILIC", which was applied to systematically evaluate the mobile phase effect on ZIC-HILIC (zwitterionic type of hydrophilic interaction chromatography) glycopeptide enrichment. The four most commonly used MS compatible organic solvents were investigated: (i) acetonitrile, (ii) methanol, (iii) ethanol and (iv) isopropanol. Glycopeptide enrichment efficiencies were evaluated for each solvent system using samples of increasing complexity ranging from well-defined synthetic glycopeptides spiked into different concentrations of tryptic BSA peptides, followed by standard glycoproteins, and a complex sample derived from human (depleted and non-depleted) serum. ZIC-HILIC glycopeptide efficiency largely relied upon the used solvent. Different organic mobile phases enriched distinct glycopeptide subsets in a peptide backbone hydrophilicity-dependant manner. Acetonitrile provided the best compromise for the retention of both hydrophilic and hydrophobic glycopeptides, whereas methanol was confirmed to be unsuitable for this purpose. The enrichment efficiency of ethanol and isopropanol towards highly hydrophobic glycopeptides was compromised as considerable co-enrichment of unmodified peptides occurred, though for some hydrophobic glycopeptides isopropanol showed the best enrichment properties. This study shows that even minor differences in the peptide backbone and solvent do significantly influence HILIC glycopeptide enrichment and need to be carefully considered when employed for glycopeptide enrichment. Graphical Abstract The organic solvent plays a

  10. A42867, a novel glycopeptide antibiotic.

    PubMed

    Riva, E; Gastaldo, L; Beretta, M G; Ferrari, P; Zerilli, L F; Cassani, G; Selva, E; Goldstein, B P; Berti, M; Parenti, F

    1989-04-01

    A42867 is a new glycopeptide antibiotic of the ristocetin-vancomycin class active against aerobic and anaerobic Gram-positive bacteria. A42867 is produced by a strain of Nocardia nov. sp. ATCC 53492. A42867 was isolated during a screening program aimed at the discovery of new members of this glycopeptide class of antibiotics, by affinity chromatography based on an acyl-D-alanyl-D-alanine probe. The structure of A42867 was elucidated by fast atom bombardment MS, high field 2D 1H NMR spectroscopy, and HPLC analysis of the hydrolyzed carbohydrates. A42867 differs from vancomycin in the sugar portion and in the presence of only one chlorine atom in the peptide core. Its biological activity on Gram-positive aerobic and anaerobic bacteria is similar to that of other antibiotics of this group.

  11. Isolation, purification, and characterization of avian antimicrobial glycopeptide from the posterior salivary gland of Sepia pharaonis.

    PubMed

    Karthik, R; Saravanan, R; Ebenezar, K Kumar; Sivamalai, T

    2015-02-01

    A proteinaceous glycopeptide was isolated from the posterior salivary gland (PSG) of Sepia pharaonis by gel (Sephadex G-100) filtration chromatography and purified by reversed-phase high-performance liquid chromatography (RP-HPLC). Among the collected fractions, fraction 12 showed a retention time (RT) of 31 min. The total protein and neutral sugar contents of the purified glycopeptide were recorded as 68.14 and 2.95 mg, respectively. The molecular weight of the purified glycopeptide was found to be ~50 kDa. The infrared (IR) and circular dichroism (CD) spectroscopy confirmed the presence of peptide and secondary structure in the purified glycopeptide. The antibacterial activity of the purified glycopeptide against avian bacterial strains was also determined. Gas chromatography-mass spectrometry (GC-MS) of the purified glycopeptide revealed the likely compounds for the antibacterial activity such as 22, 23-dibromostigmasterol acetate, 3-methyl 2-(2-oxypropyl) furan, and 2,4,4-trimethyl-3-hydroxymethyl-5A-(3-methyl-but-2-enyl)-cyclohexene. These three compounds found in the purified glycopeptide could be responsible for the antibacterial activity against the avian pathogens. The results of this study suggest that the purified glycopeptide from the PSG of S. pharaonis could be an antibacterial agent against avian bacterial pathogens.

  12. Native N-glycopeptide thioester synthesis through N→S acyl transfer

    PubMed Central

    Premdjee, Bhavesh; Adams, Anna L.; Macmillan, Derek

    2011-01-01

    Peptide thioesters are important tools for the total synthesis of proteins using native chemical ligation (NCL). Preparation of glycopeptide thioesters, that enable the assembly of homogeneously glycosylated proteins, is complicated by the perceived fragile nature of the sugar moiety. Herein, we demonstrate the compatibility of thioester formation via N→S acyl transfer with native N-glycopeptides and report observations that will aid in their preparation. PMID:21676613

  13. Glycopeptide use is associated with increased mortality in Enterococcus faecalis bacteraemia.

    PubMed

    Foo, Hong; Chater, Mathew; Maley, Michael; van Hal, Sebastiaan J

    2014-08-01

    Enterococci are an important cause of nosocomial and community-acquired infections, with bacteraemia being one of the most common presentations. Although inappropriate antimicrobial therapy has been associated with poorer outcomes in Enterococcus faecalis (EF) bacteraemia, the impact of antimicrobial choice, namely β-lactam versus glycopeptide therapy, has not been well described. We sought to determine whether choice of antibiotic affects patient outcomes in EF bacteraemia. This retrospective cohort study was conducted at Liverpool and Bankstown Lidcombe Hospitals, Sydney, Australia between 2006 and 2013. Medical records and laboratory data for consecutive EF bacteraemias were reviewed. Clinical and microbiological data were obtained for all patients who received appropriate antimicrobial therapy with either a β-lactam or a glycopeptide antibiotic. Outcomes and predictors of mortality were determined and treatment groups were compared. One hundred and seventy-two episodes of clinically significant EF bacteraemias received appropriate antimicrobial therapy with a β-lactam (n = 126) or a glycopeptide (n = 46). All-cause 30 day mortality was 15.1%, with mortality significantly higher in patients receiving glycopeptide therapy compared with β-lactam therapy (26.1% versus 11.1%, P = 0.015). Glycopeptide therapy remained an independent predictor of 30 day mortality [OR 2.46 (95% CI 1.01-6.02), P = 0.048]. APACHE II score [OR 1.10 (95% CI 1.02-1.18), P = 0.011] and malignancy [OR 2.58 (95% CI 1.03-6.49), P = 0.044] were also independent predictors of 30 day mortality. Glycopeptide use is associated with increased mortality in patients with EF bacteraemia. In the treatment of β-lactam-susceptible EF bacteraemia, β-lactams should be considered first-line therapy. © The Author 2014. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  14. Chiral Recognition with Macrocyclic Glycopeptides: Mechanisms and Applications

    NASA Astrophysics Data System (ADS)

    Berthod, Alain; Qiu, Hai Xiao; Staroverov, Sergey M.; Kuznestov, Mikhail A.; Armstrong, Daniel W.

    The macrocyclic glycopeptide chiral selectors are natural molecules produced by bacterial fermentation. Purified and bonded to silica particles, they make very useful chiral stationary phases (CSP) with a broad spectrum of applicability in enantiomeric separation. The macrocyclic glycopeptide CSPs are multimodal, the same column being able to work in normal phase mode with apolar mobile phase, in reversed-phase mode, or in polar ionic mode with 100% alcoholic mobile phase of adjusted pH. The role of the carbohydrate units is described as well as the critical charge-charge docking interaction responsible for the amino acid enantiomer recognition. The complimentary phenomenon is also exposed.

  15. Photoinduced Thiol-ene Chemistry Applied to the Synthesis of Self-Assembling Elastin-Inspired Glycopeptides.

    PubMed

    Piccirillo, Germano; Pepe, Antonietta; Bedini, Emiliano; Bochicchio, Brigida

    2017-02-21

    Synthetic (glyco)peptides inspired by proteins able to self-assemble are appealing biomaterials in the field of tissue engineering and regenerative medicine. Herein, for the first time, taking advantage of thiol-ene chemistry coupled to solid-phase peptide synthesis, a self-assembling peptide inspired by elastin protein was bioconjugated to three carbohydrates in order to obtain the corresponding glycopeptides. They were studied at the molecular and supramolecular level. The results show that the carbohydrate influences the molecular conformation of the glycopeptide and its self-aggregation properties as well. As future perspective, the results could enable us to tune the final self-aggregation properties of the glycopeptide by changing the sugar moiety. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Harnessing the synthetic capabilities of glycopeptide antibiotic tailoring enzymes: characterization of the UK-68,597 biosynthetic cluster.

    PubMed

    Yim, Grace; Kalan, Lindsay; Koteva, Kalinka; Thaker, Maulik N; Waglechner, Nicholas; Tang, Irene; Wright, Gerard D

    2014-11-24

    In this study, a draft genome sequence of Actinoplanes sp. ATCC 53533 was assembled, and an 81-kb biosynthetic cluster for the unusual sulfated glycopeptide UK-68,597 was identified. Glycopeptide antibiotics are important in the treatment of infections caused by Gram-positive bacteria. Glycopeptides contain heptapeptide backbones that are modified by many tailoring enzymes, including glycosyltransferases, sulfotransferases, methyltransferases, and halogenases, generating extensive chemical and functional diversity. Several tailoring enzymes in the cluster were examined in vitro for their ability to modify glycopeptides, resulting in the synthesis of novel molecules. Tailoring enzymes were also expressed in the producer of the glycopeptide aglycone A47934, generating additional chemical diversity. This work characterizes the biosynthetic program of UK-68,597 and demonstrates the capacity to expand glycopeptide chemical diversity by harnessing the unique chemistry of tailoring enzymes. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  17. Autoantibodies to aberrantly glycosylated MUC1 in early stage breast cancer are associated with a better prognosis

    PubMed Central

    2011-01-01

    Introduction Detection of serum biomarkers for early diagnosis of breast cancer remains an important goal. Changes in the structure of O-linked glycans occur in all breast cancers resulting in the expression of glycoproteins that are antigenically distinct. Indeed, the serum assay widely used for monitoring disease progression in breast cancer (CA15.3), detects a glycoprotein (MUC1), but elevated levels of the antigen cannot be detected in early stage patients. However, since the immune system acts to amplify the antigenic signal, antibodies can be detected in sera long before the antigen. We have exploited the change in O-glycosylation to measure autoantibody responses to cancer-associated glycoforms of MUC1 in sera from early stage breast cancer patients. Methods We used a microarray platform of 60mer MUC1 glycopeptides, to confirm the presence of autoantibodies to cancer associated glycoforms of MUC1 in a proportion of early breast cancer patients (54/198). Five positive sera were selected for detailed definition of the reactive epitopes using on chip glycosylation technology and a panel of glycopeptides based on a single MUC1 tandem repeat carrying specific glycans at specific sites. Based on these results, larger amounts of an extended repertoire of defined MUC1 glycopeptides were synthesised, printed on microarrays, and screened with sera from a large cohort of breast cancer patients (n = 395), patients with benign breast disease (n = 108) and healthy controls (n = 99). All sera were collected in the 1970s and 1980s and complete clinical follow-up of breast cancer patients is available. Results The presence and level of autoantibodies was significantly higher in the sera from cancer patients compared with the controls, and a highly significant correlation with age was observed. High levels of a subset of autoantibodies to the core3MUC1 (GlcNAcβ1-3GalNAc-MUC1) and STnMUC1 (NeuAcα2,6GalNAc-MUC1) glycoforms were significantly associated with reduced incidence

  18. Identification of epitopes recognised by mucosal CD4(+) T-cell populations from cattle experimentally colonised with Escherichia coli O157:H7.

    PubMed

    Corbishley, Alexander; Connelley, Timothy K; Wolfson, Eliza B; Ballingall, Keith; Beckett, Amy E; Gally, David L; McNeilly, Tom N

    2016-09-02

    Vaccines targeting enterohaemorrhagic Escherichia coli (EHEC) O157:H7 shedding in cattle are only partially protective. The correlates of protection of these vaccines are unknown, but it is probable that they reduce bacterial adherence at the mucosal surface via the induction of blocking antibodies. Recent studies have indicated a role for cellular immunity in cattle during colonisation, providing an impetus to understand the bacterial epitopes recognised during this response. This study mapped the epitopes of 16 EHEC O157:H7 proteins recognised by rectal lymph node CD4(+) T-cells from calves colonised with Shiga toxin producing EHEC O157:H7 strains. 20 CD4(+) T-cell epitopes specific to E. coli from 7 of the proteins were identified. The highly conserved N-terminal region of Intimin, including the signal peptide, was consistently recognised by mucosal CD4(+) T-cell populations from multiple animals of different major histocompatibility complex class II haplotypes. These T-cell epitopes are missing from many Intimin constructs used in published vaccine trials, but are relatively conserved across a range of EHEC serotypes, offering the potential to develop cross protective vaccines. Antibodies recognising H7 flagellin have been consistently identified in colonised calves; however CD4(+) T-cell epitopes from H7 flagellin were not identified in this study, suggesting that H7 flagellin may act as a T-cell independent antigen. This is the first time that the epitopes recognised by CD4(+) T-cells following colonisation with an attaching and effacing pathogen have been characterised in any species. The findings have implications for the design of antigens used in the next generation of EHEC O157:H7 vaccines.

  19. Direct fluorescence polarization assay for the detection of glycopeptide antibiotics.

    PubMed

    Yu, Linliang; Zhong, Meng; Wei, Yinan

    2010-08-15

    Glycopeptide antibiotics are widely used in the treatment of infections caused by Gram-positive bacteria. They inhibit the biosynthesis of the bacterial cell wall through binding to the D-alanyl-D-alanine (D-Ala-D-Ala) terminal peptide of the peptidoglycan precursor. Taking advantage of this highly specific interaction, we developed a direct fluorescence polarization based method for the detection of glycopeptide antibiotics. Briefly, we labeled the acetylated tripeptide Ac-L-Lys-D-Ala-D-Ala-OH with a fluorophore to create a peptide probe. Using three glycopeptide antibiotics, vancomycin, teicoplanin, and telavancin, as model compounds, we demonstrated that the fluorescence polarization of the peptide probe increased upon binding to antibiotics in a concentration dependent manner. The dissociation constants (K(d)) between the peptide probes and the antibiotics were consistent with those reported between free d-Ala-d-Ala and the antibiotics in the literature. The assay is highly reproducible and selective toward glycopeptide antibiotics. Its detection limit and work concentration range are 0.5 microM and 0.5-4 microM for vancomycin, 0.25 microM and 0.25-2 microM for teicoplanin, and 1 microM and 1-8 microM for telavancin. Furthermore, we compared our assay in parallel with a commercial fluorescence polarization immunoassay (FPIA) kit in detecting teicoplanin spiked in human blood samples. The accuracy and precision of the two methods are comparable. We expect our assay to be useful in both research and clinical laboratories.

  20. Two-Dimensional MoS2-Based Zwitterionic Hydrophilic Interaction Liquid Chromatography Material for the Specific Enrichment of Glycopeptides.

    PubMed

    Xia, Chaoshuang; Jiao, Fenglong; Gao, Fangyuan; Wang, Heping; Lv, Yayao; Shen, Yehua; Zhang, Yangjun; Qian, Xiaohong

    2018-06-05

    Mass spectrometry (MS)-based glycoproteomics research requires highly efficient sample preparation to eliminate interference from non-glycopeptides and to improve the efficiency of glycopeptide detection. In this work, a novel MoS 2 /Au-NP (gold nanoparticle)-L-cysteine nanocomposite was prepared for glycopeptide enrichment. The two-dimensional (2D) structured MoS 2 nanosheets served as a matrix that could provide a large surface area for immobilizing hydrophilic groups (such as L-cysteine) with low steric hindrance between the materials and the glycopeptides. As a result, the novel nanomaterial possessed an excellent ability to capture glycopeptides. Compared to commercial zwitterionic hydrophilic interaction liquid chromatography (ZIC-HILIC) materials, the novel nanomaterials exhibited excellent enrichment performance with ultrahigh selectivity and sensitivity (approximately 10 fmol), high binding capacity (120 mg g -1 ), high enrichment recovery (more than 93%), satisfying batch-to-batch reproducibility, and good universality for glycopeptide enrichment. In addition, its outstanding specificity and efficiency for glycopeptide enrichment was confirmed by the detection of glycopeptides from an human serum immunoglobulin G (IgG) tryptic digest in quantities as low as a 1:1250 molar ratio of IgG tryptic digest to bovine serum albumin tryptic digest. The novel nanocomposites were further used for the analysis of complex samples, and 1920 glycopeptide backbones from 775 glycoproteins were identified in three replicate analyses of 50 μg of proteins extracted from HeLa cell exosomes. The resulting highly informative mass spectra indicated that this multifunctional nanomaterial-based enrichment method could be used as a promising tool for the in-depth and comprehensive characterization of glycoproteomes in MS-based glycoproteomics.

  1. Protective B-cell epitopes of Francisella tularensis O-polysaccharide in a mouse model of respiratory tularaemia.

    PubMed

    Lu, Zhaohua; Madico, Guillermo; Roche, Marly I; Wang, Qi; Hui, Julia H; Perkins, Hillary M; Zaia, Joseph; Costello, Catherine E; Sharon, Jacqueline

    2012-07-01

    Antibodies to the lipopolysaccharide (LPS) of Francisella tularensis have been shown to be protective against respiratory tularaemia in mouse models, and we have previously described mouse monoclonal antibodies (mAbs) to non-overlapping terminal and internal epitopes of the F. tularensis LPS O-polysaccharide (OAg). In the current study, we used F. tularensis LPS oligosaccharides of defined OAg repeat length as molecular rulers in competition ELISA to demonstrate that the epitope targeted by the terminal OAg-binding mAb FB11 is contained within one tetrasaccharide repeat whereas the epitope targeted by the internal OAg-binding mAb Ab52 spans two tetrasaccharide repeats. Both mAbs conferred survival to BALB/c mice infected intranasally with the F. tularensis type B live vaccine strain and prolonged survival of BALB/c mice infected intranasally with the highly virulent F. tularensis type A strain SchuS4. The protective effects correlated with reduced bacterial burden in mAb-treated infected mice. These results indicate that an oligosaccharide with two OAg tetrasaccharide repeats covers both terminal and internal protective OAg epitopes, which may inform the design of vaccines for tularaemia. Furthermore, the FB11 and Ab52 mAbs could serve as reporters to monitor the response of vaccine recipients to protective B-cell epitopes of F. tularensis OAg. © 2012 The Authors. Immunology © 2012 Blackwell Publishing Ltd.

  2. Development of a data independent acquisition mass spectrometry workflow to enable glycopeptide analysis without predefined glycan compositional knowledge.

    PubMed

    Lin, Chi-Hung; Krisp, Christoph; Packer, Nicolle H; Molloy, Mark P

    2018-02-10

    Glycoproteomics investigates glycan moieties in a site specific manner to reveal the functional roles of protein glycosylation. Identification of glycopeptides from data-dependent acquisition (DDA) relies on high quality MS/MS spectra of glycopeptide precursors and often requires manual validation to ensure confident assignments. In this study, we investigated pseudo-MRM (MRM-HR) and data-independent acquisition (DIA) as alternative acquisition strategies for glycopeptide analysis. These approaches allow data acquisition over the full MS/MS scan range allowing data re-analysis post-acquisition, without data re-acquisition. The advantage of MRM-HR over DDA for N-glycopeptide detection was demonstrated from targeted analysis of bovine fetuin where all three N-glycosylation sites were detected, which was not the case with DDA. To overcome the duty cycle limitation of MRM-HR acquisition needed for analysis of complex samples such as plasma we trialed DIA. This allowed development of a targeted DIA method to identify N-glycopeptides without pre-defined knowledge of the glycan composition, thus providing the potential to identify N-glycopeptides with unexpected structures. This workflow was demonstrated by detection of 59 N-glycosylation sites from 41 glycoproteins from a HILIC enriched human plasma tryptic digest. 21 glycoforms of IgG1 glycopeptides were identified including two truncated structures that are rarely reported. We developed a data-independent mass spectrometry workflow to identify specific glycopeptides from complex biological mixtures. The novelty is that this approach does not require glycan composition to be pre-defined, thereby allowing glycopeptides carrying unexpected glycans to be identified. This is demonstrated through the analysis of immunoglobulins in human plasma where we detected two IgG1 glycoforms that are rarely observed. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Identification of Sialic Acid Linkages on Intact Glycopeptides via Differential Chemical Modification Using IntactGIG-HILIC

    NASA Astrophysics Data System (ADS)

    Yang, Shuang; Wu, Wells W.; Shen, Rong-Fong; Bern, Marshall; Cipollo, John

    2018-04-01

    Mass spectrometric analysis of intact glycopeptides can reveal detailed information about glycosite, glycan structural features, and their heterogeneity. Sialyl glycopeptides can be positively, negatively, or neutrally charged depending on pH of their buffer solution and ionization conditions. To detect sialoglycopeptides, a negative-ion mode mass spectrometry may be applied with a minimal loss of sialic acids, although the positively charged or neutral glycopeptides may be excluded. Alternatively, the sialyl glycopeptides can be identified using positive-ion mode analysis by doping a high concentration of sodium salts to the analytes. Although manipulation of unmodified sialoglycopeptides can be useful for analysis of samples, less than optimal ionization, facile loss of sialyl and unfavorable ionization of accompanying non-sialyl peptides make such strategies suboptimal. Currently available chemical derivatization methods, while stabilizing for sialic acid, mask sialic acid linkage configuration. Here, we report the development of a novel approach to neutralize sialic acids via sequentially chemical modification that also reveals their linkage configuration, often an important determinant in biological function. This method utilizes several components to facilitate glycopeptide identification. These include the following: solid phase derivatization, enhanced ionization of sialoglycopeptides, differentiation of sialic acid linkage, and enrichment of the modified glycopeptides by hydrophilic interaction liquid chromatography. This technology can be used as a tool for quantitative analysis of protein sialylation in diseases with determination of sialic acid linkage configuration. [Figure not available: see fulltext.

  4. [Emergence of glycopeptide resistant Enterococcus faecium in Algeria: a case report].

    PubMed

    Hamidi, Moufida; Ammari, Houria; Ghaffor, Mohamed; Benamrouche, Nabila; Tali-Maamar, Hassiba; Tala-Khir, Farida; Younsi, Mokhtar; Rahal, Kheira

    2013-01-01

    A glycopeptide-resistant Enterococcus faecium (EFRG) was isolated from a wound in a patient hospitalized in a university hospital in Algiers. This strain was resistant to several antibiotics. This patient was carrying this strain in the digestive tract which may partly explain its origin. Genotypic comparison of the two strains by pulsed field gel electrophoresis showed that it was the same strain. Glycopeptide resistance was due to the presence of the vanA gene. Vigilance is required facing the emergence of strains of EFRG in our hospitals.

  5. Comparative activity of tedizolid and glycopeptide combination therapies for the treatment of Staphylococcus aureus infections: an in vitro and in vivo evaluation against strains with reduced susceptibility to glycopeptides.

    PubMed

    Betts, J W; Abdul Momin, H F; Phee, L M; Wareham, D W

    2018-02-01

    Glycopeptides are widely used for the treatment of meticillin-resistant Staphylococcus aureus (MRSA) infections. Although difficult to detect, isolates with reduced (GISA), hetero (hGISA) or complete (GRSA) resistance to glycopeptides are increasingly reported. Optimal therapy for such strains is unknown. We compared the in vitro and in vivo activity of tedizolid (TED), a recently licensed oxazolidonone, with vancomycin (VAN) and teicoplanin (TEIC) combined with fusidic acid (FD) or rifampicin (RIF) against S. aureus (SA) with reduced susceptibility to glycopeptides. Susceptibility was determined for six (GISA, hGISA and GRSA) reference strains and 72 clinical MRSA isolates screened for hGISA/GISA-like phenotypes. Synergy and bactericidal activity were assessed using chequerboard and time-kill assays. The G. mellonella wax moth caterpillar model was used to measure the activity of TED and the combinations in vivo. Glycopeptide MICs (VAN/TEIC) ranged from 0.5-8/4 and 0.125-1 for TED. No significant synergy was noted when VAN/TEIC were combined with either RIF or FD. Time-kill assays confirmed that TED was bacteriostatic but superior to VAN and TEIC against GISA strains. In G. mellonella TED was more effective than TEIC monotherapy versus GISA strains. The combination of TEIC with RIF was the most effective combination overall, both in vitro and in vivo. TED had good in vitro activity versus MRSA including those with reduced susceptibility to glycopeptides. Although bacteriostatic, it was effective in the G. mellonella model and superior to TEIC in the treatment of GISA. Although this supports the use of TED for MRSA and GISA, the TEIC/RIF combination also warrants further study.

  6. Sulfated Glycopeptide Nanostructures for Multipotent Protein Activation

    PubMed Central

    Lee, Sungsoo S.; Fyrner, Timmy; Chen, Feng; Álvarez, Zaida; Sleep, Eduard; Chun, Danielle S.; Weiner, Joseph A.; Cook, Ralph W.; Freshman, Ryan D.; Schallmo, Michael S.; Katchko, Karina M.; Schneider, Andrew D.; Smith, Justin T.; Yun, Chawon; Singh, Gurmit; Hashmi, Sohaib Z.; McClendon, Mark T.; Yu, Zhilin; Stock, Stuart R.; Hsu, Wellington K.; Hsu, Erin L.; Stupp, Samuel I.

    2017-01-01

    Biological systems have evolved to utilize numerous proteins with capacity to bind polysaccharides for the purpose of optimizing their function. A well-known subset of these proteins with binding domains for the highly diverse sulfated polysaccharides are important growth factors involved in biological development and tissue repair. We report here on supramolecular sulfated glycopeptide nanostructures, which display a trisulfated monosaccharide on their surfaces and bind five critical proteins with very different polysaccharide binding domains. Binding does not disrupt the filamentous shape of the nanostructures or their internal β-sheet backbone, but must involve accessible adaptive configurations to interact with such different proteins. The glycopeptide nanostructures amplified signaling of bone morphogenetic protein 2 significantly more than the natural sulfated polysaccharide heparin, and promoted regeneration of bone in the spine with a protein dose that is 100-fold lower than expected. These super-bioactive nanostructures may enable many therapies in the horizon involving proteins. PMID:28650443

  7. Structure and Function of the Glycopeptide N-methyltransferase MtfA, a Tool for the Biosynthesis of Modified Glycopeptide Antibiotics

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shi, Rong; Lamb, Sherry S.; Zakeri, Bijan

    2009-06-01

    There is a considerable interest in the modification of existing antibiotics to generate new antimicrobials. Glycopeptide antibiotics (GPAs) are effective against serious Gram-positive bacterial pathogens including methicillin-resistant Staphylococcus aureus. However, resistance to these antibiotics is becoming a serious problem requiring new strategies. We show that the Amycolatopsis orientalis (S)-adenosyl-L-methionine-dependent methyltransferase MtfA, from the vancomycin-class GPA chloroeremomycin biosynthetic pathway, catalyzes in vivo and in vitro methyl transfer to generate methylated GPA derivatives of the teicoplanin class. The crystal structure of MtfA complexed with (S)-adenosyl-L-methionine, (S)-adenosylhomocysteine, or sinefungin inhibitor, coupled with mutagenesis, identified His228 as a likely general base required for methylmore » transfer to the N terminus of the glycopeptide. Computational docking and molecular dynamics simulations were used to model binding of demethyl-vancomycin aglycone to MtfA. These results demonstrate its utility as a tool for engineering methylated analogs of GPAs.« less

  8. Epithelial Mesenchymal Transition Induces Aberrant Glycosylation through Hexosamine Biosynthetic Pathway Activation.

    PubMed

    Lucena, Miguel C; Carvalho-Cruz, Patricia; Donadio, Joana L; Oliveira, Isadora A; de Queiroz, Rafaela M; Marinho-Carvalho, Monica M; Sola-Penna, Mauro; de Paula, Iron F; Gondim, Katia C; McComb, Mark E; Costello, Catherine E; Whelan, Stephen A; Todeschini, Adriane R; Dias, Wagner B

    2016-06-17

    Deregulated cellular metabolism is a hallmark of tumors. Cancer cells increase glucose and glutamine flux to provide energy needs and macromolecular synthesis demands. Several studies have been focused on the importance of glycolysis and pentose phosphate pathway. However, a neglected but very important branch of glucose metabolism is the hexosamine biosynthesis pathway (HBP). The HBP is a branch of the glucose metabolic pathway that consumes ∼2-5% of the total glucose, generating UDP-GlcNAc as the end product. UDP-GlcNAc is the donor substrate used in multiple glycosylation reactions. Thus, HBP links the altered metabolism with aberrant glycosylation providing a mechanism for cancer cells to sense and respond to microenvironment changes. Here, we investigate the changes of glucose metabolism during epithelial mesenchymal transition (EMT) and the role of O-GlcNAcylation in this process. We show that A549 cells increase glucose uptake during EMT, but instead of increasing the glycolysis and pentose phosphate pathway, the glucose is shunted through the HBP. The activation of HBP induces an aberrant cell surface glycosylation and O-GlcNAcylation. The cell surface glycans display an increase of sialylation α2-6, poly-LacNAc, and fucosylation, all known epitopes found in different tumor models. In addition, modulation of O-GlcNAc levels was demonstrated to be important during the EMT process. Taken together, our results indicate that EMT is an applicable model to study metabolic and glycophenotype changes during carcinogenesis, suggesting that cell glycosylation senses metabolic changes and modulates cell plasticity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  9. Epitope mapping: the first step in developing epitope-based vaccines.

    PubMed

    Gershoni, Jonathan M; Roitburd-Berman, Anna; Siman-Tov, Dror D; Tarnovitski Freund, Natalia; Weiss, Yael

    2007-01-01

    Antibodies are an effective line of defense in preventing infectious diseases. Highly potent neutralizing antibodies can intercept a virus before it attaches to its target cell and, thus, inactivate it. This ability is based on the antibodies' specific recognition of epitopes, the sites of the antigen to which antibodies bind. Thus, understanding the antibody/epitope interaction provides a basis for the rational design of preventive vaccines. It is assumed that immunization with the precise epitope, corresponding to an effective neutralizing antibody, would elicit the generation of similarly potent antibodies in the vaccinee. Such a vaccine would be a 'B-cell epitope-based vaccine', the implementation of which requires the ability to backtrack from a desired antibody to its corresponding epitope. In this article we discuss a range of methods that enable epitope discovery based on a specific antibody. Such a reversed immunological approach is the first step in the rational design of an epitope-based vaccine. Undoubtedly, the gold standard for epitope definition is x-ray analyses of crystals of antigen:antibody complexes. This method provides atomic resolution of the epitope; however, it is not readily applicable to many antigens and antibodies, and requires a very high degree of sophistication and expertise. Most other methods rely on the ability to monitor the binding of the antibody to antigen fragments or mutated variations. In mutagenesis of the antigen, loss of binding due to point modification of an amino acid residue is often considered an indication of an epitope component. In addition, computational combinatorial methods for epitope mapping are also useful. These methods rely on the ability of the antibody of interest to affinity isolate specific short peptides from combinatorial phage display peptide libraries. The peptides are then regarded as leads for the definition of the epitope corresponding to the antibody used to screen the peptide library. For

  10. Biomedicine of Enkephalin-Derived Glycopeptide Analgesics

    NASA Astrophysics Data System (ADS)

    Polt, Robin

    The incorporation of glycosides into peptide neurotransmitters imparts drug-like character to the neurotransmitter "message" via "membrane hopping". The importance of the glycopeptide-membrane interaction is emphasized, and the biousian theory is briefly explained. Application of this approach to enkephalins, the endogenous opioid peptides, leads to potent analgesic compounds capable of systemic delivery. The clinical applications of these compounds are advocated by the author.

  11. Exploring Site-Specific N-Glycosylation Microheterogeneity of Haptoglobin using Glycopeptide CID Tandem Mass Spectra and Glycan Database Search

    PubMed Central

    Chandler, Kevin Brown; Pompach, Petr; Goldman, Radoslav

    2013-01-01

    Glycosylation is a common protein modification with a significant role in many vital cellular processes and human diseases, making the characterization of protein-attached glycan structures important for understanding cell biology and disease processes. Direct analysis of protein N-glycosylation by tandem mass spectrometry of glycopeptides promises site-specific elucidation of N-glycan microheterogeneity, something which detached N-glycan and de-glycosylated peptide analyses cannot provide. However, successful implementation of direct N-glycopeptide analysis by tandem mass spectrometry remains a challenge. In this work, we consider algorithmic techniques for the analysis of LC-MS/MS data acquired from glycopeptide-enriched fractions of enzymatic digests of purified proteins. We implement a computational strategy which takes advantage of the properties of CID fragmentation spectra of N-glycopeptides, matching the MS/MS spectra to peptide-glycan pairs from protein sequences and glycan structure databases. Significantly, we also propose a novel false-discovery-rate estimation technique to estimate and manage the number of false identifications. We use a human glycoprotein standard, haptoglobin, digested with trypsin and GluC, enriched for glycopeptides using HILIC chromatography, and analyzed by LC-MS/MS to demonstrate our algorithmic strategy and evaluate its performance. Our software, GlycoPeptideSearch (GPS), assigned glycopeptide identifications to 246 of the spectra at false-discovery-rate 5.58%, identifying 42 distinct haptoglobin peptide-glycan pairs at each of the four haptoglobin N-linked glycosylation sites. We further demonstrate the effectiveness of this approach by analyzing plasma-derived haptoglobin, identifying 136 N-linked glycopeptide spectra at false-discovery-rate 0.4%, representing 15 distinct glycopeptides on at least three of the four N-linked glycosylation sites. The software, GlycoPeptideSearch, is available for download from http

  12. New glycoproteomics software, GlycoPep Evaluator, generates decoy glycopeptides de novo and enables accurate false discovery rate analysis for small data sets.

    PubMed

    Zhu, Zhikai; Su, Xiaomeng; Go, Eden P; Desaire, Heather

    2014-09-16

    Glycoproteins are biologically significant large molecules that participate in numerous cellular activities. In order to obtain site-specific protein glycosylation information, intact glycopeptides, with the glycan attached to the peptide sequence, are characterized by tandem mass spectrometry (MS/MS) methods such as collision-induced dissociation (CID) and electron transfer dissociation (ETD). While several emerging automated tools are developed, no consensus is present in the field about the best way to determine the reliability of the tools and/or provide the false discovery rate (FDR). A common approach to calculate FDRs for glycopeptide analysis, adopted from the target-decoy strategy in proteomics, employs a decoy database that is created based on the target protein sequence database. Nonetheless, this approach is not optimal in measuring the confidence of N-linked glycopeptide matches, because the glycopeptide data set is considerably smaller compared to that of peptides, and the requirement of a consensus sequence for N-glycosylation further limits the number of possible decoy glycopeptides tested in a database search. To address the need to accurately determine FDRs for automated glycopeptide assignments, we developed GlycoPep Evaluator (GPE), a tool that helps to measure FDRs in identifying glycopeptides without using a decoy database. GPE generates decoy glycopeptides de novo for every target glycopeptide, in a 1:20 target-to-decoy ratio. The decoys, along with target glycopeptides, are scored against the ETD data, from which FDRs can be calculated accurately based on the number of decoy matches and the ratio of the number of targets to decoys, for small data sets. GPE is freely accessible for download and can work with any search engine that interprets ETD data of N-linked glycopeptides. The software is provided at https://desairegroup.ku.edu/research.

  13. Antibodies to Both Terminal and Internal B-Cell Epitopes of Francisella tularensis O-Polysaccharide Produced by Patients with Tularemia

    PubMed Central

    Lu, Zhaohua; Perkins, Hillary M.

    2014-01-01

    Francisella tularensis, the Gram-negative bacterium that causes tularemia, is considered a potential bioterrorism threat due to its low infectivity dose and the high morbidity and mortality from respiratory disease. We previously characterized two mouse monoclonal antibodies (MAbs) specific for the O-polysaccharide (O antigen [OAg]) of F. tularensis lipopolysaccharide (LPS): Ab63, which targets a terminal epitope at the nonreducing end of OAg, and Ab52, which targets a repeating internal OAg epitope. These two MAbs were protective in a mouse model of respiratory tularemia. To determine whether these epitope types are also targeted by humans, we tested the ability of each of 18 blood serum samples from 11 tularemia patients to inhibit the binding of Ab63 or Ab52 to F. tularensis LPS in a competition enzyme-linked immunosorbent assay (ELISA). Although all serum samples had Ab63- and Ab52-inhibitory activities, the ratios of Ab63 to Ab52 inhibitory potencies varied 75-fold. However, the variation was only 2.3-fold for sequential serum samples from the same patient, indicating different distributions of terminal- versus internal-binding antibodies in different individuals. Western blot analysis using class-specific anti-human Ig secondary antibodies showed that both terminal- and internal-binding OAg antibodies were of the IgG, IgM, and IgA isotypes. These results support the use of a mouse model to discover protective B-cell epitopes for tularemia vaccines or prophylactic/therapeutic antibodies, and they present a general strategy for interrogating the antibody responses of patients and vaccinees to microbial carbohydrate epitopes that have been characterized in experimental animals. PMID:24351753

  14. Inadequate Reference Datasets Biased toward Short Non-epitopes Confound B-cell Epitope Prediction*

    PubMed Central

    Rahman, Kh. Shamsur; Chowdhury, Erfan Ullah; Sachse, Konrad; Kaltenboeck, Bernhard

    2016-01-01

    X-ray crystallography has shown that an antibody paratope typically binds 15–22 amino acids (aa) of an epitope, of which 2–5 randomly distributed amino acids contribute most of the binding energy. In contrast, researchers typically choose for B-cell epitope mapping short peptide antigens in antibody binding assays. Furthermore, short 6–11-aa epitopes, and in particular non-epitopes, are over-represented in published B-cell epitope datasets that are commonly used for development of B-cell epitope prediction approaches from protein antigen sequences. We hypothesized that such suboptimal length peptides result in weak antibody binding and cause false-negative results. We tested the influence of peptide antigen length on antibody binding by analyzing data on more than 900 peptides used for B-cell epitope mapping of immunodominant proteins of Chlamydia spp. We demonstrate that short 7–12-aa peptides of B-cell epitopes bind antibodies poorly; thus, epitope mapping with short peptide antigens falsely classifies many B-cell epitopes as non-epitopes. We also show in published datasets of confirmed epitopes and non-epitopes a direct correlation between length of peptide antigens and antibody binding. Elimination of short, ≤11-aa epitope/non-epitope sequences improved datasets for evaluation of in silico B-cell epitope prediction. Achieving up to 86% accuracy, protein disorder tendency is the best indicator of B-cell epitope regions for chlamydial and published datasets. For B-cell epitope prediction, the most effective approach is plotting disorder of protein sequences with the IUPred-L scale, followed by antibody reactivity testing of 16–30-aa peptides from peak regions. This strategy overcomes the well known inaccuracy of in silico B-cell epitope prediction from primary protein sequences. PMID:27189949

  15. A review of the current place of glycopeptides in turkish medical practice

    PubMed Central

    Erdem, Hakan; Oncul, Oral

    2007-01-01

    Background: Glycopeptide antibiotics are considered by many investigators to be the last resort in the treatment of gram-positive bacterial infections. Objective: The aim of this review was to assess the place of glycopeptides in the treatment of common gram-positive bacteria in accordance with the current epidemiologic data in Turkey. Methods: A search of both the English- and Turkish-language literature indexed on MEDLINE, Ulakbim (Turkey), and Pleksus (Turkey) was performed using the terms: vancomycin, teicoplanin, and glycopeptides, or their Turkish-language counterparts. The complete texts of the articles found in these databases were obtained from the electronic library of Gulhane Medical Academy, Ankara, Turkey. Articles from regional journals, without the support of an electronic format, were obtained by direct communication. Articles of interest were those based on studies occurring in Turkish populations, with special consideration given to publications in press after 2002. Results: Staphylococci were the most frequent gram-positive pathogens encountered in Turkish hospitals. Studies have found that ∼74% of strains were Staphylococcus aureus and the remaining strains were coagulase-negative staphylococci (CoNS). Overall methicillin resistance in staphylococci was reported as ∼60%. In Turkey, S aureus was one of the most common infectious agents found inside hospitals and is deemed a growing threat in the community. While the rate of methicillin resistance in community-acquired isolates is ∼4%, the data from hospitals suggest that reduced resistance comprises most of the isolates. In the studies reviewed, older quinolones like ciprofloxacin and ofloxacin seem to be ineffective in nearly half of the S aureus isolates. Alternatives like rifampicin, gentamicin, tetracycline, trimethoprim/sulfamethoxazole (TMP/SMX), clindamycin, and erythromycin have had substantial resistance profiles in >50% of the strains. In recent Turkish studies, in vitro profiles

  16. Specificity of Induction of Glycopeptide Antibiotic Resistance in the Producing Actinomycetes.

    PubMed

    Binda, Elisa; Cappelletti, Pamela; Marinelli, Flavia; Marcone, Giorgia Letizia

    2018-04-25

    Glycopeptide antibiotics are drugs of last resort for treating severe infections caused by Gram-positive pathogens. It is widely believed that glycopeptide-resistance determinants ( van genes) are ultimately derived from the producing actinomycetes. We hereby investigated the relationship between the antimicrobial activity of vancomycin and teicoplanins and their differential ability to induce van gene expression in Actinoplanes teichomyceticus —the producer of teicoplanin—and Nonomuraea gerenzanensis —the producer of the teicoplanin-like A40926. As a control, we used the well-characterized resistance model Streptomyces coelicolor . The enzyme activities of a cytoplasmic-soluble d,d-dipeptidase and of a membrane-associated d,d-carboxypeptidase (corresponding to VanX and VanY respectively) involved in resistant cell wall remodeling were measured in the actinomycetes grown in the presence or absence of subinhibitory concentrations of vancomycin, teicoplanin, and A40926. Results indicated that actinomycetes possess diverse self-resistance mechanisms, and that each of them responds differently to glycopeptide induction. Gene swapping among teicoplanins-producing actinomycetes indicated that cross-talking is possible and provides useful information for predicting the evolution of future resistance gene combinations emerging in pathogens.

  17. pGlyco 2.0 enables precision N-glycoproteomics with comprehensive quality control and one-step mass spectrometry for intact glycopeptide identification.

    PubMed

    Liu, Ming-Qi; Zeng, Wen-Feng; Fang, Pan; Cao, Wei-Qian; Liu, Chao; Yan, Guo-Quan; Zhang, Yang; Peng, Chao; Wu, Jian-Qiang; Zhang, Xiao-Jin; Tu, Hui-Jun; Chi, Hao; Sun, Rui-Xiang; Cao, Yong; Dong, Meng-Qiu; Jiang, Bi-Yun; Huang, Jiang-Ming; Shen, Hua-Li; Wong, Catherine C L; He, Si-Min; Yang, Peng-Yuan

    2017-09-05

    The precise and large-scale identification of intact glycopeptides is a critical step in glycoproteomics. Owing to the complexity of glycosylation, the current overall throughput, data quality and accessibility of intact glycopeptide identification lack behind those in routine proteomic analyses. Here, we propose a workflow for the precise high-throughput identification of intact N-glycopeptides at the proteome scale using stepped-energy fragmentation and a dedicated search engine. pGlyco 2.0 conducts comprehensive quality control including false discovery rate evaluation at all three levels of matches to glycans, peptides and glycopeptides, improving the current level of accuracy of intact glycopeptide identification. The N-glycoproteome of samples metabolically labeled with 15 N/ 13 C were analyzed quantitatively and utilized to validate the glycopeptide identification, which could be used as a novel benchmark pipeline to compare different search engines. Finally, we report a large-scale glycoproteome dataset consisting of 10,009 distinct site-specific N-glycans on 1988 glycosylation sites from 955 glycoproteins in five mouse tissues.Protein glycosylation is a heterogeneous post-translational modification that generates greater proteomic diversity that is difficult to analyze. Here the authors describe pGlyco 2.0, a workflow for the precise one step identification of intact N-glycopeptides at the proteome scale.

  18. High-yield production of the VP1 structural protein epitope from serotype O foot-and-mouth disease virus in Escherichia coli.

    PubMed

    Jung, Joon-Goo; Lee, Yong Jae; Velmurugan, Natarajan; Ko, Young-Joon; Lee, Hyang-Sim; Jeong, Ki Jun

    2013-07-01

    For effective control of foot-and-mouth disease (FMD), the development of rapid diagnostic systems and vaccines are required against its etiological agent, FMD virus (FMDV). To accomplish this, efficient large-scale expression of the FMDV VP1 protein, with high solubility, needs to be optimized. We attempted to produce high levels of a serotype O FMDV VP1 epitope in Escherichia coli. We identified the subtype-independent serotype O FMDV VP1 epitope sequence and used it to construct a glutathione S-transferase (GST) fusion protein. For efficient production of the FMDV VP1 epitope fused to GST (VP1e-GST), four E. coli strains and three temperatures were examined. The conditions yielding the greatest level of VP1e-GST with highest solubility were achieved with E. coli BL21(DE3) at 25 °C. For high-level production, fed-batch cultures were conducted in 5-l bioreactors. When cells were induced at a high density and complex feeding solutions were supplied, approximately 11 g of VP1e-GST was obtained from a 2.9-l culture. Following purification, the VP1 epitope was used to immunize rabbits, and we confirmed that it induced an immune response.

  19. Large-scale identification of core-fucosylated glycopeptide sites in pancreatic cancer serum using mass spectrometry.

    PubMed

    Tan, Zhijing; Yin, Haidi; Nie, Song; Lin, Zhenxin; Zhu, Jianhui; Ruffin, Mack T; Anderson, Michelle A; Simeone, Diane M; Lubman, David M

    2015-04-03

    Glycosylation has significant effects on protein function and cell metastasis, which are important in cancer progression. It is of great interest to identify site-specific glycosylation in search of potential cancer biomarkers. However, the abundance of glycopeptides is low compared to that of nonglycopeptides after trypsin digestion of serum samples, and the mass spectrometric signals of glycopeptides are often masked by coeluting nonglycopeptides due to low ionization efficiency. Selective enrichment of glycopeptides from complex serum samples is essential for mass spectrometry (MS)-based analysis. Herein, a strategy has been optimized using LCA enrichment to improve the identification of core-fucosylation (CF) sites in serum of pancreatic cancer patients. The optimized strategy was then applied to analyze CF glycopeptide sites in 13 sets of serum samples from pancreatic cancer, chronic pancreatitis, healthy controls, and a standard reference. In total, 630 core-fucosylation sites were identified from 322 CF proteins in pancreatic cancer patient serum using an Orbitrap Elite mass spectrometer. Further data analysis revealed that 8 CF peptides exhibited a significant difference between pancreatic cancer and other controls, which may be potential diagnostic biomarkers for pancreatic cancer.

  20. Evaluation of cross-protection against three topotypes of serotype O foot-and-mouth disease virus in pigs vaccinated with multi-epitope protein vaccine incorporated with poly(I:C).

    PubMed

    Cao, Yimei; Lu, Zengjun; Li, Dong; Fan, Pengju; Sun, Pu; Bao, Huifang; Fu, Yuanfang; Li, Pinghua; Bai, Xingwen; Chen, Yingli; Xie, Baoxia; Liu, Zaixin

    2014-01-31

    Epitope-based vaccines are always questioned for their cross-protection against the antigenically variable foot-and-mouth disease virus (FMDV). In this study, we proved the cross-protection effect of a multi-epitope vaccine incorporated with poly(I:C) against three topotypes of O type FMDV. A total of 45 naïve pigs were vaccinated with different doses of multi-epitope protein vaccine incorporated with poly(I:C). At 28 days post-vaccination, 45 vaccinated and 6 unvaccinated control pigs (two pigs for each group) were challenged with three topotypes of virulent O type FMDV, namely, O/Mya/98 (Southeast Asia topotype), O/HN/CHA/93 (Cathay topotype) and O/Tibet/CHA/99 (PanAsia topotype) strains. All unvaccinated pigs developed generalised FMD clinical signs. Results showed that all pigs (n=15) conferred complete protection against the O/Mya/98 and O/HN/CHA/93 FMDV strains, 11 of which were protected against the O/Tibet/CHA/99 FMDV strain. The 50% protective dose values of the vaccine against the O/Mya/98, O/HN/CHA/93 and O/Tibet/CHA/99 FMDV strains were 15.59, 15.59 and 7.05, respectively. Contact challenge experiment showed that transmission occurred from the donors to the unvaccinated but not to vaccinated pigs. These results showed that vaccination with multi-epitope protein vaccine incorporated with poly(I:C) can efficiently prevent FMD in pigs. Copyright © 2013 Elsevier B.V. All rights reserved.

  1. Highly Efficient Release of Glycopeptides from Hydrazide Beads by Hydroxylamine Assisted PNGase F Deglycosylation for N-Glycoproteome Analysis.

    PubMed

    Huang, Junfeng; Wan, Hao; Yao, Yating; Li, Jinan; Cheng, Kai; Mao, Jiawei; Chen, Jin; Wang, Yan; Qin, Hongqiang; Zhang, Weibing; Ye, Mingliang; Zou, Hanfa

    2015-10-20

    Selective enrichment of glycopeptides from complex sample followed by cleavage of N-glycans by PNGase F to expose an easily detectable mark on the former glycosylation sites has become the popular protocol for comprehensive glycoproteome analysis. On account of the high enrichment specificity, hydrazide chemistry based solid-phase extraction of N-linked glycopeptides technique has sparked numerous interests. However, the enzymatic release of glycopeptides captured by hydrazide beads through direct incubation of the beads with PNGase F is not efficient due to the inherent steric hindrance effect. In this study, we developed a hydroxylamine assisted PNGase F deglycosylation (HAPD) method using the hydroxylamine to release glycopeptides captured on the hydrazide beads through the cleavage of hydrazone bonds by transamination followed with the PNGase F deglycosylation of the released glycopeptides in the free solution. Because of the homogeneous condition for the deglycosylation, the recovery of deglycosylated peptides (deglycopeptides) was improved significantly. It was found that 27% more N-glycosylation sites were identified by the HAPD strategy compared with the conventional method. Moreover, the ratio of identified N-terminal glycosylated peptides was improved over 5-fold.

  2. Naturally Occurring Structural Isomers in Serum IgA1 O-Glycosylation

    PubMed Central

    Takahashi, Kazuo; Smith, Archer D.; Poulsen, Knud; Kilian, Mogens; Julian, Bruce A.; Mestecky, Jiri; Novak, Jan; Renfrow, Matthew B.

    2013-01-01

    IgA is the most abundantly produced antibody and plays an important role in the mucosal immune system. Human IgA is represented by two isotypes, IgA1 and IgA2. The major structural difference between these two subclasses is the presence of nine potential sites of O-glycosylation in the hinge region between the first and second constant region domains of the heavy chain. Thr225, Thr228, Ser230, Ser232 and Thr236 have been identified as the predominant sites of O-glycan attachment. The range and distribution of O-glycan chains at each site within the context of adjacent sites in this clustered region create a complex heterogeneity of surface epitopes that is incompletely defined. We previously described the analysis of IgA1 O-glycan heterogeneity by use of high resolution LC/MS and electron capture dissociation tandem MS to unambiguously localize all amino acid attachment sites in IgA1 (Ale) myeloma protein. Here, we report the identification and elucidation of IgA1 O-glycopeptide structural isomers that occur based on amino acid position of the attached glycans (positional isomers) and the structure of the O-glycan chains at individual sites (glycan isomers). These isomers are present in a model IgA1 (Mce1) myeloma protein and occur naturally in normal human serum IgA1. Variable O-glycan chains attached to Ser230, Thr233 or Thr236 produce the predominant positional isomers, including O-glycans composed of a single GalNAc residue. These findings represent the first definitive identification of structural isomeric IgA1 O-glycoforms, define the single-site heterogeneity for all O-glycan sites in a single sample, and have implications for defining epitopes based on clustered O-glycan variability. PMID:22067045

  3. Glycopeptide Susceptibility Profiles of Staphylococcus haemolyticus Bloodstream Isolates

    PubMed Central

    Biavasco, Francesca; Vignaroli, Carla; Lazzarini, Raffaella; Varaldo, Pietro E.

    2000-01-01

    Twelve clinical strains of Staphylococcus haemolyticus (eight methicillin resistant and three methicillin susceptible), isolated from blood cultures between 1982 and 1997, were investigated for teicoplanin and vancomycin susceptibility profiles. On the basis of conventional MIC tests and breakpoints, four isolates were susceptible (MICs, 1 to 8 μg/ml) and eight were resistant (MICs, 32 to 64 μg/ml) to teicoplanin while all were susceptible to vancomycin (MICs, 1 to 2 μg/ml). All four strains for which the conventional teicoplanin MICs were within the range of susceptibility expressed heterogeneous resistance to teicoplanin and homogeneous vancomycin susceptibility. Of the eight strains for which the conventional teicoplanin MICs were within the range of resistance, six expressed heterogeneous and two expressed homogeneous teicoplanin resistance while seven showed heterogeneous vancomycin resistance profiles (with subpopulations growing on 8 μg of the drug per ml at frequencies of ≥10−6 for six strains and 10−7 for one) and one demonstrated homogeneous vancomycin susceptibility. Of six bloodstream isolates of other staphylococcal species (S. aureus, S. epidermidis, and S. simulans), for all of which the conventional teicoplanin MICs were ≥4 μg/ml and the vancomycin MICs were ≤2 μg/ml, none exhibited heterogeneous susceptibility profiles for teicoplanin while three showed homogeneous and three showed heterogeneous susceptibility profiles for vancomycin (with subpopulations growing on 8 μg of the drug per ml found for only one strain). The results of this study indicate that a heterogeneous response to glycopeptides is a common feature of S. haemolyticus isolates and suggest that susceptibility to glycopeptides as determined by conventional MIC tests may not be predictive of the outcome of glycopeptide therapy. PMID:11036034

  4. High-yield secretion of recombinant proteins expressed in tobacco cell culture with a designer glycopeptide tag: Process development.

    PubMed

    Zhang, Ningning; Gonzalez, Maria; Savary, Brett; Xu, Jianfeng

    2016-03-01

    Low-yield protein production remains the most significant economic hurdle with plant cell culture technology. Fusions of recombinant proteins with hydroxyproline-O-glycosylated designer glycopeptide tags have consistently boosted secreted protein yields. This prompted us to study the process development of this technology aiming to achieve productivity levels necessary for commercial viability. We used a tobacco BY-2 cell culture expressing EGFP as fusion with a glycopeptide tag comprised of 32 repeat of "Ser-Pro" dipeptide, or (SP)32 , to study cell growth and protein secretion, culture scale-up, and establishment of perfusion cultures for continuous production. The BY-2 cells accumulated low levels of cell biomass (~7.5 g DW/L) in Schenk & Hildebrandt medium, but secreted high yields of (SP)32 -tagged EGFP (125 mg/L). Protein productivity of the cell culture has been stable for 6.0 years. The BY-2 cells cultured in a 5-L bioreactor similarly produced high secreted protein yield at 131 mg/L. Successful operation of a cell perfusion culture for 30 days was achieved under the perfusion rate of 0.25 and 0.5 day(-1) , generating a protein volumetric productivity of 17.6 and 28.9 mg/day/L, respectively. This research demonstrates the great potential of the designer glycopeptide technology for use in commercial production of valuable proteins with plant cell cultures. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  5. An accessible protocol for solid-phase extraction of N-linked glycopeptides through reductive amination by amine-functionalized magnetic nanoparticles.

    PubMed

    Zhang, Ying; Kuang, Min; Zhang, Lijuan; Yang, Pengyuan; Lu, Haojie

    2013-06-04

    In light of the significance of glycosylation for wealthy biological events, it is important to prefractionate glycoproteins/glycopeptides from complex biological samples. Herein, we reported a novel protocol of solid-phase extraction of glycopeptides through a reductive amination reaction by employing the easily accessible 3-aminopropyltriethoxysilane (APTES)-functionalized magnetic nanoparticles. The amino groups from APTES, which were assembled onto the surface of the nanoparticles through a one-step silanization reaction, could conjugate with the aldehydes from oxidized glycopeptides and, therefore, completed the extraction. To the best of our knowledge, this is the first example of applying the reductive amination reaction into the isolation of glycopeptides. Due to the elimination of the desalting step, the detection limit of glycopeptides was improved by 2 orders of magnitude, compared to the traditional hydrazide chemistry-based solid phase extraction, while the extraction time was shortened to 4 h, suggesting the high sensitivity, specificity, and efficiency for the extraction of N-linked glycopeptides by this method. In the meantime, high selectivity toward glycoproteins was also observed in the separation of Ribonuclease B from the mixtures contaminated with bovine serum albumin. What's more, this technique required significantly less sample volume, as demonstrated in the successful mapping of glycosylation of human colorectal cancer serum with the sample volume as little as 5 μL. Because of all these attractive features, we believe that the innovative protocol proposed here will shed new light on the research of glycosylation profiling.

  6. A40926, a new glycopeptide antibiotic with anti-Neisseria activity.

    PubMed Central

    Goldstein, B P; Selva, E; Gastaldo, L; Berti, M; Pallanza, R; Ripamonti, F; Ferrari, P; Denaro, M; Arioli, V; Cassani, G

    1987-01-01

    In the course of a search for glycopeptide antibiotics having novel biological properties, we isolated A40926. Produced by an actinomycete of the genus Actinomadura, A40926 is a complex of four main factors which contain a fatty acid as part of a glycolipid attached to the peptide backbone. Its activity was, in most respects, similar to that of other glycopeptides, such as vancomycin and teicoplanin. However, in addition to inhibiting gram-positive bacteria, A40926 was very active against Neisseria gonorrhoeae. A40926 was rapidly bactericidal for N. gonorrhoeae clinical isolates at concentrations equal to or slightly higher than the MIC. In mice, levels in serum were higher and more prolonged than those of an equivalent subcutaneous dose of teicoplanin. These properties suggest that A40926 may have potential in the therapy of gonorrhea. PMID:2964225

  7. Tryptic digestion of human GPIIIa. Isolation and biochemical characterization of the 23 kDa N-terminal glycopeptide carrying the antigenic determinant for a monoclonal antibody (P37) which inhibits platelet aggregation.

    PubMed Central

    Calvete, J J; Rivas, G; Maruri, M; Alvarez, M V; McGregor, J L; Hew, C L; Gonzalez-Rodriguez, J

    1988-01-01

    Early digestion of pure human platelet glycoprotein IIIa (GPIIIa) leads to a single cleavage of the molecule at 23 kDa far from one of the terminal amino acids. Automated Edman degradation demonstrates that GPIIIa and the smaller (23 kDa) tryptic fragment share the same N-terminal amino acid sequence. A further cleavage occurs in the larger fragment (80 kDa), reducing its apparent molecular mass by 10 kDa. The 23 kDa fragment remains attached to the larger ones in unreduced samples. Stepwise reduction of early digested GPIIIa with dithioerythritol selectively reduces the single disulphide bond joining the smaller (23 kDa) to the larger (80/70 kDa) fragments. Two fractions were obtained by size-exclusion chromatography of early digested GPIIIa after partial or full reduction and alkylation. The larger-size fraction contains the 80/70 kDa fragments, while the 23 kDa fragment is isolated in the smaller. The amino acid compositions of these fractions do not differ very significantly from the composition of GPIIIa; however the 23 kDa fragment contains only 10.2% by weight of sugars and is richer in neuraminic acid. Disulphide bonds are distributed four in the 23 kDa glycopeptide and 20-21 in the 80/70 kDa glycopeptide. The epitope for P37, a monoclonal antibody which inhibits platelet aggregation [Melero & González-Rodríguez (1984) Eur. J. Biochem. 141, 421-427] is situated within the first 17 kDa of the N-terminal region of GPIIIa, which gives a special functional interest to this extracellular region of GPIIIa. On the other hand, the epitopes for GPIIIa-specific monoclonal antibodies, P6, P35, P40 and P97, which do not interfere with platelet aggregation, are located within the larger tryptic fragment (80/70 kDa). Thus, the antigenic areas available in the extracellular surface of GPIIIa for these five monoclonal antibodies are now more precisely delineated. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:2455507

  8. Extracting Both Peptide Sequence and Glycan Structural Information by 157 nm Photodissociation of N-Linked Glycopeptides

    PubMed Central

    Zhang, Liangyi; Reilly, James P.

    2009-01-01

    157 nm photodissociation of N-linked glycopeptides was investigated in MALDI tandem time-of-flight (TOF) and linear ion trap mass spectrometers. Singly-charged glycopeptides yielded abundant peptide and glycan fragments. The peptide fragments included a series of x-, y-, v- and w- ions with the glycan remaining intact. These provide information about the peptide sequence and the glycosylation site. In addition to glycosidic fragments, abundant cross-ring glycan fragments that are not observed in low-energy CID were detected. These fragments provide insight into the glycan sequence and linkages. Doubly-charged glycopeptides generated by nanospray in the linear ion trap mass spectrometer also yielded peptide and glycan fragments. However, the former were dominated by low-energy fragments such as b- and y- type ions while glycan was primarily cleaved at glycosidic bonds. PMID:19113943

  9. An antibody reactive to the Gly63–Lys68 epitope of NT-proBNP exhibits O-glycosylation-independent binding

    PubMed Central

    Lee, Yujean; Kim, Hyori; Chung, Junho

    2014-01-01

    The N-terminal fragment of prohormone brain natriuretic peptide (NT-proBNP) is a commonly used biomarker for the diagnosis of congestive heart failure, although its biological function is not well known. NT-proBNP exhibits heavy O-linked glycosylation, and it is quite difficult to develop an antibody that exhibits glycosylation-independent binding. We developed an antibody that binds to the recombinant NT-proBNP protein and its deglycosylated form with similar affinities in an enzyme immunoassay. The epitope was defined as Gly63–Lys68 based on mimetic peptide screening, site-directed mutagenesis and a competition assay with a peptide mimotope. The nearest O-glycosylation residues are Thr58 and Thr71; therefore, four amino acid residues intervene between the epitope and those residues in both directions. In conclusion, we report that an antibody reactive to Gly63–Lys68 of NT-proBNP exhibits O-glycosylation-independent binding. PMID:25236766

  10. Distinctive and Complementary MS2 Fragmentation Characteristics for Identification of Sulfated Sialylated N-Glycopeptides by nanoLC-MS/MS Workflow

    NASA Astrophysics Data System (ADS)

    Kuo, Chu-Wei; Guu, Shih-Yun; Khoo, Kay-Hooi

    2018-04-01

    High sensitivity identification of sulfated glycans carried on specific sites of glycoproteins is an important requisite for investigation of molecular recognition events involved in diverse biological processes. However, aiming for resolving site-specific glycosylation of sulfated glycopeptides by direct LC-MS2 sequencing is technically most challenging. Other than the usual limiting factors such as lower abundance and ionization efficiency compared to analysis of non-glycosylated peptides, confident identification of sulfated glycopeptides among the more abundant non-sulfated glycopeptides requires additional considerations in the selective enrichment and detection strategies. Metal oxide has been applied to enrich phosphopeptides and sialylated glycopeptides, but its use to capture sulfated glycopeptides has not been investigated. Likewise, various complementary MS2 fragmentation modes have yet to be tested against sialylated and non-sialylated sulfoglycopeptides due to limited appropriate sample availability. In this study, we have investigated the feasibility of sequencing tryptic sulfated N-glycopeptide and its MS2 fragmentation characteristics by first optimizing the enrichment methods to allow efficient LC-MS detection and MS2 analysis by a combination of CID, HCD, ETD, and EThcD on hybrid and tribrid Orbitrap instruments. Characteristic sulfated glyco-oxonium ions and direct loss of sulfite from precursors were detected as evidences of sulfate modification. It is anticipated that the technical advances demonstrated in this study would allow a feasible extension of our sulfoglycomic analysis to sulfoglycoproteomics. [Figure not available: see fulltext.

  11. N-Glycan Structure Annotation of Glycopeptides Using a Linearized Glycan Structure Database (GlyDB)

    PubMed Central

    Ren, Jian Min; Rejtar, Tomas; Li, Lingyun; Karger, Barry L.

    2008-01-01

    While glycoproteins are abundant in nature, and changes in glycosylation occur in cancer and other diseases, glycoprotein characterization remains a challenge due to the structural complexity of the biopolymers. This paper presents a general strategy, termed GlyDB, for glycan structure annotation of N-linked glycopeptides from tandem mass spectra in the LC-MS analysis of proteolytic digests of glycoproteins. The GlyDB approach takes advantage of low-energy collision induced dissociation of N-linked glycopeptides that preferentially cleaves the glycosidic bonds while the peptide backbone remains intact. A theoretical glycan structure database derived from biosynthetic rules for N-linked glycans was constructed employing a novel representation of branched glycan structures consisting of multiple linear sequences. The commonly used peptide identification program, Sequest, could then be utilized to assign experimental tandem mass spectra to individual glycoforms. Analysis of synthetic glycopeptides and well-characterized glycoproteins demonstrate that the GlyDB approach can be a useful tool for annotation of glycan structures and for selection of a limited number of potential glycan structure candidates for targeted validation. PMID:17625816

  12. Complex Regulatory Networks Governing Production of the Glycopeptide A40926.

    PubMed

    Alduina, Rosa; Sosio, Margherita; Donadio, Stefano

    2018-04-05

    Glycopeptides (GPAs) are an important class of antibiotics, with vancomycin and teicoplanin being used in the last 40 years as drugs of last resort to treat infections caused by Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus . A few new GPAs have since reached the market. One of them is dalbavancin, a derivative of A40926 produced by the actinomycete Nonomuraea sp. ATCC 39727, recently classified as N. gerenzanensis . This review summarizes what we currently know on the multilevel regulatory processes governing production of the glycopeptide A40926 and the different approaches used to increase antibiotic yields. Some nutrients, e.g., valine, l-glutamine and maltodextrin, and some endogenous proteins, e.g., Dbv3, Dbv4 and RpoB R , have a positive role on A40926 biosynthesis, while other factors, e.g., phosphate, ammonium and Dbv23, have a negative effect. Overall, the results available so far point to a complex regulatory network controlling A40926 in the native producing strain.

  13. Isolation of N-linked glycopeptides by hydrazine-functionalized magnetic particles.

    PubMed

    Sun, Shisheng; Yang, Ganglong; Wang, Ting; Wang, Qinzhe; Chen, Chao; Li, Zheng

    2010-04-01

    We introduce a novel combination of magnetic particles with hydrazine chemistry, dubbed as hydrazine-functionalized magnetic particles (HFMP) for isolation of glycopeptides. Four methods have been developed and compared for the production of HFMP by hydrazine modification of the surface of the carboxyl and epoxy-silanized magnetic particles, respectively. The evaluation of the capability and specificity of HFMP as well as the optimization of the coupling condition for capturing of glycoproteins were systematically investigated. The results showed that HFMP prepared by adipic dihydrazide functionalization from carboxyl-silanized magnetic particles (HFCA) displayed the maximum capture capacity and isolated efficiency for glycoprotein. When measured with glycoproteins, the capacity of the HFCA (1 g) for coupling bovine fetuin was 130 +/- 5.3 mg. The capability of this method was also confirmed by successful isolation of all formerly glycosylated peptides from standard glycoproteins and identification of their glycosylation sites, which demonstrated the feasibility of the HFCA as an alternative solid support for isolation of glycoproteins/glycopeptides.

  14. Site-specific N-glycosylation analysis: matrix-assisted laser desorption/ionization quadrupole-quadrupole time-of-flight tandem mass spectral signatures for recognition and identification of glycopeptides.

    PubMed

    Krokhin, Oleg; Ens, Werner; Standing, Kenneth G; Wilkins, John; Perreault, Hélène

    2004-01-01

    The identification of glycosylation sites in proteins is often possible through a combination of proteolytic digestion, separation, mass spectrometry (MS) and tandem MS (MS/MS). Liquid chromatography (LC) in combination with MS/MS has been a reliable method for detecting glycopeptides in digestion mixtures, and for assigning glycosylation sites and glycopeptide sequences. Direct interfacing of LC with MS relies on electrospray ionization, which produces ions with two, three or four charges for most proteolytic peptides and glycopeptides. MS/MS spectra of such glycopeptide ions often lead to ambiguous interpretation if deconvolution to the singly charged level is not used. In contrast, the matrix-assisted laser desorption/ionization (MALDI) technique usually produces singly charged peptide and glycopeptide ions. These ions require an extended m/z range, as provided by the quadrupole-quadrupole time-of-flight (QqTOF) instrument used in these experiments, but the main advantages of studying singly charged ions are the simplicity and consistency of the MS/MS spectra. A first aim of the present study is to develop methods to recognize and use glycopeptide [M+H]+ ions as precursors for MS/MS, and thus for glycopeptide/glycoprotein identification as part of wider proteomics studies. Secondly, this article aims at demonstrating the usefulness of MALDI-MS/MS spectra of N-glycopeptides. Mixtures of diverse types of proteins, obtained commercially, were prepared and subjected to reduction, alkylation and tryptic digestion. Micro-column reversed-phase separation allowed deposition of several fractions on MALDI plates, followed by MS and MS/MS analysis of all peptides. Glycopeptide fractions were identified after MS by their specific m/z spacing patterns (162, 203, 291 u) between glycoforms, and then analyzed by MS/MS. In most cases, MS/MS spectra of [M+H]+ ions of glycopeptides featured peaks useful for determining sugar composition, peptide sequence, and thus probable

  15. Convergent Synthesis of N-Linked Glycopeptides via Aminolysis of ω-Asp p-Nitrophenyl Thioesters in Solution.

    PubMed

    Du, Jing-Jing; Gao, Xiao-Fei; Xin, Ling-Ming; Lei, Ze; Liu, Zheng; Guo, Jun

    2016-10-07

    An efficient N-linked glycosylation reaction between glycosylamines and p-nitrophenyl thioester peptides has been developed. The reaction conditions are mild and compatible with the C-terminal free carboxylic acid group and the unprotected N-linked sialyloligosaccharide. By means of this convergent strategy, a versatile N-glycopeptide fragment containing an N-terminal Thz and a C-terminal thioester was readily prepared, which is available for the synthesis of long glycopeptides and glycoproteins using the protocol of native chemical ligation.

  16. BepiPred-2.0: improving sequence-based B-cell epitope prediction using conformational epitopes

    PubMed Central

    Jespersen, Martin Closter; Peters, Bjoern

    2017-01-01

    Abstract Antibodies have become an indispensable tool for many biotechnological and clinical applications. They bind their molecular target (antigen) by recognizing a portion of its structure (epitope) in a highly specific manner. The ability to predict epitopes from antigen sequences alone is a complex task. Despite substantial effort, limited advancement has been achieved over the last decade in the accuracy of epitope prediction methods, especially for those that rely on the sequence of the antigen only. Here, we present BepiPred-2.0 (http://www.cbs.dtu.dk/services/BepiPred/), a web server for predicting B-cell epitopes from antigen sequences. BepiPred-2.0 is based on a random forest algorithm trained on epitopes annotated from antibody-antigen protein structures. This new method was found to outperform other available tools for sequence-based epitope prediction both on epitope data derived from solved 3D structures, and on a large collection of linear epitopes downloaded from the IEDB database. The method displays results in a user-friendly and informative way, both for computer-savvy and non-expert users. We believe that BepiPred-2.0 will be a valuable tool for the bioinformatics and immunology community. PMID:28472356

  17. Epitope discovery in West Nile virus infection: Identification and immune recognition of viral epitopes.

    PubMed

    McMurtrey, Curtis P; Lelic, Alina; Piazza, Paolo; Chakrabarti, Ayan K; Yablonsky, Eric J; Wahl, Angela; Bardet, Wilfried; Eckerd, Annette; Cook, Robert L; Hess, Rachael; Buchli, Rico; Loeb, Mark; Rinaldo, Charles R; Bramson, Jonathan; Hildebrand, William H

    2008-02-26

    Cytotoxic T lymphocytes (CTL) play an important role in the control and elimination of infection by West Nile virus (WNV), yet the class I human leukocyte antigen (HLA)-presented peptide epitopes that enable CTL recognition of WNV-infected cells remain uncharacterized. The goals of this work were first to discover the peptide epitopes that distinguish the class I HLA of WNV-infected cells and then to test the T cell reactivity of newly discovered WNV epitopes. To discover WNV-immune epitopes, class I HLA was harvested from WNV (NY99 strain)-infected and uninfected HeLa cells. Then peptide epitopes were eluted from affinity-purified HLA, and peptide epitopes from infected and uninfected cells were comparatively mapped by mass spectroscopy. Six virus-derived peptides from five different viral proteins (E, NS2b, NS3, NS4b, and NS5) were discovered as unique to HLA-A*0201 of infected cells, demonstrating that the peptides sampled by class I HLA are distributed widely throughout the WNV proteome. When tested with CTL from infected individuals, one dominant WNV target was apparent, two epitopes were subdominant, and three demonstrated little CTL reactivity. Finally, a sequence comparison of these epitopes with the hundreds of viral isolates shows that HLA-A*0201 presents epitopes derived from conserved regions of the virus. Detection and recovery from WNV infection are therefore functions of the ability of class I HLA molecules to reveal conserved WNV epitopes to an intact cellular immune system that subsequently recognizes infected cells.

  18. BepiPred-2.0: improving sequence-based B-cell epitope prediction using conformational epitopes.

    PubMed

    Jespersen, Martin Closter; Peters, Bjoern; Nielsen, Morten; Marcatili, Paolo

    2017-07-03

    Antibodies have become an indispensable tool for many biotechnological and clinical applications. They bind their molecular target (antigen) by recognizing a portion of its structure (epitope) in a highly specific manner. The ability to predict epitopes from antigen sequences alone is a complex task. Despite substantial effort, limited advancement has been achieved over the last decade in the accuracy of epitope prediction methods, especially for those that rely on the sequence of the antigen only. Here, we present BepiPred-2.0 (http://www.cbs.dtu.dk/services/BepiPred/), a web server for predicting B-cell epitopes from antigen sequences. BepiPred-2.0 is based on a random forest algorithm trained on epitopes annotated from antibody-antigen protein structures. This new method was found to outperform other available tools for sequence-based epitope prediction both on epitope data derived from solved 3D structures, and on a large collection of linear epitopes downloaded from the IEDB database. The method displays results in a user-friendly and informative way, both for computer-savvy and non-expert users. We believe that BepiPred-2.0 will be a valuable tool for the bioinformatics and immunology community. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Validation of VITEK 2 Version 4.01 Software for Detection, Identification, and Classification of Glycopeptide-Resistant Enterococci

    PubMed Central

    Abele-Horn, Marianne; Hommers, Leif; Trabold, René; Frosch, Matthias

    2006-01-01

    We evaluated the ability of the new VITEK 2 version 4.01 software to identify and detect glycopeptide-resistant enterococci compared to that of the reference broth microdilution method and to classify them into the vanA, vanB, vanC1, and vanC2 genotypes. Moreover, the accuracy of antimicrobial susceptibility testing with agents with improved potencies against glycopeptide-resistant enterococci was determined. A total of 121 enterococci were investigated. The new VITEK 2 software was able to identify 114 (94.2%) enterococcal strains correctly to the species level and to classify 119 (98.3%) enterococci correctly to the glycopeptide resistance genotype level. One Enterococcus casseliflavus strain and six Enterococcus faecium vanA strains with low-level resistance to vancomycin were identified with low discrimination, requiring additional tests. One of the vanA strains was misclassified as the vanB type, and one glycopeptide-susceptible E. facium wild type was misclassified as the vanA type. The overall essential agreements for antimicrobial susceptibility testing results were 94.2% for vancomycin, 95.9% for teicoplanin, 100% for quinupristin-dalfopristin and moxifloxacin, and 97.5% for linezolid. The rates of minor errors were 9% for teicoplanin and 5% for the other antibiotic agents. The identification and susceptibility data were produced within 4 h to 6 h 30 min and 8 h 15 min to 12 h 15 min. In conclusion, use of VITEK 2 version 4.01 software appears to be a reliable method for the identification and detection of glycopeptide-resistant enterococci as well as an improvement over the use of the former VITEK 2 database. However, a significant reduction in the detection time would be desirable. PMID:16390951

  20. Analysis of PNGase F-resistant N-glycopeptides using SugarQb for Proteome Discoverer 2.1 reveals cryptic substrate specificities.

    PubMed

    Stadlmann, Johannes; Hoi, David M; Taubenschmid, Jasmin; Mechtler, Karl; Penninger, Josef M

    2018-05-18

    SugarQb (www.imba.oeaw.ac.at/sugarqb) is a freely available collection of computational tools for the automated identification of intact glycopeptides from high-resolution HCD MS/MS data-sets in the Proteome Discoverer environment. We report the migration of SugarQb to the latest and free version of Proteome Discoverer 2.1, and apply it to the analysis of PNGase F-resistant N-glycopeptides from mouse embryonic stem cells. The analysis of intact glycopeptides highlights unexpected technical limitations to PNGase F-dependent glycoproteomic workflows at the proteome level, and warrants a critical re-interpretation of seminal data-sets in the context of N-glycosylation-site prediction. This article is protected by copyright. All rights reserved. This article is protected by copyright. All rights reserved.

  1. Proteomic Analysis Reveals Aberrant O-GlcNAcylation of Extracellular Proteins from Breast Cancer Cell Secretion.

    PubMed

    Netsirisawan, Pukkavadee; Chokchaichamnankit, Daranee; Srisomsap, Chantragan; Svasti, Jisnuson; Champattanachai, Voraratt

    2015-01-01

    O-GlcNAcylation is a unique intracellular protein modification; however, few extracellular O-GlcNAc-modified proteins have been discovered. We have previously demonstrated that many cellular proteins were aberrant in O-GlcNAcylation in breast cancer tissues. In the present study, therefore, we investigated whether O-GlcNAc-modified proteins were abnormally secreted from breast cancer cells. Intracellular and extracellular proteins were prepared from cell lysates of breast cancer cells (MCF-7 and MDA-MB-231) and normal breast cells (HMEC) and from their serum-free media (SFM), respectively. O-GlcNAcylation level was examined by immunoblotting. O-GlcNAc-Modified proteins were identified using two-dimensional gel electrophoresis and Liquid Chromatography-tandem Mass Spectrometry. O-GlcNAcylation level was significantly increased in the extracellular compartment of both types of cancer cells compared to normal cells. Interestingly, O-GlcNAc patterns differed between intracellular and extracellular proteins. Proteomic analysis revealed that many O-GlcNAc spots in MCF-7 secretions were abnormally increased in comparison to those in HMEC secretions. Among these, transitional endoplasmic reticulum ATPase (TER ATPase) and heat-shock 70 kDa (HSP70) were confirmed to be O-GlcNAc-modified. The levels of O-GlcNAc-HSP70 and O-GlcNAc-TER ATPase were higher in SFM from MCF-7 cells than in that from HMEC. O-GlcNAcomic study of the extracellular compartments reveals aberrant O-GlcNAc-secreted proteins, which may be of interest as potential biomarkers in breast cancer. Copyright© 2015, International Institute of Anticancer Research (Dr. John G. Delinasios), All rights reserved.

  2. Glycopeptides versus β-lactams for the prevention of surgical site infections in cardiovascular and orthopedic surgery: a meta-analysis.

    PubMed

    Saleh, Anas; Khanna, Ashish; Chagin, Kevin M; Klika, Alison K; Johnston, Douglas; Barsoum, Wael K

    2015-01-01

    To compare the efficacy of glycopeptides and β-lactams in preventing surgical site infections (SSIs) in cardiac, vascular, and orthopedic surgery. The cost-effectiveness of switching from β-lactams to glycopeptides for preoperative antibiotic prophylaxis has been controversial. β-Lactams are generally recommended in clean surgical procedures, but they are ineffective against resistant gram-positive bacteria. PubMed, International Pharmaceuticals Abstracts, Scopus, and Cochrane were searched for randomized clinical trials comparing glycopeptides and β-lactams for prophylaxis in adults undergoing cardiac, vascular, or orthopedic surgery. Abstracts and conference proceedings were included. Two independent reviewers performed study selection, data extraction, and assessment of risk of bias. Fourteen studies with a total of 8952 patients were analyzed. No difference was detected in overall SSIs between antibiotic types. However, compared with β-lactams, glycopeptides reduced the risk of resistant staphylococcal SSIs by 48% (relative risk, 0.52; 95% confidence interval, 0.29-0.93; P = 0.03) and enterococcal SSIs by 64% (relative risk, 0.36; 95% confidence interval, 0.16-0.80; P = 0.01), but increased respiratory tract infections by 54% (relative risk, 1.54; 95% confidence interval, 1.19-2.01; P ≤ 0.01). Subgroup analysis of cardiac procedures showed superiority of β-lactams in preventing superficial and deep chest SSIs, susceptible staphylococcal SSIs, and respiratory tract infections. Glycopeptides reduce the risk of resistant staphylococcal SSIs and enterococcal SSIs, but increase the risk of respiratory tract infections. Additional high-quality randomized clinical trials are needed as these results are limited by high risk of bias.

  3. Heterologous expression of glycopeptide resistance vanHAX gene clusters from soil bacteria in Enterococcus faecalis.

    PubMed

    Hasman, Henrik; Aarestrup, Frank M; Dalsgaard, Anders; Guardabassi, Luca

    2006-04-01

    The aim of the study was to determine whether glycopeptide resistance gene clusters from soil bacteria could be heterologously expressed in Enterococcus faecalis and adapt to the new host following exposure to vancomycin. The vanHAX clusters from Paenibacillus thiaminolyticus PT-2B1, Paenibacillus apiarius PA-B2B and Amycolatopsis coloradensis DSM 44225 were separately cloned in an appropriately constructed shuttle vector containing the two-component regulatory system (vanRS) of Tn1546. The complete vanA(PT) operon (vanRSHAXY) from P. thiaminolyticus PT-2B1 was cloned in the same shuttle vector lacking enterococcal vanRS. All plasmid constructs were electroporated into E. faecalis JH2-2 and the MICs of vancomycin and teicoplanin were determined for each recombinant strain before and following exposure to sublethal concentrations of vancomycin. The vanHAX clusters from P. thiaminolyticus and P. apiarius conferred high-level vancomycin resistance (MIC > or = 125 mg/L) in E. faecalis JH2-2. In contrast, cloning of the vanHAX cluster from A. coloradensis did not result in a significant increase of vancomycin resistance (MIC = 0.7 mg/L). Resistance to vancomycin was not observed after cloning the complete vanA(PT) operon from P. thiaminolyticus (MIC = 2 mg/L), but this recombinant rapidly adapted to high concentrations of vancomycin (MIC = 500 mg/L) following exposure to sub-lethal concentrations of this antibiotic. The results showed that vanA(PT) in P. thiaminolyticus is a possible ancestor of vanA-mediated glycopeptide resistance in enterococci. Experimental evidence supported the hypothesis that enterococci did not acquire glycopeptide resistance directly from glycopeptide-producing organisms such as A. coloradensis.

  4. Adamantoylated biologically active small peptides and glycopeptides structurally related to the bacterial peptidoglycan.

    PubMed

    Frkanec, Ruža; Vranešić, Branka; Tomić, Srdjanka

    2013-01-01

    A large number of novel synthetic compounds representing smaller parts of original peptidoglycan molecules have been synthesized and found to possess versatile biological activity, particularly immunomodulating properties. A series of compounds containing the adamantyl residues coupled to peptides and glycopeptides characteristic for bacterial peptidoglycan was described. The new adamantylpeptides and adamantylglycopeptides were prepared starting from N-protected racemic adamantylglycine and dipeptide L-Ala-D-isoglutamine. The adamantyl glycopeptides were obtained by coupling the adamantyltripeptides with alpha-D-mannose moiety through spacer molecule of fixed chirality. Since the starting material was D,L-(adamantyl-glycine) the condensation products with the dipeptide were mixtures of diastereoisomers. The obtained diastereoisomers were separated, characterized, and tested for immunostimulating activity. An HPLC method for purity testing was developed and adapted for the particular compounds.

  5. The fully synthetic MAG-Tn3 therapeutic vaccine containing the tetanus toxoid-derived TT830-844 universal epitope provides anti-tumor immunity.

    PubMed

    Laubreton, Daphné; Bay, Sylvie; Sedlik, Christine; Artaud, Cécile; Ganneau, Christelle; Dériaud, Edith; Viel, Sophie; Puaux, Anne-Laure; Amigorena, Sebastian; Gérard, Catherine; Lo-Man, Richard; Leclerc, Claude

    2016-03-01

    Malignant transformations are often associated with aberrant glycosylation processes that lead to the expression of new carbohydrate antigens at the surface of tumor cells. Of these carbohydrate antigens, the Tn antigen is particularly highly expressed in many carcinomas, especially in breast carcinoma. We designed MAG-Tn3, a fully synthetic vaccine based on three consecutive Tn moieties that are O-linked to a CD4+ T cell epitope, to induce anti-Tn antibody responses that could be helpful for therapeutic vaccination against cancer. To ensure broad coverage within the human population, the tetanus toxoid-derived peptide TT830-844 was selected as a T-helper epitope because it can bind to various HLA-DRB molecules. We showed that the MAG-Tn3 vaccine, which was formulated with the GSK proprietary immunostimulant AS15 and designed for human cancer therapy, is able to induce an anti-Tn antibody response in mice of various H-2 haplotypes, and this response correlates with the ability to induce a specific T cell response against the TT830-844 peptide. The universality of the TT830-844 peptide was extended to new H-2 and HLA-DRB molecules that were capable of binding this T cell epitope. Finally, the MAG-Tn3 vaccine was able to induce anti-Tn antibody responses in cynomolgus monkeys, which targeted Tn-expressing tumor cells and mediated tumor cell death both in vitro and in vivo. Thus, MAG-Tn3 is a highly promising anticancer vaccine that is currently under evaluation in a phase I clinical trial.

  6. Absolute Quantitation of Glycoforms of Two Human IgG Subclasses Using Synthetic Fc Peptides and Glycopeptides

    NASA Astrophysics Data System (ADS)

    Roy, Rini; Ang, Evelyn; Komatsu, Emy; Domalaon, Ronald; Bosseboeuf, Adrien; Harb, Jean; Hermouet, Sylvie; Krokhin, Oleg; Schweizer, Frank; Perreault, Hélène

    2018-05-01

    Immunoglobulins, such as immunoglobulin G (IgG), are of prime importance in the immune system. Polyclonal human IgG comprises four subclasses, of which IgG1 and IgG2 are the most abundant in healthy individuals. In an effort to develop an absolute MALDI-ToF-MS quantitative method for these subclasses and their Fc N-glycoforms, (glyco)peptides were synthesized using a solid-phase approach and used as internal standards. Tryptic digest glycopeptides from monoclonal IgG1 and IgG2 samples were first quantified using EEQYN(GlcNAc)STYR and EEQFN(GlcNAc)STFR standards, respectively. For IgG1, a similar glycopeptide where tyrosine (Y) was isotopically labelled was used to quantify monoclonal IgG1 that had been treated with the enzyme Endo-F2, i.e., yielding tryptic glycopeptide EEQYN(GlcNAc)STYR. The next step was to quantify single subclasses within polyclonal human IgG samples. Although ion abundances in the MALDI spectra often showed higher signals for IgG2 than IgG1, depending on the spotting solvent used, determination of amounts using the newly developed quantitative method allowed to obtain accurate concentrations where IgG1 species were predominant. It was observed that simultaneous analysis of IgG1 and IgG2 yielded non-quantitative results and that more success was obtained when subclasses were quantified one by one. More experiments served to assess the respective extraction and ionization efficiencies of EEQYNSTYR/EEQFNSTFR and EEQYN(GlcNAc)STYR/EEQFN(GlcNAc)STFR mixtures under different solvent and concentration conditions.

  7. Design and synthesis of multifunctional gold nanoparticles bearing tumor-associated glycopeptide antigens as potential cancer vaccines.

    PubMed

    Brinãs, Raymond P; Sundgren, Andreas; Sahoo, Padmini; Morey, Susan; Rittenhouse-Olson, Kate; Wilding, Greg E; Deng, Wei; Barchi, Joseph J

    2012-08-15

    The development of vaccines against specific types of cancers will offer new modalities for therapeutic intervention. Here, we describe the synthesis of a novel vaccine construction prepared from spherical gold nanoparticles of 3-5 nm core diameters. The particles were coated with both the tumor-associated glycopeptides antigens containing the cell-surface mucin MUC4 with Thomsen Friedenreich (TF) antigen attached at different sites and a 28-residue peptide from the complement derived protein C3d to act as a B-cell activating "molecular adjuvant". The synthesis entailed solid-phase glycopeptide synthesis, design of appropriate linkers, and attachment chemistry of the various molecules to the particles. Attachment to the gold surface was mediated by a novel thiol-containing 33 atom linker which was further modified to be included as a third "spacer" component in the synthesis of several three-component vaccine platforms. Groups of mice were vaccinated either with one of the nanoplatform constructs or with control particles without antigen coating. Evaluation of sera from the immunized animals in enzyme immunoassays (EIA) against each glycopeptide antigen showed a small but statistically significant immune response with production of both IgM and IgG isotypes. Vaccines with one carbohydrate antigen (B, C, and E) gave more robust responses than the one with two contiguous disaccharides (D), and vaccine E with a TF antigen attached to threonine at the 10th position of the peptide was selected for IgG over IgM suggesting isotype switching. The data suggested that this platform may be a viable delivery system for tumor-associated glycopeptide antigens.

  8. EpitopeViewer: a Java application for the visualization and analysis of immune epitopes in the Immune Epitope Database and Analysis Resource (IEDB).

    PubMed

    Beaver, John E; Bourne, Philip E; Ponomarenko, Julia V

    2007-02-21

    Structural information about epitopes, particularly the three-dimensional (3D) structures of antigens in complex with immune receptors, presents a valuable source of data for immunology. This information is available in the Protein Data Bank (PDB) and provided in curated form by the Immune Epitope Database and Analysis Resource (IEDB). With continued growth in these data and the importance in understanding molecular level interactions of immunological interest there is a need for new specialized molecular visualization and analysis tools. The EpitopeViewer is a platform-independent Java application for the visualization of the three-dimensional structure and sequence of epitopes and analyses of their interactions with antigen-specific receptors of the immune system (antibodies, T cell receptors and MHC molecules). The viewer renders both 3D views and two-dimensional plots of intermolecular interactions between the antigen and receptor(s) by reading curated data from the IEDB and/or calculated on-the-fly from atom coordinates from the PDB. The 3D views and associated interactions can be saved for future use and publication. The EpitopeViewer can be accessed from the IEDB Web site http://www.immuneepitope.org through the quick link 'Browse Records by 3D Structure.' The EpitopeViewer is designed and been tested for use by immunologists with little or no training in molecular graphics. The EpitopeViewer can be launched from most popular Web browsers without user intervention. A Java Runtime Environment (RJE) 1.4.2 or higher is required.

  9. Direct glycan structure determination of intact N-linked glycopeptides by low-energy collision-induced dissociation tandem mass spectrometry and predicted spectral library searching.

    PubMed

    Pai, Pei-Jing; Hu, Yingwei; Lam, Henry

    2016-08-31

    Intact glycopeptide MS analysis to reveal site-specific protein glycosylation is an important frontier of proteomics. However, computational tools for analyzing MS/MS spectra of intact glycopeptides are still limited and not well-integrated into existing workflows. In this work, a new computational tool which combines the spectral library building/searching tool, SpectraST (Lam et al. Nat. Methods2008, 5, 873-875), and the glycopeptide fragmentation prediction tool, MassAnalyzer (Zhang et al. Anal. Chem.2010, 82, 10194-10202) for intact glycopeptide analysis has been developed. Specifically, this tool enables the determination of the glycan structure directly from low-energy collision-induced dissociation (CID) spectra of intact glycopeptides. Given a list of possible glycopeptide sequences as input, a sample-specific spectral library of MassAnalyzer-predicted spectra is built using SpectraST. Glycan identification from CID spectra is achieved by spectral library searching against this library, in which both m/z and intensity information of the possible fragmentation ions are taken into consideration for improved accuracy. We validated our method using a standard glycoprotein, human transferrin, and evaluated its potential to be used in site-specific glycosylation profiling of glycoprotein datasets from LC-MS/MS. In addition, we further applied our method to reveal, for the first time, the site-specific N-glycosylation profile of recombinant human acetylcholinesterase expressed in HEK293 cells. For maximum usability, SpectraST is developed as part of the Trans-Proteomic Pipeline (TPP), a freely available and open-source software suite for MS data analysis. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Proteasomes generate spliced epitopes by two different mechanisms and as efficiently as non-spliced epitopes

    PubMed Central

    Ebstein, F.; Textoris-Taube, K.; Keller, C.; Golnik, R.; Vigneron, N.; Van den Eynde, B. J.; Schuler-Thurner, B.; Schadendorf, D.; Lorenz, F. K. M.; Uckert, W.; Urban, S.; Lehmann, A.; Albrecht-Koepke, N.; Janek, K.; Henklein, P.; Niewienda, A.; Kloetzel, P. M.; Mishto, M.

    2016-01-01

    Proteasome-catalyzed peptide splicing represents an additional catalytic activity of proteasomes contributing to the pool of MHC-class I-presented epitopes. We here biochemically and functionally characterized a new melanoma gp100 derived spliced epitope. We demonstrate that the gp100mel47–52/40–42 antigenic peptide is generated in vitro and in cellulo by a not yet described proteasomal condensation reaction. gp100mel47–52/40–42 generation is enhanced in the presence of the β5i/LMP7 proteasome-subunit and elicits a peptide-specific CD8+ T cell response. Importantly, we demonstrate that different gp100mel-derived spliced epitopes are generated and presented to CD8+ T cells with efficacies comparable to non-spliced canonical tumor epitopes and that gp100mel-derived spliced epitopes trigger activation of CD8+ T cells found in peripheral blood of half of the melanoma patients tested. Our data suggest that both transpeptidation and condensation reactions contribute to the frequent generation of spliced epitopes also in vivo and that their immune relevance may be comparable to non-spliced epitopes. PMID:27049119

  11. N- and O-Glycosylation in the Murine Synaptosome*

    PubMed Central

    Trinidad, Jonathan C.; Schoepfer, Ralf; Burlingame, Alma L.; Medzihradszky, Katalin F.

    2013-01-01

    We present the first large scale study characterizing both N- and O-linked glycosylation in a site-specific manner on hundreds of proteins. We demonstrate that a lectin-affinity fractionation step using wheat germ agglutinin enriches not only peptides carrying intracellular O-GlcNAc, but also those bearing ER/Golgi-derived N- and O-linked carbohydrate structures. Liquid chromatography-MS (LC/MS) analysis with high accuracy precursor mass measurements and high sensitivity ion trap electron-transfer dissociation (ETD) were utilized for structural characterization of glycopeptides. Our results reveal both the identity of the precise sites of glycosylation and information on the oligosaccharide structures possible on these proteins. We report a novel iterative approach that allowed us to interpret the ETD data set directly without making prior assumptions about the nature and distribution of oligosaccharides present in our glycopeptide mixture. Over 2500 unique N- and O-linked glycopeptides were identified on 453 proteins. The extent of microheterogeneity varied extensively, and up to 19 different oligosaccharides were attached at a given site. We describe the presence of the well-known mucin-type structures for O-glycosylation, an EGF-domain-specific fucosylation and a rare O-mannosylation on the transmembrane phosphatase Ptprz1. Finally, we identified three examples of O-glycosylation on tyrosine residues. PMID:23816992

  12. N- and O-glycosylation in the murine synaptosome.

    PubMed

    Trinidad, Jonathan C; Schoepfer, Ralf; Burlingame, Alma L; Medzihradszky, Katalin F

    2013-12-01

    We present the first large scale study characterizing both N- and O-linked glycosylation in a site-specific manner on hundreds of proteins. We demonstrate that a lectin-affinity fractionation step using wheat germ agglutinin enriches not only peptides carrying intracellular O-GlcNAc, but also those bearing ER/Golgi-derived N- and O-linked carbohydrate structures. Liquid chromatography-MS (LC/MS) analysis with high accuracy precursor mass measurements and high sensitivity ion trap electron-transfer dissociation (ETD) were utilized for structural characterization of glycopeptides. Our results reveal both the identity of the precise sites of glycosylation and information on the oligosaccharide structures possible on these proteins. We report a novel iterative approach that allowed us to interpret the ETD data set directly without making prior assumptions about the nature and distribution of oligosaccharides present in our glycopeptide mixture. Over 2500 unique N- and O-linked glycopeptides were identified on 453 proteins. The extent of microheterogeneity varied extensively, and up to 19 different oligosaccharides were attached at a given site. We describe the presence of the well-known mucin-type structures for O-glycosylation, an EGF-domain-specific fucosylation and a rare O-mannosylation on the transmembrane phosphatase Ptprz1. Finally, we identified three examples of O-glycosylation on tyrosine residues.

  13. Antigiardial activity of glycoproteins and glycopeptides from Ziziphus honey.

    PubMed

    Mohammed, Seif Eldin A; Kabashi, Ahmed S; Koko, Waleed S; Azim, M Kamran

    2015-01-01

    Natural honey contains an array of glycoproteins, proteoglycans and glycopeptides. Size-exclusion chromatography fractionated Ziziphus honey proteins into five peaks with molecular masses in the range from 10 to >200 kDa. The fractionated proteins exhibited in vitro activities against Giardia lamblia with IC50 values ≤ 25 μg/mL. Results indicated that honey proteins were more active as antiprotozoal agents than metronidazole. This study indicated the potential of honey proteins and peptides as novel antigiardial agents.

  14. Members of the Genera Paenibacillus and Rhodococcus Harbor Genes Homologous to Enterococcal Glycopeptide Resistance Genes vanA and vanB

    PubMed Central

    Guardabassi, L.; Christensen, H.; Hasman, H.; Dalsgaard, A.

    2004-01-01

    Genes homologous to enterococcal glycopeptide resistance genes vanA and vanB were found in glycopeptide-resistant Paenibacillus and Rhodococcus strains from soil. The putative d-Ala:d-Lac ligase genes in Paenibacillus thiaminolyticus PT-2B1 and Paenibacillus apiarius PA-B2B were closely related to vanA (92 and 87%) and flanked by genes homologous to vanH and vanX in vanA operons. PMID:15561881

  15. Members of the genera Paenibacillus and Rhodococcus harbor genes homologous to enterococcal glycopeptide resistance genes vanA and vanB.

    PubMed

    Guardabassi, L; Christensen, H; Hasman, H; Dalsgaard, A

    2004-12-01

    Genes homologous to enterococcal glycopeptide resistance genes vanA and vanB were found in glycopeptide-resistant Paenibacillus and Rhodococcus strains from soil. The putative D-Ala:D-Lac ligase genes in Paenibacillus thiaminolyticus PT-2B1 and Paenibacillus apiarius PA-B2B were closely related to vanA (92 and 87%) and flanked by genes homologous to vanH and vanX in vanA operons.

  16. Frequent associations between CTL and T-Helper epitopes in HIV-1 genomes and implications for multi-epitope vaccine designs

    PubMed Central

    2010-01-01

    Background Epitope vaccines have been suggested as a strategy to counteract viral escape and development of drug resistance. Multiple studies have shown that Cytotoxic T-Lymphocyte (CTL) and T-Helper (Th) epitopes can generate strong immune responses in Human Immunodeficiency Virus (HIV-1). However, not much is known about the relationship among different types of HIV epitopes, particularly those epitopes that can be considered potential candidates for inclusion in the multi-epitope vaccines. Results In this study we used association rule mining to examine relationship between different types of epitopes (CTL, Th and antibody epitopes) from nine protein-coding HIV-1 genes to identify strong associations as potent multi-epitope vaccine candidates. Our results revealed 137 association rules that were consistently present in the majority of reference and non-reference HIV-1 genomes and included epitopes of two different types (CTL and Th) from three different genes (Gag, Pol and Nef). These rules involved 14 non-overlapping epitope regions that frequently co-occurred despite high mutation and recombination rates, including in genomes of circulating recombinant forms. These epitope regions were also highly conserved at both the amino acid and nucleotide levels indicating strong purifying selection driven by functional and/or structural constraints and hence, the diminished likelihood of successful escape mutations. Conclusions Our results provide a comprehensive systematic survey of CTL, Th and Ab epitopes that are both highly conserved and co-occur together among all subtypes of HIV-1, including circulating recombinant forms. Several co-occurring epitope combinations were identified as potent candidates for inclusion in multi-epitope vaccines, including epitopes that are immuno-responsive to different arms of the host immune machinery and can enable stronger and more efficient immune responses, similar to responses achieved with adjuvant therapies. Signature of strong

  17. Analysis of Glycoproteins in Human Serum by Means of Glycospecific Magnetic Bead Separation and LC-MALDI-TOF/TOF Analysis with Automated Glycopeptide Detection

    PubMed Central

    Sparbier, Katrin; Asperger, Arndt; Resemann, Anja; Kessler, Irina; Koch, Sonja; Wenzel, Thomas; Stein, Günter; Vorwerg, Lars; Suckau, Detlev; Kostrzewa, Markus

    2007-01-01

    Comprehensive proteomic analyses require efficient and selective pre-fractionation to facilitate analysis of post-translationally modified peptides and proteins, and automated analysis workflows enabling the detection, identification, and structural characterization of the corresponding peptide modifications. Human serum contains a high number of glycoproteins, comprising several orders of magnitude in concentration. Thereby, isolation and subsequent identification of low-abundant glycoproteins from serum is a challenging task. selective capturing of glycopeptides and -proteins was attained by means of magnetic particles specifically functionalized with lectins or boronic acids that bind to various structural motifs. Human serum was incubated with differentially functionalized magnetic micro-particles (lectins or boronic acids), and isolated proteins were digested with trypsin. Subsequently, the resulting complex mixture of peptides and glycopeptides was subjected to LC-MALDI analysis and database searching. In parallel, a second magnetic bead capturing was performed on the peptide level to separate and analyze by LC-MALDI intact glycopeptides, both peptide sequence and glycan structure. Detection of glycopeptides was achieved by means of a software algorithm that allows extraction and characterization of potential glycopeptide candidates from large LC-MALDI-MS/MS data sets, based on N-glycopeptide-specific fragmentation patterns and characteristic fragment mass peaks, respectively. By means of fast and simple glycospecific capturing applied in conjunction with extensive LC-MALDI-MS/MS analysis and novel data analysis tools, a high number of low-abundant proteins were identified, comprising known or predicted glycosylation sites. According to the specific binding preferences of the different types of beads, complementary results were obtained from the experiments using either magnetic ConA-, LCA-, WGA-, and boronic acid beads, respectively. PMID:17916798

  18. Opioid glycopeptide analgesics derived from endogenous enkephalins and endorphins

    PubMed Central

    Li, Yingxue; Lefever, Mark R; Muthu, Dhanasekaran; Bidlack, Jean M; Bilsky, Edward J; Polt, Robin

    2012-01-01

    Over the past two decades, potent and selective analgesics have been developed from endogenous opioid peptides. Glycosylation provides an important means of modulating interaction with biological membranes, which greatly affects the pharmacodynamics and pharmacokinetics of the resulting glycopeptide analogues. Furthermore, manipulation of the membrane affinity allows penetration of cellular barriers that block efficient drug distribution, including the blood–brain barrier. Extremely potent and selective opiate agonists have been developed from endogenous peptides, some of which show great promise as drug candidates. PMID:22300099

  19. Design and Characterization of Epitope-Scaffold Immunogens That Present the Motavizumab Epitope from Respiratory Syncytial Virus

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    McLellan, Jason S.; Correia, Bruno E.; Chen, Man

    2012-06-28

    Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, but an effective vaccine has not yet been developed. An ideal vaccine would elicit protective antibodies while avoiding virus-specific T-cell responses, which have been implicated in vaccine-enhanced disease with previous RSV vaccines. We propose that heterologous proteins designed to present RSV-neutralizing antibody epitopes and to elicit cognate antibodies have the potential to fulfill these vaccine requirements, as they can be fashioned to be free of viral T-cell epitopes. Here we present the design and characterization of three epitope-scaffolds that present the epitope of motavizumab, a potentmore » neutralizing antibody that binds to a helix-loop-helix motif in the RSV fusion glycoprotein. Two of the epitope-scaffolds could be purified, and one epitope-scaffold based on a Staphylococcus aureus protein A domain bound motavizumab with kinetic and thermodynamic properties consistent with the free epitope-scaffold being stabilized in a conformation that closely resembled the motavizumab-bound state. This epitope-scaffold was well folded as assessed by circular dichroism and isothermal titration calorimetry, and its crystal structure (determined in complex with motavizumab to 1.9 {angstrom} resolution) was similar to the computationally designed model, with all hydrogen-bond interactions critical for binding to motavizumab preserved. Immunization of mice with this epitope-scaffold failed to elicit neutralizing antibodies but did elicit sera with F binding activity. The elicitation of F binding antibodies suggests that some of the design criteria for eliciting protective antibodies without virus-specific T-cell responses are being met, but additional optimization of these novel immunogens is required.« less

  20. Design and characterization of epitope-scaffold immunogens that present the motavizumab epitope from respiratory syncytial virus.

    PubMed

    McLellan, Jason S; Correia, Bruno E; Chen, Man; Yang, Yongping; Graham, Barney S; Schief, William R; Kwong, Peter D

    2011-06-24

    Respiratory syncytial virus (RSV) is a major cause of respiratory tract infections in infants, but an effective vaccine has not yet been developed. An ideal vaccine would elicit protective antibodies while avoiding virus-specific T-cell responses, which have been implicated in vaccine-enhanced disease with previous RSV vaccines. We propose that heterologous proteins designed to present RSV-neutralizing antibody epitopes and to elicit cognate antibodies have the potential to fulfill these vaccine requirements, as they can be fashioned to be free of viral T-cell epitopes. Here we present the design and characterization of three epitope-scaffolds that present the epitope of motavizumab, a potent neutralizing antibody that binds to a helix-loop-helix motif in the RSV fusion glycoprotein. Two of the epitope-scaffolds could be purified, and one epitope-scaffold based on a Staphylococcus aureus protein A domain bound motavizumab with kinetic and thermodynamic properties consistent with the free epitope-scaffold being stabilized in a conformation that closely resembled the motavizumab-bound state. This epitope-scaffold was well folded as assessed by circular dichroism and isothermal titration calorimetry, and its crystal structure (determined in complex with motavizumab to 1.9 Å resolution) was similar to the computationally designed model, with all hydrogen-bond interactions critical for binding to motavizumab preserved. Immunization of mice with this epitope-scaffold failed to elicit neutralizing antibodies but did elicit sera with F binding activity. The elicitation of F binding antibodies suggests that some of the design criteria for eliciting protective antibodies without virus-specific T-cell responses are being met, but additional optimization of these novel immunogens is required. Published by Elsevier Ltd.

  1. An engineered high affinity Fbs1 carbohydrate binding protein for selective capture of N-glycans and N-glycopeptides

    PubMed Central

    Chen, Minyong; Shi, Xiaofeng; Duke, Rebecca M.; Ruse, Cristian I.; Dai, Nan; Taron, Christopher H.; Samuelson, James C.

    2017-01-01

    A method for selective and comprehensive enrichment of N-linked glycopeptides was developed to facilitate detection of micro-heterogeneity of N-glycosylation. The method takes advantage of the inherent properties of Fbs1, which functions within the ubiquitin-mediated degradation system to recognize the common core pentasaccharide motif (Man3GlcNAc2) of N-linked glycoproteins. We show that Fbs1 is able to bind diverse types of N-linked glycomolecules; however, wild-type Fbs1 preferentially binds high-mannose-containing glycans. We identified Fbs1 variants through mutagenesis and plasmid display selection, which possess higher affinity and improved recovery of complex N-glycomolecules. In particular, we demonstrate that the Fbs1 GYR variant may be employed for substantially unbiased enrichment of N-linked glycopeptides from human serum. Most importantly, this highly efficient N-glycopeptide enrichment method enables the simultaneous determination of N-glycan composition and N-glycosites with a deeper coverage (compared to lectin enrichment) and improves large-scale N-glycoproteomics studies due to greatly reduced sample complexity. PMID:28534482

  2. Hexapeptide Derivatives of Glycopeptide Antibiotics: Tools for Mechanism of Action Studies

    PubMed Central

    Allen, Norris E.; LeTourneau, Deborah L.; Hobbs, Joe N.; Thompson, Richard C.

    2002-01-01

    Hexapeptide (des-N-methylleucyl) derivatives of LY264826 were prepared in order to examine further the role of N-substituted hydrophobic side chains in defining the mechanisms of action of semisynthetic glycopeptide antibiotics. The hexapeptide of LY264826 binds to the cell wall intermediate analog l-Lys-d-Ala-d-Ala with a 100-fold lower affinity than LY264826 and inhibits Micrococcus luteus almost 200-fold more poorly than LY264826 does. Alkylation of the 4-epi-vancosamine moiety of the disaccharide significantly enhanced the antibacterial activity of the hexapeptide. Alkylation did not affect the binding affinity for d-alanyl-d-alanine residues; however, it did enhance dimerization 7,000-fold and enhanced binding to bacterial membrane vesicles noticeably compared with the levels of dimerization and binding for the unsubstituted hexapeptide. The findings from this study complement those presented in an earlier report (N. E. Allen, D. L. LeTourneau, and J. N. Hobbs, Jr., J. Antibiot. 50:677-684, 1997) and are consistent with the conclusion that the enhanced antibacterial activities of semisynthetic glycopeptide antibiotics derive from the ability of the hydrophobic side chain to markedly affect both dimerization and binding to bacterial membranes. PMID:12121903

  3. A fluorescent glycosyl-imprinted polymer for pH and temperature regulated sensing of target glycopeptide antibiotic.

    PubMed

    Chen, Kuncai; He, Rong; Luo, Xiaoyan; Qin, Pengzhe; Tan, Lei; Tang, Youwen; Yang, Zhicong

    2017-08-15

    This paper demonstrates a new strategy for developing a fluorescent glycosyl-imprinted polymer for pH and temperature regulated sensing of target glycopeptide antibiotic. The technique provides amino modified Mn-doped ZnS QDs as fluorescent supports, 4-vinylphenylbronic acid as a covalent monomer, N-isopropyl acrylamide as a thermo-responsive monomer in combination with acrylamide as a non-covalent monomer, and glycosyl moiety of a glycopeptide antibiotic as a template to produce fluorescent molecularly imprinted polymer (FMIP) in aqueous solution. The FMIP can alter its functional moieties and structure with pH and temperature stimulation. This allows recognition of target molecules through control of pH and temperature. The fluorescence intensity of the FMIP was enhanced gradually as the concentration of telavancin increased, and showed selective recognition toward the target glycopeptide antibiotic preferentially among other antibiotics. Using the FMIP as a sensing material, good linear correlations were obtained over the concentration range of 3.0-300.0μg/L and with a low limit of detection of 1.0μg/L. The analysis results of telavancin in real samples were consistent with that obtained by liquid chromatography tandem mass spectrometry. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Site-specific O-Glycosylation Analysis of Human Blood Plasma Proteins*

    PubMed Central

    Hoffmann, Marcus; Marx, Kristina; Reichl, Udo; Wuhrer, Manfred; Rapp, Erdmann

    2016-01-01

    Site-specific glycosylation analysis is key to investigate structure-function relationships of glycoproteins, e.g. in the context of antigenicity and disease progression. The analysis, though, is quite challenging and time consuming, in particular for O-glycosylated proteins. In consequence, despite their clinical and biopharmaceutical importance, many human blood plasma glycoproteins have not been characterized comprehensively with respect to their O-glycosylation. Here, we report on the site-specific O-glycosylation analysis of human blood plasma glycoproteins. To this end pooled human blood plasma of healthy donors was proteolytically digested using a broad-specific enzyme (Proteinase K), followed by a precipitation step, as well as a glycopeptide enrichment and fractionation step via hydrophilic interaction liquid chromatography, the latter being optimized for intact O-glycopeptides carrying short mucin-type core-1 and -2 O-glycans, which represent the vast majority of O-glycans on human blood plasma proteins. Enriched O-glycopeptide fractions were subjected to mass spectrometric analysis using reversed-phase liquid chromatography coupled online to an ion trap mass spectrometer operated in positive-ion mode. Peptide identity and glycan composition were derived from low-energy collision-induced dissociation fragment spectra acquired in multistage mode. To pinpoint the O-glycosylation sites glycopeptides were fragmented using electron transfer dissociation. Spectra were annotated by database searches as well as manually. Overall, 31 O-glycosylation sites and regions belonging to 22 proteins were identified, the majority being acute-phase proteins. Strikingly, also 11 novel O-glycosylation sites and regions were identified. In total 23 O-glycosylation sites could be pinpointed. Interestingly, the use of Proteinase K proved to be particularly beneficial in this context. The identified O-glycan compositions most probably correspond to mono- and disialylated core-1

  5. HLA Epitopes: The Targets of Monoclonal and Alloantibodies Defined

    PubMed Central

    Nguyen, Anh

    2017-01-01

    Sensitization to human leukocyte antigens (HLA) in organ transplant patients causes graft rejection, according to the humoral theory of transplantation. Sensitization is almost ubiquitous as anti-HLA antibodies are found in almost all sera of transplant recipients. Advances in testing assays and amino acid sequencing of HLA along with computer software contributed further to the understanding of antibody-antigen reactivity. It is commonly understood that antibodies bind to HLA antigens. With current knowledge of epitopes, it is more accurate to describe that antibodies bind to their target epitopes on the surface of HLA molecular chains. Epitopes are present on a single HLA (private epitope) or shared by multiple antigens (public epitope). The phenomenon of cross-reactivity in HLA testing, often explained as cross-reactive groups (CREGs) of antigens with antibody, can be clearly explained now by public epitopes. Since 2006, we defined and reported 194 HLA class I unique epitopes, including 56 cryptic epitopes on dissociated HLA class I heavy chains, 83 HLA class II epitopes, 60 epitopes on HLA-DRB1, 15 epitopes on HLA-DQB1, 3 epitopes on HLA-DQA1, 5 epitopes on HLA-DPB1, and 7 MICA epitopes. In this paper, we provide a summary of our findings. PMID:28626773

  6. Heterogeneous resistance to vancomycin in Staphylococcus epidermidis, Staphylococcus haemolyticus and Staphylococcus warneri clinical strains: characterisation of glycopeptide susceptibility profiles and cell wall thickening.

    PubMed

    Nunes, Ana Paula Ferreira; Teixeira, Lúcia Martins; Iorio, Natália Lopes Pontes; Bastos, Carla Callegário Reis; de Sousa Fonseca, Leila; Souto-Padrón, Thaís; dos Santos, Kátia Regina Netto

    2006-04-01

    The population analysis profile (PAP) method as well as analysis of autolytic activity and cellular ultrastructure by transmission electron microscopy (TEM) were used to characterise Staphylococcus epidermidis, Staphylococcus haemolyticus and Staphylococcus warneri clinical strains with reduced susceptibility to glycopeptides. All strains showed heterogeneous profiles to vancomycin and teicoplanin by the PAP method. Subpopulations that grew in the presence of high concentrations of each drug were selected from the PAP as derivative strains. Their glycopeptide minimal inhibitory concentrations (MICs) were determined and subsequently all parental and derivative strains were grown in one-half of the MIC of vancomycin or teicoplanin. An increase in cell wall thickness of all derivative strains was seen by TEM, with statistically significant values (P<0.01) compared with their respective parental strains. In general, variable rates of autolysis among the strains were observed. Cell wall thickness is an important factor involved in glycopeptide resistance and, in association with PAP results, confirmed the Brazilian coagulase-negative staphylococci clinical isolates as being heteroresistant to glycopeptides. Detection of these heteroresistant organisms is important in order to achieve more judicious use of vancomycin and teicoplanin in hospitals.

  7. Increasing the productivity of glycopeptides analysis by using higher-energy collision dissociation-accurate mass-product-dependent electron transfer dissociation.

    PubMed

    Saba, Julian; Dutta, Sucharita; Hemenway, Eric; Viner, Rosa

    2012-01-01

    Currently, glycans are attracting attention from the scientific community as potential biomarkers or as posttranslational modifications (PTMs) of therapeutic proteins. However, structural characterization of glycoproteins and glycopeptides remains analytically challenging. Here, we report on the implementation of a novel acquisition strategy termed higher-energy collision dissociation-accurate mass-product-dependent electron transfer dissociation (HCD-PD-ETD) on a hybrid linear ion trap-orbitrap mass spectrometer. This acquisition strategy uses the complementary fragmentations of ETD and HCD for glycopeptides analysis in an intelligent fashion. Furthermore, the approach minimizes user input for optimizing instrumental parameters and enables straightforward detection of glycopeptides. ETD spectra are only acquired when glycan oxonium ions from MS/MS HCD are detected. The advantage of this approach is that it streamlines data analysis and improves dynamic range and duty cycle. Here, we present the benefits of HCD-PD-ETD relative to the traditional alternating HCD/ETD for a trainer set containing twelve-protein mixture with two glycoproteins: human serotransferrin, ovalbumin and contaminations of two other: bovine alpha 1 acid glycoprotein (bAGP) and bovine fetuin.

  8. Synthesis of hydrazide-functionalized hydrophilic polymer hybrid graphene oxide for highly efficient N-glycopeptide enrichment and identification by mass spectrometry.

    PubMed

    Bai, Haihong; Pan, Yiting; Guo, Cong; Zhao, Xinyuan; Shen, Bingquan; Wang, Xinghe; Liu, Zeyuan; Cheng, Yuanguo; Qin, Weijie; Qian, Xiaohong

    2017-08-15

    Protein N-glycosylation is one of the most important post-translational modifications, participating in many key biological and pathological processes. Large-scale and precise identification of N-glycosylated proteins and peptides is especially beneficial for understanding their biological functions and for discovery of new clinical biomarkers and therapeutic drug targets. However, protein N-glycosylation is microheterogeneous and low abundant in living organisms, therefore specific enrichment of N-glycosylated proteins/peptides before mass spectrometry analysis is a prerequisite. In this work, we developed a new type of polymer hybrid graphene oxide (GO) by in situ growth of hydrazide-functionalized hydrophilic polymer chains on the GO surface (GO-PAAH) for selective N-glycopeptide enrichment and identification by mass spectrometry. The densely attached and low steric hindrance hydrazide groups as well as the highly hydrophilic nature of GO-PAAH facilitate N-glycopeptide enrichment by the combination of hydrazide capturing and HILIC interaction. Taking advantage of the unique features of GO-PAAH, all of the three N-glycopeptides of bovine fetuin were successfully enriched and identified with significantly enhanced signal intensities from a digest mixture of bovine fetuin and bovine serum albumin at a mass ratio of 1:100, demonstrating the excellent enrichment selectivity of GO-PAAH. Furthermore, a total of 507 N-glycosylation sites and 480 N-glycopeptides in 232 N-glycoproteins were enriched and identified from 10μL of human serum by three replicates using this novel enrichment material, which is nearly two times higher than the commercial hydrazide resin based method (280 N-glycosylation sites, 261 N-glycopeptides and 144 N-glycoproteins in three experiments). Among the identified, 95 N-glycosylation sites were not reported in the Uniprot database, and 106 N-glycoproteins were disease related in the Nextprot database, indicating the potential of this new

  9. Construction and characterization of 3A-epitope-tagged foot-and-mouth disease virus.

    PubMed

    Ma, Xueqing; Li, Pinghua; Sun, Pu; Bai, Xingwen; Bao, Huifang; Lu, Zengjun; Fu, Yuanfang; Cao, Yimei; Li, Dong; Chen, Yingli; Qiao, Zilin; Liu, Zaixin

    2015-04-01

    Nonstructural protein 3A of foot-and-mouth disease virus (FMDV) is a partially conserved protein of 153 amino acids (aa) in most FMDVs examined to date. Specific deletion in the FMDV 3A protein has been associated with the inability of FMDV to grow in primary bovine cells and cause disease in cattle. However, the aa residues playing key roles in these processes are poorly understood. In this study, we constructed epitope-tagged FMDVs containing an 8 aa FLAG epitope, a 9 aa haemagglutinin (HA) epitope, and a 10 aa c-Myc epitope to substitute residues 94-101, 93-101, and 93-102 of 3A protein, respectively, using a recently developed O/SEA/Mya-98 FMDV infectious cDNA clone. Immunofluorescence assay (IFA), Western blot and sequence analysis showed that the epitope-tagged viruses stably maintained and expressed the foreign epitopes even after 10 serial passages in BHK-21 cells. The epitope-tagged viruses displayed growth properties and plaque phenotypes similar to those of the parental virus in BHK-21 cells. However, the epitope-tagged viruses exhibited lower growth rates and smaller plaque size phenotypes than those of the parental virus in primary fetal bovine kidney (FBK) cells, but similar growth properties and plaque phenotypes to those of the recombinant viruses harboring 93-102 deletion in 3A. These results demonstrate that the decreased ability of FMDV to replicate in primary bovine cells was not associated with the length of 3A, and the genetic determinant thought to play key role in decreased ability to replicate in primary bovine cells could be reduced from 93-102 residues to 8 aa residues at positions 94-101 in 3A protein. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Ultra-high performance liquid chromatography tandem mass spectrometry for the determination of five glycopeptide antibiotics in food and biological samples using solid-phase extraction.

    PubMed

    Deng, Fenfang; Yu, Hong; Pan, Xinhong; Hu, Guoyuan; Wang, Qiqin; Peng, Rongfei; Tan, Lei; Yang, Zhicong

    2018-02-23

    This paper demonstrated the development and validation of an ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) method for simultaneous determination of five glycopeptide antibiotics in food and biological samples. The target glycopeptide antibiotics were isolated from the samples by solvent extraction, and the extracts were cleaned with a tandem solid-phase extraction step using mixed strong cation exchange and hydrophilic/lipophilic balance cartridges. Subsequently, the analytes were eluted with different solvents, and then quantified by UHPLC-MS/MS in the positive ionization mode with multiple reaction monitoring. Under optimal conditions, good linear correlations were obtained for the five glycopeptide antibiotics in the concentration range of 1.0 μg/L to 20.0 μg/L, and with linear correlation coefficients >0.998. Employing this method, the target glycopeptide antibiotics in food and biological samples were identified with a recovery of 83.0-102%, and a low quantitation limit of 1.0 μg/kg in food and 2.0 μg/L in biological samples with low matrix effects. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. 3-Aminoquinoline/p-coumaric acid as a MALDI matrix for glycopeptides, carbohydrates, and phosphopeptides.

    PubMed

    Fukuyama, Yuko; Funakoshi, Natsumi; Takeyama, Kohei; Hioki, Yusaku; Nishikaze, Takashi; Kaneshiro, Kaoru; Kawabata, Shin-Ichirou; Iwamoto, Shinichi; Tanaka, Koichi

    2014-02-18

    Glycosylation and phosphorylation are important post-translational modifications in biological processes and biomarker research. The difficulty in analyzing these modifications is mainly their low abundance and dissociation of labile regions such as sialic acids or phosphate groups. One solution in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is to improve matrices for glycopeptides, carbohydrates, and phosphopeptides by increasing the sensitivity and suppressing dissociation of the labile regions. Recently, a liquid matrix 3-aminoquinoline (3-AQ)/α-cyano-4-hydroxycinnamic acid (CHCA) (3-AQ/CHCA), introduced by Kolli et al. in 1996, has been reported to increase sensitivity for carbohydrates or phosphopeptides, but it has not been systematically evaluated for glycopeptides. In addition, 3-AQ/CHCA enhances the dissociation of labile regions. In contrast, a liquid matrix 1,1,3,3-tetramethylguanidium (TMG, G) salt of p-coumaric acid (CA) (G3CA) was reported to suppress dissociation of sulfate groups or sialic acids of carbohydrates. Here we introduce a liquid matrix 3-AQ/CA for glycopeptides, carbohydrates, and phosphopeptides. All of the analytes were detected as [M + H](+) or [M - H](-) with higher or comparable sensitivity using 3-AQ/CA compared with 3-AQ/CHCA or 2,5-dihydroxybenzoic acid (2,5-DHB). The sensitivity was increased 1- to 1000-fold using 3-AQ/CA. The dissociation of labile regions such as sialic acids or phosphate groups and the fragmentation of neutral carbohydrates were suppressed more using 3-AQ/CA than using 3-AQ/CHCA or 2,5-DHB. 3-AQ/CA was thus determined to be an effective MALDI matrix for high sensitivity and the suppression of dissociation of labile regions in glycosylation and phosphorylation analyses.

  12. Linkage-specific sialic acid derivatization for MALDI-TOF-MS profiling of IgG glycopeptides.

    PubMed

    de Haan, Noortje; Reiding, Karli R; Haberger, Markus; Reusch, Dietmar; Falck, David; Wuhrer, Manfred

    2015-08-18

    Glycosylation is a common co- and post-translational protein modification, having a large influence on protein properties like conformation and solubility. Furthermore, glycosylation is an important determinant of efficacy and clearance of biopharmaceuticals such as immunoglobulin G (IgG). Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) shows potential for the site-specific glycosylation analysis of IgG at the glycopeptide level. With this approach, however, important information about glycopeptide sialylation is not duly covered because of in-source and metastable decay of the sialylated species. Here, we present a highly repeatable sialic acid derivatization method to allow subclass-specific MALDI-TOF-MS analysis of tryptic IgG glycopeptides. The method, employing dimethylamidation with the carboxylic acid activator 1-ethyl-3-(3-dimethylamino)propyl)carbodiimide (EDC) and the catalyst 1-hydroxybenzotriazole (HOBt), results in different masses for the functionally divergent α2,3- and α2,6-linked sialic acids. Respective lactonization and dimethylamidation leads to their direct discrimination in MS and importantly, both glycan and peptide moieties reacted in a controlled manner. In addition, stabilization allowed the acquisition of fragmentation spectra informative with respect to glycosylation and peptide sequence. This was in contrast to fragmentation spectra of underivatized samples, which were dominated by sialic acid loss. The method allowed the facile discrimination and relative quantitation of IgG Fc sialylation in therapeutic IgG samples. The method has considerable potential for future site- and sialic acid linkage-specific glycosylation profiling of therapeutic antibodies, as well as for subclass-specific biomarker discovery in clinical IgG samples derived from plasma.

  13. Automatic Generation of Validated Specific Epitope Sets.

    PubMed

    Carrasco Pro, Sebastian; Sidney, John; Paul, Sinu; Lindestam Arlehamn, Cecilia; Weiskopf, Daniela; Peters, Bjoern; Sette, Alessandro

    2015-01-01

    Accurate measurement of B and T cell responses is a valuable tool to study autoimmunity, allergies, immunity to pathogens, and host-pathogen interactions and assist in the design and evaluation of T cell vaccines and immunotherapies. In this context, it is desirable to elucidate a method to select validated reference sets of epitopes to allow detection of T and B cells. However, the ever-growing information contained in the Immune Epitope Database (IEDB) and the differences in quality and subjects studied between epitope assays make this task complicated. In this study, we develop a novel method to automatically select reference epitope sets according to a categorization system employed by the IEDB. From the sets generated, three epitope sets (EBV, mycobacteria and dengue) were experimentally validated by detection of T cell reactivity ex vivo from human donors. Furthermore, a web application that will potentially be implemented in the IEDB was created to allow users the capacity to generate customized epitope sets.

  14. Enhancing antibody patent protection using epitope mapping information

    PubMed Central

    Deng, Xiaoxiang; Storz, Ulrich; Doranz, Benjamin J.

    2018-01-01

    ABSTRACT As the $100B therapeutic monoclonal antibody (mAb) market continues to grow, developers of therapeutic mAbs increasingly face the need to strengthen patent protection of their products and enforce their patents in courts. In view of changes in the patent law landscape, patent applications are strategically using information on the precise binding sites of their mAbs, i.e., the epitopes, to support patent novelty, non-obviousness, subject matter, and a tightened written description requirement for broad genus antibody claims. Epitope data can also allow freedom-to-operate for second-generation mAbs by differentiation from patented first-generation mAbs. Numerous high profile court cases, including Amgen v. Sanofi over rival mAbs that block PCSK9 activity, have been centered on epitope mapping claims, highlighting the importance of epitopes in determining broad mAb patent rights. Based on these cases, epitope mapping claims must describe a sufficiently large number of mAbs that share an epitope, and each epitope must be described at amino acid resolution. Here, we review current best practices for the use of epitope information to overcome the increasing challenges of patenting mAbs, and how the quality, conformation, and resolution of epitope residue data can influence the breadth and strength of mAb patents. PMID:29120697

  15. Epitope topography controls bioactivity in supramolecular nanofibers

    PubMed Central

    Sur, Shantanu; Tantakitti, Faifan; Matson, John B.; Stupp, Samuel I.

    2015-01-01

    Incorporating bioactivity into artificial scaffolds using peptide epitopes present in the extracellular matrix (ECM) is a well-known approach. A common strategy has involved epitopes that provide cells with attachment points and external cues through interaction with integrin receptors. Although a variety of bioactive sequences have been identified so far, less is known about their optimal display in a scaffold. We report here on the use of self-assembled peptide amphiphile (PA) nanofiber matrices to investigate the impact of spatial presentation of the fibronectin derived epitope RGDS on cell response. Using one, three, or five glycine residues, RGDS epitopes were systematically spaced out from the surface of the rigid nanofibers. We found that cell morphology was strongly affected by the separation of the epitope from the nanofiber surface, with the longest distance yielding the most cell-spreading, bundling of actin filaments, and a round-to-polygonal transformation of cell shape. Cell response to this type of epitope display was also accompanied with activated integrin-mediated signaling and formation of stronger adhesions between cells and substrate. Interestingly, unlike length, changing the molecular flexibility of the linker had minimal influence on cell behavior on the substrate for reasons that remain poorly understood. The use in this study of high persistence length nanofibers rather than common flexible polymers allows us to conclude that epitope topography at the nanoscale structure of a scaffold influences its bioactive properties independent of epitope density and mechanical properties. PMID:25745558

  16. Ecdysteroid-stimulated synthesis and secretion of an N-acetyl-D-glucosamine-rich glycopeptide in a lepidopteran cell line derived from imaginal discs.

    PubMed

    Porcheron, P; Morinière, M; Coudouel, N; Oberlander, H

    1991-01-01

    Hormone-regulated processing of N-acetyl-D-glucosamine was studied in an insect cell line derived from imaginal wing discs of the Indian meal moth, Plodia interpunctella (Hübner). The cell line, IAL-PID2, responded to treatment with 20-hydroxyecdysone with increased incorporation of GlcNAc into glycoproteins. Cycloheximide and tunicamycin counteracted the action of the hormone. In particular, treatment with 20-hydroxyecdysone resulted in the secretion of a 5,000 dalton N-acetyl-D-glucosamine-rich glycopeptide by the IAL-PID2 cells. Accumulation of this peptide was prevented by the use of teflubenzuron, a potent chitin synthesis inhibitor. A glycopeptide of similar molecular weight was observed in imaginal discs of P. interpunctella treated with 20-hydroxyecdysone in vitro, under conditions that induce chitin synthesis. Although the function of the 5,000 dalton glycopeptide is not known, we believe that the PID2 cell line is a promising model for molecular analysis of ecdysteroid-regulated processing of aminosugars by epidermal cells during insect development.

  17. Dissecting linear and conformational epitopes on the native thyrotropin receptor.

    PubMed

    Ando, Takao; Latif, Rauf; Daniel, Samira; Eguchi, Katsumi; Davies, Terry F

    2004-11-01

    The TSH receptor (TSHR) is the primary antigen in Graves' disease. In this condition, autoantibodies to the TSHR that have intrinsic thyroid-stimulating activity develop. We studied the epitopes on the native TSHR using polyclonal antisera and monoclonal antibodies (mAbs) derived from an Armenian hamster model of Graves' disease. Of 14 hamster mAbs analyzed, five were shown to bind to conformational epitopes including one mAb with potent thyroid-stimulating activity. Overlapping conformational epitopes were determined by cell-binding competition assays using fluorescently labeled mAbs. We identified two distinct conformational epitopes: epitope A for both stimulating and blocking mAbs and epitope B for only blocking mAbs. Examination of an additional three mouse-derived stimulating TSHR-mAbs also showed exclusive binding to epitope A. The remaining nine hamster-derived mAbs were neutral or low-affinity blocking antibodies that recognized linear epitopes within the TSHR cleaved region (residues 316-366) (epitope C). Serum from the immunized hamsters also recognized conformational epitopes A and B but, in addition, also contained high levels of TSHR-Abs interacting within the linear epitope C region. In summary, these studies indicated that the natively conformed TSHR had a restricted set of epitopes recognized by TSHR-mAbs and that the binding site for stimulating TSHR-Abs was highly conserved. However, high-affinity TSHR-blocking antibodies recognized two conformational epitopes, one of which was indistinguishable from the thyroid-stimulating epitope. Hence, TSHR-stimulating and blocking antibodies cannot be distinguished purely on the basis of their conformational epitope recognition.

  18. Localization of functional receptor epitopes on the structure of ciliary neurotrophic factor indicates a conserved, function-related epitope topography among helical cytokines.

    PubMed

    Panayotatos, N; Radziejewska, E; Acheson, A; Somogyi, R; Thadani, A; Hendrickson, W A; McDonald, N Q

    1995-06-09

    By rational mutagenesis, receptor-specific functional analysis, and visualization of complex formation in solution, we identified individual amino acid side chains involved specifically in the interaction of ciliary neurotrophic factor (CNTF) with CNTFR alpha and not with the beta-components, gp130 and LIFR. In the crystal structure, the side chains of these residues, which are located in helix A, the AB loop, helix B, and helix D, are surface accessible and are clustered in space, thus constituting an epitope for CNTFR alpha. By the same analysis, a partial epitope for gp130 was also identified on the surface of helix A that faces away from the alpha-epitope. Superposition of the CNTF and growth hormone structures showed that the location of these epitopes on CNTF is analogous to the location of the first and second receptor epitopes on the surface of growth hormone. Further comparison with proposed binding sites for alpha- and beta-receptors on interleukin-6 and leukemia inhibitory factor indicated that this epitope topology is conserved among helical cytokines. In each case, epitope I is utilized by the specificity-conferring component, whereas epitopes II and III are used by accessory components. Thus, in addition to a common fold, helical cytokines share a conserved order of receptor epitopes that is function related.

  19. ‘Multi-Epitope-Targeted’ Immune-Specific Therapy for a Multiple Sclerosis-Like Disease via Engineered Multi-Epitope Protein Is Superior to Peptides

    PubMed Central

    Zilkha-Falb, Rina; Yosef-Hemo, Reut; Cohen, Lydia; Ben-Nun, Avraham

    2011-01-01

    Antigen-induced peripheral tolerance is potentially one of the most efficient and specific therapeutic approaches for autoimmune diseases. Although highly effective in animal models, antigen-based strategies have not yet been translated into practicable human therapy, and several clinical trials using a single antigen or peptidic-epitope in multiple sclerosis (MS) yielded disappointing results. In these clinical trials, however, the apparent complexity and dynamics of the pathogenic autoimmunity associated with MS, which result from the multiplicity of potential target antigens and “epitope spread”, have not been sufficiently considered. Thus, targeting pathogenic T-cells reactive against a single antigen/epitope is unlikely to be sufficient; to be effective, immunospecific therapy to MS should logically neutralize concomitantly T-cells reactive against as many major target antigens/epitopes as possible. We investigated such “multi-epitope-targeting” approach in murine experimental autoimmune encephalomyelitis (EAE) associated with a single (“classical”) or multiple (“complex”) anti-myelin autoreactivities, using cocktail of different encephalitogenic peptides vis-a-vis artificial multi-epitope-protein (designated Y-MSPc) encompassing rationally selected MS-relevant epitopes of five major myelin antigens, as “multi-epitope-targeting” agents. Y-MSPc was superior to peptide(s) in concomitantly downregulating pathogenic T-cells reactive against multiple myelin antigens/epitopes, via inducing more effective, longer lasting peripheral regulatory mechanisms (cytokine shift, anergy, and Foxp3+ CTLA4+ regulatory T-cells). Y-MSPc was also consistently more effective than the disease-inducing single peptide or peptide cocktail, not only in suppressing the development of “classical” or “complex EAE” or ameliorating ongoing disease, but most importantly, in reversing chronic EAE. Overall, our data emphasize that a “multi-epitope

  20. Vicilin allergens of peanut and tree nuts (walnut, hazelnut and cashew nut) share structurally related IgE-binding epitopes.

    PubMed

    Barre, Annick; Sordet, Camille; Culerrier, Raphaël; Rancé, Fabienne; Didier, Alain; Rougé, Pierre

    2008-03-01

    Surface-exposed IgE-binding epitopes of close overall conformation were characterized on the molecular surface of three-dimensional models built for the vicilin allergens of peanut (Ara h 1), walnut (Jug r 2), hazelnut (Cor a 11) and cashew nut (Ana o 1). They correspond to linear stretches of conserved amino acid sequences mainly located along the C-terminus of the polypeptide chains. A glyco-epitope corresponding to an exposed N-glycosylation site could also interfere with the IgE-binding epitopes. All these epitopic regions should participate in the IgE-binding cross-reactivity commonly reported between tree nuts or between peanut and some tree nuts in sensitized individuals. Owing to this epitopic community which constitutes a risk of cross-sensitization, the avoidance or a restricted consumption of other tree nuts should be recommended to peanut-sensitized individuals.

  1. Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

    DOE PAGES

    Ng, Simon; Lin, Edith; Kitov, Pavel I.; ...

    2015-04-10

    Here we describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10 8 glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl α-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3more » out of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.« less

  2. Genetically Encoded Fragment-Based Discovery of Glycopeptide Ligands for Carbohydrate-Binding Proteins

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ng, Simon; Lin, Edith; Kitov, Pavel I.

    Here we describe an approach to accelerate the search for competitive inhibitors for carbohydrate-recognition domains (CRDs). Genetically encoded fragment-based-discovery (GE-FBD) uses selection of phagedisplayed glycopeptides to dock a glycan fragment at the CRD and guide selection of Synergistic peptide motifs adjacent to the CRD. Starting from concanavalin A (ConA), a mannose (Man)-binding protein, as a bait, we narrowed a library of 10 8 glycopeptides to 86 leads that share a consensus motif, Man-WYD. Validation of synthetic leads yielded Man-WYDLF that exhibited 40 50-fold enhancement in affinity over methyl α-D-mannopyranoside (MeMan). Lectin array Suggested specificity: Man-WYD derivative bound only to 3more » out of 17 proteins-ConA, LcH, and PSA-that bind to Man. An X-ray structure of ConA.:Man-WYD proved that the trimannoside core and Man-WYD exhibit identical CRD docking; but their extra-CRD binding modes are significantly. different. Still, they have comparable affinity and selectivity for various Man-binding proteins. The intriguing observation provides new insight into functional mimicry :of carbohydrates by peptide ligands. GE-FBD may provide an alternative to rapidly search for competitive inhibitors for lectins.« less

  3. Differential Epitope Mapping by STD NMR Spectroscopy To Reveal the Nature of Protein-Ligand Contacts.

    PubMed

    Monaco, Serena; Tailford, Louise E; Juge, Nathalie; Angulo, Jesus

    2017-11-27

    Saturation transfer difference (STD) NMR spectroscopy is extensively used to obtain epitope maps of ligands binding to protein receptors, thereby revealing structural details of the interaction, which is key to direct lead optimization efforts in drug discovery. However, it does not give information about the nature of the amino acids surrounding the ligand in the binding pocket. Herein, we report the development of the novel method differential epitope mapping by STD NMR (DEEP-STD NMR) for identifying the type of protein residues contacting the ligand. The method produces differential epitope maps through 1) differential frequency STD NMR and/or 2) differential solvent (D 2 O/H 2 O) STD NMR experiments. The two approaches provide different complementary information on the binding pocket. We demonstrate that DEEP-STD NMR can be used to readily obtain pharmacophore information on the protein. Furthermore, if the 3D structure of the protein is known, this information also helps in orienting the ligand in the binding pocket. © 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.

  4. Measles Virus Hemagglutinin Protein Epitopes: The Basis of Antigenic Stability.

    PubMed

    Tahara, Maino; Bürckert, Jean-Philippe; Kanou, Kazuhiko; Maenaka, Katsumi; Muller, Claude P; Takeda, Makoto

    2016-08-02

    Globally eliminating measles using available vaccines is biologically feasible because the measles virus (MV) hemagglutinin (H) protein is antigenically stable. The H protein is responsible for receptor binding, and is the main target of neutralizing antibodies. The immunodominant epitope, known as the hemagglutinating and noose epitope, is located near the receptor-binding site (RBS). The RBS also contains an immunodominant epitope. Loss of receptor binding correlates with an escape from the neutralization by antibodies that target the epitope at RBS. Another neutralizing epitope is located near RBS and is shielded by an N-linked sugar in certain genotype strains. However, human sera from vaccinees and measles patients neutralized all MV strains with similar efficiencies, regardless of the N-linked sugar modification or mutations at these epitopes. Two other major epitopes exist at a distance from RBS. One has an unstructured flexible domain with a linear neutralizing epitope. When MV-H forms a tetramer (dimer of dimers), these epitopes may form the dimer-dimer interface, and one of the two epitopes may also interact with the F protein. The neutralization mechanisms of antibodies that recognize these epitopes may involve inhibiting the H-F interaction or blocking the fusion cascade after MV-H binds to its receptors.

  5. Measles Virus Hemagglutinin Protein Epitopes: The Basis of Antigenic Stability

    PubMed Central

    Tahara, Maino; Bürckert, Jean-Philippe; Kanou, Kazuhiko; Maenaka, Katsumi; Muller, Claude P.; Takeda, Makoto

    2016-01-01

    Globally eliminating measles using available vaccines is biologically feasible because the measles virus (MV) hemagglutinin (H) protein is antigenically stable. The H protein is responsible for receptor binding, and is the main target of neutralizing antibodies. The immunodominant epitope, known as the hemagglutinating and noose epitope, is located near the receptor-binding site (RBS). The RBS also contains an immunodominant epitope. Loss of receptor binding correlates with an escape from the neutralization by antibodies that target the epitope at RBS. Another neutralizing epitope is located near RBS and is shielded by an N-linked sugar in certain genotype strains. However, human sera from vaccinees and measles patients neutralized all MV strains with similar efficiencies, regardless of the N-linked sugar modification or mutations at these epitopes. Two other major epitopes exist at a distance from RBS. One has an unstructured flexible domain with a linear neutralizing epitope. When MV-H forms a tetramer (dimer of dimers), these epitopes may form the dimer-dimer interface, and one of the two epitopes may also interact with the F protein. The neutralization mechanisms of antibodies that recognize these epitopes may involve inhibiting the H-F interaction or blocking the fusion cascade after MV-H binds to its receptors. PMID:27490564

  6. Proof of principle for epitope-focused vaccine design

    PubMed Central

    Correia, Bruno E.; Bates, John T.; Loomis, Rebecca J.; Baneyx, Gretchen; Carrico, Christopher; Jardine, Joseph G.; Rupert, Peter; Correnti, Colin; Kalyuzhniy, Oleksandr; Vittal, Vinayak; Connell, Mary J.; Stevens, Eric; Schroeter, Alexandria; Chen, Man; MacPherson, Skye; Serra, Andreia M.; Adachi, Yumiko; Holmes, Margaret A.; Li, Yuxing; Klevit, Rachel E.; Graham, Barney S.; Wyatt, Richard T.; Baker, David; Strong, Roland K.; Crowe, James E.; Johnson, Philip R.; Schief, William R.

    2014-01-01

    Summary Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Multiple major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus (RSV), that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for research and development of a human RSV vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets including antigenically highly variable pathogens such as HIV and influenza. PMID:24499818

  7. Proof of principle for epitope-focused vaccine design

    NASA Astrophysics Data System (ADS)

    Correia, Bruno E.; Bates, John T.; Loomis, Rebecca J.; Baneyx, Gretchen; Carrico, Chris; Jardine, Joseph G.; Rupert, Peter; Correnti, Colin; Kalyuzhniy, Oleksandr; Vittal, Vinayak; Connell, Mary J.; Stevens, Eric; Schroeter, Alexandria; Chen, Man; MacPherson, Skye; Serra, Andreia M.; Adachi, Yumiko; Holmes, Margaret A.; Li, Yuxing; Klevit, Rachel E.; Graham, Barney S.; Wyatt, Richard T.; Baker, David; Strong, Roland K.; Crowe, James E.; Johnson, Philip R.; Schief, William R.

    2014-03-01

    Vaccines prevent infectious disease largely by inducing protective neutralizing antibodies against vulnerable epitopes. Several major pathogens have resisted traditional vaccine development, although vulnerable epitopes targeted by neutralizing antibodies have been identified for several such cases. Hence, new vaccine design methods to induce epitope-specific neutralizing antibodies are needed. Here we show, with a neutralization epitope from respiratory syncytial virus, that computational protein design can generate small, thermally and conformationally stable protein scaffolds that accurately mimic the viral epitope structure and induce potent neutralizing antibodies. These scaffolds represent promising leads for the research and development of a human respiratory syncytial virus vaccine needed to protect infants, young children and the elderly. More generally, the results provide proof of principle for epitope-focused and scaffold-based vaccine design, and encourage the evaluation and further development of these strategies for a variety of other vaccine targets, including antigenically highly variable pathogens such as human immunodeficiency virus and influenza.

  8. DRREP: deep ridge regressed epitope predictor.

    PubMed

    Sher, Gene; Zhi, Degui; Zhang, Shaojie

    2017-10-03

    The ability to predict epitopes plays an enormous role in vaccine development in terms of our ability to zero in on where to do a more thorough in-vivo analysis of the protein in question. Though for the past decade there have been numerous advancements and improvements in epitope prediction, on average the best benchmark prediction accuracies are still only around 60%. New machine learning algorithms have arisen within the domain of deep learning, text mining, and convolutional networks. This paper presents a novel analytically trained and string kernel using deep neural network, which is tailored for continuous epitope prediction, called: Deep Ridge Regressed Epitope Predictor (DRREP). DRREP was tested on long protein sequences from the following datasets: SARS, Pellequer, HIV, AntiJen, and SEQ194. DRREP was compared to numerous state of the art epitope predictors, including the most recently published predictors called LBtope and DMNLBE. Using area under ROC curve (AUC), DRREP achieved a performance improvement over the best performing predictors on SARS (13.7%), HIV (8.9%), Pellequer (1.5%), and SEQ194 (3.1%), with its performance being matched only on the AntiJen dataset, by the LBtope predictor, where both DRREP and LBtope achieved an AUC of 0.702. DRREP is an analytically trained deep neural network, thus capable of learning in a single step through regression. By combining the features of deep learning, string kernels, and convolutional networks, the system is able to perform residue-by-residue prediction of continues epitopes with higher accuracy than the current state of the art predictors.

  9. Self-Adjuvanting Glycopeptide Conjugate Vaccine against Disseminated Candidiasis

    PubMed Central

    Xin, Hong; Cartmell, Jonathan; Bailey, Justin J.; Dziadek, Sebastian; Bundle, David R.; Cutler, Jim E.

    2012-01-01

    Our research on pathogenesis of disseminated candidiasis led to the discovery that antibodies specific for Candida albicans cell surface β-1, 2–mannotriose [β-(Man)3] protect mice. A 14 mer peptide Fba, which derived from the N-terminal portion of the C. albicans cytosolic/cell surface protein fructose-bisphosphate aldolase, was used as the glycan carrier and resulted in a novel synthetic glycopeptide vaccine β-(Man)3-Fba. By a dendritic cell-based immunization approach, this conjugate induced protective antibody responses against both the glycan and peptide parts of the vaccine. In this report, we modified the β-(Man)3-Fba conjugate by coupling it to tetanus toxoid (TT) in order to improve immunogenicity and allow for use of an adjuvant suitable for human use. By new immunization procedures entirely compatible with human use, the modified β-(Man)3-Fba-TT was administered either alone or as a mixture made with alum or monophosphoryl lipid A (MPL) adjuvants and given to mice by a subcutaneous (s.c.) route. Mice vaccinated with or, surprisingly, without adjuvant responded well by making robust antibody responses. The immunized groups showed a high degree of protection against a lethal challenge with C. albicans as evidenced by increased survival times and reduced kidney fungal burden as compared to control groups that received only adjuvant or DPBS buffer prior to challenge. To confirm that induced antibodies were protective, sera from mice immunized against the β-(Man)3-Fba-TT conjugate transferred protection against disseminated candidiasis to naïve mice, whereas C. albicans-absorbed immune sera did not. Similar antibody responses and protection induced by the β-(Man)3-Fba-TT vaccine was observed in inbred BALB/c and outbred Swiss Webster mice. We conclude that addition of TT to the glycopeptide conjugate results in a self-adjuvanting vaccine that promotes robust antibody responses without the need for additional adjuvant, which is novel and represents a

  10. Characterization of neutralizing epitopes of varicella-zoster virus glycoprotein H.

    PubMed

    Akahori, Yasushi; Suzuki, Kazuhiro; Daikoku, Tohru; Iwai, Masae; Yoshida, Yoshihiro; Asano, Yoshizo; Kurosawa, Yoshikazu; Shiraki, Kimiyasu

    2009-02-01

    Varicella-zoster virus (VZV) glycoprotein H (gH) is the major neutralization target of VZV, and its neutralizing epitope is conformational. Ten neutralizing human monoclonal antibodies to gH were used to map the epitopes by immunohistochemical analysis and were categorized into seven epitope groups. The combinational neutralization efficacy of two epitope groups was not synergistic. Each epitope was partially or completely resistant to concanavalin A blocking of the glycomoiety of gH, and their antibodies inhibited the cell-to-cell spread of infection. The neutralization epitope comprised at least seven independent protein portions of gH that served as the target to inhibit cell-to-cell spread.

  11. Ligand-induced Epitope Masking

    PubMed Central

    Mould, A. Paul; Askari, Janet A.; Byron, Adam; Takada, Yoshikazu; Jowitt, Thomas A.; Humphries, Martin J.

    2016-01-01

    We previously demonstrated that Arg-Gly-Asp (RGD)-containing ligand-mimetic inhibitors of integrins are unable to dissociate pre-formed integrin-fibronectin complexes (IFCs). These observations suggested that amino acid residues involved in integrin-fibronectin binding become obscured in the ligand-occupied state. Because the epitopes of some function-blocking anti-integrin monoclonal antibodies (mAbs) lie near the ligand-binding pocket, it follows that the epitopes of these mAbs may become shielded in the ligand-occupied state. Here, we tested whether function-blocking mAbs directed against α5β1 can interact with the integrin after it forms a complex with an RGD-containing fragment of fibronectin. We showed that the anti-α5 subunit mAbs JBS5, SNAKA52, 16, and P1D6 failed to disrupt IFCs and hence appeared unable to bind to the ligand-occupied state. In contrast, the allosteric anti-β1 subunit mAbs 13, 4B4, and AIIB2 could dissociate IFCs and therefore were able to interact with the ligand-bound state. However, another class of function-blocking anti-β1 mAbs, exemplified by Lia1/2, could not disrupt IFCs. This second class of mAbs was also distinguished from 13, 4B4, and AIIB2 by their ability to induce homotypic cell aggregation. Although the epitope of Lia1/2 was closely overlapping with those of 13, 4B4, and AIIB2, it appeared to lie closer to the ligand-binding pocket. A new model of the α5β1-fibronectin complex supports our hypothesis that the epitopes of mAbs that fail to bind to the ligand-occupied state lie within, or very close to, the integrin-fibronectin interface. Importantly, our findings imply that the efficacy of some therapeutic anti-integrin mAbs could be limited by epitope masking. PMID:27484800

  12. Epitope Mapping by Phage Display.

    PubMed

    Moreira, Gustavo Marçal Schmidt Garcia; Fühner, Viola; Hust, Michael

    2018-01-01

    Among the molecules of the immune system, antibodies, particularly monoclonal antibodies (mAbs), have been shown to be interesting for many biological applications. Due to their ability to recognize only a unique part of their target, mAbs are usually very specific. These targets can have many different compositions, but the most common ones are proteins or peptides that are usually from outside the host, although self-proteins can also be targeted in autoimmune diseases, or in some types of cancer. The parts of a mAb that interact with its target compose the paratope, while the recognized parts of the target compose the epitope. Knowing the epitope is valuable for the improvement of a biological product, e.g., a diagnostic assay, a therapeutic mAb, or a vaccine, as well as for the elucidation of immune responses. The current techniques for epitope mapping rely on the presentation of the target, or parts of it, in a way that it can interact with a certain mAb. Even though there are several techniques available, each has its pros and cons. Thus, the choice for one of them is usually dependent on the preference and availability of the researcher, opening possibility for improvement, or development of alternative techniques. Phage display, for example, is a versatile technology, which allows the presentation of many different oligopeptides that can be tested against different antibodies, fitting the need for an epitope mapping approach. In this chapter, a protocol for the construction of a single-target oligopeptide phage library, as well as for the panning procedure for epitope mapping using phage display is given.

  13. Immunotherapy for Alzheimer's disease: DNA- and protein-based epitope vaccines.

    PubMed

    Davtyan, Hayk; Petrushina, Irina; Ghochikyan, Anahit

    2014-01-01

    Active immunotherapy for Alzheimer's disease (AD) is aimed to induce antibodies specific to amyloid-beta (Aβ) that are capable to reduce the level of Aβ in the CNS of Alzheimer's disease patients. First clinical trial AN-1792 that was based on vaccination with full-length Aβ42 showed that safe and effective AD vaccine should induce high titers of anti-Aβ antibodies without activation of harmful autoreactive T cells. Replacement of self-T cell epitope with foreign epitope, keeping self-B cell epitope intact, may allow to induce high titers of anti-Aβ antibodies while avoiding the activation of T cells specific to Aβ. Here we describe the protocols for evaluation of AD DNA- or multiple antigenic peptide (MAP)-based epitope vaccines composed of Aβ(1-11) B cell epitope fused to synthetic T cell epitope PADRE (Aβ(1-11)-PADRE). All protocols could be used for testing any epitope vaccine constructed in your lab and composed of other T cell epitopes using the appropriate peptides in tests for evaluation of humoral and cellular immune responses.

  14. Rapid Screening for Potential Epitopes Reactive with a Polycolonal Antibody by Solution-Phase H/D Exchange Monitored by FT-ICR Mass Spectrometry

    NASA Astrophysics Data System (ADS)

    Zhang, Qian; Noble, Kyle A.; Mao, Yuan; Young, Nicolas L.; Sathe, Shridhar K.; Roux, Kenneth H.; Marshall, Alan G.

    2013-07-01

    The potential epitopes of a recombinant food allergen protein, cashew Ana o 2, reactive to polyclonal antibodies, were mapped by solution-phase amide backbone H/D exchange (HDX) coupled with Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR MS). Ana o 2 polyclonal antibodies were purified in the serum from a goat immunized with cashew nut extract. Antibodies were incubated with recombinant Ana o 2 (rAna o 2) to form antigen:polyclonal antibody (Ag:pAb) complexes. Complexed and uncomplexed (free) rAna o 2 were then subjected to HDX-MS analysis. Four regions protected from H/D exchange upon pAb binding are identified as potential epitopes and mapped onto a homologous model.

  15. Sulfated glycopeptide nanostructures for multipotent protein activation

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Lee, Sungsoo S.; Fyrner, Timmy; Chen, Feng

    Biological systems have evolved to utilize numerous proteins with capacity to bind polysaccharides for the purpose of optimizing their function. A well-known subset of these proteins with binding domains for the highly diverse sulfated polysaccharides are important growth factors involved in biological development and tissue repair. We report here on supramolecular sulfated glycopeptide nanostructures, which display a trisulfated monosaccharide on their surfaces and bind five critical proteins with different polysaccharide-binding domains. Binding does not disrupt the filamentous shape of the nanostructures or their internal β-sheet backbone, but must involve accessible adaptive configurations to interact with such different proteins. The glycopeptidemore » nanostructures amplified signalling of bone morphogenetic protein 2 significantly more than the natural sulfated polysaccharide heparin, and promoted regeneration of bone in the spine with a protein dose that is 100-fold lower than that required in the animal model. These highly bioactive nanostructures may enable many therapies in the future involving proteins.« less

  16. Human eyes do not need monochromatic aberrations for dynamic accommodation.

    PubMed

    Bernal-Molina, Paula; Marín-Franch, Iván; Del Águila-Carrasco, Antonio J; Esteve-Taboada, Jose J; López-Gil, Norberto; Kruger, Philip B; Montés-Micó, Robert

    2017-09-01

    To determine if human accommodation uses the eye's own monochromatic aberrations to track dynamic accommodative stimuli. Wavefront aberrations were measured while subjects monocularly viewed a monochromatic Maltese cross moving sinusoidally around 2D of accommodative demand with 1D amplitude at 0.2 Hz. The amplitude and phase (delay) of the accommodation response were compared to the actual vergence of the stimulus to obtain gain and temporal phase, calculated from wavefront aberrations recorded over time during experimental trials. The tested conditions were as follows: Correction of all the subject's aberrations except defocus (C); Correction of all the subject's aberrations except defocus and habitual second-order astigmatism (AS); Correction of all the subject's aberrations except defocus and odd higher-order aberrations (HOAs); Correction of all the subject's aberrations except defocus and even HOAs (E); Natural aberrations of the subject's eye, i.e., the adaptive-optics system only corrected the optical system's aberrations (N); Correction of all the subject's aberrations except defocus and fourth-order spherical aberration (SA). The correction was performed at 20 Hz and each condition was repeated six times in randomised order. Average gain (±2 standard errors of the mean) varied little across conditions; between 0.55 ± 0.06 (SA), and 0.62 ± 0.06 (AS). Average phase (±2 standard errors of the mean) also varied little; between 0.41 ± 0.02 s (E), and 0.47 ± 0.02 s (O). After Bonferroni correction, no statistically significant differences in gain or phase were found in the presence of specific monochromatic aberrations or in their absence. These results show that the eye's monochromatic aberrations are not necessary for accommodation to track dynamic accommodative stimuli. © 2017 The Authors. Ophthalmic and Physiological Optics published by John Wiley & Sons Ltd on behalf of College of Optometrists.

  17. Antigenic structure of the herpes simplex virus type 1 glycoprotein C: demonstration of a linear epitope situated in an environment of highly conformation-dependent epitopes.

    PubMed

    Sjöblom, I; Glorioso, J C; Sjögren-Jansson, E; Olofsson, S

    1992-03-01

    A continuous epitope, situated within or in close proximity to antigenic site II of the herpes simplex virus type 1-specified glycoprotein C (gC-1), was identified. The continuous linear nature of the epitope, defined by a monoclonal antibody C2H12, was established by three independent lines of evidence: (i) The epitope was detectable by immunoblot under denaturing and reducing conditions. (ii) The epitope was detectable by RIPA of extracts from TM-treated HSV-infected cells, despite the malfolding caused by this treatment. (iii) The epitope was detected in an approximately 5,000-dalton papain fragment of gC-1. A mapping analysis, primarily based on use of mutant virus, expressing truncated gC-1 molecules, suggested that the mapping position of the epitope was delimited by amino acids 120 and 230. Other epitopes of this region of gC-1 are highly conformation-dependent, and the existence of a linear epitope, accessible on native gC-1, may facilitate the elucidation of the functional anatomy of gC-1.

  18. Meta-analysis of All Immune Epitope Data in the Flavivirus Genus: Inventory of Current Immune Epitope Data Status in the Context of Virus Immunity and Immunopathology

    PubMed Central

    Greenbaum, Jason; Blythe, Martin; Peters, Bjoern; Sette, Alessandro

    2010-01-01

    Abstract A meta-analysis was performed in order to inventory the immune epitope data related to viruses in the genus Flavivirus. Nearly 2000 epitopes were captured from over 130 individual Flavivirus-related references identified from PubMed and reported as of September 2009. This report includes all epitope structures and associated immune reactivity from the past and current literature, including: the epitope distribution among pathogens and related strains, the epitope distribution among different pathogen antigens, the number of epitopes defined in human and animal models of disease, the relationship between epitopes identified in different disease states following natural (or experimental) infection, and data from studies focused on candidate vaccines. We found that the majority of epitopes were defined for dengue virus (DENV) and West Nile virus (WNV). The prominence of DENV and WNV data in the epitope literature is likely a reflection of their overall worldwide impact on human disease, and the lack of vaccines. Conversely, the relatively smaller number of epitopes defined for the other viruses within the genus (yellow fever and Japanese encephalitis virus) most likely reflects the presence of established prophylaxis and/or their more modest impact on morbidity and mortality globally. Through this work we hope to provide useful data to those working in the area of Flavivirus research. PMID:20565291

  19. Dissecting Antibodies with Regards to Linear and Conformational Epitopes

    PubMed Central

    Forsström, Björn; Bisławska Axnäs, Barbara; Rockberg, Johan; Danielsson, Hanna; Bohlin, Anna; Uhlen, Mathias

    2015-01-01

    An important issue for the performance and specificity of an antibody is the nature of the binding to its protein target, including if the recognition involves linear or conformational epitopes. Here, we dissect polyclonal sera by creating epitope-specific antibody fractions using a combination of epitope mapping and an affinity capture approach involving both synthesized peptides and recombinant protein fragments. This allowed us to study the relative amounts of antibodies to linear and conformational epitopes in the polyclonal sera as well as the ability of each antibody-fraction to detect its target protein in Western blot assays. The majority of the analyzed polyclonal sera were found to have most of the target-specific antibodies directed towards linear epitopes and these were in many cases giving Western blot bands of correct molecular weight. In contrast, many of the antibodies towards conformational epitopes did not bind their target proteins in the Western blot assays. The results from this work have given us insights regarding the nature of the antibody response generated by immunization with recombinant protein fragments and has demonstrated the advantage of using antibodies recognizing linear epitopes for immunoassay involving wholly or partially denatured protein targets. PMID:25816293

  20. Structural analysis of B-cell epitopes in antibody:protein complexes

    PubMed Central

    Kringelum, Jens Vindahl; Nielsen, Morten; Padkjær, Søren Berg; Lund, Ole

    2012-01-01

    The binding of antigens to antibodies is one of the key events in an immune response against foreign molecules and is a critical element of several biomedical applications including vaccines and immunotherapeutics. For development of such applications, the identification of antibody binding sites (B-cell epitopes) is essential. However experimental epitope mapping is highly cost-intensive and computer-aided methods do in general have moderate performance. One major reason for this moderate performance is an incomplete understanding of what characterizes an epitope. To fill this gap, we here developed a novel framework for comparing and superimposing B-cell epitopes and applied it on a dataset of 107 non-similar antigen:antibody structures extracted from the PDB database. With the presented framework, we were able to describe the general B-cell epitope as a flat, oblong, oval shaped volume consisting of predominantly hydrophobic amino acids in the center flanked by charged residues. The average epitope was found to be made up of ~15 residues with one linear stretch of 5 or more residues constituting more than half of the epitope size. Furthermore, the epitope area is predominantly constrained to a plane above the antibody tip, in which the epitope is orientated in a −30 to 60 degree angle relative to the light to heavy chain antibody direction. Contrary to previously findings, we did not find a significant deviation between the amino acid composition in epitopes and the composition of equally exposed parts of the antigen surface. Our results, in combination with previously findings, give a detailed picture of the B-cell epitope that may be used in development of improved B-cell prediction methods. PMID:22784991

  1. Antibodies to a conformational epitope on gp41 neutralize HIV-1 by destabilizing the Env spike

    PubMed Central

    Lee, Jeong Hyun; Leaman, Daniel P.; Kim, Arthur S.; Torrents de la Peña, Alba; Sliepen, Kwinten; Yasmeen, Anila; Derking, Ronald; Ramos, Alejandra; de Taeye, Steven W.; Ozorowski, Gabriel; Klein, Florian; Burton, Dennis R.; Nussenzweig, Michel C.; Poignard, Pascal; Moore, John P.; Klasse, Per Johan; Sanders, Rogier W.; Zwick, Michael B.; Wilson, Ian A.; Ward, Andrew B.

    2015-01-01

    The recent identification of three broadly neutralizing antibodies (bnAbs) against gp120–gp41 interface epitopes has expanded the targetable surface on the HIV-1 envelope glycoprotein (Env) trimer. By using biochemical, biophysical and computational methods, we map the previously unknown trimer epitopes of two related antibodies, 3BC315 and 3BC176. A cryo-EM reconstruction of a soluble Env trimer bound to 3BC315 Fab at 9.3 Å resolution reveals that the antibody binds between two gp41 protomers, and neutralizes the virus by accelerating trimer decay. In contrast, bnAb 35O22 binding to a partially overlapping quaternary epitope at the gp120–gp41 interface does not induce decay. A conserved gp41-proximal glycan at N88 was also shown to play a role in the binding kinetics of 3BC176 and 3BC315. Finally, our data suggest that the dynamic structure of the Env trimer influences exposure of bnAb epitopes. PMID:26404402

  2. Improved Method for Linear B-Cell Epitope Prediction Using Antigen’s Primary Sequence

    PubMed Central

    Raghava, Gajendra P. S.

    2013-01-01

    One of the major challenges in designing a peptide-based vaccine is the identification of antigenic regions in an antigen that can stimulate B-cell’s response, also called B-cell epitopes. In the past, several methods have been developed for the prediction of conformational and linear (or continuous) B-cell epitopes. However, the existing methods for predicting linear B-cell epitopes are far from perfection. In this study, an attempt has been made to develop an improved method for predicting linear B-cell epitopes. We have retrieved experimentally validated B-cell epitopes as well as non B-cell epitopes from Immune Epitope Database and derived two types of datasets called Lbtope_Variable and Lbtope_Fixed length datasets. The Lbtope_Variable dataset contains 14876 B-cell epitope and 23321 non-epitopes of variable length where as Lbtope_Fixed length dataset contains 12063 B-cell epitopes and 20589 non-epitopes of fixed length. We also evaluated the performance of models on above datasets after removing highly identical peptides from the datasets. In addition, we have derived third dataset Lbtope_Confirm having 1042 epitopes and 1795 non-epitopes where each epitope or non-epitope has been experimentally validated in at least two studies. A number of models have been developed to discriminate epitopes and non-epitopes using different machine-learning techniques like Support Vector Machine, and K-Nearest Neighbor. We achieved accuracy from ∼54% to 86% using diverse s features like binary profile, dipeptide composition, AAP (amino acid pair) profile. In this study, for the first time experimentally validated non B-cell epitopes have been used for developing method for predicting linear B-cell epitopes. In previous studies, random peptides have been used as non B-cell epitopes. In order to provide service to scientific community, a web server LBtope has been developed for predicting and designing B-cell epitopes (http://crdd.osdd.net/raghava/lbtope/). PMID:23667458

  3. Epitope diversification driven by non-tumor epitope-specific Th1 and Th17 mediates potent antitumor reactivity.

    PubMed

    Ichikawa, Kosuke; Kagamu, Hiroshi; Koyama, Kenichi; Miyabayashi, Takao; Koshio, Jun; Miura, Satoru; Watanabe, Satoshi; Yoshizawa, Hirohisa; Narita, Ichiei

    2012-09-21

    MHC class I-restricted peptide-based vaccination therapies have been conducted to treat cancer patients, because CD8⁺ CTL can efficiently induce apoptosis of tumor cells in an MHC class I-restricted epitope-specific manner. Interestingly, clinical responders are known to demonstrate reactivity to epitopes other than those used for vaccination; however, the mechanism underlying how antitumor T cells with diverse specificity are induced is unclear. In this study, we demonstrated that dendritic cells (DCs) that engulfed apoptotic tumor cells in the presence of non-tumor MHC class II-restricted epitope peptides, OVA(323-339), efficiently presented tumor-associated antigens upon effector-dominant CD4⁺ T cell balance against regulatory T cells (Treg) for the OVA(323-339) epitope. Th1 and Th17 induced tumor-associated antigens presentation of DC, while Th2 ameliorated tumor-antigen presentation for CD8⁺ T cells. Blocking experiments with anti-IL-23p19 antibody and anti-IL-23 receptor indicated that an autocrine mechanism of IL-23 likely mediated the diverted tumor-associated antigens presentation of DC. Tumor-associated antigens presentation of DC induced by OVA(323-339) epitope-specific CD4⁺ T cells resulted in facilitated antitumor immunity in both priming and effector phase in vivo. Notably, this immunotherapy did not require pretreatment to reduce Treg induced by tumor. This strategy may have clinical implications for designing effective antitumor immunotherapies. Copyright © 2012 Elsevier Ltd. All rights reserved.

  4. CD18 activation epitopes induced by leukocyte activation.

    PubMed

    Beals, C R; Edwards, A C; Gottschalk, R J; Kuijpers, T W; Staunton, D E

    2001-12-01

    The cell surface adhesion molecule LFA-1 coordinates leukocyte trafficking and is a costimulatory molecule for T cell activation. We developed a panel of mAbs that recognize activation epitopes on the CD18 subunit, and show that stimulation of T lymphocytes appears to be accompanied by a conformational change in a subpopulation of LFA-1 that does not require ligand binding. Activation epitope up-regulation requires divalent cations, is sensitive to cellular signal transduction events, and correlates with cell adhesion. In addition, the stimulated appearance of these activation epitopes is absent in cell lines from patients with leukocyte adhesion deficiency-1/variant that has previously been shown to be defective in LFA-1 activation. Thus, these activation epitope Abs can be used to dissect signal transmission to CD18. Evidence suggests that these CD18 activation epitopes are induced early in cellular activation and are independent of actin rearrangement necessary for avid adhesion. We have also determined that function-blocking CD18 Abs inhibit the induction of activation epitopes. One activation epitope Ab binds to a site on CD18 distinct from that of the blocking Abs, indicating that the blocking Abs suppress a conformational change in LFA-1. We also find that these neoepitopes are present on rLFA-1 with high affinity for ICAM-1 and their binding is modulated in parallel with the affinity of LFA-1 for ICAM-1. Collectively, these neoepitope Abs identify a subpopulation of LFA-1 most likely with high affinity for ICAM-1 and necessary for LFA-1 function.

  5. Antibody Production and Th1-biased Response Induced by an Epitope Vaccine Composed of Cholera Toxin B Unit and Helicobacter pylori Lpp20 Epitopes.

    PubMed

    Li, Yan; Chen, Zhongbiao; Ye, Jianbin; Ning, Lijun; Luo, Jun; Zhang, Lili; Jiang, Yin; Xi, Yue; Ning, Yunshan

    2016-06-01

    The epitope vaccine is an attractive potential for prophylactic and therapeutic vaccination against Helicobacter pylori (H. pylori) infection. Lpp20 is one of major protective antigens which trigger immune response after H. pylori invades host and has been considered as an excellent vaccine candidate for the control of H. pylori infection. In our previous study, one B-cell epitope and two CD4(+) T-cell epitopes of Lpp20 were identified. In this study, an epitope vaccine composed of mucosal adjuvant cholera toxin B subunit (CTB) and these three identified Lpp20 epitopes were constructed to investigate the efficacy of this epitope vaccine in mice. The epitope vaccine including CTB, one B-cell, and two CD4(+) T-cell epitopes of Lpp20 was constructed and named CTB-Lpp20, which was then expressed in Escherichia coli and used for intraperitoneal immunization in BALB/c mice. The immunogenicity, specificity, and ability to induce antibodies against Lpp20 and cytokine secretion were evaluated. After that, CTB-Lpp20 was intragastrically immunized to investigate the prophylactic and therapeutic efficacy in infected mice. The results indicated that the epitope vaccine CTB-Lpp20 possessed good immunogenicity and immunoreactivity and could elicit specific high level of antibodies against Lpp20 and the cytokine of IFN-γ and IL-17. Additionally, CTB-Lpp20 significantly decreased H. pylori colonization in H. pylori challenging mice, and the protection was correlated with IgG, IgA, and sIgA antibody and Th1-type cytokines. This study will be better for understanding the protective immunity of epitope vaccine, and CTB-Lpp20 may be an alternative strategy for combating H. pylori invasion. © 2015 John Wiley & Sons Ltd.

  6. Machine learning-based methods for prediction of linear B-cell epitopes.

    PubMed

    Wang, Hsin-Wei; Pai, Tun-Wen

    2014-01-01

    B-cell epitope prediction facilitates immunologists in designing peptide-based vaccine, diagnostic test, disease prevention, treatment, and antibody production. In comparison with T-cell epitope prediction, the performance of variable length B-cell epitope prediction is still yet to be satisfied. Fortunately, due to increasingly available verified epitope databases, bioinformaticians could adopt machine learning-based algorithms on all curated data to design an improved prediction tool for biomedical researchers. Here, we have reviewed related epitope prediction papers, especially those for linear B-cell epitope prediction. It should be noticed that a combination of selected propensity scales and statistics of epitope residues with machine learning-based tools formulated a general way for constructing linear B-cell epitope prediction systems. It is also observed from most of the comparison results that the kernel method of support vector machine (SVM) classifier outperformed other machine learning-based approaches. Hence, in this chapter, except reviewing recently published papers, we have introduced the fundamentals of B-cell epitope and SVM techniques. In addition, an example of linear B-cell prediction system based on physicochemical features and amino acid combinations is illustrated in details.

  7. Epitope mapping of botulinum neurotoxins light chains

    PubMed Central

    Zdanovsky, Alexey; Zdanovsky, Denis; Zdanovskaia, Maria

    2012-01-01

    Botulinum neurotoxins (BoNTs) are listed among the most potent biothreat agents. Simultaneously, two out of seven known serotypes of these toxins are used in medicine and cosmetics. This situation calls for development of detailed epitope maps of these toxins. Such maps will help to develop new ways for decreasing damage caused by these toxins if they were to be used as weapons while retaining the therapeutic effect of these toxins used as medicine. Here, we used a library of random fragments of DNA encoding the catalytic domain of botulinum neurotoxin serotype A to identify short epitope-forming sequences. We demonstrated that knowledge of such sequences in a BoNT of one serotype can be used for identification of epitope-forming sequences in other serotypes of BoNTs. We also demonstrated a serodiagnostic value of identified sequences and their ability to retain epitope-specific structures and trigger production of corresponding antibodies, even when they are transferred into a background of a completely alien carrier protein. PMID:22922018

  8. Determination of aberration center of Ronchigram for automated aberration correctors in scanning transmission electron microscopy.

    PubMed

    Sannomiya, Takumi; Sawada, Hidetaka; Nakamichi, Tomohiro; Hosokawa, Fumio; Nakamura, Yoshio; Tanishiro, Yasumasa; Takayanagi, Kunio

    2013-12-01

    A generic method to determine the aberration center is established, which can be utilized for aberration calculation and axis alignment for aberration corrected electron microscopes. In this method, decentering induced secondary aberrations from inherent primary aberrations are minimized to find the appropriate axis center. The fitness function to find the optimal decentering vector for the axis was defined as a sum of decentering induced secondary aberrations with properly distributed weight values according to the aberration order. Since the appropriate decentering vector is determined from the aberration values calculated at an arbitrary center axis, only one aberration measurement is in principle required to find the center, resulting in /very fast center search. This approach was tested for the Ronchigram based aberration calculation method for aberration corrected scanning transmission electron microscopy. Both in simulation and in experiments, the center search was confirmed to work well although the convergence to find the best axis becomes slower with larger primary aberrations. Such aberration center determination is expected to fully automatize the aberration correction procedures, which used to require pre-alignment of experienced users. This approach is also applicable to automated aperture positioning. Copyright © 2013 Elsevier B.V. All rights reserved.

  9. Artificial-epitope mapping for CK-MB assay.

    PubMed

    Tai, Dar-Fu; Ho, Yi-Fang; Wu, Cheng-Hsin; Lin, Tzu-Chieh; Lu, Kuo-Hao; Lin, Kun-Shian

    2011-06-07

    A quantitative method using artificial antibody to detect creatine kinases was developed. Linear epitope sequences were selected based on an artificial-epitope mapping strategy. Nine different MIPs corresponding to the selected peptides were then fabricated on QCM chips. The subtle conformational changes were also recognized by these chips.

  10. Characteristic glycopeptides associated with extreme human longevity identified through plasma glycoproteomics.

    PubMed

    Miura, Yuri; Hashii, Noritaka; Ohta, Yuki; Itakura, Yoko; Tsumoto, Hiroki; Suzuki, Junya; Takakura, Daisuke; Abe, Yukiko; Arai, Yasumichi; Toyoda, Masashi; Kawasaki, Nana; Hirose, Nobuyoshi; Endo, Tamao

    2018-06-01

    Glycosylation is highly susceptible to changes of the physiological conditions, and accordingly, is a potential biomarker associated with several diseases and/or longevity. Semi-supercentenarians (SSCs; older than 105 years) are thought to be a model of human longevity. Thus, we performed glycoproteomics using plasma samples of SSCs, and identified proteins and conjugated N-glycans that are characteristic of extreme human longevity. Plasma proteins from Japanese semi-supercentenarians (SSCs, 106-109 years), aged controls (70-88 years), and young controls (20-38 years) were analysed by using lectin microarrays and liquid chromatography/mass spectrometry (LC/MS). Peak area ratios of glycopeptides to corresponding normalising peptides were subjected to orthogonal projections to latent structures discriminant analysis (OPLS-DA). Furthermore, plasma levels of clinical biomarkers were measured. We found two lectins such as Phaseolus vulgaris, and Erythrina cristagalli (ECA), of which protein binding were characteristically increased in SSCs. Peak area ratios of ECA-enriched glycopeptides were successfully discriminated between SSCs and controls using OPLS-DA, and indicated that tri-antennary and sialylated N-glycans of haptoglobin at Asn207 and Asn211 sites were characterized in SSCs. Sialylated glycans of haptoglobin are a potential biomarker of several diseases, such as hepatocellular carcinoma, liver cirrhosis, and IgA-nephritis. However, the SSCs analysed here did not suffer from these diseases. Tri-antennary and sialylated N-glycans on haptoglobin at the Asn207 and Asn211 sites were abundant in SSCs and characteristic of extreme human longevity. We found abundant glycans in SSCs, which may be associated with human longevity. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Dynamic interplay between catalytic and lectin domains of GalNAc-transferases modulates protein O-glycosylation

    NASA Astrophysics Data System (ADS)

    Lira-Navarrete, Erandi; de Las Rivas, Matilde; Compañón, Ismael; Pallarés, María Carmen; Kong, Yun; Iglesias-Fernández, Javier; Bernardes, Gonçalo J. L.; Peregrina, Jesús M.; Rovira, Carme; Bernadó, Pau; Bruscolini, Pierpaolo; Clausen, Henrik; Lostao, Anabel; Corzana, Francisco; Hurtado-Guerrero, Ramon

    2015-05-01

    Protein O-glycosylation is controlled by polypeptide GalNAc-transferases (GalNAc-Ts) that uniquely feature both a catalytic and lectin domain. The underlying molecular basis of how the lectin domains of GalNAc-Ts contribute to glycopeptide specificity and catalysis remains unclear. Here we present the first crystal structures of complexes of GalNAc-T2 with glycopeptides that together with enhanced sampling molecular dynamics simulations demonstrate a cooperative mechanism by which the lectin domain enables free acceptor sites binding of glycopeptides into the catalytic domain. Atomic force microscopy and small-angle X-ray scattering experiments further reveal a dynamic conformational landscape of GalNAc-T2 and a prominent role of compact structures that are both required for efficient catalysis. Our model indicates that the activity profile of GalNAc-T2 is dictated by conformational heterogeneity and relies on a flexible linker located between the catalytic and the lectin domains. Our results also shed light on how GalNAc-Ts generate dense decoration of proteins with O-glycans.

  12. Plant Lectins Targeting O-Glycans at the Cell Surface as Tools for Cancer Diagnosis, Prognosis and Therapy.

    PubMed

    Poiroux, Guillaume; Barre, Annick; van Damme, Els J M; Benoist, Hervé; Rougé, Pierre

    2017-06-09

    Aberrant O -glycans expressed at the surface of cancer cells consist of membrane-tethered glycoproteins (T and Tn antigens) and glycolipids (Lewis a, Lewis x and Forssman antigens). All of these O -glycans have been identified as glyco-markers of interest for the diagnosis and the prognosis of cancer diseases. These epitopes are specifically detected using T/Tn-specific lectins isolated from various plants such as jacalin from Artocarpus integrifola , and fungi such as the Agaricus bisporus lectin. These lectins accommodate T/Tn antigens at the monosaccharide-binding site; residues located in the surrounding extended binding-site of the lectins often participate in the binding of more extended epitopes. Depending on the shape and size of the extended carbohydrate-binding site, their fine sugar-binding specificity towards complex O -glycans readily differs from one lectin to another, resulting in a great diversity in their sugar-recognition capacity. T/Tn-specific lectins have been extensively used for the histochemical detection of cancer cells in biopsies and for the follow up of the cancer progression and evolution. T/Tn-specific lectins also induce a caspase-dependent apoptosis in cancer cells, often associated with a more or less severe inhibition of proliferation. Moreover, they provide another potential source of molecules adapted to the building of photosensitizer-conjugates allowing a specific targeting to cancer cells, for the photodynamic treatment of tumors.

  13. Plant Lectins Targeting O-Glycans at the Cell Surface as Tools for Cancer Diagnosis, Prognosis and Therapy

    PubMed Central

    Poiroux, Guillaume; Barre, Annick; van Damme, Els J. M.; Benoist, Hervé; Rougé, Pierre

    2017-01-01

    Aberrant O-glycans expressed at the surface of cancer cells consist of membrane-tethered glycoproteins (T and Tn antigens) and glycolipids (Lewis a, Lewis x and Forssman antigens). All of these O-glycans have been identified as glyco-markers of interest for the diagnosis and the prognosis of cancer diseases. These epitopes are specifically detected using T/Tn-specific lectins isolated from various plants such as jacalin from Artocarpus integrifola, and fungi such as the Agaricus bisporus lectin. These lectins accommodate T/Tn antigens at the monosaccharide-binding site; residues located in the surrounding extended binding-site of the lectins often participate in the binding of more extended epitopes. Depending on the shape and size of the extended carbohydrate-binding site, their fine sugar-binding specificity towards complex O-glycans readily differs from one lectin to another, resulting in a great diversity in their sugar-recognition capacity. T/Tn-specific lectins have been extensively used for the histochemical detection of cancer cells in biopsies and for the follow up of the cancer progression and evolution. T/Tn-specific lectins also induce a caspase-dependent apoptosis in cancer cells, often associated with a more or less severe inhibition of proliferation. Moreover, they provide another potential source of molecules adapted to the building of photosensitizer-conjugates allowing a specific targeting to cancer cells, for the photodynamic treatment of tumors. PMID:28598369

  14. Pathogenesis of NOD Diabetes is Initiated by Reactivity to the Insulin B Chain 9–23 Epitope and Involves Functional Epitope Spreading1

    PubMed Central

    Prasad, Suchitra; Kohm, Adam P.; McMahon, Jeffrey S.; Luo, Xunrong; Miller, Stephen D.

    2012-01-01

    Type 1 diabetes (T1D) is mediated by destruction of pancreatic β cells by CD4 and CD8 T cells specific for epitopes on numerous diabetogenic autoantigens resulting in loss of glucose homeostasis. Employing antigen-specific tolerance induced by i.v. administration of syngeneic splenocytes ECDI cross-linked to various diabetogenic antigens/epitopes (Ag-SP), we show that epitope spreading plays a functional role in the pathogenesis of T1D in NOD mice. Specifically, Ag-SP coupled with intact insulin, Ins B9–23 or Ins B15–23, but not GAD65509–528, GAD65524–543 or IGRP206–214, protected 4–6 week-old NOD mice from the eventual development of clinical disease; infiltration of immune cells to the pancreatic islets; and blocked the induction of DTH responses in a Treg-dependent, antigen-specific manner. However, tolerance induction in 19–21 week-old NOD mice was effectively accomplished only by Ins-SP, suggesting Ins B9–23 is a dominant initiating epitope, but autoimmune responses to insulin epitope(s) distinct from Ins B9–23 emerge during disease progression. PMID:22647732

  15. Determination of asparagine, glutamine and pyrrolidonecarboxylic acid in total enzymic hydrolysates of peptides and glycopeptides by gas–liquid chromatography

    PubMed Central

    Hediger, Hedy; Stevens, Richard L.; Brandenberger, Hans; Schmid, Karl

    1973-01-01

    A new procedure for the qualitative and quantitative determination of asparagine, glutamine and pyrrolidonecarboxylic acid in total enzymic hydrolysates of peptides and glycopeptides based on g.l.c. has been developed. Under the conditions of esterification and trifluoroacetylation N-trifluoroacetylaspartic acid mono-n-butyl ester was formed from asparagine and N-trifluoroacetylglutamic acid mono-n-butyl ester from both glutamine and pyrrolidonecarboxylic acid. To distinguish between the latter two compounds, the esterification was carried out at room temperature yielding 30% of esterified pyrrolidonecarboxylic acid but less than 1% of esterified glutamine. In extending the g.l.c. of amino acids, the previously unknown positions in the g.l.c. elution pattern of the following amino acids could also be reproducibly determined: carboxymethylcysteine, homoserine, hydroxylysine and ∈-methyl-lysine. Further, certain glycopeptides were investigated and the artifacts due to their carbohydrate moieties were determined. PMID:4733240

  16. Crystal Structure of StaL, A Glycopeptide Antibiotic Sulfotransferase from Streptomyces Toyocaensis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shi,R.; Lamb, S.; Bhat, S.

    2007-01-01

    Over the past decade, antimicrobial resistance has emerged as a major public health crisis. Glycopeptide antibiotics such as vanco-mycin and teicoplanin are clinically important for the treatment of Gram-positive bacterial infections. StaL is a 3'-phosphoadenosine 5'-phosphosulfate-dependent sulfotransferase capable of sulfating the cross-linked heptapeptide substrate both in vivo and in vitro, yielding the product A47934 [GenBank], unique teicoplanin-class glycopeptide antibiotic. The sulfonation reaction catalyzed by StaL constitutes the final step in A47934 [GenBank] biosynthesis. Here we report the crystal structure of StaL and its complex with the cofactor product 3'-phosphoadenosine 5'-phosphate. This is only the second prokaryotic sulfotransferase to be structurallymore » characterized. StaL belongs to the large sulfotransferase family and shows higher similarity to cytosolic sulfotransferases (ST) than to the bacterial ST (Stf0). StaL has a novel dimerization motif, different from any other STs that have been structurally characterized. We have also applied molecular modeling to investigate the binding mode of the unique substrate, desulfo-A47934. Based on the structural analysis and modeling results, a series of residues was mutated and kinetically characterized. In addition to the conserved residues (Lys{sup 12}, His{sup 67}, and Ser{sup 98}), molecular modeling, fluorescence quenching experiments, and mutagenesis studies identified several other residues essential for substrate binding and/or activity, including Trp{sup 34}, His{sup 43}, Phe{sup 77}, Trp{sup 132}, and Glu{sup 205}.« less

  17. Computer-Aided Design of an Epitope-Based Vaccine against Epstein-Barr Virus

    PubMed Central

    Alonso-Padilla, Julio

    2017-01-01

    Epstein-Barr virus is a very common human virus that infects 90% of human adults. EBV replicates in epithelial and B cells and causes infectious mononucleosis. EBV infection is also linked to various cancers, including Burkitt's lymphoma and nasopharyngeal carcinomas, and autoimmune diseases such as multiple sclerosis. Currently, there are no effective drugs or vaccines to treat or prevent EBV infection. Herein, we applied a computer-aided strategy to design a prophylactic epitope vaccine ensemble from experimentally defined T and B cell epitopes. Such strategy relies on identifying conserved epitopes in conjunction with predictions of HLA presentation for T cell epitope selection and calculations of accessibility and flexibility for B cell epitope selection. The T cell component includes 14 CD8 T cell epitopes from early antigens and 4 CD4 T cell epitopes, targeted during the course of a natural infection and providing a population protection coverage of over 95% and 81.8%, respectively. The B cell component consists of 3 experimentally defined B cell epitopes from gp350 plus 4 predicted B cell epitopes from other EBV envelope glycoproteins, all mapping in flexible and solvent accessible regions. We discuss the rationale for the formulation and possible deployment of this epitope vaccine ensemble. PMID:29119120

  18. Predicting the Retention Behavior of Specific O-Linked Glycopeptides.

    PubMed

    Badgett, Majors J; Boyes, Barry; Orlando, Ron

    2017-09-01

    O -Linked glycosylation is a common post-translational modification that can alter the overall structure, polarity, and function of proteins. Reverse-phase (RP) chromatography is the most common chromatographic approach to analyze O -glycosylated peptides and their unmodified counterparts, even though this approach often does not provide adequate separation of these two species. Hydrophilic interaction liquid chromatography (HILIC) can be a solution to this problem, as the polar glycan interacts with the polar stationary phase and potentially offers the ability to resolve the peptide from its modified form(s). In this paper, HILIC is used to separate peptides with O - N -acetylgalactosamine ( O -GalNAc), O - N -acetylglucosamine ( O -GlcNAc), and O -fucose additions from their native forms, and coefficients representing the extent of hydrophilicity were derived using linear regression analysis as a means to predict the retention times of peptides with these modifications.

  19. Predicting the Retention Behavior of Specific O-Linked Glycopeptides

    PubMed Central

    Badgett, Majors J.; Boyes, Barry; Orlando, Ron

    2017-01-01

    O-Linked glycosylation is a common post-translational modification that can alter the overall structure, polarity, and function of proteins. Reverse-phase (RP) chromatography is the most common chromatographic approach to analyze O-glycosylated peptides and their unmodified counterparts, even though this approach often does not provide adequate separation of these two species. Hydrophilic interaction liquid chromatography (HILIC) can be a solution to this problem, as the polar glycan interacts with the polar stationary phase and potentially offers the ability to resolve the peptide from its modified form(s). In this paper, HILIC is used to separate peptides with O-N-acetylgalactosamine (O-GalNAc), O-N-acetylglucosamine (O-GlcNAc), and O-fucose additions from their native forms, and coefficients representing the extent of hydrophilicity were derived using linear regression analysis as a means to predict the retention times of peptides with these modifications. PMID:28785176

  20. Permian ginkgophyte fossils from the Dolomites resemble extant O-ha-tsuki aberrant leaf-like fructifications of Ginkgo biloba L

    PubMed Central

    2010-01-01

    Background Structural elucidation and analysis of fructifications of plants is fundamental for understanding their evolution. In case of Ginkgo biloba, attention was drawn by Fujii in 1896 to aberrant fructifications of Ginkgo biloba whose seeds are attached to leaves, called O-ha-tsuki in Japan. This well-known phenomenon was now interpreted by Fujii as being homologous to ancestral sporophylls. The common fructification of Ginkgo biloba consists of 1-2 (rarely more) ovules on a dichotomously divided stalk, the ovules on top of short stalklets, with collars supporting the ovules. There is essentially no disagreement that either the whole stalk with its stalklets, collars and ovules is homologous to a sporophyll, or, alternatively, just one stalklet, collar and ovule each correspond to a sporophyll. For the transition of an ancestral sporophyll resembling extant O-ha-tsuki aberrant leaves into the common fructification with stalklet/collar/ovule, evolutionary reduction of the leaf lamina of such ancestral sporophylls has to be assumed. Furthermore, such ancestral sporophylls would be expected in the fossil record of ginkgophytes. Results From the Upper Permian of the Bletterbach gorge (Dolomites, South Tyrol, Italy) ginkgophyte leaves of the genus Sphenobaiera were discovered. Among several specimens, one shows putatively attached seeds, while other specimens, depending on their state of preservation, show seeds in positions strongly suggesting such attachment. Morphology and results of a cuticular analysis are in agreement with an affiliation of the fossil to the ginkgophytes and the cuticle of the seed is comparable to that of Triassic and Jurassic ones and to those of extant Ginkgo biloba. The Sphenobaiera leaves with putatively attached seeds closely resemble seed-bearing O-ha-tsuki leaves of extant Ginkgo biloba. This leads to the hypothesis that, at least for some groups of ginkgophytes represented by extant Ginkgo biloba, such sporophylls represent the

  1. Interpreting Chromosome Aberration Spectra

    NASA Technical Reports Server (NTRS)

    Levy, Dan; Reeder, Christopher; Loucas, Bradford; Hlatky, Lynn; Chen, Allen; Cornforth, Michael; Sachs, Rainer

    2007-01-01

    Ionizing radiation can damage cells by breaking both strands of DNA in multiple locations, essentially cutting chromosomes into pieces. The cell has enzymatic mechanisms to repair such breaks; however, these mechanisms are imperfect and, in an exchange process, may produce a large-scale rearrangement of the genome, called a chromosome aberration. Chromosome aberrations are important in killing cells, during carcinogenesis, in characterizing repair/misrepair pathways, in retrospective radiation biodosimetry, and in a number of other ways. DNA staining techniques such as mFISH ( multicolor fluorescent in situ hybridization) provide a means for analyzing aberration spectra by examining observed final patterns. Unfortunately, an mFISH observed final pattern often does not uniquely determine the underlying exchange process. Further, resolution limitations in the painting protocol sometimes lead to apparently incomplete final patterns. We here describe an algorithm for systematically finding exchange processes consistent with any observed final pattern. This algorithm uses aberration multigraphs, a mathematical formalism that links the various aspects of aberration formation. By applying a measure to the space of consistent multigraphs, we will show how to generate model-specific distributions of aberration processes from mFISH experimental data. The approach is implemented by software freely available over the internet. As a sample application, we apply these algorithms to an aberration data set, obtaining a distribution of exchange cycle sizes, which serves to measure aberration complexity. Estimating complexity, in turn, helps indicate how damaging the aberrations are and may facilitate identification of radiation type in retrospective biodosimetry.

  2. Identification of B cell epitopes of alcohol dehydrogenase allergen of Curvularia lunata.

    PubMed

    Nair, Smitha; Kukreja, Neetu; Singh, Bhanu Pratap; Arora, Naveen

    2011-01-01

    Epitope identification assists in developing molecules for clinical applications and is useful in defining molecular features of allergens for understanding structure/function relationship. The present study was aimed to identify the B cell epitopes of alcohol dehydrogenase (ADH) allergen from Curvularia lunata using in-silico methods and immunoassay. B cell epitopes of ADH were predicted by sequence and structure based methods and protein-protein interaction tools while T cell epitopes by inhibitory concentration and binding score methods. The epitopes were superimposed on a three dimensional model of ADH generated by homology modeling and analyzed for antigenic characteristics. Peptides corresponding to predicted epitopes were synthesized and immunoreactivity assessed by ELISA using individual and pooled patients' sera. The homology model showed GroES like catalytic domain joined to Rossmann superfamily domain by an alpha helix. Stereochemical quality was confirmed by Procheck which showed 90% residues in most favorable region of Ramachandran plot while Errat gave a quality score of 92.733%. Six B cell (P1-P6) and four T cell (P7-P10) epitopes were predicted by a combination of methods. Peptide P2 (epitope P2) showed E(X)(2)GGP(X)(3)KKI conserved pattern among allergens of pathogenesis related family. It was predicted as high affinity binder based on electronegativity and low hydrophobicity. The computational methods employed were validated using Bet v 1 and Der p 2 allergens where 67% and 60% of the epitope residues were predicted correctly. Among B cell epitopes, Peptide P2 showed maximum IgE binding with individual and pooled patients' sera (mean OD 0.604±0.059 and 0.506±0.0035, respectively) followed by P1, P4 and P3 epitopes. All T cell epitopes showed lower IgE binding. Four B cell epitopes of C. lunata ADH were identified. Peptide P2 can serve as a potential candidate for diagnosis of allergic diseases.

  3. High Throughput T Epitope Mapping and Vaccine Development

    PubMed Central

    Li Pira, Giuseppina; Ivaldi, Federico; Moretti, Paolo; Manca, Fabrizio

    2010-01-01

    Mapping of antigenic peptide sequences from proteins of relevant pathogens recognized by T helper (Th) and by cytolytic T lymphocytes (CTL) is crucial for vaccine development. In fact, mapping of T-cell epitopes provides useful information for the design of peptide-based vaccines and of peptide libraries to monitor specific cellular immunity in protected individuals, patients and vaccinees. Nevertheless, epitope mapping is a challenging task. In fact, large panels of overlapping peptides need to be tested with lymphocytes to identify the sequences that induce a T-cell response. Since numerous peptide panels from antigenic proteins are to be screened, lymphocytes available from human subjects are a limiting factor. To overcome this limitation, high throughput (HTP) approaches based on miniaturization and automation of T-cell assays are needed. Here we consider the most recent applications of the HTP approach to T epitope mapping. The alternative or complementary use of in silico prediction and experimental epitope definition is discussed in the context of the recent literature. The currently used methods are described with special reference to the possibility of applying the HTP concept to make epitope mapping an easier procedure in terms of time, workload, reagents, cells and overall cost. PMID:20617148

  4. Comparison of HPLC/ESI-FTICR MS Versus MALDI-TOF/TOF MS for Glycopeptide Analysis of a Highly Glycosylated HIV Envelope Glycoprotein

    PubMed Central

    Irungu, Janet; Go, Eden P.; Zhang, Ying; Dalpathado, Dilusha S.; Liao, Hua-Xin; Haynes, Barton F.; Desaire, Heather

    2013-01-01

    Defining the structures and locations of the glycans attached on secreted proteins and virus envelope proteins is important in understanding how glycosylation affects their biological properties. Glycopeptide mass spectrometry (MS)-based analysis is a very powerful, emerging approach to characterize glycoproteins, in which glycosylation sites and the corresponding glycan structures are elucidated in a single MS experiment. However, to date there is not a consensus regarding which mass spectrometric platform provides the best glycosylation coverage information. Herein, we employ two of the most widely used MS approaches, online high performance liquid chromatography-electrospray ionization mass spectrometry (HPLC/ESI-MS) and offline HPLC followed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), to determine which of the two approaches provides the best glycosylation coverage information of a complex glycoprotein, the group M consensus HIV-1 envelope, CON-S gp140ΔCFI, which has 31 potential glycosylation sites. Our results highlight differences in the informational content obtained between the two methods such as the overall number of glycosylation sites detected, the numbers of N-linked glycans present at each site, and the type of confirmatory information obtained about the glycopeptide using MS/MS experiments. The two approaches are quite complementary, both in their coverage of glycopeptides and in the information they provide in MS/MS experiments. The information in this study contributes to the field of mass spectrometry by demonstrating the strengths and limitations of two widely used MS platforms in glycoprotein analysis. PMID:18565761

  5. A high-throughput shotgun mutagenesis approach to mapping B-cell antibody epitopes.

    PubMed

    Davidson, Edgar; Doranz, Benjamin J

    2014-09-01

    Characterizing the binding sites of monoclonal antibodies (mAbs) on protein targets, their 'epitopes', can aid in the discovery and development of new therapeutics, diagnostics and vaccines. However, the speed of epitope mapping techniques has not kept pace with the increasingly large numbers of mAbs being isolated. Obtaining detailed epitope maps for functionally relevant antibodies can be challenging, particularly for conformational epitopes on structurally complex proteins. To enable rapid epitope mapping, we developed a high-throughput strategy, shotgun mutagenesis, that enables the identification of both linear and conformational epitopes in a fraction of the time required by conventional approaches. Shotgun mutagenesis epitope mapping is based on large-scale mutagenesis and rapid cellular testing of natively folded proteins. Hundreds of mutant plasmids are individually cloned, arrayed in 384-well microplates, expressed within human cells, and tested for mAb reactivity. Residues are identified as a component of a mAb epitope if their mutation (e.g. to alanine) does not support candidate mAb binding but does support that of other conformational mAbs or allows full protein function. Shotgun mutagenesis is particularly suited for studying structurally complex proteins because targets are expressed in their native form directly within human cells. Shotgun mutagenesis has been used to delineate hundreds of epitopes on a variety of proteins, including G protein-coupled receptor and viral envelope proteins. The epitopes mapped on dengue virus prM/E represent one of the largest collections of epitope information for any viral protein, and results are being used to design better vaccines and drugs. © 2014 John Wiley & Sons Ltd.

  6. Standardization of Epitopes for Human Chorionic Gonadotropin (hCG) Immunoassays.

    PubMed

    Berger, Peter; Lapthorn, Adrian J

    2016-01-01

    hCG and its variants are markers for pregnancy tests, pregnancyrelated complications, trophoblastic diseases, pre-natal screening of Down's syndrome and doping controls. Strong demands are imposed on diagnostic methods by the dynamic changes in the absolute and relative levels of hCG protein backbone variants and glycosylation isoforms in serum and urine during development of pregnancy or the progression/remission of tumors. Observed differences in the results between commercial diagnostic immunoassays reflect the unequal molar recognition of the different metabolic hCG variants, in particular the hCG beta core fragment (hCGβcf), by the diagnostic antibodies (Abs), as their epitopes are not standardized, and the fact that suboptimal hCG standards are used. To rapidly characterize Abs by their epitope recognition and specificity to evaluate their suitability for diagnostic immunoassays a procedure of comparative epitope mapping has been developed using epitope-defined reference Abs. Comparative epitope mapping of diagnostic Abs will provide the basis for the standardization of diagnostic antigenic domains/epitopes and consequently for improved reliability of hCG measurements. Diagnostic first line assays likely consist of pairs of Abs that recognize specific epitopes at the top of the neighboring peptide loops 1 and 3 (Ł1+3) and the cystine knot (ck) of hCGβ, respectively. In future, significant improvements of reliability, robustness and comparability of the results of immunoassays for complex glycoproteins such as hCG will be achieved by the use (i) of standardized diagnostic Abs against welldefined epitopes and (ii) of the new International Standards for hCG and for five hCG variants established by WHO, that are calibrated in molar (SI) units.

  7. Rapid Fine Conformational Epitope Mapping Using Comprehensive Mutagenesis and Deep Sequencing*

    PubMed Central

    Kowalsky, Caitlin A.; Faber, Matthew S.; Nath, Aritro; Dann, Hailey E.; Kelly, Vince W.; Liu, Li; Shanker, Purva; Wagner, Ellen K.; Maynard, Jennifer A.; Chan, Christina; Whitehead, Timothy A.

    2015-01-01

    Knowledge of the fine location of neutralizing and non-neutralizing epitopes on human pathogens affords a better understanding of the structural basis of antibody efficacy, which will expedite rational design of vaccines, prophylactics, and therapeutics. However, full utilization of the wealth of information from single cell techniques and antibody repertoire sequencing awaits the development of a high throughput, inexpensive method to map the conformational epitopes for antibody-antigen interactions. Here we show such an approach that combines comprehensive mutagenesis, cell surface display, and DNA deep sequencing. We develop analytical equations to identify epitope positions and show the method effectiveness by mapping the fine epitope for different antibodies targeting TNF, pertussis toxin, and the cancer target TROP2. In all three cases, the experimentally determined conformational epitope was consistent with previous experimental datasets, confirming the reliability of the experimental pipeline. Once the comprehensive library is generated, fine conformational epitope maps can be prepared at a rate of four per day. PMID:26296891

  8. Levels of HIV1 gp120 3D B-cell epitopes mutability and variability: searching for possible vaccine epitopes.

    PubMed

    Khrustalev, Vladislav Victorovich

    2010-01-01

    We used a DiscoTope 1.2 (http://www.cbs.dtu.dk/services/DiscoTope/), Epitopia (http://epitopia.tau.ac.il/) and EPCES (http://www.t38.physik.tu-muenchen.de/programs.htm) algorithms to map discontinuous B-cell epitopes in HIV1 gp120. The most mutable nucleotides in HIV genes are guanine (because of G to A hypermutagenesis) and cytosine (because of C to U and C to A mutations). The higher is the level of guanine and cytosine usage in third (neutral) codon positions and the lower is their level in first and second codon positions of the coding region, the more stable should be an epitope encoded by this region. We compared guanine and cytosine usage in regions coding for five predicted 3D B-cell epitopes of gp120. To make this comparison we used GenBank resource: 385 sequences of env gene obtained from ten HIV1-infected individuals were studied (http://www.barkovsky.hotmail.ru/Data/Seqgp120.htm). The most protected from nonsynonymous nucleotide mutations of guanine and cytosine 3D B-cell epitope is situated in the first conserved region of gp120 (it is mapped from 66th to 86th amino acid residue). We applied a test of variability to confirm this finding. Indeed, the less mutable predicted B-cell epitope is the less variable one. MEGA4 (standard PAM matrix) was used for the alignments and "VVK Consensus" algorithm (http://www.barkovsky.hotmail.ru) was used for the calculations.

  9. Cytotoxic T Lymphocyte Epitopes of HIV-1 Nef

    PubMed Central

    Lucchiari-Hartz, Maria; van Endert, Peter M.; Lauvau, Grégoire; Maier, Reinhard; Meyerhans, Andreas; Mann, Derek; Eichmann, Klaus; Niedermann, Gabriele

    2000-01-01

    Although a pivotal role of proteasomes in the proteolytic generation of epitopes for major histocompatibility complex (MHC) class I presentation is undisputed, their precise function is currently the subject of an active debate: do proteasomes generate many epitopes in definitive form, or do they merely generate the COOH termini, whereas the definitive NH2 termini are cleaved by aminopeptidases? We determined five naturally processed MHC class I ligands derived from HIV-1 Nef. Unexpectedly, the five ligands correspond to only three cytotoxic T lymphocyte (CTL) epitopes, two of which occur in two COOH-terminal length variants. Parallel analyses of proteasomal digests of a Nef fragment encompassing the epitopes revealed that all five ligands are direct products of proteasomes. Moreover, in four of the five ligands, the NH2 termini correspond to major proteasome cleavage sites, and putative NH2-terminally extended precursor fragments were detected for only one of the five ligands. All ligands are transported by the transporter associated with antigen processing (TAP). The combined results from these five ligands provide strong evidence that many definitive MHC class I ligands are precisely cleaved at both ends by proteasomes. Additional evidence supporting this conclusion is discussed, along with contrasting results of others who propose a strong role for NH2-terminal trimming with direct proteasomal epitope generation being a rare event. PMID:10637269

  10. HIV-1 subtype A gag variability and epitope evolution.

    PubMed

    Abidi, Syed Hani; Kalish, Marcia L; Abbas, Farhat; Rowland-Jones, Sarah; Ali, Syed

    2014-01-01

    The aim of this study was to examine the course of time-dependent evolution of HIV-1 subtype A on a global level, especially with respect to the dynamics of immunogenic HIV gag epitopes. We used a total of 1,893 HIV-1 subtype A gag sequences representing a timeline from 1985 through 2010, and 19 different countries in Africa, Europe and Asia. The phylogenetic relationship of subtype A gag and its epidemic dynamics was analysed through a Maximum Likelihood tree and Bayesian Skyline plot, genomic variability was measured in terms of G → A substitutions and Shannon entropy, and the time-dependent evolution of HIV subtype A gag epitopes was examined. Finally, to confirm observations on globally reported HIV subtype A sequences, we analysed the gag epitope data from our Kenyan, Pakistani, and Afghan cohorts, where both cohort-specific gene epitope variability and HLA restriction profiles of gag epitopes were examined. The most recent common ancestor of the HIV subtype A epidemic was estimated to be 1956 ± 1. A period of exponential growth began about 1980 and lasted for approximately 7 years, stabilized for 15 years, declined for 2-3 years, then stabilized again from about 2004. During the course of evolution, a gradual increase in genomic variability was observed that peaked in 2005-2010. We observed that the number of point mutations and novel epitopes in gag also peaked concurrently during 2005-2010. It appears that as the HIV subtype A epidemic spread globally, changing population immunogenetic pressures may have played a role in steering immune-evolution of this subtype in new directions. This trend is apparent in the genomic variability and epitope diversity of HIV-1 subtype A gag sequences.

  11. CD8+ T cell recognition of an endogenously processed epitope is regulated primarily by residues within the epitope

    PubMed Central

    1992-01-01

    Cytotoxic T lymphocytes (CTL) recognize short antigenic peptides associated with cell surface class I major histocompatibility complex (MHC) molecules. This association presumably occurs between newly synthesized class I MHC molecules and peptide fragments in a pre-Golgi compartment. Little is known about the factors that regulate the formation of these antigenic peptide fragments within the cell. To examine the role of residues within a core epitope and in the flanking sequences for the generation and presentation of the newly synthesized peptide fragment recognized by CD8+ CTL, we have mutagenized the coding sequence for the CTL epitope spanning residues 202-221 in the influenza A/Japan/57 hemagglutinin (HA). In this study over 60 substitution mutations in the epitope were tested for their effects on target cell sensitization using a cytoplasmic viral expression system. The HA202- 221 site contains two overlapping subsites defined by CTL clones 11-1 and 40-2. Mutations in HA residues 204-213 or residues 210-219 often abolished target cell lysis by CTL clones 11-1 and 40-2, respectively. Although residues outside the core epitope did not usually affect the ability to be lysed by CTL clones, substitution of a Gly residue for Val-214 abolished lysis by clone 11-1. These data suggest that residues within a site that affect MHC binding and T cell receptor recognition appear to play the predominant role in dictating the formation of the antigenic complex recognized by CD8+ CTL, and therefore the antigenicity of the protein antigen presented to CD8+ T cells. Most alterations in residues flanking the endogenously expressed epitope do not appreciably affect the generation and recognition of the site. PMID:1383384

  12. Computational elucidation of potential antigenic CTL epitopes in Ebola virus.

    PubMed

    Dikhit, Manas R; Kumar, Santosh; Vijaymahantesh; Sahoo, Bikash R; Mansuri, Rani; Amit, Ajay; Yousuf Ansari, Md; Sahoo, Ganesh C; Bimal, Sanjiva; Das, Pradeep

    2015-12-01

    Cell-mediated immunity is important for the control of Ebola virus infection. We hypothesized that those HLA A0201 and HLA B40 restricted epitopes derived from Ebola virus proteins, would mount a good antigenic response. Here we employed an immunoinformatics approach to identify specific 9mer amino acid which may be capable of inducing a robust cell-mediated immune response in humans. We identified a set of 28 epitopes that had no homologs in humans. Specifically, the epitopes derived from NP, RdRp, GP and VP40 share population coverage of 93.40%, 84.15%, 74.94% and 77.12%, respectively. Based on the other HLA binding specificity and population coverage, seven novel promiscuous epitopes were identified. These 7 promiscuous epitopes from NP, RdRp and GP were found to have world-wide population coverage of more than 95% indicating their potential significance as useful candidates for vaccine design. Epitope conservancy analysis also suggested that most of the peptides are highly conserved (100%) in other virulent Ebola strain (Mayinga-76, Kikwit-95 and Makona-G3816- 2014) and can therefore be further investigated for their immunological relevance and usefulness as vaccine candidates. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Analysis of epitope information related to Bacillus anthracis and Clostridium botulinum

    PubMed Central

    Zarebski, Laura M; Vaughan, Kerrie; Sidney, John; Peters, Bjoern; Grey, Howard; Janda, Kim D; Casadevall, Arturo

    2012-01-01

    We have reviewed the information about epitopes of immunological interest from Clostridium botulinum and Bacillus anthracis, by mining the Immune Epitope Database and Analysis Resource. For both pathogens, the vast majority of epitopes reported to date are derived from a single protein: the protective antigen of B. anthracis and the neurotoxin type A of C. botulinum. A detailed analysis of the data was performed to characterize the function, localization and conservancy of epitopes identified as neutralizing and/or protective. In order to broaden the scope of this analysis, we have also included data describing immune responses against defined fragments (over 50 amino acids long) of the relevant antigens. The scarce information on T-cell determinants and on epitopes from other antigens besides the toxins, highlights a gap in our knowledge and identifies areas for future research. Despite this, several distinct structures at the epitope and fragment level are described herein, which could be potential additions to future vaccines or targets of novel immunotherapeutics and diagnostic reagents. PMID:18251694

  14. Autoantibodies to MUC1 glycopeptides cannot be used as a screening assay for early detection of breast, ovarian, lung or pancreatic cancer

    PubMed Central

    Burford, B; Gentry-Maharaj, A; Graham, R; Allen, D; Pedersen, J W; Nudelman, A S; Blixt, O; Fourkala, E O; Bueti, D; Dawnay, A; Ford, J; Desai, R; David, L; Trinder, P; Acres, B; Schwientek, T; Gammerman, A; Reis, C A; Silva, L; Osório, H; Hallett, R; Wandall, H H; Mandel, U; Hollingsworth, M A; Jacobs, I; Fentiman, I; Clausen, H; Taylor-Papadimitriou, J; Menon, U; Burchell, J M

    2013-01-01

    Background: Autoantibodies have been detected in sera before diagnosis of cancer leading to interest in their potential as screening/early detection biomarkers. As we have found autoantibodies to MUC1 glycopeptides to be elevated in early-stage breast cancer patients, in this study we analysed these autoantibodies in large population cohorts of sera taken before cancer diagnosis. Methods: Serum samples from women who subsequently developed breast cancer, and aged-matched controls, were identified from UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS) and Guernsey serum banks to formed discovery and validation sets. These were screened on a microarray platform of 60mer MUC1 glycopeptides and recombinant MUC1 containing 16 tandem repeats. Additional case–control sets comprised of women who subsequently developed ovarian, pancreatic and lung cancer were also screened on the arrays. Results: In the discovery (273 cases, 273 controls) and the two validation sets (UKCTOCS 426 cases, 426 controls; Guernsey 303 cases and 606 controls), no differences were found in autoantibody reactivity to MUC1 tandem repeat peptide or glycoforms between cases and controls. Furthermore, no differences were observed between ovarian, pancreatic and lung cancer cases and controls. Conclusion: This robust, validated study shows autoantibodies to MUC1 peptide or glycopeptides cannot be used for breast, ovarian, lung or pancreatic cancer screening. This has significant implications for research on the use of MUC1 in cancer detection. PMID:23652307

  15. Elicitation of Neutralizing Antibodies Directed against CD4-Induced Epitope(s) Using a CD4 Mimetic Cross-Linked to a HIV-1 Envelope Glycoprotein

    PubMed Central

    Dey, Antu K.; Burke, Brian; Sun, Yide; Sirokman, Klara; Nandi, Avishek; Hartog, Karin; Lian, Ying; Geonnotti, Anthony R.; Montefiori, David; Franti, Michael; Martin, Grégoire; Carfi, Andrea; Kessler, Pascal; Martin, Loïc; Srivastava, Indresh K.; Barnett, Susan W.

    2012-01-01

    The identification of HIV-1 envelope glycoprotein (Env) structures that can generate broadly neutralizing antibodies (BNAbs) is pivotal to the development of a successful vaccine against HIV-1 aimed at eliciting effective humoral immune responses. To that end, the production of novel Env structure(s) that might induce BNAbs by presentation of conserved epitopes, which are otherwise occluded, is critical. Here, we focus on a structure that stabilizes Env in a conformation representative of its primary (CD4) receptor-bound state, thereby exposing highly conserved “CD4 induced” (CD4i) epitope(s) known to be important for co-receptor binding and subsequent virus infection. A CD4-mimetic miniprotein, miniCD4 (M64U1-SH), was produced and covalently complexed to recombinant, trimeric gp140 envelope glycoprotein (gp140) using site-specific disulfide linkages. The resulting gp140-miniCD4 (gp140-S-S-M64U1) complex was recognized by CD4i antibodies and the HIV-1 co-receptor, CCR5. The gp140-miniCD4 complex elicited the highest titers of CD4i binding antibodies as well as enhanced neutralizing antibodies against Tier 1 viruses as compared to gp140 protein alone following immunization of rabbits. Neutralization against HIV-27312/V434M and additional serum mapping confirm the specific elicitation of antibodies directed to the CD4i epitope(s). These results demonstrate the utility of structure-based approach in improving immunogenic response against specific region, such as the CD4i epitope(s) here, and its potential role in vaccine application. PMID:22291921

  16. Synthetic B-Cell Epitopes Eliciting Cross-Neutralizing Antibodies: Strategies for Future Dengue Vaccine

    PubMed Central

    Poh, Chit Laa; Kirk, Kristin; McBride, William John Hannan; Aaskov, John; Grollo, Lara

    2016-01-01

    Dengue virus (DENV) is a major public health threat worldwide. A key element in protection from dengue fever is the neutralising antibody response. Anti-dengue IgG purified from DENV-2 infected human sera showed reactivity against several peptides when evaluated by ELISA and epitope extraction techniques. A multi-step computational approach predicted six antigenic regions within the E protein of DENV-2 that concur with the 6 epitopes identified by the combined ELISA and epitope extraction approach. The selected peptides representing B-cell epitopes were attached to a known dengue T-helper epitope and evaluated for their vaccine potency. Immunization of mice revealed two novel synthetic vaccine constructs that elicited good humoral immune responses and produced cross-reactive neutralising antibodies against DENV-1, 2 and 3. The findings indicate new directions for epitope mapping and contribute towards the future development of multi-epitope based synthetic peptide vaccine. PMID:27223692

  17. Prediction of linear B-cell epitopes of hepatitis C virus for vaccine development

    PubMed Central

    2015-01-01

    Background High genetic heterogeneity in the hepatitis C virus (HCV) is the major challenge of the development of an effective vaccine. Existing studies for developing HCV vaccines have mainly focused on T-cell immune response. However, identification of linear B-cell epitopes that can stimulate B-cell response is one of the major tasks of peptide-based vaccine development. Owing to the variability in B-cell epitope length, the prediction of B-cell epitopes is much more complex than that of T-cell epitopes. Furthermore, the motifs of linear B-cell epitopes in different pathogens are quite different (e. g. HCV and hepatitis B virus). To cope with this challenge, this work aims to propose an HCV-customized sequence-based prediction method to identify B-cell epitopes of HCV. Results This work establishes an experimentally verified dataset comprising the B-cell response of HCV dataset consisting of 774 linear B-cell epitopes and 774 non B-cell epitopes from the Immune Epitope Database. An interpretable rule mining system of B-cell epitopes (IRMS-BE) is proposed to select informative physicochemical properties (PCPs) and then extracts several if-then rule-based knowledge for identifying B-cell epitopes. A web server Bcell-HCV was implemented using an SVM with the 34 informative PCPs, which achieved a training accuracy of 79.7% and test accuracy of 70.7% better than the SVM-based methods for identifying B-cell epitopes of HCV and the two general-purpose methods. This work performs advanced analysis of the 34 informative properties, and the results indicate that the most effective property is the alpha-helix structure of epitopes, which influences the connection between host cells and the E2 proteins of HCV. Furthermore, 12 interpretable rules are acquired from top-five PCPs and achieve a sensitivity of 75.6% and specificity of 71.3%. Finally, a conserved promising vaccine candidate, PDREMVLYQE, is identified for inclusion in a vaccine against HCV. Conclusions This work

  18. Human dengue virus serotype 2 neutralizing antibodies target two distinct quaternary epitopes

    PubMed Central

    Gallichotte, Emily N.; Baric, Thomas J.; Widman, Douglas G.; Whitehead, Steve; Baric, Ralph S.; de Silva, Aravinda M.

    2018-01-01

    Dengue virus (DENV) infection causes dengue fever, dengue hemorrhagic fever and dengue shock syndrome. It is estimated that a third of the world’s population is at risk for infection, with an estimated 390 million infections annually. Dengue virus serotype 2 (DENV2) causes severe epidemics, and the leading tetravalent dengue vaccine has lower efficacy against DENV2 compared to the other 3 serotypes. In natural DENV2 infections, strongly neutralizing type-specific antibodies provide protection against subsequent DENV2 infection. While the epitopes of some human DENV2 type-specific antibodies have been mapped, it is not known if these are representative of the polyclonal antibody response. Using structure-guided immunogen design and reverse genetics, we generated a panel of recombinant viruses containing amino acid alterations and epitope transplants between different serotypes. Using this panel of recombinant viruses in binding, competition, and neutralization assays, we have finely mapped the epitopes of three human DENV2 type-specific monoclonal antibodies, finding shared and distinct epitope regions. Additionally, we used these recombinant viruses and polyclonal sera to dissect the epitope-specific responses following primary DENV2 natural infection and monovalent vaccination. Our results demonstrate that antibodies raised following DENV2 infection or vaccination circulate as separate populations that neutralize by occupying domain III and domain I quaternary epitopes. The fraction of neutralizing antibodies directed to different epitopes differs between individuals. The identification of these epitopes could potentially be harnessed to evaluate epitope-specific antibody responses as correlates of protective immunity, potentially improving vaccine design. PMID:29481552

  19. Epitope predictions indicate the presence of two distinct types of epitope-antibody-reactivities determined by epitope profiling of intravenous immunoglobulins.

    PubMed

    Luštrek, Mitja; Lorenz, Peter; Kreutzer, Michael; Qian, Zilliang; Steinbeck, Felix; Wu, Di; Born, Nadine; Ziems, Bjoern; Hecker, Michael; Blank, Miri; Shoenfeld, Yehuda; Cao, Zhiwei; Glocker, Michael O; Li, Yixue; Fuellen, Georg; Thiesen, Hans-Jürgen

    2013-01-01

    Epitope-antibody-reactivities (EAR) of intravenous immunoglobulins (IVIGs) determined for 75,534 peptides by microarray analysis demonstrate that roughly 9% of peptides derived from 870 different human protein sequences react with antibodies present in IVIG. Computational prediction of linear B cell epitopes was conducted using machine learning with an ensemble of classifiers in combination with position weight matrix (PWM) analysis. Machine learning slightly outperformed PWM with area under the curve (AUC) of 0.884 vs. 0.849. Two different types of epitope-antibody recognition-modes (Type I EAR and Type II EAR) were found. Peptides of Type I EAR are high in tyrosine, tryptophan and phenylalanine, and low in asparagine, glutamine and glutamic acid residues, whereas for peptides of Type II EAR it is the other way around. Representative crystal structures present in the Protein Data Bank (PDB) of Type I EAR are PDB 1TZI and PDB 2DD8, while PDB 2FD6 and 2J4W are typical for Type II EAR. Type I EAR peptides share predicted propensities for being presented by MHC class I and class II complexes. The latter interaction possibly favors T cell-dependent antibody responses including IgG class switching. Peptides of Type II EAR are predicted not to be preferentially presented by MHC complexes, thus implying the involvement of T cell-independent IgG class switch mechanisms. The high extent of IgG immunoglobulin reactivity with human peptides implies that circulating IgG molecules are prone to bind to human protein/peptide structures under non-pathological, non-inflammatory conditions. A webserver for predicting EAR of peptide sequences is available at www.sysmed-immun.eu/EAR.

  20. Confirmation of a new conserved linear epitope of Lyssavirus nucleoprotein.

    PubMed

    Xinjun, Lv; Xuejun, Ma; Lihua, Wang; Hao, Li; Xinxin, Shen; Pengcheng, Yu; Qing, Tang; Guodong, Liang

    2012-05-01

    Bioinformatics analysis was used to predict potential epitopes of Lyssavirus nucleoprotein and highlighted some distinct differences in the quantity and localization of the epitopes disclosed by epitope analysis of monoclonal antibodies against Lyssavirus nucleoprotein. Bioinformatics analysis showed that the domain containing residues 152-164 of Lyssavirus nucleoprotein was a conserved linear epitope that had not been reported previously. Immunization of two rabbits with the corresponding synthetic peptide conjugated to the Keyhole Limpe hemocyanin (KLH) macromolecule resulted in a titer of anti-peptide antibody above 1:200,000 in rabbit sera as detected by indirect enzyme-linked immunosorbent assay (ELISA). Western blot analysis demonstrated that the anti-peptide antibody recognized denatured Lyssavirus nucleoprotein in sodium dodecylsulfonate-polyacrylate gel electrophoresis (SDS-PAGE). Affinity chromatography purification and FITC-labeling of the anti-peptide antibody in rabbit sera was performed. FITC-labeled anti-peptide antibody could recognize Lyssavirus nucleoprotein in BSR cells and canine brain tissues even at a 1:200 dilution. Residues 152-164 of Lyssavirus nucleoprotein were verified as a conserved linear epitope in Lyssavirus. Copyright © 2012 Elsevier B.V. All rights reserved.

  1. Effects of brevetoxins on murine myeloma SP2/O cells: Aberrant cellular division

    USGS Publications Warehouse

    Han, T.K.; Derby, M.; Martin, D.F.; Wright, S.D.; Dao, M.L.

    2003-01-01

    Massive deaths of manatees (Trichechus manatus latirostris) during the red tide seasons have been attributed to brevetoxins produced by the dinoflagellate Karenia brevis (formerly Ptychodiscus breve and Gymnodinium breve). Although these toxins have been found in macrophages and lymphocytes in the lung, liver, and secondary lymphoid tissues of these animals, the molecular mechanisms of brevetoxicosis have not yet been identified. To investigate the effects of brevetoxins on immune cells, a murine myeloma cell line (SP2/O) was used as a model for in vitro studies. By adding brevetoxins to cultures of the SP2/O cells at concentrations ranging from 20 to 600 ng/ml, an apparent increase in proliferation was observed at around 2 hours post challenge as compared to the unchallenged cell cultures. This was followed by a drop in cell number at around 3 hours, suggesting an aberrant effect of brevetoxins on cellular division, the cells generated at 2 hours being apparently short-lived. In situ immunochemical staining of the SP2/O cells at 1 and 2 hour post challenge showed an accumulation of the toxins in the nucleus. A 21-kDa protein was subsequently isolated from the SP2/O cells as having brevetoxin-binding properties, and immunologically identified as p21, a nuclear factor known to down-regulate cellular proliferation through inhibition of cyclin-dependent kinases. These data are the first on a possible effect of brevetoxins on the cell cycle via binding to p21, a phenomenon that needs to be further investigated and validated in normal immune cells.

  2. Determining Zebrafish Epitope Reactivity to Commercially Available Antibodies.

    PubMed

    Villarreal, Michael A; Biediger, Nicole M; Bonner, Natalie A; Miller, Jennifer N; Zepeda, Samantha K; Ricard, Benjamin J; García, Dana M; Lewis, Karen A

    2017-08-01

    Antibodies raised against mammalian proteins may exhibit cross-reactivity with zebrafish proteins, making these antibodies useful for fish studies. However, zebrafish may express multiple paralogues of similar sequence and size, making them difficult to distinguish by traditional Western blot analysis. To identify the zebrafish proteins that are recognized by an antimammalian antibody, we developed a system to screen putative epitopes by cloning the sequences between the yeast SUMO protein and a C-terminal 6xHis tag. The recombinant fusion protein was expressed in Escherichia coli and analyzed by Western blot to conclusively identify epitopes that exhibit cross-reactivity with the antibodies of interest. This approach can be used to determine the species cross-reactivity and epitope specificity of a wide variety of peptide antigen-derived antibodies.

  3. Quinolone and Glycopeptide Therapy for Infection in Mouse Following Exposure to Mixed-Field Neutron-Gamma-Photon Radiation

    DTIC Science & Technology

    1993-01-01

    prevents mortality, the addition of a glycopeptide can gens such as Staphylococcus spp., evaluating the enhance systemic infection by resistant...bacteria 35 10 15- 20 25 30 60 (including six E.faecalis and five Staphylococcus aureus) L IJ Days (Table 3). Treatment In the third experiment the...5.00 and 5.25Gy and most Staphylococcus spp. were of Streplococcus spp. increased. 3.3. Antimicrobial serum concentrations Antimicrobial serum

  4. Mixed Connective Tissue Disease and Epitope Spreading: An Historical Cohort Study.

    PubMed

    Escolà-Vergé, Laura; Pinal-Fernandez, Iago; Fernandez-Codina, Andreu; Callejas-Moraga, Eduardo L; Espinosa, Juan; Marin, Ana; Labrador-Horrillo, Moises; Selva-O'Callaghan, Albert

    2017-04-01

    Mixed connective tissue disease (MCTD) is characterized by the presence of anti-U1-snRNP autoantibodies and a variable set of associated clinical features. Some MCTD patients test positive over time to autoantibodies against Sm, proteins spatially related with U1-snRNP. This situation has been attributed to expanding of the autoimmune response by a phenomenon known as epitope spreading. Our aim was to study the frequency of this phenomenon in MCTD patients and the specific clinical features of those with epitope spreading. All anti-U1-RNP-positive patients (2010-2015) were retrospectively reviewed, and those meeting the MCTD criteria were included in the study. Patients showing epitope spreading were compared with the remainder of the MCTD cohort. In addition, the clinical features of patients with epitope spreading were compared before and after the phenomenon occurred. Among 72 anti-U1-RNP-positive patients, 40 (37 women) were diagnosed with MCTD. Thirteen MCTD patients (43%) presented epitope spreading, mainly during the first 2 years after the diagnosis of the disease (median, 1.4 years). Patients with epitope spreading had a significantly lower prevalence of skin sclerosis (0% vs. 44%, P = 0.004) and a greater prevalence of interstitial lung disease (46% vs. 15%, P = 0.05) than those without. Arthritis (92% vs. 25%, P = 0.02) and muscle involvement (67% vs. 17%, P = 0.02) were less frequent after epitope spreading had occurred. Epitope spreading is common in MCTD, occurring early after the diagnosis. The clinical manifestations in patients with this phenomenon differ from those without, and their clinical features change after the immunological phenomenon has occurred.

  5. Thyrotropin Receptor Epitope and Human Leukocyte Antigen in Graves’ Disease

    PubMed Central

    Inaba, Hidefumi; De Groot, Leslie J.; Akamizu, Takashi

    2016-01-01

    Graves’ disease (GD) is an organ-specific autoimmune disease, and thyrotropin (TSH) receptor (TSHR) is a major autoantigen in this condition. Since the extracellular domain of human TSHR (TSHR-ECD) is shed into the circulation, TSHR-ECD is a preferentially immunogenic portion of TSHR. Both genetic factors and environmental factors contribute to development of GD. Inheritance of human leukocyte antigen (HLA) genes, especially HLA-DR3, is associated with GD. TSHR-ECD protein is endocytosed into antigen-presenting cells (APCs), and processed to TSHR-ECD peptides. These peptide epitopes bind to HLA-class II molecules, and subsequently the complex of HLA-class II and TSHR-ECD epitope is presented to CD4+ T cells. The activated CD4+ T cells secrete cytokines/chemokines that stimulate B-cells to produce TSAb, and in turn hyperthyroidism occurs. Numerous studies have been done to identify T- and B-cell epitopes in TSHR-ECD, including (1) in silico, (2) in vitro, (3) in vivo, and (4) clinical experiments. Murine models of GD and HLA-transgenic mice have played a pivotal role in elucidating the immunological mechanisms. To date, linear or conformational epitopes of TSHR-ECD, as well as the molecular structure of the epitope-binding groove in HLA-DR, were reported to be related to the pathogenesis in GD. Dysfunction of central tolerance in the thymus, or in peripheral tolerance, such as regulatory T cells, could allow development of GD. Novel treatments using TSHR antagonists or mutated TSHR peptides have been reported to be effective. We review and update the role of immunogenic TSHR epitopes and HLA in GD, and offer perspectives on TSHR epitope specific treatments. PMID:27602020

  6. BEST: Improved Prediction of B-Cell Epitopes from Antigen Sequences

    PubMed Central

    Gao, Jianzhao; Faraggi, Eshel; Zhou, Yaoqi; Ruan, Jishou; Kurgan, Lukasz

    2012-01-01

    Accurate identification of immunogenic regions in a given antigen chain is a difficult and actively pursued problem. Although accurate predictors for T-cell epitopes are already in place, the prediction of the B-cell epitopes requires further research. We overview the available approaches for the prediction of B-cell epitopes and propose a novel and accurate sequence-based solution. Our BEST (B-cell Epitope prediction using Support vector machine Tool) method predicts epitopes from antigen sequences, in contrast to some method that predict only from short sequence fragments, using a new architecture based on averaging selected scores generated from sliding 20-mers by a Support Vector Machine (SVM). The SVM predictor utilizes a comprehensive and custom designed set of inputs generated by combining information derived from the chain, sequence conservation, similarity to known (training) epitopes, and predicted secondary structure and relative solvent accessibility. Empirical evaluation on benchmark datasets demonstrates that BEST outperforms several modern sequence-based B-cell epitope predictors including ABCPred, method by Chen et al. (2007), BCPred, COBEpro, BayesB, and CBTOPE, when considering the predictions from antigen chains and from the chain fragments. Our method obtains a cross-validated area under the receiver operating characteristic curve (AUC) for the fragment-based prediction at 0.81 and 0.85, depending on the dataset. The AUCs of BEST on the benchmark sets of full antigen chains equal 0.57 and 0.6, which is significantly and slightly better than the next best method we tested. We also present case studies to contrast the propensity profiles generated by BEST and several other methods. PMID:22761950

  7. Mast Cells Produce a Unique Chondroitin Sulfate Epitope.

    PubMed

    Farrugia, Brooke L; Whitelock, John M; O'Grady, Robert; Caterson, Bruce; Lord, Megan S

    2016-02-01

    The granules of mast cells contain a myriad of mediators that are stored and protected by the sulfated glycosaminoglycan (GAG) chains that decorate proteoglycans. Whereas heparin is the GAG predominantly associated with mast cells, mast cell proteoglycans are also decorated with heparan sulfate and chondroitin sulfate (CS). This study investigated a unique CS structure produced by mast cells that was detected with the antibody clone 2B6 in the absence of chondroitinase ABC digestion. Mast cells in rodent tissue sections were characterized using toluidine blue, Leder stain and the presence of mast cell tryptase. The novel CS epitope was identified in rodent tissue sections and localized to cells that were morphologically similar to cells chemically identified as mast cells. The rodent mast cell-like line RBL-2H3 was also shown to express the novel CS epitope. This epitope co-localized with multiple CS proteoglycans in both rodent tissue and RBL-2H3 cultured cells. These findings suggest that the novel CS epitope that decorates mast cell proteoglycans may play a role in the way these chains are structured in mast cells. © 2016 The Histochemical Society.

  8. Conformational Occlusion of Blockade Antibody Epitopes, a Novel Mechanism of GII.4 Human Norovirus Immune Evasion.

    PubMed

    Lindesmith, Lisa C; Mallory, Michael L; Debbink, Kari; Donaldson, Eric F; Brewer-Jensen, Paul D; Swann, Excel W; Sheahan, Timothy P; Graham, Rachel L; Beltramello, Martina; Corti, Davide; Lanzavecchia, Antonio; Baric, Ralph S

    2018-01-01

    Extensive antigenic diversity within the GII.4 genotype of human norovirus is a major driver of pandemic emergence and a significant obstacle to development of cross-protective immunity after natural infection and vaccination. However, human and mouse monoclonal antibody studies indicate that, although rare, antibodies to conserved GII.4 blockade epitopes are generated. The mechanisms by which these epitopes evade immune surveillance are uncertain. Here, we developed a new approach for identifying conserved GII.4 norovirus epitopes. Utilizing a unique set of virus-like particles (VLPs) representing the in vivo -evolved sequence diversity within an immunocompromised person, we identify key residues within epitope F, a conserved GII.4 blockade antibody epitope. The residues critical for antibody binding are proximal to evolving blockade epitope E. Like epitope F, antibody blockade of epitope E was temperature sensitive, indicating that particle conformation regulates antibody access not only to the conserved GII.4 blockade epitope F but also to the evolving epitope E. These data highlight novel GII.4 mechanisms to protect blockade antibody epitopes, map essential residues of a GII.4 conserved epitope, and expand our understanding of how viral particle dynamics may drive antigenicity and antibody-mediated protection by effectively shielding blockade epitopes. Our data support the notion that GII.4 particle breathing may well represent a major mechanism of humoral immune evasion supporting cyclic pandemic virus persistence and spread in human populations. IMPORTANCE In this study, we use norovirus virus-like particles to identify key residues of a conserved GII.4 blockade antibody epitope. Further, we identify an additional GII.4 blockade antibody epitope to be occluded, with antibody access governed by temperature and particle dynamics. These findings provide additional support for particle conformation-based presentation of binding residues mediated by a particle

  9. Rapid Endolysosomal Escape and Controlled Intracellular Trafficking of Cell Surface Mimetic Quantum-Dots-Anchored Peptides and Glycopeptides.

    PubMed

    Tan, Roger S; Naruchi, Kentaro; Amano, Maho; Hinou, Hiroshi; Nishimura, Shin-Ichiro

    2015-09-18

    A novel strategy for the development of a high performance nanoparticules platform was established by means of cell surface mimetic quantum-dots (QDs)-anchored peptides/glycopeptides, which was developed as a model system for nanoparticle-based drug delivery (NDD) vehicles with defined functions helping the specific intracellular trafficking after initial endocytosis. In this paper, we proposed a standardized protocol for the preparation of multifunctional QDs that allows for efficient cellular uptake and rapid escaping from the endolysosomal system and subsequent cytoplasmic molecular delivery to the target cellular compartment. Chemoselective ligation of the ketone-functionalized hexahistidine derivative facilitated both efficient endocytic entry and rapid endolysosomal escape of the aminooxy/phosphorylcholine self-assembled monolayer-coated QDs (AO/PCSAM-QDs) to the cytosol in various cell lines such as human normal and cancer cells, while modifications of these QDs with cell-penetrating arginine-rich peptides showed poor cellular uptake and induced self-aggregation of AO/PCSAM-QDs. Combined use of hexahistidylated AO/PCSAM-QDs with serglycine-like glycopeptides, namely synthetic proteoglycan initiators (PGIs), elicited the entry and controlled intracellular trafficking, Golgi localization, and also excretion of these nanoparticles, which suggested that the present approach would provide an ideal platform for the design of high performance NDD systems.

  10. Humoral Epitope Spreading in Autoimmune Bullous Diseases

    PubMed Central

    Didona, Dario; Di Zenzo, Giovanni

    2018-01-01

    Autoimmune blistering diseases are characterized by autoantibodies against structural adhesion proteins of the skin and mucous membranes. Extensive characterization of their autoantibody targets has improved understanding of pathogenesis and laid the basis for the study of antigens/epitopes diversification, a process termed epitope spreading (ES). In this review, we have reported and discussed ES phenomena in autoimmune bullous diseases and underlined their functional role in disease pathogenesis. A functional ES has been proposed: (1) in bullous pemphigoid patients and correlates with the initial phase of the disease, (2) in pemphigus vulgaris patients with mucosal involvement during the clinical transition to a mucocutaneous form, (3) in endemic pemphigus foliaceus, underlining its role in disease pathogenesis, and (4) in numerous cases of disease transition associated with an intermolecular diversification of immune response. All these findings could give useful information to better understand autoimmune disease pathogenesis and to design antigen/epitope specific therapeutic approaches. PMID:29719538

  11. A General Synthetic Approach for Designing Epitope Targeted Macrocyclic Peptide Ligands.

    PubMed

    Das, Samir; Nag, Arundhati; Liang, JingXin; Bunck, David N; Umeda, Aiko; Farrow, Blake; Coppock, Matthew B; Sarkes, Deborah A; Finch, Amethist S; Agnew, Heather D; Pitram, Suresh; Lai, Bert; Yu, Mary Beth; Museth, A Katrine; Deyle, Kaycie M; Lepe, Bianca; Rodriguez-Rivera, Frances P; McCarthy, Amy; Alvarez-Villalonga, Belen; Chen, Ann; Heath, John; Stratis-Cullum, Dimitra N; Heath, James R

    2015-11-02

    We describe a general synthetic strategy for developing high-affinity peptide binders against specific epitopes of challenging protein biomarkers. The epitope of interest is synthesized as a polypeptide, with a detection biotin tag and a strategically placed azide (or alkyne) presenting amino acid. This synthetic epitope (SynEp) is incubated with a library of complementary alkyne or azide presenting peptides. Library elements that bind the SynEp in the correct orientation undergo the Huisgen cycloaddition, and are covalently linked to the SynEp. Hit peptides are tested against the full-length protein to identify the best binder. We describe development of epitope-targeted linear or macrocycle peptide ligands against 12 different diagnostic or therapeutic analytes. The general epitope targeting capability for these low molecular weight synthetic ligands enables a range of therapeutic and diagnostic applications, similar to those of monoclonal antibodies. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. A novel multi-variant epitope ensemble vaccine against avian leukosis virus subgroup J.

    PubMed

    Wang, Xiaoyu; Zhou, Defang; Wang, Guihua; Huang, Libo; Zheng, Qiankun; Li, Chengui; Cheng, Ziqiang

    2017-12-04

    The hypervariable antigenicity and immunosuppressive features of avian leukosis virus subgroup J (ALV-J) has led to great challenges to develop effective vaccines. Epitope vaccine will be a perspective trend. Previously, we identified a variant antigenic neutralizing epitope in hypervariable region 1 (hr1) of ALV-J, N-LRDFIA/E/TKWKS/GDDL/HLIRPYVNQS-C. BLAST analysis showed that the mutation of A, E, T and H in this epitope cover 79% of all ALV-J strains. Base on this data, we designed a multi-variant epitope ensemble vaccine comprising the four mutation variants linked with glycine and serine. The recombinant multi-variant epitope gene was expressed in Escherichia coli BL21. The expressed protein of the variant multi-variant epitope gene can react with positive sera and monoclonal antibodies of ALV-J, while cannot react with ALV-J negative sera. The multi-variant epitope vaccine that conjugated Freund's adjuvant complete/incomplete showed high immunogenicity that reached the titer of 1:64,000 at 42 days post immunization and maintained the immune period for at least 126 days in SPF chickens. Further, we demonstrated that the antibody induced by the variant multi-variant ensemble epitope vaccine recognized and neutralized different ALV-J strains (NX0101, TA1, WS1, BZ1224 and BZ4). Protection experiment that was evaluated by clinical symptom, viral shedding, weight gain, gross and histopathology showed 100% chickens that inoculated the multi-epitope vaccine were well protected against ALV-J challenge. The result shows a promising multi-variant epitope ensemble vaccine against hypervariable viruses in animals. Copyright © 2017 Elsevier Ltd. All rights reserved.

  13. Mask-induced aberration in EUV lithography

    NASA Astrophysics Data System (ADS)

    Nakajima, Yumi; Sato, Takashi; Inanami, Ryoichi; Nakasugi, Tetsuro; Higashiki, Tatsuhiko

    2009-04-01

    We estimated aberrations using Zernike sensitivity analysis. We found the difference of the tolerated aberration with line direction for illumination. The tolerated aberration of perpendicular line for illumination is much smaller than that of parallel line. We consider this difference to be attributable to the mask 3D effect. We call it mask-induced aberration. In the case of the perpendicular line for illumination, there was a difference in CD between right line and left line without aberration. In this report, we discuss the possibility of pattern formation in NA 0.25 generation EUV lithography tool. In perpendicular pattern for EUV light, the dominant part of aberration is mask-induced aberration. In EUV lithography, pattern correction based on the mask topography effect will be more important.

  14. Replication-Competent Foamy Virus Vaccine Vectors as Novel Epitope Scaffolds for Immunotherapy

    PubMed Central

    Lei, Janet; Osen, Wolfram; Gardyan, Adriane; Hotz-Wagenblatt, Agnes; Wei, Guochao; Gissmann, Lutz; Eichmüller, Stefan; Löchelt, Martin

    2015-01-01

    The use of whole viruses as antigen scaffolds is a recent development in vaccination that improves immunogenicity without the need for additional adjuvants. Previous studies highlighted the potential of foamy viruses (FVs) in prophylactic vaccination and gene therapy. Replication-competent FVs can trigger immune signaling and integrate into the host genome, resulting in persistent antigen expression and a robust immune response. Here, we explored feline foamy virus (FFV) proteins as scaffolds for therapeutic B and T cell epitope delivery in vitro. Infection- and cancer-related B and T cell epitopes were grafted into FFV Gag, Env, or Bet by residue replacement, either at sites of high local sequence homology between the epitope and the host protein or in regions known to tolerate sequence alterations. Modified proviruses were evaluated in vitro for protein steady state levels, particle release, and virus titer in permissive cells. Modification of Gag and Env was mostly detrimental to their function. As anticipated, modification of Bet had no impact on virion release and affected virus titers of only some recombinants. Further evaluation of Bet as an epitope carrier was performed using T cell epitopes from the model antigen chicken ovalbumin (OVA), human tyrosinase-related protein 2 (TRP-2), and oncoprotein E7 of human papillomavirus type 16 (HPV16E7). Transfection of murine cells with constructs encoding Bet-epitope chimeric proteins led to efficient MHC-I-restricted epitope presentation as confirmed by interferon-gamma enzyme-linked immunospot assays using epitope-specific cytotoxic T lymphocyte (CTL) lines. FFV infection-mediated transduction of cells with epitope-carrying Bet also induced T-cell responses, albeit with reduced efficacy, in a process independent from the presence of free peptides. We show that primate FV Bet is also a promising T cell epitope carrier for clinical translation. The data demonstrate the utility of replication-competent and -attenuated

  15. Using a combined computational-experimental approach to predict antibody-specific B cell epitopes.

    PubMed

    Sela-Culang, Inbal; Benhnia, Mohammed Rafii-El-Idrissi; Matho, Michael H; Kaever, Thomas; Maybeno, Matt; Schlossman, Andrew; Nimrod, Guy; Li, Sheng; Xiang, Yan; Zajonc, Dirk; Crotty, Shane; Ofran, Yanay; Peters, Bjoern

    2014-04-08

    Antibody epitope mapping is crucial for understanding B cell-mediated immunity and required for characterizing therapeutic antibodies. In contrast to T cell epitope mapping, no computational tools are in widespread use for prediction of B cell epitopes. Here, we show that, utilizing the sequence of an antibody, it is possible to identify discontinuous epitopes on its cognate antigen. The predictions are based on residue-pairing preferences and other interface characteristics. We combined these antibody-specific predictions with results of cross-blocking experiments that identify groups of antibodies with overlapping epitopes to improve the predictions. We validate the high performance of this approach by mapping the epitopes of a set of antibodies against the previously uncharacterized D8 antigen, using complementary techniques to reduce method-specific biases (X-ray crystallography, peptide ELISA, deuterium exchange, and site-directed mutagenesis). These results suggest that antibody-specific computational predictions and simple cross-blocking experiments allow for accurate prediction of residues in conformational B cell epitopes. Copyright © 2014 Elsevier Ltd. All rights reserved.

  16. Epitope enhancement for immunohistochemical demonstration of tartrate-resistant acid phosphatase.

    PubMed

    Janckila, A J; Lear, S C; Martin, A W; Yam, L T

    1996-03-01

    We have developed a monoclonal antibody (9C5) for immunohistochemical localization of tartrate-resistant acid phosphatase (TRAcP). This antibody reacts with a denatured epitope of TRAcP and requires enhancement methods to promote antigenicity in paraffin-embedded tissues. We used this antibody to systematically examine proteolytic digestion and heat denaturation conditions for epitope enhancement in both paraffin sections and fixed smears. The goal was to increase the sensitivity of the immunohistochemical stain for TRAcP. Optimal conditions for proteolytic digestion were established. Denaturation in a conventional boiling water bath was compared to microwave irradiation in several commonly used solutions. Immunohistochemistry was compared directly to TRAcP cytochemistry in fixed smears from hairy cell leukemia specimens to gauge the level of sensitivity of our improved method. Attempts were made to "retrieve" the 9C5 epitope from overfixed tissues and aged smears. Maximal immunoreactivity of TRAcP was achieved by microwave irradiation in a citrate or Tris buffer of pH 6.0-8.0 without the need for a subsequent protease digestion step. With this method of epitope enhancement, immunohistochemistry with antibody 9C5 was as sensitive as direct cytochemical staining of TRAcP activity. However, once a tissue specimen had been overfixed or a smear stored for a year or more, the 9C5 epitope was no longer retrievable. The key element in epitope enhancement for 9C5 immunohistochemistry is heat denaturation of the target epitope. Immunohistochemistry of TRAcP in paraffin sections would be a great asset to the study of specialized forms of the monocyte/macrophage lineage and to the process of macrophage activation. It would also provide another means for more precise evaluation of residual disease in bone marrow of patients treated for hairy cell leukemia.

  17. Enterovirus 71 viral capsid protein linear epitopes: Identification and characterization

    PubMed Central

    2012-01-01

    Background To characterize the human humoral immune response against enterovirus 71 (EV71) infection and map human epitopes on the viral capsid proteins. Methods A series of 256 peptides spanning the capsid proteins (VP1, VP2, VP3) of BJ08 strain (genomic C4) were synthesized. An indirect enzyme-linked immunosorbent assay (ELISA) was carried out to detect anti-EV71 IgM and IgG in sera of infected children in acute or recovery phase. The partially overlapped peptides contained 12 amino acids and were coated in the plate as antigen (0.1 μg/μl). Sera from rabbits immunized with inactivated BJ08 virus were also used to screen the peptide panel. Results A total of 10 human anti-EV71 IgM epitopes (vp1-14 in VP1; vp2-6, 21, 40 and 50 in VP2 and vp3-10, 12, 15, 24 and 75 in VP3) were identified in acute phase sera. In contrast, only one anti-EV71 IgG epitope in VP1 (vp1-15) was identified in sera of recovery stage. Four rabbit anti-EV71 IgG epitopes (vp1-14, 31, 54 and 71) were identified and mapped to VP1. Conclusion These data suggested that human IgM epitopes were mainly mapped to VP2 and VP3 with multi-epitope responses occurred at acute infection, while the only IgG epitope located on protein VP1 was activated in recovery phase sera. The dynamic changes of humoral immune response at different stages of infection may have public health significance in evaluation of EV71 vaccine immunogenicity and the clinical application of diagnostic reagents. PMID:22264266

  18. Preparation of hydrophilic monolithic capillary column by in situ photo-polymerization of N-vinyl-2-pyrrolidinone and acrylamide for highly selective and sensitive enrichment of N-linked glycopeptides.

    PubMed

    Jiang, Hao; Yuan, Huiming; Qu, Yanyan; Liang, Yu; Jiang, Bo; Wu, Qi; Deng, Nan; Liang, Zhen; Zhang, Lihua; Zhang, Yukui

    2016-01-01

    In this study, a novel kind of amide functionalized hydrophilic monolith was synthesized by the in situ photo-polymerization of N-vinyl-2-pyrrolidinone (NVP), acrylamide (AM), and N, N'-methylenebisacrylamide (MBA) in a UV transparent capillary, and successfully applied for hydrophilic interaction chromatography (HILIC) based enrichment of N-linked glycopeptides. With 2 μg of the tryptic digests of IgG as the sample, after enrichment, 18 glycopeptides could be identified by MALDI-TOF/TOF MS analysis. Furthermore, with the mixture of BSA and IgG digests (10,000:1, m/m) as the sample, 6 N-linked glycopeptides were unambiguously identified after enrichment, indicating the high selectivity and good specificity of such material. Moreover, such a monolithic capillary column was also applied for the N-glycosylation sites profiling of 6 μg protein digests from HeLa cells and 1 μL human serum. In total, 530 and 262 unique N-glycosylated peptides were identified, respectively, corresponding to 282 and 124N-glycoproteins, demonstrating its great potential for the large scale glycoproteomics analysis. Copyright © 2015 Elsevier B.V. All rights reserved.

  19. Broadly neutralizing epitopes in the Plasmodium vivax vaccine candidate Duffy Binding Protein

    DOE PAGES

    Chen, Edwin; Salinas, Nichole D.; Huang, Yining; ...

    2016-05-18

    Plasmodium vivax Duffy Binding Protein (PvDBP) is the most promising vaccine candidate for P. vivax malaria. The polymorphic nature of PvDBP induces strain-specific immune responses, however, and the epitopes of broadly neutralizing antibodies are unknown. These features hamper the rational design of potent DBP-based vaccines and necessitate the identification of globally conserved epitopes. Using X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange mass spectrometry, and mutational mapping, we have defined epitopes for three inhibitory mAbs (mAbs 2D10, 2H2, and 2C6) and one noninhibitory mAb (3D10) that engage DBP. These studies expand the currently known inhibitory epitope repertoire by establishing protective motifsmore » in subdomain three outside the receptor-binding and dimerization residues of DBP, and introduce globally conserved protective targets. All of the epitopes are highly conserved among DBP alleles. In conclusion, the identification of broadly conserved epitopes of inhibitory antibodies provides critical motifs that should be retained in the next generation of potent vaccines for P. vivax malaria.« less

  20. Broadly neutralizing epitopes in the Plasmodium vivax vaccine candidate Duffy Binding Protein

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chen, Edwin; Salinas, Nichole D.; Huang, Yining

    Plasmodium vivax Duffy Binding Protein (PvDBP) is the most promising vaccine candidate for P. vivax malaria. The polymorphic nature of PvDBP induces strain-specific immune responses, however, and the epitopes of broadly neutralizing antibodies are unknown. These features hamper the rational design of potent DBP-based vaccines and necessitate the identification of globally conserved epitopes. Using X-ray crystallography, small-angle X-ray scattering, hydrogen-deuterium exchange mass spectrometry, and mutational mapping, we have defined epitopes for three inhibitory mAbs (mAbs 2D10, 2H2, and 2C6) and one noninhibitory mAb (3D10) that engage DBP. These studies expand the currently known inhibitory epitope repertoire by establishing protective motifsmore » in subdomain three outside the receptor-binding and dimerization residues of DBP, and introduce globally conserved protective targets. All of the epitopes are highly conserved among DBP alleles. In conclusion, the identification of broadly conserved epitopes of inhibitory antibodies provides critical motifs that should be retained in the next generation of potent vaccines for P. vivax malaria.« less

  1. Malondialdehyde epitopes as targets of immunity and the implications for atherosclerosis

    PubMed Central

    Binder, Christoph J.

    2018-01-01

    Accumulating evidence suggests that oxidation-specific epitopes (OSEs) constitute a novel class of damage-associated molecular patterns (DAMPs) generated during high oxidative stress but also in the physiological process of apoptosis. To deal with the potentially harmful consequences of such epitopes, the immune system has developed several mechanisms to protect from OSEs and to orchestrate their clearance, including IgM natural antibodies and both cellular and membrane-bound receptors. Here, we focus on malondialdehyde (MDA) epitopes as prominent examples of OSEs that trigger both innate and adaptive immune responses. First, we review the mechanism of MDA generation, the different types of adducts on various biomolecules and provide relevant examples for physiological carriers of MDA such as apoptotic cells, microvesicles (MV) or oxidized low-density lipoproteins (LDL). Based on recent insights, we argue that MDA epitopes contribute to the maintenance of homeostatic functions by acting as markers of elevated oxidative stress and tissue damage. We discuss multiple lines of evidence that MDA epitopes are pro-inflammatory and thus important targets of innate and adaptive immune responses. Finally, we illustrate the relevance of MDA epitopes in human pathologies by describing their capacity to drive inflammatory processes in atherosclerosis and highlighting protective mechanisms of immunity that could be exploited for therapeutic purposes. PMID:27235680

  2. In silico design of Mycobacterium tuberculosis epitope ensemble vaccines.

    PubMed

    Shah, Preksha; Mistry, Jaymisha; Reche, Pedro A; Gatherer, Derek; Flower, Darren R

    2018-05-01

    Effective control of Mycobacterium tuberculosis is a global necessity. In 2015, tuberculosis (TB) caused more deaths than HIV. Considering the increasing prevalence of multi-drug resistant forms of M. tuberculosis, the need for effective TB vaccines becomes imperative. Currently, the only licensed TB vaccine is Bacillus Calmette-Guérin (BCG). Yet, BCG has many drawbacks limiting its efficacy and applicability. We applied advanced computational procedures to derive a universal TB vaccine and one targeting East Africa. Our approach selects an optimal set of highly conserved, experimentally validated epitopes, with high projected population coverage (PPC). Through rigorous data analysis, five different potential vaccine combinations were selected each with PPC above 80% for East Africa and above 90% for the World. Two potential vaccines only contained CD8+ epitopes, while the others included both CD4+ and CD8+ epitopes. Our prime vaccine candidate was a putative seven-epitope ensemble comprising: SRGWSLIKSVRLGNA, KPRIITLTMNPALDI, AAHKGLMNIALAISA, FPAGGSTGSL, MLLAVTVSL, QSSFYSDW and KMRCGAPRY, with a 97.4% global PPC and a 92.7% East African PPC. Copyright © 2018 Elsevier Ltd. All rights reserved.

  3. High-Throughput Epitope Binning Assays on Label-Free Array-Based Biosensors Can Yield Exquisite Epitope Discrimination That Facilitates the Selection of Monoclonal Antibodies with Functional Activity

    PubMed Central

    Abdiche, Yasmina Noubia; Miles, Adam; Eckman, Josh; Foletti, Davide; Van Blarcom, Thomas J.; Yeung, Yik Andy; Pons, Jaume; Rajpal, Arvind

    2014-01-01

    Here, we demonstrate how array-based label-free biosensors can be applied to the multiplexed interaction analysis of large panels of analyte/ligand pairs, such as the epitope binning of monoclonal antibodies (mAbs). In this application, the larger the number of mAbs that are analyzed for cross-blocking in a pairwise and combinatorial manner against their specific antigen, the higher the probability of discriminating their epitopes. Since cross-blocking of two mAbs is necessary but not sufficient for them to bind an identical epitope, high-resolution epitope binning analysis determined by high-throughput experiments can enable the identification of mAbs with similar but unique epitopes. We demonstrate that a mAb's epitope and functional activity are correlated, thereby strengthening the relevance of epitope binning data to the discovery of therapeutic mAbs. We evaluated two state-of-the-art label-free biosensors that enable the parallel analysis of 96 unique analyte/ligand interactions and nearly ten thousand total interactions per unattended run. The IBIS-MX96 is a microarray-based surface plasmon resonance imager (SPRi) integrated with continuous flow microspotting technology whereas the Octet-HTX is equipped with disposable fiber optic sensors that use biolayer interferometry (BLI) detection. We compared their throughput, versatility, ease of sample preparation, and sample consumption in the context of epitope binning assays. We conclude that the main advantages of the SPRi technology are its exceptionally low sample consumption, facile sample preparation, and unparalleled unattended throughput. In contrast, the BLI technology is highly flexible because it allows for the simultaneous interaction analysis of 96 independent analyte/ligand pairs, ad hoc sensor replacement and on-line reloading of an analyte- or ligand-array. Thus, the complementary use of these two platforms can expedite applications that are relevant to the discovery of therapeutic mAbs, depending

  4. Camera processing with chromatic aberration.

    PubMed

    Korneliussen, Jan Tore; Hirakawa, Keigo

    2014-10-01

    Since the refractive index of materials commonly used for lens depends on the wavelengths of light, practical camera optics fail to converge light to a single point on an image plane. Known as chromatic aberration, this phenomenon distorts image details by introducing magnification error, defocus blur, and color fringes. Though achromatic and apochromatic lens designs reduce chromatic aberration to a degree, they are complex and expensive and they do not offer a perfect correction. In this paper, we propose a new postcapture processing scheme designed to overcome these problems computationally. Specifically, the proposed solution is comprised of chromatic aberration-tolerant demosaicking algorithm and post-demosaicking chromatic aberration correction. Experiments with simulated and real sensor data verify that the chromatic aberration is effectively corrected.

  5. Corneal higher-order aberrations and higher-order Strehl ratio after aberration-free ablation profile to treat compound myopic astigmatism.

    PubMed

    Brenner, Luis F

    2015-12-01

    To evaluate the changes in corneal higher-order aberrations (HOAs) and their impact on corneal higher-order Strehl ratio after aberration-free ablation profile. Verter Institute, H. Olhos, São Paulo, Brazil. Prospective interventional study. Eyes that had aberration-free myopic ablation were divided into 3 groups, based on the spherical equivalent (SE). The corneal HOAs and higher-order Strehl ratios were calculated before surgery and 3 months after surgery. The postoperative uncorrected-distance visual acuity, corrected-distance visual acuity, and SE did not present statistical differences among groups (88 eyes, P > .05). For a 6 mm pupil, the corneal HOA showed a mean increase of 0.17 μm (range 0.39 to 0.56 μm) (P < .001) and the corneal higher-order Strehl ratio presented a reduction of 0.03 (from 0.25 to 0.22) (P = .001). The following consistent linear predictive model was obtained: corneal HOA induction = 1.474 - 0.032 × SE - 0.225 × OZ, where OZ is the optical zone (R(2) = 0.49, adjusted R(2) = 0.48, P < .001). The corneal HOAs and the higher-order Strehl ratios deteriorated after moderate and high myopic ablations. The worsening in corneal aberrations and optical quality were related to the magnitude of the intended correction and did not affect high-contrast visual performance. The OZ was the only modifiable parameter capable to restrain the optical quality loss. The author has no financial or proprietary interest in any material or method mentioned. Copyright © 2015 ASCRS and ESCRS. Published by Elsevier Inc. All rights reserved.

  6. Accuracy of the VITEK 2 System To Detect Glycopeptide Resistance in Enterococci

    PubMed Central

    van den Braak, Nicole; Goessens, Wil; van Belkum, Alex; Verbrugh, Henri A.; Endtz, Hubert P.

    2001-01-01

    We evaluated the accuracy of the VITEK 2 fully automated system to detect and identify glycopeptide-resistant enterococci (GRE) compared to a reference agar dilution method. The sensitivity of vancomycin susceptibility testing with VITEK 2 for the detection of vanA, vanB, and vanC1 strains was 100%. The sensitivity of vancomycin susceptibility testing of vanC2 strains was 77%. The sensitivity of teicoplanin susceptibility testing of vanA strains was 90%. Of 80 vanC enterococci, 78 (98%) were correctly identified by VITEK 2 as Enterococcus gallinarum/Enterococcus casseliflavus. Since the identification and susceptibility data are produced within 3 and 8 h, respectively, VITEK 2 appears a fast and reliable method for detection of GRE in microbiology laboratories. PMID:11136798

  7. A novel Minimalist Cell-Free MHC Class II Antigen Processing System Identifies Immunodominant Epitopes

    PubMed Central

    Hartman, Isamu Z.; Kim, AeRyon; Cotter, Robert J.; Walter, Kimberly; Dalai, Sarat K.; Boronina, Tatiana; Griffith, Wendell; Schwenk, Robert; Lanar, David E.; Krzych, Urszula; Cole, Robert N.; Sadegh-Nasseri, Scheherazade

    2010-01-01

    Immunodominance is defined as restricted responsiveness of T cells to a few selected epitopes from complex antigens. Strategies currently used for elucidating CD4+ T cell epitopes are inadequate. To understand the mechanism of epitope selection for helper T cells, we established a cell-free antigen processing system composed of defined proteins: MHC class II, cathepsins, and HLA-DM. Our minimalist system successfully identified the physiologically selected immunodominant epitopes of model antigens, HA1 from influenza virus (A/Texas/1/77) and type II collagen. When applied for de novo epitope identification to a malaria antigen, or HA1 from H5N1 virus (Avian Flu), the system selected a single epitope from each protein that were confirmed to be immunodominant by their capacity to activate CD4+ T cells in HLA-DR1 positive human volunteers or transgenic mice immunized with the corresponding proteins. Thus, we provide a powerful new tool for the identification of physiologically relevant helper T cell epitopes from antigens. PMID:21037588

  8. CYTOMEGALOVIRUS VECTORS VIOLATE CD8+ T CELL EPITOPE RECOGNITION PARADIGMS

    PubMed Central

    Hansen, Scott G.; Sacha, Jonah B.; Hughes, Colette M.; Ford, Julia C.; Burwitz, Benjamin J.; Scholz, Isabel; Gilbride, Roxanne M.; Lewis, Matthew S.; Gilliam, Awbrey N.; Ventura, Abigail B.; Malouli, Daniel; Xu, Guangwu; Richards, Rebecca; Whizin, Nathan; Reed, Jason S.; Hammond, Katherine B.; Fischer, Miranda; Turner, John M.; Legasse, Alfred W.; Axthelm, Michael K.; Edlefsen, Paul T.; Nelson, Jay A.; Lifson, Jeffrey D.; Früh, Klaus; Picker, Louis J.

    2013-01-01

    CD8+ T cell responses focus on a small fraction of pathogen- or vaccine-encoded peptides, and for some pathogens, these restricted recognition hierarchies limit the effectiveness of anti-pathogen immunity. We found that simian immunodeficiency virus (SIV) protein-expressing Rhesus Cytomegalovirus (RhCMV) vectors elicit SIV-specific CD8+ T cells that recognize unusual, diverse and highly promiscuous epitopes, including dominant responses to epitopes restricted by class II major histocompatibility complex (MHC) molecules. Induction of canonical SIV epitope-specific CD8+ T cell responses is suppressed by the RhCMV-encoded Rh189 (US11) gene, and the promiscuous MHC class I- and class II-restricted CD8+ T cell responses only occur in the absence of the Rh157.4-.6 (UL128-131) genes. Thus, CMV vectors can be genetically programmed to achieve distinct patterns of CD8+ T cell epitope recognition. PMID:23704576

  9. Prediction of common epitopes on hemagglutinin of the influenza A virus (H1 subtype).

    PubMed

    Guo, Chunyan; Xie, Xin; Li, Huijin; Zhao, Penghua; Zhao, Xiangrong; Sun, Jingying; Wang, Haifang; Liu, Yang; Li, Yan; Hu, Qiaoxia; Hu, Jun; Li, Yuan

    2015-02-01

    Influenza A virus infection is a persistent threat to public health worldwide due to hemagglutinin (HA) variation. Current vaccines against influenza A virus provide immunity to viral isolates similar to vaccine strains. Antibodies against common epitopes provide immunity to diverse influenza virus strains and protect against future pandemic influenza. Therefore, it is vital to analyze common HA antigenic epitopes of influenza virus. In this study, 14 strains of monoclonal antibodies with high sensitivity to common epitopes of influenza virus antigens identified in our previous study were selected as the tool to predict common HA epitopes. The common HA antigenic epitopes were divided into four categories by ELISA blocking experiments, and separately, into three categories according to the preliminary results of computer simulation. Comparison between the results of computer simulations and ELISA blocking experiments indicated that at least two classes of common epitopes are present in influenza virus HA. This study provides experimental data for improving the prediction of HA epitopes of influenza virus (H1 subtype) and the development of a potential universal vaccine as well as a novel approach for the prediction of epitopes on other pathogenic microorganisms. Copyright © 2014 Elsevier Inc. All rights reserved.

  10. Breast Mucin Tumor-Specific Epitopes for Cancer Immunotherapy

    DTIC Science & Technology

    1998-09-01

    reactivity with tumor-specific monoclonal antibodies show that antigenicity is maximized with the 40 amino acid MUC1-mtr2. By contrast, the MUC1-mtr3...associated mucins (7). The presence of tumor-specific epitopes is evidenced by the development of many monoclonal antibodies (mAb) that recognize...P1-P5 in the tandem repeat sequence (7). This epitope was identified by competition of antibody binding to tumor- specific mucin by synthetic

  11. Developmental Regulation of a Plasma Membrane Arabinogalactan Protein Epitope in Oilseed Rape Flowers.

    PubMed Central

    Pennell, RI; Janniche, L; Kjellbom, P; Scofield, GN; Peart, JM; Roberts, K

    1991-01-01

    We have identified and characterized the temporal and spatial regulation of a plasma membrane arabinogalactan protein epitope during development of the aerial parts of oilseed rape using the monoclonal antibody JIM8. The JIM8 epitope is expressed by the first cells of the embryo and by certain cells in the sexual organs of flowers. During embryogenesis, the JIM8 epitope ceases to be expressed by the embryo proper but is still found in the suspensor. During differentiation of the stamens and carpels, expression of the JIM8 epitope progresses from one cell type to another, ultimately specifying the endothecium and sperm cells, the nucellar epidermis, synergid cells, and the egg cell. This complex temporal sequence demonstrates rapid turnover of the JIM8 epitope. There is no direct evidence for any cell-inductive process in plant development. However, if cell-cell interactions exist in plants and participate in flower development, the JIM8 epitope may be a marker for one set of them. PMID:12324592

  12. Human Antibodies that Recognize Novel Immunodominant Quaternary Epitopes on the HIV-1 Env Protein

    PubMed Central

    Hicar, Mark D.; Chen, Xuemin; Sulli, Chidananda; Barnes, Trevor; Goodman, Jason; Sojar, Hakimuddin; Briney, Bryan; Willis, Jordan; Chukwuma, Valentine U.; Kalams, Spyros A.; Doranz, Benjamin J.; Spearman, Paul; Crowe, James E.

    2016-01-01

    Numerous broadly neutralizing antibodies (Abs) target epitopes that are formed or enhanced during mature HIV envelope formation (i.e. quaternary epitopes). Generally, it is thought that Env epitopes that induce broadly neutralizing Abs are difficult to access and poorly immunogenic because of the characteristic oligomerization, conformational flexibility, sequence diversity and extensive glycosylation of Env protein. To enhance for isolation of quaternary epitope-targeting Abs (QtAbs), we previously used HIV virus-like particles (VLPs) to bind B cells from long-term non-progressor subjects to identify a panel of monoclonal Abs. When expressed as recombinant full-length Abs, a subset of these novel Abs exhibited the binding profiles of QtAbs, as they either failed to bind to monomeric Env protein or showed much higher affinity for Env trimers and VLPs. These QtAbs represented a significant proportion of the B-cell response identified with VLPs. The Ab genes of these clones were highly mutated, but they did not neutralize common HIV strains. We sought to further define the epitopes targeted by these QtAbs. Competition-binding and mapping studies revealed these Abs targeted four separate epitopes; they also failed to compete for binding by Abs to known major neutralizing epitopes. Detailed epitope mapping studies revealed that two of the four epitopes were located in the gp41 subunit of Env. These QtAbs bound pre-fusion forms of antigen and showed differential binding kinetics depending on whether oligomers were produced as recombinant gp140 trimers or as full-length Env incorporated into VLPs. Antigenic regions within gp41 present unexpectedly diverse structural epitopes, including these QtAb epitopes, which may be targeted by the naturally occurring Ab response to HIV infection. PMID:27411063

  13. Blister-inducing antibodies target multiple epitopes on collagen VII in mice

    PubMed Central

    Csorba, Kinga; Chiriac, Mircea Teodor; Florea, Florina; Ghinia, Miruna Georgiana; Licarete, Emilia; Rados, Andreea; Sas, Alexandra; Vuta, Vlad; Sitaru, Cassian

    2014-01-01

    Epidermolysis bullosa acquisita (EBA) is an autoimmune subepidermal blistering disease of mucous membranes and the skin caused by autoantibodies against collagen VII. In silico and wet laboratory epitope mapping studies revealed numerous distinct epitopes recognized by EBA patients' autoantibodies within the non-collagenous (NC)1 and NC2 domains of collagen VII. However, the distribution of pathogenic epitopes on collagen VII has not yet been described. In this study, we therefore performed an in vivo functional epitope mapping of pathogenic autoantibodies in experimental EBA. Animals (n = 10/group) immunized against fragments of the NC1 and NC2 domains of collagen VII or injected with antibodies generated against the same fragments developed to different extent experimental EBA. Our results demonstrate that antibodies targeting multiple, distinct epitopes distributed over the entire NC1, but not NC2 domain of collagen VII induce blistering skin disease in vivo. Our present findings have crucial implications for the development of antigen-specific B- and T cell-targeted therapies in EBA. PMID:25091020

  14. Identification of OppA2 Linear Epitopes as Serodiagnostic Markers for Lyme Disease

    PubMed Central

    Signorino, Giacomo; Arnaboldi, Paul M.; Petzke, Mary M.

    2014-01-01

    Laboratory diagnosis of Lyme disease is based on the serological detection of antibodies against the etiologic agent Borrelia burgdorferi. Current diagnostics are insensitive at detecting early infection, when treatment is most effective. This deficiency results from the limited number of B. burgdorferi antigens expressed in early infection and the use of an insensitive two-tier paradigm, put in place to deal with insufficient specificity associated with the use of whole-protein antigens and/or bacterial lysates as serodiagnostic targets. Whole-protein antigens contain epitopes that are unique to B. burgdorferi as well as cross-reactive epitopes found in other bacteria. One method for overcoming the limitations imposed by cross-reactive epitopes is the use of short peptides containing epitopes unique to B. burgdorferi as antigen targets. This eliminates nonspecific epitopes. Using overlapping peptide libraries, we performed epitope mapping of linear epitopes in oligopeptide permease A2 (OppA2), a member of the oligopeptide permease (Opp) family of peptide transporters, expressed during early B. burgdorferi infection. We identified 9 epitopes, synthesized peptides containing these epitopes, and screened those using panels of blood from patients with early Lyme disease, rheumatoid arthritis (RA), or syphilis or from healthy individuals. Two of the peptides, OppA2 (191-225) (amino acids comprising the peptide are shown in parentheses) and OppA2 (381-400), are highly conserved among the three major pathogenic Borrelia species responsible for most Lyme disease cases in North America and Europe. They detected antibodies in Lyme disease patient sera with sufficient sensitivity and specificity to indicate that they could have value in a serological assay for Lyme disease. PMID:24623628

  15. Localization of non-linear neutralizing B cell epitopes on ricin toxin's enzymatic subunit (RTA).

    PubMed

    O'Hara, Joanne M; Kasten-Jolly, Jane C; Reynolds, Claire E; Mantis, Nicholas J

    2014-01-01

    Efforts to develop a vaccine for ricin toxin are focused on identifying highly immunogenic, safe, and thermostable recombinant derivatives of ricin's enzymatic A subunit (RTA). As a means to guide vaccine design, we have embarked on an effort to generate a comprehensive neutralizing and non-neutralizing B cell epitope map of RTA. In a series of previous studies, we identified three spatially distinct linear (continuous), neutralizing epitopes on RTA, as defined by monoclonal antibodies (mAbs) PB10 (and R70), SyH7, and GD12. In this report we now describe a new collection of 19 toxin-neutralizing mAbs that bind non-linear epitopes on RTA. The most potent toxin-neutralizing mAbs in this new collection, namely WECB2, TB12, PA1, PH12 and IB2 each had nanamolar (or sub-nanomolar) affinities for ricin and were each capable of passively protecting mice against a 5-10xLD50 toxin challenge. Competitive binding assays by surface plasmon resonance revealed that WECB2 binds an epitope that overlaps with PB10 and R70; TB12, PA1, PH12 recognize epitope(s) close to or overlapping with SyH7's epitope; and GD12 and IB2 recognize epitopes that are spatially distinct from all other toxin-neutralizing mAbs. We estimate that we have now accounted for ∼75% of the predicted epitopes on the surface of RTA and that toxin-neutralizing mAbs are directed against a very limited number of these epitopes. Having this information provides a framework for further refinement of RTA mutagenesis and vaccine design. Copyright © 2013 Elsevier B.V. All rights reserved.

  16. Masking of antigenic epitopes by antibodies shapes the humoral immune response to influenza

    PubMed Central

    Zarnitsyna, Veronika I.; Ellebedy, Ali H.; Davis, Carl; Jacob, Joshy; Ahmed, Rafi; Antia, Rustom

    2015-01-01

    The immune responses to influenza, a virus that exhibits strain variation, show complex dynamics where prior immunity shapes the response to the subsequent infecting strains. Original antigenic sin (OAS) describes the observation that antibodies to the first encountered influenza strain, specifically antibodies to the epitopes on the head of influenza's main surface glycoprotein, haemagglutinin (HA), dominate following infection with new drifted strains. OAS suggests that responses to the original strain are preferentially boosted. Recent studies also show limited boosting of the antibodies to conserved epitopes on the stem of HA, which are attractive targets for a ‘universal vaccine’. We develop multi-epitope models to explore how pre-existing immunity modulates the immune response to new strains following immunization. Our models suggest that the masking of antigenic epitopes by antibodies may play an important role in describing the complex dynamics of OAS and limited boosting of antibodies to the stem of HA. Analysis of recently published data confirms model predictions for how pre-existing antibodies to an epitope on HA decrease the magnitude of boosting of the antibody response to this epitope following immunization. We explore strategies for boosting of antibodies to conserved epitopes and generating broadly protective immunity to multiple strains. PMID:26194761

  17. PepMapper: a collaborative web tool for mapping epitopes from affinity-selected peptides.

    PubMed

    Chen, Wenhan; Guo, William W; Huang, Yanxin; Ma, Zhiqiang

    2012-01-01

    Epitope mapping from affinity-selected peptides has become popular in epitope prediction, and correspondingly many Web-based tools have been developed in recent years. However, the performance of these tools varies in different circumstances. To address this problem, we employed an ensemble approach to incorporate two popular Web tools, MimoPro and Pep-3D-Search, together for taking advantages offered by both methods so as to give users more options for their specific purposes of epitope-peptide mapping. The combined operation of Union finds as many associated peptides as possible from both methods, which increases sensitivity in finding potential epitopic regions on a given antigen surface. The combined operation of Intersection achieves to some extent the mutual verification by the two methods and hence increases the likelihood of locating the genuine epitopic region on a given antigen in relation to the interacting peptides. The Consistency between Intersection and Union is an indirect sufficient condition to assess the likelihood of successful peptide-epitope mapping. On average from 27 tests, the combined operations of PepMapper outperformed either MimoPro or Pep-3D-Search alone. Therefore, PepMapper is another multipurpose mapping tool for epitope prediction from affinity-selected peptides. The Web server can be freely accessed at: http://informatics.nenu.edu.cn/PepMapper/

  18. Designing Probes for Immunodiagnostics: Structural Insights into an Epitope Targeting Burkholderia Infections.

    PubMed

    Capelli, Riccardo; Matterazzo, Elena; Amabili, Marco; Peri, Claudio; Gori, Alessandro; Gagni, Paola; Chiari, Marcella; Lertmemongkolchai, Ganjana; Cretich, Marina; Bolognesi, Martino; Colombo, Giorgio; Gourlay, Louise J

    2017-10-13

    Structure-based epitope prediction drives the design of diagnostic peptidic probes to reveal specific antibodies elicited in response to infections. We previously identified a highly immunoreactive epitope from the peptidoglycan-associated lipoprotein (Pal) antigen from Burkholderia pseudomallei, which could also diagnose Burkholderia cepacia infections. Here, considering the high phylogenetic conservation within Burkholderia species, we ask whether cross-reactivity can be reciprocally displayed by the synthetic epitope from B. cenocepacia. We perform comparative analyses of the conformational preferences and diagnostic performances of the corresponding epitopes from the two Burkholderia species when presented in the context of the full-length proteins or as isolated peptides. The effects of conformation on the diagnostic potential and cross-reactivity of Pal peptide epitopes are rationalized on the basis of the 1.8 Å crystal structure of B. cenocepacia Pal and through computational analyses. Our results are discussed in the context of designing new diagnostic molecules for the early detection of infectious diseases.

  19. The design and implementation of the immune epitope database and analysis resource

    PubMed Central

    Peters, Bjoern; Sidney, John; Bourne, Phil; Bui, Huynh-Hoa; Buus, Soeren; Doh, Grace; Fleri, Ward; Kronenberg, Mitch; Kubo, Ralph; Lund, Ole; Nemazee, David; Ponomarenko, Julia V.; Sathiamurthy, Muthu; Schoenberger, Stephen P.; Stewart, Scott; Surko, Pamela; Way, Scott; Wilson, Steve; Sette, Alessandro

    2016-01-01

    Epitopes are defined as parts of antigens interacting with receptors of the immune system. Knowledge about their intrinsic structure and how they affect the immune response is required to continue development of techniques that detect, monitor, and fight diseases. Their scientific importance is reflected in the vast amount of epitope-related information gathered, ranging from interactions between epitopes and major histocompatibility complex molecules determined by X-ray crystallography to clinical studies analyzing correlates of protection for epitope based vaccines. Our goal is to provide a central resource capable of capturing this information, allowing users to access and connect realms of knowledge that are currently separated and difficult to access. Here, we portray a new initiative, “The Immune Epitope Database and Analysis Resource.” We describe how we plan to capture, structure, and store this information, what query interfaces we will make available to the public, and what additional predictive and analytical tools we will provide. PMID:15895191

  20. The role of glutamic or aspartic acid in position four of the epitope binding motif and thyrotropin receptor-extracellular domain epitope selection in Graves' disease.

    PubMed

    Inaba, Hidefumi; Martin, William; Ardito, Matt; De Groot, Anne Searls; De Groot, Leslie J

    2010-06-01

    Development of Graves' disease (GD) is related to HLA-DRB1*0301 (DR3),and more specifically to arginine at position 74 of the DRB1 molecule. The extracellular domain (ECD) of human TSH receptor (hTSH-R) contains the target antigen. We analyzed the relation between hTSH-R-ECD peptides and DR molecules to determine whether aspartic acid (D) or glutamic acid (E) at position four in the binding motif influenced selection of functional epitopes. Peptide epitopes from TSH-R-ECD with D or E in position four (D/E+) had higher affinity for binding to DR3 than peptides without D/E (D/E-) (IC(50) 29.3 vs. 61.4, P = 0.0024). HLA-DR7, negatively correlated with GD, and DRB1*0302 (HLA-DR18), not associated with GD, had different profiles of epitope binding. Toxic GD patients who are DR3+ had higher responses to D/E+ peptides than D/E- peptides (stimulation index 1.42 vs. 1.22, P = 0.028). All DR3+ GD patients (toxic + euthyroid) had higher responses, with borderline significance (Sl; 1.32 vs. 1.18, P = 0.051). Splenocytes of DR3 transgenic mice immunized to TSH-R-ECD responded to D/E+ peptides more than D/E- peptides (stimulation index 1.95 vs. 1.69, P = 0.036). Seven of nine hTSH-R-ECD peptide epitopes reported to be reactive with GD patients' peripheral blood mononuclear cells contain binding motifs with D/E at position four. TSH-R-ECD epitopes with D/E in position four of the binding motif bind more strongly to DRB1*0301 than epitopes that are D/E- and are more stimulatory to GD patients' peripheral blood mononuclear cells and to splenocytes from mice immunized to hTSH-R. These epitopes appear important in immunogenicity to TSH-R due to their favored binding to HLA-DR3, thus increasing presentation to T cells.

  1. Systems analysis of singly and multiply O-glycosylated peptides in the human serum glycoproteome via EThcD and HCD mass spectrometry.

    PubMed

    Zhang, Yong; Xie, Xinfang; Zhao, Xinyuan; Tian, Fang; Lv, Jicheng; Ying, Wantao; Qian, Xiaohong

    2018-01-06

    Human serum has been intensively studied to identify biomarkers via global proteomic analysis. The altered O-glycoproteome is associated with human pathological state including cancer, inflammatory and degenerative diseases and is an attractive source of disease biomarkers. Because of the microheterogeneity and macroheterogeneity of O-glycosylation, site-specific O-glycosylation analysis in human serum is still challenging. Here, we developed a systematic strategy that combined multiple enzyme digestion, multidimensional separation for sample preparation and high-resolution tandem MS with Byonic software for intact O-glycopeptide characterization. We demonstrated that multiple enzyme digestion or multidimensional separation can make sample preparation more efficient and that EThcD is not only suitable for the identification of singly O-glycosylated peptides (50.3%) but also doubly (21.2%) and triply (28.5%) O-glycosylated peptides. Totally, with the strict scoring criteria, 499 non-redundant intact O-glycopeptides, 173 O-glycosylation sites and 6 types of O-glycans originating from 49 O-glycoprotein groups were identified in human serum, including 121 novel O-glycosylation sites. Currently, this is the largest data set of site-specific native O-glycoproteome from human serum samples. We expect that the strategies developed by this study will facilitate in-depth analyses of native O-glycoproteomes in human serum and provide opportunities to understand the functional roles of protein O-glycosylation in human health and diseases. The altered O-glycoproteome is associated with human pathological state and is an attractive source of disease biomarkers. However, site-specific O-glycosylation analysis is challenging because of the microheterogeneity (different glycoforms attached to one glycosylation site) and macroheterogeneity (site occupancy) of O-glycosylation. In this work, we developed a systematic strategy for intact O-glycopeptide characterization. This study took

  2. HIV-1 V3 loop crown epitope-focused mimotope selection by patient serum from random phage display libraries: implications for the epitope structural features.

    PubMed

    Gazarian, Karlen G; Palacios-Rodríguez, Yadira; Gazarian, Tatiana G; Huerta, Leonor

    2013-06-01

    The crown region of the V3 loop in HIV-1 that contains the conserved amino acid sequence GPGR/G is known as the principal neutralizing determinant due to the extraordinary ability of antibodies to this region to neutralize the virus. To complement the existing peptide models of this epitope, we describe a family of 18 phage-displayed peptides, which include linear 12mer and constrained 7mer peptides that was selected by screening random libraries with serum from HIV-1 subtype B-infected patients. The 7mer constrained peptides presented two conserved amino acid sequences: PR-L in N-terminus and GPG in the C-terminus. On the basis of these peptides we propose a mimotope model of the V3 crown epitope in which the PR-L and GPG sequences represent the two known epitope binding sites. The GPG, has the same function as the V3 crown GPGR sequence but without the involvement of the "R" despite its being considered as the signature of the epitope in B-subtype viruses. The PR-L contains a proline not existing in the epitope that is postulated to induce kinks in the backbones of all peptides and create a spatial element mimicking the N-terminal conformationally variable binding site. Rabbit serum to these mimotopes recognized the V3 peptides and moderately decreased the fusion between HIV-1 Env- and CD4-expressing Jurkat cells. This study proposes the efficient generation by means of patient sera of V3 epitope mimics validated by interaction with the antibodies to contemporary viruses induced in patients. The serum antibody-selectable mimotopes are sources of novel information on the fine structure-function properties of HIV-1 principal neutralizing domain and candidate anti-HIV-1 immunogens. Copyright © 2012 Elsevier Ltd. All rights reserved.

  3. ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping.

    PubMed

    Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo; Lund, Ole; Buus, Søren; Nielsen, Morten

    2017-01-01

    Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope.

  4. Ab-initio conformational epitope structure prediction using genetic algorithm and SVM for vaccine design.

    PubMed

    Moghram, Basem Ameen; Nabil, Emad; Badr, Amr

    2018-01-01

    T-cell epitope structure identification is a significant challenging immunoinformatic problem within epitope-based vaccine design. Epitopes or antigenic peptides are a set of amino acids that bind with the Major Histocompatibility Complex (MHC) molecules. The aim of this process is presented by Antigen Presenting Cells to be inspected by T-cells. MHC-molecule-binding epitopes are responsible for triggering the immune response to antigens. The epitope's three-dimensional (3D) molecular structure (i.e., tertiary structure) reflects its proper function. Therefore, the identification of MHC class-II epitopes structure is a significant step towards epitope-based vaccine design and understanding of the immune system. In this paper, we propose a new technique using a Genetic Algorithm for Predicting the Epitope Structure (GAPES), to predict the structure of MHC class-II epitopes based on their sequence. The proposed Elitist-based genetic algorithm for predicting the epitope's tertiary structure is based on Ab-Initio Empirical Conformational Energy Program for Peptides (ECEPP) Force Field Model. The developed secondary structure prediction technique relies on Ramachandran Plot. We used two alignment algorithms: the ROSS alignment and TM-Score alignment. We applied four different alignment approaches to calculate the similarity scores of the dataset under test. We utilized the support vector machine (SVM) classifier as an evaluation of the prediction performance. The prediction accuracy and the Area Under Receiver Operating Characteristic (ROC) Curve (AUC) were calculated as measures of performance. The calculations are performed on twelve similarity-reduced datasets of the Immune Epitope Data Base (IEDB) and a large dataset of peptide-binding affinities to HLA-DRB1*0101. The results showed that GAPES was reliable and very accurate. We achieved an average prediction accuracy of 93.50% and an average AUC of 0.974 in the IEDB dataset. Also, we achieved an accuracy of 95

  5. Identification of Novel Avian Influenza Virus Derived CD8+ T-Cell Epitopes

    PubMed Central

    Reemers, Sylvia S. N.; van Haarlem, Daphne A.; Sijts, Alice J. A. M.; Vervelde, Lonneke; Jansen, Christine A.

    2012-01-01

    Avian influenza virus (AIV) infection is a continuing threat to both humans and poultry. Influenza virus specific CD8+ T cells are associated with protection against homologous and heterologous influenza strains. In contrast to what has been described for humans and mice, knowledge on epitope-specific CD8+ T cells in chickens is limited. Therefore, we set out to identify AIV-specific CD8+ T-cell epitopes. Epitope predictions based on anchor residues resulted in 33 candidate epitopes. MHC I inbred chickens were infected with a low pathogenic AIV strain and sacrificed at 5, 7, 10 and 14 days post infection (dpi). Lymphocytes isolated from lung, spleen and blood were stimulated ex vivo with AIV-specific pooled or individual peptides and the production of IFNγ was determined by ELIspot. This resulted in the identification of 12 MHC B12-restricted, 3 B4-restricted and 1 B19-restricted AIV- specific CD8+ T-cell epitopes. In conclusion, we have identified novel AIV-derived CD8+ T-cell epitopes for several inbred chicken strains. This knowledge can be used to study the role of CD8+ T cells against AIV infection in a natural host for influenza, and may be important for vaccine development. PMID:22384112

  6. Graph-based optimization of epitope coverage for vaccine antigen design

    DOE PAGES

    Theiler, James Patrick; Korber, Bette Tina Marie

    2017-01-29

    Epigraph is a recently developed algorithm that enables the computationally efficient design of single or multi-antigen vaccines to maximize the potential epitope coverage for a diverse pathogen population. Potential epitopes are defined as short contiguous stretches of proteins, comparable in length to T-cell epitopes. This optimal coverage problem can be formulated in terms of a directed graph, with candidate antigens represented as paths that traverse this graph. Epigraph protein sequences can also be used as the basis for designing peptides for experimental evaluation of immune responses in natural infections to highly variable proteins. The epigraph tool suite also enables rapidmore » characterization of populations of diverse sequences from an immunological perspective. Fundamental distance measures are based on immunologically relevant shared potential epitope frequencies, rather than simple Hamming or phylogenetic distances. Here, we provide a mathematical description of the epigraph algorithm, include a comparison of different heuristics that can be used when graphs are not acyclic, and we describe an additional tool we have added to the web-based epigraph tool suite that provides frequency summaries of all distinct potential epitopes in a population. Lastly, we also show examples of the graphical output and summary tables that can be generated using the epigraph tool suite and explain their content and applications.« less

  7. Graph-based optimization of epitope coverage for vaccine antigen design

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Theiler, James Patrick; Korber, Bette Tina Marie

    Epigraph is a recently developed algorithm that enables the computationally efficient design of single or multi-antigen vaccines to maximize the potential epitope coverage for a diverse pathogen population. Potential epitopes are defined as short contiguous stretches of proteins, comparable in length to T-cell epitopes. This optimal coverage problem can be formulated in terms of a directed graph, with candidate antigens represented as paths that traverse this graph. Epigraph protein sequences can also be used as the basis for designing peptides for experimental evaluation of immune responses in natural infections to highly variable proteins. The epigraph tool suite also enables rapidmore » characterization of populations of diverse sequences from an immunological perspective. Fundamental distance measures are based on immunologically relevant shared potential epitope frequencies, rather than simple Hamming or phylogenetic distances. Here, we provide a mathematical description of the epigraph algorithm, include a comparison of different heuristics that can be used when graphs are not acyclic, and we describe an additional tool we have added to the web-based epigraph tool suite that provides frequency summaries of all distinct potential epitopes in a population. Lastly, we also show examples of the graphical output and summary tables that can be generated using the epigraph tool suite and explain their content and applications.« less

  8. IgM-mediated autoimmune responses directed against anchorage epitopes are greater in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) than in major depression.

    PubMed

    Maes, Michael; Mihaylova, Ivana; Kubera, Marta; Leunis, Jean-Claude; Twisk, Frank N M; Geffard, Michel

    2012-12-01

    Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and depression are considered to be neuro-immune disorders (Maes and Twisk, BMC Medicine 8:35, 2010). There is also evidence that depression and ME/CFS are accompanied by oxidative and nitrosative stress (O&NS) and by increased autoantibodies to a number of self-epitopes some of which have become immunogenic due to damage by O&NS. The aim of this study is to examine IgM-mediated autoimmune responses to different self-epitopes in ME/CFS versus depression. We examined serum IgM antibodies to three anchorage molecules (palmitic and myristic acid and S-farnesyl-L-cysteine); acetylcholine; and conjugated NO-modified adducts in 26 patients with major depression; 16 patients with ME/CFS, 15 with chronic fatigue; and 17 normal controls. Severity of fatigue and physio-somatic (F&S) symptoms was measured with the Fibromyalgia and Chronic Fatigue Syndrome Rating Scale. Serum IgM antibodies to the three anchorage molecules and NO-phenylalanine were significantly higher in ME/CFS than in depression. The autoimmune responses to oxidatively, but not nitrosatively, modified self-epitopes were significantly higher in ME/CFS than in depression and were associated with F&S symptoms. The autoimmune activity directed against conjugated acetylcholine did not differ significantly between ME/CFS and depression, but was greater in the patients than controls. Partially overlapping pathways, i.e. increased IgM antibodies to a multitude of neo-epitopes, underpin both ME/CFS and depression, while greater autoimmune responses directed against anchorage molecules and oxidatively modified neo-epitopes discriminate patients with ME/CFS from those with depression. These autoimmune responses directed against neoantigenic determinants may play a role in the dysregulation of key cellular functions in both disorders, e.g. intracellular signal transduction, cellular differentiation and apoptosis, but their impact may be more important in ME

  9. Antibody specific epitope prediction-emergence of a new paradigm.

    PubMed

    Sela-Culang, Inbal; Ofran, Yanay; Peters, Bjoern

    2015-04-01

    The development of accurate tools for predicting B-cell epitopes is important but difficult. Traditional methods have examined which regions in an antigen are likely binding sites of an antibody. However, it is becoming increasingly clear that most antigen surface residues will be able to bind one or more of the myriad of possible antibodies. In recent years, new approaches have emerged for predicting an epitope for a specific antibody, utilizing information encoded in antibody sequence or structure. Applying such antibody-specific predictions to groups of antibodies in combination with easily obtainable experimental data improves the performance of epitope predictions. We expect that further advances of such tools will be possible with the integration of immunoglobulin repertoire sequencing data. Copyright © 2015 Elsevier B.V. All rights reserved.

  10. Analysis of predicted B and T-cell epitopes in Der p 23, allergen from Dermatophagoides pteronyssinus.

    PubMed

    Fanuel, Songwe; Tabesh, Saeideh; Sadroddiny, Esmaeil; Kardar, Gholam Ali

    2017-01-01

    House dust mite (HDM) allergy is the leading cause of IgE-mediated hypersensitivity. Therefore identifying potential epitopes in the Dermatophagoide pteronyssinus 23 (Der p 23), a major house dust mite allergen will aid in the development of therapeutic vaccines and diagnostic kits for HDM allergy. Experimental methods of epitope discovery have been widely exploited for the mapping of potential allergens. This study sought to use immunoinformatic methods to analyze the structure of Der p 23 for potential immunoreactive B and T-cell epitopes that could be useful for AIT and allergy diagnosis. We retrieved a Der p 23 allergen sequence from Genbank database and then analyzed it using a combination of web-based sequence analysis tools including the Immune Epitope Database (IEDB), Protparam, BCPREDS, ABCpred, BepiPred, Bcepred among others to predict the physiochemical properties and epitope spectra of the Der p 23 allergen. We then built 3D models of the predicted B-cell epitopes, T cell epitopes and Der p 23 for sequence structure homology analysis. Our results identified peptides 'TRWNEDE', 'TVHPTTTEQPDDK', and 'NDDDPTT' as immunogenic linear B-cell epitopes while 'CPSRFGYFADPKDPH' and 'CPGNTRWNEDEETCT' were found to be the most suitable T-cell epitopes that interacted well with a large number of MHC II alleles. Both epitopes had high population coverage as well as showing a 100% conservancy. These five Der p 23 epitopes are useful for AIT vaccines and HDM allergy diagnosis development.

  11. In silico identification and characterization of common epitope-based peptide vaccine for Nipah and Hendra viruses.

    PubMed

    Saha, Chayan Kumar; Mahbub Hasan, Md; Saddam Hossain, Md; Asraful Jahan, Md; Azad, Abul Kalam

    2017-06-01

    To explore a common B- and T-cell epitope-based vaccine that can elicit an immune response against encephalitis causing genus Henipaviruses, Hendra virus (HeV) and Nipah virus (NiV). Membrane proteins F, G and M of HeV and NiV were retrieved from the protein database and subjected to different bioinformatics tools to predict antigenic B-cell epitopes. Best B-cell epitopes were then analyzed to predict their T-cell antigenic potentiality. Antigenic B- and T-cell epitopes that shared maximum identity with HeV and NiV were selected. Stability of the selected epitopes was predicted. Finally, the selected epitopes were subjected to molecular docking simulation with HLA-DR to confirm their antigenic potentiality in silico. One epitope from G proteins, one from M proteins and none from F proteins were selected based on their antigenic potentiality. The epitope from the G proteins was stable whereas that from M was unstable. The M-epitope was made stable by adding flanking dipeptides. The 15-mer G-epitope (VDPLRVQWRNNSVIS) showed at least 66% identity with all NiV and HeV G protein sequences, while the 15-mer M-epitope (GKLEFRRNNAIAFKG) with the dipeptide flanking residues showed 73% identity with all NiV and HeV M protein sequences available in the database. Molecular docking simulation with most frequent MHC class-II (MHC II) and class-I (MHC I) molecules showed that these epitopes could bind within HLA binding grooves to elicit an immune response. Data in our present study revealed the notion that the epitopes from G and M proteins might be the target for peptide-based subunit vaccine design against HeV and NiV. However, the biochemical analysis is necessary to experimentally validate the interaction of epitopes individually with the MHC molecules through elucidation of immunity induction. Copyright © 2017 Hainan Medical University. Production and hosting by Elsevier B.V. All rights reserved.

  12. Computational Prediction of Neutralization Epitopes Targeted by Human Anti-V3 HIV Monoclonal Antibodies

    PubMed Central

    Shmelkov, Evgeny; Krachmarov, Chavdar; Grigoryan, Arsen V.; Pinter, Abraham; Statnikov, Alexander; Cardozo, Timothy

    2014-01-01

    The extreme diversity of HIV-1 strains presents a formidable challenge for HIV-1 vaccine design. Although antibodies (Abs) can neutralize HIV-1 and potentially protect against infection, antibodies that target the immunogenic viral surface protein gp120 have widely variable and poorly predictable cross-strain reactivity. Here, we developed a novel computational approach, the Method of Dynamic Epitopes, for identification of neutralization epitopes targeted by anti-HIV-1 monoclonal antibodies (mAbs). Our data demonstrate that this approach, based purely on calculated energetics and 3D structural information, accurately predicts the presence of neutralization epitopes targeted by V3-specific mAbs 2219 and 447-52D in any HIV-1 strain. The method was used to calculate the range of conservation of these specific epitopes across all circulating HIV-1 viruses. Accurately identifying an Ab-targeted neutralization epitope in a virus by computational means enables easy prediction of the breadth of reactivity of specific mAbs across the diversity of thousands of different circulating HIV-1 variants and facilitates rational design and selection of immunogens mimicking specific mAb-targeted epitopes in a multivalent HIV-1 vaccine. The defined epitopes can also be used for the purpose of epitope-specific analyses of breakthrough sequences recorded in vaccine clinical trials. Thus, our study is a prototype for a valuable tool for rational HIV-1 vaccine design. PMID:24587168

  13. Epitope Mapping of Avian Influenza M2e Protein: Different Species Recognise Various Epitopes

    PubMed Central

    Hasan, Noor Haliza; Ignjatovic, Jagoda; Tarigan, Simson; Peaston, Anne; Hemmatzadeh, Farhid

    2016-01-01

    A common approach for developing diagnostic tests for influenza virus detection is the use of mouse or rabbit monoclonal and/or polyclonal antibodies against a target antigen of the virus. However, comparative mapping of the target antigen using antibodies from different animal sources has not been evaluated before. This is important because identification of antigenic determinants of the target antigen in different species plays a central role to ensure the efficiency of a diagnostic test, such as competitive ELISA or immunohistochemistry-based tests. Interest in the matrix 2 ectodomain (M2e) protein of avian influenza virus (AIV) as a candidate for a universal vaccine and also as a marker for detection of virus infection in vaccinated animals (DIVA) is the rationale for the selection of this protein for comparative mapping evaluation. This study aimed to map the epitopes of the M2e protein of avian influenza virus H5N1 using chicken, mouse and rabbit monoclonal or monospecific antibodies. Our findings revealed that rabbit antibodies (rAbs) recognized epitope 6EVETPTRN13 of the M2e, located at the N-terminal of the protein, while mouse (mAb) and chicken antibodies (cAbs) recognized epitope 10PTRNEWECK18, located at the centre region of the protein. The findings highlighted the difference between the M2e antigenic determinants recognized by different species that emphasized the importance of comparative mapping of antibody reactivity from different animals to the same antigen, especially in the case of multi-host infectious agents such as influenza. The findings are of importance for antigenic mapping, as well as diagnostic test and vaccine development. PMID:27362795

  14. Lessons learned from successful human vaccines: Delineating key epitopes by dissecting the capsid proteins

    PubMed Central

    Zhang, Xiao; Xin, Lu; Li, Shaowei; Fang, Mujin; Zhang, Jun; Xia, Ningshao; Zhao, Qinjian

    2015-01-01

    Recombinant VLP-based vaccines have been successfully used against 3 diseases caused by viral infections: Hepatitis B, cervical cancer and hepatitis E. The VLP approach is attracting increasing attention in vaccine design and development for human and veterinary use. This review summarizes the clinically relevant epitopes on the VLP antigens in successful human vaccines. These virion-like epitopes, which can be delineated with molecular biology, cryo-electron microscopy and x-ray crystallographic methods, are the prerequisites for these efficacious vaccines to elicit functional antibodies. The critical epitopes and key factors influencing these epitopes are discussed for the HEV, HPV and HBV vaccines. A pentamer (for HPV) or a dimer (for HEV and HBV), rather than a monomer, is the basic building block harboring critical epitopes for the assembly of VLP antigen. The processing and formulation of VLP-based vaccines need to be developed to promote the formation and stabilization of these epitopes in the recombinant antigens. Delineating the critical epitopes is essential for antigen design in the early phase of vaccine development and for critical quality attribute analysis in the commercial phase of vaccine manufacturing. PMID:25751641

  15. In Silico Analysis of Epitope-Based Vaccine Candidates against Hepatitis B Virus Polymerase Protein

    PubMed Central

    Zheng, Juzeng; Lin, Xianfan; Wang, Xiuyan; Zheng, Liyu; Lan, Songsong; Jin, Sisi; Ou, Zhanfan; Wu, Jinming

    2017-01-01

    Hepatitis B virus (HBV) infection has persisted as a major public health problem due to the lack of an effective treatment for those chronically infected. Therapeutic vaccination holds promise, and targeting HBV polymerase is pivotal for viral eradication. In this research, a computational approach was employed to predict suitable HBV polymerase targeting multi-peptides for vaccine candidate selection. We then performed in-depth computational analysis to evaluate the predicted epitopes’ immunogenicity, conservation, population coverage, and toxicity. Lastly, molecular docking and MHC-peptide complex stabilization assay were utilized to determine the binding energy and affinity of epitopes to the HLA-A0201 molecule. Criteria-based analysis provided four predicted epitopes, RVTGGVFLV, VSIPWTHKV, YMDDVVLGA and HLYSHPIIL. Assay results indicated the lowest binding energy and high affinity to the HLA-A0201 molecule for epitopes VSIPWTHKV and YMDDVVLGA and epitopes RVTGGVFLV and VSIPWTHKV, respectively. Regions 307 to 320 and 377 to 387 were considered to have the highest probability to be involved in B cell epitopes. The T cell and B cell epitopes identified in this study are promising targets for an epitope-focused, peptide-based HBV vaccine, and provide insight into HBV-induced immune response. PMID:28509875

  16. Variation at Extra-epitopic Amino Acid Residues Influences Suppression of Influenza Virus Replication by M158-66 Epitope-Specific CD8+ T Lymphocytes.

    PubMed

    van de Sandt, Carolien E; Pronk, Mark R; van Baalen, Carel A; Fouchier, Ron A M; Rimmelzwaan, Guus F

    2018-06-01

    Influenza virus-specific CD8 + T lymphocytes (CTLs) contribute to clearance of influenza virus infections and reduce disease severity. Variation at amino acid residues located in or outside CTL epitopes has been shown to affect viral recognition by virus-specific CTLs. In the present study, we investigated the effect of naturally occurring variation at residues outside the conserved immunodominant and HLA*0201-restricted M1 58-66 epitope, located in the influenza virus M1 protein, on the extent of virus replication in the presence of CTLs specific for the epitope. To this end, we used isogenic viruses with an M1 gene segment derived from either an avian or a human influenza virus, HLA-transgenic human epithelial cells, human T cell clones specific for the M1 58-66 epitope or a control epitope, and a novel, purposely developed in vitro system to coculture influenza virus-infected cells with T cells. We found that the M gene segment of a human influenza A/H3N2 virus afforded the virus the capacity to replicate better in the presence of M1 58-66 -specific CTLs than the M gene segment of avian viruses. These findings are in concordance with previously observed differential CTL activation, caused by variation at extra-epitopic residues, and may reflect an immune adaptation strategy of human influenza viruses that allows them to cope with potent CTL immunity to the M1 58-66 epitope in HLA-A*0201-positive individuals, resulting in increased virus replication and shedding and possibly increasing disease severity. IMPORTANCE Influenza viruses are among the leading causes of acute respiratory tract infections. CD8 + T lymphocytes display a high degree of cross-reactivity with influenza A viruses of various subtypes and are considered an important correlate of protection. Unraveling viral immune evasion strategies and identifying signs of immune adaptation are important for defining the role of CD8 + T lymphocytes in affording protection more accurately. Improving our insight

  17. ArrayPitope: Automated Analysis of Amino Acid Substitutions for Peptide Microarray-Based Antibody Epitope Mapping

    PubMed Central

    Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo; Lund, Ole; Buus, Søren

    2017-01-01

    Identification of epitopes targeted by antibodies (B cell epitopes) is of critical importance for the development of many diagnostic and therapeutic tools. For clinical usage, such epitopes must be extensively characterized in order to validate specificity and to document potential cross-reactivity. B cell epitopes are typically classified as either linear epitopes, i.e. short consecutive segments from the protein sequence or conformational epitopes adapted through native protein folding. Recent advances in high-density peptide microarrays enable high-throughput, high-resolution identification and characterization of linear B cell epitopes. Using exhaustive amino acid substitution analysis of peptides originating from target antigens, these microarrays can be used to address the specificity of polyclonal antibodies raised against such antigens containing hundreds of epitopes. However, the interpretation of the data provided in such large-scale screenings is far from trivial and in most cases it requires advanced computational and statistical skills. Here, we present an online application for automated identification of linear B cell epitopes, allowing the non-expert user to analyse peptide microarray data. The application takes as input quantitative peptide data of fully or partially substituted overlapping peptides from a given antigen sequence and identifies epitope residues (residues that are significantly affected by substitutions) and visualize the selectivity towards each residue by sequence logo plots. Demonstrating utility, the application was used to identify and address the antibody specificity of 18 linear epitope regions in Human Serum Albumin (HSA), using peptide microarray data consisting of fully substituted peptides spanning the entire sequence of HSA and incubated with polyclonal rabbit anti-HSA (and mouse anti-rabbit-Cy3). The application is made available at: www.cbs.dtu.dk/services/ArrayPitope. PMID:28095436

  18. Antibody protection reveals extended epitopes on the human TSH receptor.

    PubMed

    Latif, Rauf; Teixeira, Avelino; Michalek, Krzysztof; Ali, M Rejwan; Schlesinger, Max; Baliram, Ramkumarie; Morshed, Syed A; Davies, Terry F

    2012-01-01

    Stimulating, and some blocking, antibodies to the TSH receptor (TSHR) have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD). However, successful crystallization of TSHR residues 22-260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs) have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1-412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity.

  19. Antibody Protection Reveals Extended Epitopes on the Human TSH Receptor

    PubMed Central

    Latif, Rauf; Teixeira, Avelino; Michalek, Krzysztof; Ali, M. Rejwan; Schlesinger, Max; Baliram, Ramkumarie; Morshed, Syed A.; Davies, Terry F.

    2012-01-01

    Stimulating, and some blocking, antibodies to the TSH receptor (TSHR) have conformation-dependent epitopes reported to involve primarily the leucine rich repeat region of the ectodomain (LRD). However, successful crystallization of TSHR residues 22–260 has omitted important extracellular non-LRD residues including the hinge region which connects the TSHR ectodomain to the transmembrane domain and which is involved in ligand induced signal transduction. The aim of the present study, therefore, was to determine if TSHR antibodies (TSHR-Abs) have non-LRD binding sites outside the LRD. To obtain this information we employed the method of epitope protection in which we first protected TSHR residues 1–412 with intact TSHR antibodies and then enzymatically digested the unprotected residues. Those peptides remaining were subsequently delineated by mass spectrometry. Fourteen out of 23 of the reported stimulating monoclonal TSHR-Ab crystal contact residues were protected by this technique which may reflect the higher binding energies of certain residues detected in this approach. Comparing the protected epitopes of two stimulating TSHR-Abs we found both similarities and differences but both antibodies also contacted the hinge region and the amino terminus of the TSHR following the signal peptide and encompassing cysteine box 1 which has previously been shown to be important for TSH binding and activation. A monoclonal blocking TSHR antibody revealed a similar pattern of binding regions but the residues that it contacted on the LRD were again distinct. These data demonstrated that conformationally dependent TSHR-Abs had epitopes not confined to the LRDs but also incorporated epitopes not revealed in the available crystal structure. Furthermore, the data also indicated that in addition to overlapping contact regions within the LRD, there are unique epitope patterns for each of the antibodies which may contribute to their functional heterogeneity. PMID:22957097

  20. Defining epitope coverage requirements for T cell-based HIV vaccines: Theoretical considerations and practical applications

    PubMed Central

    2011-01-01

    Background HIV vaccine development must address the genetic diversity and plasticity of the virus that permits the presentation of diverse genetic forms to the immune system and subsequent escape from immune pressure. Assessment of potential HIV strain coverage by candidate T cell-based vaccines (whether natural sequence or computationally optimized products) is now a critical component in interpreting candidate vaccine suitability. Methods We have utilized an N-mer identity algorithm to represent T cell epitopes and explore potential coverage of the global HIV pandemic using natural sequences derived from candidate HIV vaccines. Breadth (the number of T cell epitopes generated) and depth (the variant coverage within a T cell epitope) analyses have been incorporated into the model to explore vaccine coverage requirements in terms of the number of discrete T cell epitopes generated. Results We show that when multiple epitope generation by a vaccine product is considered a far more nuanced appraisal of the potential HIV strain coverage of the vaccine product emerges. By considering epitope breadth and depth several important observations were made: (1) epitope breadth requirements to reach particular levels of vaccine coverage, even for natural sequence-based vaccine products is not necessarily an intractable problem for the immune system; (2) increasing the valency (number of T cell epitope variants present) of vaccine products dramatically decreases the epitope requirements to reach particular coverage levels for any epidemic; (3) considering multiple-hit models (more than one exact epitope match with an incoming HIV strain) places a significantly higher requirement upon epitope breadth in order to reach a given level of coverage, to the point where low valency natural sequence based products would not practically be able to generate sufficient epitopes. Conclusions When HIV vaccine sequences are compared against datasets of potential incoming viruses important

  1. Characterization of CTL Recognized Epitopes on Human Breast Tumors

    DTIC Science & Technology

    1996-09-01

    maturation and effector function of cellular immune cytotoxic effectors such as CTL (11). (c) The epitopes defined on tumor Ag are self-peptides of...have been reported to be expressed in breast and ovarian cancer cells (18), and they apparently function by maintaining the undifferentiated state...Body of the Report The purpose of the present work continues to be the characterization of the functional significance of the CTL epitopes as potential

  2. Correlations between corneal and total wavefront aberrations

    NASA Astrophysics Data System (ADS)

    Mrochen, Michael; Jankov, Mirko; Bueeler, Michael; Seiler, Theo

    2002-06-01

    Purpose: Corneal topography data expressed as corneal aberrations are frequently used to report corneal laser surgery results. However, the optical image quality at the retina depends on all optical elements of the eye such as the human lens. Thus, the aim of this study was to investigate the correlations between the corneal and total wavefront aberrations and to discuss the importance of corneal aberrations for representing corneal laser surgery results. Methods: Thirty three eyes of 22 myopic subjects were measured with a corneal topography system and a Tschernig-type wavefront analyzer after the pupils were dilated to at least 6 mm in diameter. All measurements were centered with respect to the line of sight. Corneal and total wavefront aberrations were calculated up to the 6th Zernike order in the same reference plane. Results: Statistically significant correlations (p < 0.05) between the corneal and total wavefront aberrations were found for the astigmatism (C3,C5) and all 3rd Zernike order coefficients such as coma (C7,C8). No statistically significant correlations were found for all 4th to 6th order Zernike coefficients except for the 5th order horizontal coma C18 (p equals 0.003). On average, all Zernike coefficients for the corneal aberrations were found to be larger compared to Zernike coefficients for the total wavefront aberrations. Conclusions: Corneal aberrations are only of limited use for representing the optical quality of the human eye after corneal laser surgery. This is due to the lack of correlation between corneal and total wavefront aberrations in most of the higher order aberrations. Besides this, the data present in this study yield towards an aberration balancing between corneal aberrations and the optical elements within the eye that reduces the aberration from the cornea by a certain degree. Consequently, ideal customized ablations have to take both, corneal and total wavefront aberrations, into consideration.

  3. Evaluation of Lipopolysaccharides and Polysaccharides of Different Epitopic Structures in the Indirect Enzyme-Linked Immunosorbent Assay for Diagnosis of Brucellosis in Small Ruminants and Cattle

    PubMed Central

    Alonso-Urmeneta, B.; Marín, C.; Aragón, V.; Blasco, J. M.; Díaz, R.; Moriyón, I.

    1998-01-01

    Brucella abortus and Brucella melitensis have surface lipopolysaccharides and polysaccharides carrying B. melitensis-type (M) and B. abortus-type (A) epitopes as well as common (C) epitopes present in all smooth Brucella biotypes. Crude lipopolysaccharides, hydrolytic O polysaccharides, and native hapten polysaccharides of MC or AC specificity were evaluated in indirect enzyme-linked immunosorbent assays with polyclonal, monoclonal, or protein G conjugates by using sera from cattle, sheep, and goats infected with AC, MC, or AMC Brucella biotypes. Regardless of the antigen, the levels of antibodies were lower in goats than in sheep and highest in cattle. The diagnostic performance of the assay was not affected by the absence of lipid A-core epitopes, the presence of contaminating outer membrane proteins, the AC or MC epitopic structure of the absorbed antigen, or the conjugate used. Moreover, with sera from cattle vaccinated with B. abortus S19 (AC) or from sheep and goats vaccinated with B. melitensis Rev 1 (MC), AC and MC antigens showed similar levels of reactivity. The results show that antibodies to the C epitopes largely dominate in infection, and this is consistent with the existence of multiple overlapping C epitopes (V. Weynants, D. Gilson, A. Cloeckaert, A. Tibor, P. A. Denoel, F. Godfroid, J. N. Limet, and J.-J. Letesson, Infect. Immun. 65:1939–1943, 1997) rather than with one or two C epitopes. It is concluded that, by adaptation to the corresponding antibody levels, brucellosis in cattle, sheep, and goats can be diagnosed by immunosorbent assay with a single combination of conjugate and antigen. PMID:9801329

  4. Discovery of novel targets for multi-epitope vaccines: Screening of HIV-1 genomes using association rule mining

    PubMed Central

    Paul, Sinu; Piontkivska, Helen

    2009-01-01

    Background Studies have shown that in the genome of human immunodeficiency virus (HIV-1) regions responsible for interactions with the host's immune system, namely, cytotoxic T-lymphocyte (CTL) epitopes tend to cluster together in relatively conserved regions. On the other hand, "epitope-less" regions or regions with relatively low density of epitopes tend to be more variable. However, very little is known about relationships among epitopes from different genes, in other words, whether particular epitopes from different genes would occur together in the same viral genome. To identify CTL epitopes in different genes that co-occur in HIV genomes, association rule mining was used. Results Using a set of 189 best-defined HIV-1 CTL/CD8+ epitopes from 9 different protein-coding genes, as described by Frahm, Linde & Brander (2007), we examined the complete genomic sequences of 62 reference HIV sequences (including 13 subtypes and sub-subtypes with approximately 4 representative sequences for each subtype or sub-subtype, and 18 circulating recombinant forms). The results showed that despite inclusion of recombinant sequences that would be expected to break-up associations of epitopes in different genes when two different genomes are recombined, there exist particular combinations of epitopes (epitope associations) that occur repeatedly across the world-wide population of HIV-1. For example, Pol epitope LFLDGIDKA is found to be significantly associated with epitopes GHQAAMQML and FLKEKGGL from Gag and Nef, respectively, and this association rule is observed even among circulating recombinant forms. Conclusion We have identified CTL epitope combinations co-occurring in HIV-1 genomes including different subtypes and recombinant forms. Such co-occurrence has important implications for design of complex vaccines (multi-epitope vaccines) and/or drugs that would target multiple HIV-1 regions at once and, thus, may be expected to overcome challenges associated with viral escape

  5. Computationally predicted IgE epitopes of walnut allergens contribute to cross-reactivity with peanuts

    PubMed Central

    Maleki, Soheila J.; Teuber, Suzanne S.; Cheng, Hsiaopo; Chen, Deliang; Comstock, Sarah S.; Ruan, Sanbao; Schein, Catherine H.

    2011-01-01

    Background Cross reactivity between peanuts and tree nuts implies that similar IgE epitopes are present in their proteins. Objective To determine whether walnut sequences similar to known peanut IgE binding sequences, according to the property distance (PD) scale implemented in the Structural Database of Allergenic Proteins (SDAP), react with IgE from sera of patients with allergy to walnut and/or peanut. Methods Patient sera were characterized by Western blotting for IgE-binding to nut protein extracts, and to peptides from walnut and peanut allergens, similar to known peanut epitopes as defined by low PD values, synthesized on membranes. Competitive ELISA was used to show that peanut and predicted walnut epitope sequences compete with purified Ara h 2 for binding to IgE in serum from a cross-reactive patient. Results Sequences from the vicilin walnut allergen Jug r 2 which had low PD values to epitopes of the peanut allergen Ara h 2, a 2s-albumin, bound IgE in sera from five patients who reacted to either walnut, peanut or both. A walnut epitope recognized by 6 patients mapped to a surface-exposed region on a model of the N-terminal pro-region of Jug r 2. A predicted walnut epitope competed for IgE binding to Ara h 2 in serum as well as the known IgE epitope from Ara h 2. Conclusions Sequences with low PD value (<8.5) to known IgE epitopes could contribute to cross-reactivity between allergens. This further validates the PD scoring method for predicting cross-reactive epitopes in allergens. PMID:21883278

  6. OF TRYPANOSOMATIDS. ENDOTRANSFORMATIONS AND ABERRATIONS].

    PubMed

    Frolov, A O; Malysheva, M N; Kostygov, A Yu

    2016-01-01

    Endotransformations and aberrations of the life cycle in the evolutionary history of trypanosomatids (Kinetoplastea: Trypanosomatidae) are analyzed. We treat the term "endotransformations" as evolutionarily fixed changes of phases and/or developmental stages of parasites. By contrast, we treat aberrations as evolutionary unstable, periodically arising deformations of developmental phases of trypanosomatids, never leading to life cycle changes. Various examples of life cycle endotransformations and aberrations in representatives of the family Trypanosomatidae are discussed.

  7. Approaching rational epitope vaccine design for hepatitis C virus with meta-server and multivalent scaffolding

    NASA Astrophysics Data System (ADS)

    He, Linling; Cheng, Yushao; Kong, Leopold; Azadnia, Parisa; Giang, Erick; Kim, Justin; Wood, Malcolm R.; Wilson, Ian A.; Law, Mansun; Zhu, Jiang

    2015-08-01

    Development of a prophylactic vaccine against hepatitis C virus (HCV) has been hampered by the extraordinary viral diversity and the poor host immune response. Scaffolding, by grafting an epitope onto a heterologous protein scaffold, offers a possible solution to epitope vaccine design. In this study, we designed and characterized epitope vaccine antigens for the antigenic sites of HCV envelope glycoproteins E1 (residues 314-324) and E2 (residues 412-423), for which neutralizing antibody-bound structures are available. We first combined six structural alignment algorithms in a “scaffolding meta-server” to search for diverse scaffolds that can structurally accommodate the HCV epitopes. For each antigenic site, ten scaffolds were selected for computational design, and the resulting epitope scaffolds were analyzed using structure-scoring functions and molecular dynamics simulation. We experimentally confirmed that three E1 and five E2 epitope scaffolds bound to their respective neutralizing antibodies, but with different kinetics. We then investigated a “multivalent scaffolding” approach by displaying 24 copies of an epitope scaffold on a self-assembling nanoparticle, which markedly increased the avidity of antibody binding. Our study thus demonstrates the utility of a multi-scale scaffolding strategy in epitope vaccine design and provides promising HCV immunogens for further assessment in vivo.

  8. Preparation and application of epitope magnetic molecularly imprinted polymers for enrichment of sulfonamide antibiotics in water.

    PubMed

    Hu, Yufeng; Wang, Cheng; Li, Xiangdao; Liu, Lifen

    2017-10-01

    Sulfonamides, which are widely used synthetic antibiotics, are hydrophilic and stable. They can easily migrate into the environment and aquatic animals, and increase the risk of cancer, drug resistance, and allergic symptoms if consumed by humans. Here, we developed an epitope magnetic imprinting approach to enrich multiple sulfonamide antibiotics from a water sample. Epitope magnetic molecularly imprinted polymers (EMMIPs) were prepared by free-radical polymerization using vinyl-functioned Fe 3 O 4 as a core, sulfanilamide (SA) as a dummy template, methacrylic acid as a functional monomer, and ethylene glycol dimethacrylate as a cross-linker. The performance of the EMMIPs was first evaluated by rebinding SA, and then an adsorption experiment was conducted to assess the extraction of multiple sulfonamide antibiotics containing the SA group. The binding experiments showed that the EMMIPs reached adsorption equilibrium in only 5 min with adsorption of SA at 2040 μg/g, compared with just 462 μg/g for the epitope magnetic non-imprinted polymers. EMMIPs were combined with HPLC for the detection of six sulfonamide antibiotics in surface water samples. The recoveries ranged from 79.3 to 92.4% and the relative standard deviations from 0.9 to 7.3%. © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  9. Identification of two novel immunodominant UreB CD4(+) T cell epitopes in Helicobacter pylori infected subjects.

    PubMed

    Yang, Wu-Chen; Chen, Li; Li, Hai-Bo; Li, Bin; Hu, Jian; Zhang, Jin-Yong; Yang, Shi-Ming; Zou, Quan-Ming; Guo, Hong; Wu, Chao

    2013-02-06

    An epitope-based vaccine is a promising option for treating Helicobacter pylori (H. pylori) infection. Epitope mapping is the first step in designing an epitope-based vaccine. A pivotal role of CD4(+) T cells in protection against H. pylori has been accepted, but few Th epitopes have been identified. In this study, two novel UreB CD4(+) T cell epitopes were identified using PBMCs obtained from two H. pylori infected subjects. We determined the restriction molecules by antibody blocking and used various Epstein-Barr virus-transformed B lymphocyte cell lines (BLCLs) with different HLA alleles as APCs to present peptides to CD4(+) T cells. These epitopes were DRB1*1404-restricted UreB(373-385) and DRB1*0803-restricted UreB(438-452). The T cells specific to these epitopes not only recognized autologous DCs loaded with recombinant UreB but also those pulsed with H. pylori whole cell lysates, suggesting that these epitope peptides are naturally processed. These epitopes have important value for designing an effective H. pylori vaccine. Copyright © 2012 Elsevier Ltd. All rights reserved.

  10. Processing of two latent membrane protein 1 MHC class I epitopes requires tripeptidyl peptidase II involvement.

    PubMed

    Diekmann, Jan; Adamopoulou, Eleni; Beck, Olaf; Rauser, Georg; Lurati, Sarah; Tenzer, Stefan; Einsele, Hermann; Rammensee, Hans-Georg; Schild, Hansjörg; Topp, Max S

    2009-08-01

    The EBV Ag latent membrane protein 1 (LMP1) has been described as a potential target for T cell immunotherapy in EBV-related malignancies. However, only a few CD8(+) T cell epitopes are known, and the benefit of LMP1-specific T cell immunotherapy has not yet been proven. In this work, we studied the processing of the two LMP1 HLA-A02-restricted epitopes, YLLEMLRWL and YLQQNWWTL. We found that target cells endogenously expressing the native LMP1 are not recognized by CTLs specific for these epitopes because the N-terminal part of LMP1 limits the efficiency of epitope generation. We further observed that the proteasome is not required for the generation of both epitopes and that the YLLEMLRWL epitope seems to be destroyed by the proteasome, because blocking of proteasomal activities enhanced specific CTL activation. Activation of LMP1-specific CTLs could be significantly reduced after inhibition of the tripeptidyl peptidase II, suggesting a role for this peptidase in the processing of both epitopes. Taken together, our results demonstrate that the MHC class I-restricted LMP1 epitopes studied in this work are two of very few epitopes known to date to be processed proteasome independently by tripeptidyl peptidase II.

  11. [Immunoreactivity of chimeric proteins carrying poliovirus epitopes on the VP6 of rotavirus as a vector].

    PubMed

    Pan, X-X; Zhao, B-X; Teng, Y-M; Xia, W-Y; Wang, J; Li, X-F; Liao, G-Y; Yang, С; Chen, Y-D

    2016-01-01

    Rotavirus and poliovirus continue to present significant risks and burden of disease to children in developing countries. Developing a combined vaccine may effectively prevent both illnesses and may be advantageous in terms of maximizing compliance and vaccine coverage at the same visit. Recently, we sought to generate a vaccine vector by incorporating multiple epitopes into the rotavirus group antigenic protein, VP6. In the present study, a foreign epitope presenting a system using VP6 as a vector was created with six sites on the outer surface of the vector that could be used for insertion of foreign epitopes, and three VP6-based PV1 epitope chimeric proteins were constructed. The chimeric proteins were confirmed by immunoblot, immunofluorescence assay, and injected into guinea pigs to analyze the epitope-specific humoral response. Results showed that these chimeric proteins reacted with anti-VP6F and -PV1 antibodies, and elicited antibodies against both proteins in guinea pigs. Antibodies against the chimeric proteins carrying PV1 epitopes neutralized rotavirus Wa and PV1 infection in vitro. Our study contributes to a better understanding of the use of VP6-based vectors as multiple-epitope delivery vehicles and the epitopes displayed in this form could be considered for development of epitope-based vaccines against rotavirus and poliovirus.

  12. A Comparison of Epitope Repertoires Associated with Myasthenia Gravis in Humans and Nonhuman Hosts

    PubMed Central

    Vaughan, Kerrie; Kim, Yohan; Sette, Alessandro

    2012-01-01

    Here we analyzed the molecular targets associated with myasthenia gravis (MG) immune responses, enabled by an immune epitope database (IEDB) inventory of approximately 600 MG-related epitopes derived from 175 references. The vast majority of epitopes were derived from the α-subunit of human AChR suggesting that other MG-associated autoantigens should be investigated further. Human α-AChR was mostly characterized in humans, whereas reactivity primarily to T. californica AChR was examined in animal models. While the fine specificity of T-cell response was similar in the two systems, substantial antibody reactivity to the C-terminus was detected in the nonhuman system, but not in humans. Further analysis showed that the reactivity of nonhuman hosts to the C-terminus was eliminated when data were restricted to hosts tested in the context of autoimmune disease (spontaneous or induced), demonstrating that the epitopes recognized in humans and animals were shared when disease was present. Finally, we provided data subsets relevant to particular applications, including those associated with HLA typing or restriction, sets of epitopes recognized by monoclonal antibodies, and epitopes associated with modulation of immunity or disease. In conclusion, this analysis highlights gaps, differences, and similarities in the epitope repertoires of humans and animal models. PMID:23243503

  13. Biochemical and structural characterisation of the second oxidative crosslinking step during the biosynthesis of the glycopeptide antibiotic A47934.

    PubMed

    Ulrich, Veronika; Brieke, Clara; Cryle, Max J

    2016-01-01

    The chemical complexity and biological activity of the glycopeptide antibiotics (GPAs) stems from their unique crosslinked structure, which is generated by the actions of cytochrome P450 (Oxy) enzymes that affect the crosslinking of aromatic side chains of amino acid residues contained within the GPA heptapeptide precursor. Given the crucial role peptide cyclisation plays in GPA activity, the characterisation of this process is of great importance in understanding the biosynthesis of these important antibiotics. Here, we report the cyclisation activity and crystal structure of StaF, the D- O -E ring forming Oxy enzyme from A47934 biosynthesis. Our results show that the specificity of StaF is reduced when compared to Oxy enzymes catalysing C- O -D ring formation and that this activity relies on interactions with the non-ribosomal peptide synthetase via the X-domain. Despite the interaction of StaF with the A47934 X-domain being weaker than for the preceding Oxy enzyme StaH, StaF retains higher levels of in vitro activity: we postulate that this is due to the ability of the StaF/X-domain complex to allow substrate reorganisation after initial complex formation has occurred. These results highlight the importance of testing different peptide/protein carrier constructs for in vitro GPA cyclisation assays and show that different Oxy homologues can display significantly different catalytic propensities despite their overall similarities.

  14. Monochromatic ocular wave aberrations in young monkeys

    PubMed Central

    Ramamirtham, Ramkumar; Kee, Chea-su; Hung, Li-Fang; Qiao-Grider, Ying; Roorda, Austin; Smith, Earl L.

    2006-01-01

    High-order monochromatic aberrations could potentially influence vision-dependent refractive development in a variety of ways. As a first step in understanding the effects of wave aberration on refractive development, we characterized the maturational changes that take place in the high-order aberrations of infant rhesus monkey eyes. Specifically, we compared the monochromatic wave aberrations of infant and adolescent animals and measured the longitudinal changes in the high-order aberrations of infant monkeys during the early period when emmetropization takes place. Our main findings were that (1) adolescent monkey eyes have excellent optical quality, exhibiting total RMS errors that were slightly better than those for adult human eyes that have the same numerical aperture and (2) shortly after birth, infant rhesus monkeys exhibited relatively larger magnitudes of high-order aberrations predominately spherical aberration, coma, and trefoil, which decreased rapidly to assume adolescent values by about 200 days of age. The results demonstrate that rhesus monkey eyes are a good model for studying the contribution of individual ocular components to the eye’s overall aberration structure, the mechanisms responsible for the improvements in optical quality that occur during early ocular development, and the effects of high-order aberrations on ocular growth and emmetropization. PMID:16750549

  15. Screening and identification of novel B cell epitopes of Toxoplasma gondii SAG1.

    PubMed

    Wang, Yanhua; Wang, Guangxiang; Zhang, Delin; Yin, Hong; Wang, Meng

    2013-04-30

    The identification of protein epitopes is useful for diagnostic purposes and for the development of peptide vaccines. In this study, the epitopes of Toxoplasma gondii SAG1 were identified using synthetic peptide techniques with the aid of bioinformatics. Eleven peptides derived from T. gondii SAG1 were assessed by ELISA using pig sera from different time points after infection. Four (PS4, PS6, PS10 and PS11), out of the eleven peptides tested were recognized by all sera. Then, shorter peptides that were derived from PS4, PS6, PS10 and PS11 were predicted using bioinformatics and tested by experimentation. Four out of nine shorter peptides were identified successfully (amino acids 106-120, 166-180, 289-300 and 313-332). We have precisely located the epitopes of T. gondii SAG1 using pig sera collected at different time points after infection. The identified epitopes may be useful for the further study of epitope-based vaccines and diagnostic reagents.

  16. The molecular relationship between antigenic domains and epitopes on hCG.

    PubMed

    Berger, Peter; Lapthorn, Adrian J

    2016-08-01

    Antigenic domains are defined to contain a limited number of neighboring epitopes recognized by antibodies (Abs) but their molecular relationship remains rather elusive. We thoroughly analyzed the antigenic surface of the important pregnancy and tumor marker human chorionic gonadotropin (hCG), a cystine knot (ck) growth factor, and set antigenic domains and epitopes in molecular relationships to each other. Antigenic domains on hCG, its free hCGα and hCGβ subunits are dependent on appropriate inherent molecular features such as molecular accessibility and protrusion indices that determine bulging structures accessible to Abs. The banana-shaped intact hCG comprises ∼7500Å(2) of antigenic surface with minimally five antigenic domains that encompass a continuum of overlapping non-linear composite epitopes, not taking into account the C-terminal peptide extension of hCGβ (hCGβCTP). Epitopes within an antigenic domain are defined by specific Abs, that bury nearly 1000Å(2) of surface accessible area on the antigen and recognize a few up to 15 amino acid (aa) residues, whereby between 2 and 5 of these provide the essential binding energy. Variability in Ab binding modes to the contact aa residues are responsible for the variation in affinity and intra- and inter-species specificity, e.g. cross-reactions with luteinizing hormone (LH). Each genetically distinct fragment antigen binding (Fab) defines its own epitope. Consequently, recognition of the same epitope by different Abs is only possible in cases of genetically identical sequences of its binding sites. Due to combinatorial V(D)J gene segment variability of heavy and light chains, Abs defining numerous epitopes within an antigenic domain can be generated by different individuals and species. Far more than hundred Abs against the immuno-dominant antigenic domains of either subunit at both ends of the hCG-molecule, the tips of peptide loops one and three (Ł1+3) protruding from the central ck, encompassing h

  17. Iteration of ultrasound aberration correction methods

    NASA Astrophysics Data System (ADS)

    Maasoey, Svein-Erik; Angelsen, Bjoern; Varslot, Trond

    2004-05-01

    Aberration in ultrasound medical imaging is usually modeled by time-delay and amplitude variations concentrated on the transmitting/receiving array. This filter process is here denoted a TDA filter. The TDA filter is an approximation to the physical aberration process, which occurs over an extended part of the human body wall. Estimation of the TDA filter, and performing correction on transmit and receive, has proven difficult. It has yet to be shown that this method works adequately for severe aberration. Estimation of the TDA filter can be iterated by retransmitting a corrected signal and re-estimate until a convergence criterion is fulfilled (adaptive imaging). Two methods for estimating time-delay and amplitude variations in receive signals from random scatterers have been developed. One method correlates each element signal with a reference signal. The other method use eigenvalue decomposition of the receive cross-spectrum matrix, based upon a receive energy-maximizing criterion. Simulations of iterating aberration correction with a TDA filter have been investigated to study its convergence properties. A weak and strong human-body wall model generated aberration. Both emulated the human abdominal wall. Results after iteration improve aberration correction substantially, and both estimation methods converge, even for the case of strong aberration.

  18. Epitope mapping of commercial antibodies that detect myocilin.

    PubMed

    Patterson-Orazem, Athéna C; Hill, Shannon E; Fautsch, Michael P; Lieberman, Raquel L

    2018-05-09

    The presence of myocilin is often used in the process of validating trabecular meshwork (TM) cells and eye tissues, but the antibody reagents used for detection are poorly characterized. Indeed, for over a century, researchers have been using antibodies to track proteins of interest in a variety of biological contexts, but many antibodies remain ill-defined at the molecular level and in their target epitope. Such issues have prompted efforts from major funding agencies to validate reagents and combat reproducibility issues across biomedical sciences. Here we characterize the epitopes recognized by four commercial myocilin antibodies, aided by structurally and biochemically characterized myocilin fragments. All four antibodies recognize enriched myocilin secreted from human TM cell media. The detection of myocilin fragments by ELISA and Western blot reveal a variety of epitopes across the myocilin polypeptide chain. A more precise understanding of myocilin antibody targets, including conformational specificity, should aid the community in standardizing protocols across laboratories and in turn, lead to a better understanding of eye physiology and disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

  19. T-Epitope Designer: A HLA-peptide binding prediction server.

    PubMed

    Kangueane, Pandjassarame; Sakharkar, Meena Kishore

    2005-05-15

    The current challenge in synthetic vaccine design is the development of a methodology to identify and test short antigen peptides as potential T-cell epitopes. Recently, we described a HLA-peptide binding model (using structural properties) capable of predicting peptides binding to any HLA allele. Consequently, we have developed a web server named T-EPITOPE DESIGNER to facilitate HLA-peptide binding prediction. The prediction server is based on a model that defines peptide binding pockets using information gleaned from X-ray crystal structures of HLA-peptide complexes, followed by the estimation of peptide binding to binding pockets. Thus, the prediction server enables the calculation of peptide binding to HLA alleles. This model is superior to many existing methods because of its potential application to any given HLA allele whose sequence is clearly defined. The web server finds potential application in T cell epitope vaccine design. http://www.bioinformation.net/ted/

  20. Electro-optical study of the exposure of Azospirillum brasilense carbohydrate epitopes.

    PubMed

    Guliy, Olga I; Matora, Larisa Yu; Dykman, Lev A; Staroverov, Sergey A; Burygin, Gennady L; Bunin, Viktor D; Burov, Andrei M; Ignatov, Oleg V

    2015-01-01

    The exposure of Azospirillum brasilense carbohydrate epitopes was investigated by electro-optical analysis of bacterial cell suspensions. To study changes in the electro-optical (EO) properties of the suspensions, we used antibodies generated to the complete lipopolysaccharide of A. brasilense type strain Sp7 and also antibodies to the smooth and rough O polysaccharides of Sp7. After 18 hr of culture growth, the EO signal of the suspension treated with antibodies to smooth O polysaccharide was approximately 20% lower than that of the suspension treated with antibodies to complete lipopolysaccharide (control). After 72 hr of culture growth, the strongest EO signal was observed for the cells treated with antibodies to rough O polysaccharide (approximately 46% greater than the control), whereas for the cells treated with antibodies to smooth O polysaccharide, it was much lower (approximately 23% of the control). These data were confirmed by electron microscopy. The results of the study may have importance for the rapid evaluation of changes in lipopolysaccharide form in microbial biotechnology, when the antigenic composition of the bacterial surface requires close control.

  1. Parallel Immunizations of Rabbits Using the Same Antigen Yield Antibodies with Similar, but Not Identical, Epitopes

    PubMed Central

    Hjelm, Barbara; Forsström, Björn; Löfblom, John; Rockberg, Johan; Uhlén, Mathias

    2012-01-01

    A problem for the generation of polyclonal antibodies is the potential difficulties for obtaining a renewable resource due to batch-to-batch variations when the same antigen is immunized into several separate animals. Here, we have investigated this issue by determining the epitopes of antibodies generated from parallel immunizations of rabbits with recombinant antigens corresponding to ten human protein targets. The epitopes were mapped by both a suspension bead array approach using overlapping synthetic 15-mer peptides and a bacterial display approach using expression of random fragments of the antigen on the surface of bacteria. Both methods determined antibody binding with the aid of fluorescent-based analysis. In addition, one polyclonal antibody was fractionated by peptide-specific affinity capture for in-depth comparison of epitopes. The results show that the same antigen immunized in several rabbits yields polyclonal antibodies with similar epitopes, but with larger differences in the relative amounts of antibodies to the different epitopes. In some cases, unique epitopes were observed for one of the immunizations. The results suggest that polyclonal antibodies generated by repeated immunizations do not display an identical epitope pattern, although many of the epitopes are similar. PMID:23284606

  2. Analysis of the epitope structure of Plum pox virus coat protein.

    PubMed

    Candresse, Thierry; Saenz, Pilar; García, Juan Antonio; Boscia, Donato; Navratil, Milan; Gorris, Maria Teresa; Cambra, Mariano

    2011-05-01

    Typing of the particular Plum pox virus (PPV) strain responsible in an outbreak has important practical implications and is frequently performed using strain-specific monoclonal antibodies (MAbs). Analysis in Western blots of the reactivity of 24 MAbs to a 112-amino-acid N-terminal fragment of the PPV coat protein (CP) expressed in Escherichia coli showed that 21 of the 24 MAbs recognized linear or denaturation-insensitive epitopes. A series of eight C-truncated CP fragments allowed the mapping of the epitopes recognized by the MAbs. In all, 14 of them reacted to the N-terminal hypervariable region, defining a minimum of six epitopes, while 7 reacted to the beginning of the core region, defining a minimum of three epitopes. Sequence comparisons allowed the more precise positioning of regions recognized by several MAbs, including those recognized by the 5B-IVIA universal MAb (amino acids 94 to 100) and by the 4DG5 and 4DG11 D serogroup-specific MAbs (amino acids 43 to 64). A similar approach coupled with infectious cDNA clone mutagenesis showed that a V74T mutation in the N-terminus of the CP abolished the binding of the M serogroup-specific AL MAb. Taken together, these results provide a detailed positioning of the epitopes recognized by the most widely used PPV detection and typing MAbs.

  3. [Monochromatic aberration in accommodation. Dynamic wavefront analysis].

    PubMed

    Fritzsch, M; Dawczynski, J; Jurkutat, S; Vollandt, R; Strobel, J

    2011-06-01

    Monochromatic aberrations may influence the visual acuity of the eye. They are not stable and can be affected by different factors. The subject of the following paper is the dynamic investigation of the changes in wavefront aberration with accommodation. Dynamic measurement of higher and lower order aberrations was performed with a WASCA Wavefront Analyzer (Carl-Zeiss-Meditec) and a specially constructed target device for aligning objects in far and near distances on 25 subjects aged from 15 to 27 years old. Wavefront aberrations showed some significant changes in accommodation. In addition to the characteristic sphere reaction accompanying miosis and changes in horizontal prism (Z(1) (1)) in the sense of a convergence movement of the eyeball also occurred. Furthermore defocus rose (Z(2) (0)) and astigmatism (Z(2) (-2)) changed. In higher-order aberrations a decrease in coma-like Zernike polynomials (Z(3) (-1), Z(3) (1)) was found. The most obvious change appeared in spherical aberration (Z(4) (0)) which increased and changed from positive to negative. In addition the secondary astigmatism (Z(4) (-2)) and quadrafoil (Z(4) (4)) rise also increased. The total root mean square (RMS), as well as the higher-order aberrations (RMS-HO) significantly increased in accommodation which is associated with a theoretical reduction of visual acuity. An analysis of the influence of pupil size on aberrations showed significant increases in defocus, spherical aberration, quadrafoil, RMS and RMS HO by increasing pupil diameter. By accommodation-associated miosis, the growing aberrations are partially compensated by focusing on near objects. Temporal analysis of the accommodation process with dynamic wavefront analysis revealed significant delays in pupil response and changing of prism in relation to the sphere reaction. In accommodation to near objects a discrete time ahead of third order aberrations in relation to the sphere response was found. Using dynamic wavefront measurement

  4. Wavefront aberrations of x-ray dynamical diffraction beams.

    PubMed

    Liao, Keliang; Hong, Youli; Sheng, Weifan

    2014-10-01

    The effects of dynamical diffraction in x-ray diffractive optics with large numerical aperture render the wavefront aberrations difficult to describe using the aberration polynomials, yet knowledge of them plays an important role in a vast variety of scientific problems ranging from optical testing to adaptive optics. Although the diffraction theory of optical aberrations was established decades ago, its application in the area of x-ray dynamical diffraction theory (DDT) is still lacking. Here, we conduct a theoretical study on the aberration properties of x-ray dynamical diffraction beams. By treating the modulus of the complex envelope as the amplitude weight function in the orthogonalization procedure, we generalize the nonrecursive matrix method for the determination of orthonormal aberration polynomials, wherein Zernike DDT and Legendre DDT polynomials are proposed. As an example, we investigate the aberration evolution inside a tilted multilayer Laue lens. The corresponding Legendre DDT polynomials are obtained numerically, which represent balanced aberrations yielding minimum variance of the classical aberrations of an anamorphic optical system. The balancing of classical aberrations and their standard deviations are discussed. We also present the Strehl ratio of the primary and secondary balanced aberrations.

  5. NetCTLpan: pan-specific MHC class I pathway epitope predictions

    PubMed Central

    Larsen, Mette Voldby; Lundegaard, Claus; Nielsen, Morten

    2010-01-01

    Reliable predictions of immunogenic peptides are essential in rational vaccine design and can minimize the experimental effort needed to identify epitopes. In this work, we describe a pan-specific major histocompatibility complex (MHC) class I epitope predictor, NetCTLpan. The method integrates predictions of proteasomal cleavage, transporter associated with antigen processing (TAP) transport efficiency, and MHC class I binding affinity into a MHC class I pathway likelihood score and is an improved and extended version of NetCTL. The NetCTLpan method performs predictions for all MHC class I molecules with known protein sequence and allows predictions for 8-, 9-, 10-, and 11-mer peptides. In order to meet the need for a low false positive rate, the method is optimized to achieve high specificity. The method was trained and validated on large datasets of experimentally identified MHC class I ligands and cytotoxic T lymphocyte (CTL) epitopes. It has been reported that MHC molecules are differentially dependent on TAP transport and proteasomal cleavage. Here, we did not find any consistent signs of such MHC dependencies, and the NetCTLpan method is implemented with fixed weights for proteasomal cleavage and TAP transport for all MHC molecules. The predictive performance of the NetCTLpan method was shown to outperform other state-of-the-art CTL epitope prediction methods. Our results further confirm the importance of using full-type human leukocyte antigen restriction information when identifying MHC class I epitopes. Using the NetCTLpan method, the experimental effort to identify 90% of new epitopes can be reduced by 15% and 40%, respectively, when compared to the NetMHCpan and NetCTL methods. The method and benchmark datasets are available at http://www.cbs.dtu.dk/services/NetCTLpan/. Electronic supplementary material The online version of this article (doi:10.1007/s00251-010-0441-4) contains supplementary material, which is available to authorized users. PMID

  6. Pep19 drives epitope spreading in periodontitis and periodontitis-associated autoimmune diseases.

    PubMed

    Kwon, E-Y; Cha, G S; Jeong, E; Lee, J-Y; Kim, S-J; Surh, C D; Choi, J

    2016-06-01

    Epitope spreading is one of valid mechanisms operating in immunopathological processes of infection-induced autoimmune diseases. We hypothesized that the peptide 19 from Porphyromonas gingivalis heat shock protein (HSP) 60 (Pep19) may be the dominant epitope from which epitope-specific immune response to subdominant epitopes may diversify sequentially into autoimmune responses directed at human neoepitopes in P. gingivalis-induced periodontitis and autoimmune diseases. However, the exact feature and mechanism on how Pep19 may drive epitope spreading into human autoantigens in chronic periodontitis or P. gingivalis-induced experimental periodontitis has not been clarified. The present study was performed with the following specific aims: (i) to delineate retrospectively the features of epitope spreading by human cross-sectional analysis; (ii) to demonstrate prospectively the epitope spreading into new antigenic determinants in an ordered, predictable and sequential manner in experimental periodontitis; and (iii) to clarify the mechanism on how immunization with Pep19 may mobilize helper T cells or elicit B-cell responses to human autoantigens and neoantigen. The study was devised for two independent investigations - a cross-sectional analysis on clinical subjects and a prospective analysis on experimental periodontitis - each being subdivided further into two additional independent observations. Cross-sectional dot immunoblot pattern against a panel of peptides of P. gingivalis HSP60 and human HSP60 was performed among age-dependent healthy subjects and between healthy subjects, patients with chronic periodontitis and patients with autoimmune disease, to identify epitope spreading. A peptide-specific T-cell line was established for phenotype analysis and for proliferation assay to an array of identical peptides. An identical prospective analysis was performed in P. gingivalis-induced experimental periodontitis or in Pep19-immunized mice. Cross-reactivity of anti

  7. On the Definition of Aberration

    NASA Astrophysics Data System (ADS)

    Xu, Minghui; Wang, Guangli

    2014-12-01

    There was a groundbreaking step in the history of astronomy in 1728 when the effect of aberration was discovered by James Bradley (1693-1762). Recently, the solar acceleration, due to the variations in the aberrational effect of extragalactic sources caused by it, has been determined from VLBI observations with an uncertainty of about 0.5 mm{\\cdot}{s^{-1}}{\\cdot}{yr^{-1}} level. As a basic concept in astrometry with a nearly 300-year history, the definition of aberration, however, is still equivocal and discordant in the literature. It has been under continuing debate whether it depends on the relative motion between the observer and the observed source or only on the motion of the observer with respect to the frame of reference. In this paper, we will review the debate and the inconsistency in the definition of the aberration since the last century, and then discuss its definition in detail, which involves the discussions on the planetary aberration, the stellar aberration, the proper motion of an object during the travel time of light from the object to the observer, and the way of selecting the reference frame to express and distinguish the motions of the source and the observer. The aberration is essentially caused by the transformation between coordinate systems, and consequently quantified by the velocity of the observer with respect to the selected reference frame, independent of the motion of the source. Obviously, this nature is totally different from that of the definition given by the IAU WG NFA (Capitaine, 2007) in 2006, which is stated as, ``the apparent angular displacement of the observed position of a celestial object from its geometric position, caused by the finite velocity of light in combination with the motions of the observer and of the observed object.''

  8. Epitope Mapping of Monoclonal Antibody PMab-48 Against Dog Podoplanin.

    PubMed

    Yamada, Shinji; Kaneko, Mika K; Itai, Shunsuke; Chang, Yao-Wen; Nakamura, Takuro; Yanaka, Miyuki; Ogasawara, Satoshi; Murata, Takeshi; Uchida, Hiroaki; Tahara, Hideaki; Harada, Hiroyuki; Kato, Yukinari

    2018-04-02

    Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is expressed on normal renal podocytes, pulmonary type I alveolar cells, and lymphatic endothelial cells. Increased expression of PDPN in cancers is associated with poor prognosis and hematogenous metastasis through interactions with C-type lectin-like receptor 2 (CLEC-2) on platelets. We previously reported a novel PMab-48 antibody, which is an anti-dog PDPN (dPDPN) monoclonal antibody (mAb) recognizing PDPN expressed in lymphatic endothelial cells. However, the binding epitope of PMab-48 is yet to be clarified. In this study, an enzyme-linked immunosorbent assay and flow cytometry were used to investigate epitopes of PMab-48. The results revealed that the critical epitope of PMab-48 comprises Asp29, Asp30, Ile31, Ile32, and Pro33 of dPDPN.

  9. In Vivo Validation of Predicted and Conserved T Cell Epitopes in a Swine Influenza Model

    PubMed Central

    Gutiérrez, Andres H.; Loving, Crystal; Moise, Leonard; Terry, Frances E.; Brockmeier, Susan L.; Hughes, Holly R.; Martin, William D.; De Groot, Anne S.

    2016-01-01

    Swine influenza is a highly contagious respiratory viral infection in pigs that is responsible for significant financial losses to pig farmers annually. Current measures to protect herds from infection include: inactivated whole-virus vaccines, subunit vaccines, and alpha replicon-based vaccines. As is true for influenza vaccines for humans, these strategies do not provide broad protection against the diverse strains of influenza A virus (IAV) currently circulating in U.S. swine. Improved approaches to developing swine influenza vaccines are needed. Here, we used immunoinformatics tools to identify class I and II T cell epitopes highly conserved in seven representative strains of IAV in U.S. swine and predicted to bind to Swine Leukocyte Antigen (SLA) alleles prevalent in commercial swine. Epitope-specific interferon-gamma (IFNγ) recall responses to pooled peptides and whole virus were detected in pigs immunized with multi-epitope plasmid DNA vaccines encoding strings of class I and II putative epitopes. In a retrospective analysis of the IFNγ responses to individual peptides compared to predictions specific to the SLA alleles of cohort pigs, we evaluated the predictive performance of PigMatrix and demonstrated its ability to distinguish non-immunogenic from immunogenic peptides and to identify promiscuous class II epitopes. Overall, this study confirms the capacity of PigMatrix to predict immunogenic T cell epitopes and demonstrate its potential for use in the design of epitope-driven vaccines for swine. Additional studies that match the SLA haplotype of animals with the study epitopes will be required to evaluate the degree of immune protection conferred by epitope-driven DNA vaccines in pigs. PMID:27411061

  10. Screening and identification of novel B cell epitopes of Toxoplasma gondii SAG1

    PubMed Central

    2013-01-01

    Background The identification of protein epitopes is useful for diagnostic purposes and for the development of peptide vaccines. In this study, the epitopes of Toxoplasma gondii SAG1 were identified using synthetic peptide techniques with the aid of bioinformatics. Findings Eleven peptides derived from T. gondii SAG1 were assessed by ELISA using pig sera from different time points after infection. Four (PS4, PS6, PS10 and PS11), out of the eleven peptides tested were recognized by all sera. Then, shorter peptides that were derived from PS4, PS6, PS10 and PS11 were predicted using bioinformatics and tested by experimentation. Four out of nine shorter peptides were identified successfully (amino acids 106–120, 166–180, 289–300 and 313–332). Conclusions We have precisely located the epitopes of T. gondii SAG1 using pig sera collected at different time points after infection. The identified epitopes may be useful for the further study of epitope-based vaccines and diagnostic reagents. PMID:23631709

  11. Prediction of Pan-Specific B-Cell Epitopes From Nucleocapsid Protein of Hantaviruses Causing Hantavirus Cardiopulmonary Syndrome.

    PubMed

    Kalaiselvan, Sagadevan; Sankar, Sathish; Ramamurthy, Mageshbabu; Ghosh, Asit Ranjan; Nandagopal, Balaji; Sridharan, Gopalan

    2017-08-01

    Hantaviruses are emerging viral pathogens that causes hantavirus cardiopulmonary syndrome (HCPS) in the Americas, a severe, sometimes fatal, respiratory disease in humans with a case fatality rate of ≥50%. IgM and IgG-based serological detection methods are the most common approaches used for laboratory diagnosis of hantaviruses. Such emerging viral pathogens emphasizes the need for improved rapid diagnostic devices and vaccines incorporating pan-specific epitopes of genotypes. We predicted linear B-cell epitopes for hantaviruses that are specific to genotypes causing HCPS in humans using in silico prediction servers. We modeled the Andes and Sin Nombre hantavirus nucleocapsid protein to locate the identified epitopes. Based on the mean percent prediction probability score, epitope IMASKSVGS/TAEEKLKKKSAF was identified as the best candidate B-cell epitope specific for hantaviruses causing HCPS. Promiscuous epitopes were identified in the C-terminal of the protein. Our study for the first time has reported pan-specific B-cell epitopes for developing immunoassays in the detection of antibodies to hantaviruses causing HCPS. Identification of epitopes with pan-specific recognition of all genotypes causing HCPS could be valuable for the development of immunodiagnositic tools toward pan-detection of hantavirus antibodies in ELISA. J. Cell. Biochem. 118: 2320-2324, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  12. Effect of monochromatic aberrations on photorefractive patterns

    NASA Astrophysics Data System (ADS)

    Campbell, Melanie C. W.; Bobier, W. R.; Roorda, A.

    1995-08-01

    Photorefractive methods have become popular in the measurement of refractive and accommodative states of infants and children owing to their photographic nature and rapid speed of measurement. As in the case of any method that measures the refractive state of the human eye, monochromatic aberrations will reduce the accuracy of the measurement. Monochromatic aberrations cannot be as easily predicted or controlled as chromatic aberrations during the measurement, and accordingly they will introduce measurement errors. This study defines this error or uncertainty by extending the existing paraxial optical analyses of coaxial and eccentric photorefraction. This new optical analysis predicts that, for the amounts of spherical aberration (SA) reported for the human eye, there will be a significant degree of measurement uncertainty introduced for all photorefractive methods. The dioptric amount of this uncertainty may exceed the maximum amount of SA present in the eye. The calculated effects on photorefractive measurement of a real eye with a mixture of spherical aberration and coma are shown to be significant. The ability, developed here, to predict photorefractive patterns corresponding to different amounts and types of monochromatic aberration may in the future lead to an extension of photorefractive methods to the dual measurement of refractive states and aberrations of individual eyes. aberration, retinal image quality,

  13. Characterizing the Interactions between a Naturally Primed Immunoglobulin A and Its Conserved Bacteroides thetaiotaomicron Species-specific Epitope in Gnotobiotic Mice*

    PubMed Central

    Peterson, Daniel A.; Planer, Joseph D.; Guruge, Janaki L.; Xue, Lai; Downey-Virgin, Whitt; Goodman, Andrew L.; Seedorf, Henning; Gordon, Jeffrey I.

    2015-01-01

    The adaptive immune response to the human gut microbiota consists of a complex repertoire of antibodies interacting with a broad range of taxa. Fusing intestinal lamina propria lymphocytes from mice monocolonized with Bacteroides thetaiotaomicron to a myeloma fusion partner allowed us to recover hybridomas that captured naturally primed, antigen-specific antibody responses representing multiple isotypes, including IgA. One of these hybridomas, 260.8, produced a monoclonal antibody that recognizes an epitope specific for B. thetaiotaomicron isolates in a large panel of hospital- and community-acquired Bacteroides. Whole genome transposon mutagenesis revealed a 19-gene locus, involved in LPS O-antigen polysaccharide synthesis and conserved among multiple B. thetaiotaomicron isolates, that is required for 260.8 epitope expression. Mutants in this locus exhibited marked fitness defects in vitro during growth in rich medium and in gnotobiotic mice colonized with defined communities of human gut symbionts. Expression of the 260.8 epitope was sustained during 10 months of daily passage in vitro and during 14 months of monocolonization of gnotobiotic wild-type, Rag1−/−, or Myd88−/− mice. Comparison of gnotobiotic Rag1−/− mice with and without subcutaneous 260.8 hybridomas disclosed that this IgA did not affect B. thetaiotaomicron population density or suppress 260.8 epitope production but did affect bacterial gene expression in ways emblematic of a diminished host innate immune response. Our study illustrates an approach for (i) generating diagnostic antibodies, (ii) characterizing IgA responses along a continuum of specificity/degeneracy that defines the IgA repertoire to gut symbionts, and (iii) identifying immunogenic epitopes that affect competitiveness and help maintain host-microbe mutualism. PMID:25795776

  14. Meta-analysis of immune epitope data for all Plasmodia: overview and applications for malarial immunobiology and vaccine-related issues

    PubMed Central

    Vaughan, K.; Blythe, M.; Greenbaum, J.; Zhang, Q.; Peters, B.; Doolan, D. L.; Sette, A.

    2012-01-01

    Summary We present a comprehensive meta-analysis of more than 500 references, describing nearly 5000 unique B cell and T cell epitopes derived from the Plasmodium genus, and detailing thousands of immunological assays. This is the first inventory of epitope data related to malaria-specific immunology, plasmodial pathogenesis, and vaccine performance. The survey included host and pathogen species distribution of epitopes, the number of antibody vs. CD4+ and CD8+ T cell epitopes, the genomic distribution of recognized epitopes, variance among epitopes from different parasite strains, and the characterization of protective epitopes and of epitopes associated with parasite evasion of the host immune response. The results identify knowledge gaps and areas for further investigation. This information has relevance to issues, such as the identification of epitopes and antigens associated with protective immunity, the design and development of candidate malaria vaccines, and characterization of immune response to strain polymorphisms. PMID:19149776

  15. Depigmented allergoids reveal new epitopes with capacity to induce IgG blocking antibodies.

    PubMed

    López-Matas, M Angeles; Gallego, Mayte; Iraola, Víctor; Robinson, Douglas; Carnés, Jerónimo

    2013-01-01

    The synthesis of allergen-specific blocking IgGs that interact with IgE after allergen immunotherapy (SIT) has been related to clinical efficacy. The objectives were to investigate the epitope specificity of IgG-antibodies induced by depigmented-polymerized (Dpg-Pol) allergoids and unmodified allergen extracts, and examine IgE-blocking activity of induced IgG-antibodies. Rabbits were immunized with native and Dpg-Pol extracts of birch pollen, and serum samples were obtained. Recognition of linear IgG-epitopes of Bet v 1 and Bet v 2 and the capacity of these IgG-antibodies to block binding of human-IgE was determined. Serum from rabbits immunized with native extracts recognised 11 linear epitopes from Bet v 1, while that from Dpg-Pol-immunized animals recognised 8. For Bet v 2, 8 epitopes were recognized by IgG from native immunized animals, and 9 from Dpg-Pol immunized one. Dpg-Pol and native immunized serum did not always recognise the same epitopes, but specific-IgG from both could block human-IgE binding sites for native extract. Depigmented-polymerized birch extract stimulates the synthesis of specific IgG-antibodies which recognize common but also novel epitopes compared with native extracts. IgG-antibodies induced by Dpg-Pol effectively inhibit human-IgE binding to allergens which may be part of the mechanism of action of SIT.

  16. Conformational B-cell epitopes prediction from sequences using cost-sensitive ensemble classifiers and spatial clustering.

    PubMed

    Zhang, Jian; Zhao, Xiaowei; Sun, Pingping; Gao, Bo; Ma, Zhiqiang

    2014-01-01

    B-cell epitopes are regions of the antigen surface which can be recognized by certain antibodies and elicit the immune response. Identification of epitopes for a given antigen chain finds vital applications in vaccine and drug research. Experimental prediction of B-cell epitopes is time-consuming and resource intensive, which may benefit from the computational approaches to identify B-cell epitopes. In this paper, a novel cost-sensitive ensemble algorithm is proposed for predicting the antigenic determinant residues and then a spatial clustering algorithm is adopted to identify the potential epitopes. Firstly, we explore various discriminative features from primary sequences. Secondly, cost-sensitive ensemble scheme is introduced to deal with imbalanced learning problem. Thirdly, we adopt spatial algorithm to tell which residues may potentially form the epitopes. Based on the strategies mentioned above, a new predictor, called CBEP (conformational B-cell epitopes prediction), is proposed in this study. CBEP achieves good prediction performance with the mean AUC scores (AUCs) of 0.721 and 0.703 on two benchmark datasets (bound and unbound) using the leave-one-out cross-validation (LOOCV). When compared with previous prediction tools, CBEP produces higher sensitivity and comparable specificity values. A web server named CBEP which implements the proposed method is available for academic use.

  17. The immune epitope database: a historical retrospective of the first decade.

    PubMed

    Salimi, Nima; Fleri, Ward; Peters, Bjoern; Sette, Alessandro

    2012-10-01

    As the amount of biomedical information available in the literature continues to increase, databases that aggregate this information continue to grow in importance and scope. The population of databases can occur either through fully automated text mining approaches or through manual curation by human subject experts. We here report our experiences in populating the National Institute of Allergy and Infectious Diseases sponsored Immune Epitope Database and Analysis Resource (IEDB, http://iedb.org), which was created in 2003, and as of 2012 captures the epitope information from approximately 99% of all papers published to date that describe immune epitopes (with the exception of cancer and HIV data). This was achieved using a hybrid model based on automated document categorization and extensive human expert involvement. This task required automated scanning of over 22 million PubMed abstracts followed by classification and curation of over 13 000 references, including over 7000 infectious disease-related manuscripts, over 1000 allergy-related manuscripts, roughly 4000 related to autoimmunity, and 1000 transplant/alloantigen-related manuscripts. The IEDB curation involves an unprecedented level of detail, capturing for each paper the actual experiments performed for each different epitope structure. Key to enabling this process was the extensive use of ontologies to ensure rigorous and consistent data representation as well as interoperability with other bioinformatics resources, including the Protein Data Bank, Chemical Entities of Biological Interest, and the NIAID Bioinformatics Resource Centers. A growing fraction of the IEDB data derives from direct submissions by research groups engaged in epitope discovery, and is being facilitated by the implementation of novel data submission tools. The present explosion of information contained in biological databases demands effective query and display capabilities to optimize the user experience. Accordingly, the

  18. Prediction and characterization of novel epitopes of serotype A foot-and-mouth disease viruses circulating in East Africa using site-directed mutagenesis

    PubMed Central

    Bari, Fufa Dawo; Parida, Satya; Asfor, Amin S.; Haydon, Daniel T.; Reeve, Richard; Paton, David J.

    2015-01-01

    Epitopes on the surface of the foot-and-mouth disease virus (FMDV) capsid have been identified by monoclonal antibody (mAb) escape mutant studies leading to the designation of four antigenic sites in serotype A FMDV. Previous work focused on viruses isolated mainly from Asia, Europe and Latin America. In this study we report on the prediction of epitopes in African serotype A FMDVs and testing of selected epitopes using reverse genetics. Twenty-four capsid amino acid residues were predicted to be of antigenic significance by analysing the capsid sequences (n = 56) using in silico methods, and six residues by correlating capsid sequence with serum–virus neutralization data. The predicted residues were distributed on the surface-exposed capsid regions, VP1–VP3. The significance of residue changes at eight of the predicted epitopes was tested by site-directed mutagenesis using a cDNA clone resulting in the generation of 12 mutant viruses involving seven sites. The effect of the amino acid substitutions on the antigenic nature of the virus was assessed by virus neutralization (VN) test. Mutations at four different positions, namely VP1-43, VP1-45, VP2-191 and VP3-132, led to significant reduction in VN titre (P value = 0.05, 0.05, 0.001 and 0.05, respectively). This is the first time, to our knowledge, that the antigenic regions encompassing amino acids VP1-43 to -45 (equivalent to antigenic site 3 in serotype O), VP2-191 and VP3-132 have been predicted as epitopes and evaluated serologically for serotype A FMDVs. This identifies novel capsid epitopes of recently circulating serotype A FMDVs in East Africa. PMID:25614587

  19. Differential Recognition of Mycobacterium tuberculosis-Specific Epitopes as a Function of Tuberculosis Disease History.

    PubMed

    Scriba, Thomas J; Carpenter, Chelsea; Pro, Sebastian Carrasco; Sidney, John; Musvosvi, Munyaradzi; Rozot, Virginie; Seumois, Grégory; Rosales, Sandy L; Vijayanand, Pandurangan; Goletti, Delia; Makgotlho, Edward; Hanekom, Willem; Hatherill, Mark; Peters, Bjoern; Sette, Alessandro; Arlehamn, Cecilia S Lindestam

    2017-09-15

    Individuals with a history of tuberculosis (TB) disease are at elevated risk of disease recurrence. The underlying cause is not known, but one explanation is that previous disease results in less-effective immunity against Mycobacterium tuberculosis (Mtb). We hypothesized that the repertoire of Mtb-derived epitopes recognized by T cells from individuals with latent Mtb infection differs as a function of previous diagnosis of active TB disease. T-cell responses to peptide pools in samples collected from an adult screening and an adolescent validation cohort were measured by IFN-γ enzyme-linked immunospot assay or intracellular cytokine staining. We identified a set of "type 2" T-cell epitopes that were recognized at 10-fold-lower levels in Mtb-infected individuals with a history of TB disease less than 6 years ago than in those without previous TB. By contrast, "type 1" epitopes were recognized equally well in individuals with or without previous TB. The differential epitope recognition was not due to differences in HLA class II binding, memory phenotypes, or gene expression in the responding T cells. Instead, "TB disease history-sensitive" type 2 epitopes were significantly (P < 0.0001) more homologous to sequences from bacteria found in the human microbiome than type 1 epitopes. Preferential loss of T-cell reactivity to Mtb epitopes that are homologous to bacteria in the microbiome in persons with previous TB disease may reflect long-term effects of antibiotic TB treatment on the microbiome.

  20. Mimotopes identify conformational epitopes on parvalbumin, the major fish allergen.

    PubMed

    Untersmayr, Eva; Szalai, Krisztina; Riemer, Angelika B; Hemmer, Wolfgang; Swoboda, Ines; Hantusch, Brigitte; Schöll, Isabella; Spitzauer, Susanne; Scheiner, Otto; Jarisch, Reinhart; Boltz-Nitulescu, George; Jensen-Jarolim, Erika

    2006-03-01

    Parvalbumin, the major fish allergen, is recognized by allergen-specific IgE of more than 90% of all fish-allergic patients. A detailed knowledge of allergenic structures is crucial for developing a vaccine inducing blocking antibodies specifically directed towards the IgE binding epitopes. In the present study we aimed to use the phage display technique to generate mimotopes, which mimic epitopes on parvalbumin. Parvalbumin-specific IgE was purified from sera of fish-allergic patients and used for screening of a constrained decamer phage library. After four rounds of biopanning using parvalbumin-specific IgE, five phage clones were selected which were specifically recognized by parvalbumin-specific IgE as well as IgG. DNA sequencing and peptide alignment revealed a high degree of sequence similarities between the mimotopes. Interestingly, on the surface of natural parvalbumin three regions could be defined by computational mimotope matching. In accordance, previously defined allergenic peptides of cod parvalbumin highlighted areas in close proximity or overlapping with the mimotope matching sites. From the presented data we conclude that our approach identified conformational epitopes of parvalbumin relevant for IgE and IgG binding. We suggest that these mimotopes are suitable candidates for an epitope-specific immunotherapy of fish-allergic patients.

  1. Metabolic engineering of Agrobacterium sp. strain ATCC 31749 for production of an α-Gal epitope

    PubMed Central

    2010-01-01

    Background Oligosaccharides containing a terminal Gal-α1,3-Gal moiety are collectively known as α-Gal epitopes. α-Gal epitopes are integral components of several medical treatments under development, including flu and HIV vaccines as well as cancer treatments. The difficulty associated with synthesizing the α-Gal epitope hinders the development and application of these treatments due to the limited availability and high cost of the α-Gal epitope. This work illustrates the development of a whole-cell biocatalyst for synthesizing the α-Gal epitope, Gal-α1,3-Lac. Results Agrobacterium sp. ATCC 31749 was engineered to produce Gal-α1,3-Lac by the introduction of a UDP-galactose 4'-epimerase:α1,3-galactosyltransferase fusion enzyme. The engineered Agrobacterium synthesized 0.4 g/L of the α-Gal epitope. Additional metabolic engineering efforts addressed the factors limiting α-Gal epitope production, namely the availability of the two substrates, lactose and UDP-glucose. Through expression of a lactose permease, the intracellular lactose concentration increased by 60 to 110%, subsequently leading to an improvement in Gal-α1,3-Lac production. Knockout of the curdlan synthase gene increased UDP-glucose availability by eliminating the consumption of UDP-glucose for synthesis of the curdlan polysaccharide. With these additional engineering efforts, the final engineered strain synthesized approximately 1 g/L of Gal-α1,3-Lac. Conclusions The Agrobacterium biocatalyst developed in this work synthesizes gram-scale quantities of α-Gal epitope and does not require expensive cofactors or permeabilization, making it a useful biocatalyst for industrial production of the α-Gal epitope. Furthermore, the engineered Agrobacterium, with increased lactose uptake and improved UDP-glucose availability, is a promising host for the production of other medically-relevant oligosaccharides. PMID:20067629

  2. Imaging characteristics of Zernike and annular polynomial aberrations.

    PubMed

    Mahajan, Virendra N; Díaz, José Antonio

    2013-04-01

    The general equations for the point-spread function (PSF) and optical transfer function (OTF) are given for any pupil shape, and they are applied to optical imaging systems with circular and annular pupils. The symmetry properties of the PSF, the real and imaginary parts of the OTF, and the modulation transfer function (MTF) of a system with a circular pupil aberrated by a Zernike circle polynomial aberration are derived. The interferograms and PSFs are illustrated for some typical polynomial aberrations with a sigma value of one wave, and 3D PSFs and MTFs are shown for 0.1 wave. The Strehl ratio is also calculated for polynomial aberrations with a sigma value of 0.1 wave, and shown to be well estimated from the sigma value. The numerical results are compared with the corresponding results in the literature. Because of the same angular dependence of the corresponding annular and circle polynomial aberrations, the symmetry properties of systems with annular pupils aberrated by an annular polynomial aberration are the same as those for a circular pupil aberrated by a corresponding circle polynomial aberration. They are also illustrated with numerical examples.

  3. Immunochemical characterization of the O antigens of two Proteus strains, O8-related antigen of Proteus mirabilis 12 B-r and O2-related antigen of Proteus genomospecies 5/6 12 B-k, infecting a hospitalized patient in Poland.

    PubMed

    Drzewiecka, Dominika; Shashkov, Alexander S; Arbatsky, Nikolay P; Knirel, Yuriy A

    2016-05-01

    A hospitalized 73-year-old woman was infected with a Proteus mirabilis strain, 12 B-r, isolated from the place of injection of a blood catheter. Another strain, 12 B-k, recognized as Proteus genomospecies 5 or 6, was isolated from the patient's faeces, which was an example of a nosocomial infection rather than an auto-infection. Serological investigation using ELISA and Western blotting showed that strain 12 B-k from faeces belonged to the Proteus O2 serogroup. Strain 12 B-r from the wound displayed cross-reactions with several Proteus O serogroups due to common epitopes on the core or O-specific parts of the lipopolysaccharide. Studies of the isolated 12 B-r O-specific polysaccharide by NMR spectroscopy revealed its close structural similarity to that of Proteus O8. The only difference in 12 B-r was the presence of an additional GlcNAc-linked phosphoethanolamine residue, which creates a putative epitope responsible for the cross-reactivity with Pt. mirabilis O16. The new O-antigen form could appear as a result of adaptation of the bacterium to a changing environment. On the basis of the data obtained, we suggest division of the O8 serogroup into two subgroups: O8a for strains of various Proteus species that have been previously classified into the O8 serogroup, and O8a,b for Pt. mirabilis 12 B-r, where 'a' is a common epitope and 'b' is a phosphoethanolamine-associated epitope. These findings further confirm serological and structural heterogeneity of O antigens of Proteus strains isolated lately from patients in Poland.

  4. Scaffold optimization in discontinuous epitope containing protein mimics of gp120 using smart libraries.

    PubMed

    Mulder, Gwenn E; Quarles van Ufford, H Linda C; van Ameijde, Jeroen; Brouwer, Arwin J; Kruijtzer, John A W; Liskamp, Rob M J

    2013-04-28

    A diversity of protein surface discontinuous epitope mimics is now rapidly and efficiently accessible. Despite the important role of protein-protein interactions involving discontinuous epitopes in a wide range of diseases, mimicry of discontinuous epitopes using peptide-based molecules remains a major challenge. Using copper(I) catalyzed azide-alkyne cycloaddition (CuAAC), we have developed a general and efficient method for the synthesis of collections of discontinuous epitope mimics. Up to three different cyclic peptides, representing discontinuous epitopes in HIV-gp120, were conjugated to a selection of scaffold molecules. Variation of the scaffold molecule, optimization of the ring size of the cyclic peptides and screening of the resulting libraries for successful protein mimics led to an HIV gp120 mimic with an IC50 value of 1.7 μM. The approach described here provides rapid and highly reproducible access to clean, smart libraries of very complex bio-molecular constructs representing protein mimics for use as synthetic vaccines and beyond.

  5. Natural variants of cytotoxic epitopes are T-cell receptor antagonists for antiviral cytotoxic T cells

    NASA Astrophysics Data System (ADS)

    Bertoletti, Antonio; Sette, Alessandro; Chisari, Francis V.; Penna, Amalia; Levrero, Massimo; Carli, Marco De; Fiaccadori, Franco; Ferrari, Carlo

    1994-06-01

    IT has been suggested that mutations within immunodominant cytotoxic T-lymphocyte (CTL) epitopes may be exploited by viruses to evade protective immune responses critical for clearance1-4. Viral escape could originate from passive mechanisms, such as mutations within crucial CTL epitopes, either affecting major histocompatibility complex binding or T-cell antigen receptor (TCR) recognition. Additionally, it has recently been shown that substitutions of TCR contact sites can yield analogue peptides that can still interact with the T-cell receptor but be unable to deliver a full stimulatory signal, thus inducing anergy5 or acting as an antagonist for the TCR6-8. We report here that hepatitis B virus isolates derived from two chronically infected patients display variant epitopes that act as natural TCR antagonists with the capacity to inhibit the CTL response to the wild-type epitope. During natural infection, TCR antagonist mutations of CTL epitopes could contribute to the development of viral persistence, especially if the antiviral CTL response is monospecific or the epitope is strongly immunodominant.

  6. A mathematical framework for the selection of an optimal set of peptides for epitope-based vaccines.

    PubMed

    Toussaint, Nora C; Dönnes, Pierre; Kohlbacher, Oliver

    2008-12-01

    Epitope-based vaccines (EVs) have a wide range of applications: from therapeutic to prophylactic approaches, from infectious diseases to cancer. The development of an EV is based on the knowledge of target-specific antigens from which immunogenic peptides, so-called epitopes, are derived. Such epitopes form the key components of the EV. Due to regulatory, economic, and practical concerns the number of epitopes that can be included in an EV is limited. Furthermore, as the major histocompatibility complex (MHC) binding these epitopes is highly polymorphic, every patient possesses a set of MHC class I and class II molecules of differing specificities. A peptide combination effective for one person can thus be completely ineffective for another. This renders the optimal selection of these epitopes an important and interesting optimization problem. In this work we present a mathematical framework based on integer linear programming (ILP) that allows the formulation of various flavors of the vaccine design problem and the efficient identification of optimal sets of epitopes. Out of a user-defined set of predicted or experimentally determined epitopes, the framework selects the set with the maximum likelihood of eliciting a broad and potent immune response. Our ILP approach allows an elegant and flexible formulation of numerous variants of the EV design problem. In order to demonstrate this, we show how common immunological requirements for a good EV (e.g., coverage of epitopes from each antigen, coverage of all MHC alleles in a set, or avoidance of epitopes with high mutation rates) can be translated into constraints or modifications of the objective function within the ILP framework. An implementation of the algorithm outperforms a simple greedy strategy as well as a previously suggested evolutionary algorithm and has runtimes on the order of seconds for typical problem sizes.

  7. Nodal aberration theory applied to freeform surfaces

    NASA Astrophysics Data System (ADS)

    Fuerschbach, Kyle; Rolland, Jannick P.; Thompson, Kevin P.

    2014-12-01

    When new three-dimensional packages are developed for imaging optical systems, the rotational symmetry of the optical system is often broken, changing its imaging behavior and making the optical performance worse. A method to restore the performance is to use freeform optical surfaces that compensate directly the aberrations introduced from tilting and decentering the optical surfaces. In order to effectively optimize the shape of a freeform surface to restore optical functionality, it is helpful to understand the aberration effect the surface may induce. Using nodal aberration theory the aberration fields induced by a freeform surface in an optical system are explored. These theoretical predications are experimentally validated with the design and implementation of an aberration generating telescope.

  8. Corneal spherical aberration in Saudi population

    PubMed Central

    Al-Sayyari, Tarfah M.; Fawzy, Samah M.; Al-Saleh, Ahmed A.

    2014-01-01

    Purpose To find out the mean corneal spherical aberration and its changes with age in Saudi population. Setting AlHokama Eye Specialist Center, Riyadh, Saudi Arabia. Methods Three hundred (300) eyes of 185 Saudi subjects (97 men and 88 women), whose age ranged from 15 to 85 years old, with matched refractive errors, were divided into three groups according to their age, 100 for each. All the subjects were included in measuring the spherical aberration (SA) using pentacam HR (OCULUS, Germany) at the 6-mm optical zone. Results The mean corneal spherical aberration (CSA) of the fourth order (Z40) of the whole groups was 0.252 ± 0.1154 μm. Patients from 15 to 35 years old have root mean square (RMS) of CSA of 0.2068 ± 0.07151 μm, 0.2370 ± 0.08023 μm was the RMS of CSA of the patients from 35 to 50 years old, while those from 50 to 85 years old have a CSA-RMS of 0.31511 ± 0.1503 μm (P < 0.0001). A positive correlation was found between the spherical aberration (Z40) and the progress of age (r = 0.3429, P < 0.0001). The high order aberration (HOA) presented 28.1% of the total corneal aberrations. While the fourth order corneal spherical aberration constituted 57% of the HOA and 16% of the total aberration. The pupil diameter shows a negative correlation with the increase in age (P = 0.0012). Conclusion Our results showed a CSA (Z40) that is varied among the population, comparable to other studies, and significantly correlates to the progress of age. PMID:25278799

  9. Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.

    PubMed

    Ofran, Yanay; Schlessinger, Avner; Rost, Burkhard

    2008-11-01

    Exact identification of complementarity determining regions (CDRs) is crucial for understanding and manipulating antigenic interactions. One way to do this is by marking residues on the antibody that interact with B cell epitopes on the antigen. This, of course, requires identification of B cell epitopes, which could be done by marking residues on the antigen that bind to CDRs, thus requiring identification of CDRs. To circumvent this vicious circle, existing tools for identifying CDRs are based on sequence analysis or general biophysical principles. Often, these tools, which are based on partial data, fail to agree on the boundaries of the CDRs. Herein we present an automated procedure for identifying CDRs and B cell epitopes using consensus structural regions that interact with the antigens in all known antibody-protein complexes. Consequently, we provide the first comprehensive analysis of all CDR-epitope complexes of known three-dimensional structure. The CDRs we identify only partially overlap with the regions suggested by existing methods. We found that the general physicochemical properties of both CDRs and B cell epitopes are rather peculiar. In particular, only four amino acids account for most of the sequence of CDRs, and several types of amino acids almost never appear in them. The secondary structure content and the conservation of B cell epitopes are found to be different than previously thought. These characteristics of CDRs and epitopes may be instrumental in choosing which residues to mutate in experimental search for epitopes. They may also assist in computational design of antibodies and in predicting B cell epitopes.

  10. An overview of bioinformatics tools for epitope prediction: implications on vaccine development.

    PubMed

    Soria-Guerra, Ruth E; Nieto-Gomez, Ricardo; Govea-Alonso, Dania O; Rosales-Mendoza, Sergio

    2015-02-01

    Exploitation of recombinant DNA and sequencing technologies has led to a new concept in vaccination in which isolated epitopes, capable of stimulating a specific immune response, have been identified and used to achieve advanced vaccine formulations; replacing those constituted by whole pathogen-formulations. In this context, bioinformatics approaches play a critical role on analyzing multiple genomes to select the protective epitopes in silico. It is conceived that cocktails of defined epitopes or chimeric protein arrangements, including the target epitopes, may provide a rationale design capable to elicit convenient humoral or cellular immune responses. This review presents a comprehensive compilation of the most advantageous online immunological software and searchable, in order to facilitate the design and development of vaccines. An outlook on how these tools are supporting vaccine development is presented. HIV and influenza have been taken as examples of promising developments on vaccination against hypervariable viruses. Perspectives in this field are also envisioned. Copyright © 2014 Elsevier Inc. All rights reserved.

  11. Reliable B Cell Epitope Predictions: Impacts of Method Development and Improved Benchmarking

    PubMed Central

    Kringelum, Jens Vindahl; Lundegaard, Claus; Lund, Ole; Nielsen, Morten

    2012-01-01

    The interaction between antibodies and antigens is one of the most important immune system mechanisms for clearing infectious organisms from the host. Antibodies bind to antigens at sites referred to as B-cell epitopes. Identification of the exact location of B-cell epitopes is essential in several biomedical applications such as; rational vaccine design, development of disease diagnostics and immunotherapeutics. However, experimental mapping of epitopes is resource intensive making in silico methods an appealing complementary approach. To date, the reported performance of methods for in silico mapping of B-cell epitopes has been moderate. Several issues regarding the evaluation data sets may however have led to the performance values being underestimated: Rarely, all potential epitopes have been mapped on an antigen, and antibodies are generally raised against the antigen in a given biological context not against the antigen monomer. Improper dealing with these aspects leads to many artificial false positive predictions and hence to incorrect low performance values. To demonstrate the impact of proper benchmark definitions, we here present an updated version of the DiscoTope method incorporating a novel spatial neighborhood definition and half-sphere exposure as surface measure. Compared to other state-of-the-art prediction methods, Discotope-2.0 displayed improved performance both in cross-validation and in independent evaluations. Using DiscoTope-2.0, we assessed the impact on performance when using proper benchmark definitions. For 13 proteins in the training data set where sufficient biological information was available to make a proper benchmark redefinition, the average AUC performance was improved from 0.791 to 0.824. Similarly, the average AUC performance on an independent evaluation data set improved from 0.712 to 0.727. Our results thus demonstrate that given proper benchmark definitions, B-cell epitope prediction methods achieve highly significant

  12. Three Immunoproteasome-Associated Subunits Cooperatively Generate a Cytotoxic T-Lymphocyte Epitope of Epstein-Barr Virus LMP2A by Overcoming Specific Structures Resistant to Epitope Liberation

    PubMed Central

    Ito, Yoshinori; Kondo, Eisei; Demachi-Okamura, Ayako; Akatsuka, Yoshiki; Tsujimura, Kunio; Tanimoto, Mitsune; Morishima, Yasuo; Takahashi, Toshitada; Kuzushima, Kiyotaka

    2006-01-01

    The precise roles of gamma interferon-inducible immunoproteasome-associated molecules in generation of cytotoxic T-lymphocyte (CTL) epitopes have yet to be fully elucidated. We describe here a unique epitope derived from the Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) presented by HLA-A*2402 molecules. Generation of the epitope, designated LMP2A222-230, from the full-length protein requires the immunoproteasome subunit low-molecular-weight protein 7 (ip-LMP7) and the proteasome activator 28-α subunit and is accelerated by ip-LMP2, as revealed by gene expression experiments using an LMP2A222-230-specific CTL clone as a responder in enzyme-linked immunospot assays. The unequivocal involvement of all three components was confirmed by RNA interference gene silencing. Interestingly, the LMP2A222-230 epitope could be efficiently generated from incomplete EBV-LMP2A fragments that were produced by puromycin treatment or gene-engineered shortened EBV-LMP2A lacking some of its hydrophobic domains. In addition, epitope generation was increased by a single amino acid substitution from leucine to alanine immediately flanking the C terminus, this being predicted by a web-accessible program to increase the cleavage strength. Taken together, the data indicate that the generation of LMP2A222-230 is influenced not only by extrinsic factors such as immunoproteasomes but also by intrinsic factors such as the length of the EBV-LMP2A protein and proteasomal cleavage strength at specific positions in the source antigen. PMID:16378990

  13. Identification of a conserved B-cell epitope on duck hepatitis A type 1 virus VP1 protein.

    PubMed

    Wu, Xiaoying; Li, Xiaojun; Zhang, Qingshan; Wulin, Shaozhou; Bai, Xiaofei; Zhang, Tingting; Wang, Yue; Liu, Ming; Zhang, Yun

    2015-01-01

    The VP1 protein of duck hepatitis A virus (DHAV) is a major structural protein that induces neutralizing antibodies in ducks; however, B-cell epitopes on the VP1 protein of duck hepatitis A genotype 1 virus (DHAV-1) have not been characterized. To characterize B-cell epitopes on VP1, we used the monoclonal antibody (mAb) 2D10 against Escherichia coli-expressed VP1 of DHAV-1. In vitro, mAb 2D10 neutralized DHAV-1 virus. By using an array of overlapping 12-mer peptides, we found that mAb 2D10 recognized phages displaying peptides with the consensus motif LPAPTS. Sequence alignment showed that the epitope 173LPAPTS178 is highly conserved among the DHAV-1 genotypes. Moreover, the six amino acid peptide LPAPTS was proven to be the minimal unit of the epitope with maximal binding activity to mAb 2D10. DHAV-1-positive duck serum reacted with the epitope in dot blotting assay, revealing the importance of the six amino acids of the epitope for antibody-epitope binding. Competitive inhibition assays of mAb 2D10 binding to synthetic LPAPTS peptides and truncated VP1 protein fragments, detected by Western blotting, also verify that LPAPTS was the VP1 epitope. We identified LPAPTS as a VP1-specific linear B-cell epitope recognized by the neutralizing mAb 2D10. Our findings have potential applications in the development of diagnostic techniques and epitope-based marker vaccines against DHAV-1.

  14. Transcranial phase aberration correction using beam simulations and MR-ARFI

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Vyas, Urvi, E-mail: urvi.vyas@gmail.com; Kaye, Elena; Pauly, Kim Butts

    2014-03-15

    Purpose: Transcranial magnetic resonance-guided focused ultrasound surgery is a noninvasive technique for causing selective tissue necrosis. Variations in density, thickness, and shape of the skull cause aberrations in the location and shape of the focal zone. In this paper, the authors propose a hybrid simulation-MR-ARFI technique to achieve aberration correction for transcranial MR-guided focused ultrasound surgery. The technique uses ultrasound beam propagation simulations with MR Acoustic Radiation Force Imaging (MR-ARFI) to correct skull-caused phase aberrations. Methods: Skull-based numerical aberrations were obtained from a MR-guided focused ultrasound patient treatment and were added to all elements of the InSightec conformal bone focusedmore » ultrasound surgery transducer during transmission. In the first experiment, the 1024 aberrations derived from a human skull were condensed into 16 aberrations by averaging over the transducer area of 64 elements. In the second experiment, all 1024 aberrations were applied to the transducer. The aberrated MR-ARFI images were used in the hybrid simulation-MR-ARFI technique to find 16 estimated aberrations. These estimated aberrations were subtracted from the original aberrations to result in the corrected images. Each aberration experiment (16-aberration and 1024-aberration) was repeated three times. Results: The corrected MR-ARFI image was compared to the aberrated image and the ideal image (image with zero aberrations) for each experiment. The hybrid simulation-MR-ARFI technique resulted in an average increase in focal MR-ARFI phase of 44% for the 16-aberration case and 52% for the 1024-aberration case, and recovered 83% and 39% of the ideal MR-ARFI phase for the 16-aberrations and 1024-aberration case, respectively. Conclusions: Using one MR-ARFI image and noa priori information about the applied phase aberrations, the hybrid simulation-MR-ARFI technique improved the maximum MR-ARFI phase of the beam's focus.« less

  15. Structure-Based Design of Hepatitis C Virus Vaccines That Elicit Neutralizing Antibody Responses to a Conserved Epitope

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Pierce, Brian G.; Boucher, Elisabeth N.; Piepenbrink, Kurt H.

    Despite recent advances in therapeutic options, hepatitis C virus (HCV) remains a severe global disease burden, and a vaccine can substantially reduce its incidence. Due to its extremely high sequence variability, HCV can readily escape the immune response; thus, an effective vaccine must target conserved, functionally important epitopes. Using the structure of a broadly neutralizing antibody in complex with a conserved linear epitope from the HCV E2 envelope glycoprotein (residues 412 to 423; epitope I), we performed structure-based design of immunogens to induce antibody responses to this epitope. This resulted in epitope-based immunogens based on a cyclic defensin protein, asmore » well as a bivalent immunogen with two copies of the epitope on the E2 surface. We solved the X-ray structure of a cyclic immunogen in complex with the HCV1 antibody and confirmed preservation of the epitope conformation and the HCV1 interface. Mice vaccinated with our designed immunogens produced robust antibody responses to epitope I, and their serum could neutralize HCV. Notably, the cyclic designs induced greater epitope-specific responses and neutralization than the native peptide epitope. Beyond successfully designing several novel HCV immunogens, this study demonstrates the principle that neutralizing anti-HCV antibodies can be induced by epitope-based, engineered vaccines and provides the basis for further efforts in structure-based design of HCV vaccines. IMPORTANCEHepatitis C virus is a leading cause of liver disease and liver cancer, with approximately 3% of the world's population infected. To combat this virus, an effective vaccine would have distinct advantages over current therapeutic options, yet experimental vaccines have not been successful to date, due in part to the virus's high sequence variability leading to immune escape. In this study, we rationally designed several vaccine immunogens based on the structure of a conserved epitope that is the target of broadly

  16. Wild-type and mutant SOD1 share an aberrant conformation and a common pathogenic pathway in ALS

    PubMed Central

    Bosco, Daryl A.; Morfini, Gerardo; Karabacak, N. Murat; Song, Yuyu; Gros-Louis, Francois; Pasinelli, Piera; Goolsby, Holly; Fontaine, Benjamin A.; Lemay, Nathan; McKenna-Yasek, Diane; Frosch, Matthew P.; Agar, Jeffery N.; Julien, Jean-Pierre; Brady, Scott T.; Brown, Robert H.

    2010-01-01

    Many mutations confer upon copper/zinc superoxide dismutase-1 (SOD1) one or more toxic function(s) that impair motor neuron viability and cause familial amyotrophic lateral sclerosis (FALS). Using a conformation-specific antibody that detects misfolded SOD1 (C4F6), we demonstrate that oxidized WT-SOD1 and mutant-SOD1 share a conformational epitope that is not present in normal WT-SOD1. In a subset of human sporadic ALS (SALS) cases, motor neurons in the lumbosacral spinal cord displayed striking C4F6 immunoreactivity, denoting the presence of aberrant WT-SOD1 species. Recombinant, oxidized WT-SOD1 and WT-SOD1 immunopurified from SALS tissues inhibited kinesin-based fast axonal transport in a manner similar to FALS-linked mutant SOD1. Studies here suggest that WT-SOD1 can be pathogenic in SALS and identifies an SOD1-dependent pathogenic mechanism common to FALS and SALS. PMID:20953194

  17. Liposomal preparations of muramyl glycopeptides as immunomodulators and adjuvants.

    PubMed

    Turánek, Jaroslav; Ledvina, Miroslav; Kasná, Andrea; Vacek, Antonín; Hríbalova, Vera; Krejcí, Josef; Miller, Andrew D

    2006-04-12

    The need for safe and structurally defined immunomodulators and adjuvants is increasing in connection with the recently observed marked increase in the prevalence of pathological conditions characterized by immunodeficiency. Important groups of such compounds are muramyl glycopeptides, analogs of muramyl dipeptide (MDP), glucosaminyl-muramyl dipeptide (GMDP), and desmuramylpeptides. We have designed and synthesized new types of analogs with changes in both the sugar and the peptide parts of the molecule that show a high immunostimulating and adjuvant activity and suppressed adverse side effects. The introduction of lipophilic residues has also improved their incorporation into liposomes, which represent a suitable drug carrier. The proliposome-liposome method is based on the conversion of the initial proliposome preparation into liposome dispersion by dilution with the aqueous phase. The description of a home-made stirred thermostated cell and its link-up with a liquid delivery system for a rapid and automated preparation of multilamellar liposomes at strictly controlled conditions (sterility, temperature, dilution rate and schedule) is presented. The cell has been designed for laboratory-scale preparation of liposomes (300-1000 mg of phospholipid per run) in a procedure taking less than 90 min. The method can be readily scaled up. Examples of adjuvant and immunostimulatory effect of liposomal preparation in mice model will be presented.

  18. Anti-forensics of chromatic aberration

    NASA Astrophysics Data System (ADS)

    Mayer, Owen; Stamm, Matthew C.

    2015-03-01

    Over the past decade, a number of information forensic techniques have been developed to identify digital image manipulation and falsification. Recent research has shown, however, that an intelligent forger can use anti-forensic countermeasures to disguise their forgeries. In this paper, an anti-forensic technique is proposed to falsify the lateral chromatic aberration present in a digital image. Lateral chromatic aberration corresponds to the relative contraction or expansion between an image's color channels that occurs due to a lens's inability to focus all wavelengths of light on the same point. Previous work has used localized inconsistencies in an image's chromatic aberration to expose cut-and-paste image forgeries. The anti-forensic technique presented in this paper operates by estimating the expected lateral chromatic aberration at an image location, then removing deviations from this estimate caused by tampering or falsification. Experimental results are presented that demonstrate that our anti-forensic technique can be used to effectively disguise evidence of an image forgery.

  19. Chimeric peptide constructs comprising linear B-cell epitopes: application to the serodiagnosis of infectious diseases.

    PubMed

    Lu, Yudong; Li, Zhong; Teng, Huan; Xu, Hongke; Qi, Songnan; He, Jian'an; Gu, Dayong; Chen, Qijun; Ma, Hongwei

    2015-08-21

    Linear B-cell epitopes are ideal biomarkers for the serodiagnosis of infectious diseases. However, the long-predicted diagnostic value of epitopes has not been realized. Here, we demonstrated a method, diagnostic epitopes in four steps (DEIFS), that delivers a combination of epitopes for the serodiagnosis of infectious diseases with a high success rate. Using DEIFS for malaria, we identified 6 epitopes from 8 peptides and combined them into 3 chimeric peptide constructs. Along with 4 other peptides, we developed a rapid diagnostic test (RDT), which is able to differentiate Plasmodium falciparum (P. falciparum) from Plasmodium vivax (P. vivax) infections with 95.6% overall sensitivity and 99.1% overall specificity. In addition to applications in diagnosis, DEIFS could also be used in the diagnosis of virus and bacterium infections, discovery of vaccine candidates, evaluation of vaccine potency, and study of disease progression.

  20. Chimeric peptide constructs comprising linear B-cell epitopes: application to the serodiagnosis of infectious diseases

    PubMed Central

    Lu, Yudong; Li, Zhong; Teng, Huan; Xu, Hongke; Qi, Songnan; He, Jian’an; Gu, Dayong; Chen, Qijun; Ma, Hongwei

    2015-01-01

    Linear B-cell epitopes are ideal biomarkers for the serodiagnosis of infectious diseases. However, the long-predicted diagnostic value of epitopes has not been realized. Here, we demonstrated a method, diagnostic epitopes in four steps (DEIFS), that delivers a combination of epitopes for the serodiagnosis of infectious diseases with a high success rate. Using DEIFS for malaria, we identified 6 epitopes from 8 peptides and combined them into 3 chimeric peptide constructs. Along with 4 other peptides, we developed a rapid diagnostic test (RDT), which is able to differentiate Plasmodium falciparum (P. falciparum) from Plasmodium vivax (P. vivax) infections with 95.6% overall sensitivity and 99.1% overall specificity. In addition to applications in diagnosis, DEIFS could also be used in the diagnosis of virus and bacterium infections, discovery of vaccine candidates, evaluation of vaccine potency, and study of disease progression. PMID:26293607

  1. Defining species-specific immunodominant B cell epitopes for molecular serology of Chlamydia species.

    PubMed

    Rahman, K Shamsur; Chowdhury, Erfan U; Poudel, Anil; Ruettger, Anke; Sachse, Konrad; Kaltenboeck, Bernhard

    2015-05-01

    Urgently needed species-specific enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against Chlamydia spp. have been elusive due to high cross-reactivity of chlamydial antigens. To identify Chlamydia species-specific B cell epitopes for such assays, we ranked the potential epitopes of immunodominant chlamydial proteins that are polymorphic among all Chlamydia species. High-scoring peptides were synthesized with N-terminal biotin, followed by a serine-glycine-serine-glycine spacer, immobilized onto streptavidin-coated microtiter plates, and tested with mono-specific mouse hyperimmune sera against each Chlamydia species in chemiluminescent ELISAs. For each of nine Chlamydia species, three to nine dominant polymorphic B cell epitope regions were identified on OmpA, CT618, PmpD, IncA, CT529, CT442, IncG, Omp2, TarP, and IncE proteins. Peptides corresponding to 16- to 40-amino-acid species-specific sequences of these epitopes reacted highly and with absolute specificity with homologous, but not heterologous, Chlamydia monospecies-specific sera. Host-independent reactivity of such epitopes was confirmed by testing of six C. pecorum-specific peptides from five proteins with C. pecorum-reactive sera from cattle, the natural host of C. pecorum. The probability of cross-reactivity of peptide antigens from closely related chlamydial species or strains correlated with percent sequence identity and declined to zero at <50% sequence identity. Thus, phylograms of B cell epitope regions predict the specificity of peptide antigens for rational use in the genus-, species-, or serovar-specific molecular serology of Chlamydia spp. We anticipate that these peptide antigens will improve chlamydial serology by providing easily accessible assays to nonspecialist laboratories. Our approach also lends itself to the identification of relevant epitopes of other microbial pathogens. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  2. Defining Species-Specific Immunodominant B Cell Epitopes for Molecular Serology of Chlamydia Species

    PubMed Central

    Rahman, K. Shamsur; Chowdhury, Erfan U.; Poudel, Anil; Ruettger, Anke; Sachse, Konrad

    2015-01-01

    Urgently needed species-specific enzyme-linked immunosorbent assays (ELISAs) for the detection of antibodies against Chlamydia spp. have been elusive due to high cross-reactivity of chlamydial antigens. To identify Chlamydia species-specific B cell epitopes for such assays, we ranked the potential epitopes of immunodominant chlamydial proteins that are polymorphic among all Chlamydia species. High-scoring peptides were synthesized with N-terminal biotin, followed by a serine-glycine-serine-glycine spacer, immobilized onto streptavidin-coated microtiter plates, and tested with mono-specific mouse hyperimmune sera against each Chlamydia species in chemiluminescent ELISAs. For each of nine Chlamydia species, three to nine dominant polymorphic B cell epitope regions were identified on OmpA, CT618, PmpD, IncA, CT529, CT442, IncG, Omp2, TarP, and IncE proteins. Peptides corresponding to 16- to 40-amino-acid species-specific sequences of these epitopes reacted highly and with absolute specificity with homologous, but not heterologous, Chlamydia monospecies-specific sera. Host-independent reactivity of such epitopes was confirmed by testing of six C. pecorum-specific peptides from five proteins with C. pecorum-reactive sera from cattle, the natural host of C. pecorum. The probability of cross-reactivity of peptide antigens from closely related chlamydial species or strains correlated with percent sequence identity and declined to zero at <50% sequence identity. Thus, phylograms of B cell epitope regions predict the specificity of peptide antigens for rational use in the genus-, species-, or serovar-specific molecular serology of Chlamydia spp. We anticipate that these peptide antigens will improve chlamydial serology by providing easily accessible assays to nonspecialist laboratories. Our approach also lends itself to the identification of relevant epitopes of other microbial pathogens. PMID:25761461

  3. Depigmented Allergoids Reveal New Epitopes with Capacity to Induce IgG Blocking Antibodies

    PubMed Central

    López-Matas, M. Angeles; Gallego, Mayte; Iraola, Víctor; Robinson, Douglas; Carnés, Jerónimo

    2013-01-01

    Background. The synthesis of allergen-specific blocking IgGs that interact with IgE after allergen immunotherapy (SIT) has been related to clinical efficacy. The objectives were to investigate the epitope specificity of IgG-antibodies induced by depigmented-polymerized (Dpg-Pol) allergoids and unmodified allergen extracts, and examine IgE-blocking activity of induced IgG-antibodies. Methods. Rabbits were immunized with native and Dpg-Pol extracts of birch pollen, and serum samples were obtained. Recognition of linear IgG-epitopes of Bet v 1 and Bet v 2 and the capacity of these IgG-antibodies to block binding of human-IgE was determined. Results. Serum from rabbits immunized with native extracts recognised 11 linear epitopes from Bet v 1, while that from Dpg-Pol-immunized animals recognised 8. For Bet v 2, 8 epitopes were recognized by IgG from native immunized animals, and 9 from Dpg-Pol immunized one. Dpg-Pol and native immunized serum did not always recognise the same epitopes, but specific-IgG from both could block human-IgE binding sites for native extract. Conclusions. Depigmented-polymerized birch extract stimulates the synthesis of specific IgG-antibodies which recognize common but also novel epitopes compared with native extracts. IgG-antibodies induced by Dpg-Pol effectively inhibit human-IgE binding to allergens which may be part of the mechanism of action of SIT. PMID:24222901

  4. A Evaluation of Optical Aberrations in Underwater Hologrammetry

    NASA Astrophysics Data System (ADS)

    Kilpatrick, J. M.

    Available from UMI in association with The British Library. An iterative ray-trace procedure is developed in conjunction with semi-analytic expressions for spherical aberration, coma, and astigmatism in the reconstructed holographic images of underwater objects. An exact expression for the astigmatic difference is obtained, based on the geometry of the caustic for refraction. The geometrical characteristics of the aberrated images associated with axial and non-axial field positions are represented by ray intersection diagrams. A third order expression for the wavefront aberration introduced at a planar air/water boundary is given. The associated third order aberration coefficients are used to obtain analytic expressions for the aberrations observed in underwater hologrammetry. The results of the third order treatment are shown to give good agreement with the results obtained by geometrical ray tracing and by direct measurement on the reconstructed real image. The third order aberration coefficients are employed to estimate the limit of resolution in the presence of the aberrations associated with reconstruction in air. In concurrence with practical observations it is found that the estimated resolution is primarily limited by astigmatism. The limitations of the planar window in underwater imaging applications are outlined and various schemes are considered to effect a reduction in the extent of aberration. The analogous problems encountered in underwater photography are examined in order to establish the grounds for a common solution based on a conventional optical corrector. The performance of one such system, the Ivanoff Corrector, is investigated. The spherical aberration associated with axial image formation is evaluated. The equivalence of the third order wavefront aberration introduced at a planar air/water boundary to that introduced upon reconstruction by an appropriate wavelength change is shown to provide a basis for the compensation of aberrations in

  5. Broad cross-reactive T cell receptor repertoires recognizing dissimilar Epstein-Barr and influenza A virus epitopes

    PubMed Central

    Clute, Shalyn C.; Naumov, Yuri N.; Watkin, Levi B.; Aslan, Nuray; Sullivan, John L.; Thorley-Lawson, David A.; Luzuriaga, Katherine; Welsh, Raymond M.; Puzone, Roberto; Celada, Franco; Selin, Liisa K.

    2013-01-01

    Memory T cells cross-reactive with epitopes encoded by related or even unrelated viruses may alter the immune response and pathogenesis of infection by a process known as heterologous immunity. Because a challenge virus epitope may react with only a subset of the T cell repertoire in a cross-reactive epitope-specific memory pool, the vigorous cross-reactive response may be narrowly focused, or oligoclonal. We show here, by examining human T cell cross-reactivity between the HLA-A2-restricted influenza A virus-encoded M158-66 epitope (GILGFVFTL) and the dissimilar Epstein-Barr virus-encoded BMLF1280-288 epitope (GLCTLVAML), that under some conditions heterologous immunity can lead to a significant broadening rather than a narrowing of the T cell receptor repertoire. We suggest that dissimilar cross-reactive epitopes might generate a broad rather than narrow T cell repertoire if there is a lack of dominant high affinity clones, and this hypothesis is supported by computer simulation. PMID:21048112

  6. Entry kinetics and mouse virulence of Ross River virus mutants altered in neutralization epitopes.

    PubMed

    Vrati, S; Kerr, P J; Weir, R C; Dalgarno, L

    1996-03-01

    Previously we identified the locations of three neutralization epitopes (a, b1 and b2) of Ross River virus (RRV) by sequencing a number of variants resistant to monoclonal antibody neutralization which were found to have single amino acid substitutions in the E2 protein (S. Vrati, C.A. Fernon, L. Dalgarno, and R.C. Weir, Virology 162:346-353, 1988). We have now studied the biological properties of these variants in BHK cells and their virulence in mice. While variants altered in epitopes a and/or b1 showed no difference, variants altered in epitope b2, including a triple variant altered in epitopes a, b1, and b2, showed rapid penetration but retarded kinetics of growth and RNA and protein synthesis in BHK cells compared with RRV T48, the parent virus. Variants altered in epitopes a and/or b1 showed no change in mouse virulence. However, two of the six epitope b2 variants examined had attenuated mouse virulence. They had a four- to fivefold-higher 50% lethal dose (LD50), although no change in the average survival time of infected mice was observed. These variants grew to titers in mouse tissues similar to those of RRV T48. The ID50 of the triple variant was unchanged, but infected mice had an increased average survival time. This variant produced lower levels of viremia in infected mice. On the basis of these findings we propose that both the receptor binding site and neutralization epitopes of RRV are nearby or in the same domain of the E2 protein.

  7. Harnessing Computational Biology for Exact Linear B-Cell Epitope Prediction: A Novel Amino Acid Composition-Based Feature Descriptor.

    PubMed

    Saravanan, Vijayakumar; Gautham, Namasivayam

    2015-10-01

    Proteins embody epitopes that serve as their antigenic determinants. Epitopes occupy a central place in integrative biology, not to mention as targets for novel vaccine, pharmaceutical, and systems diagnostics development. The presence of T-cell and B-cell epitopes has been extensively studied due to their potential in synthetic vaccine design. However, reliable prediction of linear B-cell epitope remains a formidable challenge. Earlier studies have reported discrepancy in amino acid composition between the epitopes and non-epitopes. Hence, this study proposed and developed a novel amino acid composition-based feature descriptor, Dipeptide Deviation from Expected Mean (DDE), to distinguish the linear B-cell epitopes from non-epitopes effectively. In this study, for the first time, only exact linear B-cell epitopes and non-epitopes have been utilized for developing the prediction method, unlike the use of epitope-containing regions in earlier reports. To evaluate the performance of the DDE feature vector, models have been developed with two widely used machine-learning techniques Support Vector Machine and AdaBoost-Random Forest. Five-fold cross-validation performance of the proposed method with error-free dataset and dataset from other studies achieved an overall accuracy between nearly 61% and 73%, with balance between sensitivity and specificity metrics. Performance of the DDE feature vector was better (with accuracy difference of about 2% to 12%), in comparison to other amino acid-derived features on different datasets. This study reflects the efficiency of the DDE feature vector in enhancing the linear B-cell epitope prediction performance, compared to other feature representations. The proposed method is made as a stand-alone tool available freely for researchers, particularly for those interested in vaccine design and novel molecular target development for systems therapeutics and diagnostics: https://github.com/brsaran/LBEEP.

  8. Localization of neutralization epitopes on adenovirus fiber knob from species C.

    PubMed

    Lang, Shuai; Wang, Lizheng; Wang, Zixuan; Zhu, Rui; Yan, Jingyi; Wang, Baoming; Wu, Jiaxin; Zhang, Haihong; Wu, Hui; Zhou, Yan; Kong, Wei; Yu, Bin; Yu, Xianghui

    2016-04-01

    Although potential neutralization epitopes on the fiber knob of adenovirus (AdV) serotype 2 (Ad2) and Ad5 have been revealed, few studies have been carried out to identify neutralization epitopes on the knob from a broader panel of AdV serotypes. In this study, based on sequence and structural analysis of knobs from Ad1, Ad2, Ad5 and Ad6 (all from species C), several trimeric chimeric knob proteins were expressed in Escherichia coli to identify the locations of neutralization epitopes on the knobs by analysing their reactivity with mouse and rabbit polyclonal sera raised against AdVs and human sera with natural AdV infection. The dominant neutralization epitopes were located mainly in the N-terminal part of knobs from Ad1, Ad2 and Ad5, but they seemed to be located in the C-terminal part of the Ad6 knob, with some individual differences in rabbit and human populations. Our study adds to our understanding of humoral immune responses to AdVs and will facilitate the construction of more desirable capsid-modified recombinant Ad5 vectors.

  9. In Vitro Activity of TD-1792, a Multivalent Glycopeptide-Cephalosporin Antibiotic, against 377 Strains of Anaerobic Bacteria and 34 Strains of Corynebacterium Species

    PubMed Central

    Citron, Diane M.; Warren, Yumi A.; Goldstein, Ellie J. C.

    2012-01-01

    TD-1792 is a multivalent glycopeptide-cephalosporin heterodimer antibiotic with potent activity against Gram-positive bacteria. We tested TD-1792 against 377 anaerobes and 34 strains of Corynebacterium species. Against nearly all Gram-positive strains, TD-1792 had an MIC90 of 0.25 μg/ml and was typically 3 to 7 dilutions more active than vancomycin and daptomycin. PMID:22290981

  10. Persistence of Animal and Human Glycopeptide-Resistant Enterococci on Two Norwegian Poultry Farms Formerly Exposed to Avoparcin Is Associated with a Widespread Plasmid-Mediated vanA Element within a Polyclonal Enterococcus faecium Population

    PubMed Central

    Johnsen, P. J.; Østerhus, J. I.; Sletvold, H.; Sørum, M.; Kruse, H.; Nielsen, K.; Simonsen, G. S.; Sundsfjord, A.

    2005-01-01

    The evolutionary processes responsible for the long-term persistence of glycopeptide-resistant Enterococcus faecium (GREF) in nonselective environments were addressed by genetic analyses of E. faecium populations in animals and humans on two Norwegian poultry farms that were previously exposed to avoparcin. A total of 222 fecal GREF (n = 136) and glycopeptide-susceptible (n = 86) E. faecium (GSEF) isolates were obtained from farmers and poultry on three separate occasions in 1998 and 1999. Pulsed-field gel electrophoresis (PFGE) and plasmid DNA analyses discerned 22 GREF and 32 GSEF PFGE types within shifting polyclonal animal and human E. faecium populations and indicated the presence of transferable plasmid-mediated vanA resistance, respectively. Examples of dominant, persistent GREF PFGE types supported the notion that environmentally well-adapted GREF types may counteract the reversal of resistance. PFGE analyses, sequencing of the purK housekeeping gene, and partial typing of vanA-containing Tn1546 suggested a common animal and human reservoir of glycopeptide resistance. Inverse PCR amplification and sequence analyses targeting the right end of the Tn1546-plasmid junction fragment strongly indicated the presence of a common single Tn1546-plasmid-mediated element in 20 of 22 GREF PFGE types. This observation was further strengthened by vanY-vanZ hybridization analyses of plasmid DNAs as well as the finding of a physical linkage between Tn1546 and a putative postsegregation killing system for seven GREF PFGE types. In conclusion, our observations suggest that the molecular unit of persistence of glycopeptide resistance is a common mobile plasmid-mediated vanA-containing element within a polyclonal GREF population that changes over time. In addition, we propose that “plasmid addiction systems” may contribute to the persistence of GREF in nonselective environments. PMID:15640183

  11. Mapping the B cell epitopes within the major capsid protein L1 of human papillomavirus type 16.

    PubMed

    Wang, Aiping; Li, Ning; Zhou, Jingming; Chen, Yumei; Jiang, Min; Qi, Yanhua; Liu, Hongliang; Liu, Yankai; Liu, Dongmin; Zhao, Jianguo; Wang, Yanwei; Zhang, Gaiping

    2018-06-26

    Persistent infection with human papillomavirus type16 (HPV16) has much association with the development of cervical cancer. L1 is the major capsid protein of HPV, it has been well investigated as a potential vaccine candidate. However, B cell epitopes present on L1 have not been well characterized. To identify the potential B-cell antigenic epitopes within HPV16 L1 protein, sixteen serial overlapping truncations (H1-H16) covering the whole region were expressed in E. coli and used in mice immunization. The mice antisera were tested in ELISA binding, IFA and HI assays. Finally, four fragments (H2, H4, H11, H12) were found to contain B cell epitopes of HPV16 L1 protein in ELISA and IFA assays, three fragments (H2, H3, H9) might contain neutralizing epitopes of HPV16 L1 protein in HI assay. Among them, H11 and H12 fragments contain B cell epitopes have never been reported before, and H3 was found as hemagglutination inhibition epitope for the first time. This work provides new insights to B cell epitopes on HPV16 L1 protein. Several new epitopes were identified and may provide some guidance for HPV16 subunit vaccine design. The results of this study might open new perspectives on the antibody-antigen reaction and have important implications for the development of epitopes-based protective HPV16 vaccines. Copyright © 2018. Published by Elsevier B.V.

  12. Substantial gaps in knowledge of Bordetella pertussis antibody and T cell epitopes relevant for natural immunity and vaccine efficacy

    PubMed Central

    Vaughan, Kerrie; Seymour, Emily; Peters, Bjoern; Sette, Alessandro

    2016-01-01

    The recent increase in whooping cough in vaccinated populations has been attributed to waning immunity associated with the acellular vaccine. The Immune Epitope Database (IEDB) is a repository of immune epitope data from the published literature and includes T cell and antibody epitopes for human pathogens. The IEDB conducted a review of the epitope literature, which revealed 300 Bordetella pertussis-related epitopes from 39 references. Epitope data are currently available for six virulence factors of B. pertussis: pertussis toxin, pertactin, fimbrial 2, fimbrial 3, adenylate cyclase and filamentous hemagglutinin. The majority of epitopes were defined for antibody reactivity; fewer T cell determinants were reported. Analysis of available protective correlates data revealed a number of candidate epitopes; however few are defined in humans and few have been shown to be protective. Moreover, there are a limited number of studies defining epitopes from natural infection versus whole cell or acellular/subunit vaccines. The relationship between epitope location and structural features, as well as antigenic drift (SNP analysis) was also investigated. We conclude that the cumulative data is yet insufficient to address many fundamental questions related to vaccine failure and this underscores the need for further investigation of B. pertussis immunity at the molecular level. PMID:24530743

  13. Characterizing the interactions between a naturally primed immunoglobulin A and its conserved Bacteroides thetaiotaomicron species-specific epitope in gnotobiotic mice.

    PubMed

    Peterson, Daniel A; Planer, Joseph D; Guruge, Janaki L; Xue, Lai; Downey-Virgin, Whitt; Goodman, Andrew L; Seedorf, Henning; Gordon, Jeffrey I

    2015-05-15

    The adaptive immune response to the human gut microbiota consists of a complex repertoire of antibodies interacting with a broad range of taxa. Fusing intestinal lamina propria lymphocytes from mice monocolonized with Bacteroides thetaiotaomicron to a myeloma fusion partner allowed us to recover hybridomas that captured naturally primed, antigen-specific antibody responses representing multiple isotypes, including IgA. One of these hybridomas, 260.8, produced a monoclonal antibody that recognizes an epitope specific for B. thetaiotaomicron isolates in a large panel of hospital- and community-acquired Bacteroides. Whole genome transposon mutagenesis revealed a 19-gene locus, involved in LPS O-antigen polysaccharide synthesis and conserved among multiple B. thetaiotaomicron isolates, that is required for 260.8 epitope expression. Mutants in this locus exhibited marked fitness defects in vitro during growth in rich medium and in gnotobiotic mice colonized with defined communities of human gut symbionts. Expression of the 260.8 epitope was sustained during 10 months of daily passage in vitro and during 14 months of monocolonization of gnotobiotic wild-type, Rag1-/-, or Myd88-/- mice. Comparison of gnotobiotic Rag1-/- mice with and without subcutaneous 260.8 hybridomas disclosed that this IgA did not affect B. thetaiotaomicron population density or suppress 260.8 epitope production but did affect bacterial gene expression in ways emblematic of a diminished host innate immune response. Our study illustrates an approach for (i) generating diagnostic antibodies, (ii) characterizing IgA responses along a continuum of specificity/degeneracy that defines the IgA repertoire to gut symbionts, and (iii) identifying immunogenic epitopes that affect competitiveness and help maintain host-microbe mutualism. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  14. In silico design of a DNA-based HIV-1 multi-epitope vaccine for Chinese populations

    PubMed Central

    Yang, Yi; Sun, Weilai; Guo, Jingjing; Zhao, Guangyu; Sun, Shihui; Yu, Hong; Guo, Yan; Li, Jungfeng; Jin, Xia; Du, Lanying; Jiang, Shibo; Kou, Zhihua; Zhou, Yusen

    2015-01-01

    The development of an HIV-1 vaccine that is capable of inducing effective and broadly cross-reactive humoral and cellular immune responses remains a challenging task because of the extensive diversity of HIV-1, the difference of virus subtypes (clades) in different geographical regions, and the polymorphism of human leukocyte antigens (HLA). We performed an in silico design of 3 DNA vaccines, designated pJW4303-MEG1, pJW4303-MEG2 and pJW4303-MEG3, encoding multi-epitopes that are highly conserved within the HIV-1 subtypes most prevalent in China and can be recognized through HLA alleles dominant in China. The pJW4303-MEG1-encoded protein consisted of one Th epitope in Env, and one, 2, and 6 epitopes in Pol, Env, and Gag proteins, respectively, with a GGGS linker sequence between epitopes. The pJW4303-MEG2-encoded protein contained similar epitopes in a different order, but with the same linker as pJW4303-MEG1. The pJW4303-MEG3-encoded protein contained the same epitopes in the same order as that of pJW4303-MEG2, but with a different linker sequence (AAY). To evaluate immunogenicity, mice were immunized intramuscularly with these DNA vaccines. Both pJW4303-MEG1 and pJW4303-MEG2 vaccines induced equally potent humoral and cellular immune responses in the vaccinated mice, while pJW4303-MEG3 did not induce immune responses. These results indicate that both epitope and linker sequences are important in designing effective epitope-based vaccines against HIV-1 and other viruses. PMID:25839222

  15. Aberrant laryngeal location of Onchocerca lupi in a dog.

    PubMed

    Alho, Ana Margarida; Cruz, Luís; Coelho, Ana; Martinho, Filipe; Mansinho, Mário; Annoscia, Giada; Lia, Riccardo P; Giannelli, Alessio; Otranto, Domenico; de Carvalho, Luís Madeira

    2016-06-01

    Onchocerca lupi (Spirurida, Onchocercidae) is an emerging vector-borne helminth that causes nodular lesions associated with acute or chronic ocular disease in dogs and cats. Since its first description in dogs in 1991, this zoonotic filarioid has been increasingly reported in Europe and the United States. An 8-year-old outdoor mixed-breed female dog from the Algarve (southern Portugal) was presented with a history of severe dyspnoea. Cervical and thoracic radiographs revealed a slight reduction in the diameter of the cervical trachea and a moderate increase in radiopacity of the laryngeal soft tissue. An exploratory laryngoscopy was performed, revealing filiform worms associated with stenosis of the thyroid cartilage and a purulent necrotic tissue in the larynx lumen. A single sessile nodule, protruding from the dorsal wall of the laryngeal lumen caused a severe reduction of the glottis and tracheal diameter. Fragments of the worms were morphologically and molecularly identified as O. lupi. Histological examination of the nodule showed a granulomatous reaction with sections of coiled gravid female nematodes. Following laryngoscopy, a tracheostomy tube was inserted to relieve dyspnoea and ivermectin (300 μg/kg, once a week, for 8 weeks) combined with prednisolone was prescribed. The dog showed a complete recovery. Although O. lupi has been isolated in human patients from the spinal cord, this is the first report of an aberrant migration of O. lupi in a dog. The veterinary medical community should pay attention to aberrant location of O. lupi and consider onchocercosis as a differential diagnosis for airway obstruction in dogs. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  16. Definition of natural T cell antigens with mimicry epitopes obtained from dedicated synthetic peptide libraries.

    PubMed

    Hiemstra, H S; van Veelen, P A; Schloot, N C; Geluk, A; van Meijgaarden, K E; Willemen, S J; Leunissen, J A; Benckhuijsen, W E; Amons, R; de Vries, R R; Roep, B O; Ottenhoff, T H; Drijfhout, J W

    1998-10-15

    Progress has recently been made in the use of synthetic peptide libraries for the identification of T cell-stimulating ligands. T cell epitopes identified from synthetic libraries are mimics of natural epitopes. Here we show how the mimicry epitopes obtained from synthetic peptide libraries enable unambiguous identification of natural T cell Ags. Synthetic peptide libraries were screened with Mycobacterium tuberculosis-reactive and -autoreactive T cell clones. In two cases, database homology searches with mimicry epitopes isolated from a dedicated synthetic peptide library allowed immediate identification of the natural antigenic protein. In two other cases, an amino acid pattern that reflected the epitope requirements of the T cell was determined by substitution and omission mixture analysis. Subsequently, the natural Ag was identified from databases using this refined pattern. This approach opens new perspectives for rapid and reliable Ag definition, representing a feasible alternative to the biochemical and genetic approaches described thus far.

  17. Quantitative synthesis of genetically encoded glycopeptide libraries displayed on M13 phage.

    PubMed

    Ng, Simon; Jafari, Mohammad R; Matochko, Wadim L; Derda, Ratmir

    2012-09-21

    Phage display is a powerful technology that enables the discovery of peptide ligands for many targets. Chemical modification of phage libraries have allowed the identification of ligands with properties not encountered in natural polypeptides. In this report, we demonstrated the synthesis of 2 × 10(8) genetically encoded glycopeptides from a commercially available phage-displayed peptide library (Ph.D.-7) in a two-step, one-pot reaction in <1.5 h. Unlike previous reports, we bypassed genetic engineering of phage. The glycan moiety was introduced via an oxime ligation following oxidation of an N-terminal Ser/Thr; these residues are present in the peptide libraries at 20-30% abundance. The construction of libraries was facilitated by simple characterization, which directly assessed the yield and regioselectivity of chemical reactions performed on phage. This quantification method also allowed facile yield determination of reactions in 10(9) distinct molecules. We envision that the methodology described herein will find broad application in the synthesis of custom chemically modified phage libraries.

  18. Aberrated laser beams in terms of Zernike polynomials

    NASA Astrophysics Data System (ADS)

    Alda, Javier; Alonso, Jose; Bernabeu, Eusebio

    1996-11-01

    The characterization of light beams has devoted a lot of attention in the past decade. Several formalisms have been presented to treat the problem of parameter invariance and characterization in the propagation of light beam along ideal, ABCD, optical systems. The hard and soft apertured optical systems have been treated too. Also some aberrations have been analyzed, but it has not appeared a formalism able to treat the problem as a whole. In this contribution we use a classical approach to describe the problem of aberrated, and therefore apertured, light beams. The wavefront aberration is included in a pure phase term expanded in terms of the Zernike polynomials. Then, we can use the relation between the lower order Zernike polynomia and the Seidel or third order aberrations. We analyze the astigmatism, the spherical aberration and the coma, and we show how higher order aberrations can be taken into account. We have calculated the divergence, and the radius of curvature of such aberrated beams and the influence of these aberrations in the quality of the light beam. Some numerical simulations have been done to illustrate the method.

  19. Structural centrosome aberrations promote non-cell-autonomous invasiveness.

    PubMed

    Ganier, Olivier; Schnerch, Dominik; Oertle, Philipp; Lim, Roderick Yh; Plodinec, Marija; Nigg, Erich A

    2018-05-02

    Centrosomes are the main microtubule-organizing centers of animal cells. Although centrosome aberrations are common in tumors, their consequences remain subject to debate. Here, we studied the impact of structural centrosome aberrations, induced by deregulated expression of ninein-like protein (NLP), on epithelial spheres grown in Matrigel matrices. We demonstrate that NLP-induced structural centrosome aberrations trigger the escape ("budding") of living cells from epithelia. Remarkably, all cells disseminating into the matrix were undergoing mitosis. This invasive behavior reflects a novel mechanism that depends on the acquisition of two distinct properties. First, NLP-induced centrosome aberrations trigger a re-organization of the cytoskeleton, which stabilizes microtubules and weakens E-cadherin junctions during mitosis. Second, atomic force microscopy reveals that cells harboring these centrosome aberrations display increased stiffness. As a consequence, mitotic cells are pushed out of mosaic epithelia, particularly if they lack centrosome aberrations. We conclude that centrosome aberrations can trigger cell dissemination through a novel, non-cell-autonomous mechanism, raising the prospect that centrosome aberrations contribute to the dissemination of metastatic cells harboring normal centrosomes. © 2018 The Authors. Published under the terms of the CC BY NC ND 4.0 license.

  20. Combinatorial contextualization of peptidic epitopes for enhanced cellular immunity.

    PubMed

    Ito, Masaki; Hayashi, Kazumi; Adachi, Eru; Minamisawa, Tamiko; Homma, Sadamu; Koido, Shigeo; Shiba, Kiyotaka

    2014-01-01

    Invocation of cellular immunity by epitopic peptides remains largely dependent on empirically developed protocols, such as interfusion of aluminum salts or emulsification using terpenoids and surfactants. To explore novel vaccine formulation, epitopic peptide motifs were co-programmed with structural motifs to produce artificial antigens using our "motif-programming" approach. As a proof of concept, we used an ovalbumin (OVA) system and prepared an artificial protein library by combinatorially polymerizing MHC class I and II sequences from OVA along with a sequence that tends to form secondary structures. The purified endotoxin-free proteins were then examined for their ability to activate OVA-specific T-cell hybridoma cells after being processed within dendritic cells. One clone, F37A (containing three MHC I and two MHC II OVA epitopes), possessed a greater ability to evoke cellular immunity than the native OVA or the other artificial antigens. The sensitivity profiles of drugs that interfered with the F37A uptake differed from those of the other artificial proteins and OVA, suggesting that alteration of the cross-presentation pathway is responsible for the enhanced immunogenicity. Moreover, F37A, but not an epitopic peptide, invoked cellular immunity when injected together with monophosphoryl lipid A (MPL), and retarded tumor growth in mice. Thus, an artificially synthesized protein antigen induced cellular immunity in vivo in the absence of incomplete Freund's adjuvant or aluminum salts. The method described here could be potentially used for developing vaccines for such intractable ailments as AIDS, malaria and cancer, ailments in which cellular immunity likely play a crucial role in prevention and treatment.

  1. Whole eye wavefront aberrations in Mexican male subjects.

    PubMed

    Cantú, Roberto; Rosales, Marco A; Tepichín, Eduardo; Curioca, Andrée; Montes, Victor; Bonilla, Julio

    2004-01-01

    To analyze the characteristics, incidence, and appearance of wavefront aberrations in undilated, normal, unoperated eyes. Eighty-eight eyes of 44 healthy male Mexican subjects (mean age 25.32 years, range 18 to 36 yr) were divided into three groups based on uncorrected visual acuity of greater than or equal to 20/20, 20/30, or 20/40. UCVA measurements were obtained using an Acuity Max computer screen chart. Wavefront aberrations were measured with the Nidek OPD-Scan ARK 10000, Ver. 1.11b. All measurements were carried out at the same center by the same technician during a single session, following manufacturer instructions. Background illumination was 3 Lux. Wavefront aberration measurements for each group were statistically analyzed using StatView; an average eye was characterized and the resulting aberrations were simulated using MATLAB. We obtained wavefront aberration maps for the 20/20 undilated normal unoperated eyes for total, low, and high order aberration coefficients. Wavefront maps for right eyes were practically the same as those for left eyes. Higher aberrations did not contribute substantially to total wavefront analysis. Average aberrations of this "normal eye" will be used as criteria to decide the necessity of wavefront-guided ablation in our facilities. We will focus on the nearly zero average of high order aberrations in this normal whole eye as a reference to be matched.

  2. Role for cell adhesion and glycosyl (HNK-1 and oligomannoside) recognition in the sharpening of the regenerating retinotectal projection in goldfish.

    PubMed

    Schmidt, J T; Schachner, M

    1998-12-01

    Cell-adhesion molecules (CAMs) are thought to play crucial roles in development and plasticity in the nervous system. This study tested for a role for cell adhesion and in particular, the recognition of two glycosyl epitopes (HNK-1 and oligomannoside) in the activity-driven sharpening of the retinotopic map formed by the regenerating retinal fibers of goldfish. HNK-1 is a prominent glycosyl epitope on many CAMs and extracellular matrix (ECM) molecules, including NCAM, L1, ependymin, and integrins, which have all been implicated in synaptic plasticity. To test for a role of HNK-1 in the sharpening process, we used osmotic minipumps to infuse HNK-1 antibodies for 7-21 days into the tectal ventricle starting at 18 days after optic nerve crush. Retinotopic maps recorded at 76-86 days postcrush showed a lack of sharpening similar to that seen previously with two antibodies to ependymin, an HNK-1-positive ECM component present in cerebrospinal fluid. The multiunit receptive fields at each point averaged 26 degrees versus 11-12 degrees in regenerates infused with control antibodies or Ringer's alone. The HNK-1 epitope also binds to the G2 domain of laminin to mediate neuron-ECM adhesion. To test for a role for laminin, a polyclonal antibody was similarly infused and also prevented sharpening to approximately the same degree. The results support a role for the HNK-1 epitope and laminin in retinotectal sharpening. The oligomannoside epitope (recognized by monoclonal antibody L3) on the CAM L1 interacts with NCAM on the same cell to promote stronger L1 homophilic interactions between cells. Both an L1-like molecule and NCAM are prominently reexpressed in the regenerating retinotectal system of fish. Infusion of oligomannosidic glycopeptides resulted in decreased sharpening, with multiunit receptive fields that averaged 22.7 degrees. Infusions of mannose-poor glycopeptides less prominently disrupted sharpening, with average multiunit receptive fields of 18 degrees. Thus

  3. Accommodation to wavefront vergence and chromatic aberration.

    PubMed

    Wang, Yinan; Kruger, Philip B; Li, James S; Lin, Peter L; Stark, Lawrence R

    2011-05-01

    Longitudinal chromatic aberration (LCA) provides a cue to accommodation with small pupils. However, large pupils increase monochromatic aberrations, which may obscure chromatic blur. In this study, we examined the effect of pupil size and LCA on accommodation. Accommodation was recorded by infrared optometer while observers (nine normal trichromats) viewed a sinusoidally moving Maltese cross target in a Badal stimulus system. There were two illumination conditions: white (3000 K; 20 cd/m) and monochromatic (550 nm with 10 nm bandwidth; 20 cd/m) and two artificial pupil conditions (3 and 5.7 mm). Separately, static measurements of wavefront aberration were made with the eye accommodating to targets between 0 and 4 D (COAS, Wavefront Sciences). Large individual differences in accommodation to wavefront vergence and to LCA are a hallmark of accommodation. LCA continues to provide a signal at large pupil sizes despite higher levels of monochromatic aberrations. Monochromatic aberrations may defend against chromatic blur at high spatial frequencies, but accommodation responds best to optical vergence and to LCA at 3 c/deg where blur from higher order aberrations is less.

  4. Efficient Processing of the Immunodominant, HLA-A*0201-Restricted Human Immunodeficiency Virus Type 1 Cytotoxic T-Lymphocyte Epitope despite Multiple Variations in the Epitope Flanking Sequences

    PubMed Central

    Brander, Christian; Yang, Otto O.; Jones, Norman G.; Lee, Yun; Goulder, Philip; Johnson, R. Paul; Trocha, Alicja; Colbert, David; Hay, Christine; Buchbinder, Susan; Bergmann, Cornelia C.; Zweerink, Hans J.; Wolinsky, Steven; Blattner, William A.; Kalams, Spyros A.; Walker, Bruce D.

    1999-01-01

    Immune escape from cytotoxic T-lymphocyte (CTL) responses has been shown to occur not only by changes within the targeted epitope but also by changes in the flanking sequences which interfere with the processing of the immunogenic peptide. However, the frequency of such an escape mechanism has not been determined. To investigate whether naturally occurring variations in the flanking sequences of an immunodominant human immunodeficiency virus type 1 (HIV-1) Gag CTL epitope prevent antigen processing, cells infected with HIV-1 or vaccinia virus constructs encoding different patient-derived Gag sequences were tested for recognition by HLA-A*0201-restricted, p17-specific CTL. We found that the immunodominant p17 epitope (SL9) and its variants were efficiently processed from minigene expressing vectors and from six HIV-1 Gag variants expressed by recombinant vaccinia virus constructs. Furthermore, SL9-specific CTL clones derived from multiple donors efficiently inhibited virus replication when added to HLA-A*0201-bearing cells infected with primary or laboratory-adapted strains of virus, despite the variability in the SL9 flanking sequences. These data suggest that escape from this immunodominant CTL response is not frequently accomplished by changes in the epitope flanking sequences. PMID:10559335

  5. Developmental Localization and Methylesterification of Pectin Epitopes during Somatic Embryogenesis of Banana (Musa spp. AAA)

    PubMed Central

    Xu, Chunxiang; Zhao, Lu; Pan, Xiao; Šamaj, Jozef

    2011-01-01

    Background The plant cell walls play an important role in somatic embryogenesis and plant development. Pectins are major chemical components of primary cell walls while homogalacturonan (HG) is the most abundant pectin polysaccharide. Developmental regulation of HG methyl-esterification degree is important for cell adhesion, division and expansion, and in general for proper organ and plant development. Methodology/Principal Findings Developmental localization of pectic homogalacturonan (HG) epitopes and the (1→4)-β-D-galactan epitope of rhamnogalacturonan I (RG-I) and degree of pectin methyl-esterification (DM) were studied during somatic embryogenesis of banana (Musa spp. AAA). Histological analysis documented all major developmental stages including embryogenic cells (ECs), pre-globular, globular, pear-shaped and cotyledonary somatic embryos. Histochemical staining of extracellularly secreted pectins with ruthenium red showed the most intense staining at the surface of pre-globular, globular and pear-shaped somatic embryos. Biochemical analysis revealed developmental regulation of galacturonic acid content and DM in diverse embryogenic stages. Immunodots and immunolabeling on tissue sections revealed developmental regulation of highly methyl-esterified HG epitopes recognized by JIM7 and LM20 antibodies during somatic embryogenesis. Cell walls of pre-globular/globular and late-stage embryos contained both low methyl-esterified HG epitopes as well as partially and highly methyl-esterified ones. Extracellular matrix which covered surface of early developing embryos contained pectin epitopes recognized by 2F4, LM18, JIM5, JIM7 and LM5 antibodies. De-esterification of cell wall pectins by NaOH caused a decrease or an elimination of immunolabeling in the case of highly methyl-esterified HG epitopes. However, immunolabeling of some low methyl-esterified epitopes appeared stronger after this base treatment. Conclusions/Significance These data suggest that both low

  6. Evaluation and comparison of the ability of online available prediction programs to predict true linear B-cell epitopes.

    PubMed

    Costa, Juan G; Faccendini, Pablo L; Sferco, Silvano J; Lagier, Claudia M; Marcipar, Iván S

    2013-06-01

    This work deals with the use of predictors to identify useful B-cell linear epitopes to develop immunoassays. Experimental techniques to meet this goal are quite expensive and time consuming. Therefore, we tested 5 free, online prediction methods (AAPPred, ABCpred, BcePred, BepiPred and Antigenic) widely used for predicting linear epitopes, using the primary structure of the protein as the only input. We chose a set of 65 experimentally well documented epitopes obtained by the most reliable experimental techniques as our true positive set. To compare the quality of the predictor methods we used their positive predictive value (PPV), i.e. the proportion of the predicted epitopes that are true, experimentally confirmed epitopes, in relation to all the epitopes predicted. We conclude that AAPPred and ABCpred yield the best results as compared with the other programs and with a random prediction procedure. Our results also indicate that considering the consensual epitopes predicted by several programs does not improve the PPV.

  7. Development of a polyclonal antibody with broad epitope specificity for advanced glycation endproducts and localization of these epitopes in Bruch's membrane of the aging eye.

    PubMed

    Farboud, B; Aotaki-Keen, A; Miyata, T; Hjelmeland, L M; Handa, J T

    1999-07-14

    To develop an antibody that recognizes a variety of advanced glycation endproduct (AGE) epitopes. Glycolaldehyde was used to modify bovine serum albumin and HPLC analysis was used to measure pentosidine formation as an indicator of AGE formation. A polyclonal anti-AGE antibody was synthesized by injecting glycolaldehyde-incubated keyhole limpet hemocyanin into rabbits, affinity purified using AGE modified bovine serum albumin coupled to an affinity resin column, and characterized by immunoblot analysis. HPLC analysis of glycolaldehyde treated bovine serum albumin detected high levels of pentosidine formation, suggesting that glycolaldehyde is a potent precursor for pentosidine. By immunoblot analysis, our antibody recognized carboxymethyllysine and pentosidine, two well-characterized AGEs, as well as other AGE epitopes. Immunohistochemical evaluation showed evidence of AGEs in Bruch's membrane (including basal laminar deposits and drusen), choroidal extracellular matrix, and vessel walls in an 82 year old nondiabetic globe. A similar staining pattern was observed in an age-matched diabetic control. In contrast, no staining was seen with the antibody in a 20 month old nondiabetic globe. A unique anti-AGE antibody was synthesized that recognizes a variety of AGE epitopes including carboxymethyllysine and pentosidine. Its best use might be in broad surveys of the age-dependent accumulation of a large number of AGE epitopes that might not be revealed by antibodies to pentosidine or CML.

  8. Emergence of a Norovirus GII.4 Strain Correlates with Changes in Evolving Blockade Epitopes

    PubMed Central

    Lindesmith, Lisa C.; Costantini, Verónica; Swanstrom, Jesica; Debbink, Kari; Donaldson, Eric F.; Vinjé, Jan

    2013-01-01

    The major capsid protein of norovirus GII.4 strains is evolving rapidly, resulting in epidemic strains with altered antigenicity. GII.4.2006 Minerva strains circulated at pandemic levels in 2006 and persisted at lower levels until 2009. In 2009, a new GII.4 variant, GII.4.2009 New Orleans, emerged and since then has become the predominant strain circulating in human populations. To determine whether changes in evolving blockade epitopes correlate with the emergence of the GII.4.2009 New Orleans strains, we compared the antibody reactivity of a panel of mouse monoclonal antibodies (MAbs) against GII.4.2006 and GII.4.2009 virus-like particles (VLPs). Both anti-GII.4.2006 and GII.4.2009 MAbs effectively differentiated the two strains by VLP-carbohydrate ligand blockade assay. Most of the GII.4.2006 MAbs preferentially blocked GII.4.2006, while all of the GII.4.2009 MAbs preferentially blocked GII.4.2009, although 8 of 12 tested blockade MAbs blocked both VLPs. Using mutant VLPs designed to alter predicted antigenic epitopes, binding of seven of the blockade MAbs was impacted by alterations in epitope A, identifying residues 294, 296, 297, 298, 368, and 372 as important antigenic sites in these strains. Convalescent-phase serum collected from a GII.4.2009 outbreak confirmed the immunodominance of epitope A, since alterations of epitope A affected serum reactivity by 40%. These data indicate that the GII.4.2009 New Orleans variant has evolved a key blockade epitope, possibly allowing for at least partial escape from protective herd immunity and provide epidemiological support for the utility of monitoring changes in epitope A in emergent strain surveillance. PMID:23269783

  9. Experimental validation of the RATE tool for inferring HLA restrictions of T cell epitopes.

    PubMed

    Paul, Sinu; Arlehamn, Cecilia S Lindestam; Schulten, Veronique; Westernberg, Luise; Sidney, John; Peters, Bjoern; Sette, Alessandro

    2017-06-21

    The RATE tool was recently developed to computationally infer the HLA restriction of given epitopes from immune response data of HLA typed subjects without additional cumbersome experimentation. Here, RATE was validated using experimentally defined restriction data from a set of 191 tuberculosis-derived epitopes and 63 healthy individuals with MTB infection from the Western Cape Region of South Africa. Using this experimental dataset, the parameters utilized by the RATE tool to infer restriction were optimized, which included relative frequency (RF) of the subjects responding to a given epitope and expressing a given allele as compared to the general test population and the associated p-value in a Fisher's exact test. We also examined the potential for further optimization based on the predicted binding affinity of epitopes to potential restricting HLA alleles, and the absolute number of individuals expressing a given allele and responding to the specific epitope. Different statistical measures, including Matthew's correlation coefficient, accuracy, sensitivity and specificity were used to evaluate performance of RATE as a function of these criteria. Based on our results we recommend selection of HLA restrictions with cutoffs of p-value < 0.01 and RF ≥ 1.3. The usefulness of the tool was demonstrated by inferring new HLA restrictions for epitope sets where restrictions could not be experimentally determined due to lack of necessary cell lines and for an additional data set related to recognition of pollen derived epitopes from allergic patients. Experimental data sets were used to validate RATE tool and the parameters used by the RATE tool to infer restriction were optimized. New HLA restrictions were identified using the optimized RATE tool.

  10. GPS-MBA: Computational Analysis of MHC Class II Epitopes in Type 1 Diabetes

    PubMed Central

    Ren, Jian; Ma, Chuang; Gao, Tianshun; Zhou, Yanhong; Yang, Qing; Xue, Yu

    2012-01-01

    As a severe chronic metabolic disease and autoimmune disorder, type 1 diabetes (T1D) affects millions of people world-wide. Recent advances in antigen-based immunotherapy have provided a great opportunity for further treating T1D with a high degree of selectivity. It is reported that MHC class II I-Ag7 in the non-obese diabetic (NOD) mouse and human HLA-DQ8 are strongly linked to susceptibility to T1D. Thus, the identification of new I-Ag7 and HLA-DQ8 epitopes would be of great help to further experimental and biomedical manipulation efforts. In this study, a novel GPS-MBA (MHC Binding Analyzer) software package was developed for the prediction of I-Ag7 and HLA-DQ8 epitopes. Using experimentally identified epitopes as the training data sets, a previously developed GPS (Group-based Prediction System) algorithm was adopted and improved. By extensive evaluation and comparison, the GPS-MBA performance was found to be much better than other tools of this type. With this powerful tool, we predicted a number of potentially new I-Ag7 and HLA-DQ8 epitopes. Furthermore, we designed a T1D epitope database (TEDB) for all of the experimentally identified and predicted T1D-associated epitopes. Taken together, this computational prediction result and analysis provides a starting point for further experimental considerations, and GPS-MBA is demonstrated to be a useful tool for generating starting information for experimentalists. The GPS-MBA is freely accessible for academic researchers at: http://mba.biocuckoo.org. PMID:22479466

  11. Temporal Formation and Immunolocalization of an Endospore Surface Epitope During Pasteuria penetrans Sporogenesis.

    PubMed

    Brito, J A; Preston, J F; Dickson, D W; Giblin-Davis, R M; Williams, D S; Aldrich, H C; Rice, J D

    2003-09-01

    The synthesis and localization of an endospore surface epitope associated with the development of Pasteuria penetrans was determined using a monoclonal antibody (MAb) as a probe. Nematodes, uninfected or infected with P. penetrans, were harvested at 12, 16, 24, and 38 days after inoculation (DAI) and then examined to determine the developmental stage of the bacterium. Vegetative growth of P. penetrans was observed only in infected nematodes harvested at 12 and 16 DAI, whereas cells at different stages of sporulation and mature endospores were observed at 24 and 38 DAI. ELISA and immunoblot analysis revealed that the adhesin-associated epitope was first detected at 24 DAI, and increased in the later stages of sporogenesis. These results indicate that the synthesis of adhesin-related proteins occurred at a certain developmental stage relative to the sporulation process, and was associated with endospore maturation. Immunofluorescence microscopy indicated that the distribution of the epitope is nearly uniform on the periphery of each spore, as defined by parasporal fibers. Immunocytochemistry at the ultrastructural level indicated a distribution of the epitope over the parasporal fibers. The epitope also was detected over other structures such as sporangium and exosporium during the sporogenesis process, but it was not observed over the cortex, inner-spore coat, outer-spore coat, or protoplasm. The appearance of the adhesin epitope first at stage III of sporogenesis and its presence on the parasporal fibers are consistent with an adhesin-related role in the attachment of the mature endospore to the cuticle of the nematode host.

  12. Can mutational GC-pressure create new linear B-cell epitopes in herpes simplex virus type 1 glycoprotein B?

    PubMed

    Khrustalev, Vladislav Victorovich

    2009-01-01

    We showed that GC-content of nucleotide sequences coding for linear B-cell epitopes of herpes simplex virus type 1 (HSV1) glycoprotein B (gB) is higher than GC-content of sequences coding for epitope-free regions of this glycoprotein (G + C = 73 and 64%, respectively). Linear B-cell epitopes have been predicted in HSV1 gB by BepiPred algorithm ( www.cbs.dtu.dk/services/BepiPred ). Proline is an acrophilic amino acid residue (it is usually situated on the surface of protein globules, and so included in linear B-cell epitopes). Indeed, the level of proline is much higher in predicted epitopes of gB than in epitope-free regions (17.8% versus 1.8%). This amino acid is coded by GC-rich codons (CCX) that can be produced due to nucleotide substitutions caused by mutational GC-pressure. GC-pressure will also lead to disappearance of acrophobic phenylalanine, isoleucine, methionine and tyrosine coded by GC-poor codons. Results of our "in-silico directed mutagenesis" showed that single nonsynonymous substitutions in AT to GC direction in two long epitope-free regions of gB will cause formation of new linear epitopes or elongation of previously existing epitopes flanking these regions in 25% of 539 possible cases. The calculations of GC-content and amino acid content have been performed by CodonChanges algorithm ( www.barkovsky.hotmail.ru ).

  13. Sixth-order wave aberration theory of ultrawide-angle optical systems.

    PubMed

    Lu, Lijun; Cao, Yiqing

    2017-10-20

    In this paper, we develop sixth-order wave aberration theory of ultrawide-angle optical systems like fisheye lenses. Based on the concept and approach to develop wave aberration theory of plane-symmetric optical systems, we first derive the sixth-order intrinsic wave aberrations and the fifth-order ray aberrations; second, we present a method to calculate the pupil aberration of such kind of optical systems to develop the extrinsic aberrations; third, the relation of aperture-ray coordinates between adjacent optical surfaces is fitted with the second-order polynomial to improve the calculation accuracy of the wave aberrations of a fisheye lens with a large acceptance aperture. Finally, the resultant aberration expressions are applied to calculate the aberrations of two design examples of fisheye lenses; the calculation results are compared with the ray-tracing ones with Zemax software to validate the aberration expressions.

  14. Application of phage peptide display technology for the study of food allergen epitopes.

    PubMed

    Chen, Xueni; Dreskin, Stephen C

    2017-06-01

    Phage peptide display technology has been used to identify IgE-binding mimotopes (mimics of natural epitopes) that mimic conformational epitopes. This approach is effective in the characterization of those epitopes that are important for eliciting IgE-mediated allergic responses by food allergens and those that are responsible for cross-reactivity among allergenic food proteins. Application of this technology will increase our understanding of the mechanisms whereby food allergens elicit allergic reactions, will facilitate the discovery of diagnostic reagents and may lead to mimotope-based immunotherapy. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. Identification of a serotype-independent linear epitope of foot-and-mouth disease virus.

    PubMed

    Yang, Baolin; Wang, Mingxia; Liu, Wenming; Xu, Zhiqiang; Wang, Haiwei; Yang, Decheng; Ma, Wenge; Zhou, Guohui; Yu, Li

    2017-12-01

    Foot-and-mouth disease (FMD), caused by foot-and-mouth disease virus (FMDV), is a highly contagious infectious disease that affects domestic and wild cloven-hoofed animals worldwide. VP2 is a structural protein of FMDV. In this study, an FMDV serotype-independent monoclonal antibody (MAb), 10B10, against the viral capsid protein VP2 was generated, and a series of GST fusion proteins expressing a truncated peptide of VP2 was subjected to Western blot analysis using MAb 10B10. Their results indicated that the peptide 8 TLLEDRILT 16 of VP2 is the minimal requirement of the epitope recognized by MAb 10B10. Importantly, this linear epitope was highly conserved among all seven serotypes of FMDV in a sequence alignment analysis. Subsequent alanine-scanning mutagenesis analysis revealed that the residues Thr 8 and Asp 12 of the epitope were crucial for MAb-10B10 binding. Furthermore, Western blot analysis also revealed that the MAb 10B10-directed epitope could be recognized by positive sera from FMDV-infected cattle. The discovery that MAb 10B10 recognizes a serotype-independent linear epitope of FMDV suggests potential applications for this MAb in the development of serotype-independent tests for FMDV.

  16. Immunoinformatics Approach in Designing Epitope-based Vaccine Against Meningitis-inducing Bacteria (Streptococcus pneumoniae, Neisseria meningitidis, and Haemophilus influenzae Type b).

    PubMed

    Zahroh, Hilyatuz; Ma'rup, Ahmad; Tambunan, Usman Sumo Friend; Parikesit, Arli Aditya

    2016-01-01

    Meningitis infection is one of the major threats during Hajj season in Mecca. Meningitis vaccines are available, but their uses are limited in some countries due to religious reasons. Furthermore, they only give protection to certain serogroups, not to all types of meningitis-inducing bacteria. Recently, research on epitope-based vaccines has been developed intensively. Such vaccines have potential advantages over conventional vaccines in that they are safer to use and well responded to the antibody. In this study, we developed epitope-based vaccine candidates against various meningitis-inducing bacteria, including Streptococcus pneumoniae , Neisseria meningitidis , and Haemophilus influenzae type b. The epitopes were selected from their protein of polysaccharide capsule. B-cell epitopes were predicted by using BCPred, while T-cell epitope for major histocompatibility complex (MHC) class I was predicted using PAProC, TAPPred, and Immune Epitope Database. Immune Epitope Database was also used to predict T-cell epitope for MHC class II. Population coverage and molecular docking simulation were predicted against previously generated epitope vaccine candidates. The best candidates for MHC class I- and class II-restricted T-cell epitopes were MQYGDKTTF, MKEQNTLEI, ECTEGEPDY, DLSIVVPIY, YPMAMMWRNASNRAI, TLQMTLLGIVPNLNK, ETSLHHIPGISNYFI, and SLLYILEKNAEMEFD, which showed 80% population coverage. The complexes of class I T-cell epitopes-HLA-C*03:03 and class II T-cell epitopes-HLA-DRB1*11:01 showed better affinity than standards as evaluated from their Δ G binding value and the binding interaction between epitopes and HLA molecules. These peptide constructs may further be undergone in vitro and in vivo testings for the development of targeted vaccine against meningitis infection.

  17. Usefulness of the ElliPro epitope predictor program in defining the repertoire of HLA-ABC eplets.

    PubMed

    Duquesnoy, Rene J; Marrari, Marilyn

    HLA matching at the epitope level offers new opportunities to identify suitable donors for transplant patients. The International HLA Epitope Registry (www.Epregistry.com.br) describes for the various HLA loci, repertoires of eplets including those that correspond to epitopes experimentally verified with specific antibodies. There are also many eplets which have remained as theoretical entities because no informative antibodies have been found. Which of them have immunogenic potential or conversely, might be considered as non-epitopes that cannot elicit specific antibody responses? This question is important for the application of epitope-based HLA matching in clinical transplantation. Correct predictions of B-cell epitopes on antigenic proteins are essential to the effective design of microbial vaccines and the development of specific antibodies used in immunotherapy and immunodiagnostics but prediction programs based on structural and physiochemical properties of amino acid residues are generally ineffective. Recent prediction programs based on three-dimensional structures of antigen-antibody complexes are more promising. One such program is called ElliPro developed by Ponomarenko. This report describes studies demonstrating that ElliPro can predict alloantibody responses to HLA-ABC eplets. Antibody-verified eplets have amino acid residues with much higher ElliPro scores than eplets for which no specific antibodies have been found. The latter group includes residues with very low ElliPro scores; they appear to represent eplets that might be classified as non-epitopes. In conclusion, ElliPro offers a new approach to characterize epitope repertoires that are clinically relevant in HLA matching. Copyright © 2017. Published by Elsevier Inc.

  18. Identification of Relevant Conformational Epitopes on the HER2 Oncoprotein by Using Large Fragment Phage Display (LFPD)

    PubMed Central

    Gabrielli, Federico; Salvi, Roberto; Garulli, Chiara; Kalogris, Cristina; Arima, Serena; Tardella, Luca; Monaci, Paolo; Pupa, Serenella M.; Tagliabue, Elda; Montani, Maura; Quaglino, Elena; Stramucci, Lorenzo; Curcio, Claudia

    2013-01-01

    We developed a new phage-display based approach, the Large Fragment Phage Display (LFPD), that can be used for mapping conformational epitopes on target molecules of immunological interest. LFPD uses a simplified and more effective phage-display approach in which only a limited set of larger fragments (about 100 aa in length) are expressed on the phage surface. Using the human HER2 oncoprotein as a target, we identified novel B-cell conformational epitopes. The same homologous epitopes were also detected in rat HER2 and all corresponded to the epitopes predicted by computational analysis (PEPITO software), showing that LFPD gives reproducible and accurate results. Interestingly, these newly identified HER2 epitopes seem to be crucial for an effective immune response against HER2-overexpressing breast cancers and might help discriminating between metastatic breast cancer and early breast cancer patients. Overall, the results obtained in this study demonstrated the utility of LFPD and its potential application to the detection of conformational epitopes on many other molecules of interest, as well as, the development of new and potentially more effective B-cell conformational epitopes based vaccines. PMID:23555577

  19. Spherical aberrations of human astigmatic corneas.

    PubMed

    Zhao, Huawei; Dai, Guang-Ming; Chen, Li; Weeber, Henk A; Piers, Patricia A

    2011-11-01

    To evaluate whether the average spherical aberration of human astigmatic corneas is statistically equivalent to human nonastigmatic corneas. Spherical aberrations of 445 astigmatic corneas prior to laser vision correction were retrospectively investigated to determine Zernike coefficients for central corneal areas 6 mm in diameter using CTView (Sarver and Associates). Data were divided into groups according to cylinder power (0.01 to 0.25 diopters [D], 0.26 to 0.75 D, 0.76 to 1.06 D, 1.07 to 1.53 D, 1.54 to 2.00 D, and >2.00 D) and according to age by decade. Spherical aberrations were correlated with age and astigmatic power among groups and the entire population. Statistical analyses were conducted, and P<.05 was considered statistically significant. Mean patient age was 42.6±11 years. Astigmatic corneas had an average astigmatic power of 0.78±0.58 D and mean spherical aberration was 0.25±0.13 μm for the entire population and approximately the same (0.27 μm) for individual groups, ranging from 0.23 to 0.29 μm (P>.05 for all tested groups). Mean spherical aberration of astigmatic corneas was not correlated significantly with cylinder power or age (P>.05). Spherical aberrations are similar to those of nonastigmatic corneas, permitting the use of these additional data in the design of aspheric toric intra-ocular lenses. Copyright 2011, SLACK Incorporated.

  20. Identification of a conserved B-cell epitope on the GapC protein of Streptococcus dysgalactiae.

    PubMed

    Zhang, Limeng; Zhou, Xue; Fan, Ziyao; Tang, Wei; Chen, Liang; Dai, Jian; Wei, Yuhua; Zhang, Jianxin; Yang, Xuan; Yang, Xijing; Liu, Daolong; Yu, Liquan; Zhang, Hua; Wu, Zhijun; Yu, Yongzhong; Sun, Hunan; Cui, Yudong

    2015-01-01

    Streptococcus dysgalactiae (S. dysgalactia) GapC is a highly conserved surface dehydrogenase among the streptococcus spp., which is responsible for inducing protective antibody immune responses in animals. However, the B-cell epitope of S. dysgalactia GapC have not been well characterized. In this study, a monoclonal antibody 1F2 (mAb1F2) against S. dysgalactiae GapC was generated by the hybridoma technique and used to screen a phage-displayed 12-mer random peptide library (Ph.D.-12) for mapping the linear B-cell epitope. The mAb1F2 recognized phages displaying peptides with the consensus motif TRINDLT. Amino acid sequence of the motif exactly matched (30)TRINDLT(36) of the S. dysgalactia GapC. Subsequently, site-directed mutagenic analysis further demonstrated that residues R31, I32, N33, D34 and L35 formed the core of (30)TRINDLT(36), and this core motif was the minimal determinant of the B-cell epitope recognized by the mAb1F2. The epitope (30)TRINDLT(36) showed high homology among different streptococcus species. Overall, our findings characterized a conserved B-cell epitope, which will be useful for the further study of epitope-based vaccines. Copyright © 2015 Elsevier Ltd. All rights reserved.

  1. High order aberration and straylight evaluation after cataract surgery with implantation of an aspheric, aberration correcting monofocal intraocular lens

    PubMed Central

    Kretz, Florian T A; Tandogan, Tamer; Khoramnia, Ramin; Auffarth, Gerd U

    2015-01-01

    AIM To evaluate the quality of vision in respect to high order aberrations and straylight perception after implantation of an aspheric, aberration correcting, monofocal intraocular lens (IOL). METHODS Twenty-one patients (34 eyes) aged 50 to 83y underwent cataract surgery with implantation of an aspheric, aberration correcting IOL (Tecnis ZCB00, Abbott Medical Optics). Three months after surgery they were examined for uncorrected (UDVA) and corrected distance visual acuity (CDVA), contrast sensitivity (CS) under photopic and mesopic conditions with and without glare source, ocular high order aberrations (HOA, Zywave II) and retinal straylight (C-Quant). RESULTS Postoperatively, patients achieved a postoperative CDVA of 0.0 logMAR or better in 97.1% of eyes. Mean values of high order abberations were +0.02±0.27 (primary coma components) and -0.04±0.16 (spherical aberration term). Straylight values of the C-Quant were 1.35±0.44 log which is within normal range of age matched phakic patients. The CS measurements under mesopic and photopic conditions in combination with and without glare did not show any statistical significance in the patient group observed (P≥0.28). CONCLUSION The implantation of an aspherical aberration correcting monofocal IOL after cataract surgery resulted in very low residual higher order aberration (HOA) and normal straylight. PMID:26309872

  2. Accommodation to Wavefront Vergence and Chromatic Aberration

    PubMed Central

    Wang, Yinan; Kruger, Philip B.; Li, James S.; Lin, Peter L.; Stark, Lawrence R.

    2011-01-01

    Purpose Longitudinal chromatic aberration (LCA) provides a cue to accommodation with small pupils. However, large pupils increase monochromatic aberrations, which may obscure chromatic blur. In the present study, we examined the effect of pupil size and LCA on accommodation. Methods Accommodation was recorded by infrared optometer while observers (nine normal trichromats) viewed a sinusoidally moving Maltese cross target in a Badal stimulus system. There were two illumination conditions: white (3000 K; 20 cd/m2) and monochromatic (550 nm with 10 nm bandwidth; 20 cd/m2) and two artificial pupil conditions (3 mm and 5.7 mm). Separately, static measurements of wavefront aberration were made with the eye accommodating to targets between 0 and 4 D (COAS, Wavefront Sciences). Results Large individual differences in accommodation to wavefront vergence and to LCA are a hallmark of accommodation. LCA continues to provide a signal at large pupil sizes despite higher levels of monochromatic aberrations. Conclusions Monochromatic aberrations may defend against chromatic blur at high spatial frequencies, but accommodation responds best to optical vergence and to LCA at 3 c/deg where blur from higher order aberrations is less. PMID:21317666

  3. TGF-{beta}-stimulated aberrant expression of class III {beta}-tubulin via the ERK signaling pathway in cultured retinal pigment epithelial cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chung, Eun Jee; Chun, Ji Na; Jung, Sun-Ah

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer TGF-{beta} induces aberrant expression of {beta}III in RPE cells via the ERK pathway. Black-Right-Pointing-Pointer TGF-{beta} increases O-GlcNAc modification of {beta}III in RPE cells. Black-Right-Pointing-Pointer Mature RPE cells have the capacity to express a neuron-associated gene by TGF-{beta}. -- Abstract: The class III {beta}-tubulin isotype ({beta}{sub III}) is expressed exclusively by neurons within the normal human retina and is not present in normal retinal pigment epithelial (RPE) cells in situ or in the early phase of primary cultures. However, aberrant expression of class III {beta}-tubulin has been observed in passaged RPE cells and RPE cells with dedifferentiated morphology inmore » pathologic epiretinal membranes from idiopathic macular pucker, proliferative vitreoretinopathy (PVR) and proliferative diabetic retinopathy (PDR). Transforming growth factor-{beta} (TGF-{beta}) has been implicated in dedifferentiation of RPE cells and has a critical role in the development of proliferative vitreoretinal diseases. Here, we investigated the potential effects of TGF-{beta} on the aberrant expression of class III {beta}-tubulin and the intracellular signaling pathway mediating these changes. TGF-{beta}-induced aberrant expression and O-linked-{beta}-N-acetylglucosamine (O-GlcNac) modification of class III {beta}-tubulin in cultured RPE cells as determined using Western blotting, RT-PCR and immunocytochemistry. TGF-{beta} also stimulated phosphorylation of ERK. TGF-{beta}-induced aberrant expression of class III {beta}-tubulin was significantly reduced by pretreatment with U0126, an inhibitor of ERK phosphorylation. Our findings indicate that TGF-{beta} stimulated aberrant expression of class III {beta}-tubulin via activation of the ERK signaling pathway. These data demonstrate that mature RPE cells have the capacity to express a neuron-associated gene in response to TGF-{beta} stimulation and provide useful

  4. MUC-1 Tumor Antigen Agonist Epitopes for Enhancing T-cell Responses to Human Tumors | NCI Technology Transfer Center | TTC

    Cancer.gov

    Scientists at NIH have identified 7 new agonist epitopes of the MUC-1 tumor associated antigen. Compared to their native epitope counterparts, peptides reflecting these agonist epitopes have been shown to enhance the generation of human tumor cells, which in turn have a greater ability to kill human tumor cells endogenously expressing the native MUC-1 epitope.

  5. Comparison of Aberrations After Standard and Customized Refractive Surgery

    NASA Astrophysics Data System (ADS)

    Fang, L.; He, X.; Wang, Y.

    2013-09-01

    To detect possible differences in residual wavefront aberrations between standard and customized laser refractive surgery based onmathematical modeling, the residual optical aberrations after conventional and customized laser refractive surgery were compared accordingto the ablation profile with transition zone. The results indicated that ablation profile has a significant impact on the residual aberrations.The amount of residual aberrations for conventional correction is higher than that for customized correction. Additionally, the residualaberrations for high myopia eyes are markedly larger than those for moderate myopia eyes. For a 5 mm pupil, the main residual aberrationterm is coma and yet it is spherical aberration for a 7 mm pupil. When the pupil diameter is the same as optical zone or greater, themagnitudes of residual aberrations is obviously larger than that for a smaller pupil. In addition, the magnitudes of the residual fifth orsixth order aberrations are relatively large, especially secondary coma in a 6 mm pupil and secondary spherical aberration in a 7 mm pupil.Therefore, the customized ablation profile may be superior to the conventional correction even though the transition zone and treatmentdecentration are taken into account. However, the customized ablation profile will still induce significant amount of residual aberrations.

  6. The Glycan Role in the Glycopeptide Immunogenicity Revealed by Atomistic Simulations and Spectroscopic Experiments on the Multiple Sclerosis Biomarker CSF114(Glc)

    NASA Astrophysics Data System (ADS)

    Bruno, Agostino; Scrima, Mario; Novellino, Ettore; D'Errico, Gerardino; D'Ursi, Anna Maria; Limongelli, Vittorio

    2015-03-01

    Glycoproteins are often recognized as not-self molecules by antibodies triggering the onset of severe autoimmune diseases such as Multiple Sclerosis (MS). Thus, the development of antigen-mimicking biomarkers represents an attractive strategy for an early diagnosis of the disease. An example is the synthetic glycopeptide CSF114(Glc), which was designed and tested as MS biomarker and whose clinical application was limited by its reduced ability to detect autoantibodies in MS patients. In the attempt to improve the efficacy of CSF114(Glc), we have characterized all the events leading to the final binding of the biomarker to the autoantibody using atomistic simulations, ESR and NMR experiments. The glycosydic moiety plays a primary role in the whole process. In particular, in an environment mimicking that used in the clinical tests the glycopeptide assumes a α-helix structure that is functional for the interaction with the antibody. In this conformation CSF114(Glc) binds the monoclonal antibody mAb8-18C5 similarly to the myelin oligodendrocyte glycoprotein MOG, which is a known MS auto-antigen, thus explaining its diagnostic activity. Our study offers new molecular bases to design more effective biomarkers and provides a most valid protocol to investigate other systems where the environment effect is determinant for the biological activity.

  7. Correlating low-similarity peptide sequences and allergenic epitopes.

    PubMed

    Kanduc, D

    2008-01-01

    Although a high number of allergenic peptide epitopes has been experimentally identified and defined, the molecular basis and the precise mechanisms underlying peptide allergenicity are unknown. This issue was analyzed exploring the relationship between peptide allergenicity and sequence similarity to the human proteome. The structured analysis of the data reported in literature put into evidence that the most part of IgE-binding epitopes are (or harbor) pentapeptide unit(s) with no/low similarity to the human proteome, this way suggesting that no or low sequence similarity to the host proteome might represent a minimum common denominator identifying allergenic peptides. The present literature analysis might be of relevance in devising and designing short amino acid modules to be used for blocking pathogenic IgE.

  8. The HIV hide and seek game: an immunogenomic analysis of the HIV epitope repertoire.

    PubMed

    Vider-Shalit, Tal; Almani, Michal; Sarid, Ronit; Louzoun, Yoram

    2009-07-17

    Viruses employ various means to evade immune detection. One common evasion strategy is the removal of CD8 cytotoxic T-lymphocyte (CTL) epitopes. Here, we use bioinformatic tools to compute the HIV CTL epitope repertoire presented by over 8000 HIV sequences in multiple Human Leukocyte Antigen alleles. We define the 'Size of Immune Repertoire' (SIR) score, which represents the ratio between the number of the predicted epitopes within a protein and their expected number within a scrambled version of the same protein. We show that HIV proteins present less epitopes than expected and that the number of epitopes gradually decreases from SIV to recent HIV sequences. The decrease of the SIR score of HIV is accompanied by a high frequency of replacement mutations within epitopes. The SIR score of the different HIV proteins is not uniform. The regulatory proteins, Tat and Rev, expressed early during cellular infection have a low SIR score, whereas virion-associated genes that are expressed later, such as Env, Pol and Gag, have a higher SIR score. Actually, the SIR score of Gag keeps increasing over time. We hypothesize that our results reflect an HIV immune evasion strategy. This involves the targeting of the CTL immune response to viral structural and enzyme proteins, allowing the virus a time interval to propagate before its host cells are destroyed by CTLs. An efficient anti-HIV CTL response against HIV should thus also target the regulatory genes that HIV seeks to hide from the immune system.

  9. Identification of an immunodominant region of Fel d 1 and characterization of constituent epitopes.

    PubMed

    Bateman, E A L; Ardern-Jones, M R; Ogg, G S

    2008-11-01

    Characterization of T cell epitopes restricted by common HLA alleles is a powerful tool in the understanding of the immune responses to allergens and for the identification of potential peptides for future peptide immunotherapy (PIT). One important requirement is the identification and use of peptides that will bind to HLA molecules covering a large proportion of the population. To identify commonly recognized CD4(+) T cell epitopes in Fel d 1, restricted through frequently expressed HLA molecules for potential future use in PIT. HLA matched antigen presenting cells, HLA blocking antibodies, and peptide truncations were used in ELISpot assays to establish HLA-restricted T cell epitopes. Cytokine responses were measured by ex vivo and cultured IFN-gamma, IL-4, and IL-10 ELISpots. Responses to an immunodominant region of chain 2 were identified in the majority of atopic individuals and epitopes restricted by HLA-DQB1(*)06 and -DPB1(*)0401 were characterized in detail. Significantly higher ex vivo IL-4 and lower IFN-gamma responses were observed to both epitopes in individuals with atopic dermatitis (AD) compared with those without disease. IL-10 responses were significantly lower in those with AD in the individuals with HLA-DPB1(*)0401. We have identified an immunodominant region of Fel d 1 which is frequently recognized by CD4(+) T cells from atopic individuals and contains epitopes that are restricted by very common HLA alleles.

  10. Oxidation-specific epitopes are dominant targets of innate natural antibodies in mice and humans

    PubMed Central

    Chou, Meng-Yun; Fogelstrand, Linda; Hartvigsen, Karsten; Hansen, Lotte F.; Woelkers, Douglas; Shaw, Peter X.; Choi, Jeomil; Perkmann, Thomas; Bäckhed, Fredrik; Miller, Yury I.; Hörkkö, Sohvi; Corr, Maripat; Witztum, Joseph L.; Binder, Christoph J.

    2009-01-01

    Atherosclerosis is a chronic inflammatory disease characterized by the accumulation of oxidized lipoproteins and apoptotic cells. Adaptive immune responses to various oxidation-specific epitopes play an important role in atherogenesis. However, accumulating evidence suggests that these epitopes are also recognized by innate receptors, such as scavenger receptors on macrophages, and plasma proteins, such as C-reactive protein (CRP). Here, we provide multiple lines of evidence that oxidation-specific epitopes constitute a dominant, previously unrecognized target of natural Abs (NAbs) in both mice and humans. Using reconstituted mice expressing solely IgM NAbs, we have shown that approximately 30% of all NAbs bound to model oxidation-specific epitopes, as well as to atherosclerotic lesions and apoptotic cells. Because oxidative processes are ubiquitous, we hypothesized that these epitopes exert selective pressure to expand NAbs, which in turn play an important role in mediating homeostatic functions consequent to inflammation and cell death, as demonstrated by their ability to facilitate apoptotic cell clearance. These findings provide novel insights into the functions of NAbs in mediating host homeostasis and into their roles in health and diseases, such as chronic inflammatory diseases and atherosclerosis. PMID:19363291

  11. Theoretical investigation of aberrations upon ametropic human eyes

    NASA Astrophysics Data System (ADS)

    Tan, Bo; Chen, Ying-Ling; Lewis, J. W. L.; Baker, Kevin

    2003-11-01

    The human eye aberrations are important for visual acuity and ophthalmic diagnostics and surgical procedures. Reported monochromatic aberration data of the normal 20/20 human eyes are scarce. There exist even fewer reports of the relation between ametropic conditions and aberrations. We theoretically investigate the monochromatic and chromatic aberrations of human eyes for refractive errors of -10 to +10 diopters. Schematic human eye models are employed using optical design software for axial, index, and refractive types of ametropia.

  12. B cell epitopes on infliximab identified by oligopeptide microarray with unprocessed patient sera.

    PubMed

    Homann, Arne; Röckendorf, Niels; Kromminga, Arno; Frey, Andreas; Jappe, Uta

    2015-10-29

    Autoimmune diseases like rheumatoid arthritis and inflammatory bowel disease are treated with TNF-alpha-blocking antibodies such as infliximab and adalimumab. A common side effect of therapeutic antibodies is the induction of anti-drug antibodies, which may reduce therapeutic efficacy. In order to reveal immunogenic epitopes on infliximab which are responsible for the adverse effects, sera from patients treated with infliximab were screened by ELISA for anti-infliximab antibodies. Sera containing high levels of anti-drug-antibodies (>1.25 µg/ml) were analyzed in an oligopeptide microarray system containing immobilized 15-meric oligopeptides from the infliximab amino acid sequence. Immunogenic infliximab IgG-epitopes were identified by infrared fluorescence scanning and comparison of infliximab-treated patients versus untreated controls. Six relevant epitopes on infliximab were recognized by the majority of all patient sera: 4 in the variable and 2 in the constant region. Three of the epitopes in the variable region are located in the TNF-alpha binding region of infliximab. The fourth epitope of the variable part of infliximab is located close to the TNF-alpha binding region and contains an N-glycosylation sequon. The sera positive for anti-infliximab antibodies do not contain antibodies against adalimumab as determined by ELISA. Thus, there is no infliximab-adalimumab cross-reactivity as determined by these systems. Our data shall contribute to a knowledge-based recommendation for a potentially necessary therapy switch from infliximab to another type of TNF-alpha-blocker. The characterization of immunogenic epitopes on therapeutic monoclonal antibodies using unprocessed patient sera shall lead to direct translational aspects for the development of less immunogenic therapeutic antibodies. Patients benefit from less adverse events and longer lasting drug effects.

  13. Anterior Corneal, Posterior Corneal, and Lenticular Contributions to Ocular Aberrations.

    PubMed

    Atchison, David A; Suheimat, Marwan; Mathur, Ankit; Lister, Lucas J; Rozema, Jos

    2016-10-01

    To determine the corneal surfaces and lens contributions to ocular aberrations. There were 61 healthy participants with ages ranging from 20 to 55 years and refractions -8.25 diopters (D) to +3.25 D. Anterior and posterior corneal topographies were obtained with an Oculus Pentacam, and ocular aberrations were obtained with an iTrace aberrometer. Raytracing through models of corneas provided total corneal and surface component aberrations for 5-mm-diameter pupils. Lenticular contributions were given as differences between ocular and corneal aberrations. Theoretical raytracing investigated influence of object distance on aberrations. Apart from defocus, the highest aberration coefficients were horizontal astigmatism, horizontal coma, and spherical aberration. Most correlations between lenticular and ocular parameters were positive and significant, with compensation of total corneal aberrations by lenticular aberrations for 5/12 coefficients. Anterior corneal aberrations were approximately three times higher than posterior corneal aberrations and usually had opposite signs. Corneal topographic centers were displaced from aberrometer pupil centers by 0.32 ± 0.19 mm nasally and 0.02 ± 0.16 mm inferiorly; disregarding corneal decentration relative to pupil center was significant for oblique astigmatism, horizontal coma, and horizontal trefoil. An object at infinity, rather than at the image in the anterior cornea, gave incorrect aberration estimates of the posterior cornea. Corneal and lenticular aberration magnitudes are similar, and aberrations of the anterior corneal surface are approximately three times those of the posterior surface. Corneal decentration relative to pupil center has significant effects on oblique astigmatism, horizontal coma, and horizontal trefoil. When estimating component aberrations, it is important to use correct object/image conjugates and heights at surfaces.

  14. Role of T-cell epitope-based vaccine in prophylactic and therapeutic applications

    PubMed Central

    Testa, James S; Philip, Ramila

    2013-01-01

    Prophylactic and therapeutic vaccines against viral infections have advanced in recent years from attenuated live vaccines to subunit-based vaccines. An ideal prophylactic vaccine should mimic the natural immunity induced by an infection, in that it should generate long-lasting adaptive immunity. To complement subunit vaccines, which primarily target an antibody response, different methodologies are being investigated to develop vaccines capable of driving cellular immunity. T-cell epitope discovery is central to this concept. In this review, the significance of T-cell epitope-based vaccines for prophylactic and therapeutic applications is discussed. Additionally, methodologies for the discovery of T-cell epitopes, as well as recent developments in the clinical testing of these vaccines for various viral infections, are explained. PMID:23630544

  15. Elucidation of the TMab-6 Monoclonal Antibody Epitope Against Telomerase Reverse Transcriptase.

    PubMed

    Kaneko, Mika K; Yamada, Shinji; Itai, Shunsuke; Chang, Yao-Wen; Nakamura, Takuro; Yanaka, Miyuki; Harada, Hiroyuki; Suzuki, Hiroyoshi; Kato, Yukinari

    2018-05-03

    Telomerase reverse transcriptase (TERT) and mutations of the TERT promoter are significant in the pathogenesis of 1p/19q-codeleted oligodendrogliomas and isocitrate dehydrogenase gene wild-type glioblastomas, as well as melanomas and squamous cell carcinomas. We previously developed an antihuman TERT monoclonal antibody (mAb), TMab-6, which is applicable in immunohistochemistry for human tissues. However, the binding epitope of TMab-6 against TERT is yet to be elucidated. In this study, enzyme-linked immunosorbent assay and immunohistochemistry were utilized for investigating the epitope of TMab-6. The findings revealed that the critical epitope of TMab-6 is the TERT sequence PSTSRPPRPWD; Thr310 and Ser311 of TERT are especially significant amino acids for TMab-6 recognition.

  16. Nodal aberration theory for wild-filed asymmetric optical systems

    NASA Astrophysics Data System (ADS)

    Chen, Yang; Cheng, Xuemin; Hao, Qun

    2016-10-01

    Nodal Aberration Theory (NAT) was used to calculate the zero field position in Full Field Display (FFD) for the given aberration term. Aiming at wide-filed non-rotational symmetric decentered optical systems, we have presented the nodal geography behavior of the family of third-order and fifth-order aberrations. Meanwhile, we have calculated the wavefront aberration expressions when one optical element in the system is tilted, which was not at the entrance pupil. By using a three-piece-cellphone lens example in optical design software CodeV, the nodal geography is testified under several situations; and the wavefront aberrations are calculated when the optical element is tilted. The properties of the nodal aberrations are analyzed by using Fringe Zernike coefficients, which are directly related with the wavefront aberration terms and usually obtained by real ray trace and wavefront surface fitting.

  17. Quantitative analysis of core fucosylation of serum proteins in liver diseases by LC-MS-MRM.

    PubMed

    Ma, Junfeng; Sanda, Miloslav; Wei, Renhuizi; Zhang, Lihua; Goldman, Radoslav

    2018-02-07

    Aberrant core fucosylation of proteins has been linked to liver diseases. In this study, we carried out multiple reaction monitoring (MRM) quantification of core fucosylated N-glycopeptides of serum proteins partially deglycosylated by a combination of endoglycosidases (endoF1, endoF2, and endoF3). To minimize variability associated with the preparatory steps, the analysis was performed without enrichment of glycopeptides or fractionation of serum besides the nanoRP chromatography. Specifically, we quantified core fucosylation of 22 N-glycopeptides derived from 17 proteins together with protein abundance of these glycoproteins in a cohort of 45 participants (15 disease-free control, 15 fibrosis and 15 cirrhosis patients) using a multiplex nanoUPLC-MS-MRM workflow. We find increased core fucosylation of 5 glycopeptides at the stage of liver fibrosis (i.e., N630 of serotransferrin, N107 of alpha-1-antitrypsin, N253 of plasma protease C1 inhibitor, N397 of ceruloplasmin, and N86 of vitronectin), increase of additional 6 glycopeptides at the stage of cirrhosis (i.e., N138 and N762 of ceruloplasmin, N354 of clusterin, N187 of hemopexin, N71 of immunoglobulin J chain, and N127 of lumican), while the degree of core fucosylation of 10 glycopeptides did not change. Interestingly, although we observe an increase in the core fucosylation at N86 of vitronectin in liver fibrosis, core fucosylation decreases on the N169 glycopeptide of the same protein. Our results demonstrate that the changes in core fucosylation are protein and site specific during the progression of fibrotic liver disease and independent of the changes in the quantity of N-glycoproteins. It is expected that the fully optimized multiplex LC-MS-MRM assay of core fucosylated glycopeptides will be useful for the serologic assessment of the fibrosis of liver. We have quantified the difference in core fucosylation among three comparison groups (healthy control, fibrosis and cirrhosis patients) using a sensitive and

  18. [Study on the DNA vaccine against foot-and-mouth disease virus using the heavy chain constant region of swine IgG as the carrier for peptide epitopes].

    PubMed

    Li, G J; Yan, W Y; Xu, Q X; Sheng, Z T; Zheng, Z X

    2001-05-01

    The peptide of amino acids 141-160 of VP1 protein of foot-and-mouth disease virus (FMDV) is a major B cell epitope and the peptide of amino acids 21-40 is an important T cell epitope. In this study, the DNA fragments of 141-160 and 21-40 peptide epitopes of a strain of type O FMDV was chemically synthesized and arranged into a tandem repeat 141-160 (20AA)-21-40 (20AA)-141-160 (20AA). This tandem sequence was fused to the 3' end of the heavy chain constant region gene of swine immunoglobulin G and was then cloned into mammalian expression vector pCDM8 to form a recombinant plasmid pCDM8FZ3. After pCDM8FZ3 was inoculated intramuscularly into guinea pigs, it elicited a neutralizing antibody response and a specific spleen T cell proliferative response, and 66% of the vaccinated animals were protected from viral challenge. Our study indicated that the heavy chain constant region of swine IgG can act as the carrier protein for FMDV peptide epitopes, and pC-DM8FZ3 is a potential DNA vaccine candidate to prevent FMDV infection.

  19. Aberration corrected STEM by means of diffraction gratings

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Linck, Martin; Ercius, Peter A.; Pierce, Jordan S.

    In the past 15 years, the advent of aberration correction technology in electron microscopy has enabled materials analysis on the atomic scale. This is made possible by precise arrangements of multipole electrodes and magnetic solenoids to compensate the aberrations inherent to any focusing element of an electron microscope. In this paper, we describe an alternative method to correct for the spherical aberration of the objective lens in scanning transmission electron microscopy (STEM) using a passive, nanofabricated diffractive optical element. This holographic device is installed in the probe forming aperture of a conventional electron microscope and can be designed to removemore » arbitrarily complex aberrations from the electron's wave front. In this work, we show a proof-of-principle experiment that demonstrates successful correction of the spherical aberration in STEM by means of such a grating corrector (GCOR). Our GCOR enables us to record aberration-corrected high-resolution high-angle annular dark field (HAADF-) STEM images, although yet without advancement in probe current and resolution. Finally, improvements in this technology could provide an economical solution for aberration-corrected high-resolution STEM in certain use scenarios.« less

  20. Aberration corrected STEM by means of diffraction gratings

    DOE PAGES

    Linck, Martin; Ercius, Peter A.; Pierce, Jordan S.; ...

    2017-06-12

    In the past 15 years, the advent of aberration correction technology in electron microscopy has enabled materials analysis on the atomic scale. This is made possible by precise arrangements of multipole electrodes and magnetic solenoids to compensate the aberrations inherent to any focusing element of an electron microscope. In this paper, we describe an alternative method to correct for the spherical aberration of the objective lens in scanning transmission electron microscopy (STEM) using a passive, nanofabricated diffractive optical element. This holographic device is installed in the probe forming aperture of a conventional electron microscope and can be designed to removemore » arbitrarily complex aberrations from the electron's wave front. In this work, we show a proof-of-principle experiment that demonstrates successful correction of the spherical aberration in STEM by means of such a grating corrector (GCOR). Our GCOR enables us to record aberration-corrected high-resolution high-angle annular dark field (HAADF-) STEM images, although yet without advancement in probe current and resolution. Finally, improvements in this technology could provide an economical solution for aberration-corrected high-resolution STEM in certain use scenarios.« less

  1. Mapping HLA-A2, -A3 and -B7 supertype-restricted T-cell epitopes in the ebolavirus proteome.

    PubMed

    Lim, Wan Ching; Khan, Asif M

    2018-01-19

    Ebolavirus (EBOV) is responsible for one of the most fatal diseases encountered by mankind. Cellular T-cell responses have been implicated to be important in providing protection against the virus. Antigenic variation can result in viral escape from immune recognition. Mapping targets of immune responses among the sequence of viral proteins is, thus, an important first step towards understanding the immune responses to viral variants and can aid in the identification of vaccine targets. Herein, we performed a large-scale, proteome-wide mapping and diversity analyses of putative HLA supertype-restricted T-cell epitopes of Zaire ebolavirus (ZEBOV), the most pathogenic species among the EBOV family. All publicly available ZEBOV sequences (14,098) for each of the nine viral proteins were retrieved, removed of irrelevant and duplicate sequences, and aligned. The overall proteome diversity of the non-redundant sequences was studied by use of Shannon's entropy. The sequences were predicted, by use of the NetCTLpan server, for HLA-A2, -A3, and -B7 supertype-restricted epitopes, which are relevant to African and other ethnicities and provide for large (~86%) population coverage. The predicted epitopes were mapped to the alignment of each protein for analyses of antigenic sequence diversity and relevance to structure and function. The putative epitopes were validated by comparison with experimentally confirmed epitopes. ZEBOV proteome was generally conserved, with an average entropy of 0.16. The 185 HLA supertype-restricted T-cell epitopes predicted (82 (A2), 37 (A3) and 66 (B7)) mapped to 125 alignment positions and covered ~24% of the proteome length. Many of the epitopes showed a propensity to co-localize at select positions of the alignment. Thirty (30) of the mapped positions were completely conserved and may be attractive for vaccine design. The remaining (95) positions had one or more epitopes, with or without non-epitope variants. A significant number (24) of the

  2. Structure of a protective epitope of group B Streptococcus type III capsular polysaccharide.

    PubMed

    Carboni, Filippo; Adamo, Roberto; Fabbrini, Monica; De Ricco, Riccardo; Cattaneo, Vittorio; Brogioni, Barbara; Veggi, Daniele; Pinto, Vittoria; Passalacqua, Irene; Oldrini, Davide; Rappuoli, Rino; Malito, Enrico; Margarit, Immaculada Y Ros; Berti, Francesco

    2017-05-09

    Despite substantial progress in the prevention of group B Streptococcus (GBS) disease with the introduction of intrapartum antibiotic prophylaxis, this pathogen remains a leading cause of neonatal infection. Capsular polysaccharide conjugate vaccines have been tested in phase I/II clinical studies, showing promise for further development. Mapping of epitopes recognized by protective antibodies is crucial for understanding the mechanism of action of vaccines and for enabling antigen design. In this study, we report the structure of the epitope recognized by a monoclonal antibody with opsonophagocytic activity and representative of the protective response against type III GBS polysaccharide. The structure and the atomic-level interactions were determined by saturation transfer difference (STD)-NMR and X-ray crystallography using oligosaccharides obtained by synthetic and depolymerization procedures. The GBS PSIII epitope is made by six sugars. Four of them derive from two adjacent repeating units of the PSIII backbone and two of them from the branched galactose-sialic acid disaccharide contained in this sequence. The sialic acid residue establishes direct binding interactions with the functional antibody. The crystal structure provides insight into the molecular basis of antibody-carbohydrate interactions and confirms that the conformational epitope is not required for antigen recognition. Understanding the structural basis of immune recognition of capsular polysaccharide epitopes can aid in the design of novel glycoconjugate vaccines.

  3. Aberration correction for charged particle lithography

    NASA Astrophysics Data System (ADS)

    Munro, Eric; Zhu, Xieqing; Rouse, John A.; Liu, Haoning

    2001-12-01

    At present, the throughput of projection-type charge particle lithography systems, such as PREVAIL and SCALPEL, is limited primarily by the combined effects of field curvature in the projection lenses and Coulomb interaction in the particle beam. These are fundamental physical limitations, inherent in charged particle optics, so there seems little scope for significantly improving the design of such systems, using conventional rotationally symmetric electron lenses. This paper explores the possibility of overcoming the field aberrations of round electron lense, by using a novel aberration corrector, proposed by Professor H. Rose of University of Darmstadt, called a hexapole planator. In this scheme, a set of round lenses is first used to simultaneously correct distortion and coma. The hexapole planator is then used to correct the field curvature and astigmatism, and to create a negative spherical aberration. The size of the transfer lenses around the planator can then be adjusted to zero the residual spherical aberration. In a way, an electron optical projection system is obtained that is free of all primary geometrical aberrations. In this paper, the feasibility of this concept has been studied with a computer simulation. The simulations verify that this scheme can indeed work, for both electrostatic and magnetic projection systems. Two design studies have been carried out. The first is for an electrostatic system that could be used for ion beam lithography, and the second is for a magnetic projection system for electron beam lithography. In both cases, designs have been achieved in which all primary third-order geometrical aberrations are totally eliminated.

  4. Aberration Compensation in Aplanatic Solid Immersion Lens Microscopy

    DTIC Science & Technology

    2013-11-08

    model and ray tracing software ( Zemax ) to understand how much aberrations are in the system and how much can be compensated by the DM. Subsequently...aberration. Table 2 shows the Zemax simulation on this particular case. With aberration compensation, the finest resolvable group is at 252 nm

  5. [Prediction and evolution of B cell epitopes of hemagglutinin in human-infecting H6N1 avian influenza virus].

    PubMed

    Yang, Jianke; Yuan, Jian; Gao, Jiguang; Zhu, Xiaolei; Lin, Aiqin

    2015-01-01

    To predict B cell epitopes of hemagglutinin (HA) of human-infecting H6N1 avian influenza virus and analyze their evolutionary characteristics. The dataset was downloaded from GISAID and GenBank databases. And the linear and conformational B cell epitopes of HA were predicted separately by various bioinformatic software. Furthermore, the conservation, adaptation and other evolutionary characteristics were also analyzed by some bioinformatic means. Four linear epitopes (A, B, C and D) and two conformational epitopes (E and F) were obtained after consideration of multiple factors. And the C epitope and sites ( 41, 157, 186, 187) mutated easily, but the other epitopes were very conservative and the D epitope was the most conservative. Interestingly, the site 157 was identified under positive selection, suggesting that it may be a particularly important site to make the virus evade the attack from the host immune system. The HA of human-infecting H6N1 avian influenza virus has five conservative B cell epitopes (three linear and two conformational) and one site under positive selection. The findings would facilitate the vaccine development, virus control and pathogenesis understanding.

  6. Development of a multi-epitope peptide vaccine inducing robust T cell responses against brucellosis using immunoinformatics based approaches.

    PubMed

    Saadi, Mahdiye; Karkhah, Ahmad; Nouri, Hamid Reza

    2017-07-01

    Current investigations have demonstrated that a multi-epitope peptide vaccine targeting multiple antigens could be considered as an ideal approach for prevention and treatment of brucellosis. According to the latest findings, the most effective immunogenic antigens of brucella to induce immune responses are included Omp31, BP26, BLS, DnaK and L7-L12. Therefore, in the present study, an in silico approach was used to design a novel multi-epitope vaccine to elicit a desirable immune response against brucellosis. First, five novel T-cell epitopes were selected from Omp31, BP26, BLS, DnaK and L7-L12 proteins using different servers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Selected epitopes were fused together by GPGPG linkers to facilitate the immune processing and epitope presentation. Moreover, cholera toxin B (CTB) was linked to N terminal of vaccine construct as an adjuvant by using EAAAK linker. A multi-epitope vaccine was designed based on predicted epitopes which was 377 amino acid residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this multi-epitope vaccine were assessed using immunoinformatics tools and servers. Based on obtained results, a soluble, and non-allergic protein with 40.59kDa molecular weight was constructed. Expasy ProtParam classified this chimeric protein as a stable protein and also 89.8% residues of constructed vaccine were located in favored regions of the Ramachandran plot. Furthermore, this multi-epitope peptide vaccine was able to strongly induce T cell and B-cell mediated immune responses. In conclusion, immunoinformatics analysis indicated that this multi-epitope peptide vaccine can be effectively expressed and potentially be used for prophylactic or therapeutic usages against brucellosis. Copyright © 2017 Elsevier B.V. All

  7. Greater epitope recognition of shrimp allergens by children than by adults suggests that shrimp sensitization decreases with age.

    PubMed

    Ayuso, Rosalía; Sánchez-Garcia, Silvia; Lin, Jing; Fu, Zhiyan; Ibáñez, María Dolores; Carrillo, Teresa; Blanco, Carlos; Goldis, Marina; Bardina, Ludmila; Sastre, Joaquín; Sampson, Hugh A

    2010-06-01

    Shellfish allergy is a long-lasting disorder typically affecting adults. Despite its high prevalence, there is limited information about allergenic shrimp proteins and the epitopes implicated in such allergic reactions. We sought to identify the IgE-binding epitopes of the 4 shrimp allergens and to characterize epitope recognition profiles of children and adults with shrimp allergy. Fifty-three subjects, 34 children and 19 adults, were selected with immediate allergic reactions to shrimp, increased shrimp-specific serum IgE levels, and positive immunoblot binding to shrimp. Study subjects and 7 nonatopic control subjects were tested by means of peptide microarray for IgE binding with synthetic overlapping peptides spanning the sequences of Litopenaeus vannamei shrimp tropomyosin, arginine kinase (AK), myosin light chain (MLC), and sarcoplasmic calcium-binding protein (SCP). The Wilcoxon test was used to determine significant differences in z scores between patients and control subjects. The median shrimp IgE level was 4-fold higher in children than in adults (47 vs 12.5 kU(A)/L). The frequency of allergen recognition was higher in children (tropomyosin, 81% [94% for children and 61% for adults]; MLC, 57% [70% for children and 31% for adults]; AK, 51% [67% for children and 21% for adults]; and SCP, 45% [59% for children and 21% for adults]), whereas control subjects showed negligible binding. Seven IgE-binding regions were identified in tropomyosin by means of peptide microarray, confirming previously identified shrimp epitopes. In addition, 3 new epitopes were identified in tropomyosin (epitopes 1, 3, and 5b-c), 5 epitopes were identified in MLC, 3 epitopes were identified in SCP, and 7 epitopes were identified in AK. Interestingly, frequency of individual epitope recognition, as well as intensity of IgE binding, was significantly greater in children than in adults for all 4 proteins. Children with shrimp allergy have greater shrimp-specific IgE antibody levels and

  8. Epitope-Specific Suppression of IgG Responses by Passively Administered Specific IgG: Evidence of Epitope Masking.

    PubMed

    Bergström, Joakim J E; Xu, Hui; Heyman, Birgitta

    2017-01-01

    Specific IgG, passively administered together with particulate antigen, can completely prevent induction of antibody responses to this antigen. The ability of IgG to suppress antibody responses to sheep red blood cells (SRBCs) is intact in mice lacking FcγRs, complement factor 1q, C3, or complement receptors 1 and 2, suggesting that Fc-dependent effector functions are not involved. Two of the most widely discussed explanations for the suppressive effect are increased clearance of IgG-antigen complexes and/or that IgG "hides" the antigen from recognition by specific B cells, so-called epitope masking. The majority of data on how IgG induces suppression was obtained through studies of the effects on IgM-secreting single spleen cells during the first week after immunization. Here, we show that IgG also suppresses antigen-specific extrafollicular antibody-secreting cells, germinal center B-cells, long-lived plasma cells, long-term IgG responses, and induction of memory antibody responses. IgG anti-SRBC reduced the amount of SRBC in the spleens of wild-type, but not of FcγR-deficient mice. However, no correlation between suppression and the amount of SRBC in the spleen was observed, suggesting that increased clearance does not explain IgG-mediated suppression. Instead, we found compelling evidence for epitope masking because IgG anti-NP administered with NP-SRBC suppressed the IgG anti-NP, but not the IgG anti-SRBC response. Vice versa, IgG anti-SRBC administered with NP-SRBC, suppressed only the IgG anti-SRBC response. In conclusion, passively transferred IgG suppressed all measured parameters of an antigen-specific antibody/B cell response and an important mechanism of action is likely to be epitope masking.

  9. Identification and characterization of haemagglutinin epitopes of Avibacterium paragallinarum serovar C.

    PubMed

    Noro, Taichi; Oishi, Eiji; Kaneshige, Takahiro; Yaguchi, Kazuhiko; Amimoto, Katsuhiko; Shimizu, Mitsugu

    2008-10-15

    The objectives of this study were to identify haemagglutinin (HA) epitopes of Avibacterium paragallinarum serovar C that are capable of eliciting haemagglutination inhibition (HI) antibody, and to investigate their immunogenic role. Three conformational epitopes were detected on HA by blocking ELISA and immuno-dot blot analysis using a panel of five monoclonal antibodies (MAbs) with HI activity, designated 8C1C, 4G8B, 24E4D, 11E11B, and 10D1A. The minimum DNA regions coding these three epitopes were 3195, 2862, and 807bp in size, and mapped within a gene with 6117bp. Nine DNA fragments of various lengths were prepared, and their recombinant proteins were generated in E. coli. One recombinant protein, designated HPC5.5, was recognized by MAb 8C1C, and had strong ability to adsorb HI antibody to Av. paragallinarum serovar C. Other recombinant proteins designated HPC5.1, HPC4.8, and HPC2.5 did not react with MAb 8C1C and only slightly adsorbed HI antibody. All chickens immunized once with HPC5.5 did not show any typical clinical signs such as nasal discharge or facial edema against challenge inoculation with Av. paragallinarum serovar C. However, HPC5.1, which was recognized by four MAbs (not including MAb 8C1C), showed only partial protective immunity in five of eight immunized chickens. The results suggest that the HA epitope recognized by MAb 8C1C is the major epitope responsible for eliciting HI antibody, and HPC5.5 is a practical candidate protein to develop a new vaccine against avian infectious coryza caused by Av. paragallinarum serovar C.

  10. Analysis of ChimeriVax Japanese Encephalitis Virus envelope for T-cell epitopes and comparison to circulating strain sequences.

    PubMed

    De Groot, Anne S; Martin, William; Moise, Leonard; Guirakhoo, Farshad; Monath, Thomas

    2007-11-19

    T-cell epitope variability is associated with viral immune escape and may influence the outcome of vaccination against the highly variable Japanese Encephalitis Virus (JEV). We computationally analyzed the ChimeriVax-JEV vaccine envelope sequence for T helper epitopes that are conserved in 12 circulating JEV strains and discovered 75% conservation among putative epitopes. Among non-identical epitopes, only minor amino acid changes that would not significantly affect HLA-binding were present. Therefore, in most cases, circulating strain epitopes could be restricted by the same HLA and are likely to stimulate a cross-reactive T-cell response. Based on this analysis, we predict no significant abrogation of ChimeriVax-JEV-conferred protection against circulating JEV strains.

  11. Identification and Localization of Minimal MHC-restricted CD8+ T Cell Epitopes within the Plasmodium falciparum AMA1 Protein

    DTIC Science & Technology

    2010-08-24

    A01/A02 B44/B44 002 A01/A02 B08/B44 005 A01/A02 B08/ B27 008 A02/A03 B27 / B27 012 A01/A03 B44/B58 Low resolution molecular HLA typing permitted...Fourteen 8-10-mer epitopes were predicted to bind to HLA supertypes A01 (3 epitopes), A02 (4 epitopes), B08 (2 epitopes) and B44 (5 epitopes). Nine...of seven HLA alleles. These HLA alleles belong to four HLA supertypes that have a phenotypic frequency between 23% - 100% in different human

  12. Pectic homogalacturonan masks abundant sets of xyloglucan epitopes in plant cell walls.

    PubMed

    Marcus, Susan E; Verhertbruggen, Yves; Hervé, Cécile; Ordaz-Ortiz, José J; Farkas, Vladimir; Pedersen, Henriette L; Willats, William G T; Knox, J Paul

    2008-05-22

    Molecular probes are required to detect cell wall polymers in-situ to aid understanding of their cell biology and several studies have shown that cell wall epitopes have restricted occurrences across sections of plant organs indicating that cell wall structure is highly developmentally regulated. Xyloglucan is the major hemicellulose or cross-linking glycan of the primary cell walls of dicotyledons although little is known of its occurrence or functions in relation to cell development and cell wall microstructure. Using a neoglycoprotein approach, in which a XXXG heptasaccharide of tamarind seed xyloglucan was coupled to BSA to produce an immunogen, we have generated a rat monoclonal antibody (designated LM15) to the XXXG structural motif of xyloglucans. The specificity of LM15 has been confirmed by the analysis of LM15 binding using glycan microarrays and oligosaccharide hapten inhibition of binding studies. The use of LM15 for the analysis of xyloglucan in the cell walls of tamarind and nasturtium seeds, in which xyloglucan occurs as a storage polysaccharide, indicated that the LM15 xyloglucan epitope occurs throughout the thickened cell walls of the tamarind seed and in the outer regions, adjacent to middle lamellae, of the thickened cell walls of the nasturtium seed. Immunofluorescence analysis of LM15 binding to sections of tobacco and pea stem internodes indicated that the xyloglucan epitope was restricted to a few cell types in these organs. Enzymatic removal of pectic homogalacturonan from equivalent sections resulted in the abundant detection of distinct patterns of the LM15 xyloglucan epitope across these organs and a diversity of occurrences in relation to the cell wall microstructure of a range of cell types. These observations support ideas that xyloglucan is associated with pectin in plant cell walls. They also indicate that documented patterns of cell wall epitopes in relation to cell development and cell differentiation may need to be re

  13. Distinct Mechanisms Regulate Exposure of Neutralizing Epitopes in the V2 and V3 Loops of HIV-1 Envelope

    PubMed Central

    Upadhyay, Chitra; Mayr, Luzia M.; Zhang, Jing; Kumar, Rajnish; Gorny, Miroslaw K.; Nádas, Arthur; Zolla-Pazner, Susan

    2014-01-01

    ABSTRACT Broadly neutralizing antibodies targeting the HIV-1 envelope (Env) are key components for protection against HIV-1. However, many cross-reactive epitopes are often occluded. This study investigates the mechanisms contributing to the masking of V2i (variable loop V2 integrin) epitopes compared to the accessibility of V3 epitopes. V2i are conformation-dependent epitopes encompassing the integrin α4β7-binding motif on the V1V2 loop of HIV-1 Env gp120. The V2i monoclonal antibodies (MAbs) display extensive cross-reactivity with gp120 monomers from many subtypes but neutralize only few viruses, indicating V2i's cryptic nature. First, we asked whether CD4-induced Env conformational changes affect V2i epitopes similarly to V3. CD4 treatment of BaL and JRFL pseudoviruses increased their neutralization sensitivity to V3 MAbs but not to the V2i MAbs. Second, the contribution of N-glycans in masking V2i versus V3 epitopes was evaluated by testing the neutralization of pseudoviruses produced in the presence of a glycosidase inhibitor, kifunensine. Viruses grown in kifunensine were more sensitive to neutralization by V3 but not V2i MAbs. Finally, we evaluated the time-dependent dynamics of the V2i and V3 epitopes. Extending the time of virus-MAb interaction to 18 h before adding target cells increased virus neutralization by some V2i MAbs and all V3 MAbs tested. Consistent with this, V2i MAb binding to Env on the surface of transfected cells also increased in a time-dependent manner. Hence, V2i and V3 epitopes are highly dynamic, but distinct factors modulate the antibody accessibility of these epitopes. The study reveals the importance of the structural dynamics of V2i and V3 epitopes in determining HIV-1 neutralization by antibodies targeting these sites. IMPORTANCE Conserved neutralizing epitopes are present in the V1V2 and V3 regions of HIV-1 Env, but these epitopes are often occluded from Abs. This study reveals that distinct mechanisms contribute to the masking

  14. Towards Identifying Protective B-Cell Epitopes: The PspA Story.

    PubMed

    Khan, Naeem; Jan, Arif T

    2017-01-01

    Pneumococcal surface protein A (PspA) is one of the most abundant cell surface protein of Streptococcus pneumoniae ( S. pneumoniae ). PspA variants are structurally and serologically diverse and help evade complement-mediated phagocytosis of S. pneumoniae , which is essential for its survival in the host. PspA is currently been screened for employment in the generation of more effective (serotype independent) vaccine to overcome the limitations of polysaccharide based vaccines, providing serotype specific immune responses. The cross-protection eliciting regions of PspA localize to the α-helical and proline rich regions. Recent data indicate significant variation in the ability of antibodies induced against the recombinant PspA variants to recognize distinct S. pneumoniae strains. Hence, screening for the identification of the topographical repertoire of B-cell epitopes that elicit cross-protective immune response seems essential in the engineering of a superior PspA-based vaccine. Herein, we revisit epitope identification in PspA and the utility of hybridoma technology in directing the identification of protective epitope regions of PspA that can be used in vaccine research.

  15. P. falciparum and P. vivax Epitope-Focused VLPs Elicit Sterile Immunity to Blood Stage Infections.

    PubMed

    Whitacre, David C; Espinosa, Diego A; Peters, Cory J; Jones, Joyce E; Tucker, Amy E; Peterson, Darrell L; Zavala, Fidel P; Milich, David R

    2015-01-01

    In order to design P. falciparum preerythrocytic vaccine candidates, a library of circumsporozoite (CS) T and B cell epitopes displayed on the woodchuck hepatitis virus core antigen (WHcAg) VLP platform was produced. To test the protective efficacy of the WHcAg-CS VLPs, hybrid CS P. berghei/P. falciparum (Pb/Pf) sporozoites were used to challenge immunized mice. VLPs carrying 1 or 2 different CS repeat B cell epitopes and 3 VLPs carrying different CS non-repeat B cell epitopes elicited high levels of anti-insert antibodies (Abs). Whereas, VLPs carrying CS repeat B cell epitopes conferred 98% protection of the liver against a 10,000 Pb/Pf sporozoite challenge, VLPs carrying the CS non-repeat B cell eptiopes were minimally-to-non-protective. One-to-three CS-specific CD4/CD8 T cell sites were also fused to VLPs, which primed CS-specific as well as WHcAg-specific T cells. However, a VLP carrying only the 3 T cell domains failed to protect against a sporozoite challenge, indicating a requirement for anti-CS repeat Abs. A VLP carrying 2 CS repeat B cell epitopes and 3 CS T cell sites in alum adjuvant elicited high titer anti-CS Abs (endpoint dilution titer >1x10(6)) and provided 80-100% protection against blood stage malaria. Using a similar strategy, VLPs were constructed carrying P. vivax CS repeat B cell epitopes (WHc-Pv-78), which elicited high levels of anti-CS Abs and conferred 99% protection of the liver against a 10,000 Pb/Pv sporozoite challenge and elicited sterile immunity to blood stage infection. These results indicate that immunization with epitope-focused VLPs carrying selected B and T cell epitopes from the P. falciparum and P. vivax CS proteins can elicit sterile immunity against blood stage malaria. Hybrid WHcAg-CS VLPs could provide the basis for a bivalent P. falciparum/P. vivax malaria vaccine.

  16. Rooting Out Aberrant Behavior in Training.

    ERIC Educational Resources Information Center

    Kokalis, Jerry, Jr.; Paquin, Dave

    1989-01-01

    Discusses aberrant, or disruptive, behavior in an industrial/business, classroom-based, instructor-led training setting. Three examples of aberrant behavior are described, typical case studies are provided for each, and preventive (long-term) and corrective (on-the-spot) strategies for dealing with the problems are discussed. (LRW)

  17. Ara h 1 CD4+ T cell epitope-based peptides: candidates for a peanut allergy therapeutic.

    PubMed

    Prickett, S R; Voskamp, A L; Phan, T; Dacumos-Hill, A; Mannering, S I; Rolland, J M; O'Hehir, R E

    2013-06-01

    Peanut allergy is a life-threatening condition; there is currently no cure. While whole allergen extracts are used for specific immunotherapy for many allergies, they can cause severe reactions and even fatalities in peanut allergy. To identify short, HLA-degenerate CD4(+) T cell epitope-based peptides of the major peanut allergen Ara h 1 that target allergen-specific T cells without causing IgE-mediated inflammatory cell activation, as candidates for safe peanut-specific immunotherapy. Ara h 1-specific CD4(+) T cell lines (TCL) were generated from peripheral blood mononuclear cells (PBMC) of peanut-allergic subjects using CFSE-based methodology. T cell epitopes were identified using CFSE and thymidine-based proliferation assays. Epitope HLA-restriction was investigated using blocking antibodies, HLA-genotyping and epitope prediction algorithms. Functional peanut-specific IgE reactivity to peptides was assessed by basophil activation assay. A total of 145 Ara h 1-specific TCL were generated from 18 HLA-diverse peanut-allergic subjects. The TCL recognized 20-mer peptides throughout Ara h 1. Nine 20-mers containing the most frequently recognized epitopes were selected and their recognition confirmed in 18 additional peanut-allergic subjects. Ten core epitopes were mapped within these 20-mers. These were HLA-DQ and/or HLA-DR restricted, with each presented on at least two different HLA-molecules. Seven short (≤ 20 aa) non-basophil-reactive peptides encompassing all core epitopes were designed and validated in peanut-allergic donor PBMC T cell assays. Short CD4(+) T cell epitope-based Ara h 1 peptides were identified as novel candidates for a safe, T cell targeted peanut-specific immunotherapy for HLA-diverse populations. © 2013 John Wiley & Sons Ltd.

  18. Knowing prior methicillin-resistant Staphylococcus aureus (MRSA) infection or colonization status increases the empirical use of glycopeptides in MRSA bacteraemia and may decrease mortality.

    PubMed

    Robinson, J O; Phillips, M; Christiansen, K J; Pearson, J C; Coombs, G W; Murray, R J

    2014-06-01

    To compare the management and outcome of methicillin-resistant Staphylococcus aureus (MRSA) bacteraemia in patients known to be MRSA-colonized/infected (C-patients) with the management and outcome in those not known to be colonized/infected (NC-patients), we conducted a 10-year retrospective review of MRSA bacteraemia in an adult tertiary hospital. Clinical data were obtained by chart review, and mortality data from linked databases. Prior MRSA colonization/infection status was available to treating clinicians at the time of the bacteraemia as a 'Micro-Alert' tag on the patient's labels, in medical charts, and in electronic information systems. C-patients accounted for 35.4% of all MRSA bacteraemia episodes. C-patients were more likely to be indigenous, to be diabetic, or to have a history of previous S. aureus infection. Markers of illness severity (Simplified Acute Physiology Score (SAPS)-II, need for admission to the intensive-care unit, length of stay, and metastatic seeding) were similar in both groups. Empirical therapy included a glycopeptide in 49.3% of C-patients vs. 18.9% of NC-patients (p <0.01), and contained an antibiotic to which the MRSA isolate tested susceptible in vitro in 56.7% of C-patients vs. 45.1% of NC-patients (p 0.13). All-cause 7-day and 30-day mortality were 7.5% vs. 18.9% (p 0.04), and 22.4% vs. 31.1% (p 0.20), in the C-patient and NC-patient groups, respectively. Knowing MRSA colonization status was significantly associated with lower 30-day mortality in Cox regression analysis (p <0.01). These data suggest that mortality from MRSA bacteraemia is lower in C-patients, which may reflect the earlier use of glycopeptides. The low use of empirical glycopeptides in septic patients known to be previously MRSA-colonized/infected may represent a missed opportunity for infection control to positively impact on clinical management. © 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and

  19. Thyrotropin receptor autoantibodies (TSHRAbs): epitopes, origins and clinical significance.

    PubMed

    Kohn, Leonard D; Harii, Norikazu

    2003-01-01

    Epitopes for > 95% stimulating thyrotropin receptor autoantibodies (TSHRAbs) causally implicated in Graves' disease (Basedow's disease or primary hyperthyroidism) have been identified on on the N-terminal portion of the TSHR extracellular domain, residues 8-165. If the stimulating TSHRAb activity is solely dependent on this region, it is termed homogeneous; if its activity is only largely related to this region, it is termed heterogeneous. The presence of a heterogeneous stimulating TSHRAb in a patient is associated with rapid responses to propylthiouracil or methimazole and may be predictive of long term remission with these oral immunosuppressives. Epitopes for two different Graves' autoantibodies that inhibit TSH binding, TSH binding inhibition immunoglobulins or TBIIs, have also been identified on this region of the TSHR. They do not increase cAMP levels, although one may activate the inositol phosphate, Ca++, arachidonate release signal system. The epitope of blocking TSHRAbs with the ability to inhibit TSH binding (TBII activity), TSH activity, and stimulating TSHRAb activity, and that are causally implicated in the primary hypothyroidism of patients with idiopathic myxedema or some patients with Hashimoto's disease have, in contrast, been largely identified largely on the C-terminal portion of the TSHR extracellular domain, residues 270-395. They have been implicated as important in pregnancy where they attenuate the signs and symptoms of Graves' hyperthyroidism. The appearance of these blocking TSHRAbs during pregnancy in Graves' patients might cause overt or occult hypothyroidism, with resultant effects on fetal development and postnatal intelligence levels. The different TSHRAbs can exist in the same patient at any moment in time, potentially making disease expression a sum of their activities. Assays taking advantage of the epitope mapping findings enable us to detect individual TSHRAbs within a single patient and to better understand their clinical

  20. Group A Streptococcal vaccine candidate: contribution of epitope to size, antigen presenting cell interaction and immunogenicity.

    PubMed

    Zaman, Mehfuz; Chandrudu, Saranya; Giddam, Ashwini K; Reiman, Jennifer; Skwarczynski, Mariusz; McPhun, Virginia; Moyle, Peter M; Batzloff, Michael R; Good, Michael F; Toth, Istvan

    2014-12-01

    Utilize lipopeptide vaccine delivery system to develop a vaccine candidate against Group A Streptococcus. Lipopeptides synthesized by solid-phase peptide synthesis-bearing carboxyl (C)-terminal and amino (N)-terminal Group A Streptococcus peptide epitopes. Nanoparticles formed were evaluated in vivo. Immune responses were induced in mice without additional adjuvant. We demonstrated for the first time that incorporation of the C-terminal epitope significantly enhanced the N-terminal epitope-specific antibody response and correlated with forming smaller nanoparticles. Antigen-presenting cells had increased uptake and maturation by smaller, more immunogenic nanoparticles. Antibodies raised by vaccination recognized isolates. Demonstrated the lipopeptidic nanoparticles to induce an immune response which can be influenced by the combined effect of epitope choice and size.

  1. Clinical Control of HIV-1 by Cytotoxic T Cells Specific for Multiple Conserved Epitopes.

    PubMed

    Murakoshi, Hayato; Akahoshi, Tomohiro; Koyanagi, Madoka; Chikata, Takayuki; Naruto, Takuya; Maruyama, Rie; Tamura, Yoshiko; Ishizuka, Naoki; Gatanaga, Hiroyuki; Oka, Shinichi; Takiguchi, Masafumi

    2015-05-01

    Identification and characterization of CD8(+) T cells effectively controlling HIV-1 variants are necessary for the development of AIDS vaccines and for studies of AIDS pathogenesis, although such CD8(+) T cells have been only partially identified. In this study, we sought to identify CD8(+) T cells controlling HIV-1 variants in 401 Japanese individuals chronically infected with HIV-1 subtype B, in which protective alleles HLA-B*57 and HLA-B*27 are very rare, by using comprehensive and exhaustive methods. We identified 13 epitope-specific CD8(+) T cells controlling HIV-1 in Japanese individuals, though 9 of these epitopes were not previously reported. The breadths of the T cell responses to the 13 epitopes were inversely associated with plasma viral load (P = 2.2 × 10(-11)) and positively associated with CD4 count (P = 1.2 × 10(-11)), indicating strong synergistic effects of these T cells on HIV-1 control in vivo. Nine of these epitopes were conserved among HIV-1 subtype B-infected individuals, whereas three out of four nonconserved epitopes were cross-recognized by the specific T cells. These findings indicate that these 12 epitopes are strong candidates for antigens for an AIDS vaccine. The present study highlighted a strategy to identify CD8(+) T cells controlling HIV-1 and demonstrated effective control of HIV-1 by those specific for 12 conserved or cross-reactive epitopes. HLA-B*27-restricted and HLA-B*57-restricted cytotoxic T lymphocytes (CTLs) play a key role in controlling HIV-1 in Caucasians and Africans, whereas it is unclear which CTLs control HIV-1 in Asian countries, where HLA-B*57 and HLA-B*27 are very rare. A recent study showed that HLA-B*67:01 and HLA-B*52:01-C*12:02 haplotypes were protective alleles in Japanese individuals, but it is unknown whether CTLs restricted by these alleles control HIV-1. In this study, we identified 13 CTLs controlling HIV-1 in Japan by using comprehensive and exhaustive methods. They included 5 HLA-B*52:01-restricted

  2. Clinical Control of HIV-1 by Cytotoxic T Cells Specific for Multiple Conserved Epitopes

    PubMed Central

    Murakoshi, Hayato; Akahoshi, Tomohiro; Koyanagi, Madoka; Chikata, Takayuki; Naruto, Takuya; Maruyama, Rie; Tamura, Yoshiko; Ishizuka, Naoki; Gatanaga, Hiroyuki; Oka, Shinichi

    2015-01-01

    ABSTRACT Identification and characterization of CD8+ T cells effectively controlling HIV-1 variants are necessary for the development of AIDS vaccines and for studies of AIDS pathogenesis, although such CD8+ T cells have been only partially identified. In this study, we sought to identify CD8+ T cells controlling HIV-1 variants in 401 Japanese individuals chronically infected with HIV-1 subtype B, in which protective alleles HLA-B*57 and HLA-B*27 are very rare, by using comprehensive and exhaustive methods. We identified 13 epitope-specific CD8+ T cells controlling HIV-1 in Japanese individuals, though 9 of these epitopes were not previously reported. The breadths of the T cell responses to the 13 epitopes were inversely associated with plasma viral load (P = 2.2 × 10−11) and positively associated with CD4 count (P = 1.2 × 10−11), indicating strong synergistic effects of these T cells on HIV-1 control in vivo. Nine of these epitopes were conserved among HIV-1 subtype B-infected individuals, whereas three out of four nonconserved epitopes were cross-recognized by the specific T cells. These findings indicate that these 12 epitopes are strong candidates for antigens for an AIDS vaccine. The present study highlighted a strategy to identify CD8+ T cells controlling HIV-1 and demonstrated effective control of HIV-1 by those specific for 12 conserved or cross-reactive epitopes. IMPORTANCE HLA-B*27-restricted and HLA-B*57-restricted cytotoxic T lymphocytes (CTLs) play a key role in controlling HIV-1 in Caucasians and Africans, whereas it is unclear which CTLs control HIV-1 in Asian countries, where HLA-B*57 and HLA-B*27 are very rare. A recent study showed that HLA-B*67:01 and HLA-B*52:01-C*12:02 haplotypes were protective alleles in Japanese individuals, but it is unknown whether CTLs restricted by these alleles control HIV-1. In this study, we identified 13 CTLs controlling HIV-1 in Japan by using comprehensive and exhaustive methods. They included 5 HLA-B*52

  3. Epitope mapping of anti-interleukin-13 neutralizing antibody CNTO607.

    PubMed

    Teplyakov, Alexey; Obmolova, Galina; Wu, Sheng-Jiun; Luo, Jinquan; Kang, James; O'Neil, Karyn; Gilliland, Gary L

    2009-05-29

    CNTO607 is a neutralizing anti-interleukin-13 (IL-13) human monoclonal antibody obtained from a phage display library. To determine how this antibody inhibits the biological effect of IL-13, we determined the binding epitope by X-ray crystallography. The crystal structure of the complex between CNTO607 Fab and IL-13 reveals the antibody epitope at the surface formed by helices A and D of IL-13. This epitope overlaps with the IL-4Ralpha/IL-13Ralpha1 receptor-binding site, which explains the neutralizing effect of CNTO607. The extensive antibody interface covers an area of 1000 A(2), which is consistent with the high binding affinity. The key features of the interface are the charge and shape complementarity of the molecules that include two hydrophobic pockets on IL-13 that accommodate Phe32 [complementarity-determining region (CDR) L2] and Trp100a (CDR H3) and a number of salt bridges between basic residues of IL-13 and acidic residues of the antibody. Comparison with the structure of the free Fab shows that the CDR residues do not change their conformation upon complex formation, with the exception of two residues in CDR H3, Trp100a and Asp100b, which change rotamer conformations. To evaluate the relative contribution of the epitope residues to CNTO607 binding, we performed alanine-scanning mutagenesis of the A-D region of IL-13. This study confirmed the primary role of electrostatic interactions for antigen recognition.

  4. Third-rank chromatic aberrations of electron lenses.

    PubMed

    Liu, Zhixiong

    2018-02-01

    In this paper the third-rank chromatic aberration coefficients of round electron lenses are analytically derived and numerically calculated by Mathematica. Furthermore, the numerical results are cross-checked by the differential algebraic (DA) method, which verifies that all the formulas for the third-rank chromatic aberration coefficients are completely correct. It is hoped that this work would be helpful for further chromatic aberration correction in electron microscopy. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Bioinformatics and immunologic investigation on B and T cell epitopes of Cur l 3, a major allergen of Curvularia lunata.

    PubMed

    Sharma, Vidhu; Singh, Bhanu P; Gaur, Shailendra N; Pasha, Santosh; Arora, Naveen

    2009-06-01

    The knowledge on epitopes of proteins can help in devising new therapeutic modalities for allergic disorders. In the present study, five B (P1-P5) and five T cell (P6-P10) epitopes were predicted in silico based on sequence homology model of Cur l 3, a major allergen of Curvularia lunata. Peptides (epitopes) were synthesized and assessed for biological activity by ELISA, competitive ELISA, lymphoproliferation and cytokine profiling using Curvularia allergic patients' sera. B cell peptides showed higher IgE binding by ELISA than T cell epitopes except P6. Peptides P1-P6 achieved EC(50) at 100 ng, whereas P7-P10 required 10 mug in inhibition assays. Peripheral blood mononuclear cells from Curvularia allergic patients (n = 20) showed higher lymphoproliferation for T cell epitopes than B cell epitopes except P6 confirming the properties of B and T cell prediction. The supernatant from these patients show highest interleukin-4 release on stimulation with P6 followed by B cell peptides. P4 and P6 together identified 35/37 of Curvularia positive patients by skin tests. In summary, experimental analysis confirmed in silico predicted epitopes containing important antigenic regions of Cur l 3. P6, a predicted T cell epitope, showed the presence of a cryptic B cell epitope. Peptides P4 and P6 have potential for clinical application. The approach used here is relevant and may be used to delineate epitopes of other proteins.

  6. The Distribution of Chromosomal Aberrations in Human Cells Predicted by a Generalized Time-Dependent Model of Radiation-Induced Formation of Aberrations

    NASA Technical Reports Server (NTRS)

    Ponomarev, Artem L.; George, K.; Cucinotta, F. A.

    2011-01-01

    New experimental data show how chromosomal aberrations for low- and high-LET radiation are dependent on DSB repair deficiencies in wild-type, AT and NBS cells. We simulated the development of chromosomal aberrations in these cells lines in a stochastic track-structure-dependent model, in which different cells have different kinetics of DSB repair. We updated a previously formulated model of chromosomal aberrations, which was based on a stochastic Monte Carlo approach, to consider the time-dependence of DSB rejoining. The previous version of the model had an assumption that all DSBs would rejoin, and therefore we called it a time-independent model. The chromosomal-aberrations model takes into account the DNA and track structure for low- and high-LET radiations, and provides an explanation and prediction of the statistics of rare and more complex aberrations. We compared the program-simulated kinetics of DSB rejoining to the experimentally-derived bimodal exponential curves of the DSB kinetics. We scored the formation of translocations, dicentrics, acentric and centric rings, deletions, and inversions. The fraction of DSBs participating in aberrations was studied in relation to the rejoining time. Comparisons of simulated dose dependence for simple aberrations to the experimental dose-dependence for HF19, AT and NBS cells will be made.

  7. Determination of critical epitope of PcMab-47 against human podocalyxin.

    PubMed

    Itai, Shunsuke; Yamada, Shinji; Kaneko, Mika K; Kato, Yukinari

    2018-07-01

    Podocalyxin (PODXL) is a type I transmembrane protein, which is highly glycosylated. PODXL is expressed in some types of human cancer tissues including oral, breast, and lung cancer tissues and may promote tumor growth, invasion, and metastasis. We previously produced PcMab-47, a novel anti-PODXL monoclonal antibody (mAb) which reacts with endogenous PODXL-expressing cancer cell lines and normal cells independently of glycosylation in Western blot, flow cytometry, and immunohistochemical analysis. In this study, we used enzyme-linked immunosorbent assay (ELISA), flow cytometry, and immunohistochemical analysis to determine the epitope of PcMab-47. The minimum epitope of PcMab-47 was found to be Asp207, His208, Leu209, and Met210. A blocking peptide containing this minimum epitope completely neutralized PcMab-47 reaction against oral cancer cells by flow cytometry and immunohistochemical analysis. These findings could lead to the production of more functional anti-PODXL mAbs, which are advantageous for antitumor activities.

  8. Efficient chemo-enzymatic gluten detoxification: reducing toxic epitopes for celiac patients improving functional properties

    PubMed Central

    Ribeiro, Miguel; Nunes, Fernando M.; Guedes, Sofia; Domingues, Pedro; Silva, Amélia M.; Carrillo, Jose Maria; Rodriguez-Quijano, Marta; Branlard, Gérard; Igrejas, Gilberto

    2015-01-01

    Protein engineering of gluten, the exogenous effector in celiac disease, seeking its detoxification by selective chemical modification of toxic epitopes is a very attractive strategy and promising technology when compared to pharmacological treatment or genetic engineering of wheat. Here we present a simple and efficient chemo-enzymatic methodology that decreases celiac disease toxic epitopes of gluten proteins improving its technological value through microbial transglutaminase-mediated transamidation of glutamine with n-butylamine under reducing conditions. First, we found that using low concentrations of amine-nucleophile under non-reducing conditions, the decrease in toxic epitopes is mainly due to transglutaminase-mediated cross-linking. Second, using high amine nucleophile concentrations protein cross-linking is substantially reduced. Third, reducing conditions increase 7-fold the transamidation reaction further decreasing toxic epitopes amount. Fourth, using n-butylamine improves gluten hydrophobicity that strengthens the gluten network. These results open the possibility of tailoring gluten for producing hypoallergenic flours while still taking advantage of the unique viscoelastic properties of gluten. PMID:26691232

  9. Phage display peptide libraries in molecular allergology: from epitope mapping to mimotope-based immunotherapy.

    PubMed

    Luzar, J; Štrukelj, B; Lunder, M

    2016-11-01

    Identification of allergen epitopes is a key component in proper understanding of the pathogenesis of type I allergies, for understanding cross-reactivity and for the development of mimotope immunotherapeutics. Phage particles have garnered recognition in the field of molecular allergology due to their value not only in competitive immunoscreening of peptide libraries but also as immunogenic carriers of allergen mimotopes. They integrate epitope discovery technology and immunization functions into a single platform. This article provides an overview of allergen mimotopes identified through the phage display technique. We discuss the contribution of phage display peptide libraries in determining dominant B-cell epitopes of allergens, in developing mimotope immunotherapy, in understanding cross-reactivity, and in determining IgE epitope profiles of individual patients to improve diagnostics and individualize immunotherapy. We also discuss the advantages and pitfalls of the methodology used to identify and validate the mimotopes. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  10. A Synthetic Glycan Microarray Enables Epitope Mapping of Plant Cell Wall Glycan-Directed Antibodies.

    PubMed

    Ruprecht, Colin; Bartetzko, Max P; Senf, Deborah; Dallabernadina, Pietro; Boos, Irene; Andersen, Mathias C F; Kotake, Toshihisa; Knox, J Paul; Hahn, Michael G; Clausen, Mads H; Pfrengle, Fabian

    2017-11-01

    In the last three decades, more than 200 monoclonal antibodies have been raised against most classes of plant cell wall polysaccharides by different laboratories worldwide. These antibodies are widely used to identify differences in plant cell wall components in mutants, organ and tissue types, and developmental stages. Despite their importance and broad use, the precise binding epitope has been determined for only a few of these antibodies. Here, we use a plant glycan microarray equipped with 88 synthetic oligosaccharides to comprehensively map the epitopes of plant cell wall glycan-directed antibodies. Our results reveal the binding epitopes for 78 arabinogalactan-, rhamnogalacturonan-, xylan-, and xyloglucan-directed antibodies. We demonstrate that, with knowledge of the exact epitopes recognized by individual antibodies, specific glycosyl hydrolases can be implemented into immunological cell wall analyses, providing a framework to obtain structural information on plant cell wall glycans with unprecedented molecular precision. © 2017 American Society of Plant Biologists. All Rights Reserved.

  11. Harmonic oscillator states in aberration optics

    NASA Technical Reports Server (NTRS)

    Wolf, Kurt Bernardo

    1993-01-01

    The states of the three-dimensional quantum harmonic oscillator classify optical aberrations of axis-symmetric systems due to the isomorphism between the two mathematical structures. Cartesian quanta and angular momentum classifications have their corresponding aberration classifications. The operation of concatenation of optical elements introduces a new operation between harmonic oscillator states.

  12. Recombinant Mip-PilE-FlaA dominant epitopes vaccine candidate against Legionella pneumophila.

    PubMed

    He, Jinlei; Huang, Fan; Chen, Han; Chen, Qiwei; Zhang, Junrong; Li, Jiao; Chen, Dali; Chen, Jianping

    2017-06-01

    Legionella pneumophila is the main causative agent of Legionnaires' disease, which is a severe multi-system disease with pneumonia as the primary manifestation. We designed a recombinant Mip-PilE-FlaA dominant epitopes vaccine against Legionella pneumophila to prevent the disease and evaluated its immunogenicity and protective immunity. The protein structures of Mip, PilE and FlaA were analyzed using a computer, and the gene sequences of the dominant epitopes of the three proteins were selected to construct and optimize the vaccine. The optimized mip, pilE, flaA and recombinant mip-pilE-flaA gene sequences were cloned, expressed and purified. The purified proteins were used as dominant epitopes vaccines to immunize BALB/c mice and determine the protective immunity and immunogenicity of these purified proteins. The identification confirmed that the recombinant mip-pilE-flaA was successfully cloned and expressed. ELISA revealed that the Mip-PilE-FlaA group produced the highest IgG response, and this protein may considerably improve the production of some cytokines in BALB/c mice. Histopathology analyses of lungs from mice immunized with Mip-PilE-FlaA revealed a certain protective effect. Our work demonstrated that the recombinant dominant epitopes of Mip-PilE-FlaA exhibited strong immunogenicity and immune protection, and this protein may be an efficient epitopes vaccine candidate against Legionella pneumophila. Copyright © 2017 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.

  13. Optimal selection of epitopes for TXP-immunoaffinity mass spectrometry.

    PubMed

    Planatscher, Hannes; Supper, Jochen; Poetz, Oliver; Stoll, Dieter; Joos, Thomas; Templin, Markus F; Zell, Andreas

    2010-06-25

    Mass spectrometry (MS) based protein profiling has become one of the key technologies in biomedical research and biomarker discovery. One bottleneck in MS-based protein analysis is sample preparation and an efficient fractionation step to reduce the complexity of the biological samples, which are too complex to be analyzed directly with MS. Sample preparation strategies that reduce the complexity of tryptic digests by using immunoaffinity based methods have shown to lead to a substantial increase in throughput and sensitivity in the proteomic mass spectrometry approach. The limitation of using such immunoaffinity-based approaches is the availability of the appropriate peptide specific capture antibodies. Recent developments in these approaches, where subsets of peptides with short identical terminal sequences can be enriched using antibodies directed against short terminal epitopes, promise a significant gain in efficiency. We show that the minimal set of terminal epitopes for the coverage of a target protein list can be found by the formulation as a set cover problem, preceded by a filtering pipeline for the exclusion of peptides and target epitopes with undesirable properties. For small datasets (a few hundred proteins) it is possible to solve the problem to optimality with moderate computational effort using commercial or free solvers. Larger datasets, like full proteomes require the use of heuristics.

  14. Monoclonal antibodies to molluskan hemocyanin from Concholepas concholepas demonstrate common and specific epitopes among subunits.

    PubMed

    Oliva, Harold; Moltedo, Bruno; De Ioannes, Pablo; Faunes, Fernando; De Ioannes, Alfredo E; Becker, María Inés

    2002-10-01

    We studied the reactivity of mouse monoclonal antibodies (MAbs) against the hemocyanin from the Chilean marine gastropod Concholepas concholepas (CCH). This protein has been successfully used as a carrier to produce antibodies to haptens and peptides. All MAbs (13) belonging to IgG subclass exhibit dissociation constants (K(d)) from 1 x 10(-7) M to 1 x 10(-9) M. MAbs were characterized by enzyme-linked immunosorbant assay (ELISA) using CCH treated with different procedures, including dissociation into CCH-A and CCH-B subunits, Western blot, enzymatic digestion, chemical deglycosylation, and thermal denaturation. MAbs were classified into three categories, according to subunit specificity by ELISA. The epitope distribution shows that CCH subunits display common epitopes (group I, 5 MAbs, 1H5, 2A8, 3A5, 3B3, and 3E3), as well as specific epitopes for CCH-A subunits (group II, 3 MAbs, 1B8, 4D8, and 8E5) and for CCH-B subunits (group III, 5 MAbs, 1A4, 1E4, 2H10, 3B7, and 7B4). The results can be summarized as follows: (1). six antibodies react with thermal denatured CCH, suggesting that they recognize linear epitopes, whereas seven recognize conformational epitopes; (2). oxidation of carbohydrate moieties does not affect the binding of the MAbs; (3). enzymatic digestion of CCH decreases the reactivity of all antibodies irrespective of the protease used (elastase or trypsin); (4). bringing together the above data, in addition to epitopic complementarity analysis, we identified 12 different epitopes on the CCH molecule recognized by these MAbs. The anti-CCH MAbs presented here can be useful tools to understand the subunit organization of the CCH and its complex structure, which can explain its immunogenic and immunostimulating properties in mammals.

  15. High-performance liquid chromatographic enantioseparation of 2-aminomono- and dihydroxycyclopentanecarboxylic and 2-aminodihydroxycyclohexanecarboxylic acids on macrocyclic glycopeptide-based phases.

    PubMed

    Berkecz, Róbert; Ilisz, István; Benedek, Gabriella; Fülöp, Ferenc; Armstrong, Daniel W; Péter, Antal

    2009-02-06

    The direct separation of the enantiomers of four 2-aminomono- or dihydroxycyclopentanecarboxylic acids and four 2-aminodihydroxycyclohexanecarboxylic acids was performed on chiral stationary phases containing macrocyclic glycopeptide antibiotics such as teicoplanin (Astec Chirobiotic T and T2), teicoplanin aglycone (Chirobiotic TAG) or ristocetin A (Chirobiotic R) as chiral selectors. The effects of the nature of organic modifiers, the pH, the mobile phase composition and the structures of the analytes on the separation were investigated. Chirobiotic TAG, and in some cases Chirobiotic T, proved to be the most useful of these columns. The elution sequence was determined in most cases.

  16. Molecular fingerprinting of complex grass allergoids: size assessments reveal new insights in epitope repertoires and functional capacities.

    PubMed

    Starchenka, S; Bell, A J; Mwange, J; Skinner, M A; Heath, M D

    2017-01-01

    Subcutaneous allergen immunotherapy (SCIT) is a well-documented treatment for allergic disease which involves injections of native allergen or modified (allergoid) extracts. The use of allergoid vaccines is a growing sector of the allergy immunotherapy market, associated with shorter-course therapy. The aim of this study was the structural and immunological characterisation of group 1 (Lol p 1) IgG-binding epitopes within a complex mix grass allergoid formulation containing rye grass. HP-SEC was used to resolve a mix grass allergoid preparation of high molecular weight into several distinct fractions with defined molecular weight and elution profiles. Allergen verification of the HP-SEC allergoid fractions was confirmed by mass spectrometry analysis. IgE and IgG immunoreactivity of the allergoid preparations was explored and Lol p 1 specific IgG-binding epitopes mapped by SPOT synthesis technology (PepSpot™) with structural analysis based on a Lol p 1 homology model. Grass specific IgE reactivity of the mix grass modified extract (allergoid) was diminished in comparison with the mix grass native extract. A difference in IgG profiles was observed between an intact mix grass allergoid preparation and HP-SEC allergoid fractions, which indicated enhancement of accessible reactive IgG epitopes across size distribution profiles of the mix grass allergoid formulation. Detailed analysis of the epitope specificity showed retention of six Lol p 1 IgG-binding epitopes in the mix grass modified extract. The structural and immunological changes which take place following the grass allergen modification process was further unravelled revealing distinct IgG immunological profiles. All epitopes were mapped on the solvent exposed area of Lol p 1 homology model accessible for IgG binding. One of the epitopes was identified as an 'immunodominant' Lol p 1 IgG-binding epitope (62-IFKDGRGCGSCFEIK-76) and classified as a novel epitope. The results from this study support the concept

  17. Identification of proteins with the CDw75 epitope in human colorectal cancer

    PubMed Central

    Mariño-Crespo, Óscar; Fernández-Briera, Almudena; Gil-Martín, Emilio

    2018-01-01

    The CDw75 epitope is an α(2,6) sialylated antigen overexpressed in colorectal cancer (CRC), where its expression correlates with the progression of the disease. The CDw75 epitope is located mainly in N-glycoproteins, whose identity remains unknown. The aim of the present study was to identify proteins with the CDw75 epitope as a strategy to deepen the understanding of molecular pathogenesis of CRC and to identify novel biomarkers for this disease. For this purpose, a two-dimensional electrophoresis approach was employed. Protein spots in the gels were matched to the corresponding CDw75 positive spots in the immunoblotted polyvinylidene difluoride membranes, and further identification of the protein species was performed by mass spectrometry. Additionally, one-dimensional western blotting experiments were performed to verify the expression of these candidate proteins in the colorectal tissue and their coincidence in molecular mass with the CDw75-positive bands. The findings of the present study indicate that haptoglobin and the keratins 8 (K8) and 18 (K18) are proteins with the CDw75 epitope in the colorectal tissue from CRC patients and also suggest novel functions and cellular locations for these proteins in the colorectal tissue and in relation to CRC. PMID:29391890

  18. Viral evolution in HLA-B27-restricted CTL epitopes in human immunodeficiency virus type 1-infected individuals.

    PubMed

    Setiawan, Laurentia C; Gijsbers, Esther F; van Nuenen, Adrianus C; Kootstra, Neeltje A

    2015-08-01

    The HLA-B27 allele is over-represented among human immunodeficiency virus type 1-infected long-term non-progressors. In these patients, strong CTL responses targeting HLA-B27-restricted viral epitopes have been associated with long-term asymptomatic survival. Indeed, loss of control of viraemia in HLA-B27 patients has been associated with CTL escape at position 264 in the immunodominant KK10 epitope. This CTL escape mutation in the viral Gag protein has been associated with severe viral attenuation and may require the presence of compensatory mutations before emerging. Here, we studied sequence evolution within HLA-B27-restricted CTL epitopes in the viral Gag protein during the course of infection of seven HLA-B27-positive patients. Longitudinal gag sequences obtained at different time points around the time of AIDS diagnosis were obtained and analysed for the presence of mutations in epitopes restricted by HLA-B27, and for potential compensatory mutations. Sequence variations were observed in the HLA-B27-restricted CTL epitopes IK9 and DR11, and the immunodominant KK10 epitope. However, the presence of sequence variations in the HLA-B27-restricted CTL epitopes could not be associated with an increase in viraemia in the majority of the patients studied. Furthermore, we observed low genetic diversity in the gag region of the viral variants throughout the course of infection, which is indicative of low viral replication and corresponds to the low viral load observed in the HLA-B27-positive patients. These data indicated that control of viral replication can be maintained in HLA-B27-positive patients despite the emergence of viral mutations in HLA-B27-restricted epitopes.

  19. Distortion of ultrashort pulses caused by aberrations

    NASA Astrophysics Data System (ADS)

    Horváth, Z. L.; Kovács, A. P.; Bor, Zs.

    The effect of the primary wave aberrations (spherical aberration, astigmatism and coma) on ultrashort pulses is studied by the Nijboer-Zernike theory. The results of the geometrical and the wave optical treatments are compared.

  20. Aberration-free intraocular lenses - What does this really mean?

    PubMed

    Langenbucher, Achim; Schröder, Simon; Cayless, Alan; Eppig, Timo

    2017-09-01

    So-called aberration-free intraocular lenses (IOLs) are well established in modern cataract surgery. Usually, they are designed to perfectly refract a collimated light beam onto the focal point. We show how much aberration can be expected with such an IOL in a convergent light beam such as that found anterior to the human cornea. Additionally, the aberration in a collimated beam is estimated for an IOL that has no aberrations in the convergent beam. The convergent beam is modelled as the pencil of rays corresponding to the spherical wavefront resulting from a typical corneal power of 43m -1 . The IOLs are modelled as infinitely thin phase plates with 20m -1 optical power placed 5mm behind the cornea. Their aberrations are reported in terms of optical path length difference and longitudinal spherical aberration (LSA) of the marginal rays, as well as nominal spherical aberration (SA) calculated based on a Zernike representation of the wavefront-error at the corneal plane within a 6mm aperture. The IOL designed to have no aberrations in a collimated light beam has an optical path length difference of -1.8μm, and LSA of 0.15m -1 in the convergent beam of a typical eye. The corresponding nominal SA is 0.065μm. The IOL designed to have no aberrations in a convergent light beam has an optical path length difference of 1.8μm, and LSA of -0.15m -1 in the collimated beam. An IOL designed to have no aberrations in a collimated light beam will increase the SA of a patient's eye after implantation. Copyright © 2017. Published by Elsevier GmbH.

  1. [The preliminary analysis of the recognition epitopes of anti-HEV monoclonal antibodies on HEV ORF2].

    PubMed

    Xiong, Jun-Hui; Guo, Qing-Shun; Ge, Sheng-Xiang; Gu, Ying; Chen, Yi-Xin; Miao, Ji; Du, Hai-Lian; Shi, Wei-Guo; Zhang, Jun; Xia, Ning-Shao

    2008-06-01

    Western blot, capture-PCR, blocking ELISA and synthetic polypeptides were used to systematically study the recognition epitopes on HEV ORF2 of 23 anti-HEV monoclonal antibodies(McAbs) which were previously generated in our laboratory directed against HEV ORF2. Results showed that seven McAbs recognized linear epitopes that located at aa408-458 of HEV ORF2 and 16 conformation-dependent McAbs, most of which recognized the surface epitopes of native HEV, located at aa459-606 of HEV ORF2. The systematical study of the recognition epitopes of anti-HEV McAbs on HEV ORF2 provides important information for the investigation of virus receptor and HEV infection mechanism, as well as its vaccine and diagnostics development.

  2. Highly specific enrichment of N-glycoproteome through a nonreductive amination reaction using Fe3O4@SiO2-aniline nanoparticles.

    PubMed

    Zhang, Ying; Yu, Meng; Zhang, Cheng; Wang, Yali; Di, Yi; Wang, Changchun; Lu, Haojie

    2015-04-07

    A novel method based on the conjunction of aldehydes from oxidized glycopeptides to aniline groups on magnetic nanoparticles via nonreductive amination is reported for the highly selective enrichment of N-glycopeptides. For the first time, a nonreductive amination reaction has been introduced into N-glycoproteome extraction, and correspondingly a new type of aniline-functionalized nanoparticle has been designed and synthesized.

  3. Human umbilical cord-derived mesenchymal stem cells protect from hyperoxic lung injury by ameliorating aberrant elastin remodeling in the lung of O2-exposed newborn rat.

    PubMed

    Hou, Chen; Peng, Danyi; Gao, Li; Tian, Daiyin; Dai, Jihong; Luo, Zhengxiu; Liu, Enmei; Chen, Hong; Zou, Lin; Fu, Zhou

    2018-01-08

    The incidence and mortality rates of bronchopulmonary dysplasia (BPD) remain very high. Therefore, novel therapies are imminently needed to improve the outcome of this disease. Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) show promising therapeutic effects on oxygen-induced model of BPD. In our experiment, UC-MSCs were intratracheally delivered into the newborn rats exposed to hyperoxia, a well-established BPD model. This study demonstrated that UC-MSCs reduce elastin expression stimulated by 90% O 2 in human lung fibroblasts-a (HLF-a), and inhibit HLF-a transdifferentiation into myofibroblasts. In addition, the therapeutic effects of UC-MSCs in neonatal rats with BPD, UC-MSCs could inhibit lung elastase activity and reduce aberrant elastin expression and deposition in the lung of BPD rats. Overall, this study suggested that UC-MSCs could ameliorate aberrant elastin expression in the lung of hyperoxia-induced BPD model which may be associated with suppressing increased TGFβ1 activation. Copyright © 2017. Published by Elsevier Inc.

  4. Effect of aberrations in human eye on contrast sensitivity function

    NASA Astrophysics Data System (ADS)

    Quan, Wei; Wang, Feng-lin; Wang, Zhao-qi

    2011-06-01

    The quantitative analysis of the effect of aberrations in human eye on vision has important clinical value in the correction of aberrations. The wave-front aberrations of human eyes were measured with the Hartmann-Shack wave-front sensor and modulation transfer function (MTF) was computed from the wave-front aberrations. Contrast sensitivity function (CSF) was obtained from MTF and the retinal aerial image modulation (AIM). It is shown that the 2nd, 3rd, 4th, 5th, 6th Zernike aberrations deteriorate contrast sensitivity function. When the 2nd, 3rd, 4th, 5th, 6th Zernike aberrations are corrected high contrast sensitivity function can be obtained.

  5. Epitope Specificity Delimits the Functional Capabilities of Vaccine-Induced CD8 T Cell Populations

    PubMed Central

    Hill, Brenna J.; Darrah, Patricia A.; Ende, Zachary; Ambrozak, David R.; Quinn, Kylie M.; Darko, Sam; Gostick, Emma; Wooldridge, Linda; van den Berg, Hugo A.; Venturi, Vanessa; Larsen, Martin; Davenport, Miles P.; Seder, Robert A.

    2014-01-01

    Despite progress toward understanding the correlates of protective T cell immunity in HIV infection, the optimal approach to Ag delivery by vaccination remains uncertain. We characterized two immunodominant CD8 T cell populations generated in response to immunization of BALB/c mice with a replication-deficient adenovirus serotype 5 vector expressing the HIV-derived Gag and Pol proteins at equivalent levels. The Gag-AI9/H-2Kd epitope elicited high-avidity CD8 T cell populations with architecturally diverse clonotypic repertoires that displayed potent lytic activity in vivo. In contrast, the Pol-LI9/H-2Dd epitope elicited motif-constrained CD8 T cell repertoires that displayed lower levels of physical avidity and lytic activity despite equivalent measures of overall clonality. Although low-dose vaccination enhanced the functional profiles of both epitope-specific CD8 T cell populations, greater polyfunctionality was apparent within the Pol-LI9/H-2Dd specificity. Higher proportions of central memory-like cells were present after low-dose vaccination and at later time points. However, there were no noteworthy phenotypic differences between epitope-specific CD8 T cell populations across vaccine doses or time points. Collectively, these data indicate that the functional and phenotypic properties of vaccine-induced CD8 T cell populations are sensitive to dose manipulation, yet constrained by epitope specificity in a clonotype-dependent manner. PMID:25348625

  6. Mechanism of degradation of immunogenic gluten epitopes from Triticum turgidum L. var. durum by sourdough lactobacilli and fungal proteases.

    PubMed

    De Angelis, Maria; Cassone, Angela; Rizzello, Carlo G; Gagliardi, Francesca; Minervini, Fabio; Calasso, Maria; Di Cagno, Raffaella; Francavilla, Ruggero; Gobbetti, Marco

    2010-01-01

    As shown by R5 antibody-based sandwich and competitive enzyme-linked immunosorbent assay (ELISA), selected sourdough lactobacilli, in combination with fungal proteases, hydrolyzed gluten (72 h at 37 degrees C) of various cultivars of Triticum turgidum L. var. durum to less than 20 ppm. Complementary electrophoretic, chromatography, and mass spectrometry techniques were used to characterize the gluten and epitope hydrolysis. Nine peptidases were partially purified from the pooled cytoplasmic extract of the sourdough lactobacilli and used to hydrolyze the 33-mer epitope, the most immunogenic peptide generated during digestion of Triticum species. At least three peptidases (general aminopeptidase type N [PepN], X-prolyl dipeptidyl aminopeptidase [PepX], and endopeptidase PepO) were necessary to detoxify the 33-mer without generation of related immunogenic epitopes. After 14 h of incubation, the combination of all or at least six different peptidases totally hydrolyzed the 33-mer (200 mM) into free amino acids. The same results were found for other immunogenic epitopes, such as fragments 57-68 of alpha 9-gliadin, 62-75 of A-gliadin, and 134-153 of gamma-gliadin. When peptidases were used for fermentation of durum wheat semolina, they caused the hydrolysis of gluten to ca. 2 ppm. The in vivo digestion was simulated, and proteins/peptides extracted from pepsin-trypsin (PT) digestion of durum wheat semolina fermented with selected sourdough lactobacilli induced the expression of gamma interferon and interleukin 2 at levels comparable to those of the negative control. Durum wheat semolina fermented with sourdough lactobacilli was freeze-dried and used for making Italian-type pasta. The scores for cooking and sensory properties for this pasta were higher that those of conventional gluten-free pasta.

  7. Mechanism of Degradation of Immunogenic Gluten Epitopes from Triticum turgidum L. var. durum by Sourdough Lactobacilli and Fungal Proteases▿

    PubMed Central

    De Angelis, Maria; Cassone, Angela; Rizzello, Carlo G.; Gagliardi, Francesca; Minervini, Fabio; Calasso, Maria; Di Cagno, Raffaella; Francavilla, Ruggero; Gobbetti, Marco

    2010-01-01

    As shown by R5 antibody-based sandwich and competitive enzyme-linked immunosorbent assay (ELISA), selected sourdough lactobacilli, in combination with fungal proteases, hydrolyzed gluten (72 h at 37°C) of various cultivars of Triticum turgidum L. var. durum to less than 20 ppm. Complementary electrophoretic, chromatography, and mass spectrometry techniques were used to characterize the gluten and epitope hydrolysis. Nine peptidases were partially purified from the pooled cytoplasmic extract of the sourdough lactobacilli and used to hydrolyze the 33-mer epitope, the most immunogenic peptide generated during digestion of Triticum species. At least three peptidases (general aminopeptidase type N [PepN], X-prolyl dipeptidyl aminopeptidase [PepX], and endopeptidase PepO) were necessary to detoxify the 33-mer without generation of related immunogenic epitopes. After 14 h of incubation, the combination of all or at least six different peptidases totally hydrolyzed the 33-mer (200 mM) into free amino acids. The same results were found for other immunogenic epitopes, such as fragments 57-68 of α9-gliadin, 62-75 of A-gliadin, and 134-153 of γ-gliadin. When peptidases were used for fermentation of durum wheat semolina, they caused the hydrolysis of gluten to ca. 2 ppm. The in vivo digestion was simulated, and proteins/peptides extracted from pepsin-trypsin (PT) digestion of durum wheat semolina fermented with selected sourdough lactobacilli induced the expression of gamma interferon and interleukin 2 at levels comparable to those of the negative control. Durum wheat semolina fermented with sourdough lactobacilli was freeze-dried and used for making Italian-type pasta. The scores for cooking and sensory properties for this pasta were higher that those of conventional gluten-free pasta. PMID:19948868

  8. Detecting independent and recurrent copy number aberrations using interval graphs.

    PubMed

    Wu, Hsin-Ta; Hajirasouliha, Iman; Raphael, Benjamin J

    2014-06-15

    Somatic copy number aberrations SCNAS: are frequent in cancer genomes, but many of these are random, passenger events. A common strategy to distinguish functional aberrations from passengers is to identify those aberrations that are recurrent across multiple samples. However, the extensive variability in the length and position of SCNA: s makes the problem of identifying recurrent aberrations notoriously difficult. We introduce a combinatorial approach to the problem of identifying independent and recurrent SCNA: s, focusing on the key challenging of separating the overlaps in aberrations across individuals into independent events. We derive independent and recurrent SCNA: s as maximal cliques in an interval graph constructed from overlaps between aberrations. We efficiently enumerate all such cliques, and derive a dynamic programming algorithm to find an optimal selection of non-overlapping cliques, resulting in a very fast algorithm, which we call RAIG (Recurrent Aberrations from Interval Graphs). We show that RAIG outperforms other methods on simulated data and also performs well on data from three cancer types from The Cancer Genome Atlas (TCGA). In contrast to existing approaches that employ various heuristics to select independent aberrations, RAIG optimizes a well-defined objective function. We show that this allows RAIG to identify rare aberrations that are likely functional, but are obscured by overlaps with larger passenger aberrations. http://compbio.cs.brown.edu/software. © The Author 2014. Published by Oxford University Press.

  9. Mapping of melanin-concentrating hormone receptor 1 B cell epitopes predicts two major binding sites for vitiligo patient autoantibodies.

    PubMed

    Gavalas, Nikos G; Gottumukkala, Raju V S R K; Gawkrodger, David J; Watson, Philip F; Weetman, Anthony P; Kemp, E Helen

    2009-05-01

    The melanin-concentrating hormone receptor 1 (MCHR1) has been identified as a B cell autoantigen in vitiligo with antibodies to the receptor detectable in binding and function-blocking assays. Two epitope domains (amino acids 1-138 and 139-298) have been previously identified. In this study, we aimed to further define the epitope specificity of MCHR1 antibodies using phage-display technology and to identify the epitopes recognised by receptor antibodies detected in MCHR1 function-blocking assays. Antibody reactivity to MCHR1 peptides 51-80, 85-98, 154-158 and 254-260 was identified by phage-display and subsequently confirmed in phage ELISA in 2/12, 5/12, 3/12 and 6/12 of vitiligo patients, respectively. The results suggest that major autoantibody epitopes are localised in the 85-98 and 254-260 amino acid regions of MCHR1 with minor epitopes in amino acid sequences 51-80 and 154-158. Antibodies with MCHR1 function-blocking activity were determined to recognise epitope 254-260, this being the first epitope to be reported as a target site for antibodies that block the function of the receptor.

  10. Linear phase conjugation for atmospheric aberration compensation

    NASA Astrophysics Data System (ADS)

    Grasso, Robert J.; Stappaerts, Eddy A.

    1998-01-01

    Atmospheric induced aberrations can seriously degrade laser performance, greatly affecting the beam that finally reaches the target. Lasers propagated over any distance in the atmosphere suffer from a significant decrease in fluence at the target due to these aberrations. This is especially so for propagation over long distances. It is due primarily to fluctuations in the atmosphere over the propagation path, and from platform motion relative to the intended aimpoint. Also, delivery of high fluence to the target typically requires low beam divergence, thus, atmospheric turbulence, platform motion, or both results in a lack of fine aimpoint control to keep the beam directed at the target. To improve both the beam quality and amount of laser energy delivered to the target, Northrop Grumman has developed the Active Tracking System (ATS); a novel linear phase conjugation aberration compensation technique. Utilizing a silicon spatial light modulator (SLM) as a dynamic wavefront reversing element, ATS undoes aberrations induced by the atmosphere, platform motion or both. ATS continually tracks the target as well as compensates for atmospheric and platform motion induced aberrations. This results in a high fidelity, near-diffraction limited beam delivered to the target.

  11. Wave aberrations in rhesus monkeys with vision-induced ametropias

    PubMed Central

    Ramamirtham, Ramkumar; Kee, Chea-su; Hung, Li-Fang; Qiao-Grider, Ying; Huang, Juan; Roorda, Austin; Smith, Earl L.

    2007-01-01

    The purpose of this study was to investigate the relationship between refractive errors and high-order aberrations in infant rhesus monkeys. Specifically, we compared the monochromatic wave aberrations measured with a Shack-Hartman wavefront sensor between normal monkeys and monkeys with vision-induced refractive errors. Shortly after birth, both normal monkeys and treated monkeys reared with optically induced defocus or form deprivation showed a decrease in the magnitude of high-order aberrations with age. However, the decrease in aberrations was typically smaller in the treated animals. Thus, at the end of the lens-rearing period, higher than normal amounts of aberrations were observed in treated eyes, both hyperopic and myopic eyes and treated eyes that developed astigmatism, but not spherical ametropias. The total RMS wavefront error increased with the degree of spherical refractive error, but was not correlated with the degree of astigmatism. Both myopic and hyperopic treated eyes showed elevated amounts of coma and trefoil and the degree of trefoil increased with the degree of spherical ametropia. Myopic eyes also exhibited a much higher prevalence of positive spherical aberration than normal or treated hyperopic eyes. Following the onset of unrestricted vision, the amount of high-order aberrations decreased in the treated monkeys that also recovered from the experimentally induced refractive errors. Our results demonstrate that high-order aberrations are influenced by visual experience in young primates and that the increase in high-order aberrations in our treated monkeys appears to be an optical byproduct of the vision-induced alterations in ocular growth that underlie changes in refractive error. The results from our study suggest that the higher amounts of wave aberrations observed in ametropic humans are likely to be a consequence, rather than a cause, of abnormal refractive development. PMID:17825347

  12. Recognition of three epitopic regions on invasion plasmid antigen C by immune sera of rhesus monkeys infected with Shigella flexneri 2a.

    PubMed Central

    Turbyfill, K R; Joseph, S W; Oaks, E V

    1995-01-01

    The invasive ability of Shigella spp. is correlated with the expression of several plasmid-encoded proteins, including invasion plasmid antigen C (IpaC). By characterizing the antigenic structure of IpaC with monoclonal antibodies and convalescent-phase sera, it may be possible to determine the physical location of specific epitopes as well as the involvement of epitopes in a protective immune response or the host's susceptibility to disease. By using overlapping octameric synthetic peptides, which together represent the entire IpaC protein, the precise linear sequence of four surface-exposed epitopes was defined for four IpaC monoclonal antibodies. Furthermore, 17 unique peptide epitopes of IpaC were mapped by using 9-day-postinfection serum samples from 13 rhesus monkeys challenged with Shigella flexneri 2a. Each individual recognized a somewhat different array of IpaC peptide epitopes after infection with shigellae. However, the epitopes were clustered within three regions of the protein: region I (between amino acid residues 1 and 61), region II (between amino acid residues 177 and 258), and region III (between amino acid residues 298 and 307). Region II was recognized by 92% of S. flexneri-infected individuals and was considered to be a highly immunogenic region. Animals asymptomatic for shigellosis after challenge with S. flexneri recognized peptide epitopes within all three epitopic regions of IpaC, whereas symptomatic animals recognized peptides in only one or two of the epitopic regions. Antibody from monkeys challenged with S. sonnei recognized IpaC peptide epitopes which fell within and outside the three S. flexneri epitopic regions. While numerous potential epitopes exist on the IpaC protein, the identification of three regions in which epitopes are clustered suggests that these regions are significant with respect to the immune response and to subsequent pathogenesis postinfection. PMID:7558301

  13. Aberrations in stimulated emission depletion (STED) microscopy

    NASA Astrophysics Data System (ADS)

    Antonello, Jacopo; Burke, Daniel; Booth, Martin J.

    2017-12-01

    Like all methods of super-resolution microscopy, stimulated emission depletion (STED) microscopy can suffer from the effects of aberrations. The most important aspect of a STED microscope is that the depletion focus maintains a minimum, ideally zero, intensity point that is surrounded by a region of higher intensity. It follows that aberrations that cause a non-zero value of this minimum intensity are the most detrimental, as they inhibit fluorescence emission even at the centre of the depletion focus. We present analysis that elucidates the nature of these effects in terms of the different polarisation components at the focus for two-dimensional and three-dimensional STED resolution enhancement. It is found that only certain low-order aberration modes can affect the minimum intensity at the Gaussian focus. This has important consequences for the design of adaptive optics aberration correction systems.

  14. 3D resolved mapping of optical aberrations in thick tissues

    PubMed Central

    Zeng, Jun; Mahou, Pierre; Schanne-Klein, Marie-Claire; Beaurepaire, Emmanuel; Débarre, Delphine

    2012-01-01

    We demonstrate a simple method for mapping optical aberrations with 3D resolution within thick samples. The method relies on the local measurement of the variation in image quality with externally applied aberrations. We discuss the accuracy of the method as a function of the signal strength and of the aberration amplitude and we derive the achievable resolution for the resulting measurements. We then report on measured 3D aberration maps in human skin biopsies and mouse brain slices. From these data, we analyse the consequences of tissue structure and refractive index distribution on aberrations and imaging depth in normal and cleared tissue samples. The aberration maps allow the estimation of the typical aplanetism region size over which aberrations can be uniformly corrected. This method and data pave the way towards efficient correction strategies for tissue imaging applications. PMID:22876353

  15. Primary aberrations in focused radially polarized vortex beams

    NASA Astrophysics Data System (ADS)

    Biss, David P.; Brown, T. G.

    2004-02-01

    We study the effect of primary aberrations on the 3-D polarization of the electric field in a focused lowest order radially polarized beam. A full vector diffraction treatment of the focused beams is used. Attention is given to the effects of primary spherical, astigmatic, and comatic aberrations on the local polarization, Strehl ratio, and aberration induced degradation of the longitudinal field at focus

  16. Computational approach for predicting the conserved B-cell epitopes of hemagglutinin H7 subtype influenza virus.

    PubMed

    Wang, Xiangyu; Sun, Qi; Ye, Zhonghua; Hua, Ying; Shao, Na; Du, Yanli; Zhang, Qiwei; Wan, Chengsong

    2016-10-01

    An avian-origin influenza H7N9 virus epidemic occurred in China in 2013-2014, in which >422 infected people suffered from pneumonia, respiratory distress syndrome and septic shock. H7N9 viruses belong to the H7 subtype of avian-origin influenza viruses (AIV-H7). Hemagglutinin (HA) is a vital membrane protein of AIV that has an important role in host recognition and infection. The epitopes of HA are significant determinants of the regularity of epidemic and viral mutation and recombination mechanisms. The present study aimed to predict the conserved B-cell epitopes of AIV-H7 HA using a bioinformatics approach, including the three most effective epitope prediction softwares available online: Artificial Neural Network based B-cell Epitope Prediction (ABCpred), B-cell Epitope Prediction (BepiPred) and Linear B-cell Epitope Prediction (LBtope). A total of 24 strains of Euro-Asiatic AIV-H7 that had been associated with a serious poultry pandemic or had infected humans in the past 30 years were selected to identify the conserved regions of HA. Sequences were obtained from the National Center for Biotechnology Information and Global Initiative on Sharing Avian Influenza Data databases. Using a combination of software prediction and sequence comparisons, the conserved epitopes of AIV-H7 were predicted and clarified. A total of five conserved epitopes [amino acids (aa) 37-52, 131-142, 215-234, 465-484 and 487-505] with a suitable length, high antigenicity and minimal variation were predicted and confirmed. Each obtained a score of >0.80 in ABCpred, 60% in LBtope and a level of 0.35 in Bepipred. In addition, a representative amino acid change (glutamine 235 -to-leucine 235 ) in the HA protein of the 2013 AIV-H7N9 was discovered. The strategy adopted in the present study may have profound implications on the rapid diagnosis and control of infectious disease caused by H7N9 viruses, as well as by other virulent viruses, such as the Ebola virus.

  17. Prediction of B-cell linear epitopes with a combination of support vector machine classification and amino acid propensity identification.

    PubMed

    Wang, Hsin-Wei; Lin, Ya-Chi; Pai, Tun-Wen; Chang, Hao-Teng

    2011-01-01

    Epitopes are antigenic determinants that are useful because they induce B-cell antibody production and stimulate T-cell activation. Bioinformatics can enable rapid, efficient prediction of potential epitopes. Here, we designed a novel B-cell linear epitope prediction system called LEPS, Linear Epitope Prediction by Propensities and Support Vector Machine, that combined physico-chemical propensity identification and support vector machine (SVM) classification. We tested the LEPS on four datasets: AntiJen, HIV, a newly generated PC, and AHP, a combination of these three datasets. Peptides with globally or locally high physicochemical propensities were first identified as primitive linear epitope (LE) candidates. Then, candidates were classified with the SVM based on the unique features of amino acid segments. This reduced the number of predicted epitopes and enhanced the positive prediction value (PPV). Compared to four other well-known LE prediction systems, the LEPS achieved the highest accuracy (72.52%), specificity (84.22%), PPV (32.07%), and Matthews' correlation coefficient (10.36%).

  18. The Art of Optical Aberrations

    NASA Astrophysics Data System (ADS)

    Wylde, Clarissa Eileen Kenney

    Art and optics are inseparable. Though seemingly opposite disciplines, the combination of art and optics has significantly impacted both culture and science as they are now known. As history has run its course, in the sciences, arts, and their fruitful combinations, optical aberrations have proved to be a problematic hindrance to progress. In an effort to eradicate aberrations the simple beauty of these aberrational forms has been labeled as undesirable and discarded. Here, rather than approach aberrations as erroneous, these beautiful forms are elevated to be the photographic subject in a new body of work, On the Bright Side. Though many recording methods could be utilized, this work was composed on classic, medium-format, photographic film using white-light, Michelson interferometry. The resulting images are both a representation of the true light rays that interacted on the distorted mirror surfaces (data) and the artist's compositional eye for what parts of the interferogram are chosen and displayed. A detailed description of the captivating interdisciplinary procedure is documented and presented alongside the final artwork, CCD digital reference images, and deformable mirror contour maps. This alluring marriage between the arts and sciences opens up a heretofore minimally explored aspect of the inextricable art-optics connection. It additionally provides a fascinating new conversation on the importance of light and optics in photographic composition.

  19. Sanger and Next-Generation Sequencing data for characterization of CTL epitopes in archived HIV-1 proviral DNA.

    PubMed

    Tumiotto, Camille; Riviere, Lionel; Bellecave, Pantxika; Recordon-Pinson, Patricia; Vilain-Parce, Alice; Guidicelli, Gwenda-Line; Fleury, Hervé

    2017-01-01

    One of the strategies for curing viral HIV-1 is a therapeutic vaccine involving the stimulation of cytotoxic CD8-positive T cells (CTL) that are Human Leucocyte Antigen (HLA)-restricted. The lack of efficiency of previous vaccination strategies may have been due to the immunogenic peptides used, which could be different from a patient's virus epitopes and lead to a poor CTL response. To counteract this lack of specificity, conserved epitopes must be targeted. One alternative is to gather as many data as possible from a large number of patients on their HIV-1 proviral archived epitope variants, taking into account their genetic background to select the best presented CTL epitopes. In order to process big data generated by Next-Generation Sequencing (NGS) of the DNA of HIV-infected patients, we have developed a software package called TutuGenetics. This tool combines an alignment derived either from Sanger or NGS files, HLA typing, target gene and a CTL epitope list as input files. It allows automatic translation after correction of the alignment obtained between the HxB2 reference and the reads, followed by automatic calculation of the MHC IC50 value for each epitope variant and the HLA allele of the patient by using NetMHCpan 3.0, resulting in a csv file as output result. We validated this new tool by comparing Sanger and NGS (454, Roche) sequences obtained from the proviral DNA of patients at success of ART included in the Provir Latitude 45 study and showed a 90% correlation between the quantitative results of NGS and Sanger. This automated analysis combined with complementary samples should yield more data regarding the archived CTL epitopes according to the patients' HLA alleles and will be useful for screening epitopes that in theory are presented efficiently to the HLA groove, thus constituting promising immunogenic peptides for a therapeutic vaccine.

  20. Latent NOTCH3 epitopes unmasked in CADASIL and regulated by protein redox state.

    PubMed

    Zhang, Xiaojie; Lee, Soo Jung; Young, Kelly Z; Josephson, David A; Geschwind, Michael D; Wang, Michael M

    2014-10-02

    Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy CADASIL is caused by more than a hundred NOTCH3 mutations. Virtually all encoded mutant proteins contain an odd number of cysteines. As such, structural changes in NOTCH3 may be the primary molecular abnormality in CADASIL. Thus, we sought evidence for structurally altered NOTCH3 protein in CADASIL tissue. Four antibodies were raised in rabbits against two non-overlapping N-terminal NOTCH3 sequences. These reagents were used in immunohistochemical experiments to detect epitopes in post-mortem CADASIL brains (n=8), control brains, and cells overexpressing NOTCH3. To determine the biochemical nature of NOTCH3 epitopes, we used these antibodies to probe pure NOTCH3-Fc fusion proteins treated with acid, urea, guanidinium, ionic detergents, acrylamide, and thiol- and phosphorus-based reductants. All antibodies avidly stained arteries in 8 of 8 CADASIL brain samples. The most prominent staining was in degenerating media of leptomeningeal arteries and sclerotic penetrating vessels. Normal appearing vessels from control brains were not reactive. Antibodies did not react with cultured cells overexpressing NOTCH3 or with purified NOTCH3-Fc protein. Furthermore, treatment of pure protein with acid, chaotropic denaturants, alkylators, and detergents failed to unmask N-terminal NOTCH3 epitopes. Antibodies, however, recognized novel N-terminal epitopes in purified NOTCH3-Fc protein treated with three different reductants (DTT, beta-mercaptoethanol, and TCEP). We conclude that CADASIL arteries feature latent N-terminal NOTCH3 epitopes, suggesting the first evidence in vivo of NOTCH3 structural alterations. Published by Elsevier B.V.

  1. Mapping of epitopes and structural analysis of antigenic sites in the nucleoprotein of rabies virus.

    PubMed

    Goto, H; Minamoto, N; Ito, H; Ito, N; Sugiyama, M; Kinjo, T; Kawai, A

    2000-01-01

    Linear epitopes on the rabies virus nucleoprotein (N) recognized by six MAbs raised against antigenic sites I (MAbs 6-4, 12-2 and 13-27) and IV (MAbs 6-9, 7-12 and 8-1) were investigated. Based on our previous studies on sites I and IV, 24 consecutively overlapping octapeptides and N- and C-terminal-deleted mutant N proteins were prepared. Results showed that all three site I epitopes studied and two site IV epitopes (for MAbs 8-1 and 6-9) mapped to aa 358-367, and that the other site IV epitope of MAb 7-12 mapped to aa 375-383. Tests using chimeric and truncated proteins showed that MAb 8-1 also requires the N-terminal sequence of the N protein to recognize its binding region more efficiently. Immunofluorescence studies demonstrated that all three site I-specific MAbs and one site IV-specific MAb (7-12) stained the N antigen that was diffusely distributed in the whole cytoplasm; the other two site IV-specific MAbs (6-9 and 8-1) detected only the N antigen in the cytoplasmic inclusion bodies (CIB). An antigenic site II-specific MAb (6-17) also detected CIB-associated N antigen alone. Furthermore, the level of diffuse N antigens decreased after treatment of infected cells with cycloheximide. These results suggest that epitopes at site I are expressed on the immature form of the N protein, but epitope structures of site IV MAbs 6-9 and 8-1 are created and/or exposed only after maturation of the N protein.

  2. Epitope-based peptide vaccine design and target site depiction against Ebola viruses: an immunoinformatics study.

    PubMed

    Khan, M A; Hossain, M U; Rakib-Uz-Zaman, S M; Morshed, M N

    2015-07-01

    Ebola viruses (EBOVs) have been identified as an emerging threat in recent year as it causes severe haemorrhagic fever in human. Epitope-based vaccine design for EBOVs remains a top priority because a mere progress has been made in this regard. Another reason is the lack of antiviral drug and licensed vaccine although there is a severe outbreak in Central Africa. In this study, we aimed to design an epitope-based vaccine that can trigger a significant immune response as well as to prognosticate inhibitor that can bind with potential drug target sites using various immunoinformatics and docking simulation tools. The capacity to induce both humoral and cell-mediated immunity by T cell and B cell was checked for the selected protein. The peptide region spanning 9 amino acids from 42 to 50 and the sequence TLASIGTAF were found as the most potential B and T cell epitopes, respectively. This peptide could interact with 12 HLAs and showed high population coverage up to 80.99%. Using molecular docking, the epitope was further appraised for binding against HLA molecules to verify the binding cleft interaction. In addition with this, the allergenicity of the epitopes was also evaluated. In the post-therapeutic strategy, docking study of predicted 3D structure identified suitable therapeutic inhibitor against targeted protein. However, this computational epitope-based peptide vaccine designing and target site prediction against EBOVs open up a new horizon which may be the prospective way in Ebola viruses research; the results require validation by in vitro and in vivo experiments. © 2015 John Wiley & Sons Ltd.

  3. 1D Self-Assembly and Ice Recrystallization Inhibition Activity of Antifreeze Glycopeptide-Functionalized Perylene Bisimides.

    PubMed

    Adam, Madeleine K; Jarrett-Wilkins, Charles; Beards, Michael; Staykov, Emiliyan; MacFarlane, Liam R; Bell, Toby D M; Matthews, Jacqueline M; Manners, Ian; Faul, Charl F J; Moens, Pierre D J; Ben, Robert N; Wilkinson, Brendan L

    2018-06-04

    Antifreeze glycoproteins (AFGPs) are polymeric natural products that have drawn considerable interest in diverse research fields owing to their potent ice recrystallization inhibition (IRI) activity. Self-assembled materials have emerged as a promising class of biomimetic ice growth inhibitor, yet the development of AFGP-based supramolecular materials that emulate the aggregative behavior of AFGPs have not yet been reported. This work reports the first example of the 1D self-assembly and IRI activity of AFGP-functionalized perylene bisimides (AFGP-PBIs). Glycopeptide-functionalized PBIs underwent 1D self-assembly in water and showed modest IRI activity, which could be tuned through substitution of the PBI core. This work presents essential proof-of-principle for the development of novel IRIs as potential supramolecular cryoprotectants and glycoprotein mimics. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Population dynamics of aberrant chromosome 1 in mice.

    PubMed

    Sabantsev, I; Spitsin, O; Agulnik, S; Ruvinsky, A

    1993-05-01

    Natural populations of two semispecies of house mouse, Mus musculus domesticus and M.m. musculus, were found to be polymorphic for an aberrant chromosome 1 bearing a large inserted block of homogeneously staining heterochromatin. Strong meiotic drive for the aberrant chromosome from M.m. musculus was previously observed in heterozygous female mice. There are at least three meiotic drive levels determined by different allelic variants of distorter. Homozygotes had low viability and females showed low fertility. Both homo- and heterozygous males had normal fertility and their segregation patterns did not deviate from normal. Computer simulations were performed of the dynamics of aberrant chromosome 1 in demes and populations. The data demonstrate that a spontaneous mutation (inversion) of an aberrant chromosome 1, once arisen, has a high probability of spreading in a population at high coefficients of meiotic drive and migration. In the long-term, the population attains a stationary state which is determined by the drive level and migration intensity. The state of stable genotypic equilibrium is independent of deme and population size, as well as of the initial concentration of the aberrant chromosome. As populations initially polymorphic for the distorters approach the stationary state, the stronger distorter is eliminated. The frequencies of the aberrant chromosome determined by computer analysis agree well with those obtained for the studied Asian M.m. musculus populations. The evolutionary pathways for the origin and fixation of the aberrant chromosome in natural populations are considered.

  5. Beta-glucan-depleted, glycopeptide-rich extracts from Brewer's and Baker's yeast (Saccharomyces cerevisiae) lower interferon-gamma production by stimulated human blood cells in vitro.

    PubMed

    Williams, Roderick; Dias, Daniel A; Jayasinghe, Nirupama; Roessner, Ute; Bennett, Louise E

    2016-04-15

    Regulation of the human immune system requires controlled pro- and anti-inflammatory responses for host defence against infection and disease states. Yeasts (Saccharomyces cerevisiae), as used in brewing and baking, are mostly known for ability to stimulate the human immune-system predominantly reflecting the pro-inflammatory cell wall β-glucans. However, in this study, using food-compatible processing methods, glycopeptide-enriched and β-glucan-depleted products were each prepared from Brewer's and Baker's yeasts, which suppressed production of interferon-γ (IFN-γ) in human whole blood cell assay, signifying that anti-inflammatory factors are also present in yeast. Anti-inflammatory bioactivities of products prepared from Brewer's and Baker's yeast were compared with the commercial yeast product, Epicor®. While unfractionated Epicor was inactive, the C18 resin-binding fractions of Brewer's and Baker's yeast products and Epicor dose-dependently lowered IFN-γ, demonstrating that Epicor also contained both pro-inflammatory (β-glucans) and anti-inflammatory components. Anti-inflammatory activity was attributed to C18 resin-binding species glyco-peptides in Epicor and experimental yeast products. This study demonstrated that pro- and anti-inflammatory factors could be resolved and enriched in yeasts by suitable processing, with potential to improve specific activities. Crown Copyright © 2015. Published by Elsevier Ltd. All rights reserved.

  6. Aberration design of zoom lens systems using thick lens modules.

    PubMed

    Zhang, Jinkai; Chen, Xiaobo; Xi, Juntong; Wu, Zhuoqi

    2014-12-20

    A systematic approach for the aberration design of a zoom lens system using a thick lens module is presented. Each component is treated as a thick lens module at the beginning of the design. A thick lens module refers to a thick lens component with a real lens structure, like lens materials, lens curvatures, lens thicknesses, and lens interval distances. All nine third-order aberrations of a thick lens component are considered during the design. The relationship of component aberrations in different zoom positions can be approximated from the aberration shift. After minimizing the aberrations of the zoom lens system, the nine third-order aberrations of every lens component can be determined. Then the thick lens structure of every lens component can be determined after optimization according to their first-order properties and third-order aberration targets. After a third optimization for minimum practical third-order aberrations of a zoom lens system, the aberration design using the thick lens module is complete, which provides a practical zoom lens system with thick lens structures. A double-sided telecentric zoom lens system is designed using the thick lens module in this paper, which shows that this method is practical for zoom lens design.

  7. Thyroid peroxidase autoantibody epitopic 'fingerprints' in juvenile Hashimoto's thyroiditis: evidence for conservation over time and in families.

    PubMed

    Jaume, J C; Burek, C L; Hoffman, W H; Rose, N R; McLachlan, S M; Rapoport, B

    1996-04-01

    In Hashimoto's thyroiditis, the humoral component is manifest by autoantibodies to thyroid peroxidase (TPO). Epitopic 'fingerprinting' of polyclonal serum TPO autoantibodies has been facilitated by the molecular cloning and expression as Fab of a repertoire of human TPO autoantibody genes. To investigate whether TPO autoantibody fingerprints are (i) stable over long periods of time (approximately 15 years), and (ii) inherited, we studied a cohort of nine patients with juvenile Hashimoto's thyroiditis and 21 first degree relatives of four of these patients. Fingerprints were determined by competition between four selected FAB and serum autoantibodies for binding to 125I-TPO. Regardless of titre, the TPO epitopic profile was stable in 10/12 individuals whose TPO autoantibody levels were sufficient for analysis on two or three occasions over 12-15 years. Although the TPO epitopic fingerprint profiles in two families raised the possibility of inheritance, overall the data from all four families did not reveal an obvious pattern of genetic control. In no family was the TPO epitopic fingerprint associated with HLA A, B or DR. In conclusion, TPO autoantibody epitopic fingerprints are frequently conserved over many years. Studies on additional families are necessary to establish whether or not the epitopic profiles of TPO autoantibodies are inherited.

  8. Influence of High Hydrostatic Pressure on Epitope Mapping of Tobacco Mosaic Virus Coat Protein

    PubMed Central

    Bonafe, Carlos Francisco Sampaio; Arns, Clarice Weis

    2014-01-01

    Abstract In this study, we investigated the effect of high hydrostatic pressure (HHP) on tobacco mosaic virus (TMV), a model virus in immunology and one of the most studied viruses to date. Exposure to HHP significantly altered the recognition epitopes when compared to sera from mice immunized with native virus. These alterations were studied further by combining HHP with urea or low temperature and then inoculating the altered virions into Balb-C mice. The antibody titers and cross-reactivity of the resulting sera were determined by ELISA. The antigenicity of the viral particles was maintained, as assessed by using polyclonal antibodies against native virus. The antigenicity of canonical epitopes was maintained, although binding intensities varied among the treatments. The patterns of recognition determined by epitope mapping were cross checked with the prediction algorithms for the TMVcp amino acid sequence to infer which alterations had occurred. These findings suggest that different cleavage sites were exposed after the treatments and this was confirmed by epitope mapping using sera from mice immunized with virus previously exposed to HHP. PMID:24605789

  9. Synthesis of ganglioside epitopes for oligosaccharide specific immunoadsorption therapy of Guillian-Barré syndrome.

    PubMed

    Andersen, Søren M; Ling, Chang-Chun; Zhang, Ping; Townson, Kate; Willison, Hugh J; Bundle, David R

    2004-04-21

    Guillain-Barré syndrome is a postinfectious, autoimmune neuropathy resulting in neuromuscular paralysis. Auto-antibodies, often induced by bacterial infection, bind to human gangliosides possessing monosialoside and diasialoside epitopes and impair the function of nerve junctions, where these ganglioside structures are highly enriched. Truncated gangliosides representive of GD3, GQ1b and GM2 epitopes have been synthesized as methyl glycosides and as a glycosides of an eleven carbon tether. The synthetic oligosaccharide ligands are structural mimics of these highly complex ganglioside epitopes and via their ability to neutralize or remove auto-antibodies have the potential for therapy, either as soluble blocking ligands administered systemically, or as immuno-affinity ligands for use as extracorporeal immunoadsorbents.

  10. Definition of Human Epitopes Recognized in Tetanus Toxoid and Development of an Assay Strategy to Detect Ex Vivo Tetanus CD4+ T Cell Responses

    PubMed Central

    da Silva Antunes, Ricardo; Paul, Sinu; Sidney, John; Weiskopf, Daniela; Dan, Jennifer M.; Phillips, Elizabeth; Mallal, Simon; Crotty, Shane; Sette, Alessandro; Lindestam Arlehamn, Cecilia S.

    2017-01-01

    Despite widespread uses of tetanus toxoid (TT) as a vaccine, model antigen and protein carrier, TT epitopes have been poorly characterized. Herein we defined the human CD4+ T cell epitope repertoire by reevaluation of previously described epitopes and evaluation of those derived from prediction of HLA Class II binding. Forty-seven epitopes were identified following in vitro TT stimulation, with 28 epitopes accounting for 90% of the total response. Despite this diverse range of epitopes, individual responses were associated with only a few immunodominant epitopes, with each donor responding on average to 3 epitopes. For the top 14 epitopes, HLA restriction could be inferred based on HLA typing of the responding donors. HLA binding predictions re-identified the vast majority of known epitopes, and identified 24 additional novel epitopes. With these epitopes, we created a TT epitope pool, which allowed us to characterize TT responses directly ex vivo using a cytokine-independent Activation Induced Marker (AIM) assay. These TT responses were highly Th1 or Th2 polarized, which was dependent upon the original priming vaccine, either the cellular DTwP or acellular DTaP formulation. This polarization remained despite the original priming having occurred decades past and a recent booster immunization with a reduced acellular vaccine formulation. While TT responses following booster vaccination were not durably increased in magnitude, they were associated with a relative expansion of CD4+ effector memory T cells. PMID:28081174

  11. Definition of Human Epitopes Recognized in Tetanus Toxoid and Development of an Assay Strategy to Detect Ex Vivo Tetanus CD4+ T Cell Responses.

    PubMed

    da Silva Antunes, Ricardo; Paul, Sinu; Sidney, John; Weiskopf, Daniela; Dan, Jennifer M; Phillips, Elizabeth; Mallal, Simon; Crotty, Shane; Sette, Alessandro; Lindestam Arlehamn, Cecilia S

    2017-01-01

    Despite widespread uses of tetanus toxoid (TT) as a vaccine, model antigen and protein carrier, TT epitopes have been poorly characterized. Herein we defined the human CD4+ T cell epitope repertoire by reevaluation of previously described epitopes and evaluation of those derived from prediction of HLA Class II binding. Forty-seven epitopes were identified following in vitro TT stimulation, with 28 epitopes accounting for 90% of the total response. Despite this diverse range of epitopes, individual responses were associated with only a few immunodominant epitopes, with each donor responding on average to 3 epitopes. For the top 14 epitopes, HLA restriction could be inferred based on HLA typing of the responding donors. HLA binding predictions re-identified the vast majority of known epitopes, and identified 24 additional novel epitopes. With these epitopes, we created a TT epitope pool, which allowed us to characterize TT responses directly ex vivo using a cytokine-independent Activation Induced Marker (AIM) assay. These TT responses were highly Th1 or Th2 polarized, which was dependent upon the original priming vaccine, either the cellular DTwP or acellular DTaP formulation. This polarization remained despite the original priming having occurred decades past and a recent booster immunization with a reduced acellular vaccine formulation. While TT responses following booster vaccination were not durably increased in magnitude, they were associated with a relative expansion of CD4+ effector memory T cells.

  12. Mapping epitopes and antigenicity by site-directed masking

    NASA Astrophysics Data System (ADS)

    Paus, Didrik; Winter, Greg

    2006-06-01

    Here we describe a method for mapping the binding of antibodies to the surface of a folded antigen. We first created a panel of mutant antigens (-lactamase) in which single surface-exposed residues were mutated to cysteine. We then chemically tethered the cysteine residues to a solid phase, thereby masking a surface patch centered on each cysteine residue and blocking the binding of antibodies to this region of the surface. By these means we mapped the epitopes of several mAbs directed to -lactamase. Furthermore, by depleting samples of polyclonal antisera to the masked antigens and measuring the binding of each depleted sample of antisera to unmasked antigen, we mapped the antigenicity of 23 different epitopes. After immunization of mice and rabbits with -lactamase in Freund's adjuvant, we found that the antisera reacted with both native and denatured antigen and that the antibody response was mainly directed to an exposed and flexible loop region of the native antigen. By contrast, after immunization in PBS, we found that the antisera reacted only weakly with denatured antigen and that the antibody response was more evenly distributed over the antigenic surface. We suggest that denatured antigen (created during emulsification in Freund's adjuvant) elicits antibodies that bind mainly to the flexible regions of the native protein and that this explains the correlation between antigenicity and backbone flexibility. Denaturation of antigen during vaccination or natural infections would therefore be expected to focus the antibody response to the flexible loops. backbone flexibility | Freund's adjuvant | conformational epitope | antisera

  13. Aberration of a negative ion beam caused by space charge effect.

    PubMed

    Miyamoto, K; Wada, S; Hatayama, A

    2010-02-01

    Aberrations are inevitable when the charged particle beams are extracted, accelerated, transmitted, and focused with electrostatic and magnetic fields. In this study, we investigate the aberration of a negative ion accelerator for a neutral beam injector theoretically, especially the spherical aberration caused by the negative ion beam expansion due to the space charge effect. The negative ion current density profiles with the spherical aberration are compared with those without the spherical aberration. It is found that the negative ion current density profiles in a log scale are tailed due to the spherical aberration.

  14. Defining the expression hierarchy of latent T-cell epitopes in Epstein-Barr virus infection with TCR-like antibodies

    PubMed Central

    Sim, Adrian Chong Nyi; Too, Chien Tei; Oo, Min Zin; Lai, Junyun; Eio, Michelle Yating; Song, Zhenying; Srinivasan, Nalini; Tan, Diane Ai Lin; Pang, Shyue Wei; Gan, Shu Uin; Lee, Kok Onn; Loh, Thomas Kwok Seng; Chen, Jianzhu; Chan, Soh Ha; MacAry, Paul Anthony

    2013-01-01

    Epstein-Barr virus (EBV) is a gamma herpesvirus that causes a life-long latent infection in human hosts. The latent gene products LMP1, LMP2A and EBNA1 are expressed by EBV-associated tumors and peptide epitopes derived from these can be targeted by CD8 Cytotoxic T-Lymphocyte (CTL) lines. Whilst CTL-based methodologies can be utilized to infer the presence of specific latent epitopes, they do not allow a direct visualization or quantitation of these epitopes. Here, we describe the characterization of three TCR-like monoclonal antibodies (mAbs) targeting the latent epitopes LMP1125–133, LMP2A426–434 or EBNA1562–570 in association with HLA-A0201. These are employed to map the expression hierarchy of endogenously generated EBV epitopes. The dominance of EBNA1562–570 in association with HLA-A0201 was consistently observed in cell lines and EBV-associated tumor biopsies. These data highlight the discordance between MHC-epitope density and frequencies of associated CTL with implications for cell-based immunotherapies and/or vaccines for EBV-associated disease. PMID:24240815

  15. Epitope analysis of the malaria surface antigen pfs48/45 identifies a subdomain that elicits transmission blocking antibodies.

    PubMed

    Outchkourov, Nikolay; Vermunt, Adriaan; Jansen, Josephine; Kaan, Anita; Roeffen, Will; Teelen, Karina; Lasonder, Edwin; Braks, Anneke; van de Vegte-Bolmer, Marga; Qiu, Li Yan; Sauerwein, Robert; Stunnenberg, Hendrik G

    2007-06-08

    Pfs48/45, a member of a Plasmodium-specific protein family, displays conformation-dependent epitopes and is an important target for malaria transmission-blocking (TB) immunity. To design a recombinant Pfs48/45-based TB vaccine, we analyzed the conformational TB epitopes of Pfs48/45. The Pfs48/45 protein was found to consist of a C-terminal six-cysteine module recognized by anti-epitope I antibodies, a middle four-cysteine module recognized by anti-epitopes IIb and III, and an N-terminal module recognized by anti-epitope V antibodies. Refolding assays identified that a fragment of 10 cysteines (10C), comprising the middle four-cysteine and the C-terminal six-cysteine modules, possesses superior refolding capacity. The refolded and partially purified 10C conformer elicited antibodies in mice that targeted at least two of the TB epitopes (I and III). The induced antibodies could block the fertilization of Plasmodium falciparum gametes in vivo in a concentration-dependent manner. Our results provide important insight into the structural organization of the Pfs48/45 protein and experimental support for a Pfs48/45-based subunit vaccine.

  16. Analysis of Epitopes on Dengue Virus Envelope Protein Recognized by Monoclonal Antibodies and Polyclonal Human Sera by a High Throughput Assay

    PubMed Central

    Lin, Hong-En; Tsai, Wen-Yang; Liu, I-Ju; Li, Pi-Chun; Liao, Mei-Ying; Tsai, Jih-Jin; Wu, Yi-Chieh; Lai, Chih-Yun; Lu, Chih-Hsuan; Huang, Jyh-Hsiung; Chang, Gwong-Jen; Wu, Han-Chung; Wang, Wei-Kung

    2012-01-01

    Background The envelope (E) protein of dengue virus (DENV) is the major target of neutralizing antibodies and vaccine development. While previous studies on domain III or domain I/II alone have reported several epitopes of monoclonal antibodies (mAbs) against DENV E protein, the possibility of interdomain epitopes and the relationship between epitopes and neutralizing potency remain largely unexplored. Methodology/Principal Findings We developed a dot blot assay by using 67 alanine mutants of predicted surface-exposed E residues as a systematic approach to identify epitopes recognized by mAbs and polyclonal sera, and confirmed our findings using a capture-ELISA assay. Of the 12 mouse mAbs tested, three recognized a novel epitope involving residues (Q211, D215, P217) at the central interface of domain II, and three recognized residues at both domain III and the lateral ridge of domain II, suggesting a more frequent presence of interdomain epitopes than previously appreciated. Compared with mAbs generated by traditional protocols, the potent neutralizing mAbs generated by a new protocol recognized multiple residues in A strand or residues in C strand/CC′ loop of DENV2 and DENV1, and multiple residues in BC loop and residues in DE loop, EF loop/F strand or G strand of DENV1. The predominant epitopes of anti-E antibodies in polyclonal sera were found to include both fusion loop and non-fusion residues in the same or adjacent monomer. Conclusions/Significance Our analyses have implications for epitope-specific diagnostics and epitope-based dengue vaccines. This high throughput method has tremendous application for mapping both intra and interdomain epitopes recognized by human mAbs and polyclonal sera, which would further our understanding of humoral immune responses to DENV at the epitope level. PMID:22235356

  17. Antibodies to Polymorphic Invasion-Inhibitory and Non-Inhibitory Epitopes of Plasmodium falciparum Apical Membrane Antigen 1 in Human Malaria

    PubMed Central

    Mugyenyi, Cleopatra K.; Elliott, Salenna R.; McCallum, Fiona J.; Anders, Robin F.; Marsh, Kevin; Beeson, James G.

    2013-01-01

    Background Antibodies to P. falciparum apical membrane protein 1 (AMA1) may contribute to protective immunity against clinical malaria by inhibiting blood stage growth of P. falciparum, and AMA1 is a leading malaria vaccine candidate. Currently, there is limited knowledge of the acquisition of strain-specific and cross-reactive antibodies to AMA1 in humans, or the acquisition of invasion-inhibitory antibodies to AMA1. Methodology/Findings We examined the acquisition of human antibodies to specific polymorphic invasion-inhibitory and non-inhibitory AMA1 epitopes, defined by the monoclonal antibodies 1F9 and 2C5, respectively. Naturally acquired antibodies were measured in cohorts of Kenyan children and adults. Antibodies to the invasion-inhibitory 1F9 epitope and non-inhibitory 2C5 epitope were measured indirectly by competition ELISA. Antibodies to the 1F9 and 2C5 epitopes were acquired by children and correlated with exposure, and higher antibody levels and prevalence were observed with increasing age and with active P. falciparum infection. Of note, the prevalence of antibodies to the inhibitory 1F9 epitope was lower than antibodies to AMA1 or the 2C5 epitope. Antibodies to AMA1 ectodomain, the 1F9 or 2C5 epitopes, or a combination of responses, showed some association with protection from P. falciparum malaria in a prospective longitudinal study. Furthermore, antibodies to the invasion-inhibitory 1F9 epitope were positively correlated with parasite growth-inhibitory activity of serum antibodies. Conclusions/Significance Individuals acquire antibodies to functional, polymorphic epitopes of AMA1 that may contribute to protective immunity, and these findings have implications for AMA1 vaccine development. Measuring antibodies to the 1F9 epitope by competition ELISA may be a valuable approach to assessing human antibodies with invasion-inhibitory activity in studies of acquired immunity and vaccine trials of AMA1. PMID:23861883

  18. Epitope-cavities generated by molecularly imprinted films measure the coincident response to anthrax protective antigen and its segments.

    PubMed

    Tai, Dar-Fu; Jhang, Ming-Hong; Chen, Guan-Yu; Wang, Sue-Chen; Lu, Kuo-Hao; Lee, Yu-Der; Liu, Hsin-Tzu

    2010-03-15

    A molecularly imprinted film was fabricated, in the presence of epitope-peptides, onto a quartz crystal microbalance (QCM) chip. These five peptides are known linear or conformational epitopes of the anthrax protective antigen PA(83). Imprinting resulted in an epitope-cavity with affinity for the corresponding template. With the use of a basic monomer, the binding-effect was further enhanced increasing the affinity to nanomolar levels. The affinities of the peptide to their corresponding molecularly induced polymers (MIPs) were more closely related to the molecular weight of the analyte than to the number of residues. All epitope-cavities differentiated their epitope region on the protective antigen PA(83) as well as the corresponding furin cleavage fragments PA(63) and PA(20). The QCM chip differential response to the protective antigen fragment was observed in the picomolar range, thus demonstrating a method to manipulate protein on the surface with defined orientation.

  19. Aberrant regeneration of the third cranial nerve.

    PubMed

    Shrestha, U D; Adhikari, S

    2012-01-01

    Aberrant regeneration of the third cranial nerve is most commonly due to its damage by trauma. A ten-month old child presented with the history of a fall from a four-storey building. She developed traumatic third nerve palsy and eventually the clinical features of aberrant regeneration of the third cranial nerve. The adduction of the eye improved over time. She was advised for patching for the strabismic amblyopia as well. Traumatic third nerve palsy may result in aberrant regeneration of the third cranial nerve. In younger patients, motility of the eye in different gazes may improve over time. © NEPjOPH.

  20. RB4CD12 epitope expression and heparan sulfate disaccharide composition in brain vasculature.

    PubMed

    Hosono-Fukao, Tomomi; Ohtake-Niimi, Shiori; Nishitsuji, Kazuchika; Hossain, Md Motarab; van Kuppevelt, Toin H; Michikawa, Makoto; Uchimura, Kenji

    2011-11-01

    RB4CD12 is a phage display antibody that recognizes a heparan sulfate (HS) glycosaminoglycan epitope. The epitope structure is proposed to contain a trisulfated disaccharide, [-IdoA(2-OSO(3))-GlcNSO(3) (6-OSO(3))-], which supports HS binding to various macromolecules such as growth factors and cytokines in central nervous tissues. Chemically modified heparins that lack the trisulfated disaccharides failed to inhibit the RB4CD12 recognition of HS chains. To determine the localization of the RB4CD12 anti-HS epitope in the brain, we performed an immunohistochemical analysis for cryocut sections of mouse brain. The RB4CD12 staining signals were colocalized with laminin and were detected abundantly in the vascular basement membrane. Bacterial heparinases eliminated the RB4CD12 staining signals. The RB4CD12 epitope localization was confirmed by immunoelectron microscopy. Western blotting analysis revealed that the size of a major RB4CD12-positive molecule is ∼460 kDa in a vessel-enriched fraction of the mouse brain. Disaccharide analysis with reversed-phase ion-pair HPLC showed that [-IdoA(2-OSO(3))-GlcNSO(3) (6-OSO(3))-] trisulfated disaccharide residues are present in HS purified from the vessel-enriched brain fraction. These results indicated that the RB4CD12 anti-HS epitope exists in large quantities in the brain vascular basement membrane. Copyright © 2011 Wiley-Liss, Inc.

  1. Anti-cooperative ligand binding and dimerisation in the glycopeptide antibiotic dalbavancin† †Electronic supplementary information (ESI) available. See DOI: 10.1039/C3OB42428F Click here for additional data file.

    PubMed Central

    Cheng, Mu; Ziora, Zyta M.; Hansford, Karl A.; Blaskovich, Mark A.; Butler, Mark S.

    2014-01-01

    Dalbavancin, a semi-synthetic glycopeptide with enhanced antibiotic activity compared to vancomycin and teicoplanin, binds to the C-terminal lysyl-d-alanyl-d-alanine subunit of Lipid II, inhibiting peptidoglycan biosynthesis. In this study, micro-calorimetry and electrospray ionization (ESI)-MS have been used to investigate the relationship between oligomerisation of dalbavancin and binding of a Lipid II peptide mimic, diacetyl-Lys-d-Ala-d-Ala (Ac2-Kaa). Dalbavancin dimerised strongly in an anti-cooperative manner with ligand-binding, as was the case for ristocetin A, but not for vancomycin and teicoplanin. Dalbavancin and ristocetin A both adopt an ‘closed’ conformation upon ligand binding, suggesting anti-cooperative dimerisation with ligand-binding may be a general feature of dalbavancin/ristocetin A-like glycopeptides. Understanding these effects may provide insight into design of novel dalbavancin derivatives with cooperative ligand-binding and dimerisation characteristics that could enhance antibiotic activity. PMID:24608916

  2. Influence of flanking sequences on presentation efficiency of a CD8+ cytotoxic T-cell epitope delivered by parvovirus-like particles.

    PubMed

    Rueda, P; Morón, G; Sarraseca, J; Leclerc, C; Casal, J I

    2004-03-01

    We have previously developed an antigen-delivery system based on hybrid recombinant porcine parvovirus-like particles (PPV-VLPs) formed by the self-assembly of the VP2 protein of PPV carrying a foreign epitope at its N terminus. In this study, different constructs were made containing a CD8(+) T-cell epitope of chicken ovalbumin (OVA) to analyse the influence of the sequence inserted into VP2 on the correct processing of VLPs by antigen-presenting cells. We analysed the presentation of the OVA epitope inserted without flanking sequences or with either different natural flanking sequences or with the natural flanking sequences of a CD8(+) T-cell epitope from the lymphocytic choriomeningitis virus nucleoprotein, and as a dimer with or without linker sequences. All constructs were studied in terms of level of expression, assembly of VLPs and ability to deliver the inserted epitope into the MHC I pathway. The presentation of the OVA epitope was considerably improved by insertion of short natural flanking sequences, which indicated the relevance of the flanking sequences on the processing of PPV-VLPs. Only PPV-VLPs carrying two copies of the OVA epitope linked by two glycines were able to be properly processed, suggesting that the introduction of flexible residues between the two consecutive OVA epitopes may be necessary for the correct presentation of these dimers by PPV-VLPs. These results provide information to improve the insertion of epitopes into PPV-VLPs to facilitate their processing and presentation by MHC class I molecules.

  3. Identification of linear B-cell epitopes on goose parvovirus non-structural protein.

    PubMed

    Yu, Tian-Fei; Ma, Bo; Wang, Jun-Wei

    2016-10-15

    Goose parvovirus (GPV) infection can cause a highly contagious and lethal disease in goslings and muscovy ducklings which is widespread in all major goose (Anser anser) and Muscovy duck (Cairina moschata) farming countries, leading to a huge economic loss. Humoral immune responses play a major role in GPV immune protection during GPV infection. However, it is still unknown for the localization and immunological characteristics of B-cell epitopes on GPV non-structural protein (NSP). Therefore, in this study, the epitopes on the NSP of GPV were identified by means of overlapping peptides expressed in Escherichia coli in combination with Western blot. The results showed that the antigenic epitopes on the GPV NSP were predominantly localized in the C-terminal (aa 485-627), and especially, the fragment NS (498-532) was strongly positive. These results may facilitate future investigations on the function of NSP of GPV and the development of immunoassays for the diagnosis of GPV infection. Copyright © 2016. Published by Elsevier B.V.

  4. Identification of broadly reactive epitopes targeting major glycoproteins of Herpes simplex virus (HSV) 1 and 2 - An immunoinformatics analysis.

    PubMed

    Chauhan, Varun; Goyal, Kapil; Singh, Mini P

    2018-07-01

    Infections due to both HSV-1 and HSV-2 constitute an enormous health burden worldwide. Development of vaccine against herpes infections is a WHO supported public health priority. The viral glycoproteins have always been the major hotspots for vaccine designing. The present study was aimed to identify the conserved T and B cell epitopes in the major glycoproteins of both HSV-1 and HSV-2 via rigorous computational approaches. Identification of promiscuous T cell epitopes is of utmost importance in vaccine designing as such epitopes are capable of binding to several allelic forms of HLA and could generate effective immune response in the host. The criteria designed for identification of T and B cell epitopes was that it should be conserved in both HSV-1 and 2, promiscuous, have high affinity towards HLA alleles, should be located on the surface of glycoproteins and not be present in the glycosylation sites. This study led to the identification of 17 HLA Class II and 26 HLA Class I T cell epitopes, 9 linear and some conformational B cell epitopes. The identified T cell epitopes were further subjected to molecular docking analysis to analyze their binding patterns. Altogether we have identified 4 most promising regions in glycoproteins (2-gB, 1-gD, 1-gH) of HSV-1 and 2 which are promiscuous to HLA Class II alleles and have overlapping HLA Class I and B cell epitopes, which could be very useful in generating both arms of immune response in the host i.e. adaptive as well as humoral immunity. Further the authors propose the cross-validation of the identified epitopes in experimental settings for confirming their immunogenicity to support the present findings. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. [The polymorphism of catechol-O-methyltransferase (COMT) and hemochromatosis (HFE) genes in the radiocontaminated regions residents with different chromosome aberration frequency].

    PubMed

    Ivanova, T I; Kondrashova, T V; Krikunova, L I; Smirnova, I A; Shentereva, N I; Sychenkova, N I; Rykova, E V; Zharikova, I A; Khorokhorina, V A; Riabchenko, N I; Zamulaeva, I A

    2010-01-01

    The association between polymorphisms in genes COMT, HFE that takes part in oxidative stress regulation, and chromosome aberration frequency in lymphocytes was assessed in 278 female residents of radiation polluted regions of Central Russia: Bryansk (322 kBk/m2) and Tula Districts (137Cs - 171 kBk/m2). The C187G, G845A genotyping of HFE and G1947A (H/L) of COMT was done by means of polymerase chain reaction-restriction fragment length polymorphism. Studied population was divided into 3 subgroups by level of chromosome aberrations per cell (0-2, 3-4, >5). There was shown statistically significant difference in distribution of COMTand HFE genotypes between the groups. The high frequency of chromosome aberrations (> or = 5%) was associated with homozygotes of the high activity COMT G/G and HFE CC. Heterozygotes for G1947A COMT and C187G HFE reveal negative association with the high frequency of chromosome aberrations and correspond to "resistance factors".

  6. Limited Variation in BK Virus T-Cell Epitopes Revealed by Next-Generation Sequencing

    PubMed Central

    Sahoo, Malaya K.; Tan, Susanna K.; Chen, Sharon F.; Kapusinszky, Beatrix; Concepcion, Katherine R.; Kjelson, Lynn; Mallempati, Kalyan; Farina, Heidi M.; Fernández-Viña, Marcelo; Tyan, Dolly; Grimm, Paul C.; Anderson, Matthew W.; Concepcion, Waldo

    2015-01-01

    BK virus (BKV) infection causing end-organ disease remains a formidable challenge to the hematopoietic cell transplant (HCT) and kidney transplant fields. As BKV-specific treatments are limited, immunologic-based therapies may be a promising and novel therapeutic option for transplant recipients with persistent BKV infection. Here, we describe a whole-genome, deep-sequencing methodology and bioinformatics pipeline that identify BKV variants across the genome and at BKV-specific HLA-A2-, HLA-B0702-, and HLA-B08-restricted CD8 T-cell epitopes. BKV whole genomes were amplified using long-range PCR with four inverse primer sets, and fragmentation libraries were sequenced on the Ion Torrent Personal Genome Machine (PGM). An error model and variant-calling algorithm were developed to accurately identify rare variants. A total of 65 samples from 18 pediatric HCT and kidney recipients with quantifiable BKV DNAemia underwent whole-genome sequencing. Limited genetic variation was observed. The median number of amino acid variants identified per sample was 8 (range, 2 to 37; interquartile range, 10), with the majority of variants (77%) detected at a frequency of <5%. When normalized for length, there was no statistical difference in the median number of variants across all genes. Similarly, the predominant virus population within samples harbored T-cell epitopes similar to the reference BKV strain that was matched for the BKV genotype. Despite the conservation of epitopes, low-level variants in T-cell epitopes were detected in 77.7% (14/18) of patients. Understanding epitope variation across the whole genome provides insight into the virus-immune interface and may help guide the development of protocols for novel immunologic-based therapies. PMID:26202116

  7. Hydronephrosis by an Aberrant Renal Artery: A Case Report

    PubMed Central

    Park, Byoung Seok; Jeong, Taek Kyun; Ma, Seong Kwon; Kim, Soo Wan; Kim, Nam Ho; Choi, Ki Chul; Jeong, Yong Yeon

    2003-01-01

    Ureteropelvic junction obstruction is usually intrinsic and is most common in children. Aberrant renal arteries are present in about 30% of individuals. Aberrant renal arteries to the inferior pole cross anteriorly to the ureter and may cause hydronephrosis. To the best of our knowledge, although there are some papers about aberrant renal arteries producing ureteropelvic junction obstruction, there is no report of a case which is diagnosed by the new modalities, such as computed tomography angiogram (CTA) or magnetic resonance angiogram (MRA). We describe a 36-year-old woman with right hydronephrosis. Kidney ultrasonogram and excretory urogram revealed right hydronephrosis. CTA and MRA clearly displayed an aberrant renal artery and hydronephrosis. The patient underwent surgical exploration. For the evaluation of hydronephrosis by an aberrant renal artery, use of CTA and MRA is advocated. PMID:12760271

  8. Ocular higher-order aberrations in a school children population.

    PubMed

    Papamastorakis, George; Panagopoulou, Sophia; Tsilimbaris, Militadis K; Pallikaris, Ioannis G; Plainis, Sotiris

    2015-01-01

    The primary objective of the study was to explore the statistics of ocular higher-order aberrations in a population of primary and secondary school children. A sample of 557 children aged 10-15 years were selected from two primary and two secondary schools in Heraklion, Greece. Children were classified by age in three subgroups: group I (10.7±0.5 years), group II (12.4±0.5 years) and group III (14.5±0.5 years). Ocular aberrations were measured using a wavefront aberrometer (COAS, AMO Wavefront Sciences, USA) at mesopic light levels (illuminance at cornea was 4lux). Wavefront analysis was achieved for a 5mm pupil. Statistical analysis was carried out for the right eye only. The average coefficient of most high-order aberrations did not differ from zero with the exception of vertical (0.076μm) and horizontal (0.018μm) coma, oblique trefoil (-0.055μm) and spherical aberration (0.018μm). The most prominent change between the three groups was observed for the spherical aberration, which increased from 0.007μm (SE 0.005) in group I to 0.011μm (SE 0.004) in group II and 0.030μm (SE 0.004) in group III. Significant differences were also found for the oblique astigmatism and the third-order coma aberrations. Differences in the low levels of ocular spherical aberration in young children possibly reflect differences in lenticular spherical aberration and relate to the gradient refractive index of the lens. The evaluation of spherical aberration at certain stages of eye growth may help to better understand the underlying mechanisms of myopia development. Copyright © 2014 Spanish General Council of Optometry. Published by Elsevier Espana. All rights reserved.

  9. Ocular higher-order aberrations in a school children population

    PubMed Central

    Papamastorakis, George; Panagopoulou, Sophia; Tsilimbaris, Militadis K.; Pallikaris, Ioannis G.; Plainis, Sotiris

    2014-01-01

    Purpose The primary objective of the study was to explore the statistics of ocular higher-order aberrations in a population of primary and secondary school children. Methods A sample of 557 children aged 10–15 years were selected from two primary and two secondary schools in Heraklion, Greece. Children were classified by age in three subgroups: group I (10.7 ± 0.5 years), group II (12.4 ± 0.5 years) and group III (14.5 ± 0.5 years). Ocular aberrations were measured using a wavefront aberrometer (COAS, AMO Wavefront Sciences, USA) at mesopic light levels (illuminance at cornea was 4 lux). Wavefront analysis was achieved for a 5 mm pupil. Statistical analysis was carried out for the right eye only. Results The average coefficient of most high-order aberrations did not differ from zero with the exception of vertical (0.076 μm) and horizontal (0.018 μm) coma, oblique trefoil (−0.055 μm) and spherical aberration (0.018 μm). The most prominent change between the three groups was observed for the spherical aberration, which increased from 0.007 μm (SE 0.005) in group I to 0.011 μm (SE 0.004) in group II and 0.030 μm (SE 0.004) in group III. Significant differences were also found for the oblique astigmatism and the third-order coma aberrations. Conclusions Differences in the low levels of ocular spherical aberration in young children possibly reflect differences in lenticular spherical aberration and relate to the gradient refractive index of the lens. The evaluation of spherical aberration at certain stages of eye growth may help to better understand the underlying mechanisms of myopia development. PMID:25288226

  10. Chemical Control over T-Cell Activation in Vivo Using Deprotection of trans-Cyclooctene-Modified Epitopes.

    PubMed

    van der Gracht, Anouk M F; de Geus, Mark A R; Camps, Marcel G M; Ruckwardt, Tracy J; Sarris, Alexi J C; Bremmers, Jessica; Maurits, Elmer; Pawlak, Joanna B; Posthoorn, Michelle M; Bonger, Kimberly M; Filippov, Dmitri V; Overkleeft, Herman S; Robillard, Marc S; Ossendorp, Ferry; van Kasteren, Sander I

    2018-06-15

    Activation of a cytotoxic T-cell is a complex multistep process, and tools to study the molecular events and their dynamics that result in T-cell activation in situ and in vivo are scarce. Here, we report the design and use of conditional epitopes for time-controlled T-cell activation in vivo. We show that trans-cyclooctene-protected SIINFEKL (with the lysine amine masked) is unable to elicit the T-cell response characteristic for the free SIINFEKL epitope. Epitope uncaging by means of an inverse-electron demand Diels-Alder (IEDDA) event restored T-cell activation and provided temporal control of T-cell proliferation in vivo.

  11. Efficient induction of CD25- iTreg by co-immunization requires strongly antigenic epitopes for T cells.

    PubMed

    Geng, Shuang; Yu, Yang; Kang, Youmin; Pavlakis, George; Jin, Huali; Li, Jinyao; Hu, Yanxin; Hu, Weibin; Wang, Shuang; Wang, Bin

    2011-05-05

    We previously showed that co-immunization with a protein antigen and a DNA vaccine coding for the same antigen induces CD40 low IL-10 high tolerogenic DCs, which in turn stimulates the expansion of antigen-specific CD4+CD25-Foxp3+ regulatory T cells (CD25- iTreg). However, it was unclear how to choose the antigen sequence to maximize tolerogenic antigen presentation and, consequently, CD25- iTreg induction. In the present study, we demonstrated the requirement of highly antigenic epitopes for CD25- iTreg induction. Firstly, we showed that the induction of CD25- iTreg by tolerogenic DC can be blocked by anti-MHC-II antibody. Next, both the number and the suppressive activity of CD25- iTreg correlated positively with the overt antigenicity of an epitope to activate T cells. Finally, in a mouse model of dermatitis, highly antigenic epitopes derived from a flea allergen not only induced more CD25- iTreg, but also more effectively prevented allergenic reaction to the allergen than did weakly antigenic epitopes. Our data thus indicate that efficient induction of CD25- iTreg requires highly antigenic peptide epitopes. This finding suggests that highly antigenic epitopes should be used for efficient induction of CD25- iTreg for clinical applications such as flea allergic dermatitis.

  12. Re-evaluating the generation of a "proteasome-independent" MHC class I-restricted CD8 T cell epitope.

    PubMed

    Wherry, E John; Golovina, Tatiana N; Morrison, Susan E; Sinnathamby, Gomathinayagam; McElhaugh, Michael J; Shockey, David C; Eisenlohr, Laurence C

    2006-02-15

    The proteasome is primarily responsible for the generation of MHC class I-restricted CTL epitopes. However, some epitopes, such as NP(147-155) of the influenza nucleoprotein (NP), are presented efficiently in the presence of proteasome inhibitors. The pathways used to generate such apparently "proteasome-independent" epitopes remain poorly defined. We have examined the generation of NP(147-155) and a second proteasome-dependent NP epitope, NP(50-57), using cells adapted to growth in the presence of proteasome inhibitors and also through protease overexpression. We observed that: 1) Ag processing and presentation proceeds in proteasome-inhibitor adapted cells but may become more dependent, at least in part, on nonproteasomal protease(s), 2) tripeptidyl peptidase II does not substitute for the proteasome in the generation of NP(147-155), 3) overexpression of leucine aminopeptidase, thymet oligopeptidase, puromycin-sensitive aminopeptidase, and bleomycin hydrolase, has little impact on the processing and presentation of NP(50-57) or NP(147-155), and 4) proteasome-inhibitor treatment altered the specificity of substrate cleavage by the proteasome using cell-free digests favoring NP(147-155) epitope preservation. Based on these results, we propose a central role for the proteasome in epitope generation even in the presence of proteasome inhibitors, although such inhibitors will likely alter cleavage patterns and may increase the dependence of the processing pathway on postproteasomal enzymes.

  13. Study on oxidative metabolism of S180 cells induced by meretrix glycopeptide

    NASA Astrophysics Data System (ADS)

    Wu, Jielian; Wang, Ping; Kang, Huizhu

    2017-03-01

    Previous in vitro researches have showed that MGP0501, a natural glycopeptide isolated from Meretrix meretrix, can inhibit proliferation or induce apoptosis in human gastric carcinoma, lung cance (A549), Leukemia K562, mouse melanoma B16, hepatoma or breast cancer cells (MDA-MB-231). In this study, we performed an in vivo study to investigate the anti-tumor effect and mechanisms of MGP0501 on xenografted sarcoma 180 (S180) in mice. Results revealed that the inhibition rates of S180 on solid tumors were 69.72%, with a concentration of 6 mg/kg MGP0501,which was significantly higher than that of CTX. In addition, the biochemical metabolism analysis showed that MGP0501 could enhance the activities of glutathione tablets (GSH-Px) and catalase (CAT) and supersxide dismutase (SOD) in liver of mice. The content of malondialdehyde (MDA) in liver, on the contrary, was decreased. The promotion to antioxidation and the elimination of free radical in liver also attribute the antitumor activity of MGP0501. These results indicated that in vivo antitumor activity is associated with enhanced antioxidant capacity in S180 xenografts-bearing mice.

  14. Glycoform Analysis of Recombinant and Human Immunodeficiency Virus Envelope Protein gp120 via Higher Energy Collisional Dissociation and Spectral-Aligning Strategy

    PubMed Central

    2015-01-01

    Envelope protein gp120 of human immunodeficiency virus (HIV) is armored with a dense glycan shield, which plays critical roles in envelope folding, immune-evasion, infectivity, and immunogenicity. Site-specific glycosylation profiling of recombinant gp120 is very challenging. Therefore, glycoproteomic analysis of native viral gp120 is still formidable to date. This challenge promoted us to employ a Q-Exactive mass spectrometer to identify low abundant glycopeptides from virion-associated gp120. To search the HCD-MS data for glycopeptides, a novel spectral-aligning strategy was developed. This strategy depends on the observation that glycopeptides and the corresponding deglycosylated peptides share very similar MS/MS pattern in terms of b- and y-ions that do not contain the site of glycosylation. Moreover, glycopeptides with an identical peptide backbone show nearly resembling spectra regardless of the attached glycan structures. For the recombinant gp120, this “copy–paste” spectral pattern of glycopeptides facilitated identification of 2224 spectra using only 18 spectral templates, and after precursor mass correction, 1268 (57%) spectra were assigned to 460 unique glycopeptides accommodating 19 N-linked and one O-linked glycosylation sites (glycosites). Strikingly, we were able to observe five N- and one O-linked glycosites in native gp120. We further revealed that except for Asn276 in the C2 region, glycans were processed to contain both high mannose and hybrid/complex glycans; an additional four N-linked glycosites were decorated with high mannose type. Core 1 O-linked glycan Gal1GalNAc1 was seen for the O-linked glycosite at Thr499. This direct observation of site-specific glycosylation of virion-derived gp120 has implications in HIV glycobiology and vaccine design. PMID:24941220

  15. Epitope mapping of the domains of human angiotensin converting enzyme.

    PubMed

    Kugaevskaya, Elena V; Kolesanova, Ekaterina F; Kozin, Sergey A; Veselovsky, Alexander V; Dedinsky, Ilya R; Elisseeva, Yulia E

    2006-06-01

    Somatic angiotensin converting enzyme (sACE), contains in its single chain two homologous domains (called N- and C-domains), each bearing a functional zinc-dependent active site. The present study aims to define the differences between two sACE domains and to localize experimentally revealed antigenic determinants (B-epitopes) in the recently determined three-dimensional structure of testicular tACE. The predicted linear antigenic determinants of human sACE were determined by peptide scanning ("PEPSCAN") approach. Essential difference was demonstrated between locations of the epitopes in the N- and C-domains. Comparison of arrangement of epitopes in the human domains with the corresponding sequences of some mammalian sACEs enabled to classify the revealed antigenic determinants as variable or conserved areas. The location of antigenic determinants with respect to various structural elements and to functionally important sites of the human sACE C-domain was estimated. The majority of antigenic sites of the C-domain were located at the irregular elements and at the boundaries of secondary structure elements. The data show structural differences between the sACE domains. The experimentally revealed antigenic determinants were in agreement with the recently determined crystal tACE structure. New potential applications are open to successfully produce mono-specific and group-specific antipeptide antibodies.

  16. Varied distribution of RhD epitopes in the Indian population.

    PubMed

    Kulkarni, S S; Gupte, S C; Vasantha, K; Mohanty, D; Ghosh, K

    2007-01-01

    Inhabited by more than 4000 caste and tribal groups, India has an extremely heterogenous population. For thousands of years many tribal groups have practised endogamy and are practically genetically isolated. Traditionally, polyclonal anti-D reagent has been used for RhD typing; though monoclonal antibodies are increasingly being used. As a result, blood banks find it difficult to assign the RhD status to an increasing number of people. As monoclonal anti-D typing reagents may not detect all RhD antigen epitopes, we studied the RhD antigen epitope heterogeneity in different population groups in India. Red cells of 5315 RhD-positive individuals belonging to different castes and tribes of India were tested with 30 different epitope-specific monoclonal anti-D antibodies. No single monoclonal antibody could detect all RhD-positive red cells detected by polyclonal antisera. The highest proportion of D antigen was detected by LHM 76/55 and BRAD-8 (98%) monoclonal antibodies. We need to determine the correct mix of monoclonal antibodies that will detect nearly all RhD antigens detected by polyclonal anti-D sera. Similarly, before accepting monoclonal anti-D for therapeutic use, it would be necessary to determine the appropriate ones for use in the Indian population.

  17. Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422

    PubMed Central

    Sandomenico, Annamaria; Leonardi, Antonio; Berisio, Rita; Sanguigno, Luca; Focà, Giuseppina; Focà, Annalia; Ruggiero, Alessia; Doti, Nunzianna; Muscariello, Livio; Barone, Daniela; Farina, Claudio; Owsianka, Ania; Vitagliano, Luigi

    2016-01-01

    ABSTRACT The hepatitis C virus (HCV) E2 envelope glycoprotein is crucial for virus entry into hepatocytes. A conserved region of E2 encompassing amino acids 412 to 423 (epitope I) and containing Trp420, a residue critical for virus entry, is recognized by several broadly neutralizing antibodies. Peptides embodying this epitope I sequence adopt a β-hairpin conformation when bound to neutralizing monoclonal antibodies (MAbs) AP33 and HCV1. We therefore generated new mouse MAbs that were able to bind to a cyclic peptide containing E2 residues 412 to 422 (C-epitope I) but not to the linear counterpart. These MAbs bound to purified E2 with affinities of about 50 nM, but they were unable to neutralize virus infection. Structural analysis of the complex between C-epitope I and one of our MAbs (C2) showed that the Trp420 side chain is largely buried in the combining site and that the Asn417 side chain, which is glycosylated in E2 and solvent exposed in other complexes, is slightly buried upon C2 binding. Also, the orientation of the cyclic peptide in the antibody-combining site is rotated by 180° compared to the orientations of the other complexes. All these structural features, however, do not explain the lack of neutralization activity. This is instead ascribed to the high degree of selectivity of the new MAbs for the cyclic epitope and to their inability to interact with the epitope in more flexible and extended conformations, which recent data suggest play a role in the mechanisms of neutralization escape. IMPORTANCE Hepatitis C virus (HCV) remains a major health care burden, affecting almost 3% of the global population. The conserved epitope comprising residues 412 to 423 of the viral E2 glycoprotein is a valid vaccine candidate because antibodies recognizing this region exhibit potent neutralizing activity. This epitope adopts a β-hairpin conformation when bound to neutralizing MAbs. We explored the potential of cyclic peptides mimicking this structure to elicit

  18. Generalized Alvarez lens for correction of laser aberrations

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    LaFortune, K N

    2004-12-02

    The Alvarez lens (US Patent No. 3,305,294 [1]) is a compact aberration corrector. The original design emphasized in the patent consists of a pair of adjacent optical elements that provide a variable focus. A lens system with a variable effective focal length is nothing new. Such systems are widely used in cameras, for example. It is the compactness and simplicity of operation that is the key advantage of the Alvarez lens. All of the complexity is folded into the design and fabrication of the optical elements. As mentioned in the Alvarez patent [1] and elaborated upon in Palusinski et al.more » [2], if one is willing to fold even more complexity into the optical elements, it is possible to correct higher-order aberrations as well. There is no theoretical limit to the number or degree of wavefront distortions that can be corrected. The only limitation is that there must be a fixed relative magnitude of the aberrations. Independent correction of each component of the higher-order aberrations can not be performed without additional elements and degrees of freedom [3]. Under some circumstances, coupling may be observed between different aberrations. This can be mitigated with the appropriate choice of design parameters. New methods are available today that increase the practicality of making higher-order aberration correctors [4,5,6].« less

  19. Statistical estimation of ultrasonic propagation path parameters for aberration correction.

    PubMed

    Waag, Robert C; Astheimer, Jeffrey P

    2005-05-01

    Parameters in a linear filter model for ultrasonic propagation are found using statistical estimation. The model uses an inhomogeneous-medium Green's function that is decomposed into a homogeneous-transmission term and a path-dependent aberration term. Power and cross-power spectra of random-medium scattering are estimated over the frequency band of the transmit-receive system by using closely situated scattering volumes. The frequency-domain magnitude of the aberration is obtained from a normalization of the power spectrum. The corresponding phase is reconstructed from cross-power spectra of subaperture signals at adjacent receive positions by a recursion. The subapertures constrain the receive sensitivity pattern to eliminate measurement system phase contributions. The recursion uses a Laplacian-based algorithm to obtain phase from phase differences. Pulse-echo waveforms were acquired from a point reflector and a tissue-like scattering phantom through a tissue-mimicking aberration path from neighboring volumes having essentially the same aberration path. Propagation path aberration parameters calculated from the measurements of random scattering through the aberration phantom agree with corresponding parameters calculated for the same aberrator and array position by using echoes from the point reflector. The results indicate the approach describes, in addition to time shifts, waveform amplitude and shape changes produced by propagation through distributed aberration under realistic conditions.

  20. A 12-residue epitope displayed on phage T7 reacts strongly with antibodies against foot-and-mouth disease virus.

    PubMed

    Wong, Chuan Loo; Yong, Chean Yeah; Muhamad, Azira; Syahir, Amir; Omar, Abdul Rahman; Sieo, Chin Chin; Tan, Wen Siang

    2018-05-01

    Foot-and-mouth disease (FMD) is a major threat to the livestock industry worldwide. Despite constant surveillance and effective vaccination, the perpetual mutations of the foot-and-mouth disease virus (FMDV) pose a huge challenge to FMD diagnosis. The immunodominant region of the FMDV VP1 protein (residues 131-170) displayed on phage T7 has been used to detect anti-FMDV in bovine sera. In the present study, the functional epitope was further delineated using amino acid sequence alignment, homology modelling and phage display. Two highly conserved regions (VP1 145-152 and VP1 159-170 ) were identified among different FMDV serotypes. The coding regions of these two epitopes were fused separately to the T7 genome and displayed on the phage particles. Interestingly, chimeric phage displaying the VP1 159-170 epitope demonstrated a higher antigenicity than that displaying the VP1 131-170 epitope. By contrast, phage T7 displaying the VP1 145-152 epitope did not react significantly with the anti-FMDV antibodies in vaccinated bovine sera. This study has successfully identified a smaller functional epitope, VP1 159-170 , located at the C-terminal end of the structural VP1 protein. The phage T7 displaying this shorter epitope is a promising diagnostic reagent to detect anti-FMDV antibodies in vaccinated animals.

  1. Antibodies Targeting Closely Adjacent or Minimally Overlapping Epitopes Can Displace One Another

    PubMed Central

    Abdiche, Yasmina Noubia; Yeung, Andy Yik; Ni, Irene; Stone, Donna; Miles, Adam; Morishige, Winse; Rossi, Andrea; Strop, Pavel

    2017-01-01

    Here we describe how real-time label-free biosensors can be used to identify antibodies that compete for closely adjacent or minimally overlapping epitopes on their specific antigen via a mechanism of antibody displacement. By kinetically perturbing one another’s binding towards their antigen via the formation of a transient trimolecular complex, antibodies can displace one another in a fully reversible and dose-dependent manner. Displacements can be readily identified when epitope binning assays are performed in a classical sandwich assay format whereby a solution antibody (analyte) is tested for binding to its antigen that is first captured via an immobilized antibody (ligand) because an inverted sandwiching response is observed when an analyte displaces a ligand, signifying the antigen’s unusually rapid dissociation from its ligand. In addition to classifying antibodies within a panel in terms of their ability to block or sandwich pair with one another, displacement provides a hybrid mechanism of competition. Using high-throughput epitope binning studies we demonstrate that displacements can be observed on any target, if the antibody panel contains appropriate epitope diversity. Unidirectional displacements occurring between disparate-affinity antibodies can generate apparent asymmetries in a cross-blocking experiment, confounding their interpretation. However, examining competition across a wide enough concentration range will often reveal that these displacements are reversible. Displacement provides a gentle and efficient way of eluting antigen from an otherwise high affinity binding partner which can be leveraged in designing reagents or therapeutic antibodies with unique properties. PMID:28060885

  2. Antibodies Targeting Closely Adjacent or Minimally Overlapping Epitopes Can Displace One Another.

    PubMed

    Abdiche, Yasmina Noubia; Yeung, Andy Yik; Ni, Irene; Stone, Donna; Miles, Adam; Morishige, Winse; Rossi, Andrea; Strop, Pavel

    2017-01-01

    Here we describe how real-time label-free biosensors can be used to identify antibodies that compete for closely adjacent or minimally overlapping epitopes on their specific antigen via a mechanism of antibody displacement. By kinetically perturbing one another's binding towards their antigen via the formation of a transient trimolecular complex, antibodies can displace one another in a fully reversible and dose-dependent manner. Displacements can be readily identified when epitope binning assays are performed in a classical sandwich assay format whereby a solution antibody (analyte) is tested for binding to its antigen that is first captured via an immobilized antibody (ligand) because an inverted sandwiching response is observed when an analyte displaces a ligand, signifying the antigen's unusually rapid dissociation from its ligand. In addition to classifying antibodies within a panel in terms of their ability to block or sandwich pair with one another, displacement provides a hybrid mechanism of competition. Using high-throughput epitope binning studies we demonstrate that displacements can be observed on any target, if the antibody panel contains appropriate epitope diversity. Unidirectional displacements occurring between disparate-affinity antibodies can generate apparent asymmetries in a cross-blocking experiment, confounding their interpretation. However, examining competition across a wide enough concentration range will often reveal that these displacements are reversible. Displacement provides a gentle and efficient way of eluting antigen from an otherwise high affinity binding partner which can be leveraged in designing reagents or therapeutic antibodies with unique properties.

  3. Fast photochemical oxidation of proteins (FPOP) maps the epitope of EGFR binding to adnectin.

    PubMed

    Yan, Yuetian; Chen, Guodong; Wei, Hui; Huang, Richard Y-C; Mo, Jingjie; Rempel, Don L; Tymiak, Adrienne A; Gross, Michael L

    2014-12-01

    Epitope mapping is an important tool for the development of monoclonal antibodies, mAbs, as therapeutic drugs. Recently, a class of therapeutic mAb alternatives, adnectins, has been developed as targeted biologics. They are derived from the 10th type III domain of human fibronectin ((10)Fn3). A common approach to map the epitope binding of these therapeutic proteins to their binding partners is X-ray crystallography. Although the crystal structure is known for Adnectin 1 binding to human epidermal growth factor receptor (EGFR), we seek to determine complementary binding in solution and to test the efficacy of footprinting for this purpose. As a relatively new tool in structural biology and complementary to X-ray crystallography, protein footprinting coupled with mass spectrometry is promising for protein-protein interaction studies. We report here the use of fast photochemical oxidation of proteins (FPOP) coupled with MS to map the epitope of EGFR-Adnectin 1 at both the peptide and amino-acid residue levels. The data correlate well with the previously determined epitopes from the crystal structure and are consistent with HDX MS data, which are presented in an accompanying paper. The FPOP-determined binding interface involves various amino-acid and peptide regions near the N terminus of EGFR. The outcome adds credibility to oxidative labeling by FPOP for epitope mapping and motivates more applications in the therapeutic protein area as a stand-alone method or in conjunction with X-ray crystallography, NMR, site-directed mutagenesis, and other orthogonal methods.

  4. Fast Photochemical Oxidation of Proteins (FPOP) Maps the Epitope of EGFR Binding to Adnectin

    NASA Astrophysics Data System (ADS)

    Yan, Yuetian; Chen, Guodong; Wei, Hui; Huang, Richard Y.-C.; Mo, Jingjie; Rempel, Don L.; Tymiak, Adrienne A.; Gross, Michael L.

    2014-12-01

    Epitope mapping is an important tool for the development of monoclonal antibodies, mAbs, as therapeutic drugs. Recently, a class of therapeutic mAb alternatives, adnectins, has been developed as targeted biologics. They are derived from the 10th type III domain of human fibronectin (10Fn3). A common approach to map the epitope binding of these therapeutic proteins to their binding partners is X-ray crystallography. Although the crystal structure is known for Adnectin 1 binding to human epidermal growth factor receptor (EGFR), we seek to determine complementary binding in solution and to test the efficacy of footprinting for this purpose. As a relatively new tool in structural biology and complementary to X-ray crystallography, protein footprinting coupled with mass spectrometry is promising for protein-protein interaction studies. We report here the use of fast photochemical oxidation of proteins (FPOP) coupled with MS to map the epitope of EGFR-Adnectin 1 at both the peptide and amino-acid residue levels. The data correlate well with the previously determined epitopes from the crystal structure and are consistent with HDX MS data, which are presented in an accompanying paper. The FPOP-determined binding interface involves various amino-acid and peptide regions near the N terminus of EGFR. The outcome adds credibility to oxidative labeling by FPOP for epitope mapping and motivates more applications in the therapeutic protein area as a stand-alone method or in conjunction with X-ray crystallography, NMR, site-directed mutagenesis, and other orthogonal methods.

  5. NEP: web server for epitope prediction based on antibody neutralization of viral strains with diverse sequences

    PubMed Central

    Chuang, Gwo-Yu; Liou, David; Kwong, Peter D.; Georgiev, Ivelin S.

    2014-01-01

    Delineation of the antigenic site, or epitope, recognized by an antibody can provide clues about functional vulnerabilities and resistance mechanisms, and can therefore guide antibody optimization and epitope-based vaccine design. Previously, we developed an algorithm for antibody-epitope prediction based on antibody neutralization of viral strains with diverse sequences and validated the algorithm on a set of broadly neutralizing HIV-1 antibodies. Here we describe the implementation of this algorithm, NEP (Neutralization-based Epitope Prediction), as a web-based server. The users must supply as input: (i) an alignment of antigen sequences of diverse viral strains; (ii) neutralization data for the antibody of interest against the same set of antigen sequences; and (iii) (optional) a structure of the unbound antigen, for enhanced prediction accuracy. The prediction results can be downloaded or viewed interactively on the antigen structure (if supplied) from the web browser using a JSmol applet. Since neutralization experiments are typically performed as one of the first steps in the characterization of an antibody to determine its breadth and potency, the NEP server can be used to predict antibody-epitope information at no additional experimental costs. NEP can be accessed on the internet at http://exon.niaid.nih.gov/nep. PMID:24782517

  6. Epitope Dampening Monotypic Measles Virus Hemagglutinin Glycoprotein Results in Resistance to Cocktail of Monoclonal Antibodies

    PubMed Central

    Lech, Patrycja J.; Tobin, Gregory J.; Bushnell, Ruth; Gutschenritter, Emily; Pham, Linh D.; Nace, Rebecca; Verhoeyen, Els; Cosset, François-Loïc; Muller, Claude P.; Russell, Stephen J.; Nara, Peter L.

    2013-01-01

    The measles virus (MV) is serologically monotypic. Life-long immunity is conferred by a single attack of measles or following vaccination with the MV vaccine. This is contrary to viruses such as influenza, which readily develop resistance to the immune system and recur. A better understanding of factors that restrain MV to one serotype may allow us to predict if MV will remain monotypic in the future and influence the design of novel MV vaccines and therapeutics. MV hemagglutinin (H) glycoprotein, binds to cellular receptors and subsequently triggers the fusion (F) glycoprotein to fuse the virus into the cell. H is also the major target for neutralizing antibodies. To explore if MV remains monotypic due to a lack of plasticity of the H glycoprotein, we used the technology of Immune Dampening to generate viruses with rationally designed N-linked glycosylation sites and mutations in different epitopes and screened for viruses that escaped monoclonal antibodies (mAbs). We then combined rationally designed mutations with naturally selected mutations to generate a virus resistant to a cocktail of neutralizing mAbs targeting four different epitopes simultaneously. Two epitopes were protected by engineered N-linked glycosylations and two epitopes acquired escape mutations via two consecutive rounds of artificial selection in the presence of mAbs. Three of these epitopes were targeted by mAbs known to interfere with receptor binding. Results demonstrate that, within the epitopes analyzed, H can tolerate mutations in different residues and additional N-linked glycosylations to escape mAbs. Understanding the degree of change that H can tolerate is important as we follow its evolution in a host whose immunity is vaccine induced by genotype A strains instead of multiple genetically distinct wild-type MVs. PMID:23300970

  7. Identification of NY-BR-1-specific CD4(+) T cell epitopes using HLA-transgenic mice.

    PubMed

    Gardyan, Adriane; Osen, Wolfram; Zörnig, Inka; Podola, Lilli; Agarwal, Maria; Aulmann, Sebastian; Ruggiero, Eliana; Schmidt, Manfred; Halama, Niels; Leuchs, Barbara; von Kalle, Christof; Beckhove, Philipp; Schneeweiss, Andreas; Jäger, Dirk; Eichmüller, Stefan B

    2015-06-01

    Breast cancer represents the second most common cancer type worldwide and has remained the leading cause of cancer-related deaths among women. The differentiation antigen NY-BR-1 appears overexpressed in invasive mammary carcinomas compared to healthy breast tissue, thus representing a promising target antigen for T cell based tumor immunotherapy approaches. Since efficient immune attack of tumors depends on the activity of tumor antigen-specific CD4(+) effector T cells, NY-BR-1 was screened for the presence of HLA-restricted CD4(+) T cell epitopes that could be included in immunological treatment approaches. Upon NY-BR-1-specific DNA immunization of HLA-transgenic mice and functional ex vivo analysis, a panel of NY-BR-1-derived library peptides was determined that specifically stimulated IFNγ secretion among splenocytes of immunized mice. Following in silico analyses, four candidate epitopes were determined which were successfully used for peptide immunization to establish NY-BR-1-specific, HLA-DRB1*0301- or HLA-DRB1*0401-restricted CD4(+) T cell lines from splenocytes of peptide immunized HLA-transgenic mice. Notably, all four CD4(+) T cell lines recognized human HLA-DR-matched dendritic cells (DC) pulsed with lysates of NY-BR-1 expressing human tumor cells, demonstrating natural processing of these epitopes also within the human system. Finally, CD4(+) T cells specific for all four CD4(+) T cell epitopes were detectable among PBMC of breast cancer patients, showing that CD4(+) T cell responses against the new epitopes are not deleted nor inactivated by self-tolerance mechanisms. Our results present the first NY-BR-1-specific HLA-DRB1*0301- and HLA-DRB1*0401-restricted T cell epitopes that could be exploited for therapeutic intervention against breast cancer. © 2014 UICC.

  8. Influence of Misalignment on High-Order Aberration Correction for Normal Human Eyes

    NASA Astrophysics Data System (ADS)

    Zhao, Hao-Xin; Xu, Bing; Xue, Li-Xia; Dai, Yun; Liu, Qian; Rao, Xue-Jun

    2008-04-01

    Although a compensation device can correct aberrations of human eyes, the effect will be degraded by its misalignment, especially for high-order aberration correction. We calculate the positioning tolerance of correction device for high-order aberrations, and within what degree the correcting effect is better than low-order aberration (defocus and astigmatism) correction. With fixed certain misalignment within the positioning tolerance, we calculate the residual wavefront rms aberration of the first-6 to first-35 terms along with the 3rd-5th terms of aberrations corrected, and the combined first-13 terms of aberrations are also studied under the same quantity of misalignment. However, the correction effect of high-order aberrations does not meliorate along with the increase of the high-order terms under some misalignment, moreover, some simple combined terms correction can achieve similar result as complex combinations. These results suggest that it is unnecessary to correct too much the terms of high-order aberrations which are difficult to accomplish in practice, and gives confidence to correct high-order aberrations out of the laboratory.

  9. Orthonormal aberration polynomials for anamorphic optical imaging systems with rectangular pupils.

    PubMed

    Mahajan, Virendra N

    2010-12-20

    The classical aberrations of an anamorphic optical imaging system, representing the terms of a power-series expansion of its aberration function, are separable in the Cartesian coordinates of a point on its pupil. We discuss the balancing of a classical aberration of a certain order with one or more such aberrations of lower order to minimize its variance across a rectangular pupil of such a system. We show that the balanced aberrations are the products of two Legendre polynomials, one for each of the two Cartesian coordinates of the pupil point. The compound Legendre polynomials are orthogonal across a rectangular pupil and, like the classical aberrations, are inherently separable in the Cartesian coordinates of the pupil point. They are different from the balanced aberrations and the corresponding orthogonal polynomials for a system with rotational symmetry but a rectangular pupil.

  10. Mapping of human autoantibody epitopes on aromatic L-amino acid decarboxylase.

    PubMed

    Candeloro, Paola; Voltattorni, Carla Borri; Perniola, Roberto; Bertoldi, Mariarita; Betterle, Corrado; Mannelli, Massimo; Giordano, Roberta; De Bellis, Annamaria; Tiberti, Claudio; Laureti, Stefano; Santeusanio, Fausto; Falorni, Alberto

    2007-03-01

    Aromatic l-amino acid decarboxylase (AADC) is target of autoantibodies in autoimmune polyendocrine syndrome I (APS I), especially in patients with autoimmune hepatitis. Little information is currently available on AADC autoantibody epitopes and on the interrelation between autoantibody-mediated inhibition of enzymatic activity and epitope specificity. We tested the immunoreactivity of full-length porcine AADC and of eight fragments of the enzyme with human serum from 18 patients with APS I, 199 with non-APS I autoimmune Addison's disease, 124 with type 1 diabetes mellitus, 36 with Graves' disease, and 141 healthy control subjects, and we evaluated the autoantibody-mediated enzymatic inhibition. AADC antibodies (Ab) were detected in 12 of 18 (67%) APS I patients and in six of 199 (3%) autoimmune Addison's disease patients. Four patients with autoimmune hepatitis were all positive for AADCAb. None of the 141 healthy control subjects, 82 patients with nonautoimmune adrenal insufficiency, 124 with type 1 diabetes mellitus, and 36 with Graves' disease were found positive. Two epitope regions, corresponding to amino acids 274-299 (E1) and 380-471 (E2) were identified. Localization of E1 was confirmed by displacement studies with synthetic peptides corresponding to peptides of porcine AADC. All 12 AADCAb-positive APS I sera reacted with E1, and seven of 12 (58%) reacted also with E2. E2-specific, but not E1-specific, autoantibodies were associated with a significant inhibition of in vitro AADC enzymatic activity. We mapped the human AADCAb epitopes to the middle and COOH-terminal regions of the enzyme. Autoantibodies to the COOH-terminal region induce a significant inhibition of enzymatic activity.

  11. Availability of the B beta(15-21) epitope on cross-linked human fibrin and its plasmic degradation products

    NASA Technical Reports Server (NTRS)

    Chen, F.; Haber, E.; Matsueda, G. R.

    1992-01-01

    The binding of radiolabeled monoclonal antifibrin antibody 59D8 (specific for fibrin but not fibrinogen) to a series of degraded fibrin clots showed that the availability of the B beta(15-21) epitope (against which 59D8 had been raised) was inversely proportional to the extent of clot lysis. Examination of digest supernatants revealed that the B beta(15-21) epitope was released from clots as a high molecular weight degradation product in the presence of calcium ions but that the generation of low molecular weight peptides occurred in the absence of calcium ions. To address the question of epitope accessibility, we compared levels of fibrin clot binding among four radioactively labeled antibodies: antifibrin monoclonal antibody 59D8, two antifibrinogen monoclonal antibodies that cross-reacted with fibrin, and an affinity-purified polyclonal antifibrinogen antibody. We expected that the antifibrinogen antibodies would show enhanced binding to clots in comparison with the antifibrin antibody. However, the epitope accessibility experiments showed that all four antibody preparations bound fibrin clots at comparable levels. Taken together, these studies demonstrated that one fibrin-specific epitope, B beta(15-21), remains available on clots as they undergo degradation by plasmin and, importantly, that the epitope is not solubilized at a rate faster than the rate at which the clot is itself solubilized. The availability of the B beta(15-21) epitope during the course of plasminolysis assures the potential utility of antifibrin antibodies such as 59D8 for detecting thrombi and targeting plasminogen activators.

  12. Epitope mapping and targeted quantitation of the cardiac biomarker troponin by SID-MRM mass spectrometry.

    PubMed

    Zhao, Cheng; Trudeau, Beth; Xie, Helen; Prostko, John; Fishpaugh, Jeffrey; Ramsay, Carol

    2014-06-01

    The absolute quantitation of the targeted protein using MS provides a promising method to evaluate/verify biomarkers used in clinical diagnostics. In this study, a cardiac biomarker, troponin I (TnI), was used as a model protein for method development. The epitope peptide of TnI was characterized by epitope excision followed with LC/MS/MS method and acted as the surrogate peptide for the targeted protein quantitation. The MRM-based MS assay using a stable internal standard that improved the selectivity, specificity, and sensitivity of the protein quantitation. Also, plasma albumin depletion and affinity enrichment of TnI by anti-TnI mAb-coated microparticles reduced the sample complexity, enhanced the dynamic range, and further improved the detecting sensitivity of the targeted protein in the biological matrix. Therefore, quantitation of TnI, a low abundant protein in human plasma, has demonstrated the applicability of the targeted protein quantitation strategy through its epitope peptide determined by epitope mapping method. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. [Construction and characterization of an epitope-mutated Asia 1 type foot-and-mouth disease virus].

    PubMed

    Zhang, Yan; Hu, Yonghao; Yang, Fan; Yang, Bo; Wang, Songhao; Zhu, Zixiang; Zheng, Haixue

    2015-01-01

    To generate an epitope-mutated foot-and-mouth disease virus (FMDV) as a marker vaccine, the infectious clone pAsia 1-FMDV containing the complete genomic cDNA of Asia 1 type FMDV was used as backbone, the residues at positions 27 and 31 in the 3D gene were mutated (H27Y and N31R). The resulting plasmid pAsia 1-FMDV-3DM encoding a mutated epitope was transfected into BHK-21 cells and the recombinant virus rAsia 1-3DM was rescued. The recombinant virus showed similar biological characteristics comparable with the parental virus. In serological neutralization test the antisera against recombine virus have a good reactivity with parental virus. The antisera against the mutant virus were shown to be reactive with the mutated epitope but not the wild-type one. The results indicated that the two virus strains could be distinguished by western blotting using synthetic peptides. This epitope-mutated FMDV strain will be evaluated as a potential marker vaccine against FMDV infections.

  14. New insights into non-conventional epitopes as T cell targets: The missing link for breaking immune tolerance in autoimmune disease?

    PubMed

    Harbige, James; Eichmann, Martin; Peakman, Mark

    2017-11-01

    The mechanism by which immune tolerance is breached in autoimmune disease is poorly understood. One possibility is that post-translational modification of self-antigens leads to peripheral recognition of neo-epitopes against which central and peripheral tolerance is inadequate. Accumulating evidence points to multiple mechanisms through which non-germline encoded sequences can give rise to these non-conventional epitopes which in turn engage the immune system as T cell targets. In particular, where these modifications alter the rules of epitope engagement with MHC molecules, such non-conventional epitopes offer a persuasive explanation for associations between specific HLA alleles and autoimmune diseases. In this review article, we discuss current understanding of mechanisms through which non-conventional epitopes may be generated, focusing on several recently described pathways that can transpose germline-encoded sequences. We contextualise these discoveries around type 1 diabetes, the prototypic organ-specific autoimmune disease in which specific HLA-DQ molecules confer high risk. Non-conventional epitopes have the potential to act as tolerance breakers or disease drivers in type 1 diabetes, prompting a timely re-evaluation of models of a etiopathogenesis. Future studies are required to elucidate the disease-relevance of a range of potential non-germline epitopes and their relationship to the natural peptide repertoire. Copyright © 2017 Elsevier Ltd. All rights reserved.

  15. Mismatch in epitope specificities between IFNγ inflamed and uninflamed conditions leads to escape from T lymphocyte killing in melanoma.

    PubMed

    Woods, Katherine; Knights, Ashley J; Anaka, Matthew; Schittenhelm, Ralf B; Purcell, Anthony W; Behren, Andreas; Cebon, Jonathan

    2016-01-01

    A current focus in cancer treatment is to broaden responses to immunotherapy. One reason these therapies may prove inadequate is that T lymphocytes fail to recognize the tumor due to differences in immunogenic epitopes presented by the cancer cells under inflammatory or non-inflammatory conditions. The antigen processing machinery of the cell, the proteasome, cleaves proteins into peptide epitopes for presentation on MHC complexes. Immunoproteasomes in inflammatory melanomas, and in antigen presenting cells of the immune system, are enzymatically different to standard proteasomes expressed by tumors with no inflammation. This corresponds to alterations in protein cleavage between proteasome subtypes, and a disparate repertoire of MHC-presented epitopes. We assessed steady state and IFNγ-induced immunoproteasome expression in melanoma cells. Using epitope specific T-lymphocyte clones, we studied processing and presentation of three NY-ESO-1 HLA-Cw3 restricted epitopes by melanoma cell lines. Our experimental model allowed comparison of the processing of three distinct epitopes from a single antigen presented on the same HLA complex. We further investigated processing of these epitopes by direct inhibition, or siRNA mediated knockdown, of the immunoproteasome catalytic subunit LMP7. Our data demonstrated a profound difference in the way in which immunogenic T-lymphocyte epitopes are presented by melanoma cells under IFNγ inflammatory versus non-inflammatory conditions. These alterations led to significant changes in the ability of T-lymphocytes to recognize and target melanoma cells. Our results illustrate a little-studied mechanism of immune escape by tumor cells which, with appropriate understanding and treatment, may be reversible. These data have implications for the design of cancer vaccines and adoptive T cell therapies.

  16. Diverse specificity and effector function among human antibodies to HIV-1 envelope glycoprotein epitopes exposed by CD4 binding

    DOE PAGES

    Guan, Yongjun; Pazgier, Marzena; Sajadi, Mohammad M.; ...

    2012-12-13

    The HIV-1 envelope glycoprotein (Env) undergoes conformational transitions consequent to CD4 binding and coreceptor engagement during viral entry. The physical steps in this process are becoming defined, but less is known about their significance as targets of antibodies potentially protective against HIV-1 infection. Here we probe the functional significance of transitional epitope exposure by characterizing 41 human mAbs specific for epitopes exposed on trimeric Env after CD4 engagement. These mAbs recognize three epitope clusters: cluster A, the gp120 face occluded by gp41 in trimeric Env; cluster B, a region proximal to the coreceptor-binding site (CoRBS) and involving the V1/V2 domain;more » and cluster C, the coreceptor-binding site. The mAbs were evaluated functionally by antibody-dependent, cell-mediated cytotoxicity (ADCC) and for neutralization of Tiers 1 and 2 pseudoviruses. All three clusters included mAbs mediating ADCC. However, there was a strong potency bias for cluster A, which harbors at least three potent ADCC epitopes whose cognate mAbs have electropositive paratopes. Cluster A epitopes are functional ADCC targets during viral entry in an assay format using virion-sensitized target cells. In contrast, only cluster C contained epitopes that were recognized by neutralizing mAbs. There was significant diversity in breadth and potency that correlated with epitope fine specificity. In contrast, ADCC potency had no relationship with neutralization potency or breadth for any epitope cluster. In conclusion, Fc-mediated effector function and neutralization coselect with specificity in anti-Env antibody responses, but the nature of selection is distinct for these two antiviral activities.« less

  17. Sex influences on the penetrance of HLA shared-epitope genotypes for rheumatoid arthritis

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Meyer, J.M.

    The association between rheumatoid arthritis (RA) and HLA DRB1 alleles may arise through linkage disequilibrium with a disease locus or the direct involvement of HLA alleles in RA. In support of the latter possibility, the shared-epitope hypothesis has been postulated, stating that conformationally similar DR{beta} chains encoded by several DRB1 alleles confer disease susceptibility. To examine these alternative hypotheses of marker-disease association and to investigate gender differences in RA susceptibility, we analyzed the distributions of PCR-based DRB1 genotypes of 309 Caucasian RA patients and 283 Caucasian controls. Initially, the marker-association-segregation {chi}{sup 2} method was used to evaluate evidence for linkagemore » disequilibrium and the direct involvement of markers DR4 Dw4, DR4 Dw14, and DR1 in RA susceptibility. Additional shared-epitope models that grouped DRB1 alleles into five classes (*0401, *0404/*0102, *0405/*0408/*0101, *1001, and all others) and postulated relationships between genotypes and RA susceptibility were also fitted to observed genotypic distributions by the method of minimal {chi}{sup 2}. For females, a linkage-disequilibrium model provided a good fit to the data, as did a shared-epitope model with RA most penetrant among individuals with the *0401, *0401 genotype. For males, the best model indicated highest RA penetrance among shared-epitope compound heterozygotes. Clinically, male RA patients had more subcutaneous nodules and greater use of slowly acting antirheumatic drugs, while female RA patients had earlier disease onset. This study therefore suggests that sex-related factors influence the RA penetrance associated with DRB1 shared-epitope genotypes and that DRB1 effects on RA prognosis and pathogenesis should be considered separately for men and women. 67 refs., 7 tabs.« less

  18. Ara h 2 peptides containing dominant CD4+ T-cell epitopes: candidates for a peanut allergy therapeutic.

    PubMed

    Prickett, Sara R; Voskamp, Astrid L; Dacumos-Hill, April; Symons, Karen; Rolland, Jennifer M; O'Hehir, Robyn E

    2011-03-01

    Peanut allergy is a life-threatening condition; there is currently no cure. Although whole allergen extracts are used for specific immunotherapy for many allergies, they can cause severe reactions, and even fatalities, in peanut allergy. This study aimed to identify short, T-cell epitope-based peptides that target allergen-specific CD4(+) T cells but do not bind IgE as candidates for safe peanut-specific immunotherapy. Multiple CD4(+) T-cell lines specific for the major peanut allergen Ara h 2 were generated from PBMCs of 16 HLA-diverse subjects with peanut allergy by using 5,6-carboxyfluorescein diacetate succinimidylester-based methodology. Proliferation and ELISPOT assays were used to identify dominant epitopes recognized by T-cell lines and to confirm recognition by peripheral blood T cells of epitope-based peptides modified for therapeutic production. HLA restriction of core epitope recognition was investigated by using anti-HLA blocking antibodies and HLA genotyping. Serum-IgE peptide-binding was assessed by dot-blot. Five dominant CD4(+) T-cell epitopes were identified in Ara h 2. In combination, these were presented by HLA-DR, HLA-DP, and HLA-DQ molecules and recognized by T cells from all 16 subjects. Three short peptide variants containing these T-cell epitopes were designed with cysteine-to-serine substitutions to facilitate stability and therapeutic production. Variant peptides showed HLA-binding degeneracy, did not bind peanut-specific serum IgE, and could directly target T(H)2-type T cells in peripheral blood of subjects with allergy. Short CD4(+) T-cell epitope-based Ara h 2 peptides were identified as novel candidates for a T-cell-targeted peanut-specific immunotherapy for an HLA-diverse population. Copyright © 2010 American Academy of Allergy, Asthma & Immunology. Published by Mosby, Inc. All rights reserved.

  19. In Silico Identification of Epitopes in Mycobacterium avium subsp. paratuberculosis Proteins That Were Upregulated under Stress Conditions

    PubMed Central

    Gurung, Ratna B.; Purdie, Auriol C.; Begg, Douglas J.

    2012-01-01

    Johne's disease in ruminants is caused by Mycobacterium avium subsp. paratuberculosis. Diagnosis of M. avium subsp. paratuberculosis infection is difficult, especially in the early stages. To date, ideal antigen candidates are not available for efficient immunization or immunodiagnosis. This study reports the in silico selection and subsequent analysis of epitopes of M. avium subsp. paratuberculosis proteins that were found to be upregulated under stress conditions as a means to identify immunogenic candidate proteins. Previous studies have reported differential regulation of proteins when M. avium subsp. paratuberculosis is exposed to stressors which induce a response similar to dormancy. Dormancy may be involved in evading host defense mechanisms, and the host may also mount an immune response against these proteins. Twenty-five M. avium subsp. paratuberculosis proteins that were previously identified as being upregulated under in vitro stress conditions were analyzed for B and T cell epitopes by use of the prediction tools at the Immune Epitope Database and Analysis Resource. Major histocompatibility complex class I T cell epitopes were predicted using an artificial neural network method, and class II T cell epitopes were predicted using the consensus method. Conformational B cell epitopes were predicted from the relevant three-dimensional structure template for each protein. Based on the greatest number of predicted epitopes, eight proteins (MAP2698c [encoded by desA2], MAP2312c [encoded by fadE19], MAP3651c [encoded by fadE3_2], MAP2872c [encoded by fabG5_2], MAP3523c [encoded by oxcA], MAP0187c [encoded by sodA], and the hypothetical proteins MAP3567 and MAP1168c) were identified as potential candidates for study of antibody- and cell-mediated immune responses within infected hosts. PMID:22496492

  20. Recognition of Porphyromonas gingivalis Gingipain Epitopes by Natural IgM Binding to Malondialdehyde Modified Low-Density Lipoprotein

    PubMed Central

    Turunen, S. Pauliina; Kummu, Outi; Harila, Kirsi; Veneskoski, Marja; Soliymani, Rabah; Baumann, Marc; Pussinen, Pirkko J.; Hörkkö, Sohvi

    2012-01-01

    Objective Increased risk for atherosclerosis is associated with infectious diseases including periodontitis. Natural IgM antibodies recognize pathogen-associated molecular patterns on bacteria, and oxidized lipid and protein epitopes on low-density lipoprotein (LDL) and apoptotic cells. We aimed to identify epitopes on periodontal pathogen Porphyromonas gingivalis recognized by natural IgM binding to malondialdehyde (MDA) modified LDL. Methods and Results Mouse monoclonal IgM (MDmAb) specific for MDA-LDL recognized epitopes on P. gingivalis on flow cytometry and chemiluminescence immunoassays. Immunization of C57BL/6 mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and apoptotic cells. Immunization of LDLR−/− mice with P. gingivalis induced IgM, but not IgG, immune response to MDA-LDL and diminished aortic lipid deposition. On Western blot MDmAb bound to P. gingivalis fragments identified as arginine-specific gingipain (Rgp) by mass spectrometry. Recombinant domains of Rgp produced in E. coli were devoid of phosphocholine epitopes but contained epitopes recognized by MDmAb and human serum IgM. Serum IgM levels to P. gingivalis were associated with anti-MDA-LDL levels in humans. Conclusion Gingipain of P. gingivalis is recognized by natural IgM and shares molecular identity with epitopes on MDA-LDL. These findings suggest a role for natural antibodies in the pathogenesis of two related inflammatory diseases, atherosclerosis and periodontitis. PMID:22496875