Design and synthesis of an antigenic mimic of the Ebola glycoprotein

OpenAIRE

Rutledge, Ryan D.; Huffman, Brian J.; Cliffel, David E.; Wright, David W.

2008-01-01

An antigenic mimic of the Ebola glycoprotein was synthesized and tested for its ability to be recognized by an anti-Ebola glycoprotein antibody. Epitope-mapping procedures yielded a suitable epitope that, when presented on the surface of a nanoparticle, forms a structure that is recognized by an antibody specific for the native protein. This mimic-antibody interaction has been quantitated through ELISA and QCM-based methods and yielded an affinity (Kd = 12 × 10−6 M) within two orders of magni...

Viral O-GalNAc peptide epitopes

DEFF Research Database (Denmark)

Olofsson, Sigvard; Blixt, Klas Ola; Bergström, Tomas

2016-01-01

Viral envelope glycoproteins are major targets for antibodies that bind to and inactivate viral particles. The capacity of a viral vaccine to induce virus-neutralizing antibodies is often used as a marker for vaccine efficacy. Yet the number of known neutralization target epitopes is restricted o...

Histidine-rich glycoprotein protects from systemic Candida infection.

Directory of Open Access Journals (Sweden)

Victoria Rydengård

2008-08-01

Full Text Available Fungi, such as Candida spp., are commonly found on the skin and at mucosal surfaces. Yet, they rarely cause invasive infections in immunocompetent individuals, an observation reflecting the ability of our innate immune system to control potentially invasive microbes found at biological boundaries. Antimicrobial proteins and peptides are becoming increasingly recognized as important effectors of innate immunity. This is illustrated further by the present investigation, demonstrating a novel antifungal role of histidine-rich glycoprotein (HRG, an abundant and multimodular plasma protein. HRG bound to Candida cells, and induced breaks in the cell walls of the organisms. Correspondingly, HRG preferentially lysed ergosterol-containing liposomes but not cholesterol-containing ones, indicating a specificity for fungal versus other types of eukaryotic membranes. Both antifungal and membrane-rupturing activities of HRG were enhanced at low pH, and mapped to the histidine-rich region of the protein. Ex vivo, HRG-containing plasma as well as fibrin clots exerted antifungal effects. In vivo, Hrg(-/- mice were susceptible to infection by C. albicans, in contrast to wild-type mice, which were highly resistant to infection. The results demonstrate a key and previously unknown antifungal role of HRG in innate immunity.

Vaccine-induced antibodies to herpes simplex virus glycoprotein D epitopes involved in virus entry and cell-to-cell spread correlate with protection against genital disease in guinea pigs.

Science.gov (United States)

Hook, Lauren M; Cairns, Tina M; Awasthi, Sita; Brooks, Benjamin D; Ditto, Noah T; Eisenberg, Roselyn J; Cohen, Gary H; Friedman, Harvey M

2018-05-01

Herpes simplex virus type 2 (HSV-2) glycoprotein D (gD2) subunit antigen is included in many preclinical candidate vaccines. The rationale for including gD2 is to produce antibodies that block crucial gD2 epitopes involved in virus entry and cell-to-cell spread. HSV-2 gD2 was the only antigen in the Herpevac Trial for Women that protected against HSV-1 genital infection but not HSV-2. In that trial, a correlation was detected between gD2 ELISA titers and protection against HSV-1, supporting the importance of antibodies. A possible explanation for the lack of protection against HSV-2 was that HSV-2 neutralization titers were low, four-fold lower than to HSV-1. Here, we evaluated neutralization titers and epitope-specific antibody responses to crucial gD2 epitopes involved in virus entry and cell-to-cell spread as correlates of immune protection against genital lesions in immunized guinea pigs. We detected a strong correlation between neutralizing antibodies and protection against genital disease. We used a high throughput biosensor competition assay to measure epitope-specific responses to seven crucial gD2 linear and conformational epitopes involved in virus entry and spread. Some animals produced antibodies to most crucial epitopes while others produced antibodies to few. The number of epitopes recognized by guinea pig immune serum correlated with protection against genital lesions. We confirmed the importance of antibodies to each crucial epitope using monoclonal antibody passive transfer that improved survival and reduced genital disease in mice after HSV-2 genital challenge. We re-evaluated our prior study of epitope-specific antibody responses in women in the Herpevac Trial. Humans produced antibodies that blocked significantly fewer crucial gD2 epitopes than guinea pigs, and antibody responses in humans to some linear epitopes were virtually absent. Neutralizing antibody titers and epitope-specific antibody responses are important immune parameters to

Insights into the Evolution of Hydroxyproline-Rich Glycoproteins from 1000 Plant Transcriptomes.

Science.gov (United States)

Johnson, Kim L; Cassin, Andrew M; Lonsdale, Andrew; Wong, Gane Ka-Shu; Soltis, Douglas E; Miles, Nicholas W; Melkonian, Michael; Melkonian, Barbara; Deyholos, Michael K; Leebens-Mack, James; Rothfels, Carl J; Stevenson, Dennis W; Graham, Sean W; Wang, Xumin; Wu, Shuangxiu; Pires, J Chris; Edger, Patrick P; Carpenter, Eric J; Bacic, Antony; Doblin, Monika S; Schultz, Carolyn J

2017-06-01

The carbohydrate-rich cell walls of land plants and algae have been the focus of much interest given the value of cell wall-based products to our current and future economies. Hydroxyproline-rich glycoproteins (HRGPs), a major group of wall glycoproteins, play important roles in plant growth and development, yet little is known about how they have evolved in parallel with the polysaccharide components of walls. We investigate the origins and evolution of the HRGP superfamily, which is commonly divided into three major multigene families: the arabinogalactan proteins (AGPs), extensins (EXTs), and proline-rich proteins. Using motif and amino acid bias, a newly developed bioinformatics pipeline, we identified HRGPs in sequences from the 1000 Plants transcriptome project (www.onekp.com). Our analyses provide new insights into the evolution of HRGPs across major evolutionary milestones, including the transition to land and the early radiation of angiosperms. Significantly, data mining reveals the origin of glycosylphosphatidylinositol (GPI)-anchored AGPs in green algae and a 3- to 4-fold increase in GPI-AGPs in liverworts and mosses. The first detection of cross-linking (CL)-EXTs is observed in bryophytes, which suggests that CL-EXTs arose though the juxtaposition of preexisting SP n EXT glycomotifs with refined Y-based motifs. We also detected the loss of CL-EXT in a few lineages, including the grass family (Poaceae), that have a cell wall composition distinct from other monocots and eudicots. A key challenge in HRGP research is tracking individual HRGPs throughout evolution. Using the 1000 Plants output, we were able to find putative orthologs of Arabidopsis pollen-specific GPI-AGPs in basal eudicots. © 2017 American Society of Plant Biologists. All Rights Reserved.

Bioinformatics Analysis of Envelope Glycoprotein E epitopes of ...

African Journals Online (AJOL)

User

2011-05-02

May 2, 2011 ... 1National Centre of Excellence in Molecular Biology, University of the Punjab Lahore, Pakistan. 2Department of ..... E glycoprotein and its interaction with antibody with the method of molecular dynamics and molecular model ...

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

Science.gov (United States)

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.

Identification of continuous human B-cell epitopes in the VP35, VP40, nucleoprotein and glycoprotein of Ebola virus.

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Pierre Becquart

Full Text Available Ebola virus (EBOV is a highly virulent human pathogen. Recovery of infected patients is associated with efficient EBOV-specific immunoglobulin G (IgG responses, whereas fatal outcome is associated with defective humoral immunity. As B-cell epitopes on EBOV are poorly defined, we sought to identify specific epitopes in four EBOV proteins (Glycoprotein (GP, Nucleoprotein (NP, and matrix Viral Protein (VP40 and VP35. For the first time, we tested EBOV IgG+ sera from asymptomatic individuals and symptomatic Gabonese survivors, collected during the early humoral response (seven days after the end of symptoms and the late memory phase (7-12 years post-infection. We also tested sera from EBOV-seropositive patients who had never had clinical signs of hemorrhagic fever or who lived in non-epidemic areas (asymptomatic subjects. We found that serum from asymptomatic individuals was more strongly reactive to VP40 peptides than to GP, NP or VP35. Interestingly, anti-EBOV IgG from asymptomatic patients targeted three immunodominant regions of VP40 reported to play a crucial role in virus assembly and budding. In contrast, serum from most survivors of the three outbreaks, collected a few days after the end of symptoms, reacted mainly with GP peptides. However, in asymptomatic subjects the longest immunodominant domains were identified in GP, and analysis of the GP crystal structure revealed that these domains covered a larger surface area of the chalice bowl formed by three GP1 subunits. The B-cell epitopes we identified in the EBOV VP35, VP40, NP and GP proteins may represent important tools for understanding the humoral response to this virus and for developing new antibody-based therapeutics or detection methods.

Identification of continuous human B-cell epitopes in the VP35, VP40, nucleoprotein and glycoprotein of Ebola virus.

Science.gov (United States)

Becquart, Pierre; Mahlakõiv, Tanel; Nkoghe, Dieudonné; Leroy, Eric M

2014-01-01

Ebola virus (EBOV) is a highly virulent human pathogen. Recovery of infected patients is associated with efficient EBOV-specific immunoglobulin G (IgG) responses, whereas fatal outcome is associated with defective humoral immunity. As B-cell epitopes on EBOV are poorly defined, we sought to identify specific epitopes in four EBOV proteins (Glycoprotein (GP), Nucleoprotein (NP), and matrix Viral Protein (VP)40 and VP35). For the first time, we tested EBOV IgG+ sera from asymptomatic individuals and symptomatic Gabonese survivors, collected during the early humoral response (seven days after the end of symptoms) and the late memory phase (7-12 years post-infection). We also tested sera from EBOV-seropositive patients who had never had clinical signs of hemorrhagic fever or who lived in non-epidemic areas (asymptomatic subjects). We found that serum from asymptomatic individuals was more strongly reactive to VP40 peptides than to GP, NP or VP35. Interestingly, anti-EBOV IgG from asymptomatic patients targeted three immunodominant regions of VP40 reported to play a crucial role in virus assembly and budding. In contrast, serum from most survivors of the three outbreaks, collected a few days after the end of symptoms, reacted mainly with GP peptides. However, in asymptomatic subjects the longest immunodominant domains were identified in GP, and analysis of the GP crystal structure revealed that these domains covered a larger surface area of the chalice bowl formed by three GP1 subunits. The B-cell epitopes we identified in the EBOV VP35, VP40, NP and GP proteins may represent important tools for understanding the humoral response to this virus and for developing new antibody-based therapeutics or detection methods.

Design and evaluation of a multi-epitope assembly Peptide (MEAP against herpes simplex virus type 2 infection in BALB/c mice

Directory of Open Access Journals (Sweden)

Pan Mingjie

2011-05-01

Full Text Available Abstract Background Human herpes simplex virus (HSV 1 and 2 causes oral, ocular, or genital infections, which remains a significant health problem worldwide. HSV-1 and -2 infections in humans range from localized skin infections of the oral, ocular, and genital regions to severe and often disseminated infections in immunocompromised hosts. Epitope based vaccination is a promising mean to achieve protective immunity and to avoid infections with Human herpes simplex virus type 2 (HSV-2. Methods The twelve selected epitopes, six B cell epitopes from different glycoprotein of HSV-2 (amino acid residues 466-473 (EQDRKPRN from envelope glycoprotein B, 216-223 (GRTDRPSA from C, 6-18 (DPSLKMADPNRFR from D, 483-491 (DPPERPDSP from E, 572-579 (EPPDDDDS from G and 286-295 (CRRRYRRPRG from I glycoprotein of HSV-2, four CD4+ T cell epitopes (amino acid residues 21-28 (NLPVLDQL from D, 162-177 (KDVTVSQVWFGHRYSQ from B, 205-224 (KAYQQGVTVDSIGMLPRFIP from D and 245-259 (KPPYTSTLLPPELSD from D and two CD8+ T cell epitopes (amino acid residues 10-20 (KMADPNRFRGK from D and 268-276 (ALLEDPAGT from D, are responsible for the elicitation of the neutralizing antibodies and cytotoxic T lymphocytes (CTLs that impart protective immunity to the host. In this study, all above epitopes were inserted into the extracellular fragment (amino acid residues 1-290 of HSV-2 glycoprotein D to construct multi-epitope assembly peptides (MEAPs by replacing some non-epitope amino acid sequences. The epitope independency of the MEAPs was predicted by three-dimensional software algorithms. The gene of the selected MEAP was expressed in E.coli BL21(DE3, and its protective efficacy against HSV-2 infection was assessed in BALB/c mice. Results The MEAP, with each inserted epitopes independently displayed on the molecule surface, was selected as candidate proteins. The results showed that the MEAP was highly immunogenic and could elicit high titer neutralizing antibodies and cell

Monoclonal antibody to an external epitope of the human mdr1 P-glycoprotein

NARCIS (Netherlands)

Arceci, R. J.; Stieglitz, K.; Bras, J.; Schinkel, A.; Baas, F.; Croop, J.

1993-01-01

A membrane glycoprotein, termed P-glycoprotein, has been shown to be responsible for cross-resistance to a broad range of structurally and functionally distinct cytotoxic agents. P-glycoprotein, encoded in humans by the mdr1 gene, functions as an energy-dependent efflux pump to exclude these

The peanut lectin-binding glycoproteins of human epidermal keratinocytes

International Nuclear Information System (INIS)

Morrison, A.I.; Keeble, S.; Watt, F.M.

1988-01-01

The peanut lectin (PNA) is known to bind more strongly to keratinocytes that are undergoing terminal differentiation than to proliferating keratinocytes. In order to investigate the significance of this change in cell-surface carbohydrate authors have identified the PNA-binding glycoproteins of cultured human keratinocytes and antibodies against them. Two heavily glycosylated bands of 110 and 250 kDa were resolved by PAGE of [ 14 C]galactose- or [ 14 C]mannose- and [ 14 C]glucosamine-labeled cell extracts eluted with galactose from PNA affinity columns. The higher molecular weight band was also detected on PNA blots of unlabeled cell extracts transferred to nitrocellulose. Both bands were sensitive to pronase digestion, but only the 250-kDa band was digested with trypsin. A rabbit antiserum that we prepared (anti-PNA-gp) immunoprecipitated both bands from cell extracts. In contrast to PNA, anti-PNA-gp bound equally to proliferating and terminally differentiating cells, indicating that some epitope(s) of the PNA-binding glycoproteins is present on the cell surface prior to terminal differentiation. When keratinocytes grown as a monolayer in low-calcium medium were switched to medium containing 2 mM calcium ions in order to induce desmosome formation and stratification, there was a dramatic redistribution of the PNA-binding glycoproteins, which became concentrated at the boundaries between cells. This may suggest a role for the glycoproteins in cell-cell interactions during stratification

The Lyssavirus glycoprotein: A key to cross-immunity

International Nuclear Information System (INIS)

Buthelezi, Sindisiwe G.; Dirr, Heini W.; Chakauya, Ereck; Chikwamba, Rachel; Martens, Lennart; Tsekoa, Tsepo L.; Stoychev, Stoyan H.; Vandermarliere, Elien

2016-01-01

Rabies is an acute viral encephalomyelitis in warm-blooded vertebrates, caused by viruses belonging to Rhabdovirus family and genus Lyssavirus. Although rabies is categorised as a neglected disease, the rabies virus (RABV) is the most studied amongst Lyssaviruses which show nearly identical infection patterns. In efforts to improving post-exposure prophylaxis, several anti-rabies monoclonal antibodies (mAbs) targeting the glycoprotein (G protein) sites I, II, III and G5 have been characterized. To explore cross-neutralization capacity of available mAbs and discover new possible B-cell epitopes, we have analyzed all available glycoprotein sequences from Lyssaviruses with a focus on sequence variation and conservation. This information was mapped on the structure of a representative G protein. We proposed several possible cross-neutralizing B-cell epitopes (GUVTTTF, WLRTV, REECLD and EHLVVEEL) in complement to the already well-characterized antigenic sites. The research could facilitate development of novel cross-reactive mAbs against RABV and even more broad, against possibly all Lyssavirus members. -- Highlights: •The current PEP has raised safety and availability concerns. •Cocktails of mAbs have been proposed as alternative treatment. •Amino acid conservation amongst Lyssavirus G proteins was studied. •Possible cross-neutralizing B-cell epitopes were proposed.

The Lyssavirus glycoprotein: A key to cross-immunity

Energy Technology Data Exchange (ETDEWEB)

Buthelezi, Sindisiwe G. [Council for Scientific and Industrial Research, Biosciences Unit, Pretoria (South Africa); Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg (South Africa); Dirr, Heini W. [Protein Structure-Function Research Unit, School of Molecular and Cell Biology, University of the Witwatersrand, Johannesburg (South Africa); Chakauya, Ereck; Chikwamba, Rachel [Council for Scientific and Industrial Research, Biosciences Unit, Pretoria (South Africa); Martens, Lennart [Unit for Computational Omics and Systems Biology, Medical Biotechnology Center, VIB, Ghent (Belgium); Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Ghent (Belgium); Tsekoa, Tsepo L. [Council for Scientific and Industrial Research, Biosciences Unit, Pretoria (South Africa); Stoychev, Stoyan H., E-mail: Sstoychev@csir.co.za [Council for Scientific and Industrial Research, Biosciences Unit, Pretoria (South Africa); Vandermarliere, Elien [Unit for Computational Omics and Systems Biology, Medical Biotechnology Center, VIB, Ghent (Belgium); Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Ghent (Belgium); Bioinformatics Institute Gent, Ghent University, Ghent (Belgium)

2016-11-15

Rabies is an acute viral encephalomyelitis in warm-blooded vertebrates, caused by viruses belonging to Rhabdovirus family and genus Lyssavirus. Although rabies is categorised as a neglected disease, the rabies virus (RABV) is the most studied amongst Lyssaviruses which show nearly identical infection patterns. In efforts to improving post-exposure prophylaxis, several anti-rabies monoclonal antibodies (mAbs) targeting the glycoprotein (G protein) sites I, II, III and G5 have been characterized. To explore cross-neutralization capacity of available mAbs and discover new possible B-cell epitopes, we have analyzed all available glycoprotein sequences from Lyssaviruses with a focus on sequence variation and conservation. This information was mapped on the structure of a representative G protein. We proposed several possible cross-neutralizing B-cell epitopes (GUVTTTF, WLRTV, REECLD and EHLVVEEL) in complement to the already well-characterized antigenic sites. The research could facilitate development of novel cross-reactive mAbs against RABV and even more broad, against possibly all Lyssavirus members. -- Highlights: •The current PEP has raised safety and availability concerns. •Cocktails of mAbs have been proposed as alternative treatment. •Amino acid conservation amongst Lyssavirus G proteins was studied. •Possible cross-neutralizing B-cell epitopes were proposed.

Human broadly neutralizing antibodies to the envelope glycoprotein complex of hepatitis C virus

DEFF Research Database (Denmark)

Giang, Erick; Dorner, Marcus; Prentoe, Jannick C

2012-01-01

, and an effective vaccine should target conserved T- and B-cell epitopes of the virus. Conserved B-cell epitopes overlapping the CD81 receptor-binding site (CD81bs) on the E2 viral envelope glycoprotein have been reported previously and provide promising vaccine targets. In this study, we isolated 73 human m......Abs recognizing five distinct antigenic regions on the virus envelope glycoprotein complex E1E2 from an HCV-immune phage-display antibody library by using an exhaustive-panning strategy. Many of these mAbs were broadly neutralizing. In particular, the mAb AR4A, recognizing a discontinuous epitope outside the CD81......bs on the E1E2 complex, has an exceptionally broad neutralizing activity toward diverse HCV genotypes and protects against heterologous HCV challenge in a small animal model. The mAb panel will be useful for the design and development of vaccine candidates to elicit broadly neutralizing antibodies...

Rational design and synthesis of altered peptide ligands based on human myelin oligodendrocyte glycoprotein 35-55 epitope: inhibition of chronic experimental autoimmune encephalomyelitis in mice.

Science.gov (United States)

Tselios, Theodore; Aggelidakis, Mihalis; Tapeinou, Anthi; Tseveleki, Vivian; Kanistras, Ioannis; Gatos, Dimitrios; Matsoukas, John

2014-11-04

Experimental autoimmune encephalomyelitis (EAE) is a demyelinating disease of the central nervous system and is an animal model of multiple sclerosis (MS). Although the etiology of MS remains unclear, there is evidence T-cell recognition of immunodominant epitopes of myelin proteins, such as the 35-55 epitope of myelin oligodendrocyte glycoprotein (MOG), plays a pathogenic role in the induction of chronic EAE. Cyclization of peptides is of great interest since the limited stability of linear peptides restricts their potential use as therapeutic agents. Herein, we have designed and synthesized a number of linear and cyclic peptides by mutating crucial T cell receptor (TCR) contact residues of the human MOG35-55 epitope. In particular, we have designed and synthesized cyclic altered peptide ligands (APLs) by mutating Arg41 with Ala or Arg41 and Arg46 with Ala. The peptides were synthesized in solid phase on 2-chlorotrityl chloride resin (CLTR-Cl) using the Fmoc/t-Bu methodology. The purity of final products was verified by RP-HPLC and their identification was achieved by ESI-MS. It was found that the substitutions of Arg at positions 41 and 46 with Ala results in peptide analogues that reduce the severity of MOG-induced EAE clinical symptoms in C57BL/6 mice when co-administered with mouse MOG35-55 peptide at the time of immunization.

Human platelet glycoprotein IX: An adhesive prototype of leucine-rich glycoproteins with flank-center-flank structures

International Nuclear Information System (INIS)

Hickey, M.J.; Williams, S.A.; Roth, G.J.

1989-01-01

The glycoprotein (GP) Ib-IX complex on the surface of human platelets functions as the von Willebrand factor receptor and mediates von Willebrand factor-dependent platelet adhesion to blood vessels. GPIX is a relatively small (M r , 17,000) protein that may provide for membrane insertion and orientation of the larger component of the complex. GPIb (M r , 165,000). Using antibody screening, the authors cloned a cDNA encoding GPIX from a human erythroleukemia cell cDNA library constructed in phage λgt11. Lacking a 5' untranslated region and start codon, the cDNA sequence includes 604 nucleotides, beginning with 495 bases at the 5' end coding for 165 amino acids, followed by a stop codon and 106 noncoding bases at the 3' end. By Northern blot analysis, the GPIX cDNA hybridizes with a single 1.0-kilobase species of platelet poly(A) + RNA. Translation of the cDNA sequence gives a predicted protein sequence beginning with a truncated putative signal sequence of 5 amino acids followed by a sequence of 17 amino acids matching that determined directly by Edman degradation of intact GPIX. GPIX contains a leucine-rich glycoprotein (LRG) sequence of 24 amino acids similar to conserved LRG sequences in GPIb and other proteins from humans, Drosophila, and yeast. The role of the flank-LRG center-flank structure in the evolution and function of the LRG proteins remains to be defined

In silico-based vaccine design against Ebola virus glycoprotein

Directory of Open Access Journals (Sweden)

Dash R

2017-03-01

Full Text Available Raju Dash,1 Rasel Das,2 Md Junaid,3 Md Forhad Chowdhury Akash,4 Ashekul Islam,5 SM Zahid Hosen1 1Molecular Modeling and Drug Design Laboratory (MMDDL, Pharmacology Research Division, Bangladesh Council of Scientific and Industrial Research (BCSIR, Chittagong, Bangladesh; 2Nanotechnology and Catalysis Research Center, University of Malaya, Kuala Lumpur, Malaysia; 3Department of Pharmaceutical Sciences, North South University, Dhaka, Bangladesh; 4Department of Pharmacy, BGC Trust University Bangladesh, Chittagong, Bangladesh; 5Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong, Bangladesh Abstract: Ebola virus (EBOV is one of the lethal viruses, causing more than 24 epidemic outbreaks to date. Despite having available molecular knowledge of this virus, no definite vaccine or other remedial agents have been developed yet for the management and avoidance of EBOV infections in humans. Disclosing this, the present study described an epitope-based peptide vaccine against EBOV, using a combination of B-cell and T-cell epitope predictions, followed by molecular docking and molecular dynamics simulation approach. Here, protein sequences of all glycoproteins of EBOV were collected and examined via in silico methods to determine the most immunogenic protein. From the identified antigenic protein, the peptide region ranging from 186 to 220 and the sequence HKEGAFFLY from the positions of 154–162 were considered the most potential B-cell and T-cell epitopes, correspondingly. Moreover, this peptide (HKEGAFFLY interacted with HLA-A*32:15 with the highest binding energy and stability, and also a good conservancy of 83.85% with maximum population coverage. The results imply that the designed epitopes could manifest vigorous enduring defensive immunity against EBOV. Keywords: Ebola virus, epitope, glycoprotein, vaccine design

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

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Julio Alonso-Padilla

2017-01-01

Full Text Available 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.

Old and New World arenaviruses share a highly conserved epitope in the fusion domain of the glycoprotein 2, which is recognized by Lassa virus-specific human CD4+ T-cell clones

International Nuclear Information System (INIS)

Meulen, Jan ter; Badusche, Marlis; Satoguina, Judith; Strecker, Thomas; Lenz, Oliver; Loeliger, Cornelius; Sakho, Mohamed; Koulemou, Kekoura; Koivogui, Lamine; Hoerauf, Achim

2004-01-01

Data from human studies and animal experiments indicate a dominant role of T-cells over antibodies in controlling acute Lassa virus infection and providing immunity to reinfection. Knowledge of the epitopes recognized by T-cells may therefore be crucial to the development of a recombinant Lassa virus vaccine. In order to study human T-cell reactivity to the most conserved structural protein of Lassa virus, the glycoprotein 2 (GP2), seven GP2-specific CD4+ T-cell clones (TCCs) were generated from the lymphocytes of a Lassa antibody positive individual. All TCC displayed high specific proliferation, showed DR-restriction, and produced IFN-γ upon stimulation with recombinant GP2. The epitope of four of the clones was localized to a short stretch of 13 amino acids located in the N-terminal part of GP2 (aa 289-301, numbering according to sequence of GPC). This epitope is conserved in all strains of Lassa virus and lymphocytic choriomeningitis virus (LCMV), shows >90% similarity in all New World arenaviruses of clade B, and overlaps with the proposed fusion domain of GP2. Peptides with conservative aa exchanges, as they naturally occur in the epitope 289-301 of the Old World arenavirus Mopeia and some New World arenaviruses, continued to effectively stimulate the Lassa-GP2-specific T-cell clones tested. The finding of a human T-helper cell epitope, which is highly conserved between Old and New World arenaviruses, is of importance for the design of arenavirus vaccines

Insights into the Evolution of Hydroxyproline-Rich Glycoproteins from 1000 Plant Transcriptomes1[OPEN

Science.gov (United States)

Cassin, Andrew M.; Soltis, Douglas E.; Miles, Nicholas W.; Melkonian, Michael; Melkonian, Barbara; Wu, Shuangxiu; Edger, Patrick P.; Carpenter, Eric J.

2017-01-01

The carbohydrate-rich cell walls of land plants and algae have been the focus of much interest given the value of cell wall-based products to our current and future economies. Hydroxyproline-rich glycoproteins (HRGPs), a major group of wall glycoproteins, play important roles in plant growth and development, yet little is known about how they have evolved in parallel with the polysaccharide components of walls. We investigate the origins and evolution of the HRGP superfamily, which is commonly divided into three major multigene families: the arabinogalactan proteins (AGPs), extensins (EXTs), and proline-rich proteins. Using motif and amino acid bias, a newly developed bioinformatics pipeline, we identified HRGPs in sequences from the 1000 Plants transcriptome project (www.onekp.com). Our analyses provide new insights into the evolution of HRGPs across major evolutionary milestones, including the transition to land and the early radiation of angiosperms. Significantly, data mining reveals the origin of glycosylphosphatidylinositol (GPI)-anchored AGPs in green algae and a 3- to 4-fold increase in GPI-AGPs in liverworts and mosses. The first detection of cross-linking (CL)-EXTs is observed in bryophytes, which suggests that CL-EXTs arose though the juxtaposition of preexisting SPn EXT glycomotifs with refined Y-based motifs. We also detected the loss of CL-EXT in a few lineages, including the grass family (Poaceae), that have a cell wall composition distinct from other monocots and eudicots. A key challenge in HRGP research is tracking individual HRGPs throughout evolution. Using the 1000 Plants output, we were able to find putative orthologs of Arabidopsis pollen-specific GPI-AGPs in basal eudicots. PMID:28446636

Selection of SARS-Coronavirus-specific B cell epitopes by phage peptide library screening and evaluation of the immunological effect of epitope-based peptides on mice

International Nuclear Information System (INIS)

Yu Hua; Jiang Lifang; Fang Danyun; Yan Huijun; Zhou Jingjiao; Zhou Junmei; Liang Yu; Gao Yang; Zhao, Wei; Long Beiguo

2007-01-01

Antibodies to SARS-Coronavirus (SARS-CoV)-specific B cell epitopes might recognize the pathogen and interrupt its adherence to and penetration of host cells. Hence, these epitopes could be useful for diagnosis and as vaccine constituents. Using the phage-displayed peptide library screening method and purified Fab fragments of immunoglobulin G (IgG Fab) from normal human sera and convalescent sera from SARS-CoV-infected patients as targets, 11 B cell epitopes of SARS-CoV spike glycoprotein (S protein) and membrane protein (M protein) were screened. After a bioinformatics tool was used to analyze these epitopes, four epitope-based S protein dodecapeptides corresponding to the predominant epitopes were chosen for synthesis. Their antigenic specificities and immunogenicities were studied in vitro and in vivo. Flow cytometry and ELISPOT analysis of lymphocytes as well as a serologic analysis of antibody showed that these peptides could trigger a rapid, highly effective, and relatively safe immune response in BALB/c mice. These findings might aid development of SARS diagnostics and vaccines. Moreover, the role of S and M proteins as important surface antigens is confirmed

The Lyssavirus glycoprotein: A key to cross-immunity.

Science.gov (United States)

Buthelezi, Sindisiwe G; Dirr, Heini W; Chakauya, Ereck; Chikwamba, Rachel; Martens, Lennart; Tsekoa, Tsepo L; Stoychev, Stoyan H; Vandermarliere, Elien

2016-11-01

Rabies is an acute viral encephalomyelitis in warm-blooded vertebrates, caused by viruses belonging to Rhabdovirus family and genus Lyssavirus. Although rabies is categorised as a neglected disease, the rabies virus (RABV) is the most studied amongst Lyssaviruses which show nearly identical infection patterns. In efforts to improving post-exposure prophylaxis, several anti-rabies monoclonal antibodies (mAbs) targeting the glycoprotein (G protein) sites I, II, III and G5 have been characterized. To explore cross-neutralization capacity of available mAbs and discover new possible B-cell epitopes, we have analyzed all available glycoprotein sequences from Lyssaviruses with a focus on sequence variation and conservation. This information was mapped on the structure of a representative G protein. We proposed several possible cross-neutralizing B-cell epitopes (GUVTTTF, WLRTV, REECLD and EHLVVEEL) in complement to the already well-characterized antigenic sites. The research could facilitate development of novel cross-reactive mAbs against RABV and even more broad, against possibly all Lyssavirus members. Copyright © 2016 Elsevier Inc. All rights reserved.

Partial characterization of the cross-reacting determinant, a carbohydrate epitope shared by decay accelerating factor and the variant surface glycoprotein of the African Trypanosoma brucei.

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Shak, S; Davitz, M A; Wolinsky, M L; Nussenzweig, V; Turner, M J; Gurnett, A

1988-03-15

The variant surface glycoprotein (VSG) of the African trypanosome is anchored in the cell membrane by a complex glycan attached to phosphatidylinositol. The carboxyl terminal portion of VSG contains a cryptic carbohydrate epitope, the cross-reacting determinant (CRD), that is revealed only after removal of the diacylglycerol by phosphatidylinositol-specific phospholipase C (PIPLC) or VSG lipase. Recently, we have shown that after hydrolysis by PIPLC, decay-accelerating factor (DAF)--a mammalian phosphatidylinositol-anchored protein--also contains the CRD epitope. Using a two site immunoradiometric assay in which the capturing antibody is a monoclonal antibody to DAF and the revealing antibody is anti-CRD, we now show that sugar phosphates significantly inhibited the binding of anti-CRD antibody to DAF released by PIPLC. DL-myo-inositol 1,2-cyclic phosphate was the most potent inhibitor of binding (IC50 less than 10(-8) M). Other sugar phosphates, such as alpha-D-glucose-1-phosphate, which also possess adjacent hydroxyl and phosphate moieties in cis also inhibited binding at low concentrations (IC50 = 10(-5) to 10(-4) M). In contrast, sugar phosphates which do not possess adjacent hydroxyl and phosphate moieties in cis and simple sugars weakly inhibited binding (IC50 greater than 10(-3) M). These results suggest that myo-inositol 1,2-cyclic phosphate contributes significantly to the epitope recognized by the anti-CRD antibody and is consistent with analysis of the carboxyl terminus of VSG, which also suggested the presence of the cyclic inositol phosphate. In light of the recent findings that human serum contains a glycan-phosphatidyl-inositol-specific phospholipase D, which converts DAF from a hydrophobic to a hydrophilic form lacking the CRD, the observation that the phosphate is crucial for expression of the epitope may be relevant in understanding the origin of CRD-negative DAF in urine and plasma.

Cell wall O-glycoproteins and N-glycoproteins: biosynthesis and some functional aspects.

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Eric eNguema-Ona

2014-10-01

Full Text Available Cell wall O-glycoproteins and N-glycoproteins are two types of glycomolecules whose glycans are structurally complex. They are both assembled and modified within the endomembrane system, i.e., the endoplasmic reticulum (ER and the Golgi apparatus, before their transport to their final locations within or outside the cell. In contrast to extensin, the O-glycan chains of arabinogalactan proteins are highly heterogeneous consisting mostly of (i a short oligo-arabinoside chain of three to four residues, and (ii a larger -1,3-linked galactan backbone with -1,6-linked side chains containing galactose, arabinose and, often, fucose, rhamnose or glucuronic acid. The fine structure of arabinogalactan chains varies between, and within plant species, and is important for the functional activities of the glycoproteins. With regards to N-glycans, ER-synthesizing events are highly conserved in all eukaryotes studied so far since they are essential for efficient protein folding. In contrast, evolutionary adaptation of N-glycan processing in the Golgi apparatus has given rise to a variety of organism-specific complex structures. Therefore, plant complex-type N-glycans contain specific glyco-epitopes such as core 1,2-xylose, core 1,3-fucose residues and Lewisa substitutions on the terminal position of the antenna. Like O-glycans, N-glycans of proteins are essential for their stability and function. Mutants affected in the glycan metabolic pathways have provided valuable information on the role of N-/O-glycoproteins in the control of growth, morphogenesis and adaptation to biotic and abiotic stresses. With regards to O-glycoproteins only extensin and arabinogalactan proteins are considered herein. The biosynthesis of these glycoproteins and functional aspects are presented and discussed in this review.

Directed Selection of Recombinant Human Monoclonal Antibodies to Herpes Simplex Virus Glycoproteins from Phage Display Libraries

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Sanna, Pietro Paolo; Williamson, R. Anthony; de Logu, Alessandro; Bloom, Floyd E.; Burton, Dennis R.

1995-07-01

Human monoclonal antibodies have considerable potential in the prophylaxis and treatment of viral disease. However, only a few such antibodies suitable for clinical use have been produced to date. We have previously shown that large panels of human recombinant monoclonal antibodies against a plethora of infectious agents, including herpes simplex virus types 1 and 2, can be established from phage display libraries. Here we demonstrate that facile cloning of recombinant Fab fragments against specific viral proteins in their native conformation can be accomplished by panning phage display libraries against viral glycoproteins "captured" from infected cell extracts by specific monoclonal antibodies immobilized on ELISA plates. We have tested this strategy by isolating six neutralizing recombinant antibodies specific for herpes simplex glycoprotein gD or gB, some of which are against conformationally sensitive epitopes. By using defined monoclonal antibodies for the antigen-capture step, this method can be used for the isolation of antibodies to specific regions and epitopes within the target viral protein. For instance, monoclonal antibodies to a nonneutralizing epitope can be used in the capture step to clone antibodies to neutralizing epitopes, or antibodies to a neutralizing epitope can be used to clone antibodies to a different neutralizing epitope. Furthermore, by using capturing antibodies to more immunodominant epitopes, one can direct the cloning to less immunogenic ones. This method should be of value in generating antibodies to be used both in the prophylaxis and treatment of viral infections and in the characterization of the mechanisms of antibody protective actions at the molecular level.

Asymptomatic HLA-A*02:01–Restricted Epitopes from Herpes Simplex Virus Glycoprotein B Preferentially Recall Polyfunctional CD8+ T Cells from Seropositive Asymptomatic Individuals and Protect HLA Transgenic Mice against Ocular Herpes

Science.gov (United States)

Dervillez, Xavier; Qureshi, Huma; Chentoufi, Aziz A.; Khan, Arif A.; Kritzer, Elizabeth; Yu, David C.; Diaz, Oscar R.; Gottimukkala, Chetan; Kalantari, Mina; Villacres, Maria C.; Scarfone, Vanessa M.; McKinney, Denise M.; Sidney, John; Sette, Alessandro; Nesburn, Anthony B.; Wechsler, Steven L.; BenMohamed, Lbachir

2014-01-01

Evidence from C57BL/6 mice suggests that CD8+ T cells, specific to the immunodominant HSV-1 glycoprotein B (gB) H-2b–restricted epitope (gB498–505), protect against ocular herpes infection and disease. However, the possible role of CD8+ T cells, specific to HLA-restricted gB epitopes, in protective immunity seen in HSV-1–seropositive asymptomatic (ASYMP) healthy individuals (who have never had clinical herpes) remains to be determined. In this study, we used multiple prediction algorithms to identify 10 potential HLA-A*02:01–restricted CD8+ T cell epitopes from the HSV-1 gB amino acid sequence. Six of these epitopes exhibited high-affinity binding to HLA-A*02:01 molecules. In 10 sequentially studied HLA-A*02:01–positive, HSV-1–seropositive ASYMP individuals, the most frequent, robust, and polyfunctional CD8+ T cell responses, as assessed by a combination of tetramer, IFN-γ-ELISPOT, CFSE proliferation, CD107a/b cytotoxic degranulation, and multiplex cytokine assays, were directed mainly against epitopes gB342–350 and gB561–569. In contrast, in 10 HLA-A*02:01–positive, HSV-1–seropositive symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent clinical herpes disease) frequent, but less robust, CD8+ T cell responses were directed mainly against nonoverlapping epitopes (gB183–191 and gB441–449). ASYMP individuals had a significantly higher proportion of HSV-gB–specific CD8+ T cells expressing CD107a/b degranulation marker and producing effector cytokines IL-2, IFN-γ, and TNF-α than did SYMP individuals. Moreover, immunization of a novel herpes-susceptible HLA-A*02:01 transgenic mouse model with ASYMP epitopes, but not with SYMP epitopes, induced strong CD8+ T cell–dependent protective immunity against ocular herpes infection and disease. These findings should guide the development of a safe and effective T cell–based herpes vaccine. PMID:24101547

The membrane-proximal tryptophan-rich region in the transmembrane glycoprotein ectodomain of feline immunodeficiency virus is important for cell entry

International Nuclear Information System (INIS)

Giannecchini, Simone; Bonci, Francesca; Pistello, Mauro; Matteucci, Donatella; Sichi, Olimpia; Rovero, Paolo; Bendinelli, Mauro

2004-01-01

The mechanisms whereby feline immunodeficiency virus (FIV) adsorbs and enters into susceptible cells are poorly understood. Here, we investigated the role exerted in such functions by the tryptophan (Trp)-rich motif present membrane-proximally in the ectodomain of the FIV transmembrane glycoprotein. Starting from p34TF10, which encodes the entire genome of FIV Petaluma, we produced 11 mutated clones having the Trp-rich motif scrambled or variously deleted or substituted. All mutated progenies adsorbed normally to cells, but the ones with severe disruptions of the motif failed to generate proviral DNA. In the latter mutants, proviral DNA formation was restored by providing an independent source of intact FIV envelope glycoproteins or by addition of the fusing agent polyethylene glycol, thus clearly indicating that their defect resided primarily at the level of cell entry. In addition, the replication-competent mutants exhibited a generally enhanced susceptibility to selected entry inhibitory synthetic peptides, suggestive of a reduced efficiency of the entry step

Human monoclonal antibodies to a novel cluster of conformational epitopes on HCV E2 with resistance to neutralization escape in a genotype 2a isolate

DEFF Research Database (Denmark)

Keck, Zhen-yong; Xia, Jinming; Wang, Yong

2012-01-01

The majority of broadly neutralizing antibodies to hepatitis C virus (HCV) are against conformational epitopes on the E2 glycoprotein. Many of them recognize overlapping epitopes in a cluster, designated as antigenic domain B, that contains residues G530 and D535. To gain information on other reg...

Histidine-rich glycoprotein can prevent development of mouse experimental glioblastoma.

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Maria Kärrlander

Full Text Available Extensive angiogenesis, formation of new capillaries from pre-existing blood vessels, is an important feature of malignant glioma. Several antiangiogenic drugs targeting vascular endothelial growth factor (VEGF or its receptors are currently in clinical trials as therapy for high-grade glioma and bevacizumab was recently approved by the FDA for treatment of recurrent glioblastoma. However, the modest efficacy of these drugs and emerging problems with anti-VEGF treatment resistance welcome the development of alternative antiangiogenic therapies. One potential candidate is histidine-rich glycoprotein (HRG, a plasma protein with antiangiogenic properties that can inhibit endothelial cell adhesion and migration. We have used the RCAS/TV-A mouse model for gliomas to investigate the effect of HRG on brain tumor development. Tumors were induced with platelet-derived growth factor-B (PDGF-B, in the presence or absence of HRG. We found that HRG had little effect on tumor incidence but could significantly inhibit the development of malignant glioma and completely prevent the occurrence of grade IV tumors (glioblastoma.

Prediction of exposed domains of envelope glycoprotein in Indian HIV-1 isolates and experimental confirmation of their immunogenicity in humans

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Mohabatkar H.

2004-01-01

Full Text Available We describe the impact of subtype differences on the seroreactivity of linear antigenic epitopes in envelope glycoprotein of HIV-1 isolates from different geographical locations. By computer analysis, we predicted potential antigenic sites of envelope glycoprotein (gp120 and gp4l of this virus. For this purpose, after fetching sequences of proteins of interest from data banks, values of hydrophilicity, flexibility, accessibility, inverted hydrophobicity, and secondary structure were considered. We identified several potential antigenic epitopes in a B subtype strain of envelope glycoprotein of HIV-1 (IIIB. Solid- phase peptide synthesis methods of Merrifield and Fmoc chemistry were used for synthesizing peptides. These synthetic peptides corresponded mainly to the C2, V3 and CD4 binding sites of gp120 and some parts of the ectodomain of gp41. The reactivity of these peptides was tested by ELISA against different HIV-1-positive sera from different locations in India. For two of these predicted epitopes, the corresponding Indian consensus sequences (LAIERYLKQQLLGWG and DIIGDIRQAHCNISEDKWNET (subtype C were also synthesized and their reactivity was tested by ELISA. These peptides also distinguished HIV-1-positive sera of Indians with C subtype infections from sera from HIV-negative subjects.

Palmitoylation of the cysteine-rich endodomain of the SARS-coronavirus spike glycoprotein is important for spike-mediated cell fusion

International Nuclear Information System (INIS)

Petit, Chad M.; Chouljenko, Vladimir N.; Iyer, Arun; Colgrove, Robin; Farzan, Michael; Knipe, David M.; Kousoulas, K.G.

2007-01-01

The SARS-coronavirus (SARS-CoV) is the etiological agent of the severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. The cytoplasmic portion of the S glycoprotein contains four cysteine-rich amino acid clusters. Individual cysteine clusters were altered via cysteine-to-alanine amino acid replacement and the modified S glycoproteins were tested for their transport to cell-surfaces and ability to cause cell fusion in transient transfection assays. Mutagenesis of the cysteine cluster I, located immediately proximal to the predicted transmembrane, domain did not appreciably reduce cell-surface expression, although S-mediated cell fusion was reduced by more than 50% in comparison to the wild-type S. Similarly, mutagenesis of the cysteine cluster II located adjacent to cluster I reduced S-mediated cell fusion by more than 60% compared to the wild-type S, while cell-surface expression was reduced by less than 20%. Mutagenesis of cysteine clusters III and IV did not appreciably affect S cell-surface expression or S-mediated cell fusion. The wild-type S was palmitoylated as evidenced by the efficient incorporation of 3 H-palmitic acid in wild-type S molecules. S glycoprotein palmitoylation was significantly reduced for mutant glycoproteins having cluster I and II cysteine changes, but was largely unaffected for cysteine cluster III and IV mutants. These results show that the S cytoplasmic domain is palmitoylated and that palmitoylation of the membrane proximal cysteine clusters I and II may be important for S-mediated cell fusion

Epitope Mapping of Monoclonal Antibody PMab-52 Against Cat Podoplanin.

Science.gov (United States)

Chang, Yao-Wen; Kaneko, Mika K; Yamada, Shinji; Kato, Yukinari

2018-02-02

The mucin-type membrane glycoprotein podoplanin (PDPN) is frequently overexpressed in numerous malignant cancers, including squamous cell carcinoma, germinal neoplasia, mesothelioma, lung cancer, oral cancer, and brain tumor. PDPN expression is strongly associated with cancer progression and poor prognosis. Furthermore, PDPN binds to C-type lectin-like receptor 2 (CLEC-2) on platelets, followed by PDPN-mediated platelet aggregation to facilitate tumor metastasis. We have previously reported a novel anti-cat PDPN (cPDPN) monoclonal antibody (mAb), PMab-52, which specifically detects cPDPN using flow cytometry analysis and successfully identifies cPDPN in feline squamous cell carcinomas. However, the specific binding epitope of cPDPN for PMab-52 remains unelucidated. In this study, a series of deletion or point mutants of cPDPN were utilized for investigating the binding epitopes of PMab-52 using flow cytometry and Western blotting. The findings of this study revealed that the critical epitopes of platelet aggregation-stimulating domain 4 (PLAG4) of cPDPN are responsible for the binding of PMab-52 to cPDPN.

Human MHC-II with Shared Epitope Motifs Are Optimal Epstein-Barr Virus Glycoprotein 42 Ligands—Relation to Rheumatoid Arthritis

Science.gov (United States)

Trier, Nicole; Izarzugaza, Jose; Chailyan, Anna; Marcatili, Paolo; Houen, Gunnar

2018-01-01

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disorder of unknown etiology, which is characterized by inflammation in the synovium and joint damage. Although the pathogenesis of RA remains to be determined, a combination of environmental (e.g., viral infections) and genetic factors influence disease onset. Especially genetic factors play a vital role in the onset of disease, as the heritability of RA is 50–60%, with the human leukocyte antigen (HLA) alleles accounting for at least 30% of the overall genetic risk. Some HLA-DR alleles encode a conserved sequence of amino acids, referred to as the shared epitope (SE) structure. By analyzing the structure of a HLA-DR molecule in complex with Epstein-Barr virus (EBV), the SE motif is suggested to play a vital role in the interaction of MHC II with the viral glycoprotein (gp) 42, an essential entry factor for EBV. EBV has been repeatedly linked to RA by several lines of evidence and, based on several findings, we suggest that EBV is able to induce the onset of RA in predisposed SE-positive individuals, by promoting entry of B-cells through direct contact between SE and gp42 in the entry complex. PMID:29361739

Cryptic nature of a conserved, CD4-inducible V3 loop neutralization epitope in the native envelope glycoprotein oligomer of CCR5-restricted, but not CXCR4-using, primary human immunodeficiency virus type 1 strains.

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Lusso, Paolo; Earl, Patricia L; Sironi, Francesca; Santoro, Fabio; Ripamonti, Chiara; Scarlatti, Gabriella; Longhi, Renato; Berger, Edward A; Burastero, Samuele E

2005-06-01

The external subunit of the human immunodeficiency virus type 1 (HIV-1) envelope glycoprotein (Env), gp120, contains conserved regions that mediate sequential interactions with two cellular receptor molecules, CD4 and a chemokine receptor, most commonly CCR5 or CXCR4. However, antibody accessibility to such regions is hindered by diverse protective mechanisms, including shielding by variable loops, conformational flexibility and extensive glycosylation. For the conserved neutralization epitopes hitherto described, antibody accessibility is reportedly unrelated to the viral coreceptor usage phenotype. Here, we characterize a novel, conserved gp120 neutralization epitope, recognized by a murine monoclonal antibody (MAb), D19, which is differentially accessible in the native HIV-1 Env according to its coreceptor specificity. The D19 epitope is contained within the third variable (V3) domain of gp120 and is distinct from those recognized by other V3-specific MAbs. To study the reactivity of MAb D19 with the native oligomeric Env, we generated a panel of PM1 cells persistently infected with diverse primary HIV-1 strains. The D19 epitope was conserved in the majority (23/29; 79.3%) of the subtype-B strains tested, as well as in selected strains from other genetic subtypes. Strikingly, in CCR5-restricted (R5) isolates, the D19 epitope was invariably cryptic, although it could be exposed by addition of soluble CD4 (sCD4); epitope masking was dependent on the native oligomeric structure of Env, since it was not observed with the corresponding monomeric gp120 molecules. By contrast, in CXCR4-using strains (X4 and R5X4), the epitope was constitutively accessible. In accordance with these results, R5 isolates were resistant to neutralization by MAb D19, becoming sensitive only upon addition of sCD4, whereas CXCR4-using isolates were neutralized regardless of the presence of sCD4. Other V3 epitopes examined did not display a similar divergence in accessibility based on

Characterization of an immunodominant cancer-specific O-glycopeptide epitope in murine podoplanin (OTS8)

DEFF Research Database (Denmark)

Steentoft, Catharina; Schjoldager, Katrine T; Cló, Emiliano

2010-01-01

antibody 237, developed to a spontaneous murine fibrosarcoma, was shown to be directed to murine podoplanin (OTS8) with truncated Tn O-glycans. Our understanding of such cancer-specific auto-antibodies to truncated glycoforms of glycoproteins is limited. Here we have investigated immunogenicity...... of a chemoenzymatically produced Tn-glycopeptide derived from the putative murine podoplanin O-glycopeptide epitope. We found that the Tn O-glycopeptide was highly immunogenic in mice and produced a Tn-glycoform specific response with no reactivity against unglycosylated peptides or the O-glycopeptide with extended O......-glycan (STn and T glycoforms). The immunodominant epitope was strictly dependent on the peptide sequence, required Tn at a specific single Thr residue (Thr(77)), and antibodies to the epitope were not found in naive mice. We further tested a Tn O-glycopeptide library derived from human podoplanin...

O-acetylation of the serine-rich repeat glycoprotein GspB is coordinated with accessory Sec transport.

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Ravin Seepersaud

2017-08-01

Full Text Available The serine-rich repeat (SRR glycoproteins are a family of adhesins found in many Gram-positive bacteria. Expression of the SRR adhesins has been linked to virulence for a variety of infections, including streptococcal endocarditis. The SRR preproteins undergo intracellular glycosylation, followed by export via the accessory Sec (aSec system. This specialized transporter is comprised of SecA2, SecY2 and three to five accessory Sec proteins (Asps that are required for export. Although the post-translational modification and transport of the SRR adhesins have been viewed as distinct processes, we found that Asp2 of Streptococcus gordonii also has an important role in modifying the SRR adhesin GspB. Biochemical analysis and mass spectrometry indicate that Asp2 is an acetyltransferase that modifies N-acetylglucosamine (GlcNAc moieties on the SRR domains of GspB. Targeted mutations of the predicted Asp2 catalytic domain had no effect on transport, but abolished acetylation. Acetylated forms of GspB were only detected when the protein was exported via the aSec system, but not when transport was abolished by secA2 deletion. In addition, GspB variants rerouted to export via the canonical Sec pathway also lacked O-acetylation, demonstrating that this modification is specific to export via the aSec system. Streptococci expressing GspB lacking O-acetylated GlcNAc were significantly reduced in their ability bind to human platelets in vitro, an interaction that has been strongly linked to virulence in the setting of endocarditis. These results demonstrate that Asp2 is a bifunctional protein involved in both the post-translational modification and transport of SRR glycoproteins. In addition, these findings indicate that these processes are coordinated during the biogenesis of SRR glycoproteins, such that the adhesin is optimally modified for binding. This requirement for the coupling of modification and export may explain the co-evolution of the SRR

Platelet Glycoprotein Ib-IX and Malignancy

Science.gov (United States)

2010-09-01

provide a unique microenvironment supporting the accumulation of more platelets and the elaboration of a fibrin - rich network produced by coagulation...process and can initiate the formation of a platelet - rich thrombus by tethering the platelet to a thrombogenic surface. Several ligands binding to GP Ib... Platelet Glycoprotein Ib-IX and Malignancy PRINCIPAL INVESTIGATOR: Jerry Ware, Ph.D

Analysis of Conformational B-Cell Epitopes in the Antibody-Antigen Complex Using the Depth Function and the Convex Hull.

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Wei Zheng

Full Text Available The prediction of conformational b-cell epitopes plays an important role in immunoinformatics. Several computational methods are proposed on the basis of discrimination determined by the solvent-accessible surface between epitopes and non-epitopes, but the performance of existing methods is far from satisfying. In this paper, depth functions and the k-th surface convex hull are used to analyze epitopes and exposed non-epitopes. On each layer of the protein, we compute relative solvent accessibility and four different types of depth functions, i.e., Chakravarty depth, DPX, half-sphere exposure and half space depth, to analyze the location of epitopes on different layers of the proteins. We found that conformational b-cell epitopes are rich in charged residues Asp, Glu, Lys, Arg, His; aliphatic residues Gly, Pro; non-charged residues Asn, Gln; and aromatic residue Tyr. Conformational b-cell epitopes are rich in coils. Conservation of epitopes is not significantly lower than that of exposed non-epitopes. The average depths (obtained by four methods for epitopes are significantly lower than that of non-epitopes on the surface using the Wilcoxon rank sum test. Epitopes are more likely to be located in the outer layer of the convex hull of a protein. On the benchmark dataset, the cumulate 10th convex hull covers 84.6% of exposed residues on the protein surface area, and nearly 95% of epitope sites. These findings may be helpful in building a predictor for epitopes.

Generation and Characterization of Monoclonal Antibodies against a Cyclic Variant of Hepatitis C Virus E2 Epitope 412-422

Science.gov (United States)

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

Phenotypic and Functional Characterization of Herpes Simplex Virus Glycoprotein B Epitope-Specific Effector and Memory CD8+ T Cells from Symptomatic and Asymptomatic Individuals with Ocular Herpes

Science.gov (United States)

Khan, Arif A.; Srivastava, Ruchi; Spencer, Doran; Garg, Sumit; Fremgen, Daniel; Vahed, Hawa; Lopes, Patricia P.; Pham, Thanh T.; Hewett, Charlie; Kuang, Jasmine; Ong, Nicolas; Huang, Lei; Scarfone, Vanessa M.; Nesburn, Anthony B.

2015-01-01

ABSTRACT Herpes simplex virus 1 (HSV-1) glycoprotein B (gB)-specific CD8+ T cells protect mice from herpes infection and disease. However, whether and which HSV-1 gB-specific CD8+ T cells play a key role in the “natural” protection seen in HSV-1-seropositive healthy asymptomatic (ASYMP) individuals (who have never had clinical herpes disease) remain to be determined. In this study, we have dissected the phenotypes and the functions of HSV-1 gB-specific CD8+ T cells from HLA-A*02:01 positive, HSV-1 seropositive ASYMP and symptomatic (SYMP) individuals (with a history of numerous episodes of recurrent ocular herpes disease). We found the following. (i) Healthy ASYMP individuals maintained a significantly higher proportion of differentiated HSV-1 gB-specific effector memory CD8+ T cells (TEM cells) (CD45RAlow CCR7low CD44high CD62Llow). In contrast, SYMP patients had frequent less-differentiated central memory CD8+ T cells (TCM cells) (CD45RAlow CCR7high CD44low CD62Lhigh). (ii) ASYMP individuals had significantly higher proportions of multifunctional effector CD8+ T cells which responded mainly to gB342–350 and gB561–569 “ASYMP” epitopes, and simultaneously produced IFN-γ, CD107a/b, granzyme B, and perforin. In contrast, effector CD8+ T cells from SYMP individuals were mostly monofunctional and were directed mainly against nonoverlapping gB17–25 and gB183–191 “SYMP” epitopes. (iii) Immunization of an HLA-A*02:01 transgenic mouse model of ocular herpes with “ASYMP” CD8+ TEM cell epitopes, but not with “SYMP” CD8+ TCM cell epitopes, induced a strong CD8+ T cell-dependent protective immunity against ocular herpes infection and disease. Our findings provide insights into the role of HSV-specific CD8+ TEM cells in protection against herpes and should be considered in the development of an effective vaccine. IMPORTANCE A significantly higher proportion of differentiated and multifunctional HSV-1 gB-specific effector memory CD8+ T cells (TEM

Indirect detection of an epitope-specific response to HIV-1 gp120 immunization in human subjects.

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Evgeny Shmelkov

Full Text Available A specific response of human serum neutralizing antibodies (nAb to a conformational epitope as a result of vaccination of human subjects with the surface envelope glycoprotein (gp120 of HIV-1 has not previously been documented. Here, we used computational analysis to assess the epitope-specific responses of human subjects, which were immunized with recombinant gp120 immunogens in the VAX003 and VAX004 clinical trials. Our computational methodology--a variation of sieve analysis--compares the occurrence of specific nAb targeted conformational 3D epitopes on viruses from infected individuals who received vaccination to the occurrence of matched epitopes in the viruses infecting placebo subjects. We specifically studied seven crystallographically defined nAb targeted conformational epitopes in the V3 loop, an immunogenic region of gp120. Of the six epitopes present in the immunogens and targeted by known monoclonal neutralizing antibodies, only the one targeted by the anti-V3 nAb 2219 exhibited a significant reduction in occurrence in vaccinated subjects compared to the placebo group. This difference occurred only in the VAX003 Thailand cohort. No difference was seen between vaccinated and placebo groups for the occurrence of an epitope that was not present in the immunogen. Thus, it can be theorized that a specific 2219-like human neutralizing antibody immune response to AIDSVAX immunization occurred in the VAX003 cohort, and that this response protected subjects from a narrow subset of HIV-1 viruses circulating in Thailand in the 1990s and bearing the conformational epitope targeted by the neutralizing antibody 2219.

Epitope Sequences in Dengue Virus NS1 Protein Identified by Monoclonal Antibodies

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Leticia Barboza Rocha

2017-10-01

Full Text Available Dengue nonstructural protein 1 (NS1 is a multi-functional glycoprotein with essential functions both in viral replication and modulation of host innate immune responses. NS1 has been established as a good surrogate marker for infection. In the present study, we generated four anti-NS1 monoclonal antibodies against recombinant NS1 protein from dengue virus serotype 2 (DENV2, which were used to map three NS1 epitopes. The sequence 193AVHADMGYWIESALNDT209 was recognized by monoclonal antibodies 2H5 and 4H1BC, which also cross-reacted with Zika virus (ZIKV protein. On the other hand, the sequence 25VHTWTEQYKFQPES38 was recognized by mAb 4F6 that did not cross react with ZIKV. Lastly, a previously unidentified DENV2 NS1-specific epitope, represented by the sequence 127ELHNQTFLIDGPETAEC143, is described in the present study after reaction with mAb 4H2, which also did not cross react with ZIKV. The selection and characterization of the epitope, specificity of anti-NS1 mAbs, may contribute to the development of diagnostic tools able to differentiate DENV and ZIKV infections.

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

Science.gov (United States)

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

Salivary agglutinin and lung scavenger receptor cysteine-rich glycoprotein 340 have broad anti-influenza activities and interactions with surfactant protein D that vary according to donor source and sialylation

DEFF Research Database (Denmark)

Hartshorn, Kevan L.; Ligtenberg, Antoon; White, Mitchell R.

2006-01-01

We previously found that scavenger receptor cysteine-rich gp-340 (glycoprotein-340), isolated from lung or saliva, directly inhibits human IAVs (influenza A viruses). We now show that salivary gp-340 has broad antiviral activity against human, equine and porcine IAV strains. Although lung...

Major role for carbohydrate epitopes preferentially recognized by chronically infected mice in the determination of Schistosoma mansoni schistosomulum surface antigenicity

International Nuclear Information System (INIS)

Omer-ali, P.; Magee, A.I.; Kelly, C.; Simpson, A.J.G.

1986-01-01

A radioimmunoassay that makes use of whole Schistosomula and 125 I-labeled protein A has been used to characterize and to quantify the binding of antisera to the surface of 3 hr mechanically transformed schistosomula of Schistosoma mansoni. This technique facilitates the determination of epitopes on the schistosomula in addition to those detected by surface labeling and immunoprecipitation. By using this technique, it has been demonstrated that there is a much greater binding to the parasite surface of antibodies from chronically infected mice (CMS) than of antibodies from mice infected with highly irradiated cercariae (VMS), and CMS recognizes epitopes that VMS does not. Treatment of the surface of the schistosomula with trifluoromethanesulphonic acid and sodium metaperiodate has suggested that the discrepancy of the binding between the two sera is due to the recognition of a large number of additional epitopes by CMS, which are carbohydrate in nature. Some of the carbohydrate epitopes are expressed on the previously described surface glycoprotein antigens of M/sub r/ 200,000, 38,000, and 17,000

Monoclonal antibodies directed to E1 glycoprotein of rubella virus

International Nuclear Information System (INIS)

Umino, Y.; Sato, A.; Katow, S.; Matsuno, T.; Sugiura, A.

1985-01-01

We have prepared four monoclonal antibodies to rubella virus E1 glycoprotein. Three nonoverlapping antigenic sites were delineated on E1 protein by competitive binding assays. Antibodies binding to one site were characterized by high hemagglutination inhibition (HI) titer but poor neutralizing activity. The addition of antiglobulin conferred neutralizing activity. Antibodies directed to two other antigenic sites had modest hemolysis inhibition but little or no HI and neutralizing activities. The addition of antiglobulin markedly augmented HI activity but had little effect on neutralizing activity. Epitopes defined by three antibodies were conserved among four rubella virus strains examined. (Author)

Structure-Based Design of Hepatitis C Virus Vaccines That Elicit Neutralizing Antibody Responses to a Conserved Epitope

Energy Technology Data Exchange (ETDEWEB)

Pierce, Brian G.; Boucher, Elisabeth N.; Piepenbrink, Kurt H.; Ejemel, Monir; Rapp, Chelsea A.; Thomas, William D.; Sundberg, Eric J.; Weng, Zhiping; Wang, Yang; Diamond, Michael S.

2017-08-09

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, as 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

Accumulation of hydroxyproline-rich glycoprotein mRNAs in response to fungal elicitor and infection.

Science.gov (United States)

Showalter, A M; Bell, J N; Cramer, C L; Bailey, J A; Varner, J E; Lamb, C J

1985-10-01

Hydroxyproline-rich glycoproteins (HRGPs) are important structural components of plant cell walls and also accumulate in response to infection as an apparent defense mechanism. Accumulation of HRGP mRNA in biologically stressed bean (Phaseolus vulgaris L.) cells was monitored by blot hybridization with (32)P-labeled tomato genomic HRGP sequences. Elicitor treatment of suspension-cultured cells caused a marked increase in hybridizable HRGP mRNA. The response was less rapid but more prolonged than that observed for mRNAs encoding enzymes of phytoalexin biosynthesis. HRGP mRNA also accumulated during race:cultivar-specific interactions between bean hypocotyls and the partially biotrophic fungus Colletotrichum lindemuthianum, the causal agent of anthracnose. In an incompatible interaction (host resistant) there was an early increase in HRGP mRNA correlated with expression of hypersensitive resistance; whereas, in a compatible interaction (host susceptible), marked accumulation of HRGP mRNA occurred as a delayed response at the onset of lesion formation. In both interactions, mRNA accumulation was observed in uninfected cells distant from the site of fungal inoculation, indicating intercellular transmission of an elicitation signal.

Identification of an immunodominant epitope in glycoproteins B and G of herpes simplex viruses (HSVs) using synthetic peptides as antigens in assay of antibodies to HSV in herpes simplex encephalitis patients.

Science.gov (United States)

Bhullar, S S; Chandak, N H; Baheti, N N; Purohit, H J; Taori, G M; Daginawala, H F; Kashyap, R S

2014-01-01

Herpes simplex encephalitis (HSE) is a severe viral infection of the central nervous system (CNS). Assay of antibody response is widely used in diagnostics of HSE. The aim of this study was to identify an immunodominant epitope determining the antibody response to herpes simplex viruses (HSVs) in cerebrospinal fluid (CSF) of HSE patients. The synthetic peptides that resembled type-common as well as type-specific domains of glycoproteins B (gB) and G (gG) of these viruses were evaluated for binding with IgM and IgG antibodies in CSF samples from HSE and non-HSE patients in ELISA. The QLHDLRF peptide, derived from gB of HSV was found to be an immunodominant epitope in the IgM and IgG antibody response. The patients with confirmed and suspected HSE showed in ELISA against this peptide 26% and 23% positivities for IgM, 43% and 37% positivities for IgG and 17% and 15% for both IgM and IgG antibodies, respectively. The total positivities of 86% and 75% for both IgM and IgG antibodies were obtained in the patients with confirmed and suspected HSE, respectively. These results demonstrate that a synthetic peptide-based diagnostics of HSE can be an efficient and easily accessible alternative. This is the first report describing the use of synthetic peptides derived from HSVs in diagnostics of HSE using patientsʹ CSF samples.

The first human epitope map of the alphaviral E1 and E2 proteins reveals a new E2 epitope with significant virus neutralizing activity.

Directory of Open Access Journals (Sweden)

Ann R Hunt

2010-07-01

Full Text Available Venezuelan equine encephalitis virus (VEEV is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion and E2 (binds receptor and elicits virus neutralizing antibodies. Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs. Six E2 epitopes (E2(c,d,e,f,g,h bound VEEV-neutralizing antibody and mapped to amino acids (aa 182-207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE.We used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants.Using 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115-119. Using a 9 A resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope.The VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically. Administration

A Highly Conserved GEQYQQLR Epitope Has Been Identified in the Nucleoprotein of Ebola Virus by Using an In Silico Approach

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Mohammad Tuhin Ali

2015-01-01

Full Text Available Ebola virus (EBOV is a deadly virus that has caused several fatal outbreaks. Recently it caused another outbreak and resulted in thousands afflicted cases. Effective and approved vaccine or therapeutic treatment against this virus is still absent. In this study, we aimed to predict B-cell epitopes from several EBOV encoded proteins which may aid in developing new antibody-based therapeutics or viral antigen detection method against this virus. Multiple sequence alignment (MSA was performed for the identification of conserved region among glycoprotein (GP, nucleoprotein (NP, and viral structural proteins (VP40, VP35, and VP24 of EBOV. Next, different consensus immunogenic and conserved sites were predicted from the conserved region(s using various computational tools which are available in Immune Epitope Database (IEDB. Among GP, NP, VP40, VP35, and VP30 protein, only NP gave a 100% conserved GEQYQQLR B-cell epitope that fulfills the ideal features of an effective B-cell epitope and could lead a way in the milieu of Ebola treatment. However, successful in vivo and in vitro studies are prerequisite to determine the actual potency of our predicted epitope and establishing it as a preventing medication against all the fatal strains of EBOV.

A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes

Science.gov (United States)

Florentino, Pilar T. V.; Real, Fernando; Orikaza, Cristina M.; da Cunha, Julia P. C.; Vitorino, Francisca N. L.; Cordero, Esteban M.; Sobreira, Tiago J. P.; Mortara, Renato A.

2018-01-01

Trypanosoma cruzi is the etiologic agent of Chagas’ disease. It is known that amastigotes derived from trypomastigotes in the extracellular milieu are infective in vitro and in vivo. Extracellular amastigotes (EAs) have a stage-specific surface antigen called Ssp-4, a GPI-anchored glycoprotein that is secreted by the parasites. By immunoprecipitation with the Ssp-4-specific monoclonal antibodies (mAb) 2C2 and 1D9, we isolated the glycoprotein from EAs. By mass spectrometry, we identified the core protein of Ssp-4 and evaluated mRNA expression and the presence of Ssp-4 carbohydrate epitopes recognized by mAb1D9. We demonstrated that the carbohydrate epitope recognized by mAb1D9 could promote host cell invasion by EAs. Although infectious EAs express lower amounts of Ssp-4 compared with less-infectious EAs (at the mRNA and protein levels), it is the glycosylation of Ssp-4 (identified by mAb1D9 staining only in infectious strains and recognized by galectin-3 on host cells) that is the determinant of EA invasion of host cells. Furthermore, Ssp-4 is secreted by EAs, either free or associated with parasite vesicles, and can participate in host-cell interactions. The results presented here describe the possible role of a carbohydrate moiety of T. cruzi surface glycoproteins in host cell invasion by EA forms, highlighting the potential of these moieties as therapeutic and vaccine targets for the treatment of Chagas’ disease. PMID:29692765

Isolation and characterization of broadly neutralizing human monoclonal antibodies to the e1 glycoprotein of hepatitis C virus

DEFF Research Database (Denmark)

Meunier, Jean-Christophe; Russell, Rodney S.; Goossens, Vera

2008-01-01

The relative importance of humoral and cellular immunity in the prevention or clearance of hepatitis C virus (HCV) infection is poorly understood. However, there is considerable evidence that neutralizing antibodies are involved in disease control. Here we describe the detailed analysis of human...... monoclonal antibodies (MAbs) directed against HCV glycoprotein E1, which may have the potential to control HCV infection. We have identified two MAbs that can strongly neutralize HCV-pseudotyped particles (HCVpp) bearing the envelope glycoproteins of genotypes 1a, 1b, 4a, 5a, and 6a and less strongly...... neutralize HCVpp bearing the envelope glycoproteins of genotype 2a. Genotype 3a was not neutralized. The epitopes for both MAbs were mapped to the region encompassing amino acids 313 to 327. In addition, robust neutralization was also observed against cell culture-adapted viruses of genotypes 1a and 2a...

The O-Linked Glycome and Blood Group Antigens ABO on Mucin-Type Glycoproteins in Mucinous and Serous Epithelial Ovarian Tumors.

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Varvara Vitiazeva

Full Text Available Mucins are heavily O-glycosylated proteins where the glycosylation has been shown to play an important role in cancer. Normal epithelial ovarian cells do not express secreted mucins, but their abnormal expression has previously been described in epithelial ovarian cancer and may relate to tumor formation and progression. The cyst fluids were shown to be a rich source for acidic glycoproteins. The study of these proteins can potentially lead to the identification of more effective biomarkers for ovarian cancer.In this study, we analyzed the expression of the MUC5AC and the O-glycosylation of acidic glycoproteins secreted into ovarian cyst fluids. The samples were obtained from patients with serous and mucinous ovarian tumors of different stages (benign, borderline, malignant and grades. The O-linked oligosaccharides were released and analyzed by negative-ion graphitized carbon Liquid Chromatography (LC coupled to Electrospray Ionization tandem Mass Spectrometry (ESI-MSn. The LC-ESI-MSn of the oligosaccharides from ovarian cyst fluids displayed differences in expression of fucose containing structures such as blood group ABO antigens and Lewis-type epitopes.The obtained data showed that serous and mucinous benign adenomas, mucinous low malignant potential carcinomas (LMPs, borderline and mucinous low-grade carcinomas have a high level of blood groups and Lewis type epitopes. In contrast, this type of fucosylated structures were low abundant in the high-grade mucinous carcinomas or in serous carcinomas. In addition, the ovarian tumors that showed a high level of expression of blood group antigens also revealed a strong reactivity towards the MUC5AC antibody. To visualize the differences between serous and mucinous ovarian tumors based on the O-glycosylation, a hierarchical cluster analysis was performed using mass spectrometry average compositions (MSAC.Mucinous benign and LMPs along with mucinous low-grade carcinomas appear to be different from

Mutations increasing exposure of a receptor binding site epitope in the soluble and oligomeric forms of the caprine arthritis-encephalitis lentivirus envelope glycoprotein

International Nuclear Information System (INIS)

Hoetzel, Isidro; Cheevers, William P.

2005-01-01

The caprine arthritis-encephalitis (CAEV) and ovine maedi-visna (MVV) viruses are resistant to antibody neutralization, a feature shared with all other lentiviruses. Whether the CAEV gp135 receptor binding site(s) (RBS) in the functional surface envelope glycoprotein (Env) is protected from antibody binding, allowing the virus to resist neutralization, is not known. Two CAEV gp135 regions were identified by extrapolating a gp135 structural model that could affect binding of antibodies to the RBS: the V1 region and a short sequence analogous in position to the human immunodeficiency virus type 1 gp120 loop B postulated to be located between two major domains of CAEV gp135. Mutation of isoleucine-166 to alanine in the putative loop B of gp135 increased the affinity of soluble gp135 for the CAEV receptor(s) and goat monoclonal antibody (Mab) F7-299 which recognizes an epitope overlapping the gp135 RBS. The I166A mutation also stabilized or exposed the F7-299 epitope in anionic detergent buffers, indicating that the I166A mutation induces conformational changes and stabilizes the RBS of soluble gp135 and enhances Mab F7-299 binding. In contrast, the affinity of a V1 deletion mutant of gp135 for the receptor and Mab F7-299 and its structural stability did not differ from that of the wild-type gp135. However, both the I166A mutation and the V1 deletion of gp135 increased cell-to-cell fusion activity and binding of Mab F7-299 to the oligomeric Env. Therefore, the CAEV gp135 RBS is protected from antibody binding by mechanisms both dependent and independent of Env oligomerization which are disrupted by the V1 deletion and the I166A mutation, respectively. In addition, we found a correlation between side-chain β-branching at amino acid position 166 and binding of Mab F7-299 to oligomeric Env and cell-to-cell fusion, suggesting local secondary structure constraints in the region around isoleucine-166 as one determinant of gp135 RBS exposure and antibody binding

Critical epitopes in the nucleocapsid protein of SFTS virus recognized by a panel of SFTS patients derived human monoclonal antibodies.

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Li Yu

Full Text Available BACKGROUND: SFTS virus (SFTSV is a newly discovered pathogen to cause severe fever with thrombocytopenia syndrome (SFTS in human. Successful control of SFTSV epidemic requires better understanding of the antigen target in humoral immune responses to the new bunyavirus infection. METHODOLOGY/PRINCIPAL FINDINGS: We have generated a combinatorial Fab antibody phage library from two SFTS patients recovered from SFTSV infection. To date, 94 unique human antibodies have been generated and characterized from over 1200 Fab antibody clones obtained by screening the library with SFTS purified virions. All those monoclonal antibodies (MAbs recognized the nucleocapsid (N protein of SFTSV while none of them were reactive to the viral glycoproteins Gn or Gc. Furthermore, over screening 1000 mouse monoclonal antibody clones derived from SFTSV virions immunization, 462 clones reacted with N protein, while only 16 clones were reactive to glycoprotein. Furthermore, epitope mapping of SFTSV N protein was performed through molecular simulation, site mutation and competitive ELISA, and we found that at least 4 distinct antigenic epitopes within N protein were recognized by those human and mouse MAbs, in particular mutation of Glu10 to Ala10 abolished or significantly reduced the binding activity of nearly most SFTS patients derived MAbs. CONCLUSIONS/SIGNIFICANCE: The large number of human recombinant MAbs derived from SFTS patients recognized the viral N protein indicated the important role of the N protein in humoral responses to SFTSV infection, and the critical epitopes we defined in this study provided molecular basis for detection and diagnosis of SFTSV infection.

Structure of an antibody in complex with its mucin domain linear epitope that is protective against Ebola virus.

Science.gov (United States)

Olal, Daniel; Kuehne, Ana I; Bale, Shridhar; Halfmann, Peter; Hashiguchi, Takao; Fusco, Marnie L; Lee, Jeffrey E; King, Liam B; Kawaoka, Yoshihiro; Dye, John M; Saphire, Erica Ollmann

2012-03-01

Antibody 14G7 is protective against lethal Ebola virus challenge and recognizes a distinct linear epitope in the prominent mucin-like domain of the Ebola virus glycoprotein GP. The structure of 14G7 in complex with its linear peptide epitope has now been determined to 2.8 Å. The structure shows that this GP sequence forms a tandem β-hairpin structure that binds deeply into a cleft in the antibody-combining site. A key threonine at the apex of one turn is critical for antibody interaction and is conserved among all Ebola viruses. This work provides further insight into the mechanism of protection by antibodies that target the protruding, highly accessible mucin-like domain of Ebola virus and the structural framework for understanding and characterizing candidate immunotherapeutics.

An update on post-translational modifications of hydroxyproline-rich glycoproteins: Towards a model highlighting their contribution to plant cell wall architecture

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May eHijazi

2014-08-01

Full Text Available Plant cell walls are composite structures mainly composed of polysaccharides, also containing a large set of proteins involved in diverse functions such as growth, environmental sensing, signaling, and defense. Research on cell wall proteins (CWPs is a challenging field since present knowledge of their role into the structure and function of cell walls is very incomplete. Among CWPs, hydroxyproline (Hyp-rich O-glycoproteins (HRGPs were classified into three categories: (i moderately glycosylated extensins (EXTs able to form covalent scaffolds; (ii hyperglycosylated arabinogalactan proteins (AGPs; and (iii Hyp/proline (Pro-Rich proteins (H/PRPs that may be non-, weakly- or highly-glycosylated. In this review, we provide a description of the main features of their post-translational modifications (PTMs, biosynthesis, structure and function. We propose a new model integrating HRGPs and their partners in cell walls. Altogether, they could form a continuous glyco-network with non-cellulosic polysaccharides via covalent bonds or non-covalent interactions, thus strongly contributing to cell wall architecture.

Cooperativity in virus neutralization by human monoclonal antibodies to two adjacent regions located at the amino terminus of hepatitis C virus E2 glycoprotein

DEFF Research Database (Denmark)

Keck, Zhenyong; Wang, Wenyan; Wang, Yong

2013-01-01

A challenge for hepatitis C virus (HCV) vaccine development is defining conserved epitopes that induce protective antibodies against this highly diverse virus. An envelope glycoprotein (E2) segment located at amino acids (aa) 412 to 423 contains highly conserved neutralizing epitopes. While...... at higher concentrations. However, the overall effect was additive neutralization. A similar pattern was observed when these antibodies were combined to block E2 binding to the HCV coreceptor, CD81. These findings demonstrate that both of these E2 regions participate in epitopes mediating virus...... (HCVcc) with various activities. Although nonneutralizing HC33 HMAbs were isolated, they had lower binding affinities than neutralizing HC33 HMAbs. These antibodies could be converted to neutralizing antibodies by affinity maturation. Unidirectional competition for binding to E2 was observed between HC33...

Induction of antibodies against epitopes inaccessible on the HIV type 1 envelope oligomer by immunization with recombinant monomeric glycoprotein 120

DEFF Research Database (Denmark)

Schønning, Kristian; Bolmstedt, A; Novotny, J

1998-01-01

An N-glycan (N306) at the base of the V3 loop of HIV-BRU gp120 is shielding a linear neutralization epitope at the tip of the V3 loop on oligomeric Env. In contrast, this epitope is readily antigenic on monomeric gp120. Immunization with recombinant monomeric HIV-BRU gp120 may thus be expected...... immunogenic structures inaccessible on the envelope oligomer. The limited ability of recombinant gp120 vaccines to induce neutralizing antibodies against primary isolates may thus not exclusively reflect genetic variation....

A human PrM antibody that recognizes a novel cryptic epitope on dengue E glycoprotein.

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Annie Hoi Yi Chan

Full Text Available Dengue virus (DENV is a major mosquito-borne pathogen infecting up to 100 million people each year; so far no effective treatment or vaccines are available. Recently, highly cross-reactive and infection-enhancing pre-membrane (prM-specific antibodies were found to dominate the anti-DENV immune response in humans, raising concern over vaccine candidates that contain native dengue prM sequences. In this study, we have isolated a broadly cross-reactive prM-specific antibody, D29, during a screen with a non-immunized human Fab-phage library against the four serotypes of DENV. The antibody is capable of restoring the infectivity of virtually non-infectious immature DENV (imDENV in FcγR-bearing K562 cells. Remarkably, D29 also cross-reacted with a cryptic epitope on the envelope (E protein located to the DI/DII junction as evidenced by site-directed mutagenesis. This cryptic epitope, while inaccessible to antibody binding in a native virus particle, may become exposed if E is not properly folded. These findings suggest that generation of anti-prM antibodies that enhance DENV infection may not be completely avoided even with immunization strategies employing E protein alone or subunits of E proteins.

Mechanism of Binding to Ebola Virus Glycoprotein by the ZMapp, ZMAb, and MB-003 Cocktail Antibodies

OpenAIRE

Davidson, Edgar; Bryan, Christopher; Fong, Rachel H.; Barnes, Trevor; Pfaff, Jennifer M.; Mabila, Manu; Rucker, Joseph B.; Doranz, Benjamin J.

2015-01-01

Cocktails of monoclonal antibodies (MAbs) that target the surface glycoprotein (GP) of Ebola virus (EBOV) are effective in nonhuman primate models and have been used under emergency compassionate-treatment protocols in human patients. However, the amino acids that form the detailed binding epitopes for the MAbs in the ZMapp, ZMAb, and the related MB-003 cocktails have yet to be identified. Other binding properties that define how each MAb functionally interacts with GP—such as affinity, epito...

A Carbohydrate Moiety of Secreted Stage-Specific Glycoprotein 4 Participates in Host Cell Invasion by Trypanosoma cruzi Extracellular Amastigotes

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Pilar T. V. Florentino

2018-04-01

Full Text Available Trypanosoma cruzi is the etiologic agent of Chagas’ disease. It is known that amastigotes derived from trypomastigotes in the extracellular milieu are infective in vitro and in vivo. Extracellular amastigotes (EAs have a stage-specific surface antigen called Ssp-4, a GPI-anchored glycoprotein that is secreted by the parasites. By immunoprecipitation with the Ssp-4-specific monoclonal antibodies (mAb 2C2 and 1D9, we isolated the glycoprotein from EAs. By mass spectrometry, we identified the core protein of Ssp-4 and evaluated mRNA expression and the presence of Ssp-4 carbohydrate epitopes recognized by mAb1D9. We demonstrated that the carbohydrate epitope recognized by mAb1D9 could promote host cell invasion by EAs. Although infectious EAs express lower amounts of Ssp-4 compared with less-infectious EAs (at the mRNA and protein levels, it is the glycosylation of Ssp-4 (identified by mAb1D9 staining only in infectious strains and recognized by galectin-3 on host cells that is the determinant of EA invasion of host cells. Furthermore, Ssp-4 is secreted by EAs, either free or associated with parasite vesicles, and can participate in host-cell interactions. The results presented here describe the possible role of a carbohydrate moiety of T. cruzi surface glycoproteins in host cell invasion by EA forms, highlighting the potential of these moieties as therapeutic and vaccine targets for the treatment of Chagas’ disease.

Cytomegalovirus (CMV) Epitope-Specific CD4+ T Cells Are Inflated in HIV+ CMV+ Subjects.

Science.gov (United States)

Abana, Chike O; Pilkinton, Mark A; Gaudieri, Silvana; Chopra, Abha; McDonnell, Wyatt J; Wanjalla, Celestine; Barnett, Louise; Gangula, Rama; Hager, Cindy; Jung, Dae K; Engelhardt, Brian G; Jagasia, Madan H; Klenerman, Paul; Phillips, Elizabeth J; Koelle, David M; Kalams, Spyros A; Mallal, Simon A

2017-11-01

Select CMV epitopes drive life-long CD8 + T cell memory inflation, but the extent of CD4 memory inflation is poorly studied. CD4 + T cells specific for human CMV (HCMV) are elevated in HIV + HCMV + subjects. To determine whether HCMV epitope-specific CD4 + T cell memory inflation occurs during HIV infection, we used HLA-DR7 (DRB1*07:01) tetramers loaded with the glycoprotein B DYSNTHSTRYV (DYS) epitope to characterize circulating CD4 + T cells in coinfected HLA-DR7 + long-term nonprogressor HIV subjects with undetectable HCMV plasma viremia. DYS-specific CD4 + T cells were inflated among these HIV + subjects compared with those from an HIV - HCMV + HLA-DR7 + cohort or with HLA-DR7-restricted CD4 + T cells from the HIV-coinfected cohort that were specific for epitopes of HCMV phosphoprotein-65, tetanus toxoid precursor, EBV nuclear Ag 2, or HIV gag protein. Inflated DYS-specific CD4 + T cells consisted of effector memory or effector memory-RA + subsets with restricted TCRβ usage and nearly monoclonal CDR3 containing novel conserved amino acids. Expression of this near-monoclonal TCR in a Jurkat cell-transfection system validated fine DYS specificity. Inflated cells were polyfunctional, not senescent, and displayed high ex vivo levels of granzyme B, CX 3 CR1, CD38, or HLA-DR but less often coexpressed CD38 + and HLA-DR + The inflation mechanism did not involve apoptosis suppression, increased proliferation, or HIV gag cross-reactivity. Instead, the findings suggest that intermittent or chronic expression of epitopes, such as DYS, drive inflation of activated CD4 + T cells that home to endothelial cells and have the potential to mediate cytotoxicity and vascular disease. Copyright © 2017 by The American Association of Immunologists, Inc.

Epitope prediction methods

DEFF Research Database (Denmark)

Karosiene, Edita

Analysis. The chapter provides detailed explanations on how to use different methods for T cell epitope discovery research, explaining how input should be given as well as how to interpret the output. In the last chapter, I present the results of a bioinformatics analysis of epitopes from the yellow fever...... peptide-MHC interactions. Furthermore, using yellow fever virus epitopes, we demonstrated the power of the %Rank score when compared with the binding affinity score of MHC prediction methods, suggesting that this score should be considered to be used for selecting potential T cell epitopes. In summary...... immune responses. Therefore, it is of great importance to be able to identify peptides that bind to MHC molecules, in order to understand the nature of immune responses and discover T cell epitopes useful for designing new vaccines and immunotherapies. MHC molecules in humans, referred to as human...

Rifampicin-dependent antibodies bind a similar or identical epitope to glycoprotein IX-specific quinine-dependent antibodies

NARCIS (Netherlands)

Burgess, Janette K.; Lopez, Jose A.; Gaudry, Leonie E.; Chong, Beng H.

2000-01-01

The drug-dependent antibody of a patient with rifampicin-induced thrombocytopenia was characterized using the antigen-capture enzyme-linked immunosorbent assay (MAIPA assay), flow cytometry, and immunoprecipitation. The antibody was found to bind glycoprotein (GP) Ib-IX but not GPIIb-IIIa because

Roles of tyrosine-rich precursor glycoproteins and dityrosine- and 3,4-dihydroxyphenylalanine-mediated protein cross-linking in development of the oocyst wall in the coccidian parasite Eimeria maxima

DEFF Research Database (Denmark)

Belli, Sabina I; Wallach, Michael G; Luxford, Catherine

2003-01-01

infection by several organisms of medical and veterinary importance such as Eimeria, Plasmodium, Toxoplasma, Cyclospora, and Neospora could be developed. Here, we show that two tyrosine-rich precursor glycoproteins, gam56 and gam82, found in specialized organelles (wall-forming bodies) in the sexual stage...... (macrogamete) of Eimeria maxima are proteolytically processed into smaller glycoproteins, which are then incorporated into the developing oocyst wall. The identification of high concentrations of dityrosine and 3,4-dihydroxyphenylalanine (DOPA) in oocyst extracts by high-pressure liquid chromatography......-mediated cross-linking might be an enzyme-catalyzed event. As such, the mechanism of oocyst wall formation in Eimeria, is analogous to the underlying mechanisms involved in the stabilization of extracellular matrices in a number of organisms, widely distributed in nature, including insect resilin, nematode...

Modification of P-selectin glycoprotein ligand-1 with a natural killer cell-restricted sulfated lactosamine creates an alternate ligand for L-selectin

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André, Pascale; Spertini, Olivier; Guia, Sophie; Rihet, Pascal; Dignat-George, Françoise; Brailly, Hervé; Sampol, José; Anderson, Paul J.; Vivier, Eric

2000-01-01

Natural killer (NK) cells are components of the innate immune system that can recognize and kill virally infected cells, tumor cells, and allogeneic cells without prior sensitization. NK cells also elaborate cytokines (e.g., interferon-γ and tumor necrosis factor-α) and chemokines (e.g., macrophage inflammatory protein-1α) that promote the acquisition of antigen-specific immunity. NK cell differentiation is accompanied by the cell surface expression of a mucin-like glycoprotein bearing an NK cell-restricted keratan sulfate-related lactosamine carbohydrate, the PEN5 epitope. Here, we report that PEN5 is a post-translational modification of P-selectin glycoprotein ligand-1 (PSGL-1). The PEN5 epitope creates on PSGL-1 a unique binding site for L-selectin, which is independent of PSGL-1 tyrosine sulfation. On the surface of NK cells, the expression of PEN5 is coordinated with the disappearance of L-selectin and the up-regulation of Killer cell Ig-like Receptors (KIR). These results indicate that NK cell differentiation is accompanied by the acquisition of a unique carbohydrate, PEN5, that can serve as part of a combination code to deliver KIR+ NK cells to specific tissues. PMID:10725346

Multiple genes encode the major surface glycoprotein of Pneumocystis carinii

DEFF Research Database (Denmark)

Kovacs, J A; Powell, F; Edman, J C

1993-01-01

hydrophobic region at the carboxyl terminus. The presence of multiple related msg genes encoding the major surface glycoprotein of P. carinii suggests that antigenic variation is a possible mechanism for evading host defenses. Further characterization of this family of genes should allow the development......The major surface antigen of Pneumocystis carinii, a life-threatening opportunistic pathogen in human immunodeficiency virus-infected patients, is an abundant glycoprotein that functions in host-organism interactions. A monoclonal antibody to this antigen is protective in animals, and thus...... blot studies using chromosomal or restricted DNA, the major surface glycoproteins are the products of a multicopy family of genes. The predicted protein has an M(r) of approximately 123,000, is relatively rich in cysteine residues (5.5%) that are very strongly conserved, and contains a well conserved...

Shared epitopes of glycoprotein A and protein 4.1 defined by antibody NaM10-3C10.

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Rasamoelisolo, M; Czerwinski, M; Willem, C; Blanchard, D

1998-06-01

We have produced the murine monoclonal antibody (MAb) NaM70-3C10 (IgM) from splenocytes of mice immunized with human red blood cells (RBCs). The MAb agglutinated untreated as well as trypsin, chymotrypsin, neuraminidase, or ficin-treated RBCs from controls. In contrast, control RBCs treated with papaine or bromelaine were not agglutinated. On immunoblots, the MAb bound to glycophorin A (GPA) and to a 80 kDa protein identified as protein 4.1. Analysis by agglutination of variant RBCs carrying hybrid glycophorins made of the N-terminus (amino acids 1-58) of GPA and of the C-terminus (amino acids 27-72) of glycophorin B (GPB) and competition-inhibition test using purified GPA and a synthetic peptide corresponding to the amino acid sequence 48-58 of GPA demonstrated that the epitope is located within residues 48-58 of GPA. Epitope analysis with immobilized peptides showed that the MAb recognizes the sequence 53Pro-Pro-Glu-Glu-GIu58 of GPA. A homologous sequence is also present within amino acids 395 to 405 of protein 4.1. Finally, the MAb bound to 16 kDa chymotryptic peptide of protein 4.1, which carries the above amino acid sequence. In conclusion, it may be assumed that NaM70-3C10 specifically recognizes a common epitope on the extracellular domain of GPA and on the intracellular protein 4.1; this specificity explains the persistence of the 80 kDa band on blots when RBCs are treated with papain.

Adjuvant-Mediated Epitope Specificity and Enhanced Neutralizing Activity of Antibodies Targeting Dengue Virus Envelope Protein

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Denicar Lina Nascimento Fabris Maeda

2017-09-01

Full Text Available The heat-labile toxins (LT produced by enterotoxigenic Escherichia coli display adjuvant effects to coadministered antigens, leading to enhanced production of serum antibodies. Despite extensive knowledge of the adjuvant properties of LT derivatives, including in vitro-generated non-toxic mutant forms, little is known about the capacity of these adjuvants to modulate the epitope specificity of antibodies directed against antigens. This study characterizes the role of LT and its non-toxic B subunit (LTB in the modulation of antibody responses to a coadministered antigen, the dengue virus (DENV envelope glycoprotein domain III (EDIII, which binds to surface receptors and mediates virus entry into host cells. In contrast to non-adjuvanted or alum-adjuvanted formulations, antibodies induced in mice immunized with LT or LTB showed enhanced virus-neutralization effects that were not ascribed to a subclass shift or antigen affinity. Nonetheless, immunosignature analyses revealed that purified LT-adjuvanted EDIII-specific antibodies display distinct epitope-binding patterns with regard to antibodies raised in mice immunized with EDIII or the alum-adjuvanted vaccine. Notably, the analyses led to the identification of a specific EDIII epitope located in the EF to FG loop, which is involved in the entry of DENV into eukaryotic cells. The present results demonstrate that LT and LTB modulate the epitope specificity of antibodies generated after immunization with coadministered antigens that, in the case of EDIII, was associated with the induction of neutralizing antibody responses. These results open perspectives for the more rational development of vaccines with enhanced protective effects against DENV infections.

Antigenic properties of the human immunodeficiency virus envelope glycoprotein gp120 on virions bound to target cells.

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Meron Mengistu

2015-03-01

Full Text Available The HIV-1 envelope glycoprotein, gp120, undergoes multiple molecular interactions and structural rearrangements during the course of host cell attachment and viral entry, which are being increasingly defined at the atomic level using isolated proteins. In comparison, antigenic markers of these dynamic changes are essentially unknown for single HIV-1 particles bound to target cells. Such markers should indicate how neutralizing and/or non-neutralizing antibodies might interdict infection by either blocking infection or sensitizing host cells for elimination by Fc-mediated effector function. Here we address this deficit by imaging fluorescently labeled CCR5-tropic HIV-1 pseudoviruses using confocal and superresolution microscopy to track the exposure of neutralizing and non-neutralizing epitopes as they appear on single HIV-1 particles bound to target cells. Epitope exposure was followed under conditions permissive or non-permissive for viral entry to delimit changes associated with virion binding from those associated with post-attachment events. We find that a previously unexpected array of gp120 epitopes is exposed rapidly upon target cell binding. This array comprises both neutralizing and non-neutralizing epitopes, the latter being hidden on free virions yet capable of serving as potent targets for Fc-mediated effector function. Under non-permissive conditions for viral entry, both neutralizing and non-neutralizing epitope exposures were relatively static over time for the majority of bound virions. Under entry-permissive conditions, epitope exposure patterns changed over time on subsets of virions that exhibited concurrent variations in virion contents. These studies reveal that bound virions are distinguished by a broad array of both neutralizing and non-neutralizing gp120 epitopes that potentially sensitize a freshly engaged target cell for destruction by Fc-mediated effector function and/or for direct neutralization at a post-binding step

Structural delineation of a quaternary, cleavage-dependent epitope at the gp41-gp120 interface on intact HIV-1 Env trimers.

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Blattner, Claudia; Lee, Jeong Hyun; Sliepen, Kwinten; Derking, Ronald; Falkowska, Emilia; de la Peña, Alba Torrents; Cupo, Albert; Julien, Jean-Philippe; van Gils, Marit; Lee, Peter S; Peng, Wenjie; Paulson, James C; Poignard, Pascal; Burton, Dennis R; Moore, John P; Sanders, Rogier W; Wilson, Ian A; Ward, Andrew B

2014-05-15

All previously characterized broadly neutralizing antibodies to the HIV-1 envelope glycoprotein (Env) target one of four major sites of vulnerability. Here, we define and structurally characterize a unique epitope on Env that is recognized by a recently discovered family of human monoclonal antibodies (PGT151-PGT158). The PGT151 epitope is comprised of residues and glycans at the interface of gp41 and gp120 within a single protomer and glycans from both subunits of a second protomer and represents a neutralizing epitope that is dependent on both gp120 and gp41. Because PGT151 binds only to properly formed, cleaved trimers, this distinctive property, and its ability to stabilize Env trimers, has enabled the successful purification of mature, cleaved Env trimers from the cell surface as a complex with PGT151. Here we compare the structural and functional properties of membrane-extracted Env trimers from several clades with those of the soluble, cleaved SOSIP gp140 trimer. Copyright © 2014 Elsevier Inc. All rights reserved.

Histochemical and structural analysis of mucous glycoprotein secreted by the gill of Mytilus edulis

International Nuclear Information System (INIS)

Ahn, Hae-Young.

1988-01-01

Studies were carried out to characterized various mucous cells in the gill filament, to ascertain structural characteristics of the secreted mucous glycoproteins, and to determine the ability of the gill epithelium to incorporate [ 14 C]glucosamine as a precursor in the biosynthesis and secretion of mucous glycoproteins. Using histochemical staining techniques, mucous cells containing neutral and acidic mucins were found in the lateral region, whereas mucous cells containing primarily neutral or sulfated mucins were found in the postlateral region. Serotonin, but not dopamine, stimulated the mucous secretion. In tissues pretreated with [ 14 C]glucosamine, the secreted glycoproteins contain incorporated radiolabel. Analysis by column chromatography using Bio-Gel P-2 and P-6 shows that the secretion contains two glycoprotein populations. Glycoprotein II has a molecular weight of 2.3 x 10 4 daltons. Upon alkaline reductive borohydride cleavage of the O-glycosidic linkages of glycoprotein I, about 70% of the radiolabel was removed from the protein. Gas chromatographic analysis of the carbohydrate composition shows that the glycoproteins contains N-acetylglucosamine (GluNAc), N-acetylgalactosamine (GalNAc), and galactose, fucose and mannose. Amino acid analysis shows that the glycoproteins are rich in serine, threonine and proline

Antigenicity of peptides comprising the immunosuppressive domain of the retroviral envelope glycoprotein [version 1; referees: 2 approved

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Bryony Jenkins

2016-12-01

Full Text Available To achieve persistent infection of the host, viruses often subvert or suppress host immunity through mechanisms that are not entirely understood. The envelope glycoprotein of several retroviruses is thought to possess potent immunosuppressive activity, mapped to a 17-amino acid residue conserved domain. Synthetic peptides corresponding to this immunosuppressive domain can inhibit lymphocyte activation, whereas mutation of key domain residues can increase the lymphocyte response to linked antigenic epitopes. Using three T cell receptors (TCRs of defined specificity, we examine the effect of the immunosuppressive domain on the T cell response to their respective antigenic peptides. We find that fusion of a T cell epitope to the immunosuppressive domain can greatly modulate its potency. However, the effects heavily depend on the particular combination of TCR and peptide-major histocompatibility complex class II (pMHC II, and are mimicked by sequence-scrambled peptides of similar length, suggesting they operate at the level of TCR-pMHC interaction. These results offer an alternative explanation for the immunogenicity of T cell epitopes comprising the putative immunosuppressive domain, which is more consistent with an effect on peptide antigenicity than true immunosuppressive activity.

Antigenicity of peptides comprising the immunosuppressive domain of the retroviral envelope glycoprotein [version 2; referees: 2 approved

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Bryony Jenkins

2017-02-01

Full Text Available To achieve persistent infection of the host, viruses often subvert or suppress host immunity through mechanisms that are not entirely understood. The envelope glycoprotein of several retroviruses is thought to possess potent immunosuppressive activity, mapped to a 17-amino acid residue conserved domain. Synthetic peptides corresponding to this immunosuppressive domain can inhibit lymphocyte activation, whereas mutation of key domain residues can increase the lymphocyte response to linked antigenic epitopes. Using three T cell receptors (TCRs of defined specificity, we examine the effect of the immunosuppressive domain on the T cell response to their respective antigenic peptides. We find that fusion of a T cell epitope to the immunosuppressive domain can greatly modulate its potency. However, the effects heavily depend on the particular combination of TCR and peptide-major histocompatibility complex class II (pMHC II, and are mimicked by sequence-scrambled peptides of similar length, suggesting they operate at the level of pMHC formation or TCR-pMHC interaction. These results offer an alternative explanation for the immunogenicity of T cell epitopes comprising the putative immunosuppressive domain, which is more consistent with an effect on peptide antigenicity than true immunosuppressive activity.

Microbiota epitope similarity either dampens or enhances the immunogenicity of disease-associated antigenic epitopes.

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Sebastian Carrasco Pro

Full Text Available The microbiome influences adaptive immunity and molecular mimicry influences T cell reactivity. Here, we evaluated whether the sequence similarity of various antigens to the microbiota dampens or increases immunogenicity of T cell epitopes. Sets of epitopes and control sequences derived from 38 antigenic categories (infectious pathogens, allergens, autoantigens were retrieved from the Immune Epitope Database (IEDB. Their similarity to microbiome sequences was calculated using the BLOSUM62 matrix. We found that sequence similarity was associated with either dampened (tolerogenic; e.g. most allergens or increased (inflammatory; e.g. Dengue and West Nile viruses likelihood of a peptide being immunogenic as a function of epitope source category. Ten-fold cross-validation and validation using sets of manually curated epitopes and non-epitopes derived from allergens were used to confirm these initial observations. Furthermore, the genus from which the microbiome homologous sequences were derived influenced whether a tolerogenic versus inflammatory modulatory effect was observed, with Fusobacterium most associated with inflammatory influences and Bacteroides most associated with tolerogenic influences. We validated these effects using PBMCs stimulated with various sets of microbiome peptides. "Tolerogenic" microbiome peptides elicited IL-10 production, "inflammatory" peptides elicited mixed IL-10/IFNγ production, while microbiome epitopes homologous to self were completely unreactive for both cytokines. We also tested the sequence similarity of cockroach epitopes to specific microbiome sequences derived from households of cockroach allergic individuals and non-allergic controls. Microbiomes from cockroach allergic households were less likely to contain sequences homologous to previously defined cockroach allergens. These results are compatible with the hypothesis that microbiome sequences may contribute to the tolerization of T cells for allergen

Demonstration of immunochemical identity between the nerve growth factor-inducible large external (NILE) glycoprotein and the cell adhesion molecule L1

DEFF Research Database (Denmark)

Bock, E; Richter-Landsberg, C; Faissner, A

1985-01-01

The nerve growth factor-inducible large external (NILE) glycoprotein and the neural cell adhesion molecule L1 were shown to be immunochemically identical. Immunoprecipitation with L1 and NILE antibodies of [3H]fucose-labeled material from culture supernatants and detergent extracts of NGF......-treated rat PC12 pheochromocytoma cells yielded comigrating bands by SDS-PAGE. NILE antibodies reacted with immunopurified L1 antigen, but not with N-CAM and other L2 epitope-bearing glycoproteins from adult mouse brain. Finally, by sequential immunoprecipitation from detergent extracts of [35S......]methionine-labeled early post-natal cerebellar cell cultures or [3H]fucose-labeled NGF-treated PC12 cells, all immunoreactivity for NILE antibody could be removed by pre-clearing with L1 antibody and vice versa....

Virion Glycoprotein-Mediated Immune Evasion by Human Cytomegalovirus: a Sticky Virus Makes a Slick Getaway

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Gardner, Thomas J.

2016-01-01

SUMMARY The prototypic herpesvirus human cytomegalovirus (CMV) exhibits the extraordinary ability to establish latency and maintain a chronic infection throughout the life of its human host. This is even more remarkable considering the robust adaptive immune response elicited by infection and reactivation from latency. In addition to the ability of CMV to exist in a quiescent latent state, its persistence is enabled by a large repertoire of viral proteins that subvert immune defense mechanisms, such as NK cell activation and major histocompatibility complex antigen presentation, within the cell. However, dissemination outside the cell presents a unique existential challenge to the CMV virion, which is studded with antigenic glycoprotein complexes targeted by a potent neutralizing antibody response. The CMV virion envelope proteins, which are critical mediators of cell attachment and entry, possess various characteristics that can mitigate the humoral immune response and prevent viral clearance. Here we review the CMV glycoprotein complexes crucial for cell attachment and entry and propose inherent properties of these proteins involved in evading the CMV humoral immune response. These include viral glycoprotein polymorphism, epitope competition, Fc receptor-mediated endocytosis, glycan shielding, and cell-to-cell spread. The consequences of CMV virion glycoprotein-mediated immune evasion have a major impact on persistence of the virus in the population, and a comprehensive understanding of these evasion strategies will assist in designing effective CMV biologics and vaccines to limit CMV-associated disease. PMID:27307580

Noncanonical Expression of a Murine Cytomegalovirus Early Protein CD8 T-Cell Epitope as an Immediate Early Epitope Based on Transcription from an Upstream Gene

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Annette Fink

2014-02-01

Full Text Available Viral CD8 T-cell epitopes, represented by viral peptides bound to major histocompatibility complex class-I (MHC-I glycoproteins, are often identified by “reverse immunology”, a strategy not requiring biochemical and structural knowledge of the actual viral protein from which they are derived by antigen processing. Instead, bioinformatic algorithms predicting the probability of C-terminal cleavage in the proteasome, as well as binding affinity to the presenting MHC-I molecules, are applied to amino acid sequences deduced from predicted open reading frames (ORFs based on the genomic sequence. If the protein corresponding to an antigenic ORF is known, it is usually inferred that the kinetic class of the protein also defines the phase in the viral replicative cycle during which the respective antigenic peptide is presented for recognition by CD8 T cells. We have previously identified a nonapeptide from the predicted ORFm164 of murine cytomegalovirus that is presented by the MHC-I allomorph H-2 Dd and that is immunodominant in BALB/c (H-2d haplotype mice. Surprisingly, although the ORFm164 protein gp36.5 is expressed as an Early (E phase protein, the m164 epitope is presented already during the Immediate Early (IE phase, based on the expression of an upstream mRNA starting within ORFm167 and encompassing ORFm164.

Cell wall glycoproteins at interaction sites between parasitic giant dodder (Cuscuta reflexa) and its host Pelargonium zonale.

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Striberny, Bernd; Krause, Kirsten

2015-01-01

The process of host plant penetration by parasitic dodder (genus Cuscuta) is accompanied by molecular and structural changes at the host/parasite interface. Recently, changes in pectin methyl esterification levels in the host cell walls abutting parasitic cells in established infection sites were reported. In addition to that, we show here that the composition of cell wall glycoproteins in Cuscuta-infected Pelargonium zonale undergoes substantial changes. While several arabinogalactan protein epitopes exhibit decreased abundances in the vicinity of the Cuscuta reflexa haustorium, extensins tend to increase in the infected areas.

Hepatitis C Virus E2 Envelope Glycoprotein Core Structure

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Kong, Leopold; Giang, Erick; Nieusma, Travis; Kadam, Rameshwar U.; Cogburn, Kristin E.; Hua, Yuanzi; Dai, Xiaoping; Stanfield, Robyn L.; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.; Law, Mansun

2014-08-26

Hepatitis C virus (HCV), a Hepacivirus, is a major cause of viral hepatitis, liver cirrhosis, and hepatocellular carcinoma. HCV envelope glycoproteins E1 and E2 mediate fusion and entry into host cells and are the primary targets of the humoral immune response. The crystal structure of the E2 core bound to broadly neutralizing antibody AR3C at 2.65 angstroms reveals a compact architecture composed of a central immunoglobulin-fold β sandwich flanked by two additional protein layers. The CD81 receptor binding site was identified by electron microscopy and site-directed mutagenesis and overlaps with the AR3C epitope. The x-ray and electron microscopy E2 structures differ markedly from predictions of an extended, three-domain, class II fusion protein fold and therefore provide valuable information for HCV drug and vaccine design.

Characterization of canine herpesvirus glycoprotein C expressed by a recombinant baculovirus in insect cells.

Science.gov (United States)

Xuan, X; Maeda, K; Mikami, T; Otsuka, H

1996-12-01

The gene encoding the canine herpesvirus (CHV) glycoprotein C (gC) homologue has been identified by sequence homology analyses with other well studied herpesviruses. Previously, we have identified three CHV glycoproteins, gp145/112, gp80 and gp47 using a panel of monoclonal antibodies (MAbs). To determine which CHV glycoprotein corresponds to gC, a recombinant baculovirus which contains the putative CHV gC structural gene under the baculovirus polyhedrin promoter was constructed. The recombinant baculovirus expressed gC-related polypeptides (44-62 kDa), which reacted only with MAbs against CHV gp80, indicating that the previously identified CHV gp80 is the translation product of the gC gene. The baculovirus expressed gC was glycosylated and transported to the surface of infected cells. At least seven neutralizing epitopes were conserved on the gC produced in insect cells. It was found that the recombinant baculovirus infected cells adsorbed murine erythrocytes as is the case for CHV-infected cells. The hemadsorption activity was inhibited by heparin, indicating that the CHV gC binds to heparan sulfate on the surface of murine erythrocytes. Mice immunized with the recombinant gC produced strong neutralizing antibodies. Our results suggest that CHV gC produced in insect cells may be useful as a subunit vaccine to control CHV infections.

Functional Regulation of the Plasma Protein Histidine-Rich Glycoprotein by Zn2+ in Settings of Tissue Injury

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Kristin M. Priebatsch

2017-03-01

Full Text Available Divalent metal ions are essential nutrients for all living organisms and are commonly protein-bound where they perform important roles in protein structure and function. This regulatory control from metals is observed in the relatively abundant plasma protein histidine-rich glycoprotein (HRG, which displays preferential binding to the second most abundant transition element in human systems, Zinc (Zn2+. HRG has been proposed to interact with a large number of protein ligands and has been implicated in the regulation of various physiological and pathological processes including the formation of immune complexes, apoptotic/necrotic and pathogen clearance, cell adhesion, antimicrobial activity, angiogenesis, coagulation and fibrinolysis. Interestingly, these processes are often associated with sites of tissue injury or tumour growth, where the concentration and distribution of Zn2+ is known to vary. Changes in Zn2+ levels have been shown to modify HRG function by altering its affinity for certain ligands and/or providing protection against proteolytic disassembly by serine proteases. This review focuses on the molecular interplay between HRG and Zn2+, and how Zn2+ binding modifies HRG-ligand interactions to regulate function in different settings of tissue injury.

Glycoprotein component of plant cell walls

International Nuclear Information System (INIS)

Cooper, J.B.; Chen, J.A.; Varner, J.E.

1984-01-01

The primary wall surrounding most dicotyledonous plant cells contains a hydroxyproline-rich glycoprotein (HRGP) component named extensin. A small group of glycopeptides solubilized from isolated cell walls by proteolysis contained a repeated pentapeptide glycosylated by tri- and tetraarabinosides linked to hydroxyproline and, by galactose, linked to serine. Recently, two complementary approaches to this problem have provided results which greatly increase the understanding of wall extensin. In this paper the authors describe what is known about the structure of soluble extensin secreted into the walls of the carrot root cells

Structural and Antigenic Definition of Hepatitis C Virus E2 Glycoprotein Epitopes Targeted by Monoclonal Antibodies

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Giuseppe Sautto

2013-01-01

Full Text Available Hepatitis C virus (HCV is the major cause of chronic liver disease as well as the major indication for liver transplantation worldwide. Current standard of care is not completely effective, not administrable in grafted patients, and burdened by several side effects. This incomplete effectiveness is mainly due to the high propensity of the virus to continually mutate under the selective pressure exerted by the host immune response as well as currently administered antiviral drugs. The E2 envelope surface glycoprotein of HCV (HCV/E2 is the main target of the host humoral immune response and for this reason one of the major variable viral proteins. However, broadly cross-neutralizing monoclonal antibodies (mAbs directed against HCV/E2 represent a promising tool for the study of virus-host interplay as well as for the development of effective prophylactic and therapeutic approaches. In the last few years many anti-HCV/E2 mAbs have been evaluated in preclinical and clinical trials as possible candidate antivirals, particularly for administration in pre- and post-transplant settings. In this review we summarize the antigenic and structural characteristics of HCV/E2 determined through the use of anti-HCV/E2 mAbs, which, given the absence of a crystal structure of this glycoprotein, represent currently the best tool available.

The Immune Epitope Database 2.0

DEFF Research Database (Denmark)

Hoof, Ilka; Vita, R; Zarebski, L

2010-01-01

The Immune Epitope Database (IEDB, www.iedb.org) provides a catalog of experimentally characterized B and T cell epitopes, as well as data on Major Histocompatibility Complex (MHC) binding and MHC ligand elution experiments. The database represents the molecular structures recognized by adaptive...... immune receptors and the experimental contexts in which these molecules were determined to be immune epitopes. Epitopes recognized in humans, nonhuman primates, rodents, pigs, cats and all other tested species are included. Both positive and negative experimental results are captured. Over the course...

Fraction A of armadillo submandibular glycoprotein and its desialylated product as sialyl-Tn and Tn receptors for lectins.

Science.gov (United States)

Wu, A M; Shen, F; Herp, A; Song, S C; Wu, J H

1995-02-27

Fraction A of the armadillo submandibular glycoprotein (ASG-A) is one of the simplest glycoproteins among mammalian salivary mucins. The carbohydrate side chains of this mucous glycoprotein have one-third of the NeuAc alpha 2-->6GalNAc (sialyl-Tn) sequence and two thirds of Tn (GalNAc alpha-->Ser/Thr) residues. Those of the desialylated product (ASG-Tn) are almost exclusively unsubstituted GalNAc residues (Tn determinant). When the binding properties of these glycoproteins were tested by a precipitin assay with Gal, GalNAc and GlcNAc specific lectins, it was found that ASG-Tn reacted strongly with all of the Tn-active lectins and completely precipitated Vicia villosa (VVL both B4 and mixture of A and B), Maclura pomifera (MPA), and Artocarpus integrifolia (jacalin) lectins. However, it precipitated poorly or negligibly with Ricinus communis (RCA1); Dolichos biflorus (DBA); Viscum album, ML-I; Arachis hypogaea (PNA), and Triticum vulgaris (WGA). The reactivity of ASG-A (sialyl-Tn) was as active as that of ASG-Tn with MPA and less or slightly less active than that of ASG-Tn with VVL-A+B, VVL-B4, HPA, WFA, and jacalin, as one-third of its Tn was sialylated. These findings indicate that ASG-A and its desialylated product (ASG-Tn) are highly useful reagents for the differentiation of Tn, T (Gal beta 1-->3GalNAc), A (GalNAc alpha 1-->3Gal) or Gal specific lectins and monoclonal antibodies against such epitopes.

Strategies for induction of catalytic antibodies toward HIV-1 glycoprotein gp120 in autoimmune prone mice.

Science.gov (United States)

Durova, Oxana M; Vorobiev, Ivan I; Smirnov, Ivan V; Reshetnyak, Andrew V; Telegin, Georgy B; Shamborant, Olga G; Orlova, Nadezda A; Genkin, Dmitry D; Bacon, Andrew; Ponomarenko, Natalia A; Friboulet, Alain; Gabibov, Alexander G

2009-11-01

Tremendous efforts to produce an efficient vaccine for HIV infection have been unsuccessful. The ability of HIV to utilize sophisticated mechanisms to escape killing by host immune system rises dramatic problems in the development of antiviral therapeutics. The HIV infection proceeds by interaction of coat viral glycoprotein gp120 trimer with CD4(+) receptor of the lymphocyte. Thus this surface antigen may be regarded as a favorable target for immunotherapy. In the present study, we have developed three different strategies to produce gp120-specific response in autoimmune prone mice (SJL strain) as potential tools for production "catalytic vaccine". Therefore (i) reactive immunization by peptidylphosphonate, structural part of the coat glycoprotein, (ii) immunization by engineered fused epitopes of gp120 and encephalogenic peptide, a part of myelin basic protein, and (iii) combined vaccination by DNA and corresponding gp120 fragments incorporated into liposomes were investigated. In the first two cases monoclonal antibodies and their recombinant fragments with amidolytic and gp120-specific proteolytic activities were characterized. In the last case, catalytic antibodies with virus neutralizing activity proved in cell line models were harvested.

Affinity selection of Nipah and Hendra virus-related vaccine candidates from a complex random peptide library displayed on bacteriophage virus-like particles

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Peabody, David S.; Chackerian, Bryce; Ashley, Carlee; Carnes, Eric; Negrete, Oscar

2017-01-24

The invention relates to virus-like particles of bacteriophage MS2 (MS2 VLPs) displaying peptide epitopes or peptide mimics of epitopes of Nipah Virus envelope glycoprotein that elicit an immune response against Nipah Virus upon vaccination of humans or animals. Affinity selection on Nipah Virus-neutralizing monoclonal antibodies using random sequence peptide libraries on MS2 VLPs selected peptides with sequence similarity to peptide sequences found within the envelope glycoprotein of Nipah itself, thus identifying the epitopes the antibodies recognize. The selected peptide sequences themselves are not necessarily identical in all respects to a sequence within Nipah Virus glycoprotein, and therefore may be referred to as epitope mimics VLPs displaying these epitope mimics can serve as vaccine. On the other hand, display of the corresponding wild-type sequence derived from Nipah Virus and corresponding to the epitope mapped by affinity selection, may also be used as a vaccine.

Encoding asymmetry of the N-glycosylation motif facilitates glycoprotein evolution.

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Ryan Williams

Full Text Available Protein N-glycosylation is found in all domains of life and has a conserved role in glycoprotein folding and stability. In animals, glycoproteins transit through the Golgi where the N-glycans are trimmed and rebuilt with sequences that bind lectins, an innovation that greatly increases structural diversity and redundancy of glycoprotein-lectin interaction at the cell surface. Here we ask whether the natural tension between increasing diversity (glycan-protein interactions and site multiplicity (backup and status quo might be revealed by a phylogenic examination of glycoproteins and NXS/T(X ≠ P N-glycosylation sites. Site loss is more likely by mutation at Asn encoded by two adenosine (A-rich codons, while site gain is more probable by generating Ser or Thr downstream of an existing Asn. Thus mutations produce sites at novel positions more frequently than the reversal of recently lost sites, and therefore more paths though sequence space are made available to natural selection. An intra-species comparison of secretory and cytosolic proteins revealed a departure from equilibrium in sequences one-mutation-away from NXS/T and in (A content, indicating strong selective pressures and exploration of N-glycosylation positions during vertebrate evolution. Furthermore, secretory proteins have evolved at rates proportional to N-glycosylation site number, indicating adaptive interactions between the N-glycans and underlying protein. Given the topology of the genetic code, mutation of (A is more often nonsynonomous, and Lys, another target of many PTMs, is also encoded by two (A-rich codons. An examination of acetyl-Lys sites in proteins indicated similar evolutionary dynamics, consistent with asymmetry of the target and recognition portions of modified sites. Our results suggest that encoding asymmetry is an ancient mechanism of evolvability that increases diversity and experimentation with PTM site positions. Strong selective pressures on PTMs may have

De novo design of peptide immunogens that mimic the coiled coil region of human T-cell leukemia virus type-1 glycoprotein 21 transmembrane subunit for induction of native protein reactive neutralizing antibodies.

Science.gov (United States)

Sundaram, Roshni; Lynch, Marcus P; Rawale, Sharad V; Sun, Yiping; Kazanji, Mirdad; Kaumaya, Pravin T P

2004-06-04

Peptide vaccines able to induce high affinity and protective neutralizing antibodies must rely in part on the design of antigenic epitopes that mimic the three-dimensional structure of the corresponding region in the native protein. We describe the design, structural characterization, immunogenicity, and neutralizing potential of antibodies elicited by conformational peptides derived from the human T-cell leukemia virus type 1 (HTLV-1) gp21 envelope glycoprotein spanning residues 347-374. We used a novel template design and a unique synthetic approach to construct two peptides (WCCR2T and CCR2T) that would each assemble into a triple helical coiled coil conformation mimicking the gp21 crystal structure. The peptide B-cell epitopes were grafted onto the epsilon side chains of three lysyl residues on a template backbone construct consisting of the sequence acetyl-XGKGKGKGCONH2 (where X represents the tetanus toxoid promiscuous T cell epitope (TT) sequence 580-599). Leucine substitutions were introduced at the a and d positions of the CCR2T sequence to maximize helical character and stability as shown by circular dichroism and guanidinium hydrochloride studies. Serum from an HTLV-1-infected patient was able to recognize the selected epitopes by enzyme-linked immunosorbent assay (ELISA). Mice immunized with the wild-type sequence (WCCR2T) and the mutant sequence (CCR2T) elicited high antibody titers that were capable of recognizing the native protein as shown by flow cytometry and whole virus ELISA. Sera and purified antibodies from immunized mice were able to reduce the formation of syncytia induced by the envelope glycoprotein of HTLV-1, suggesting that antibodies directed against the coiled coil region of gp21 are capable of disrupting cell-cell fusion. Our results indicate that these peptides represent potential candidates for use in a peptide vaccine against HTLV-1.

Defining glycoprotein cancer biomarkers by MS in conjunction with glycoprotein enrichment.

Science.gov (United States)

Song, Ehwang; Mechref, Yehia

2015-01-01

Protein glycosylation is an important and common post-translational modification. More than 50% of human proteins are believed to be glycosylated to modulate the functionality of proteins. Aberrant glycosylation has been correlated to several diseases, such as inflammatory skin diseases, diabetes mellitus, cardiovascular disorders, rheumatoid arthritis, Alzheimer's and prion diseases, and cancer. Many approved cancer biomarkers are glycoproteins which are not highly abundant proteins. Therefore, effective qualitative and quantitative assessment of glycoproteins entails enrichment methods. This chapter summarizes glycoprotein enrichment methods, including lectin affinity, immunoaffinity, hydrazide chemistry, hydrophilic interaction liquid chromatography, and click chemistry. The use of these enrichment approaches in assessing the qualitative and quantitative changes of glycoproteins in different types of cancers are presented and discussed. This chapter highlights the importance of glycoprotein enrichment techniques for the identification and characterization of new reliable cancer biomarkers.

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

DEFF Research Database (Denmark)

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

2017-01-01

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 t...

'Multi-epitope-targeted' immune-specific therapy for a multiple sclerosis-like disease via engineered multi-epitope protein is superior to peptides.

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Nathali Kaushansky

Full Text Available 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-targeting" strategy is required for

Glycoprotein on cell surfaces

International Nuclear Information System (INIS)

Muramatsu, T.

1975-01-01

There are conjugated polysaccharides in cell membranes and outside of animal cells, and they play important role in the control of cell behavior. In this paper, the studies on the glycoprotein on cell surfaces are reported. It was found that the glycoprotein on cell surfaces have both N-glycoside type and O-glycoside type saccharic chains. Therefore it can be concluded that the basic structure of the saccharic chains in the glycoprotein on cell surfaces is similar to that of blood serum and body fluid. The main glycoprotein in the membranes of red blood corpuscles has been studied most in detail, and it also has both types of saccharic chains. The glycoprotein in liver cell membranes was found to have only the saccharic chains of acid type and to be in different pattern from that in endoplasmic reticula and nuclear membranes, which also has the saccharic chains of neutral type. The structure of the saccharic chains of H-2 antigen, i.e. the peculiar glycoprotein on the surfaces of lymph system cells, has been studied, and it is similar to the saccharic chains of glycoprotein in blood serum. The saccharic chain structures of H-2 antigen and TL antigen are different. TL, H-2 (D), Lna and H-2 (K) are the glycoprotein on cell surfaces, and are independent molecules. The analysis of the saccharic chain patterns on cell surfaces was carried out, and it was shown that the acid type saccharic chains were similar to those of ordinary glycoprotein, because the enzyme of pneumococci hydrolyzed most of the acid type saccharic chains. The change of the saccharic chain patterns of glycoprotein on cell surfaces owing to canceration and multiplication is complex matter. (Kako, I.)

Automatic Generation of Validated Specific Epitope Sets

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Sebastian Carrasco Pro

2015-01-01

Full Text Available 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.

Epitope diversity of N-glycans from bovine peripheral myelin glycoprotein P0 revealed by mass spectrometry and nano probe magic angle spinning 1H NMR spectroscopy

NARCIS (Netherlands)

Vliegenthart, J.F.G.; Gutiérrez Gallego, R.; Jiménez Blanco, J.L.; Thijssen-van Zuylen, C.W.E.M.; Gotfredsen, C.H.; Voshol, H.; Duus, J.Ø.; Schachner, M.

2001-01-01

The carbohydrate structures present on the glycoproteins in the central and peripheral nerve systems are essential in many cell adhesion processes. The P0 glycoprotein, expressed by myelinating Schwann cells, plays an important role during the formation and maintenance of myelin, and it is the most

Immune epitope database analysis resource

DEFF Research Database (Denmark)

Kim, Yohan; Ponomarenko, Julia; Zhu, Zhanyang

2012-01-01

The immune epitope database analysis resource (IEDB-AR: http://tools.iedb.org) is a collection of tools for prediction and analysis of molecular targets of T- and B-cell immune responses (i.e. epitopes). Since its last publication in the NAR webserver issue in 2008, a new generation of peptide......, and the homology mapping tool was updated to enable mapping of discontinuous epitopes onto 3D structures. Furthermore, to serve a wider range of users, the number of ways in which IEDB-AR can be accessed has been expanded. Specifically, the predictive tools can be programmatically accessed using a web interface...

Identification, characterization, and synthesis of peptide epitopes and a recombinant six-epitope protein for Trichomonas vaginalis serodiagnosis

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Alderete JF

2013-08-01

Full Text Available J F Alderete, Calvin J NeaceSchool of Molecular Biosciences, College of Veterinary Medicine, Washington State University, Pullman, WA, USAAbstract: There is a need for a rapid, accurate serodiagnostic test useful for both women and men infected by Trichomonas vaginalis, which causes the number one sexually transmitted infection (STI. Women and men exposed to T. vaginalis make serum antibody to fructose-1,6-bisphosphate aldolase (ALD, α-enolase (ENO, and glyceraldehyde-3-phosphate dehydrogenase (GAP. We identified, by epitope mapping, the common and distinct epitopes of each protein detected by the sera of women patients with trichomonosis and by the sera of men highly seropositive to the immunogenic protein α-actinin (positive control sera. We analyzed the amino acid sequences to determine the extent of identity of the epitopes of each protein with other proteins in the databanks. This approach identified epitopes unique to T. vaginalis, indicating these peptide-epitopes as possible targets for a serodiagnostic test. Individual or combinations of 15-mer peptide epitopes with low to no identity with other proteins were reactive with positive control sera from both women and men but were unreactive with negative control sera. These analyses permitted the synthesis of a recombinant His6 fusion protein of 111 amino acids with an Mr of ~13.4 kDa, which consisted of 15-mer peptides of two distinct epitopes each for ALD, ENO, and GAP. This recombinant protein was purified by affinity chromatography. This composite protein was detected by enzyme-linked immunosorbent assay (ELISA, dot blots, and immunoblots, using positive control sera from women and men. These data indicate that it is possible to identify epitopes and that either singly, in combination, or as a composite protein represent targets for a point-of-care serodiagnostic test for T. vaginalis.Keywords: diagnostics, point-of-care, targets, trichomonosis

Limited naturally occurring escape in broadly neutralizing antibody epitopes in hepatitis C glycoprotein E2 and constrained sequence usage in acute infection

NARCIS (Netherlands)

Rodrigo, Chaturaka; Walker, Melanie R.; Leung, Preston; Eltahla, Auda A.; Grebely, Jason; Dore, Gregory J.; Applegate, Tanya; Page, Kimberly; Dwivedi, Sunita; Bruneau, Julie; Morris, Meghan D.; Cox, Andrea L.; Osburn, William; Kim, Arthur Y.; Schinkel, Janke; Shoukry, Naglaa H.; Lauer, Georg M.; Maher, Lisa; Hellard, Margaret; Prins, Maria; Luciani, Fabio; Lloyd, Andrew R.; Bull, Rowena A.

2017-01-01

Broadly neutralizing antibodies have been associated with spontaneous clearance of the hepatitis C infection as well as viral persistence by immune escape. Further study of neutralizing antibody epitopes is needed to unravel pathways of resistance to virus neutralization, and to identify conserved

Epitope discovery with phylogenetic hidden Markov models.

LENUS (Irish Health Repository)

Lacerda, Miguel

2010-05-01

Existing methods for the prediction of immunologically active T-cell epitopes are based on the amino acid sequence or structure of pathogen proteins. Additional information regarding the locations of epitopes may be acquired by considering the evolution of viruses in hosts with different immune backgrounds. In particular, immune-dependent evolutionary patterns at sites within or near T-cell epitopes can be used to enhance epitope identification. We have developed a mutation-selection model of T-cell epitope evolution that allows the human leukocyte antigen (HLA) genotype of the host to influence the evolutionary process. This is one of the first examples of the incorporation of environmental parameters into a phylogenetic model and has many other potential applications where the selection pressures exerted on an organism can be related directly to environmental factors. We combine this novel evolutionary model with a hidden Markov model to identify contiguous amino acid positions that appear to evolve under immune pressure in the presence of specific host immune alleles and that therefore represent potential epitopes. This phylogenetic hidden Markov model provides a rigorous probabilistic framework that can be combined with sequence or structural information to improve epitope prediction. As a demonstration, we apply the model to a data set of HIV-1 protein-coding sequences and host HLA genotypes.

Rabies virus glycoprotein as a carrier for anthrax protective antigen

International Nuclear Information System (INIS)

Smith, Mary Ellen; Koser, Martin; Xiao Sa; Siler, Catherine; McGettigan, James P.; Calkins, Catherine; Pomerantz, Roger J.; Dietzschold, Bernhard; Schnell, Matthias J.

2006-01-01

Live viral vectors expressing foreign antigens have shown great promise as vaccines against viral diseases. However, safety concerns remain a major problem regarding the use of even highly attenuated viral vectors. Using the rabies virus (RV) envelope protein as a carrier molecule, we show here that inactivated RV particles can be utilized to present Bacillus anthracis protective antigen (PA) domain-4 in the viral membrane. In addition to the RV glycoprotein (G) transmembrane and cytoplasmic domains, a portion of the RV G ectodomain was required to express the chimeric RV G anthrax PA on the cell surface. The novel antigen was also efficiently incorporated into RV virions. Mice immunized with the inactivated recombinant RV virions exhibited seroconversion against both RV G and anthrax PA, and a second inoculation greatly increased these responses. These data demonstrate that a viral envelope protein can carry a bacterial protein and that a viral carrier can display whole polypeptides compared to the limited epitope presentation of previous viral systems

High-Throughput Tools for Characterization of Antibody Epitopes

DEFF Research Database (Denmark)

Christiansen, Anders

mapping. In Chapter 1, it was examined whether combining phage display, a traditional epitope mapping approach, with HTS would improve the method. The developed approach was successfully used to map Ara h 1 epitopes in sera from patients with peanut allergy. Notably, the sera represented difficult...... proliferation advantages. Finally, in Chapter 4, a different emerging technology, next-generation peptide microarrays, was applied for epitope mapping of major peanut allergens using sera from allergic patients. New developments in the peptide microarray have enabled a greatly increased throughput....... In this study, these improvements were utilized to characterize epitopes at high resolution, i.e. determine the importance of each residue for antibody binding, for all major peanut allergens. Epitope reactivity among patients often converged on known epitope hotspots, however the binding patterns were somewhat...

Silencing ß1,2-xylosyltransferase in transgenic tomato fruits reveals xylose as constitutive component of IgE binding epitopes

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Kathrin Elisabeth Paulus

2011-08-01

Full Text Available Complex plant N-glycans containing β1,2-xylose and core α1,3-fucose are regarded as the major class of the so-called ‘carbohydrate cross-reactive determinants’ reactive with IgE antibodies in sera of many allergic patients, but their clinical relevance is still under debate. Plant glycosyltransferases, β1,2-xylosyltransferase (XylT and core α1,3-fucosyltransferase (FucT are responsible for the transfer of β1,2-linked xylose and core α1,3-linked fucose residues to N-glycans of glycoproteins, respectively. To test the clinical relevance of ß 1,2-xylose containing epitopes, expression of the tomato β1,2-xylosyltransferase was down-regulated by RNA interference (RNAi in transgenic plants. Fruits harvested from these transgenic plants were analysed for accumulation of XylT mRNA, abundance of ß1,2-xylose epitopes and their allergenic potential. Based on qPCR analysis XylT mRNA levels were reduced up to 10-fold in independent transgenic lines as compared to untransformed control, whereas no xylosylated N-glycans could be revealed by MS analysis. Immunoblotting using anti-xylose-specific IgG antibodies revealed a strong reduction of ß1,2-xylose containing epitopes. Incubating protein extracts from untransformed controls and XylT_RNAi plants with sera from tomato allergic patients showed a patient-specific reduction in IgE binding, indicating a reduced allergenic potential of XylT_RNAi tomato fruits, in vitro. To elucidate the clinical relevance of ß1,2-xylose containing complex N-glycans skin prick tests were performed demonstrating a reduced responsiveness of tomato allergic patients, in vivo. This study provides strong evidence for the clinical relevance of ß1,2-xylose containing epitopes in vivo.

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

Science.gov (United States)

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.

Thiolated polymers: evidence for the formation of disulphide bonds with mucus glycoproteins.

Science.gov (United States)

Leitner, Verena M; Walker, Greg F; Bernkop-Schnürch, Andreas

2003-09-01

Disulphide bonds between thiolated polymers (thiomers) and cysteine-rich subdomains of mucus glycoproteins are supposed to be responsible for the enhanced mucoadhesive properties of thiomers. This study set out to provide evidence for these covalent interactions using poly(acrylic acid)-cysteine conjugates of 2 and 450 kDa (PAA2-Cys, PAA450-Cys) displaying 402.5-776.0 micromol thiol groups per gram polymer. The effect of the disulphide bond breaker cysteine on thiomer-mucin disulphide bonds was monitored by (1) mucoadhesion studies and (2) rheological studies. Furthermore, (3) diffusion studies and (4) gel filtration studies were performed with thiomer-mucus mixtures. The addition of cysteine significantly (Ppolymer. Gel filtration studies showed that PAA2-Cys was able to form disulphide bonds with mucin glycoproteins resulting in an altered elution profile of the mucin/PAA2-Cys mixture in comparison to mucin alone or mucin/PAA2 mixture. According to these results, the study provides evidence for the formation of covalent bonds between thiomer and mucus glycoproteins.

Structures of phlebovirus glycoprotein Gn and identification of a neutralizing antibody epitope.

Science.gov (United States)

Wu, Yan; Zhu, Yaohua; Gao, Feng; Jiao, Yongjun; Oladejo, Babayemi O; Chai, Yan; Bi, Yuhai; Lu, Shan; Dong, Mengqiu; Zhang, Chang; Huang, Guangmei; Wong, Gary; Li, Na; Zhang, Yanfang; Li, Yan; Feng, Wen-Hai; Shi, Yi; Liang, Mifang; Zhang, Rongguang; Qi, Jianxun; Gao, George F

2017-09-05

Severe fever with thrombocytopenia syndrome virus (SFTSV) and Rift Valley fever virus (RVFV) are two arthropod-borne phleboviruses in the Bunyaviridae family, which cause severe illness in humans and animals. Glycoprotein N (Gn) is one of the envelope proteins on the virus surface and is a major antigenic component. Despite its importance for virus entry and fusion, the molecular features of the phleboviruse Gn were unknown. Here, we present the crystal structures of the Gn head domain from both SFTSV and RVFV, which display a similar compact triangular shape overall, while the three subdomains (domains I, II, and III) making up the Gn head display different arrangements. Ten cysteines in the Gn stem region are conserved among phleboviruses, four of which are responsible for Gn dimerization, as revealed in this study, and they are highly conserved for all members in Bunyaviridae Therefore, we propose an anchoring mode on the viral surface. The complex structure of the SFTSV Gn head and human neutralizing antibody MAb 4-5 reveals that helices α6 in subdomain III is the key component for neutralization. Importantly, the structure indicates that domain III is an ideal region recognized by specific neutralizing antibodies, while domain II is probably recognized by broadly neutralizing antibodies. Collectively, Gn is a desirable vaccine target, and our data provide a molecular basis for the rational design of vaccines against the diseases caused by phleboviruses and a model for bunyavirus Gn embedding on the viral surface.

High epitope expression levels increase competition between T cells.

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Almut Scherer

2006-08-01

Full Text Available Both theoretical predictions and experimental findings suggest that T cell populations can compete with each other. There is some debate on whether T cells compete for aspecific stimuli, such as access to the surface on antigen-presenting cells (APCs or for specific stimuli, such as their cognate epitope ligand. We have developed an individual-based computer simulation model to study T cell competition. Our model shows that the expression level of foreign epitopes per APC determines whether T cell competition is mainly for specific or aspecific stimuli. Under low epitope expression, competition is mainly for the specific epitope stimuli, and, hence, different epitope-specific T cell populations coexist readily. However, if epitope expression levels are high, aspecific competition becomes more important. Such between-specificity competition can lead to competitive exclusion between different epitope-specific T cell populations. Our model allows us to delineate the circumstances that facilitate coexistence of T cells of different epitope specificity. Understanding mechanisms of T cell coexistence has important practical implications for immune therapies that require a broad immune response.

Glycosylation of Recombinant Antigenic Proteins from Mycobacterium tuberculosis: In Silico Prediction of Protein Epitopes and Ex Vivo Biological Evaluation of New Semi-Synthetic Glycoconjugates.

Science.gov (United States)

Bavaro, Teodora; Tengattini, Sara; Piubelli, Luciano; Mangione, Francesca; Bernardini, Roberta; Monzillo, Vincenzina; Calarota, Sandra; Marone, Piero; Amicosante, Massimo; Pollegioni, Loredano; Temporini, Caterina; Terreni, Marco

2017-06-29

Tuberculosis is still one of the most deadly infectious diseases worldwide, and the use of conjugated antigens, obtained by combining antigenic oligosaccharides, such as the lipoarabinomannane (LAM), with antigenic proteins from Mycobacterium tuberculosis (MTB), has been proposed as a new strategy for developing efficient vaccines. In this work, we investigated the effect of the chemical glycosylation on two recombinant MTB proteins produced in E. coli with an additional seven-amino acid tag (recombinant Ag85B and TB10.4). Different semi-synthetic glycoconjugated derivatives were prepared, starting from mannose and two disaccharide analogs. The glycans were activated at the anomeric position with a thiocyanomethyl group, as required for protein glycosylation by selective reaction with lysines. The glycosylation sites and the ex vivo evaluation of the immunogenic activity of the different neo- glycoproteins were investigated. Glycosylation does not modify the immunological activity of the TB10.4 protein. Similarly, Ag85B maintains its B-cell activity after glycosylation while showing a significant reduction in the T-cell response. The results were correlated with the putative B- and T-cell epitopes, predicted using a combination of in silico systems. In the recombinant TB10.4, the unique lysine is not included in any T-cell epitope. Lys30 of Ag85B, identified as the main glycosylation site, proved to be the most important site involved in the formation of T-cell epitopes, reasonably explaining why its glycosylation strongly influenced the T-cell activity. Furthermore, additional lysines included in different epitopes (Lys103, -123 and -282) are also glycosylated. In contrast, B-cell epitopic lysines of Ag85B were found to be poorly glycosylated and, thus, the antibody interaction of Ag85B was only marginally affected after coupling with mono- or disaccharides.

Proof of principle for epitope-focused vaccine design

Science.gov (United States)

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.

Receptor-Targeted Nipah Virus Glycoproteins Improve Cell-Type Selective Gene Delivery and Reveal a Preference for Membrane-Proximal Cell Attachment.

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Ruben R Bender

2016-06-01

Full Text Available Receptor-targeted lentiviral vectors (LVs can be an effective tool for selective transfer of genes into distinct cell types of choice. Moreover, they can be used to determine the molecular properties that cell surface proteins must fulfill to act as receptors for viral glycoproteins. Here we show that LVs pseudotyped with receptor-targeted Nipah virus (NiV glycoproteins effectively enter into cells when they use cell surface proteins as receptors that bring them closely enough to the cell membrane (less than 100 Å distance. Then, they were flexible in receptor usage as demonstrated by successful targeting of EpCAM, CD20, and CD8, and as selective as LVs pseudotyped with receptor-targeted measles virus (MV glycoproteins, the current standard for cell-type specific gene delivery. Remarkably, NiV-LVs could be produced at up to two orders of magnitude higher titers compared to their MV-based counterparts and were at least 10,000-fold less effectively neutralized than MV glycoprotein pseudotyped LVs by pooled human intravenous immunoglobulin. An important finding for NiV-LVs targeted to Her2/neu was an about 100-fold higher gene transfer activity when particles were targeted to membrane-proximal regions as compared to particles binding to a more membrane-distal epitope. Likewise, the low gene transfer activity mediated by NiV-LV particles bound to the membrane distal domains of CD117 or the glutamate receptor subunit 4 (GluA4 was substantially enhanced by reducing receptor size to below 100 Å. Overall, the data suggest that the NiV glycoproteins are optimally suited for cell-type specific gene delivery with LVs and, in addition, for the first time define which parts of a cell surface protein should be targeted to achieve optimal gene transfer rates with receptor-targeted LVs.

Ineffective Degradation of Immunogenic Gluten Epitopes by Currently Available Digestive Enzyme Supplements

Science.gov (United States)

Janssen, George; Christis, Chantal; Kooy-Winkelaar, Yvonne; Edens, Luppo; Smith, Drew

2015-01-01

Background Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP). Methods Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1) enzyme assays and 2) mass spectrometric identification. Gluten epitope degradation was monitored by 1) R5 ELISA, 2) mass spectrometric analysis of the degradation products and 3) T cell proliferation assays. Findings The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data. Conclusion Currently available digestive enzyme

Ineffective degradation of immunogenic gluten epitopes by currently available digestive enzyme supplements.

Directory of Open Access Journals (Sweden)

George Janssen

Full Text Available Due to the high proline content of gluten molecules, gastrointestinal proteases are unable to fully degrade them leaving large proline-rich gluten fragments intact, including an immunogenic 33-mer from α-gliadin and a 26-mer from γ-gliadin. These latter peptides can trigger pro-inflammatory T cell responses resulting in tissue remodeling, malnutrition and a variety of other complications. A strict lifelong gluten-free diet is currently the only available treatment to cope with gluten intolerance. Post-proline cutting enzymes have been shown to effectively degrade the immunogenic gluten peptides and have been proposed as oral supplements. Several existing digestive enzyme supplements also claim to aid in gluten degradation. Here we investigate the effectiveness of such existing enzyme supplements in comparison with a well characterized post-proline cutting enzyme, Prolyl EndoPeptidase from Aspergillus niger (AN-PEP.Five commercially available digestive enzyme supplements along with purified digestive enzymes were subjected to 1 enzyme assays and 2 mass spectrometric identification. Gluten epitope degradation was monitored by 1 R5 ELISA, 2 mass spectrometric analysis of the degradation products and 3 T cell proliferation assays.The digestive enzyme supplements showed comparable proteolytic activities with near neutral pH optima and modest gluten detoxification properties as determined by ELISA. Mass spectrometric analysis revealed the presence of many different enzymes including amylases and a variety of different proteases with aminopeptidase and carboxypeptidase activity. The enzyme supplements leave the nine immunogenic epitopes of the 26-mer and 33-mer gliadin fragments largely intact. In contrast, the pure enzyme AN-PEP effectively degraded all nine epitopes in the pH range of the stomach at much lower dose. T cell proliferation assays confirmed the mass spectrometric data.Currently available digestive enzyme supplements are ineffective in

Genetic analysis of the SARS-coronavirus spike glycoprotein functional domains involved in cell-surface expression and cell-to-cell fusion

International Nuclear Information System (INIS)

Petit, Chad M.; Melancon, Jeffrey M.; Chouljenko, Vladimir N.; Colgrove, Robin; Farzan, Michael; Knipe, David M.; Kousoulas, K.G.

2005-01-01

The SARS-coronavirus (SARS-CoV) is the etiological agent of severe acute respiratory syndrome (SARS). The SARS-CoV spike (S) glycoprotein mediates membrane fusion events during virus entry and virus-induced cell-to-cell fusion. To delineate functional domains of the SARS-CoV S glycoprotein, single point mutations, cluster-to-lysine and cluster-to-alanine mutations, as well as carboxyl-terminal truncations were investigated in transient expression experiments. Mutagenesis of either the coiled-coil domain of the S glycoprotein amino terminal heptad repeat, the predicted fusion peptide, or an adjacent but distinct region, severely compromised S-mediated cell-to-cell fusion, while intracellular transport and cell-surface expression were not adversely affected. Surprisingly, a carboxyl-terminal truncation of 17 amino acids substantially increased S glycoprotein-mediated cell-to-cell fusion suggesting that the terminal 17 amino acids regulated the S fusogenic properties. In contrast, truncation of 26 or 39 amino acids eliminating either one or both of the two endodomain cysteine-rich motifs, respectively, inhibited cell fusion in comparison to the wild-type S. The 17 and 26 amino-acid deletions did not adversely affect S cell-surface expression, while the 39 amino-acid truncation inhibited S cell-surface expression suggesting that the membrane proximal cysteine-rich motif plays an essential role in S cell-surface expression. Mutagenesis of the acidic amino-acid cluster in the carboxyl terminus of the S glycoprotein as well as modification of a predicted phosphorylation site within the acidic cluster revealed that this amino-acid motif may play a functional role in the retention of S at cell surfaces. This genetic analysis reveals that the SARS-CoV S glycoprotein contains extracellular domains that regulate cell fusion as well as distinct endodomains that function in intracellular transport, cell-surface expression, and cell fusion

DRREP: deep ridge regressed epitope predictor.

Science.gov (United States)

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.

Role of the serine-rich surface glycoprotein Srr1 of Streptococcus agalactiae in the pathogenesis of infective endocarditis.

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Ho Seong Seo

Full Text Available The binding of bacteria to fibrinogen and platelets are important events in the pathogenesis of infective endocarditis. Srr1 is a serine-rich repeat glycoprotein of Streptococcus agalactiae that binds directly to the Aα chain of human fibrinogen. To assess the impact of Srr1 on the pathogenesis of endocarditis due to S. agalactiae, we first examined the binding of this organism to immobilized human platelets. Strains expressing Srr1 had significantly higher levels of binding to human platelets in vitro, as compared with isogenic Δsrr1 mutants. In addition, platelet binding was inhibited by pretreatment with anti-fibrinogen IgG or purified Srr1 binding region. To assess the contribution of Srr1 to pathogenicity, we compared the relative virulence of S. agalactiae NCTC 10/84 strain and its Δsrr1 mutant in a rat model of endocarditis, where animals were co-infected with the WT and the mutant strains at a 1:1 ratio. At 72 h post-infection, bacterial densities (CFU/g of the WT strain within vegetations, kidneys, and spleens were significantly higher, as compared with the Δsrr1 mutant. These results indicate that Srr1 contributes to the pathogenesis of endocarditis due to S. agalactiae, at least in part through its role in fibrinogen-mediated platelet binding.

Relationship between the 99mTc-MIBI and expression of P-glycoprotein in lung cancer

International Nuclear Information System (INIS)

Meng Zili; Shen Zhenya

2002-01-01

Objective: To correlate the imaging of 99m Tc-MIBI with the expression of P-glycoprotein (PGP) in lung cancer. Methods: All patients, undergoing early (30 min) and delayed (3h) 99m Tc-MIBI imaging before initiation of chemo-or radiotherapy, were diagnosed pathologically. Immunohistochemical studies were performed using a monoclonal antibody, P-170, developed against the internal epitope of PGP. Normal tissue and tumor washout rate and tumor-to-background ratio were correlated with the level of PGP expression. Results: There was an inverse correlation between tumor-to-background ratio and the density of PGP (P 0.1). Conclusion: The reduced ability for the tumors to accumulate MIBI correlates well with the increased levels of PGP expression, tumor washout rate of MIBI does not correlate with the density of PGP in tumor tissues

IgE epitopes of intact and digested Ara h 1

DEFF Research Database (Denmark)

Bøgh, Katrine Lindholm; Nielsen, H.; Madsen, Charlotte Bernhard

2012-01-01

epitopes have been suggested to be of great importance. ObjectiveThe aim of this study was to identify IgE specific epitopes of intact and digested Ara h 1, and to compare epitope patterns between humans and rats. MethodsSera from five peanut allergic patients and five Brown Norway rats were used...... to identify intact and digested Ara h 1-specific IgE epitopes by competitive immunoscreening of a phage-displayed random hepta-mer peptide library using polyclonal IgE from the individual sera. The resulting peptide sequences were mapped on the surface of a three-dimensional structure of the Ara h 1 molecule...... to mimic epitopes using a computer-based algorithm. ResultsPatients as well as rats were shown to have individual IgE epitope patterns. All epitope mimics were conformational and found to cluster into three different areas of the Ara h 1 molecule. Five epitope motifs were identified by patient IgE, which...

The leukocyte common antigen (CD45) on human pre-B leukemia cells: variant glycoprotein form expression during the cell exposure to phorbol ester is blocked by a nonselective protein kinase inhibitor H7

International Nuclear Information System (INIS)

Duraj, J.; Sedlak, J.; Chorvath, B.; Rauko, P.

1997-01-01

The human pre-B acute lymphoblastic leukemia cell line REH6 was utilized for characterization of CD45 glycoprotein by monoclonal antibodies (mAb) recognizing four distinct CD45 antigen specificities, i.e. nonrestricted CD45, restricted, CD45RA, CD45RB and CD45R0. Immunoprecipitation revealed two antigen specificities on REH6 cells of m.w. 220 kDa and 190 kDa, both presenting wide range of isoelectric point pI∼6.0-7.5. Nonrestricted CD45 epitopes were not affected by the sialyl acid cleavage with sodium meta-periodate or neuraminidase, but were sensitive to both, tunicamycin, the N-glycosylation inhibitor and monensin, an inhibitor of protein transport through the Golgi compartment. O-sialoglycoprotein endopeptidase from Pasteurella haemolytica A1 partially cleaved CD45RA and CD45RB epitopes, while nonrestricted CD45 determinants were not affected by this enzyme. Limited proteolysis of this antigen resulted in the appearance of 160-180 kDa peptide domains which retained CD45 epitopes. Further, the treatment of cells with phorbol myristate acetate (PMA) induced marked down-regulation of 220 and 190 kDa isoforms and the appearance of new 210, 180 and 170 kDa variant glycoprotein forms which were not found on parental cells. This PMA effect was not accompanied by the programmed cell death and was markedly blocked by a nonselective protein kinase (PK) inhibitor iso-quinoline sulfonamide H7. Modulation of CD45 by phorbol esters might serve as an in vitro model for an additional insight into the function of CD45 in hematopoietic cells. (author)

Immune Epitope Database and Analysis Resource (IEDB)

Data.gov (United States)

U.S. Department of Health & Human Services — This repository contains antibody/B cell and T cell epitope information and epitope prediction and analysis tools for use by the research community worldwide. Immune...

High-throughput epitope identification for snakebite antivenom

DEFF Research Database (Denmark)

Engmark, Mikael; De Masi, Federico; Laustsen, Andreas Hougaard

Insight into the epitopic recognition pattern for polyclonal antivenoms is a strong tool for accurate prediction of antivenom cross-reactivity and provides a basis for design of novel antivenoms. In this work, a high-throughput approach was applied to characterize linear epitopes in 966 individua...... toxins from pit vipers (Crotalidae) using the ICP Crotalidae antivenom. Due to an abundance of snake venom metalloproteinases and phospholipase A2s in the venoms used for production of the investigated antivenom, this study focuses on these toxin families.......Insight into the epitopic recognition pattern for polyclonal antivenoms is a strong tool for accurate prediction of antivenom cross-reactivity and provides a basis for design of novel antivenoms. In this work, a high-throughput approach was applied to characterize linear epitopes in 966 individual...

Expression of bovine herpesvirus 1 glycoproteins gI and gIII in transfected murine cells

International Nuclear Information System (INIS)

Fitzpatrick, D.R.; Zamb, T.; Parker, M.D.; van Drunen Littel-van den Hurk, S.; Babiuk, L.A.; Lawman, M.J.P.

1988-01-01

Genes encoding two of the major glycoproteins of bovine herpesvirus 1 (BHV-1), gI and gIII, were cloned into the eucaryotic expression vectors pRSVcat and pSV2neo and transfected into murine LMTK - cells, and cloned cell lines were established. The relative amounts of gI or gIII expressed from the two vectors were similar. Expression of gI was cell associated and localized predominantly in the perinuclear region, but nuclear and plasma membrane staining was also observed. Expression of gI was additionally associated with cell fusion and the formation of polykaryons and giant cells. Expression of gIII was localized predominantly in the nuclear and plasma membranes. Radioimmunoprecipitation in the presence or absence of tunicamycin revealed that the recombinant glycoproteins were proteolytically processed and glycosylated and had molecular weights similar to those of the forms of gI and gIII expressed in BHV-1 infected bovine cells. However, both recombinant glycoproteins were glycosylated to a lesser extent than were the forms found in BHV-1 infected bovine cells. For gI, a deficiency in N-linked glycosylated of the amino-terminal half of the protein was identified; for gIII, a deficiency in O-linked glycosylation was implicated. The reactivity pattern of a panel of gI- and gIII-specific monoclonal antibodies, including six which recognize conformation-dependent epitopes, was found to be unaffected by the glycosylation differences and was identical for transfected of BHV-1-infected murine cells. Use of the transfected cells as targets in immune-mediated cytotoxicity assays demonstrated the functional recognition of recombinant gI and gIII by murine antibody and cytotoxic T lymphocytes

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

Science.gov (United States)

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.

Recent Progress in Electrochemical Biosensors for Glycoproteins

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Uichi Akiba

2016-12-01

Full Text Available This review provides an overview of recent progress in the development of electrochemical biosensors for glycoproteins. Electrochemical glycoprotein sensors are constructed by combining metal and carbon electrodes with glycoprotein-selective binding elements including antibodies, lectin, phenylboronic acid and molecularly imprinted polymers. A recent trend in the preparation of glycoprotein sensors is the successful use of nanomaterials such as graphene, carbon nanotube, and metal nanoparticles. These nanomaterials are extremely useful for improving the sensitivity of glycoprotein sensors. This review focuses mainly on the protocols for the preparation of glycoprotein sensors and the materials used. Recent improvements in glycoprotein sensors are discussed by grouping the sensors into several categories based on the materials used as recognition elements.

Sustained expression of human cytomegalovirus glycoprotein B (UL55) in the seeds of homozygous rice plants.

Science.gov (United States)

Tackaberry, Eilleen S; Prior, Fiona A; Rowlandson, Karen; Tocchi, Monika; Mehic, Jelica; Porter, Suzanne; Walsh, Mike; Schleiss, Mark R; Ganz, Peter R; Sardana, Ravinder K; Altosaar, Illimar; Dudani, Anil K

2008-09-01

Production of recombinant subunit vaccines in transgenic plants may be a means of reducing vaccine costs while increasing availability and safety. A plant-derived product found safe and effective for oral administration would provide additional advantages when used as a vaccine. Outstanding issues with the technology include transgene stability through successive generations and consistent bioproduction. We previously reported expression of glycoprotein B (gB) of human cytomegalovirus in seeds of transgenic tobacco. Here the goal was to determine if gB could be similarly expressed in rice, and if so, to examine expression over several plant generations. Results show that immunoreactive gB was successfully expressed in transgenic rice seeds, with sustained expression over three generations. The gB contained several neutralizing epitopes and was stable over 27 months.

A Sulfated Glycosaminoglycan Linkage Region is a Novel Type of Human Natural Killer-1 (HNK-1 Epitope Expressed on Aggrecan in Perineuronal Nets.

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Keiko Yabuno

Full Text Available Human natural killer-1 (HNK-1 carbohydrate (HSO3-3GlcAβ1-3Galβ1-4GlcNAc-R is highly expressed in the brain and required for learning and neural plasticity. We previously demonstrated that expression of the HNK-1 epitope is mostly abolished in knockout mice for GlcAT-P (B3gat1, a major glucuronyltransferase required for HNK-1 biosynthesis, but remained in specific regions such as perineuronal nets (PNNs in these mutant mice. Considering PNNs are mainly composed of chondroitin sulfate proteoglycans (CSPGs and regulate neural plasticity, GlcAT-P-independent expression of HNK-1 in PNNs is suggested to play a role in neural plasticity. However, the function, structure, carrier glycoprotein and biosynthetic pathway for GlcAT-P-irrelevant HNK-1 epitope remain unclear. In this study, we identified a unique HNK-1 structure on aggrecan in PNNs. To determine the biosynthetic pathway for the novel HNK-1, we generated knockout mice for GlcAT-S (B3gat2, the other glucuronyltransferase required for HNK-1 biosynthesis. However, GlcAT-P and GlcAT-S double-knockout mice did not exhibit reduced HNK-1 expression compared with single GlcAT-P-knockout mice, indicating an unusual biosynthetic pathway for the HNK-1 epitope in PNNs. Aggrecan was purified from cultured cells in which GlcAT-P and -S are not expressed and we determined the structure of the novel HNK-1 epitope using liquid chromatography/mass spectrometry (LC/MS as a sulfated linkage region of glycosaminoglycans (GAGs, HSO3-GlcA-Gal-Gal-Xyl-R. Taken together, we propose a hypothetical model where GlcAT-I, the sole glucuronyltransferase required for synthesis of the GAG linkage, is also responsible for biosynthesis of the novel HNK-1 on aggrecan. These results could lead to discovery of new roles of the HNK-1 epitope in neural plasticity.

Epitope selection from an uncensored peptide library displayed on avian leukosis virus

International Nuclear Information System (INIS)

Khare, Pranay D.; Rosales, Ana G.; Bailey, Kent R.; Russell, Stephen J.; Federspiel, Mark J.

2003-01-01

Phage display libraries have provided an extraordinarily versatile technology to facilitate the isolation of peptides, growth factors, single chain antibodies, and enzymes with desired binding specificities or enzymatic activities. The overall diversity of peptides in phage display libraries can be significantly limited by Escherichia coli protein folding and processing machinery, which result in sequence censorship. To achieve an optimal diversity of displayed eukaryotic peptides, the library should be produced in the endoplasmic reticulum of eukaryotic cells using a eukaryotic display platform. In the accompanying article, we presented experiments that demonstrate that polypeptides of various sizes could be efficiently displayed on the envelope glycoproteins of a eukaryotic virus, avian leukosis virus (ALV), and the displayed polypeptides could efficiently attach to cognate receptors without interfering with viral attachment and entry into susceptible cells. In this study, methods were developed to construct a model library of randomized eight amino acid peptides using the ALV eukaryotic display platform and screen the library for specific epitopes using immobilized antibodies. A virus library with approximately 2 x 10 6 different members was generated from a plasmid library of approximately 5 x 10 6 diversity. The sequences of the randomized 24 nucleotide/eight amino acid regions of representatives of the plasmid and virus libraries were analyzed. No significant sequence censorship was observed in producing the virus display library from the plasmid library. Different populations of peptide epitopes were selected from the virus library when different monoclonal antibodies were used as the target. The results of these two studies clearly demonstrate the potential of ALV as a eukaryotic platform for the display and selection of eukaryotic polypeptides libraries

EpiJen: a server for multistep T cell epitope prediction

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Guan Pingping

2006-03-01

Full Text Available Abstract Background The main processing pathway for MHC class I ligands involves degradation of proteins by the proteasome, followed by transport of products by the transporter associated with antigen processing (TAP to the endoplasmic reticulum (ER, where peptides are bound by MHC class I molecules, and then presented on the cell surface by MHCs. The whole process is modeled here using an integrated approach, which we call EpiJen. EpiJen is based on quantitative matrices, derived by the additive method, and applied successively to select epitopes. EpiJen is available free online. Results To identify epitopes, a source protein is passed through four steps: proteasome cleavage, TAP transport, MHC binding and epitope selection. At each stage, different proportions of non-epitopes are eliminated. The final set of peptides represents no more than 5% of the whole protein sequence and will contain 85% of the true epitopes, as indicated by external validation. Compared to other integrated methods (NetCTL, WAPP and SMM, EpiJen performs best, predicting 61 of the 99 HIV epitopes used in this study. Conclusion EpiJen is a reliable multi-step algorithm for T cell epitope prediction, which belongs to the next generation of in silico T cell epitope identification methods. These methods aim to reduce subsequent experimental work by improving the success rate of epitope prediction.

Development of glycoprotein capture-based label-free method for the high-throughput screening of differential glycoproteins in hepatocellular carcinoma.

Science.gov (United States)

Chen, Rui; Tan, Yexiong; Wang, Min; Wang, Fangjun; Yao, Zhenzhen; Dong, Liwei; Ye, Mingliang; Wang, Hongyang; Zou, Hanfa

2011-07-01

A robust, reproducible, and high throughput method was developed for the relative quantitative analysis of glycoprotein abundances in human serum. Instead of quantifying glycoproteins by glycopeptides in conventional quantitative glycoproteomics, glycoproteins were quantified by nonglycosylated peptides derived from the glycoprotein digest, which consists of the capture of glycoproteins in serum samples and the release of nonglycopeptides by trypsin digestion of captured glycoproteins followed by two-dimensional liquid chromatography-tandem MS analysis of released peptides. Protein quantification was achieved by comparing the spectrum counts of identified nonglycosylated peptides of glycoproteins between different samples. This method was demonstrated to have almost the same specificity and sensitivity in glycoproteins quantification as capture at glycopeptides level. The differential abundance of proteins present at as low as nanogram per milliliter levels was quantified with high confidence. The established method was applied to the analysis of human serum samples from healthy people and patients with hepatocellular carcinoma (HCC) to screen differential glycoproteins in HCC. Thirty eight glycoproteins were found with substantial concentration changes between normal and HCC serum samples, including α-fetoprotein, the only clinically used marker for HCC diagnosis. The abundance changes of three glycoproteins, i.e. galectin-3 binding protein, insulin-like growth factor binding protein 3, and thrombospondin 1, which were associated with the development of HCC, were further confirmed by enzyme-linked immunosorbent assay. In conclusion, the developed method was an effective approach to quantitatively analyze glycoproteins in human serum and could be further applied in the biomarker discovery for HCC and other cancers.

Novel CTL epitopes identified through a Y. pestis proteome-wide analysis in the search for vaccine candidates against plague.

Science.gov (United States)

Zvi, Anat; Rotem, Shahar; Zauberman, Ayelet; Elia, Uri; Aftalion, Moshe; Bar-Haim, Erez; Mamroud, Emanuelle; Cohen, Ofer

2017-10-20

The causative agent of Plague, Yersinia pestis, is a highly virulent pathogen and a potential bioweapon. Depending on the route of infection, two prevalent occurrences of the disease are known, bubonic and pneumonic. The latter has a high fatality rate. In the absence of a licensed vaccine, intense efforts to develop a safe and efficacious vaccine have been conducted, and humoral-driven subunit vaccines containing the F1 and LcrV antigens are currently under clinical trials. It is well known that a cellular immune response might have an essential additive value to immunity and protection against Y. pestis infection. Nevertheless, very few documented epitopes eliciting a protective T-cell response have been reported. Here, we present a combined high throughput computational and experimental effort towards identification of CD8 T-cell epitopes. All 4067 proteins of Y. pestis were analyzed with state-of-the-art recently developed prediction algorithms aimed at mapping potential MHC class I binders. A compilation of the results obtained from several prediction methods revealed a total of 238,000 peptide candidates, which necessitated downstream filtering criteria. Our previously established and proven approach for enrichment of true positive CTL epitopes, which relies on mapping clusters rich in tandem or overlapping predicted MHC binders ("hotspots"), was applied, as well as considerations of predicted binding affinity. A total of 1532 peptides were tested for their ability to elicit a specific T-cell response by following the production of IFNγ from splenocytes isolated from vaccinated mice. Altogether, the screen resulted in 178 positive responders (11.8%), all novel Y. pestis CTL epitopes. These epitopes span 113 Y. pestis proteins. Substantial enrichment of membrane-associated proteins was detected for epitopes selected from hotspots of predicted MHC binders. These results considerably expand the repertoire of known CTL epitopes in Y. pestis and pave the way to

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

Science.gov (United States)

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.

In vivo imaging and specific targeting of P-glycoprotein expression in multidrug resistant nude mice xenografts with [125I]MRK-16 monoclonal antibody

International Nuclear Information System (INIS)

Scott, Andrew M.; Rosa, Eddie; Mehta, Bippin M.; Divgi, Chaitanya R.; Finn, Ronald D.; Biedler, June L.; Tsuruo, Takashi; Kalaigian, Hovannes; Larson, Steven M.

1995-01-01

Multidrug resistance (MDR) in tumors is associated with P-glycoprotein (Pgp) expression. In vivo quantitation of Pgp may allow MDR to be evaluated noninvasively prior to treatment planning. The purpose of this study was to radiolabel MRK-16, a monoclonal antibody that targets an external epitope of P-glycoprotein, and perform in vivo quantitation of P-glycoprotein in a MDR xenograft nude mouse model. MRK-16 was labeled with 125 I by the iodogen method, with subsequent purification by size exclusion chromatography. Groups of 10 Balb/c mice were each xenografted with colchicine-resistant or -sensitive neuroblastoma cell lines, respectively. Whole body clearance and tumor uptake over time was quantitated by gamma camera imaging, and biodistribution studies were performed with [ 125 ]MRK-16 and an isotype matched control antibody, A33. Quantitative autoradiography and immunohistochemistry analysis of tumors was also evaluated to confirm specific targeting of [ 125 I]MRK-16. Peak tumor uptake was at 2-3 days post-injection, and was significantly greater in resistance compared to sensitive tumors (mean % injected dose/g ± SD) (18.76 ± 2.94 vs 10.93 ± 0.96; p 125 I]MRK-16 was confirmed by comparison to [ 131 I]A33 in biodistribution studies, and localized to cellular components of tissue stroma by comparison of histologic and autoradiographic sections of sensitive and resistant tumors. Immunoblot analysis demonstrated a 4.5-fold difference in P-glycoprotein expression between sensitive and resistant cell lines without colchicine selective pressure. We conclude that in vivo quantitation of P-glycoprotein in MDR tumors can be performed with [ 125 I]MRK-16. These findings suggest a potential clinical application for radiolabeled MRK-16 in the in vivo evaluation of multidrug resistance in tumors

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

Science.gov (United States)

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.

Localization of immunodominant linear B-cell epitopes of Vibrio ...

African Journals Online (AJOL)

Outer membrane protein U (OmpU), an adhesion protein of Vibrio mimicus, is a good antigen, but its epitopes are still unclear. In order to locate the epitopes of OmpU protein, epitope prediction was performed using the amino acid sequence of OmpU protein of V. mimicus HX4 strain that was isolated from the diseased ...

Immune epitope database analysis resource (IEDB-AR)

DEFF Research Database (Denmark)

Zhang, Qing; Wang, Peng; Kim, Yohan

2008-01-01

We present a new release of the immune epitope database analysis resource (IEDB-AR, http://tools.immuneepitope.org), a repository of web-based tools for the prediction and analysis of immune epitopes. New functionalities have been added to most of the previously implemented tools, and a total...

Monoclonal Antibodies Directed toward the Hepatitis C Virus Glycoprotein E2 Detect Antigenic Differences Modulated by the N-Terminal Hypervariable Region 1 (HVR1), HVR2, and Intergenotypic Variable Region.

Science.gov (United States)

Alhammad, Yousef; Gu, Jun; Boo, Irene; Harrison, David; McCaffrey, Kathleen; Vietheer, Patricia T; Edwards, Stirling; Quinn, Charles; Coulibaly, Fásseli; Poumbourios, Pantelis; Drummer, Heidi E

2015-12-01

Hepatitis C virus (HCV) envelope glycoproteins E1 and E2 form a heterodimer and mediate receptor interactions and viral fusion. Both E1 and E2 are targets of the neutralizing antibody (NAb) response and are candidates for the production of vaccines that generate humoral immunity. Previous studies demonstrated that N-terminal hypervariable region 1 (HVR1) can modulate the neutralization potential of monoclonal antibodies (MAbs), but no information is available on the influence of HVR2 or the intergenotypic variable region (igVR) on antigenicity. In this study, we examined how the variable regions influence the antigenicity of the receptor binding domain of E2 spanning HCV polyprotein residues 384 to 661 (E2661) using a panel of MAbs raised against E2661 and E2661 lacking HVR1, HVR2, and the igVR (Δ123) and well-characterized MAbs isolated from infected humans. We show for a subset of both neutralizing and nonneutralizing MAbs that all three variable regions decrease the ability of MAbs to bind E2661 and reduce the ability of MAbs to inhibit E2-CD81 interactions. In addition, we describe a new MAb directed toward the region spanning residues 411 to 428 of E2 (MAb24) that demonstrates broad neutralization against all 7 genotypes of HCV. The ability of MAb24 to inhibit E2-CD81 interactions is strongly influenced by the three variable regions. Our data suggest that HVR1, HVR2, and the igVR modulate exposure of epitopes on the core domain of E2 and their ability to prevent E2-CD81 interactions. These studies suggest that the function of HVR2 and the igVR is to modulate antibody recognition of glycoprotein E2 and may contribute to immune evasion. This study reveals conformational and antigenic differences between the Δ123 and intact E2661 glycoproteins and provides new structural and functional data about the three variable regions and their role in occluding neutralizing and nonneutralizing epitopes on the E2 core domain. The variable regions may therefore function to

An assessment on epitope prediction methods for protozoa genomes

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Resende Daniela M

2012-11-01

Full Text Available Abstract Background Epitope prediction using computational methods represents one of the most promising approaches to vaccine development. Reduction of time, cost, and the availability of completely sequenced genomes are key points and highly motivating regarding the use of reverse vaccinology. Parasites of genus Leishmania are widely spread and they are the etiologic agents of leishmaniasis. Currently, there is no efficient vaccine against this pathogen and the drug treatment is highly toxic. The lack of sufficiently large datasets of experimentally validated parasites epitopes represents a serious limitation, especially for trypanomatids genomes. In this work we highlight the predictive performances of several algorithms that were evaluated through the development of a MySQL database built with the purpose of: a evaluating individual algorithms prediction performances and their combination for CD8+ T cell epitopes, B-cell epitopes and subcellular localization by means of AUC (Area Under Curve performance and a threshold dependent method that employs a confusion matrix; b integrating data from experimentally validated and in silico predicted epitopes; and c integrating the subcellular localization predictions and experimental data. NetCTL, NetMHC, BepiPred, BCPred12, and AAP12 algorithms were used for in silico epitope prediction and WoLF PSORT, Sigcleave and TargetP for in silico subcellular localization prediction against trypanosomatid genomes. Results A database-driven epitope prediction method was developed with built-in functions that were capable of: a removing experimental data redundancy; b parsing algorithms predictions and storage experimental validated and predict data; and c evaluating algorithm performances. Results show that a better performance is achieved when the combined prediction is considered. This is particularly true for B cell epitope predictors, where the combined prediction of AAP12 and BCPred12 reached an AUC value

Prediction of antigenic epitopes on protein surfaces by consensus scoring

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Zhang Chi

2009-09-01

Full Text Available Abstract Background Prediction of antigenic epitopes on protein surfaces is important for vaccine design. Most existing epitope prediction methods focus on protein sequences to predict continuous epitopes linear in sequence. Only a few structure-based epitope prediction algorithms are available and they have not yet shown satisfying performance. Results We present a new antigen Epitope Prediction method, which uses ConsEnsus Scoring (EPCES from six different scoring functions - residue epitope propensity, conservation score, side-chain energy score, contact number, surface planarity score, and secondary structure composition. Applied to unbounded antigen structures from an independent test set, EPCES was able to predict antigenic eptitopes with 47.8% sensitivity, 69.5% specificity and an AUC value of 0.632. The performance of the method is statistically similar to other published methods. The AUC value of EPCES is slightly higher compared to the best results of existing algorithms by about 0.034. Conclusion Our work shows consensus scoring of multiple features has a better performance than any single term. The successful prediction is also due to the new score of residue epitope propensity based on atomic solvent accessibility.

Catalase epitopes vaccine design for Helicobacter pylori: A ...

African Journals Online (AJOL)

Jane

2011-08-15

Aug 15, 2011 ... colored region and P1 anchor or the starting residue of each ..... score of MVNKDVKQTT; * 1, MVNKDVKQTT is a composition of two epitopes: MVNKDVKQT and .... Therapeutic efficacy of a multi-epitope vaccine against.

Secretion of hepatitis C virus envelope glycoproteins depends on assembly of apolipoprotein B positive lipoproteins.

Directory of Open Access Journals (Sweden)

Vinca Icard

Full Text Available The density of circulating hepatitis C virus (HCV particles in the blood of chronically infected patients is very heterogeneous. The very low density of some particles has been attributed to an association of the virus with apolipoprotein B (apoB positive and triglyceride rich lipoproteins (TRL likely resulting in hybrid lipoproteins known as lipo-viro-particles (LVP containing the viral envelope glycoproteins E1 and E2, capsid and viral RNA. The specific infectivity of these particles has been shown to be higher than the infectivity of particles of higher density. The nature of the association of HCV particles with lipoproteins remains elusive and the role of apolipoproteins in the synthesis and assembly of the viral particles is unknown. The human intestinal Caco-2 cell line differentiates in vitro into polarized and apoB secreting cells during asymmetric culture on porous filters. By using this cell culture system, cells stably expressing E1 and E2 secreted the glycoproteins into the basal culture medium after one week of differentiation concomitantly with TRL secretion. Secreted glycoproteins were only detected in apoB containing density fractions. The E1-E2 and apoB containing particles were unique complexes bearing the envelope glycoproteins at their surface since apoB could be co-immunoprecipitated with E2-specific antibodies. Envelope protein secretion was reduced by inhibiting the lipidation of apoB with an inhibitor of the microsomal triglyceride transfer protein. HCV glycoproteins were similarly secreted in association with TRL from the human liver cell line HepG2 but not by Huh-7 and Huh-7.5 hepatoma cells that proved deficient for lipoprotein assembly. These data indicate that HCV envelope glycoproteins have the intrinsic capacity to utilize apoB synthesis and lipoprotein assembly machinery even in the absence of the other HCV proteins. A model for LVP assembly is proposed.

Evidence that muscle cells do not express the histidine-rich glycoprotein associated with AMP deaminase but can internalise the plasma protein

Directory of Open Access Journals (Sweden)

A.R.M. Sabbatini

2011-02-01

Full Text Available Histidine-rich glycoprotein (HRG is synthesized by liver and is present at relatively high concentration in the plasma of vertebrates. We have previously described the association of a HRG-like molecule to purified rabbit skeletal muscle AMP deaminase (AMPD. We also provided the first evidence for the presence of a HRG-like protein in human skeletal muscle where a positive correlation between HRG content and total determined AMPD activity has been shown. In the present paper we investigate the origin of skeletal muscle HRG. The screening of a human skeletal muscle cDNA expression library using an anti-HRG antibody failed to reveal any positive clone. The RT-PCR analysis, performed on human skeletal muscle RNA as well as on RNA from the rhabdomyosarcoma (RD cell line, failed to show any mRNA specific for the plasma HRG or for the putative muscle variant. When the RD cells were incubated with human plasma HRG, a time-dependent increase of the HRG immunoreactivity was detected both at the plasma membrane level and intracellularly. The internalisation of HRG was inhibited by the addition of heparin. The above data strongly suggest that skeletal muscle cells do not synthesize the muscle variant of HRG but instead can actively internalise it from plasma.

Public epitopes and the antigenic structure of the HLA molecules.

Science.gov (United States)

Rodey, G E; Fuller, T C

1987-01-01

Simplified procedures for determining amino acid sequences in proteins and nucleotide sequences in DNA have rapidly expanded the number of MHC molecules for which primary amino acid structure is known. These molecules will be especially valuable as tools to study the structure-function relationships of globular proteins because of the extensive polymorphism of genes coding the MHC genes products. The general three-dimensional structure of class I MHC molecules was recently deduced, but the more subtle topographical microconformations are still undefined. Definition and topographical mapping of epitopes, defined by serological or cellular immune effector products, will be critical probes for these three-dimensional studies. Comparative studies of amino acid sequences among various MHC and molecules have revealed distinct regions of hypervariability in the alpha-1 and -2 domains of class I heavy chains and the alpha-1 and beta-1 domains of most class II molecules. Mutant MHC molecules that differ from each other by no more than one to three amino acids can have structural changes which may result in a loss of the private epitopes that defined the allelic gene product. On the basis of these studies, the private epitopes are thought to be determined by one or more of the hypervariable regions. Similar studies of the relationships between specific regions of the molecule and public epitopes are not fully explored. Because public epitopes are partially conserved structures, one might expect that their structure is not principally determined by hypervariable region. In fact, however, some public epitopes, such as A2/B17 and BW4/Bw6, do map to diversity regions. Epitope mapping as a means of identifying specific topographic sites and relating these sites to specific functional regions of the molecule will be difficult unless the epitopes themselves are better defined. Thus, the capacity to distinguish spatially distinct public epitopes from cross-reactive homologous

Leptospira borgpetersenii hybrid leucine-rich repeat protein: Cloning and expression, immunogenic identification and molecular docking evaluation.

Science.gov (United States)

Sritrakul, Tepyuda; Nitipan, Supachai; Wajjwalku, Worawidh; La-Ard, Anchalee; Suphatpahirapol, Chattip; Petkarnjanapong, Wimol; Ongphiphadhanakul, Boonsong; Prapong, Siriwan

2017-11-01

Leptospirosis is an important zoonotic disease, and the major outbreak of this disease in Thailand in 1999 was due largely to the Leptospira borgpetersenii serovar Sejroe. Identification of the leucine-rich repeat (LRR) LBJ_2271 protein containing immunogenic epitopes and the discovery of the LBJ_2271 ortholog in Leptospira serovar Sejroe, KU_Sej_R21_2271, led to further studies of the antigenic immune properties of KU_Sej_LRR_2271. The recombinant hybrid (rh) protein was created and expressed from a hybrid PCR fragment of KU_Sej_R21_2271 fused with DNA encoding the LBJ_2271 signal sequence for targeting protein as a membrane-anchoring protein. The fusion DNA was cloned into pET160/GW/D-TOPO® to form the pET160_hKU_R21_2271 plasmid. The plasmid was used to express the rhKU_Sej_LRR_2271 protein in Escherichia coli BL21 Star™ (DE3). The expressed protein was immunologically detected by Western blotting and immunoreactivity detection with hyperimmune sera, T cell epitope prediction by HLA allele and epitope peptide binding affinity, and potential T cell reactivity analysis. The immunogenic epitopes of the protein were evaluated and verified by HLA allele and epitope peptide complex structure molecular docking. Among fourteen best allele epitopes of this protein, binding affinity values of 12 allele epitopes remained unchanged compared to LBJ_2271. Two epitopes for alleles HLA-A0202 and -A0301 had higher IC 50 values, while T cell reactivity values of these peptides were better than values from LBJ_2271 epitopes. Eight of twelve epitope peptides had positive T-cell reactivity scores. Although the molecular docking of two epitopes, 3FPLLKEFLV11/47FPLLKEFLV55 and 50KLSTVPEGV58, into an HLA-A0202 model revealed a good fit in the docked structures, 50KLSTVPEGV58 and 94KLSTVPEEV102 are still considered as the proteins' best epitopes for allele HLA-A0202. The results of this study showed that rhKU_Sej_LRR_2271 protein contained natural immunological properties that should

Dimers of beta 2-glycoprotein I mimic the in vitro effects of beta 2-glycoprotein I-anti-beta 2-glycoprotein I antibody complexes

NARCIS (Netherlands)

Lutters, B. C.; Meijers, J. C.; Derksen, R. H.; Arnout, J.; de Groot, P. G.

2001-01-01

Anti-beta(2)-glycoprotein I antibodies are thought to cause lupus anticoagulant activity by forming bivalent complexes with beta(2)-glycoprotein I (beta(2)GPI). To test this hypothesis, chimeric fusion proteins were constructed of the dimerization domain (apple 4) of factor XI and beta(2)GPI. Both a

Flow shear stress differentially regulates endothelial uptake of nanocarriers targeted to distinct epitopes of PECAM-1.

Science.gov (United States)

Han, Jingyan; Shuvaev, Vladimir V; Davies, Peter F; Eckmann, David M; Muro, Silvia; Muzykantov, Vladimir R

2015-07-28

Targeting nanocarriers (NC) to endothelial cell adhesion molecules including Platelet-Endothelial Cell Adhesion Molecule-1 (PECAM-1 or CD31) improves drug delivery and pharmacotherapy of inflammation, oxidative stress, thrombosis and ischemia in animal models. Recent studies unveiled that hydrodynamic conditions modulate endothelial endocytosis of NC targeted to PECAM-1, but the specificity and mechanism of effects of flow remain unknown. Here we studied the effect of flow on endocytosis by human endothelial cells of NC targeted by monoclonal antibodies Ab62 and Ab37 to distinct epitopes on the distal extracellular domain of PECAM. Flow in the range of 1-8dyn/cm(2), typical for venous vasculature, stimulated the uptake of spherical Ab/NC (~180nm diameter) carrying ~50 vs 200 Ab62 and Ab37 per NC, respectively. Effect of flow was inhibited by disruption of cholesterol-rich plasmalemma domains and deletion of PECAM-1 cytosolic tail. Flow stimulated endocytosis of Ab62/NC and Ab37/NC via eliciting distinct signaling pathways mediated by RhoA/ROCK and Src Family Kinases, respectively. Therefore, flow stimulates endothelial endocytosis of Ab/NC in a PECAM-1 epitope specific manner. Using ligands of binding to distinct epitopes on the same target molecule may enable fine-tuning of intracellular delivery based on the hemodynamic conditions in the vascular area of interest. Copyright © 2015 Elsevier B.V. All rights reserved.

Some epitopes conservation in non structural 3 protein dengue virus serotype 4

Directory of Open Access Journals (Sweden)

Tegar A. P. Siregar

2016-03-01

Full Text Available AbstrakLatar belakang: Protein Non Struktural 3 (NS3 virus dengue menginduksi respon antibodi netralisasidan respon sel T CD4+ dan CD8+, serta berperan dalam replikasi virus. Protein NS3 memiliki epitopepitopsel T dan B yang terdapat perbedaan kelestarian pada berbagai strain virus dengue serotipe 4(DENV-4. Penelitian ini bertujuan untuk mengetahui kelestarian epitop sel T dan B pada protein NS3DENV-4 strain-strain dunia dan keempat serotipe virus dengue strain Indonesia.Metode: Penelitian ini dilakukan di Departemen Mikrobiologi Fakultas Kedokteran UI sejak Juni 2013 - April2014. Sekuens asam amino NS3 DENV-4 strain 081 didapatkan setelah produk PCR gen NS3 DENV-4 081disekuensing. Epitop-epitop sel T dan sel B protein NS3 DENV-4 081 dianalisis dan dibandingkan dengansekuens asam amino protein NS3 dari 124 strain DENV-4 di dunia dan keempat serotipe DENV strain Indonesia.Strain-strain dunia merupakan strain yang ada di benua Amerika (Venezuela, Colombia, dll dan Asia (Cina,Singapura, dll. Referensi posisi epitop sel T dan B protein NS3 diperoleh dari laporan penelitian terdahulu.Hasil: Delapan epitop sel T dan 2 epitop sel B dari protein NS3 DENV-4 081 ternyata identik dan lestaripada protein NS3 dari 124 strain DENV-4 dunia. Epitop sel B di posisi asam amino 537-544 pada proteinNS3 DENV-4 081 ternyata identik dan lestari dengan epitop sel B protein NS3 dari keempat serotipeDENV strain Indonesia.Kesimpulan: Kelestarian yang luas dari epitop sel T dan B pada hampir seluruh strain DENV-4 dunia danserotipe-serotipe DENV strain Indonesia. (Health Science Journal of Indonesia 2015;6:126-31Kata kunci: virus dengue, protein NS3, epitop sel T, epitop sel B AbstractBackground: Non Structural 3 (NS3 protein of dengue virus (DENV is known to induce antibody, CD4+and CD8+ T cell responses, and playing role in viral replication. NS3 protein has T and B cell epitopes,which has conservation difference between DENV-4 strains. This study aimed to identify

Diagnosis of Herpes Simplex Encephalitis by ELISA Using Antipeptide Antibodies Against Type-Common Epitopes of Glycoprotein B of Herpes Simplex Viruses.

Science.gov (United States)

Bhullar, Shradha S; Chandak, Nitin H; Baheti, Neeraj N; Purohit, Hemant J; Taori, Girdhar M; Daginawala, Hatim F; Kashyap, Rajpal S

2016-01-01

Herpes simplex encephalitis (HSE) represents one of the most severe infectious diseases of the central nervous system (CNS). As effective antiviral drugs are available, an early, rapid, and reliable diagnosis has become important. The objective of this article was to develop a sensitive ELISA protocol for herpes simplex viruses (HSV) antigen detection and quantitation by assessing the usefulness of antipeptide antibodies against potential peptides of HSV glycoprotein B (gB). A total of 180 cerebrospinal fluid (CSF) samples of HSE and non-HSE patients were analyzed using a panel of antipeptide antibodies against synthetic peptides of HSV glycoprotein gB. The cases of confirmed and suspected HSE showed 80% and 51% positivity for antipeptide against synthetic peptide QLHDLRF and 77% and 53% positivity for antipeptide against synthetic peptide MKALYPLTT, respectively for the detection of HSV antigen in CSF. The concentration of HSV antigen was found to be higher in confirmed HSE as compared to suspected HSE group and the viral load correlated well with antigen concentration obtained using the two antipeptides in CSF of confirmed HSE group. This is the first article describing the use of antibodies obtained against synthetic peptides derived from HSV in diagnostics of HSE using patients' CSF samples.

In vitro neutralization of HCV by goat antibodies against peptides encompassing regions downstream of HVR-1 of E2 glycoprotein.

Science.gov (United States)

Tabll, Ashraf A; Atef, Khaled; Bader El Din, Noha G; El Abd, Yasmine S; Salem, Ahmed; Sayed, Ahmed A; Dawood, Reham M; Omran, Moataza H; El-Awady, Mostafa K

2014-01-01

This article aims at testing several in vitro systems with various viral sources and cell lines for propagation of HCV to evaluate goat antibodies raised against three E2 epitopes in viral neutralization experiments. Four human cell lines (Huh-7, Huh-7.5, HepG2, and CaCo2) were tested using two different HCV viral sources; Genotype 4 infected sera and J6/JFH HCV cc particles. Neutralization capacity of goat Abs against conserved E2 epitopes; p412 (a.a 412-419), p517 (a.a 517-531), and p430 (a.a 430-447) were examined in the above mentioned in vitro systems. Although infection with patients' sera seems to mimic the in vitro situation, it has limited replication rates as compared with HCV cc particularly in Huh7.5 cells. Non-HCV adapted Huh-7 cells were also found susceptible for transfection with J6/JFH virus but at much slower kinetics. The results of the neutralization assay showed that anti p412 and anti p517 were highly neutralizing to HCVcc. Our data demonstrate that antibodies directed against the viral surface glycoprotein E2 reduced the infectivity of the J6/JFH virus and are promising agents for immunotherapy and HCV vaccine development.

High Throughput T Epitope Mapping and Vaccine Development

Directory of Open Access Journals (Sweden)

Giuseppina Li Pira

2010-01-01

Full Text Available 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.

Glycoprotein biosynthesis by human normal platelets

International Nuclear Information System (INIS)

Rodriguez, P.; Bello, O.; Apitz-Castro, R.

1987-01-01

Incorporation of radioactive Man, Gal, Fuc, Glc-N, and NANA into washed human normal platelets and endogenous glycoproteins has been found. Both parameters were time dependent. Analysis of hydrolyzed labeled glycoproteins by paper chromatography revealed that the radioactive monosaccharide incubated with the platelets had not been converted into other sugars. Acid hydrolysis demonstrates the presence of a glycosidic linkage. All the effort directed to the demonstration of the existence of a lipid-sugar intermediate in intact human platelets yielded negative results for Man and Glc-N used as precursors. The incorporation of these sugars into glycoproteins is insensitive to bacitracin, suggesting no involvement of lipid-linked saccharides in the synthesis of glycoproteins in human blood platelets. The absence of inhibition of the glycosylation process in the presence of cycloheximide suggests that the sugars are added to proteins present in the intact platelets. These results support the contention that glycoprotein biosynthesis in human blood platelets observed under our experimental conditions is effected through direct sugar nucleotide glycosylation

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

Science.gov (United States)

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.

Stepwise identification of HLA-A*0201-restricted CD8+ T-cell epitope peptides from herpes simplex virus type 1 genome boosted by a StepRank scheme.

Science.gov (United States)

Bi, Jianjun; Song, Rengang; Yang, Huilan; Li, Bingling; Fan, Jianyong; Liu, Zhongrong; Long, Chaoqin

2011-01-01

Identification of immunodominant epitopes is the first step in the rational design of peptide vaccines aimed at T-cell immunity. To date, however, it is yet a great challenge for accurately predicting the potent epitope peptides from a pool of large-scale candidates with an efficient manner. In this study, a method that we named StepRank has been developed for the reliable and rapid prediction of binding capabilities/affinities between proteins and genome-wide peptides. In this procedure, instead of single strategy used in most traditional epitope identification algorithms, four steps with different purposes and thus different computational demands are employed in turn to screen the large-scale peptide candidates that are normally generated from, for example, pathogenic genome. The steps 1 and 2 aim at qualitative exclusion of typical nonbinders by using empirical rule and linear statistical approach, while the steps 3 and 4 focus on quantitative examination and prediction of the interaction energy profile and binding affinity of peptide to target protein via quantitative structure-activity relationship (QSAR) and structure-based free energy analysis. We exemplify this method through its application to binding predictions of the peptide segments derived from the 76 known open-reading frames (ORFs) of herpes simplex virus type 1 (HSV-1) genome with or without affinity to human major histocompatibility complex class I (MHC I) molecule HLA-A*0201, and find that the predictive results are well compatible with the classical anchor residue theory and perfectly match for the extended motif pattern of MHC I-binding peptides. The putative epitopes are further confirmed by comparisons with 11 experimentally measured HLA-A*0201-restrcited peptides from the HSV-1 glycoproteins D and K. We expect that this well-designed scheme can be applied in the computational screening of other viral genomes as well.

Localization of immunodominant linear B-cell epitopes of Vibrio ...

African Journals Online (AJOL)

AJL

2012-05-01

May 1, 2012 ... In order to locate the epitopes of OmpU protein, epitope prediction was performed ... enzyme linked immunosorbent assay; OmpU, outer membrane protein U .... recombinant plasmids were extracted and identified by PCR,.

Epitope-dependent functional effects of celiac disease autoantibodies on transglutaminase 2

DEFF Research Database (Denmark)

Hnida, Kathrin; Stamnaes, Jorunn; du Pré, M Fleur

2016-01-01

Transglutaminase 2 (TG2) is a Ca(2+)-dependent cross-linking enzyme involved in the pathogenesis of CD. We have previously characterized a panel of anti-TG2 mAbs generated from gut plasma cells of celiac patients and identified four epitopes (epitopes 1-4) located in the N-terminal part of TG2...... of epitope 1-targeting B cells to keep TG2 active and protected from oxidation might explain why generation of epitope 1-targeting plasma cells seems to be favored in celiac patients....

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

Science.gov (United States)

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.

HIV-1 subtype A gag variability and epitope evolution.

Science.gov (United States)

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.

HIV-1 subtype A gag variability and epitope evolution.

Directory of Open Access Journals (Sweden)

Syed Hani Abidi

Full Text Available OBJECTIVE: 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. METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Salivary agglutinin, which binds Streptococcus mutans and Helicobacter pylori, is the lung scavenger receptor cysteine-rich protein gp-340.

Science.gov (United States)

Prakobphol, A; Xu, F; Hoang, V M; Larsson, T; Bergstrom, J; Johansson, I; Frängsmyr, L; Holmskov, U; Leffler, H; Nilsson, C; Borén, T; Wright, J R; Strömberg, N; Fisher, S J

2000-12-22

Salivary agglutinin is a high molecular mass component of human saliva that binds Streptococcus mutans, an oral bacterium implicated in dental caries. To study its protein sequence, we isolated the agglutinin from human parotid saliva. After trypsin digestion, a portion was analyzed by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), which gave the molecular mass of 14 unique peptides. The remainder of the digest was subjected to high performance liquid chromatography, and the separated peptides were analyzed by MALDI-TOF/post-source decay; the spectra gave the sequences of five peptides. The molecular mass and peptide sequence information showed that salivary agglutinin peptides were identical to sequences in lung (lavage) gp-340, a member of the scavenger receptor cysteine-rich protein family. Immunoblotting with antibodies that specifically recognized either lung gp-340 or the agglutinin confirmed that the salivary agglutinin was gp-340. Immunoblotting with an antibody specific to the sialyl Le(x) carbohydrate epitope detected expression on the salivary but not the lung glycoprotein, possible evidence of different glycoforms. The salivary agglutinin also interacted with Helicobacter pylori, implicated in gastritis and peptic ulcer disease, Streptococcus agalactiae, implicated in neonatal meningitis, and several oral commensal streptococci. These results identify the salivary agglutinin as gp-340 and suggest it binds bacteria that are important determinants of either the oral ecology or systemic diseases.

Epitope finding in Zika virus molecule:The first world report

Institute of Scientific and Technical Information of China (English)

Somsri Wiwanitkit; Viroj Wiwanitkit

2017-01-01

Zika virus infection is a new problematic virus infection that becomes the present public health problem. Now this mosquito borne infectious disease can be seen worldwide and can cause dengue-like infection. In addition, it can also induce trans-placental infection and result in congenital neurological defect. To prevent this infec-tion, there is still no specific vaccine. To find a new vaccine, finding epitope is the first step. Here, the authors report the study to find epitope within Zika virus molecule. According to this study, the appropriate epitopes can be seen. This is the first world report on epitope finding for Zika virus. The data can be useful for further vaccine development.

Identification of murine T-cell epitopes in Ebola virus nucleoprotein

International Nuclear Information System (INIS)

Simmons, Graham; Lee, Anee; Rennekamp, Andrew J.; Fan Xin; Bates, Paul; Shen Hao

2004-01-01

CD8 T cells play an important role in controlling Ebola infection and in mediating vaccine-induced protective immunity, yet little is known about antigenic targets in Ebola that are recognized by CD8 T cells. Overlapping peptides were used to identify major histocompatibility complex class I-restricted epitopes in mice immunized with vectors encoding Ebola nucleoprotein (NP). CD8 T-cell responses were mapped to a H-2 d -restricted epitope (NP279-288) and two H-2 b -restricted epitopes (NP44-52 and NP288-296). The identification of these epitopes will facilitate studies of immune correlates of protection and the evaluation of vaccine strategies in murine models of Ebola infection

File list: Oth.Adl.50.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Adl.50.Epitope_tags.AllCell dm3 TFs and others Epitope tags Adult SRX181419,SRX...ttp://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Adl.50.Epitope_tags.AllCell.bed ...

File list: Oth.Myo.50.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Myo.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Muscle SRX344965,SR...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Myo.50.Epitope_tags.AllCell.bed ...

File list: Oth.Adl.20.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Adl.20.Epitope_tags.AllCell dm3 TFs and others Epitope tags Adult SRX181427,SRX...ttp://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Adl.20.Epitope_tags.AllCell.bed ...

File list: Oth.Dig.10.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

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File list: Oth.Dig.20.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Dig.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags Digestive tract SRX...365692 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Dig.20.Epitope_tags.AllCell.bed ...

File list: Oth.CDV.10.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.CDV.10.Epitope_tags.AllCell mm9 TFs and others Epitope tags Cardiovascular SRX1...304813 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.CDV.10.Epitope_tags.AllCell.bed ...

File list: Oth.Unc.10.Epitope_tags.AllCell [Chip-atlas[Archive

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Full Text Available Oth.Unc.10.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags Unclassified ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.Unc.10.Epitope_tags.AllCell.bed ...

File list: Oth.Brs.50.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Brs.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Breast SRX667411,S...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Brs.50.Epitope_tags.AllCell.bed ...

File list: Oth.Adl.05.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Adl.05.Epitope_tags.AllCell dm3 TFs and others Epitope tags Adult SRX181427,SRX...ttp://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.Adl.05.Epitope_tags.AllCell.bed ...

File list: Oth.CDV.20.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.CDV.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags Cardiovascular SRX1...304813 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.CDV.20.Epitope_tags.AllCell.bed ...

File list: Oth.Myo.10.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Myo.10.Epitope_tags.AllCell mm9 TFs and others Epitope tags Muscle SRX039346,SR...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Myo.10.Epitope_tags.AllCell.bed ...

File list: Oth.CDV.50.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.CDV.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Cardiovascular SRX1...304813 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.CDV.50.Epitope_tags.AllCell.bed ...

File list: Oth.Unc.20.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Unc.20.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags Unclassified ht...tp://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.Unc.20.Epitope_tags.AllCell.bed ...

File list: Oth.Myo.05.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Myo.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags Muscle SRX039346,SR...http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Myo.05.Epitope_tags.AllCell.bed ...

Computational elucidation of potential antigenic CTL epitopes in Ebola virus.

Science.gov (United States)

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.

File list: Oth.Myo.20.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Myo.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Muscle SRX1470542,...SRX1470544 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Myo.20.Epitope_tags.AllCell.bed ...

File list: Oth.CDV.50.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.CDV.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Cardiovascular SRX...096360,SRX096362 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.CDV.50.Epitope_tags.AllCell.bed ...

File list: Oth.CDV.10.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.CDV.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Cardiovascular SRX...096360,SRX096362 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.CDV.10.Epitope_tags.AllCell.bed ...

File list: Oth.Gon.05.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Gon.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags Gonad SRX153152,SRX...153153,SRX153151 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Gon.05.Epitope_tags.AllCell.bed ...

File list: Oth.Prs.50.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Prs.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Prostate SRX084527...,SRX084528,SRX084524 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Prs.50.Epitope_tags.AllCell.bed ...

File list: Oth.Neu.10.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Neu.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Neural SRX367452,S...RX367451 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Neu.10.Epitope_tags.AllCell.bed ...

File list: Oth.YSt.50.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.YSt.50.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags Yeast strain SR...3939 http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.YSt.50.Epitope_tags.AllCell.bed ...

File list: Oth.PSC.10.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.PSC.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Pluripotent stem c...ell SRX555489 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.PSC.10.Epitope_tags.AllCell.bed ...

File list: Oth.Prs.20.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Prs.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Prostate SRX084527...,SRX084528,SRX084524 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Prs.20.Epitope_tags.AllCell.bed ...

File list: Oth.Gon.10.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Gon.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Gonad SRX204898,SR...X204899 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Gon.10.Epitope_tags.AllCell.bed ...

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

Directory of Open Access Journals (Sweden)

Babu Ramanathan

Full Text Available 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.

File list: Oth.Emb.05.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Emb.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags Embryo SRX663359,SR...SRX967653,SRX139878 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Emb.05.Epitope_tags.AllCell.bed ...

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

Science.gov (United States)

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

File list: Oth.Oth.10.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Oth.10.Epitope_tags.AllCell mm9 TFs and others Epitope tags Others SRX228677,SR...X228676,SRX228679,SRX228678 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Oth.10.Epitope_tags.AllCell.bed ...

File list: Oth.ALL.50.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.ALL.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags All cell types SRX1...995,SRX275809,SRX275811 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.ALL.50.Epitope_tags.AllCell.bed ...

File list: Oth.Kid.10.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Kid.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Kidney SRX065541,S...RX644719,SRX170375,SRX644723 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Kid.10.Epitope_tags.AllCell.bed ...

File list: Oth.Kid.50.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Kid.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Kidney SRX065541,S...RX170376,SRX065542,SRX065543 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Kid.50.Epitope_tags.AllCell.bed ...

File list: Oth.ALL.05.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.ALL.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags All cell types SRX1...460,ERX320411,SRX695808 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.ALL.05.Epitope_tags.AllCell.bed ...

Effect of partial and complete variable loop deletions of the human immunodeficiency virus type 1 envelope glycoprotein on the breadth of gp160-specific immune responses

International Nuclear Information System (INIS)

Gzyl, Jaroslaw; Bolesta, Elizabeth; Wierzbicki, Andrew; Kmieciak, Dariusz; Naito, Toshio; Honda, Mitsuo; Komuro, Katsutoshi; Kaneko, Yutaro; Kozbor, Danuta

2004-01-01

Induction of cross-reactive cellular and humoral responses to the HIV-1 envelope (env) glycoprotein was examined after DNA immunization of BALB/c mice with gp140 89.6 -derived constructs exhibiting partial or complete deletions of the V1, V2, and V3 domains. It was demonstrated that specific modification of the V3 loop (mV3) in combination with the V2-modified (mV2) or V1/V2-deleted (ΔV1/V2) region elicited increased levels of cross-reactive CD8 + T cell responses. Mice immunized with the mV2/mV3 or ΔV1/V2/mV3 gp140 89.6 plasmid DNA were greater than 50-fold more resistant to challenge with recombinant vaccinia virus (rVV) expressing heterologous env gene products than animals immunized with the wild-type (WT) counterpart. Sera from mV2/mV3- and ΔV1/V2/mV3-immunized mice exhibited the highest cross-neutralizing activity and displayed intermediate antibody avidity values which were further enhanced by challenge with rVV expressing the homologous gp160 glycoprotein. In contrast, complete deletion of the variable regions had little or no effect on the cross-reactive antibody responses. The results of these experiments indicate that the breadth of antibody responses to the HIV-1 env glycoprotein may not be increased by removal of the variable domains. Instead, partial deletions within these regions may redirect specific responses toward conserved epitopes and facilitate approaches for boosting cross-reactive cellular and antibody responses to the env glycoprotein

Confirmation of a new conserved linear epitope of Lyssavirus nucleoprotein.

Science.gov (United States)

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.

Oral Immunization with a Multivalent Epitope-Based Vaccine, Based on NAP, Urease, HSP60, and HpaA, Provides Therapeutic Effect on H. pylori Infection in Mongolian gerbils.

Science.gov (United States)

Guo, Le; Yang, Hua; Tang, Feng; Yin, Runting; Liu, Hongpeng; Gong, Xiaojuan; Wei, Jun; Zhang, Ying; Xu, Guangxian; Liu, Kunmei

2017-01-01

Epitope-based vaccine is a promising strategy for therapeutic vaccination against Helicobacter pylori ( H. pylori ) infection. A multivalent subunit vaccine containing various antigens from H. pylori is superior to a univalent subunit vaccine. However, whether a multivalent epitope-based vaccine is superior to a univalent epitope-based vaccine in therapeutic vaccination against H. pylori , remains unclear. In this study, a multivalent epitope-based vaccine named CWAE against H. pylori urease, neutrophil-activating protein (NAP), heat shock protein 60 (HSP60) and H. pylori adhesin A (HpaA) was constructed based on mucosal adjuvant cholera toxin B subunit (CTB), Th1-type adjuvant NAP, multiple copies of selected B and Th cell epitopes (UreA 27-53 , UreA 183-203 , HpaA 132-141 , and HSP60 189-203 ), and also the epitope-rich regions of urease B subunit (UreB 158-251 and UreB 321-385 ) predicted by bioinformatics. Immunological properties of CWAE vaccine were characterized in BALB/c mice model. Its therapeutic effect was evaluated in H. pylori -infected Mongolian gerbil model by comparing with a univalent epitope-based vaccine CTB-UE against H. pylori urease that was constructed in our previous studies. Both CWAE and CTB-UE could induce similar levels of specific antibodies against H. pylori urease, and had similar inhibition effect of H. pylori urease activity. However, only CWAE could induce high levels of specific antibodies to NAP, HSP60, HpaA, and also the synthetic peptides epitopes (UreB 158-172 , UreB 181-195 , UreB 211-225 , UreB 349-363 , HpaA 132-141 , and HSP60 189-203 ). In addition, oral therapeutic immunization with CWAE significantly reduced the number of H. pylori colonies in the stomach of Mongolian gerbils, compared with oral immunization using CTB-UE or H. pylori urease. The protection of CWAE was associated with higher levels of mixed CD4 + T cell (Th cell) response, IgG, and secretory IgA (sIgA) antibodies to H. pylori . These results indic ate

Oral Immunization with a Multivalent Epitope-Based Vaccine, Based on NAP, Urease, HSP60, and HpaA, Provides Therapeutic Effect on H. pylori Infection in Mongolian gerbils

Directory of Open Access Journals (Sweden)

Le Guo

2017-08-01

Full Text Available Epitope-based vaccine is a promising strategy for therapeutic vaccination against Helicobacter pylori (H. pylori infection. A multivalent subunit vaccine containing various antigens from H. pylori is superior to a univalent subunit vaccine. However, whether a multivalent epitope-based vaccine is superior to a univalent epitope-based vaccine in therapeutic vaccination against H. pylori, remains unclear. In this study, a multivalent epitope-based vaccine named CWAE against H. pylori urease, neutrophil-activating protein (NAP, heat shock protein 60 (HSP60 and H. pylori adhesin A (HpaA was constructed based on mucosal adjuvant cholera toxin B subunit (CTB, Th1-type adjuvant NAP, multiple copies of selected B and Th cell epitopes (UreA27–53, UreA183–203, HpaA132–141, and HSP60189–203, and also the epitope-rich regions of urease B subunit (UreB158–251 and UreB321–385 predicted by bioinformatics. Immunological properties of CWAE vaccine were characterized in BALB/c mice model. Its therapeutic effect was evaluated in H. pylori-infected Mongolian gerbil model by comparing with a univalent epitope-based vaccine CTB-UE against H. pylori urease that was constructed in our previous studies. Both CWAE and CTB-UE could induce similar levels of specific antibodies against H. pylori urease, and had similar inhibition effect of H. pylori urease activity. However, only CWAE could induce high levels of specific antibodies to NAP, HSP60, HpaA, and also the synthetic peptides epitopes (UreB158–172, UreB181–195, UreB211–225, UreB349–363, HpaA132–141, and HSP60189–203. In addition, oral therapeutic immunization with CWAE significantly reduced the number of H. pylori colonies in the stomach of Mongolian gerbils, compared with oral immunization using CTB-UE or H. pylori urease. The protection of CWAE was associated with higher levels of mixed CD4+ T cell (Th cell response, IgG, and secretory IgA (sIgA antibodies to H. pylori. These results indic

Ammonia transport in the kidney by Rhesus glycoproteins

Science.gov (United States)

Verlander, Jill W.

2014-01-01

Renal ammonia metabolism is a fundamental element of acid-base homeostasis, comprising a major component of both basal and physiologically altered renal net acid excretion. Over the past several years, a fundamental change in our understanding of the mechanisms of renal epithelial cell ammonia transport has occurred, replacing the previous model which was based upon diffusion equilibrium for NH3 and trapping of NH4+ with a new model in which specific and regulated transport of both NH3 and NH4+ across renal epithelial cell membranes via specific membrane proteins is required for normal ammonia metabolism. A major advance has been the recognition that members of a recently recognized transporter family, the Rhesus glycoprotein family, mediate critical roles in renal and extrarenal ammonia transport. The erythroid-specific Rhesus glycoprotein, Rh A Glycoprotein (Rhag), was the first Rhesus glycoprotein recognized as an ammonia-specific transporter. Subsequently, the nonerythroid Rh glycoproteins, Rh B Glycoprotein (Rhbg) and Rh C Glycoprotein (Rhcg), were cloned and identified as ammonia transporters. They are expressed in specific cell populations and membrane domains in distal renal epithelial cells, where they facilitate ammonia secretion. In this review, we discuss the distribution of Rhbg and Rhcg in the kidney, the regulation of their expression and activity in physiological disturbances, the effects of genetic deletion on renal ammonia metabolism, and the molecular mechanisms of Rh glycoprotein-mediated ammonia transport. PMID:24647713

File list: Oth.PSC.20.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.PSC.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags Pluripotent stem ce...822,SRX266828,SRX352996,ERX320411,SRX204802 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.PSC.20.Epitope_tags.AllCell.bed ...

File list: Oth.PSC.05.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.PSC.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags Pluripotent stem ce...821,ERX320410,SRX266822,SRX352996,ERX320411 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.PSC.05.Epitope_tags.AllCell.bed ...

File list: Oth.ALL.05.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.ALL.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags All cell types SRX...644715,SRX555489,SRX644719,SRX527876,SRX644723 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.ALL.05.Epitope_tags.AllCell.bed ...

Identification and characterization of survivin-derived H-2Kb-restricted CTL epitopes

DEFF Research Database (Denmark)

Hofmann, Uta B; Voigt, Heike; Andersen, Mads H

2009-01-01

for potential binding K(b)-restricted octamer peptide epitopes. Two epitopes, which bind strongly to K(b), were selected to test their immunogenicity in vivo. Spleen cells from mice vaccinated by intradermal injection of mature DC pulsed with these peptides displayed reactivity to the respective epitopes...

Confirmation of antibodies against L-tryptophan-like epitope in ...

African Journals Online (AJOL)

Confirmation of antibodies against L-tryptophan-like epitope in human African trypanosomosis serological diagnostic. ... number of patients in Congo. A diagnostic test based on this synthetic epitope, especially in combination with other tests, might improve the HAT diagnostic test in field conditions. Key words: Tryptophan ...

Prediction of residues in discontinuous B-cell epitopes using protein 3D structures

DEFF Research Database (Denmark)

Andersen, P.H.; Nielsen, Morten; Lund, Ole

2006-01-01

. We show that the new structure-based method has a better performance for predicting residues of discontinuous epitopes than methods based solely on sequence information, and that it can successfully predict epitope residues that have been identified by different techniques. DiscoTope detects 15...... experimental epitope mapping in both rational vaccine design and development of diagnostic tools, and may lead to more efficient epitope identification....

Chimeric rhinoviruses displaying MPER epitopes elicit anti-HIV neutralizing responses.

Directory of Open Access Journals (Sweden)

Guohua Yi

Full Text Available The development of an effective AIDS vaccine has been a formidable task, but remains a critical necessity. The well conserved membrane-proximal external region (MPER of the HIV-1 gp41 glycoprotein is one of the crucial targets for AIDS vaccine development, as it has the necessary attribute of being able to elicit antibodies capable of neutralizing diverse isolates of HIV.Guided by X-ray crystallography, molecular modeling, combinatorial chemistry, and powerful selection techniques, we designed and produced six combinatorial libraries of chimeric human rhinoviruses (HRV displaying the MPER epitopes corresponding to mAbs 2F5, 4E10, and/or Z13e1, connected to an immunogenic surface loop of HRV via linkers of varying lengths and sequences. Not all libraries led to viable chimeric viruses with the desired sequences, but the combinatorial approach allowed us to examine large numbers of MPER-displaying chimeras. Among the chimeras were five that elicited antibodies capable of significantly neutralizing HIV-1 pseudoviruses from at least three subtypes, in one case leading to neutralization of 10 pseudoviruses from all six subtypes tested.Optimization of these chimeras or closely related chimeras could conceivably lead to useful components of an effective AIDS vaccine. While the MPER of HIV may not be immunodominant in natural infection by HIV-1, its presence in a vaccine cocktail could provide critical breadth of protection.

Mast Cells Produce a Unique Chondroitin Sulfate Epitope.

Science.gov (United States)

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.

Bioinformatics Tools for the Prediction of T-Cell Epitopes

DEFF Research Database (Denmark)

Andreatta, Massimo; Nielsen, Morten

2018-01-01

T-cell responses are activated by specific peptides, called epitopes, presented on the cell surface by MHC molecules. Binding of peptides to the MHC is the most selective step in T-cell antigen presentation and therefore an essential factor in the selection of potential epitopes. Several in-vitro...

File list: Oth.Utr.05.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Utr.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Uterus SRX248763,S...,SRX735140,SRX735139,SRX210703,SRX210702,SRX095386,SRX968127 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Utr.05.Epitope_tags.AllCell.bed ...

File list: Oth.Utr.50.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Utr.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Uterus SRX188854,S...,SRX210703,SRX968127,SRX610673,SRX610674,SRX610672,SRX095386 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Utr.50.Epitope_tags.AllCell.bed ...

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

Science.gov (United States)

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

The inositol-1,2-cyclic phosphate moiety of the cross-reacting determinant, carbohydrate chains, and proteinaceous components are all responsible for the cross-reactivity of trypanosome variant surface glycoproteins.

Science.gov (United States)

Escalona, José L; Uzcanga, Graciela L; Carrasquel, Liomary M; Bubis, José

2018-01-24

Salivarian trypanosomes evade the host immune system by continually swapping their protective variant surface glycoprotein (VSG) coat. Given that VSGs from various trypanosome stocks exhibited cross-reactivity (Camargo et al., Vet. Parasitol. 207, 17-33, 2015), we analyzed here which components are the antigenic determinants for this cross-reaction. Soluble forms of VSGs were purified from four Venezuelan animal trypanosome isolates: TeAp-N/D1, TeAp-ElFrio01, TeAp-Mantecal01, and TeGu-Terecay323. By using the VSG soluble form from TeAp-N/D1, we found that neither the inositol-1,2-cyclic phosphate moiety of the cross-reacting determinant nor the carbohydrate chains were exclusively responsible for its cross-reactivity. Then, all four purified glycoproteins were digested with papain and the resulting peptides were separated by high-performance liquid chromatography. Dot blot evaluation of the fractions using sera from trypanosome-infected animals yielded peptides that possessed cross-reaction activity, demonstrating for the first time that proteinaceous epitopes are also responsible for the cross-reactivity of trypanosome VSGs.

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

Science.gov (United States)

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.

File list: Oth.Epd.05.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Epd.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Epidermis SRX51236...8,SRX512367,SRX718420,SRX512372,SRX512366,SRX512373,SRX807621,SRX807620 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Epd.05.Epitope_tags.AllCell.bed ...

File list: Oth.Epd.20.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Epd.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Epidermis SRX71842...0,SRX512368,SRX512366,SRX807621,SRX512367,SRX512372,SRX512373,SRX807620 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Epd.20.Epitope_tags.AllCell.bed ...

Mechanism of feline immunodeficiency virus envelope glycoprotein-mediated fusion

International Nuclear Information System (INIS)

Garg, Himanshu; Fuller, Frederick J.; Tompkins, Wayne A.F.

2004-01-01

Feline immunodeficiency virus (FIV) shares remarkable homology to primate lentiviruses, human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV). The process of lentiviral env glycoprotein-mediated fusion of membranes is essential for viral entry and syncytia formation. A detailed understanding of this phenomenon has helped identify new targets for antiviral drug development. Using a model based on syncytia formation between FIV env-expressing cells and a feline CD4+ T cell line we have studied the mechanism of FIV env-mediated fusion. Using this model we show that FIV env-mediated fusion mechanism and kinetics are similar to HIV env. Syncytia formation could be blocked by CXCR4 antagonist AMD3100, establishing the importance of this receptor in FIV gp120 binding. Interestingly, CXCR4 alone was not sufficient to allow fusion by a primary isolate of FIV, as env glycoprotein from FIV-NCSU 1 failed to induce syncytia in several feline cell lines expressing CXCR4. Syncytia formation could be inhibited at a post-CXCR4 binding step by synthetic peptide T1971, which inhibits interaction of heptad repeat regions of gp41 and formation of the hairpin structure. Finally, using site-directed mutagenesis, we also show that a conserved tryptophan-rich region in the membrane proximal ectodomain of gp41 is critical for fusion, possibly at steps post hairpin structure formation

Enterovirus 71 viral capsid protein linear epitopes: Identification and characterization

Directory of Open Access Journals (Sweden)

Gao Fan

2012-01-01

Full Text Available Abstract 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.

The extracellular domain of myelin oligodendrocyte glycoprotein elicits atypical experimental autoimmune encephalomyelitis in rat and Macaque species.

Directory of Open Access Journals (Sweden)

Alan D Curtis

Full Text Available Atypical models of experimental autoimmune encephalomyelitis (EAE are advantageous in that the heterogeneity of clinical signs appears more reflective of those in multiple sclerosis (MS. Conversely, models of classical EAE feature stereotypic progression of an ascending flaccid paralysis that is not a characteristic of MS. The study of atypical EAE however has been limited due to the relative lack of suitable models that feature reliable disease incidence and severity, excepting mice deficient in gamma-interferon signaling pathways. In this study, atypical EAE was induced in Lewis rats, and a related approach was effective for induction of an unusual neurologic syndrome in a cynomolgus macaque. Lewis rats were immunized with the rat immunoglobulin variable (IgV-related extracellular domain of myelin oligodendrocyte glycoprotein (IgV-MOG in complete Freund's adjuvant (CFA followed by one or more injections of rat IgV-MOG in incomplete Freund's adjuvant (IFA. The resulting disease was marked by torticollis, unilateral rigid paralysis, forelimb weakness, and high titers of anti-MOG antibody against conformational epitopes of MOG, as well as other signs of atypical EAE. A similar strategy elicited a distinct atypical form of EAE in a cynomolgus macaque. By day 36 in the monkey, titers of IgG against conformational epitopes of extracellular MOG were evident, and on day 201, the macaque had an abrupt onset of an unusual form of EAE that included a pronounced arousal-dependent, transient myotonia. The disease persisted for 6-7 weeks and was marked by a gradual, consistent improvement and an eventual full recovery without recurrence. These data indicate that one or more boosters of IgV-MOG in IFA represent a key variable for induction of atypical or unusual forms of EAE in rat and Macaca species. These studies also reveal a close correlation between humoral immunity against conformational epitopes of MOG, extended confluent demyelinating plaques in

The extracellular domain of myelin oligodendrocyte glycoprotein elicits atypical experimental autoimmune encephalomyelitis in rat and Macaque species.

Science.gov (United States)

Curtis, Alan D; Taslim, Najla; Reece, Shaun P; Grebenciucova, Elena; Ray, Richard H; Rosenbaum, Matthew D; Wardle, Robert L; Van Scott, Michael R; Mannie, Mark D

2014-01-01

Atypical models of experimental autoimmune encephalomyelitis (EAE) are advantageous in that the heterogeneity of clinical signs appears more reflective of those in multiple sclerosis (MS). Conversely, models of classical EAE feature stereotypic progression of an ascending flaccid paralysis that is not a characteristic of MS. The study of atypical EAE however has been limited due to the relative lack of suitable models that feature reliable disease incidence and severity, excepting mice deficient in gamma-interferon signaling pathways. In this study, atypical EAE was induced in Lewis rats, and a related approach was effective for induction of an unusual neurologic syndrome in a cynomolgus macaque. Lewis rats were immunized with the rat immunoglobulin variable (IgV)-related extracellular domain of myelin oligodendrocyte glycoprotein (IgV-MOG) in complete Freund's adjuvant (CFA) followed by one or more injections of rat IgV-MOG in incomplete Freund's adjuvant (IFA). The resulting disease was marked by torticollis, unilateral rigid paralysis, forelimb weakness, and high titers of anti-MOG antibody against conformational epitopes of MOG, as well as other signs of atypical EAE. A similar strategy elicited a distinct atypical form of EAE in a cynomolgus macaque. By day 36 in the monkey, titers of IgG against conformational epitopes of extracellular MOG were evident, and on day 201, the macaque had an abrupt onset of an unusual form of EAE that included a pronounced arousal-dependent, transient myotonia. The disease persisted for 6-7 weeks and was marked by a gradual, consistent improvement and an eventual full recovery without recurrence. These data indicate that one or more boosters of IgV-MOG in IFA represent a key variable for induction of atypical or unusual forms of EAE in rat and Macaca species. These studies also reveal a close correlation between humoral immunity against conformational epitopes of MOG, extended confluent demyelinating plaques in spinal cord and

File list: Oth.NoD.10.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.NoD.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags No description htt...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.NoD.10.Epitope_tags.AllCell.bed ...

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Full Text Available Oth.NoD.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags No description htt...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.NoD.50.Epitope_tags.AllCell.bed ...

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Full Text Available Oth.NoD.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags No description htt...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.NoD.05.Epitope_tags.AllCell.bed ...

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Full Text Available Oth.NoD.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags No description htt...p://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.NoD.20.Epitope_tags.AllCell.bed ...

Solubilization of glycoproteins of envelope viruses by detergents

International Nuclear Information System (INIS)

Berezin, V.E.; Zaides, V.M.; Artamsnov, A.F.; Isaeva, E.S.; Zhdanov, V.M.

1986-01-01

The action of a number of known ionic and nonionic detergents, as well as the new nonionic detergent MESK, on envelope viruses was investigated. It was shown that the nonionic detergents MESK, Triton X-100, and octyl-β-D-glucopyranoside selectively solubilize the outer glycoproteins of the virus particles. The nonionic detergent MESK has the mildest action. Using MESK, purified glycoproteins of influenza, parainfluenza, Venezuelan equine encephalomyelitis, vesicular stomatitis, rabies, and herpes viruses were obtained. The procedure for obtaining glycoproteins includes incubation of the virus suspension with the detergent MESK, removal of subvirus structures by centrifuging, and purification of glycoproteins from detergents by dialysis. Isolated glycoproteins retain a native structure and biological activity and possess high immunogenicity. The detergent MESK is promising for laboratory tests and with respect to the production of subunit vaccines

Anaplasma marginale major surface protein 2 CD4+-T-cell epitopes are evenly distributed in conserved and hypervariable regions (HVR), whereas linear B-cell epitopes are predominantly located in the HVR.

Science.gov (United States)

Abbott, Jeffrey R; Palmer, Guy H; Howard, Chris J; Hope, Jayne C; Brown, Wendy C

2004-12-01

Organisms in the genus Anaplasma express an immunodominant major surface protein 2 (MSP2), composed of a central hypervariable region (HVR) flanked by highly conserved regions. Throughout Anaplasma marginale infection, recombination results in the sequential appearance of novel MSP2 variants and subsequent control of rickettsemia by the immune response, leading to persistent infection. To determine whether immune evasion and selection for variant organisms is associated with a predominant response against HVR epitopes, T-cell and linear B-cell epitopes were localized by measuring peripheral blood gamma interferon-secreting cells, proliferation, and antibody binding to 27 overlapping peptides spanning MSP2 in 16 cattle. Similar numbers of MSP2-specific CD4(+) T-cell epitopes eliciting responses of similar magnitude were found in conserved and hypervariable regions. T-cell epitope clusters recognized by the majority of animals were identified in the HVR (amino acids [aa] 171 to 229) and conserved regions (aa 101 to 170 and 272 to 361). In contrast, linear B-cell epitopes were concentrated in the HVR, residing within hydrophilic sequences. The pattern of recognition of epitope clusters by T cells and of HVR epitopes by B cells is consistent with the influence of protein structure on epitope recognition.

File list: Oth.Liv.10.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.Liv.10.Epitope_tags.AllCell hg19 TFs and others Epitope tags Liver SRX1165095,S...RX1165103,SRX1165100,SRX1165096,SRX1165104,SRX1165101,SRX1165090,SRX1165102,SRX1165091 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Liv.10.Epitope_tags.AllCell.bed ...

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Full Text Available Oth.Liv.50.Epitope_tags.AllCell hg19 TFs and others Epitope tags Liver SRX1165103,S...RX1165095,SRX1165100,SRX1165101,SRX1165104,SRX1165102,SRX1165090,SRX1165091,SRX1165096 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Liv.50.Epitope_tags.AllCell.bed ...

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Full Text Available Oth.Liv.05.Epitope_tags.AllCell hg19 TFs and others Epitope tags Liver SRX1165095,S...RX1165103,SRX1165096,SRX1165104,SRX1165100,SRX1165101,SRX1165102,SRX1165090,SRX1165091 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Liv.05.Epitope_tags.AllCell.bed ...

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Full Text Available Oth.Liv.20.Epitope_tags.AllCell hg19 TFs and others Epitope tags Liver SRX1165103,S...RX1165095,SRX1165100,SRX1165101,SRX1165090,SRX1165104,SRX1165102,SRX1165096,SRX1165091 http://dbarchive.biosciencedbc.jp/kyushu-u/hg19/assembled/Oth.Liv.20.Epitope_tags.AllCell.bed ...

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Full Text Available Oth.NoD.10.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags No description ...http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.NoD.10.Epitope_tags.AllCell.bed ...

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Full Text Available Oth.CeL.50.Epitope_tags.AllCell dm3 TFs and others Epitope tags Cell line SRX099638...099636 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.CeL.50.Epitope_tags.AllCell.bed ...

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Full Text Available Oth.NoD.20.Epitope_tags.AllCell sacCer3 TFs and others Epitope tags No description ...http://dbarchive.biosciencedbc.jp/kyushu-u/sacCer3/assembled/Oth.NoD.20.Epitope_tags.AllCell.bed ...

File list: Oth.CeL.20.Epitope_tags.AllCell [Chip-atlas[Archive

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Full Text Available Oth.CeL.20.Epitope_tags.AllCell dm3 TFs and others Epitope tags Cell line SRX099638...099636 http://dbarchive.biosciencedbc.jp/kyushu-u/dm3/assembled/Oth.CeL.20.Epitope_tags.AllCell.bed ...

In silico-accelerated identification of conserved and immunogenic variola/vaccinia T-cell epitopes

DEFF Research Database (Denmark)

Moise, Leonard; McMurry, Julie A; Buus, Søren

2009-01-01

Epitopes shared by the vaccinia and variola viruses underlie the protective effect of vaccinia immunization against variola infection. We set out to identify a subset of cross-reactive epitopes using bioinformatics and immunological methods. Putative T-cell epitopes were computationally predicted...

File list: Oth.Neu.20.Epitope_tags.AllCell [Chip-atlas[Archive

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Full Text Available Oth.Neu.20.Epitope_tags.AllCell mm9 TFs and others Epitope tags Neural SRX275807,SR...SRX691799,SRX691794,SRX759286,SRX691798,SRX691797,SRX275809,SRX275811,SRX691795,SRX022866 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.Neu.20.Epitope_tags.AllCell.bed ...

Characterization of the disassembly and reassembly of the HBV glycoprotein surface antigen, a pliable nanoparticle vaccine platform

International Nuclear Information System (INIS)

Gallagher, John R.; Torian, Udana; McCraw, Dustin M.; Harris, Audray K.

2017-01-01

While nanoparticle vaccine technology is gaining interest due to the success of vaccines like those for the human papillomavirus that is based on viral capsid nanoparticles, little information is available on the disassembly and reassembly of viral surface glycoprotein-based nanoparticles. One such particle is the hepatitis B virus surface antigen (sAg) that exists as nanoparticles. Here we show, using biochemical analysis coupled with electron microscopy, that sAg nanoparticle disassembly requires both reducing agent to disrupt intermolecular disulfide bonds, and detergent to disrupt hydrophobic interactions that stabilize the nanoparticle. Particles were otherwise resistant to salt and urea, suggesting the driving mechanism of particle formation involves hydrophobic interactions. We reassembled isolated sAg protein into nanoparticles by detergent removal and reassembly resulted in a wider distribution of particle diameters. Knowledge of these driving forces of nanoparticle assembly and stability should facilitate construction of epitope-displaying nanoparticles that can be used as immunogens in vaccines.

Characterization of the disassembly and reassembly of the HBV glycoprotein surface antigen, a pliable nanoparticle vaccine platform

Energy Technology Data Exchange (ETDEWEB)

Gallagher, John R.; Torian, Udana; McCraw, Dustin M.; Harris, Audray K., E-mail: harrisau@mail.nih.gov

2017-02-15

While nanoparticle vaccine technology is gaining interest due to the success of vaccines like those for the human papillomavirus that is based on viral capsid nanoparticles, little information is available on the disassembly and reassembly of viral surface glycoprotein-based nanoparticles. One such particle is the hepatitis B virus surface antigen (sAg) that exists as nanoparticles. Here we show, using biochemical analysis coupled with electron microscopy, that sAg nanoparticle disassembly requires both reducing agent to disrupt intermolecular disulfide bonds, and detergent to disrupt hydrophobic interactions that stabilize the nanoparticle. Particles were otherwise resistant to salt and urea, suggesting the driving mechanism of particle formation involves hydrophobic interactions. We reassembled isolated sAg protein into nanoparticles by detergent removal and reassembly resulted in a wider distribution of particle diameters. Knowledge of these driving forces of nanoparticle assembly and stability should facilitate construction of epitope-displaying nanoparticles that can be used as immunogens in vaccines.

Plasticity and Epitope Exposure of the HIV-1 Envelope Trimer.

Science.gov (United States)

Powell, Rebecca L R; Totrov, Maxim; Itri, Vincenza; Liu, Xiaomei; Fox, Alisa; Zolla-Pazner, Susan

2017-09-01

We recently showed that mutations in the HIV-1 envelope (Env) destabilize the V3 loop, rendering neutralization-resistant viruses sensitive to V3-directed monoclonal antibodies (MAbs). Here, we investigated the propagation of this effect on other Env epitopes, with special emphasis on V2 loop exposure. Wild-type JR-FL and 19 mutant JR-FL pseudoviruses were tested for neutralization sensitivity to 21 MAbs specific for epitopes in V2, the CD4 binding site (CD4bs), and the CD4-induced (CD4i) region. Certain glycan mutants, mutations in the gp120 hydrophobic core, and mutations in residues involved in intraprotomer interactions exposed epitopes in the V2i region (which overlies the α4β7 integrin binding site) and the V3 crown, suggesting general destabilization of the distal region of the trimer apex. In contrast, other glycan mutants, mutations affecting interprotomer interactions, and mutations affecting the CD4bs exposed V3 but not V2i epitopes. These data indicate for the first time that V3 can move independently of V2, with V3 pivoting out from its "tucked" position in the trimer while apparently leaving the V2 apex intact. Notably, none of the mutations exposed V2 epitopes without also exposing V3, suggesting that movement of V2 releases V3. Most mutations increased sensitivity to CD4bs-directed MAbs without exposure of the CD4i epitope, implying these mutations facilitate the trimers' maintenance of an intermediate energy state between open and closed conformations. Taken together, these data indicate that several transient Env epitopes can be rendered more accessible to antibodies (Abs) via specific mutations, and this may facilitate the design of V1V2-targeting immunogens. IMPORTANCE Many epitopes of the HIV envelope (Env) spike are relatively inaccessible to antibodies (Abs) compared to their exposure in the open Env conformation induced by receptor binding. However, the reduced infection rate that resulted from the vaccine used in the RV144 HIV-1 vaccine

Nucleic acid-binding glycoproteins which solubilize nucleic acids in dilute acid: re-examination of the Ustilago maydis glycoproteins

Energy Technology Data Exchange (ETDEWEB)

Unrau, P.; Champ, D.R.; Young, J.L.; Grant, C.E.

1980-01-01

Holloman reported the isolation from Ustilago maydis of a glycoprotein which prevented the precipitation of nucleic acids in cold 5% trichloroacetic acid. Two glycoprotein fractions from U. maydis with this nucleic acid-solubilizing activity were isolated in our laboratory using improved purification procedures. The activity was not due to nuclease contamination. The glycoproteins are distinguished by: their ability to bind to concanavalin A-Sepharose; their differential binding to double- and single-stranded deoxyribonucleic acid, and to ribonucleic acid; their molecular weights (46,000 and 69,000); and the relative amounts present in growing versus nongrowing cells. Both fractions required sulfhydryl-reducing conditions for optimal yields, specific activity, and stability. Nucleic acid binding was cooperative, the minimum number of glycoproteins required to make a native T7 DNA molecule soluble in dilute acid being estimated at 2 and 15, respectively.

A xylogalacturonan epitope is specifically associated with plant cell detachment

DEFF Research Database (Denmark)

Willats, William George Tycho; McCartney, L.; Steele-King, C.G.

2004-01-01

A monoclonal antibody (LM8) was generated with specificity for xyloglacturonan (XGA) isolated from pea (Pisum sativum L.) testae. Characterization of the LM8 epitope indicates that it is a region of XGA that is highly substituted with xylose. Immunocytochemical analysis indicates that this epitop...

Multiple epitopes in a dodecapeptide of myelin basic protein determined bymonoclonal antibodies

International Nuclear Information System (INIS)

Price, J.O.; Whitaker, J.N.; Vasu, R.I.; Metzger, D.W.

1986-01-01

Three custom synthesized myelin basic protein (MBP) peptides, bovine peptide 79-88, human peptide 80-89, and human peptide 82-91, were used to produce four murine monoclonal antibodies (MAb) that were selected on the basis of reaction in a solid phase radioimmunoassay (SRIA) with human MBP. The MAb were compared with respect to antigen specificity against intact MBP and 10 overlapping MBP peptides. One MAb recognized an epitope near the amino-terminus of bovine MBP peptide 79-88. A second MAb was directed towards an epitope that is more reactive in human MBP peptide 45-89 than in intact MBP, but is not recognized in any of the small MBP peptides examined. The third MAb detected an epitope near the middle of human MBP peptide 80-89, whereas the fourth MAb reacted with the carboxyl-terminal portion of human MBP peptide 82-91. Epitopes recognized in SRIA were sometimes not detected by the same MAb in a fluid phase double antibody radioimmunoassay. These results demonstrate the multiplicity of potential epitopes in a dodecapeptide of MBP and do not support the concept of a single, dominant epitope in the region of MBP peptide 80-89

File list: Oth.EmF.05.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.EmF.05.Epitope_tags.AllCell mm9 TFs and others Epitope tags Embryonic fibroblas...RX542102,SRX204644,SRX204643,SRX255462,SRX255460 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.EmF.05.Epitope_tags.AllCell.bed ...

File list: Oth.EmF.50.Epitope_tags.AllCell [Chip-atlas[Archive

Lifescience Database Archive (English)

Full Text Available Oth.EmF.50.Epitope_tags.AllCell mm9 TFs and others Epitope tags Embryonic fibroblas...RX255460,SRX204644,SRX542102,SRX204643,SRX204642 http://dbarchive.biosciencedbc.jp/kyushu-u/mm9/assembled/Oth.EmF.50.Epitope_tags.AllCell.bed ...

Epitope Identification and Application for Diagnosis of Duck Tembusu Virus Infections in Ducks

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Chenxi Li

2016-11-01

Full Text Available Duck Tembusu virus (DTMUV causes substantial egg drop disease. DTMUV was first identified in China and rapidly spread to Malaysia and Thailand. The antigenicity of the DTMUV E protein has not yet been characterized. Here, we investigated antigenic sites on the E protein using the non-neutralizing monoclonal antibodies (mAbs 1F3 and 1A5. Two minimal epitopes were mapped to 221LD/NLPW225 and 87YAEYI91 by using phage display and mutagenesis. DTMUV-positive duck sera reacted with the epitopes, thus indicating the importance of the minimal amino acids of the epitopes for antibody-epitope binding. The performance of the dot blotting assay with the corresponding positive sera indicated that YAEYI was DTMUV type-specific, whereas 221LD/NLPW225 was a cross-reactive epitope for West Nile virus (WNV, dengue virus (DENV, and Japanese encephalitis virus (JEV and corresponded to conserved and variable amino acid sequences among these strains. The structure model of the E protein revealed that YAEYI and LD/NLPW were located on domain (D II, which confirmed that DII might contain a type-specific non-neutralizing epitope. The YAEYI epitope-based antigen demonstrated its diagnostic potential by reacting with high specificity to serum samples obtained from DTMUV-infected ducks. Based on these observations, a YAEYI-based serological test could be used for DTMUV surveillance and could differentiate DTMUV infections from JEV or WNV infections. These findings provide new insights into the organization of epitopes on flavivirus E proteins that might be valuable for the development of epitope-based serological diagnostic tests for DTMUV.

Glycan shield and fusion activation of a deltacoronavirus spike glycoprotein fine-tuned for enteric infections.

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Xiong, Xiaoli; Tortorici, M Alejandra; Snijder, Joost; Yoshioka, Craig; Walls, Alexandra C; Li, Wentao; McGuire, Andrew T; Rey, Félix A; Bosch, Berend-Jan; Veesler, David

2017-11-01

Coronaviruses recently emerged as major human pathogens causing outbreaks of severe acute respiratory syndrome and Middle-East respiratory syndrome. They utilize the spike (S) glycoprotein anchored in the viral envelope to mediate host attachment and fusion of the viral and cellular membranes to initiate infection. The S protein is a major determinant of the zoonotic potential of coronaviruses and is also the main target of the host humoral immune response. We report here the 3.5 Å resolution cryo-electron microscopy structure of the S glycoprotein trimer from the pathogenic porcine deltacoronavirus (PDCoV), which belongs to the recently identified delta genus. Structural and glycoproteomics data indicate that the glycans of PDCoV S are topologically conserved when compared with the human respiratory coronavirus HCoV-NL63 S, resulting in similar surface areas being shielded from neutralizing antibodies and implying that both viruses are under comparable immune pressure in their respective hosts. The structure further reveals a shortened S 2 ' activation loop, containing a reduced number of basic amino acids, which participates to rendering the spike largely protease-resistant. This property distinguishes PDCoV S from recently characterized betacoronavirus S proteins and suggests that the S protein of enterotropic PDCoV has evolved to tolerate the protease-rich environment of the small intestine and to fine-tune its fusion activation to avoid premature triggering and reduction of infectivity. IMPORTANCE Coronaviruses use transmembrane spike (S) glycoprotein trimers to promote host attachment and fusion of the viral and cellular membranes. We determined a near-atomic resolution cryo-electron microscopy structure of the S ectodomain trimer from the pathogenic porcine deltacoronavirus (PDCoV), which is responsible for diarrhea in piglets and has had devastating consequences for the swine industry worldwide. Structural and glycoproteomics data reveal that PDCoV S is

Activation of the classical pathway of complement by tobacco glycoprotein (TGP).

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Koethe, S M; Nelson, K E; Becker, C G

1995-07-15

Tobacco glycoprotein (TGP), a polyphenol-rich glycoprotein isolated from tobacco leaves, activates the classical complement pathway through a mechanism that appears to involve direct interaction with C1q. A binding site on C1q for TGP can be localized by competitive inhibition with DNA to a region located in the junction between the collagen-like and globular regions of the molecule. A protein with activity similar to TGP has also been isolated from cigarette smoke condensate (TGP-S); it shares a binding site on C1q with TGP and has similar functional activity, with the exception that complement activation does not proceed to formation of a C3 cleaving enzyme. The ability of TGP and TGP-S to activate complement can be partially duplicated using polyphenols associated with tobacco leaf and smoke, i.e., chlorogenic acid and rutin. These polyphenols also compete with TGP for a binding site on immobilized C1q, suggesting that the polyphenol portion of TGP is critical for activation of complement. These results provide an additional mechanism for complement activation by cigarette products that, in vivo, could result in a localized complement depletion, generation of biologically active complement cleavage products, and initiation of an inflammatory response.

An improved radioimmunoassay for urinary Tamm-Horsfall glycoprotein

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Dawnay, A.B. St. J.; Thornley, C.; Cattell, W.R.

1982-01-01

A rapid specific radioimmunoassay has been used to measure Tamm-Horsfall glycoprotein (TH glycoprotein) in urine, and the method described. The apparent concentration increased with increasing dilution of urine in water, reaching a plateau at 1 in 20. This increase was greater the higher the osmolality and TH glycoprotein concentration and the lower the pH of the original sample. The apparent concentration of TH glycoprotein in neat or diluted urine was not affected by freezing or by storage at 4 0 C or room temperature for at least 2 days. A physiological range for the urinary excretion rate was established as 22-56 mg/24h, (considerably higher than the amount present in serum) based on samples from 29 individuals with normal renal function, as defined by their creatinine clearance. There was no significant correlation between serum concentrations of TH glycoprotein and its urinary excretion rate, nor between urinary excretion rate and creatinine clearance. (author)

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

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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.

Identification and fine mapping of a linear B cell epitope of human vimentin

DEFF Research Database (Denmark)

Dam, Catharina Essendrup; Houen, Gunnar; Hansen, Paul R.

2014-01-01

Knowledge about antibody-antigen interactions is important for the understanding of the immune system mechanisms and for supporting development of drugs and biomarkers. A tool for identification of these antigenic epitopes of specific antibodies is epitope mapping. In this study, a modified enzyme......-linked immunosorbent assay was applied for epitope mapping of a mouse monoclonal vimentin antibody using overlapping resin-bound peptides covering the entire vimentin protein. The minimal epitope required for binding was identified as the LDSLPLVD sequence using N- and C-terminally truncated peptides. The peptide...... sequence LDSLPLVDTH was identified as the complete epitope, corresponding to amino acids 428-437 in the C-terminal end of the human vimentin protein. Alanine scanning and functionality scanning applying substituted peptides were used to identify amino acids essential for antibody reactivity. In particular...

Progranulin, a Glycoprotein Deficient in Frontotemporal Dementia, Is a Novel Substrate of Several Protein Disulfide Isomerase Family Proteins

OpenAIRE

Almeida, Sandra; Zhou, Lijuan; Gao, Fen-Biao

2011-01-01

The reduced production or activity of the cysteine-rich glycoprotein progranulin is responsible for about 20% of cases of familial frontotemporal dementia. However, little is known about the molecular mechanisms that govern the level and secretion of progranulin. Here we show that progranulin is expressed in mouse cortical neurons and more prominently in mouse microglia in culture and is abundant in the endoplasmic reticulum (ER) and Golgi. Using chemical crosslinking, immunoprecipitation, an...

High-resolution mapping of linear antibody epitopes using ultrahigh-density peptide microarrays

DEFF Research Database (Denmark)

Buus, Søren; Rockberg, Johan; Forsström, Björn

2012-01-01

Antibodies empower numerous important scientific, clinical, diagnostic, and industrial applications. Ideally, the epitope(s) targeted by an antibody should be identified and characterized, thereby establishing antibody reactivity, highlighting possible cross-reactivities, and perhaps even warning...... against unwanted (e.g. autoimmune) reactivities. Antibodies target proteins as either conformational or linear epitopes. The latter are typically probed with peptides, but the cost of peptide screening programs tends to prohibit comprehensive specificity analysis. To perform high-throughput, high......-resolution mapping of linear antibody epitopes, we have used ultrahigh-density peptide microarrays generating several hundred thousand different peptides per array. Using exhaustive length and substitution analysis, we have successfully examined the specificity of a panel of polyclonal antibodies raised against...

EPMLR: sequence-based linear B-cell epitope prediction method using multiple linear regression.

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Lian, Yao; Ge, Meng; Pan, Xian-Ming

2014-12-19

B-cell epitopes have been studied extensively due to their immunological applications, such as peptide-based vaccine development, antibody production, and disease diagnosis and therapy. Despite several decades of research, the accurate prediction of linear B-cell epitopes has remained a challenging task. In this work, based on the antigen's primary sequence information, a novel linear B-cell epitope prediction model was developed using the multiple linear regression (MLR). A 10-fold cross-validation test on a large non-redundant dataset was performed to evaluate the performance of our model. To alleviate the problem caused by the noise of negative dataset, 300 experiments utilizing 300 sub-datasets were performed. We achieved overall sensitivity of 81.8%, precision of 64.1% and area under the receiver operating characteristic curve (AUC) of 0.728. We have presented a reliable method for the identification of linear B cell epitope using antigen's primary sequence information. Moreover, a web server EPMLR has been developed for linear B-cell epitope prediction: http://www.bioinfo.tsinghua.edu.cn/epitope/EPMLR/ .

CD4+ T cells targeting dominant and cryptic epitopes from Bacillus anthracis Lethal Factor

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Stephanie eAscough

2016-01-01

Full Text Available Anthrax is an endemic infection in many countries, particularly in the developing world. The causative agent, Bacillus anthracis, mediates disease through the secretion of binary exotoxins. Until recently, research into adaptive immunity targeting this bacterial pathogen has largely focused on the humoral response to these toxins. There is, however, growing recognition that cellular immune responses involving IFNγ producing CD4+ T cells also contribute significantly to a protective memory response. An established concept in adaptive immunity to infection is that during infection of host cells, new microbial epitopes may be revealed, leading to immune recognition of so called ‘cryptic’ or ‘subdominant’ epitopes. We analysed the response to both cryptic and immunodominant T cell epitopes derived from the toxin component lethal factor and presented by a range of HLA-DR alleles. Using IFNγ-ELISPOT assays we characterised epitopes that elicited a response following immunisation with synthetic peptide and the whole protein and tested their capacities to bind purified HLA-DR molecules in vitro. We found that DR1 transgenics demonstrated T cell responses to a greater number of domain III cryptic epitopes than other HLA-DR transgenics, and that this pattern was repeated with the immunodominant epitopes, a greater proportion of these epitopes induced a T cell response when presented within the context of the whole protein. Immunodominant epitopes LF457-476 and LF467-487 were found to induce a T cell response to the peptide, as well as to the whole native LF protein in DR1 and DR15, but not in DR4 trangenics. The analysis of Domain I revealed the presence of several unique cryptic epitopes all of which showed a strong to moderate relative binding affinity to HLA-DR4 molecules. However, none of the cryptic epitopes from either domain III or I displayed notably high binding affinities across all HLA-DR alleles assayed. These responses were

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

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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

Orthobunyavirus ultrastructure and the curious tripodal glycoprotein spike.

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Thomas A Bowden

Full Text Available The genus Orthobunyavirus within the family Bunyaviridae constitutes an expanding group of emerging viruses, which threaten human and animal health. Despite the medical importance, little is known about orthobunyavirus structure, a prerequisite for understanding virus assembly and entry. Here, using electron cryo-tomography, we report the ultrastructure of Bunyamwera virus, the prototypic member of this genus. Whilst Bunyamwera virions are pleomorphic in shape, they display a locally ordered lattice of glycoprotein spikes. Each spike protrudes 18 nm from the viral membrane and becomes disordered upon introduction to an acidic environment. Using sub-tomogram averaging, we derived a three-dimensional model of the trimeric pre-fusion glycoprotein spike to 3-nm resolution. The glycoprotein spike consists mainly of the putative class-II fusion glycoprotein and exhibits a unique tripod-like arrangement. Protein-protein contacts between neighbouring spikes occur at membrane-proximal regions and intra-spike contacts at membrane-distal regions. This trimeric assembly deviates from previously observed fusion glycoprotein arrangements, suggesting a greater than anticipated repertoire of viral fusion glycoprotein oligomerization. Our study provides evidence of a pH-dependent conformational change that occurs during orthobunyaviral entry into host cells and a blueprint for the structure of this group of emerging pathogens.

Analysis of potato virus Y coat protein epitopes recognized by three commercial monoclonal antibodies.

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Tian, Yan-Ping; Hepojoki, Jussi; Ranki, Harri; Lankinen, Hilkka; Valkonen, Jari P T

2014-01-01

Potato virus Y (PVY, genus Potyvirus) causes substantial economic losses in solanaceous plants. Routine screening for PVY is an essential part of seed potato certification, and serological assays are often used. The commercial, commonly used monoclonal antibodies, MAb1128, MAb1129, and MAb1130, recognize the viral coat protein (CP) of PVY and distinguish PVYN strains from PVYO and PVYC strains, or detect all PVY strains, respectively. However, the minimal epitopes recognized by these antibodies have not been identified. SPOT peptide array was used to map the epitopes in CP recognized by MAb1128, MAb1129, and MAb1130. Then alanine replacement as well as N- and C-terminal deletion analysis of the identified peptide epitopes was done to determine critical amino acids for antibody recognition and the respective minimal epitopes. The epitopes of all antibodies were located within the 30 N-terminal-most residues. The minimal epitope of MAb1128 was 25NLNKEK30. Replacement of 25N or 27N with alanine weakened the recognition by MAb1128, and replacement of 26L, 29E, or 30K nearly precluded recognition. The minimal epitope for MAb1129 was 16RPEQGSIQSNP26 and the most critical residues for recognition were 22I and 23Q. The epitope of MAb1130 was defined by residues 5IDAGGS10. Mutation of residue 6D abrogated and mutation of 9G strongly reduced recognition of the peptide by MAb1130. Amino acid sequence alignment demonstrated that these epitopes are relatively conserved among PVY strains. Finally, recombinant CPs were produced to demonstrate that mutations in the variable positions of the epitope regions can affect detection with the MAbs. The epitope data acquired can be compared with data on PVY CP-encoding sequences produced by laboratories worldwide and utilized to monitor how widely the new variants of PVY can be detected with current seed potato certification schemes or during the inspection of imported seed potatoes as conducted with these MAbs.

Leucine-rich repeat-containing G protein-coupled receptor 4 facilitates vesicular stomatitis virus infection by binding vesicular stomatitis virus glycoprotein.

Science.gov (United States)

Zhang, Na; Huang, Hongjun; Tan, Binghe; Wei, Yinglei; Xiong, Qingqing; Yan, Yan; Hou, Lili; Wu, Nannan; Siwko, Stefan; Cimarelli, Andrea; Xu, Jianrong; Han, Honghui; Qian, Min; Liu, Mingyao; Du, Bing

2017-10-06

Vesicular stomatitis virus (VSV) and rabies and Chandipura viruses belong to the Rhabdovirus family. VSV is a common laboratory virus to study viral evolution and host immune responses to viral infection, and recombinant VSV-based vectors have been widely used for viral oncolysis, vaccination, and gene therapy. Although the tropism of VSV is broad, and its envelope glycoprotein G is often used for pseudotyping other viruses, the host cellular components involved in VSV infection remain unclear. Here, we demonstrate that the host protein leucine-rich repeat-containing G protein-coupled receptor 4 (Lgr4) is essential for VSV and VSV-G pseudotyped lentivirus (VSVG-LV) to infect susceptible cells. Accordingly, Lgr4-deficient mice had dramatically decreased VSV levels in the olfactory bulb. Furthermore, Lgr4 knockdown in RAW 264.7 cells also significantly suppressed VSV infection, and Lgr4 overexpression in RAW 264.7 cells enhanced VSV infection. Interestingly, only VSV infection relied on Lgr4, whereas infections with Newcastle disease virus, influenza A virus (A/WSN/33), and herpes simplex virus were unaffected by Lgr4 status. Of note, assays of virus entry, cell ELISA, immunoprecipitation, and surface plasmon resonance indicated that VSV bound susceptible cells via the Lgr4 extracellular domain. Pretreating cells with an Lgr4 antibody, soluble LGR4 extracellular domain, or R-spondin 1 blocked VSV infection by competitively inhibiting VSV binding to Lgr4. Taken together, the identification of Lgr4 as a VSV-specific host factor provides important insights into understanding VSV entry and its pathogenesis and lays the foundation for VSV-based gene therapy and viral oncolytic therapeutics. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Large-scale validation of methods for cytotoxic T-lymphocyte epitope prediction

DEFF Research Database (Denmark)

Larsen, Mette Voldby; Lundegaard, Claus; Lamberth, K.

2007-01-01

BACKGROUND: Reliable predictions of Cytotoxic T lymphocyte (CTL) epitopes are essential for rational vaccine design. Most importantly, they can minimize the experimental effort needed to identify epitopes. NetCTL is a web-based tool designed for predicting human CTL epitopes in any given protein....... of the other methods achieved a sensitivity of 0.64. The NetCTL-1.2 method is available at http://www.cbs.dtu.dk/services/NetCTL.All used datasets are available at http://www.cbs.dtu.dk/suppl/immunology/CTL-1.2.php....

Antibody specific epitope prediction-emergence of a new paradigm.

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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.

Identification of a variant antigenic neutralizing epitope in hypervariable region 1 of avian leukosis virus subgroup J.

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Hou, Minbo; Zhou, Defang; Li, Gen; Guo, Huijun; Liu, Jianzhu; Wang, Guihua; Zheng, Qiankun; Cheng, Ziqiang

2016-03-08

Avian leukosis virus subgroup J (ALV-J) is a hypervariable oncogenic retrovirus that causes great economic loss in poultry. Antigenic variations in the variable regions make the development of an effective vaccine a challenging task. In the present study, we identified a variant antigenic neutralizing epitope using reverse vaccinology methods. First, we predicted the B-cell epitopes in gp85 gene of ALV-J strains by DNAman and bioinformatics. Fourteen candidate epitopes were selected and linked in tandem with glycines or serines as a multi-epitope gene. The expressed protein of multi-epitope gene can induce high-titer antibody that can recognize nature ALV-J and neutralize the infectivity of ALV-J strains. Next, we identified a high effective epitope using eight overlapping fragments of gp85 gene reacting with mAb 2D5 and anti-multi-epitope sera. The identified epitope contained one of the predicted epitopes and localized in hyervariable region 1 (hr1), indicating a variant epitope. To better understand if the variants of the epitope have a good antigenicity, we synthesized four variants to react with mAb 2D5 and anti-ALV-J sera. The result showed that all variants could react with the two kinds of antibodies though they showed different antigenicity, while could not react with ALV-J negative sera. Thus, the variant antigenic neutralizing epitope was determined as 137-LRDFIA/E/TKWKS/GDDL/HLIRPYVNQS-158. The result shows a potential use of this variant epitopes as a novel multi-epitope vaccine against ALV-J in poultry. Copyright © 2016 Elsevier Ltd. All rights reserved.

The glycoprotein of measles virus

International Nuclear Information System (INIS)

Anttonen, O.; Jokinen, M.; Salmi, A.; Vainionpaeae, R.; Gahmberg, C.G.

1980-01-01

Measles virus was propagated in VERO cells and purified from the culture supernatants by two successive tartrate-density-gradient centrifugations. Surface carbohydrates were labelled both in vitro and in vivo with 3 H after treatment with galactose oxidase/NaB 3 H 4 or with [ 3 H]glucosamine. The major labelled glycoprotein in measles virions had a mol.wt. of 79000. After labelling with periodate/NaB 3 H 4 , which would result in specific labelling of sialic acid residues, the 79000-mol.wt. glycoprotein was very weakly labelled. This suggested that there is no or a very low amount of sialic acid in the virions. Further analysis of the glycoprotein showed that galactose is the terminal carbohydrate unit in the oligosaccharide, and the molecular weight of the glycopeptide obtained after Pronase digestion is about 3000. The oligosaccharide is attached to the polypeptide through an alkali-stable bond, indicating a N-glycosidic asparagine linkage. (author)

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

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Wenhan Chen

Full Text Available 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/

IDENTIFICATION OF THE CORE RESIDUES OF THE EPITOPE OF A MONOCLONAL-ANTIBODY RAISED AGAINST GLYCOPROTEIN-D OF HERPES-SIMPLEX VIRUS TYPE-1 BY SCREENING OF A RANDOM PEPTIDE LIBRARY

NARCIS (Netherlands)

SCHELLEKENS, GA; LASONDER, E; FEIJLBRIEF, M; KOEDIJK, DGAM; DRIJFHOUT, JW; SCHEFFER, AJ; WELLINGWESTER, S; WELLING, GW

1994-01-01

Random peptide libraries (RPL) displayed on the surface of a filamentous bacteriophage can be used to identify peptide ligands that interact with target molecules. We have used a 15-amino acid residue RPL displayed on bacteriophage M13 to identify the core residues within the epitope of a monoclonal

The use of HPLC-MS in T-cell epitope identification.

Science.gov (United States)

Lemmel, Claudia; Stevanović, Stefan

2003-03-01

The hunt for T-cell epitopes is going on because hopes are set on such peptide sequences for diagnosis and vaccine development in the fight against infectious and tumor diseases. In addition to a variety of other techniques used in T-cell epitope identification, mass spectrometers coupled to microcapillary liquid chromatography have now become an important and sensitive tool in separation, detection, and sequence analysis of highly complex natural major histocompatibility complex (MHC) ligand mixtures. In this article, we review the basics of mass spectrometric techniques and their on-line coupling to microcapillary liquid chromatography (microcap-LC). Furthermore, we introduce current strategies for the identification of new T-cell epitopes using microcapillary liquid chromatography-mass spectrometry (microcap-LC-MS).

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

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Piontkivska Helen

2009-07-01

Full Text Available Abstract 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

Designing and overproducing a tandem epitope of gp350/220 that shows a potential to become an EBV vaccine

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Widodo

2018-03-01

Full Text Available Background: Epstein-Barr virus (EBV can cause cancer in people from around the world. There is no EBV vaccine available for use on a global scale. However, emerging evidence suggests that the epitope on the gp350/220 capsid protein may be developed into an EBV vaccine. Nevertheless, the production of small, single epitope is challenging of stability issues and possible alteration of peptide structure. In this study, a tandem epitope was developed consisting of three single epitopes, aimed to improve stability, antigenicity and preserve epitope structure. Materials and methods: A tandem epitope was designed using bioinformatics based on the epitope structure of the gp350/220 protein. The tandem epitope structure was analyzed using a protein folding method with Abalone software, which was further refined via YASARA force field and molecular repairing using a FoldX method. Immunogenicity was examined with Epitopia software, whereas allergen properties were tested using AlgPred. The pattern of the tandem epitope binding with anti-gp350/220 antibodies was performed using Z-dock and snugDock. The tandem epitope was then overproduced in E. coli strain BL21 as a host cell. Result: Our model demonstrated a successfully designed and overproduced tandem epitope. The tandem epitope demonstrated a similar structure compared with the epitope of whole protein gp350/220. Our epitope also demonstrated non-allergen and antigenicity properties, and possessed antibody binding patterns consistent with whole protein gp350/220. Conclusion and recommendation: These data suggest a novel tandem epitope composed of three similar epitopes demonstrates antigenicity, structure, and binding properties consistent with whole protein gp350/220. We also demonstrate successful production of the tandem epitope using E. coli strain BL21 as a host. Future in vivo experimental animal research is necessary to test the ability of this tandem epitope to stimulate antibody production

Chimeric Lyssavirus Glycoproteins with Increased Immunological Potential

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Jallet, Corinne; Jacob, Yves; Bahloul, Chokri; Drings, Astrid; Desmezieres, Emmanuel; Tordo, Noël; Perrin, Pierre

1999-01-01

The rabies virus glycoprotein molecule (G) can be divided into two parts separated by a flexible hinge: the NH2 half (site II part) containing antigenic site II up to the linear region (amino acids [aa] 253 to 275 encompassing epitope VI [aa 264]) and the COOH half (site III part) containing antigenic site III and the transmembrane and cytoplasmic domains. The structural and immunological roles of each part were investigated by cell transfection and mouse DNA-based immunization with homogeneous and chimeric G genes formed by fusion of the site II part of one genotype (GT) with the site III part of the same or another GT. Various site II-site III combinations between G genes of PV (Pasteur virus strain) rabies (GT1), Mokola (GT3), and EBL1 (European bat lyssavirus 1 [GT5]) viruses were tested. Plasmids pGPV-PV, pGMok-Mok, pGMok-PV, and pGEBL1-PV induced transient expression of correctly transported and folded antigens in neuroblastoma cells and virus-neutralizing antibodies against parental viruses in mice, whereas, pG-PVIII (site III part only) and pGPV-Mok did not. The site III part of PV (GT1) was a strong inducer of T helper cells and was very effective at presenting the site II part of various GTs. Both parts are required for correct folding and transport of chimeric G proteins which have a strong potential value for immunological studies and development of multivalent vaccines. Chimeric plasmid pGEBL1-PV broadens the spectrum of protection against European lyssavirus genotypes (GT1, GT5, and GT6). PMID:9847325

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

Science.gov (United States)

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.

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

DEFF Research Database (Denmark)

Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo

2017-01-01

-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...

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

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

Science.gov (United States)

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.

Reliable B cell epitope predictions: impacts of method development and improved benchmarking

DEFF Research Database (Denmark)

Kringelum, Jens Vindahl; Lundegaard, Claus; Lund, Ole

2012-01-01

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...... 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 predictive performances suggesting these tools to be a powerful asset in rational epitope discovery. The updated version...

Docking of B-cell epitope antigen to specific hepatitis B antibody

Indian Academy of Sciences (India)

The interaction of pres1 region of hepatitis B virus B-cell epitope antigen with specific hepatitis B neutralizing monoclonal antibody was examined by docking study. We modelled the 3D complex structure of B-cell epitope antigen residues CTTPAQGNSMFPSCCCTKPTDGNCY by homology modelling and docked it with the ...

Positive-unlabeled learning for the prediction of conformational B-cell epitopes

Science.gov (United States)

2015-01-01

Background The incomplete ground truth of training data of B-cell epitopes is a demanding issue in computational epitope prediction. The challenge is that only a small fraction of the surface residues of an antigen are confirmed as antigenic residues (positive training data); the remaining residues are unlabeled. As some of these uncertain residues can possibly be grouped to form novel but currently unknown epitopes, it is misguided to unanimously classify all the unlabeled residues as negative training data following the traditional supervised learning scheme. Results We propose a positive-unlabeled learning algorithm to address this problem. The key idea is to distinguish between epitope-likely residues and reliable negative residues in unlabeled data. The method has two steps: (1) identify reliable negative residues using a weighted SVM with a high recall; and (2) construct a classification model on the positive residues and the reliable negative residues. Complex-based 10-fold cross-validation was conducted to show that this method outperforms those commonly used predictors DiscoTope 2.0, ElliPro and SEPPA 2.0 in every aspect. We conducted four case studies, in which the approach was tested on antigens of West Nile virus, dihydrofolate reductase, beta-lactamase, and two Ebola antigens whose epitopes are currently unknown. All the results were assessed on a newly-established data set of antigen structures not bound by antibodies, instead of on antibody-bound antigen structures. These bound structures may contain unfair binding information such as bound-state B-factors and protrusion index which could exaggerate the epitope prediction performance. Source codes are available on request. PMID:26681157

Comparative characteristic of the methods of protein antigens epitope mapping

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O. Yu. Galkin

2014-08-01

Full Text Available Comparative analysis of experimental methods of epitope mapping of protein antigens has been carried out. The vast majority of known techniques are involved in immunochemical study of the interaction of protein molecules or peptides with antibodies of corresponding specifici­ty. The most effective and widely applicable metho­dological techniques are those that use synthetic and genetically engineered peptides. Over the past 30 years, these groups of methods have travelled a notable evolutionary path up to the maximum automation and the detection of antigenic determinants of various types (linear and conformational epitopes, and mimotopes. Most of epitope searching algorithms were integrated into a computer program, which greatly facilitates the analysis of experimental data and makes it possible to create spatial models. It is possible to use comparative epitope mapping for solving the applied problems; this less time-consuming method is based on the analysis of competition between different antibodies interactions with the same antigen. The physical method of antigenic structure study is X-ray analysis of antigen-antibody complexes, which may be applied only to crystallizing­ proteins, and nuclear magnetic resonance.

Glutamic acid decarboxylase-derived epitopes with specific domains expand CD4(+CD25(+ regulatory T cells.

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Guojiang Chen

Full Text Available BACKGROUND: CD4(+CD25(+ regulatory T cell (Treg-based immunotherapy is considered a promising regimen for controlling the progression of autoimmune diabetes. In this study, we tested the hypothesis that the therapeutic effects of Tregs in response to the antigenic epitope stimulation depend on the structural properties of the epitopes used. METHODOLOGY/PRINCIPAL FINDINGS: Splenic lymphocytes from nonobese diabetic (NOD mice were stimulated with different glutamic acid decarboxylase (GAD-derived epitopes for 7-10 days and the frequency and function of Tregs was analyzed. We found that, although all expanded Tregs showed suppressive functions in vitro, only p524 (GAD524-538-expanded CD4(+CD25(+ T cells inhibited diabetes development in the co-transfer models, while p509 (GAD509-528- or p530 (GAD530-543-expanded CD4(+CD25(+ T cells had no such effects. Using computer-guided molecular modeling and docking methods, the differences in structural characteristics of these epitopes and the interaction mode (including binding energy and identified domains in the epitopes between the above-mentioned epitopes and MHC class II I-A(g7 were analyzed. The theoretical results showed that the epitope p524, which induced protective Tregs, possessed negative surface-electrostatic potential and bound two chains of MHC class II I-A(g7, while the epitopes p509 and p530 which had no such ability exhibited positive surface-electrostatic potential and bound one chain of I-A(g7. Furthermore, p524 bound to I-A(g7 more stably than p509 and p530. Of importance, we hypothesized and subsequently confirmed experimentally that the epitope (GAD570-585, p570, which displayed similar characteristics to p524, was a protective epitope by showing that p570-expanded CD4(+CD25(+ T cells suppressed the onset of diabetes in NOD mice. CONCLUSIONS/SIGNIFICANCE: These data suggest that molecular modeling-based structural analysis of epitopes may be an instrumental tool for prediction of

A novel monoclonal antibody to a defined peptide epitope in MUC16

DEFF Research Database (Denmark)

Marcos-Silva, Lara; Ricardo, Sara; Chen, Kowa

2015-01-01

with the tandem-repeat region, their epitopes appear to be conformational dependent and not definable by a short peptide. Aberrant glycoforms of MUC16 may constitute promising targets for diagnostic and immunotherapeutic intervention, and it is important to develop well-defined immunogens for induction of potent...... immunodominant linear peptide epitopes within the tandem repeat. We developed one monoclonal antibody, 5E11, reactive with a minimum epitope with the sequence FNTTER. This sequence contains potential N- and O-glycosylation sites and, interestingly, glycosylation blocked binding of 5E11. In immunochemistry...

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

Science.gov (United States)

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.

The hydroxyapatite-binding regions of a rat salivary glycoprotein.

Science.gov (United States)

Embery, G; Green, D R

1989-09-01

The regions of a salivary sulphated glycoprotein which are involved in its attachment to hydroxyapatite (Biogel HTP) have been characterised. The sulphated glycoprotein, a 35S-labelled preparation from mixed palatal and buccal minor gland secretions of the rat was bound onto hydroxyapatite and the resultant glycoprotein-hydroxyapatite complex was sequentially digested with pronase E and alpha-L-fucosidase, a treatment which released 86.8% +/- 1.7% of the radioactivity of the initially bound glycoprotein. The fragments which remained attached to the hydroxyapatite after enzymic digestion were fractionated on Sephadex G-25 and analysed for carbohydrate and amino acid components. A range of amino acids were detected which could reflect both glycosylated and non-glycosylated-binding regions. Sialic acid, although considered to be involved in the attachment process was not detected in any of the fragments remaining after enzymic digestion, a finding which provides indirect evidence that the enzymically liberated products do not subsequently re-attach to the hydroxyapatite surface. The notable feature of the fractions with average Mr estimated at 1000 or less is the high proportion of N-acetylhexosamine and N-acetylgalactosamine. It is apparent that the hexosamine residues, which normally bear the ester sulphate moieties of sulphated glycoproteins, play an important role in the attachment of sulphated glycoproteins to hydroxyapatite.

Isolation of allergenically active glycoprotein from Prosopis juliflora pollen.

Science.gov (United States)

Thakur, I S

1989-03-01

An allergenically active glycoprotein was homogeneously isolated from the aqueous extract of Prosopis juliflora pollen by ConA-Sepharose affinity chromatography. The molecular weight of this glycoprotein was 20,000 dalton, determined by gel filtration and SDS-PAGE. This fraction showed a total carbohydrate concentration of 25%. The purified glycoprotein revealed immunochemically most antigenic or allergenic and demonstrated homogeneous after reaction with P. juliflora pollen antiserum, characterized by gel diffusion, Immunoelectrophoresis and Radioallergosorbent test.

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

Science.gov (United States)

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.

A Novel Multi-Epitope Vaccine For Cross Protection Against Hepatitis C Virus (HCV: An Immunoinformatics Approach

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Mokhtar Nosrati

2017-02-01

Full Text Available Background: Hepatitis C virus (HCV causes acute and chronic human hepatitis infections. Due to the high genetic diversity and high rates of mutations in the genetic material so far there is no approved vaccine against HCV. Materials and Methods: The aim of this study was to determination B and T cell conserved epitopes of E1 and E2 proteins from HCV and construction of a chimeric peptide as a novel epitope based vaccine for cross-protection against the virus. For this, one B and T-cell epitope from both E1 and E2 which was predicted by EPMLR and Propred-1 server and had the highest score and antigenicity in VaxiJen 2.0 and PAP servers were selected for construction of chimeric protein as a multi-epitope vaccine. Results: The results of this study showed that the chimeric peptide had high antigenicity score and stability.Results also showed that most epitopes of E1 were located in two spectra consist of (45-65,88-107 and 148-182 while the results about B-cell epitopes of E2 showed that this protein had much less epitope than E1. The most epitope predicted for E2 were located in (12-24 and 35-54 spectra Conclusion:  In conclusion, epitope based vaccine which was designed by immunoinformatics methods could be considered as a novel and effective vaccine for cross-protection against HCV infection.

An Analysis of Trafficking Receptors Shows that CD44 and P-Selectin Glycoprotein Ligand-1 Collectively Control the Migration of Activated Human T-Cells

KAUST Repository

Ali, Amal J.

2017-05-03

Selectins guide the traffic of activated T-cells through the blood stream by mediating their tethering and rolling onto inflamed endothelium, in this way acting as beacons to help navigate them to sites of inflammation. Here, we present a comprehensive analysis of E-selectin ligands expressed on activated human T-cells. We identified several novel glycoproteins that function as E-selectin ligands. Specifically, we compared the role of P-selectin glycoprotein ligand-1 (PSGL-1) and CD43, known E-selectin ligands, to CD44, a ligand that has not previously been characterized as an E-selectin ligand on activated human T-cells. We showed that CD44 acts as a functional E-selectin ligand when expressed on both CD4+ and CD8+ T-cells. Moreover, the CD44 protein carries a binding epitope identifying it as hematopoietic cell E- and/or L-selectin ligand (HCELL). Furthermore, by knocking down these ligands individually or together in primary activated human T-cells, we demonstrated that CD44/HCELL, and not CD43, cooperates with PSGL-1 as a major E-selectin ligand. Additionally, we demonstrated the relevance of our findings to chronic autoimmune disease, by showing that CD44/HCELL and PSGL-1, but not CD43, from T-cells isolated from psoriasis patients, bind E-selectin.

Albumin is synthesized in epididymis and aggregates in a high molecular mass glycoprotein complex involved in sperm-egg fertilization.

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Kélen Fabíola Arroteia

Full Text Available The epididymis has an important role in the maturation of sperm for fertilization, but little is known about the epididymal molecules involved in sperm modifications during this process. We have previously described the expression pattern for an antigen in epididymal epithelial cells that reacts with the monoclonal antibody (mAb TRA 54. Immunohistochemical and immunoblotting analyses suggest that the epitope of the epididymal antigen probably involves a sugar moiety that is released into the epididymal lumen in an androgen-dependent manner and subsequently binds to luminal sperm. Using column chromatography, SDS-PAGE with in situ digestion and mass spectrometry, we have identified the protein recognized by mAb TRA 54 in mouse epididymal epithelial cells. The ∼65 kDa protein is part of a high molecular mass complex (∼260 kDa that is also present in the sperm acrosomal vesicle and is completely released after the acrosomal reaction. The amino acid sequence of the protein corresponded to that of albumin. Immunoprecipitates with anti-albumin antibody contained the antigen recognized by mAb TRA 54, indicating that the epididymal molecule recognized by mAb TRA 54 is albumin. RT-PCR detected albumin mRNA in the epididymis and fertilization assays in vitro showed that the glycoprotein complex containing albumin was involved in the ability of sperm to recognize and penetrate the egg zona pellucida. Together, these results indicate that epididymal-derived albumin participates in the formation of a high molecular mass glycoprotein complex that has an important role in egg fertilization.

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

Science.gov (United States)

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.

Analysis of glycoprotein-derived oligosaccharides in glycoproteins detected on two-dimensional gel by capillary electrophoresis using on-line concentration method.

Science.gov (United States)

Kamoda, Satoru; Nakanishi, Yasuharu; Kinoshita, Mitsuhiro; Ishikawa, Rika; Kakehi, Kazuaki

2006-02-17

Capillary electrophoresis (CE) is an effective tool to analyze carbohydrate mixture derived from glycoproteins with high resolution. However, CE has a disadvantage that a few nanoliters of a sample solution are injected to a narrow capillary. Therefore, we have to prepare a sample solution of high concentration for CE analysis. In the present study, we applied head column field-amplified sample stacking method to the analysis of N-linked oligosaccharides derived from glycoprotein separated by two-dimensional gel electrophoresis. Model studies demonstrated that we achieved 60-360 times concentration effect on the analysis of carbohydrate chains labeled with 3-aminobenzoic acid (3-AA). The method was applied to the analysis of N-linked oligosaccharides from glycoproteins separated and detected on PAGE gel. Heterogeneity of alpha1-acid glycoprotein (AGP), i.e. glycoforms, was examined by 2D-PAGE and N-linked oligosaccharides were released by in-gel digestion with PNGase F. The released oligosaccharides were derivatized with 3-AA and analyzed by CE. The results showed that glycoforms having lower pI values contained a larger amount of tetra- and tri-antennary oligosaccharides. In contrast, glycoforms having higher pI values contained bi-antennary oligosaccharides abundantly. The result clearly indicated that the spot of a glycoprotein glycoform detected by Coomassie brilliant blue staining on 2D-PAGE gel is sufficient for quantitative profiling of oligosaccharides.

MHC class I epitope binding prediction trained on small data sets

DEFF Research Database (Denmark)

Lundegaard, Claus; Nielsen, Morten; Lamberth, K.

2004-01-01

The identification of potential T-cell epitopes is important for development of new human or vetenary vaccines, both considering single protein/subunit vaccines, and for epitope/peptide vaccines as such. The highly diverse MHC class I alleles bind very different peptides, and accurate binding pre...... in situations where only very limited data are available for training....

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

Science.gov (United States)

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

Differential Recognition of Mycobacterium tuberculosis-Specific Epitopes as a Function of Tuberculosis Disease History.

Science.gov (United States)

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.

Humanizing recombinant glycoproteins from Chinese hamster ovary cells

DEFF Research Database (Denmark)

Hansen, Anders Holmgaard; Amann, Thomas; Kol, Stefan

With new tools for gene-editing like zinc-fingers, TALENS and CRISPR, it is now feasible totailor-make the N-Glycoforms for therapeutic glycoproteins that have previously been almost impossible. We here demonstrate a case of humanizing a recombinant human glycoprotein that in Wild type (WT) Chinese...

Determination of P-Glycoprotein Expression by Flow Cytometry in Hematological Malignancies

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Berkay Saraymen

2016-03-01

Full Text Available Objective: Determination the expression of P-glycoprotein is especially problematic for normal tissues because immuno­logical methods are limited in terms of sensitivity. We aimed to determine the expression of P-glycoprotein and CD34 by flow cytometry, and to evaluate the level of expression of P-glycoprotein and CD34 with unresponsive to treatment in pa­tients diagnosed with hematologic malignancy. Methods: Our study included fifty patients diagnosed with acute myeloblastic leukemia and acute lymphoblastic leuke­mia, and twenty healthy controls who were admitted to Erci­yes University Hematology-Oncology Hospital. The suspend­ed cells from bone marrow samples of patients and the pe­ripheral blood samples of healthy people were marked with P-glycoprotein phycoerythrin and CD34 FITC or PerCP Cy 5.5; and then surface expression was measured by means of flow cytometry. Results: In 6 of 30 acute myeloblastic leukemia patients P-glycoprotein and CD34 expression, in 6 of 20 acute lympho­blastic leukemia patients P-glycoprotein, in 5 of them CD34 expression were determined. A significant relation between P-glycoprotein and CD34 expressions in acute myeloblas­tic leukemia and acute lymphoblastic leukemia bone marrow samples was reported. Conclusion: Our data indicate that flow cytometry is more reliable, precise and faster than molecular methods for mea­suring P-glycoprotein expression and suggests the pos­sibility of a significant relationship between P-glycoprotein and CD34 expressions in acute myeloblastic leukemia and acute lymphoblastic leukemia bone marrow samples. The blast cells expressing CD34 on their surface along with P-glycoprotein simultaneously show that multi drug resistance 1 gene is mostly active in immature cells.

Alfa-2-glicoproteína rica en leucina urinaria en pacientes con apendicitis aguda (Urinary leucine-rich alpha-2-glycoprotein in patients with acute appendicitis

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Heberto Machado-Montero

2015-04-01

Full Text Available The objective of research was to determine diagnostic efficacy of urinary concentrations of leucine-rich alpha-2-glycoprotein (A2GRL in patients with acute appendicitis. There were included patients with high suspicion of acute appendicitis (group A; cases, n = 30. In group B (controls, n = 30 were included patients with non-surgical abdominal pain who attended to Hospital Central “Dr. Urquinaona”, Maracaibo, Zulia. General characteristics, urinary concentrations of A2GRL and diagnostic efficacy of assay were evaluated. Mean age of patients in group A was 36.3 +/- 8.8 years-old and in group B was 35.8 +/- 9.6 years-old (p = ns. There were not found significant differences in sex distribution, weight and height between groups (p = ns. Urinary concentrations of A2GRL were significantly higher in group A (1543.8 +/- 762.7 ng/mL compared with mean value in patients of group B (774.1 +/- 356.1 ng/mL; p < 0.0001. A2GRL presented a value below curve 0.81. A cut-off value of 1000 ng/mL, showed sensivity 73.3%, specificity 70.9%, positive predictive value 72.4% and negative predictive value 72.4%. It is concluded that urinary concentrations of A2GRL have a high diagnostic efficacy in patients with acute appendicitis

Glycoproteins of mouse vaginal epithelium: differential expression related to estrous cyclicity

DEFF Research Database (Denmark)

Horvat, B; Multhaupt, H A; Damjanov, I

1993-01-01

We used lectin overlay blotting and SDS-PAGE to analyze the estrous cycle-specific expression of mouse vaginal epithelial glycoproteins. Seven lectins chosen for their differential carbohydrate-binding specificity revealed 15 glycoproteins that showed cycle-related expression. Each lectin had...... in proestrus, coincident with the transformation of two superficial layers of vaginal squamous epithelium into mucinous cuboidal cells. Electron microscopic lectin histochemistry revealed the glycoproteins in the mucinous granules of surface cuboidal cells and in the lumen of the vagina. Our results illustrate...... the complexity of glycoconjugate synthesis in mouse vagina and reveal the distinct cycle-specific patterns of individual glycoprotein expression. These cyclic glycoproteins could serve as vaginal biochemical markers for the specific phases of the estrous cycle....

Conservation and diversity of influenza A H1N1 HLA-restricted T cell epitope candidates for epitope-based vaccines.

Directory of Open Access Journals (Sweden)

Paul Thiamjoo Tan

2010-01-01

Full Text Available The immune-related evolution of influenza viruses is exceedingly complex and current vaccines against influenza must be reformulated for each influenza season because of the high degree of antigenic drift among circulating influenza strains. Delay in vaccine production is a serious problem in responding to a pandemic situation, such as that of the current H1N1 strain. Immune escape is generally attributed to reduced antibody recognition of the viral hemagglutinin and neuraminidase proteins whose rate of mutation is much greater than that of the internal non-structural proteins. As a possible alternative, vaccines directed at T cell epitope domains of internal influenza proteins, that are less susceptible to antigenic variation, have been investigated.HLA transgenic mouse strains expressing HLA class I A*0201, A*2402, and B*0702, and class II DRB1*1501, DRB1*0301 and DRB1*0401 were immunized with 196 influenza H1N1 peptides that contained residues of highly conserved proteome sequences of the human H1N1, H3N2, H1N2, H5N1, and avian influenza A strains. Fifty-four (54 peptides that elicited 63 HLA-restricted peptide-specific T cell epitope responses were identified by IFN-gamma ELISpot assay. The 54 peptides were compared to the 2007-2009 human H1N1 sequences for selection of sequences in the design of a new candidate H1N1 vaccine, specifically targeted to highly-conserved HLA-restricted T cell epitopes.Seventeen (17 T cell epitopes in PB1, PB2, and M1 were selected as vaccine targets based on sequence conservation over the past 30 years, high functional avidity, non-identity to human peptides, clustered localization, and promiscuity to multiple HLA alleles. These candidate vaccine antigen sequences may be applicable to any avian or human influenza A virus.

Automated identification of complementarity determining regions (CDRs) reveals peculiar characteristics of CDRs and B cell epitopes.

Science.gov (United States)

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.

Herpesvirus glycoproteins undergo multiple antigenic changes before membrane fusion.

Directory of Open Access Journals (Sweden)

Daniel L Glauser

Full Text Available Herpesvirus entry is a complicated process involving multiple virion glycoproteins and culminating in membrane fusion. Glycoprotein conformation changes are likely to play key roles. Studies of recombinant glycoproteins have revealed some structural features of the virion fusion machinery. However, how the virion glycoproteins change during infection remains unclear. Here using conformation-specific monoclonal antibodies we show in situ that each component of the Murid Herpesvirus-4 (MuHV-4 entry machinery--gB, gH/gL and gp150--changes in antigenicity before tegument protein release begins. Further changes then occurred upon actual membrane fusion. Thus virions revealed their final fusogenic form only in late endosomes. The substantial antigenic differences between this form and that of extracellular virions suggested that antibodies have only a limited opportunity to block virion membrane fusion.

Soluble cysteine-rich tick saliva proteins Salp15 and Iric-1 from E. coli.

Science.gov (United States)

Kolb, Philipp; Vorreiter, Jolanta; Habicht, Jüri; Bentrop, Detlef; Wallich, Reinhard; Nassal, Michael

2015-01-01

Ticks transmit numerous pathogens, including borreliae, which cause Lyme disease. Tick saliva contains a complex mix of anti-host defense factors, including the immunosuppressive cysteine-rich secretory glycoprotein Salp15 from Ixodes scapularis ticks and orthologs like Iric-1 from Ixodes ricinus. All tick-borne microbes benefit from the immunosuppression at the tick bite site; in addition, borreliae exploit the binding of Salp15 to their outer surface protein C (OspC) for enhanced transmission. Hence, Salp15 proteins are attractive targets for anti-tick vaccines that also target borreliae. However, recombinant Salp proteins are not accessible in sufficient quantity for either vaccine manufacturing or for structural characterization. As an alternative to low-yield eukaryotic systems, we investigated cytoplasmic expression in Escherichia coli, even though this would not result in glycosylation. His-tagged Salp15 was efficiently expressed but insoluble. Among the various solubility-enhancing protein tags tested, DsbA was superior, yielding milligram amounts of soluble, monomeric Salp15 and Iric-1 fusions. Easily accessible mutants enabled epitope mapping of two monoclonal antibodies that, importantly, cross-react with glycosylated Salp15, and revealed interaction sites with OspC. Free Salp15 and Iric-1 from protease-cleavable fusions, despite limited solubility, allowed the recording of (1)H-(15)N 2D NMR spectra, suggesting partial folding of the wild-type proteins but not of Cys-free variants. Fusion to the NMR-compatible GB1 domain sufficiently enhanced solubility to reveal first secondary structure elements in (13)C/(15)N double-labeled Iric-1. Together, E. coli expression of appropriately fused Salp15 proteins may be highly valuable for the molecular characterization of the function and eventually the 3D structure of these medically relevant tick proteins.

Three-Dimensionally Functionalized Reverse Phase Glycoprotein Array for Cancer Biomarker Discovery and Validation.

Science.gov (United States)

Pan, Li; Aguilar, Hillary Andaluz; Wang, Linna; Iliuk, Anton; Tao, W Andy

2016-11-30

Glycoproteins have vast structural diversity that plays an important role in many biological processes and have great potential as disease biomarkers. Here, we report a novel functionalized reverse phase protein array (RPPA), termed polymer-based reverse phase glycoprotein array (polyGPA), to capture and profile glycoproteomes specifically, and validate glycoproteins. Nitrocellulose membrane functionalized with globular hydroxyaminodendrimers was used to covalently capture preoxidized glycans on glycoproteins from complex protein samples such as biofluids. The captured glycoproteins were subsequently detected using the same validated antibodies as in RPPA. We demonstrated the outstanding specificity, sensitivity, and quantitative capabilities of polyGPA by capturing and detecting purified as well as endogenous α-1-acid glycoprotein (AGP) in human plasma. We further applied quantitative N-glycoproteomics and the strategy to validate a panel of glycoproteins identified as potential biomarkers for bladder cancer by analyzing urine glycoproteins from bladder cancer patients or matched healthy individuals.

Glycoprotein Enrichment Analytical Techniques: Advantages and Disadvantages.

Science.gov (United States)

Zhu, R; Zacharias, L; Wooding, K M; Peng, W; Mechref, Y

2017-01-01

Protein glycosylation is one of the most important posttranslational modifications. Numerous biological functions are related to protein glycosylation. However, analytical challenges remain in the glycoprotein analysis. To overcome the challenges associated with glycoprotein analysis, many analytical techniques were developed in recent years. Enrichment methods were used to improve the sensitivity of detection, while HPLC and mass spectrometry methods were developed to facilitate the separation of glycopeptides/proteins and enhance detection, respectively. Fragmentation techniques applied in modern mass spectrometers allow the structural interpretation of glycopeptides/proteins, while automated software tools started replacing manual processing to improve the reliability and throughput of the analysis. In this chapter, the current methodologies of glycoprotein analysis were discussed. Multiple analytical techniques are compared, and advantages and disadvantages of each technique are highlighted. © 2017 Elsevier Inc. All rights reserved.

Characterization of a linear epitope on Chlamydia trachomatis serovar L2 DnaK-like protein

DEFF Research Database (Denmark)

Ozkokmen, D; Birkelund, Svend; Christiansen, Gunna

1994-01-01

A cytoplasmic 75-kDa immunogen from Chlamydia trachomatis serovar L2 has previously been characterized as being similar to the Escherichia coli heat shock protein DnaK. We have localized a linear epitope for one monoclonal antibody specific for C. trachomatis DnaK. By use of a recombinant DNA...... technique, the epitope was limited to 14 amino acids. With synthetic peptides, the epitope was further limited to eight amino acids. Six of these amino acids are conserved in bovine HSP70, which has a known three-dimensional structure. The amino acid sequence homologous to the epitope is located in a linear...

Chimeric peptide constructs comprising linear B-cell epitopes: application to the serodiagnosis of infectious diseases.

Science.gov (United States)

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.

Depigmented Allergoids Reveal New Epitopes with Capacity to Induce IgG Blocking Antibodies

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M. Angeles López-Matas

2013-01-01

Full Text Available 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.

Developing baculovirus-insect cell expression systems for humanized recombinant glycoprotein production

International Nuclear Information System (INIS)

Jarvis, Donald L.

2003-01-01

The baculovirus-insect cell expression system is widely used to produce recombinant glycoproteins for many different biomedical applications. However, due to the fundamental nature of insect glycoprotein processing pathways, this system is typically unable to produce recombinant mammalian glycoproteins with authentic oligosaccharide side chains. This minireview summarizes our current understanding of insect protein glycosylation pathways and our recent efforts to address this problem. These efforts have yielded new insect cell lines and baculoviral vectors that can produce recombinant glycoproteins with humanized oligosaccharide side chains

Identification of a novel linear B-cell epitope using a monoclonal antibody against the carboxy terminus of the canine distemper virus nucleoprotein and sequence analysis of the identified epitope in different CDV isolates.

Science.gov (United States)

Yi, Li; Cao, Zhigang; Tong, Mingwei; Cheng, Yuening; Yang, Yong; Li, Shuang; Wang, Jianke; Lin, Peng; Sun, Yaru; Zhang, Miao; Cheng, Shipeng

2017-09-29

The Nucleoprotein (NP) is the most abundant and highly immunogenic protein in canine distemper virus (CDV), playing an important role in CDV viral replication and assembly. In this study, a specific monoclonal antibody, named C8, was produced against the NP protein C terminal (amino acids 401-523). A linear N protein epitope was identified by subjecting a series of partially overlapping synthesized peptides to enzyme-linked immunosorbent assay (ELISA) analysis.The results indicated that 444 GDKYPIHFNDER 455 was the minimal linear epitope that could be recognized by mAb C8. Sequence alignments demonstrated that this linear epitope is less conserved among three CDV genotypes. We next analyzed the level of conservation of the defined epitope in19 Chinese CDV clinical isolates, and it has one site variation in amino acid among these CDV isolations. 2 isolates have the amino acid mutations F451L, while one has P448Ssubstitution.Phylogenetic analysis showed the two isolates with F451Lsubstitution had a closer relationship in a virulent strain ZJ-7, so the epitope may be a significant tag associated with virus virulence. This collection of mAb along with defined linear epitope may provide useful reagents for investigations of NP protein function and the development of CDV specific diagnostics.

Epitope-Specific Tolerance Modes Differentially Specify Susceptibility to Proteolipid Protein-Induced Experimental Autoimmune Encephalomyelitis

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Lei Wang

2017-11-01

Full Text Available Immunization with myelin components can elicit experimental autoimmune encephalomyelitis (EAE. EAE susceptibility varies between mouse strains, depending on the antigen employed. BL/6 mice are largely resistant to EAE induction with proteolipid protein (PLP, probably a reflection of antigen-specific tolerance. However, the extent and mechanism(s of tolerance to PLP remain unclear. Here, we identified three PLP epitopes in PLP-deficient BL/6 mice. PLP-sufficient mice did not respond against two of these, whereas tolerance was “leaky” for an epitope with weak predicted MHCII binding, and only this epitope was encephalitogenic. In TCR transgenic mice, the “EAE-susceptibility-associated” epitope was “ignored” by specific CD4 T cells, whereas the “resistance-associated” epitope induced clonal deletion and Treg induction in the thymus. Central tolerance was autoimmune regulator dependent and required expression and presentation of PLP by thymic epithelial cells (TECs. TEC-specific ablation of PLP revealed that peripheral tolerance, mediated by dendritic cells through recessive tolerance mechanisms (deletion and anergy, could largely compensate for a lack of central tolerance. However, adoptive EAE was exacerbated in mice lacking PLP in TECs, pointing toward a non-redundant role of the thymus in dominant tolerance to PLP. Our findings reveal multiple layers of tolerance to a central nervous system autoantigen that vary among epitopes and thereby specify disease susceptibility. Understanding how different modalities of tolerance apply to distinct T cell epitopes of a target in autoimmunity has implications for antigen-specific strategies to therapeutically interfere with unwanted immune reactions against self.

CD4+ T-cell epitope prediction using antigen processing constraints.

Science.gov (United States)

Mettu, Ramgopal R; Charles, Tysheena; Landry, Samuel J

2016-05-01

T-cell CD4+ epitopes are important targets of immunity against infectious diseases and cancer. State-of-the-art methods for MHC class II epitope prediction rely on supervised learning methods in which an implicit or explicit model of sequence specificity is constructed using a training set of peptides with experimentally tested MHC class II binding affinity. In this paper we present a novel method for CD4+ T-cell eptitope prediction based on modeling antigen-processing constraints. Previous work indicates that dominant CD4+ T-cell epitopes tend to occur adjacent to sites of initial proteolytic cleavage. Given an antigen with known three-dimensional structure, our algorithm first aggregates four types of conformational stability data in order to construct a profile of stability that allows us to identify regions of the protein that are most accessible to proteolysis. Using this profile, we then construct a profile of epitope likelihood based on the pattern of transitions from unstable to stable regions. We validate our method using 35 datasets of experimentally measured CD4+ T cell responses of mice bearing I-Ab or HLA-DR4 alleles as well as of human subjects. Overall, our results show that antigen processing constraints provide a significant source of predictive power. For epitope prediction in single-allele systems, our approach can be combined with sequence-based methods, or used in instances where little or no training data is available. In multiple-allele systems, sequence-based methods can only be used if the allele distribution of a population is known. In contrast, our approach does not make use of MHC binding prediction, and is thus agnostic to MHC class II genotypes. Copyright © 2016 Elsevier B.V. All rights reserved.

Soluble cysteine-rich tick saliva proteins Salp15 and Iric-1 from E. coli

OpenAIRE

Kolb, Philipp; Vorreiter, Jolanta; Habicht, J?ri; Bentrop, Detlef; Wallich, Reinhard; Nassal, Michael

2014-01-01

Ticks transmit numerous pathogens, including borreliae, which cause Lyme disease. Tick saliva contains a complex mix of anti-host defense factors, including the immunosuppressive cysteine-rich secretory glycoprotein Salp15 from Ixodes scapularis ticks and orthologs like Iric-1 from Ixodes ricinus. All tick-borne microbes benefit from the immunosuppression at the tick bite site; in addition, borreliae exploit the binding of Salp15 to their outer surface protein C (OspC) for enhanced transmissi...

Proteomic analysis of coronary sinus serum reveals leucine-rich alpha2-glycoprotein as a novel biomarker of ventricular dysfunction and heart failure.

LENUS (Irish Health Repository)

Watson, Chris J

2012-02-01

BACKGROUND: Heart failure (HF) prevention strategies require biomarkers that identify disease manifestation. Increases in B-type natriuretic peptide (BNP) correlate with increased risk of cardiovascular events and HF development. We hypothesize that coronary sinus serum from a high BNP hypertensive population reflects an active pathological process and can be used for biomarker exploration. Our aim was to discover differentially expressed disease-associated proteins that identify patients with ventricular dysfunction and HF. METHODS AND RESULTS: Coronary sinus serum from 11 asymptomatic, hypertensive patients underwent quantitative differential protein expression analysis by 2-dimensional difference gel electrophoresis. Proteins were identified using mass spectrometry and then studied by enzyme-linked immunosorbent assay in sera from 40 asymptomatic, hypertensive patients and 105 patients across the spectrum of ventricular dysfunction (32 asymptomatic left ventricular diastolic dysfunction, 26 diastolic HF, and 47 systolic HF patients). Leucine-rich alpha2-glycoprotein (LRG) was consistently overexpressed in high BNP serum. LRG levels correlate significantly with BNP in hypertensive, asymptomatic left ventricular diastolic dysfunction, diastolic HF, and systolic HF patient groups (P<\\/=0.05). LRG levels were able to identify HF independent of BNP. LRG correlates with coronary sinus serum levels of tumor necrosis factor-alpha (P=0.009) and interleukin-6 (P=0.021). LRG is expressed in myocardial tissue and correlates with transforming growth factor-betaR1 (P<0.001) and alpha-smooth muscle actin (P=0.025) expression. CONCLUSIONS: LRG was identified as a serum biomarker that accurately identifies patients with HF. Multivariable modeling confirmed that LRG is a stronger identifier of HF than BNP and this is independent of age, sex, creatinine, ischemia, beta-blocker therapy, and BNP.

Isolation of monoclonal antibodies with predetermined conformational epitope specificity.

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Anton M Sholukh

Full Text Available Existing technologies allow isolating antigen-specific monoclonal antibodies (mAbs from B cells. We devised a direct approach to isolate mAbs with predetermined conformational epitope specificity, using epitope mimetics (mimotopes that reflect the three-dimensional structure of given antigen subdomains. We performed differential biopanning using bacteriophages encoding random peptide libraries and polyclonal antibodies (Abs that had been affinity-purified with either native or denatured antigen. This strategy yielded conformational mimotopes. We then generated mimotope-fluorescent protein fusions, which were used as baits to isolate single memory B cells from rhesus monkeys (RMs. To amplify RM immunoglobulin variable regions, we developed RM-specific PCR primers and generated chimeric simian-human mAbs with predicted epitope specificity. We established proof-of-concept of our strategy by isolating mAbs targeting the conformational V3 loop crown of HIV Env; the new mAbs cross-neutralized viruses of different clades. The novel technology allows isolating mAbs from RMs or other hosts given experimental immunogens or infectious agents.

Regulation of glycoprotein synthesis in yeast by mating pheromones

International Nuclear Information System (INIS)

Tanner, W.

1984-01-01

In Saccharomyces cerevisiae, glycosylated proteins amount to less than 2% of the cell protein. Two intensively studied examples of yeast glycoproteins are the external cell wall - associated invertase and the vacuolar carboxypeptidase Y. Recently, it was shown that the mating pheromone, alpha factor, specifically and strongly inhibits the synthesis of N-glycosylated proteins in haploid a cells, whereas O-glycosylated proteins are not affected. In this paper, the pathways of glycoprotein biosynthesis are summarized briefly, and evidence is presented that mating pheomones have a regulatory function in glycoprotein synthesis

Substantial gaps in knowledge of Bordetella pertussis antibody and T cell epitopes relevant for natural immunity and vaccine efficacy

Science.gov (United States)

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

Engineered CHO cells for production of diverse, homogeneous glycoproteins

DEFF Research Database (Denmark)

Yang, Zhang; Wang, Shengjun; Halim, Adnan

2015-01-01

Production of glycoprotein therapeutics in Chinese hamster ovary (CHO) cells is limited by the cells' generic capacity for N-glycosylation, and production of glycoproteins with desirable homogeneous glycoforms remains a challenge. We conducted a comprehensive knockout screen of glycosyltransferas...

The Purification of a Blood Group A Glycoprotein: An Affinity Chromatography Experiment.

Science.gov (United States)

Estelrich, J.; Pouplana, R.

1988-01-01

Describes a purification process through affinity chromatography necessary to obtain specific blood group glycoproteins from erythrocytic membranes. Discusses the preparation of erythrocytic membranes, extraction of glycoprotein from membranes, affinity chromatography purification, determination of glycoproteins, and results. (CW)

Definition of natural T cell antigens with mimicry epitopes obtained from dedicated synthetic peptide libraries.

Science.gov (United States)

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.

Developmental Localization and Methylesterification of Pectin Epitopes during Somatic Embryogenesis of Banana (Musa spp. AAA)

Science.gov (United States)

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

Solution structure of the twelfth cysteine-rich ligand-binding repeat in rat megalin

International Nuclear Information System (INIS)

Wolf, Christian A.; Dancea, Felician; Shi Meichen; Bade-Noskova, Veronika; Rueterjans, Heinz; Kerjaschki, Dontscho; Luecke, Christian

2007-01-01

Megalin, an approx. 600 kDa transmembrane glycoprotein that acts as multi-ligand transporter, is a member of the low density lipoprotein receptor gene family. Several cysteine-rich repeats, each consisting of about 40 residues, are responsible for the multispecific binding of ligands. The solution structure of the twelfth cysteine-rich ligand-binding repeat with class A motif found in megalin features two short β-strands and two helical turns, yielding the typical fold with a I-III, II-V and IV-VI disulfide bridge connectivity pattern and a calcium coordination site at the C-terminal end. The resulting differences in electrostatic surface potential compared to other ligand-binding modules of this gene family, however, may be responsible for the functional divergence

Identification and characterization of two linear epitope motifs in hepatitis E virus ORF2 protein.

Directory of Open Access Journals (Sweden)

Heng Wang

Full Text Available Hepatitis E virus (HEV is responsible for hepatitis E, which represents a global public health problem. HEV genotypes 3 and 4 are reported to be zoonotic, and animals are monitored for HEV infection in the interests of public hygiene and food safety. The development of novel diagnostic methods and vaccines for HEV in humans is thus important topics of research. Opening reading frame (ORF 2 of HEV includes both linear and conformational epitopes and is regarded as the primary candidate for vaccines and diagnostic tests. We investigated the precise location of the HEV epitopes in the ORF2 protein. We prepared four monoclonal antibodies (mAbs against genotype 4 ORF2 protein and identified two linear epitopes, G438IVIPHD444 and Y457DNQH461, corresponding to two of these mAbs using phage display biopanning technology. Both these epitopes were speculated to be universal to genotypes 1, 2, 3, 4, and avian HEVs. We also used two 12-mer fragments of ORF2 protein including these two epitopes to develop a peptide-based enzyme-linked immunosorbent assay (ELISA to detect HEV in serum. This assay demonstrated good specificity but low sensitivity compared with the commercial method, indicating that these two epitopes could serve as potential candidate targets for diagnosis. Overall, these results further our understanding of the epitope distribution of HEV ORF2, and provide important information for the development of peptide-based immunodiagnostic tests to detect HEV in serum.

Temporal Formation and Immunolocalization of an Endospore Surface Epitope During Pasteuria penetrans Sporogenesis.

Science.gov (United States)

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.

GPS-MBA: computational analysis of MHC class II epitopes in type 1 diabetes.

Science.gov (United States)

Cai, Ruikun; Liu, Zexian; 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-A(g7) in the non-obese diabetic (NOD) mouse and human HLA-DQ8 are strongly linked to susceptibility to T1D. Thus, the identification of new I-A(g7) 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-A(g7) 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-A(g7) 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.

Virus-Like Particles of Chimeric Recombinant Porcine Circovirus Type 2 as Antigen Vehicle Carrying Foreign Epitopes

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Huawei Zhang

2014-12-01

Full Text Available Virus-like particles (VLPs of chimeric porcine circovirus type 2 (PCV2 were generated by replacing the nuclear localization signal (NLS; at 1–39 aa of PCV2 capsid protein (Cap with classical swine fever virus (CSFV T-cell epitope (1446–1460 aa, CSFV B-cell epitope (693–716 aa and CSFV T-cell epitope conjugated with B-cell epitope. The recombinant proteins were expressed using the baculovirus expression system and detected by immunoblotting and indirect immunofluorescence assay. The abilities to form PCV2 VLPs were confirmed by transmission electron microscopy. Immunogenicities of the three recombinant proteins were evaluated in mice. Our Results indicated that Cap protein NLS deletion or substitution with CSFV epitopes did not affect the VLPs assembly. Three chimeric Cap proteins could form VLPs and induce efficient humoral and cellular immunity against PCV2 and CSFV in mice. Results show that PCV2 VLPs can be used as an efficient antigen carrier for delivery of foreign epitopes, and a potential novel vaccine.

Epitope Mapping of Monoclonal Antibody PMab-38 Against Dog Podoplanin.

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Chang, Yao-Wen; Yamada, Shinji; Kaneko, Mika K; Kato, Yukinari

2017-12-01

Podoplanin (PDPN), a type I transmembrane sialoglycoprotein, is extensively expressed by normal lymphatic endothelial cells, renal podocytes, and pulmonary type I alveolar cells. Nevertheless, increased expression of PDPN in malignant tumors not only associates with poor prognosis but also facilitates hematogenous metastasis through interaction with C-type lectin-like receptor-2 presented on platelets, followed by PDPN-mediated platelet activation. We previously reported a novel PMab-38 antibody, an anti-dog PDPN (dPDPN) monoclonal antibody, which specifically recognizes PDPN in squamous cell carcinomas melanomas and cancer-associated fibroblasts in canine cancer tissues. However, the specific binding with the epitope of PMab-38 remains undefined. In this study, flow cytometry was utilized to investigate the epitope of PMab-38, which was determined using a series of deletion or point mutants of dPDPN. The results revealed that the critical epitope of PMab-38 is Tyr67 and Glu68 of dPDPN.

Application of phage peptide display technology for the study of food allergen epitopes.

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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.

A synthetic glycan microarray enables epitope mapping of plant cell wall glycan-directed antibodies

DEFF Research Database (Denmark)

Ruprecht, Colin; Bartetzko, Max P; Senf, Deborah

2017-01-01

In the last three decades, more than 200 monoclonal antibodies have been raised against most classes of plant cell wall polysaccharides by different laboratories world-wide. 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 for only a few of these antibodies has been determined. 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...

Identification of a serotype-independent linear epitope of foot-and-mouth disease virus.

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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.

CTL escape mediated by proteasomal destruction of an HIV-1 cryptic epitope.

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Sylvain Cardinaud

2011-05-01

Full Text Available Cytotoxic CD8+ T cells (CTLs play a critical role in controlling viral infections. HIV-infected individuals develop CTL responses against epitopes derived from viral proteins, but also against cryptic epitopes encoded by viral alternative reading frames (ARF. We studied here the mechanisms of HIV-1 escape from CTLs targeting one such cryptic epitope, Q9VF, encoded by an HIVgag ARF and presented by HLA-B*07. Using PBMCs of HIV-infected patients, we first cloned and sequenced proviral DNA encoding for Q9VF. We identified several polymorphisms with a minority of proviruses encoding at position 5 an aspartic acid (Q9VF/5D and a majority encoding an asparagine (Q9VF/5N. We compared the prevalence of each variant in PBMCs of HLA-B*07+ and HLA-B*07- patients. Proviruses encoding Q9VF/5D were significantly less represented in HLA-B*07+ than in HLA-B*07- patients, suggesting that Q9FV/5D encoding viruses might be under selective pressure in HLA-B*07+ individuals. We thus analyzed ex vivo CTL responses directed against Q9VF/5D and Q9VF/5N. Around 16% of HLA-B*07+ patients exhibited CTL responses targeting Q9VF epitopes. The frequency and the magnitude of CTL responses induced with Q9VF/5D or Q9VF/5N peptides were almost equal indicating a possible cross-reactivity of the same CTLs on the two peptides. We then dissected the cellular mechanisms involved in the presentation of Q9VF variants. As expected, cells infected with HIV strains encoding for Q9VF/5D were recognized by Q9VF/5D-specific CTLs. In contrast, Q9VF/5N-encoding strains were neither recognized by Q9VF/5N- nor by Q9VF/5D-specific CTLs. Using in vitro proteasomal digestions and MS/MS analysis, we demonstrate that the 5N variation introduces a strong proteasomal cleavage site within the epitope, leading to a dramatic reduction of Q9VF epitope production. Our results strongly suggest that HIV-1 escapes CTL surveillance by introducing mutations leading to HIV ARF-epitope destruction by proteasomes.

Thyroid Autoantibodies Display both “Original Antigenic Sin” and Epitope Spreading

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Sandra M. McLachlan

2017-12-01

Full Text Available Evidence for original antigenic sin in spontaneous thyroid autoimmunity is revealed by autoantibody interactions with immunodominant regions on thyroid autoantigens, thyroglobulin (Tg, thyroid peroxidase (TPO, and the thyrotropin receptor (TSHR A-subunit. In contrast, antibodies induced by immunization of rabbits or mice recognize diverse epitopes. Recognition of immunodominant regions persists despite fluctuations in autoantibody levels following treatment or over time. The enhancement of spontaneously arising pathogenic TSHR antibodies in transgenic human thyrotropin receptor/NOD.H2h4 mice by injecting a non-pathogenic form of TSHR A-subunit protein also provides evidence for original antigenic sin. From other studies, antigen presentation by B cells, not dendritic cells, is likely responsible for original antigenic sin. Recognition of restricted epitopes on the large glycosylated thyroid autoantigens (60-kDa A-subunit, 100-kDa TPO, and 600-kDa Tg facilitates exploring the amino acid locations in the immunodominant regions. Epitope spreading has also been revealed by autoantibodies in thyroid autoimmunity. In humans, and in mice that spontaneously develop autoimmunity to all three thyroid autoantigens, autoantibodies develop first to Tg and later to TPO and the TSHR A-subunit. The pattern of intermolecular epitope spreading is related in part to the thyroidal content of Tg, TPO and TSHR A-subunit and to the molecular sizes of these proteins. Importantly, the epitope spreading pattern provides a rationale for future antigen-specific manipulation to block the development of all thyroid autoantibodies by inducing tolerance to Tg, first in the autoantigen cascade. Because of its abundance, Tg may be the autoantigen of choice to explore antigen-specific treatment, preventing the development of pathogenic TSHR antibodies.

Isolation and characterization of two antigenic glycoproteins from the pollen of Prosopis juliflora.

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Thakur, I S

1991-03-01

Two antigenically active glycoprotein fractions were isolated from crude extract of the pollen of Prosopis juliflora using DEAE-cellulose ion exchange chromatography. The glycoproteins gave single band on polyacrylamide gel electrophoresis. The molecular weight of these two glycoprotein was 20,000 and 10,000 as determined by gel filtration on Sephadex G-75. With the help of crossed immunoelectrophoresis and gel diffusion crude extract exhibited twelve and three precipitating antigens suggesting its heterogeneous nature; and the purified glycoprotein fractions however formed single precipitin band on gel diffusion test and immunoelectrophoresis. As tested by ELISA the polyclonal antisera raised in rabbit showed strong binding affinity with glycoprotein of MW 20,000. These result indicates that the two glycoprotein fractions are not antigenically identical.

Identification of a conserved B-cell epitope on the GapC protein of Streptococcus dysgalactiae.

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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.

Purification and characterization of the glycoprotein allergen from Prosopis juliflora pollen.

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Thakur, I S

1991-02-01

Highly active glycoprotein allergens have been isolated from pollen of Prosopis juliflora by a combination of Sephadex G-100 gel filtration and Sodium dodecyl sulphate-Poly-acrylamide gel electrophoresis. The glycoprotein fraction was homogeneous, and had molecular weight 20,000. The purified glycoprotein allergen contained 20% carbohydrate, mainly arabinose and galactose. Enzymatic digestion of glycoprotein with protease released glycopeptides of molecular weight ranging from less than 1,000 to more than 5,000 on Sephadex G-25 gel filtration. Antigenicity or allergenicity testing of these glycopeptides by immunodiffusion, immunoelectrophoresis, and radioallergosorbent test indicated complete loss of allergenic activity after digestion with protease whereas incubation with beta-D-galactosidase and periodate oxidation had little affect on the allergenic activity of the glycoprotein fraction. But incubation with alpha-D-glucosidase did not affect the allergenic activity significantly. All these tests indicated that protein played significant role in allergenicity of P. juliflora pollen.

Strategies to overcome or circumvent P-glycoprotein mediated multidrug resistance.

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Yuan, Hongyu; Li, Xun; Wu, Jifeng; Li, Jinpei; Qu, Xianjun; Xu, Wenfang; Tang, Wei

2008-01-01

Cancer patients who receive chemotherapy often experience intrinsic or acquired resistance to a broad spectrum of chemotherapeutic agents. The phenomenon, termed multidrug resistance (MDR), is often associated with the over-expression of P-glycoprotein, a transmembrane protein pump, which can enhance efflux of a various chemicals structurally unrelated at the expense of ATP depletion, resulting in decrease of the intracellular cytotoxic drug accumulation. The MDR has been a big threaten to the human health and the war fight for it continues. Although several other mechanisms for MDR are elucidated in recent years, considerable efforts attempting to inverse MDR are involved in exploring P-glycoprotein modulators and suppressing P-glycoprotein expression. In this review, we will report on the recent advances in various strategies for overcoming or circumventing MDR mediated by P-glycoprotein.

Elucidation of the TMab-6 Monoclonal Antibody Epitope Against Telomerase Reverse Transcriptase.

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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.

IgE versus IgG4 epitopes of the peanut allergen Ara h 1 in patients with severe allergy

DEFF Research Database (Denmark)

Bøgh, Katrine Lindholm; Nielsen, H.; Eiwegger, T.

2014-01-01

epitopes. Objective: The aim of this study was to compare Ara h 1-specific IgE and IgG4 epitope recognition patterns in patients with severe peanut allergy, applying a method allowing for identification of both linear and conformational epitopes. Methods: Polyclonal sera from three individual patients......, suffering from severe allergic reaction to peanuts, including anaphylaxis, were used to analyse the IgE and IgG4 epitope recognition patterns of the major peanut allergen Ara h 1. Epitope identification was conducted by competitive immuno-screening of a phage-displayed random heptamer peptide library...

Functionality of Chimeric E2 Glycoproteins of BVDV and CSFV in Virus Replication

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H.G.P. van Gennip

2008-01-01

Full Text Available An intriguing difference between the E2 glycoprotein of CSFV and the other groups of pestiviruses (nonCSFV is a lack of two cysteine residues on positions cysteine 751 and 798. Other groups of pestivirus are not restricted to one species as swine, whereas CSFV is restricted to swine and wild boar. We constructed chimeric CSFV/BVDV E2 genes based on a 2D model of E2 proposed by van Rijn et al. (van Rijn et al. 1994, J Virol 68, 3934–42 and confirmed their expression by immunostaining of plasmid-transfected SK6 cells. No equivalents for the antigenic units B/C and A were found on E2 of BVDVII. This indicates major structural differences in E2. However, the immunodominant BVDVII domain A, containing epitopes with essential amino acids between position 760–764, showed to be dependent on the presence of the region defined by amino acids 684 to 796. As for the A domain of CSFV, the BVDVII A-like domain seemed to function as a separate unit. These combined domains in E2 proved to be the only combination which was functional in viral background of CSFV C-strain. The fitness of this virus (vfl c36BVDVII 684–796 seemed to be reduced compared to vfl c9 (with the complete antigenic region of BVDVII.

Functionality of Chimeric E2 Glycoproteins of BVDV and CSFV in Virus Replication

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H.G.P. Van Gennip

2008-01-01

Full Text Available An intriguing difference between the E2 glycoprotein of CSFV and the other groups of pestiviruses (nonCSFV is a lack of two cysteine residues on positions cysteine 751 and 798. Other groups of pestivirus are not restricted to one species as swine, whereas CSFV is restricted to swine and wild boar. We constructed chimeric CSFV/BVDV E2 genes based on a 2D model of E2 proposed by van Rijn et al. (van Rijn et al. 1994, J Virol 68, 3934–42 and confirmed their expression by immunostaining of plasmid-transfected SK6 cells. No equivalents for the antigenic units B/C and A were found on E2 of BVDVII. This indicates major structural differences in E2. However, the immunodominant BVDVII domain A, containing epitopes with essential amino acids between position 760–764, showed to be dependent on the presence of the region defined by amino acids 684 to 796. As for the A domain of CSFV, the BVDVII A-like domain seemed to function as a separate unit. These combined domains in E2 proved to be the only combination which was functional in viral background of CSFV C-strain. The fitness of this virus (vflc36 BVDVII 684–796 seemed to be reduced compared to vflc9 (with the complete antigenic region of BVDVII.

A novel function of N-linked glycoproteins, alpha-2-HS-glycoprotein and hemopexin: Implications for small molecule compound-mediated neuroprotection.

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Takuya Kanno

Full Text Available Therapeutic agents to the central nervous system (CNS need to be efficiently delivered to the target site of action at appropriate therapeutic levels. However, a limited number of effective drugs for the treatment of neurological diseases has been developed thus far. Further, the pharmacological mechanisms by which such therapeutic agents can protect neurons from cell death have not been fully understood. We have previously reported the novel small-molecule compound, 2-[mesityl(methylamino]-N-[4-(pyridin-2-yl-1H-imidazol-2-yl] acetamide trihydrochloride (WN1316, as a unique neuroprotectant against oxidative injury and a highly promising remedy for the treatment of amyotrophic lateral sclerosis (ALS. One of the remarkable characteristics of WN1316 is that its efficacious doses in ALS mouse models are much less than those against oxidative injury in cultured human neuronal cells. It is also noted that the WN1316 cytoprotective activity observed in cultured cells is totally dependent upon the addition of fetal bovine serum in culture medium. These findings led us to postulate some serum factors being tightly linked to the WN1316 efficacy. In this study, we sieved through fetal bovine serum proteins and identified two N-linked glycoproteins, alpha-2-HS-glycoprotein (AHSG and hemopexin (HPX, requisites to exert the WN1316 cytoprotective activity against oxidative injury in neuronal cells in vitro. Notably, the removal of glycan chains from these molecules did not affect the WN1316 cytoprotective activity. Thus, two glycoproteins, AHSG and HPX, represent a pivotal glycoprotein of the cytoprotective activity for WN1316, showing a concrete evidence for the novel glycan-independent function of serum glycoproteins in neuroprotective drug efficacy.

Changes in cell wall properties coincide with overexpression of extensin fusion proteins in suspension cultured tobacco cells.

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Tan, Li; Pu, Yunqiao; Pattathil, Sivakumar; Avci, Utku; Qian, Jin; Arter, Allison; Chen, Liwei; Hahn, Michael G; Ragauskas, Arthur J; Kieliszewski, Marcia J

2014-01-01

Extensins are one subfamily of the cell wall hydroxyproline-rich glycoproteins, containing characteristic SerHyp4 glycosylation motifs and intermolecular cross-linking motifs such as the TyrXaaTyr sequence. Extensins are believed to form a cross-linked network in the plant cell wall through the tyrosine-derivatives isodityrosine, pulcherosine, and di-isodityrosine. Overexpression of three synthetic genes encoding different elastin-arabinogalactan protein-extensin hybrids in tobacco suspension cultured cells yielded novel cross-linking glycoproteins that shared features of the extensins, arabinogalactan proteins and elastin. The cell wall properties of the three transgenic cell lines were all changed, but in different ways. One transgenic cell line showed decreased cellulose crystallinity and increased wall xyloglucan content; the second transgenic cell line contained dramatically increased hydration capacity and notably increased cell wall biomass, increased di-isodityrosine, and increased protein content; the third transgenic cell line displayed wall phenotypes similar to wild type cells, except changed xyloglucan epitope extractability. These data indicate that overexpression of modified extensins may be a route to engineer plants for bioenergy and biomaterial production.

A Functional Henipavirus Envelope Glycoprotein Pseudotyped Lentivirus Assay System

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Broder Christopher C

2010-11-01

Full Text Available Abstract Background Hendra virus (HeV and Nipah virus (NiV are newly emerged zoonotic paramyxoviruses discovered during outbreaks in Queensland, Australia in 1994 and peninsular Malaysia in 1998/9 respectively and classified within the new Henipavirus genus. Both viruses can infect a broad range of mammalian species causing severe and often-lethal disease in humans and animals, and repeated outbreaks continue to occur. Extensive laboratory studies on the host cell infection stage of HeV and NiV and the roles of their envelope glycoproteins have been hampered by their highly pathogenic nature and restriction to biosafety level-4 (BSL-4 containment. To circumvent this problem, we have developed a henipavirus envelope glycoprotein pseudotyped lentivirus assay system using either a luciferase gene or green fluorescent protein (GFP gene encoding human immunodeficiency virus type-1 (HIV-1 genome in conjunction with the HeV and NiV fusion (F and attachment (G glycoproteins. Results Functional retrovirus particles pseudotyped with henipavirus F and G glycoproteins displayed proper target cell tropism and entry and infection was dependent on the presence of the HeV and NiV receptors ephrinB2 or B3 on target cells. The functional specificity of the assay was confirmed by the lack of reporter-gene signals when particles bearing either only the F or only G glycoprotein were prepared and assayed. Virus entry could be specifically blocked when infection was carried out in the presence of a fusion inhibiting C-terminal heptad (HR-2 peptide, a well-characterized, cross-reactive, neutralizing human mAb specific for the henipavirus G glycoprotein, and soluble ephrinB2 and B3 receptors. In addition, the utility of the assay was also demonstrated by an examination of the influence of the cytoplasmic tail of F in its fusion activity and incorporation into pseudotyped virus particles by generating and testing a panel of truncation mutants of NiV and HeV F

IgE vs IgG4 epitopes of the peanut allergen Ara h 1 in patients with severe allergy

DEFF Research Database (Denmark)

Bøgh, Katrine Lindholm; Nielsen, H.; Eiwegger, T.

2013-01-01

to the allergen. However, recent studies have demonstrated the very importance of the IgG4-epitope affinity for the blocking ability. Studies comparing IgE and IgG4 binding epitopes mainly focus on the identification of linear epitopes. Peanut allergy is one of the most severe and persistent forms of food allergy....... The importance of conformational epitopes, of the major peanut allergen Ara h 1, has been demonstrated. The aim of this study was to compare Ara h 1-specific epitope patterns for IgE and IgG4 in patients with severe peanut allergy applying a method suitable to identify both linear and conformational epitopes....... Methods: Ara h 1-specific IgE and IgG4 epitope patterns were examined by competitive immunoscreening of a phage-displayed random 7-mer peptide library using polyclonal IgE and IgG4 from three individual patients suffering from severe peanut allergy. The resulting peptide sequences were mapped...

Labelled antibody techniques in glycoprotein estimation

International Nuclear Information System (INIS)

Hazra, D.K.; Ekins, R.P.; Edwards, R.; Williams, E.S.

1977-01-01

The problems in the radioimmunoassay of the glycoprotein hormones (pituitary LH, FSH and TSH and human chlorionic gonadotrophin HGG) are reviewed viz: limited specificity and sensitivity in the clinical context, interpretation of disparity between bioassay and radioimmunoassay, and interlaboratory variability. The advantages and limitations of the labelled antibody techniques - classical immonoradiometric methods and 2-site or 125 I-anti-IgG indirect labelling modifications are reviewed in general, and their theoretical potential in glycoprotein assays examined in the light of previous work. Preliminary experiments in the development of coated tube 2-site assay for glycoproteins using 125 I anti-IgG labelling are described, including conditions for maximizing solid phase extraction of the antigen, iodination of anti-IgG, and assay conditions such as effects of temperature of incubation with antigen 'hormonefree serum', heterologous serum and detergent washing. Experiments with extraction and antigen-specific antisera raised in the same or different species are described as exemplified by LH and TSH assay systems, the latter apparently promising greater sensitivity than radioimmunoassay. Proposed experimental and mathematical optimisation and validation of the method as an assay system is outlined, and the areas for further work delineated. (orig.) [de

Greater epitope recognition of shrimp allergens by children than by adults suggests that shrimp sensitization decreases with age.

Science.gov (United States)

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

Development of a multi-epitope peptide vaccine inducing robust T cell responses against brucellosis using immunoinformatics based approaches.

Science.gov (United States)

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

Structures and Functions of Pestivirus Glycoproteins: Not Simply Surface Matters.

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Wang, Fun-In; Deng, Ming-Chung; Huang, Yu-Liang; Chang, Chia-Yi

2015-06-29

Pestiviruses, which include economically important animal pathogens such as bovine viral diarrhea virus and classical swine fever virus, possess three envelope glycoproteins, namely Erns, E1, and E2. This article discusses the structures and functions of these glycoproteins and their effects on viral pathogenicity in cells in culture and in animal hosts. E2 is the most important structural protein as it interacts with cell surface receptors that determine cell tropism and induces neutralizing antibody and cytotoxic T-lymphocyte responses. All three glycoproteins are involved in virus attachment and entry into target cells. E1-E2 heterodimers are essential for viral entry and infectivity. Erns is unique because it possesses intrinsic ribonuclease (RNase) activity that can inhibit the production of type I interferons and assist in the development of persistent infections. These glycoproteins are localized to the virion surface; however, variations in amino acids and antigenic structures, disulfide bond formation, glycosylation, and RNase activity can ultimately affect the virulence of pestiviruses in animals. Along with mutations that are driven by selection pressure, antigenic differences in glycoproteins influence the efficacy of vaccines and determine the appropriateness of the vaccines that are currently being used in the field.

Nanoallergens: A multivalent platform for studying and evaluating potency of allergen epitopes in cellular degranulation

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Deak, Peter E; Vrabel, Maura R; Pizzuti, Vincenzo J; Kiziltepe, Tanyel

2016-01-01

Degranulation caused by type I hypersensitivity (allergies) is a complex biophysical process, and available experimental models for studying relevant immunoglobulin E binding epitopes on allergen proteins lack the ability to adequately evaluate, rank, and associate these epitopes individually and with each other. In this study, we propose a new allergy model system for studying potential allergen epitopes using nanoallergens, liposomes modified to effectively display IgE binding epitopes/haptens. By utilizing the covalently conjugated lipid tails on two hapten molecules (dinitrophenol and dansyl), hapten molecules were successfully incorporated into liposomes with high precision to form nanoallergens. Nanoallergens, with precisely controlled high-particle valency, can trigger degranulation with much greater sensitivity than commonly used bovine serum albumin conjugates. In rat basophil leukemia cell experiments, nanoallergens with only 2% hapten loading were able to trigger degranulation in vitro at concentrations as low as 10 pM. Additionally, unlike bovine serum albumin-hapten conjugates, nanoallergens allow exact control over particle size and valency. By varying the nanoallergen parameters such as size, valency, monovalent affinity of hapten, and specific IgE ratios, we exposed the importance of these variables on degranulation intensity while demonstrating nanoallergens’ potential for evaluating both high- and low-affinity epitopes. The data presented in this article establish nanoallergen platform as a reliable and versatile allergy model to study and evaluate allergen epitopes in mast cell degranulation. PMID:27188517

Specific immunotherapy modifies allergen-specific CD4+ T cell responses in an epitope-dependent manner

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Wambre, Erik; DeLong, Jonathan H.; James, Eddie A.; Torres-Chinn, Nadia; Pfützner, Wolfgang; Möbs, Christian; Durham, Stephen R.; Till, Stephen J.; Robinson, David; Kwok, William W.

2014-01-01

Background Understanding the mechanisms by which the immune system induces and controls allergic inflammation at the T cell epitope level is critical for the design of new allergy vaccine strategies. Objective To characterize allergen-specific T cell responses linked with allergy or peripheral tolerance and to determine how CD4+ T cell responses to individual allergen-derived epitopes change over allergen-specific immunotherapy (ASIT). Methods Timothy grass pollen (TGP) allergy was used as a model for studying grass pollen allergies. The breadth, magnitude, epitope hierarchy and phenotype of the DR04:01-restricted TGP-specific T cell responses in ten grass pollen allergic, five non-atopic and six allergy vaccine-treated individuals was determined using an ex vivo pMHCII-tetramer approach. Results CD4+ T cells in allergic individuals are directed to a broad range of TGP epitopes characterized by defined immunodominance hierarchy patterns and with distinct functional profiles that depend on the epitope recognized. Epitopes that are restricted specifically to either TH2 or TH1/TR1 responses were identified. ASIT was associated with preferential deletion of allergen-specific TH2 cells and without significant change in frequency of TH1/TR1 cells. Conclusions Preferential allergen-specific TH2-cells deletion after repeated high doses antigen stimulation can be another independent mechanism to restore tolerance to allergen during immunotherapy. PMID:24373351

Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

International Nuclear Information System (INIS)

Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga; Luque, Daniel; Terrón, María C.; Calder, Lesley J.; Melero, José A.

2014-01-01

Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV F occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV F , we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV F at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy

Polyclonal and monoclonal antibodies specific for the six-helix bundle of the human respiratory syncytial virus fusion glycoprotein as probes of the protein post-fusion conformation

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Palomo, Concepción; Mas, Vicente; Vázquez, Mónica; Cano, Olga [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Luque, Daniel; Terrón, María C. [Unidad de Microscopía Electrónica y Confocal, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain); Calder, Lesley J. [National Institute for Medical Research, MRC, Mill Hill, London NW7 1AA (United Kingdom); Melero, José A., E-mail: jmelero@isciii.es [Unidad de Biología Viral, Centro Nacional de Microbiología, Madrid (Spain); CIBER de Enfermedades Respiratorias, Instituto de Salud Carlos III, Majadahonda, 28220 Madrid (Spain)

2014-07-15

Human respiratory syncytial virus (hRSV) has two major surface glycoproteins (G and F) anchored in the lipid envelope. Membrane fusion promoted by hRSV{sub F} occurs via refolding from a pre-fusion form to a highly stable post-fusion state involving large conformational changes of the F trimer. One of these changes results in assembly of two heptad repeat sequences (HRA and HRB) into a six-helix bundle (6HB) motif. To assist in distinguishing pre- and post-fusion conformations of hRSV{sub F}, we have prepared polyclonal (α-6HB) and monoclonal (R145) rabbit antibodies specific for the 6HB. Among other applications, these antibodies were used to explore the requirements of 6HB formation by isolated protein segments or peptides and by truncated mutants of the F protein. Site-directed mutagenesis and electron microscopy located the R145 epitope in the post-fusion hRSV{sub F} at a site distantly located from previously mapped epitopes, extending the repertoire of antibodies that can decorate the F molecule. - Highlights: • Antibodies specific for post-fusion respiratory syncytial virus fusion protein are described. • Polyclonal antibodies were obtained in rabbit inoculated with chimeric heptad repeats. • Antibody binding required assembly of a six-helix bundle in the post-fusion protein. • A monoclonal antibody with similar structural requirements is also described. • Binding of this antibody to the post-fusion protein was visualized by electron microscopy.

Conserved B-cell epitopes among human bocavirus species indicate potential diagnostic targets.

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Zhuo Zhou

Full Text Available BACKGROUND: Human bocavirus species 1-4 (HBoV1-4 have been associated with respiratory and enteric infections in children. However, the immunological mechanisms in response to HBoV infections are not fully understood. Though previous studies have shown cross-reactivities between HBoV species, the epitopes responsible for this phenomenon remain unknown. In this study, we used genomic and immunologic approaches to identify the reactive epitopes conserved across multiple HBoV species and explored their potential as the basis of a novel diagnostic test for HBoVs. METHODOLOGY/PRINCIPAL FINDINGS: We generated HBoV1-3 VP2 gene fragment phage display libraries (GFPDLs and used these libraries to analyze mouse antisera against VP2 protein of HBoV1, 2, and 3, and human sera positive for HBoVs. Using this approach, we mapped four epitope clusters of HBoVs and identified two immunodominant peptides--P1 (¹MSDTDIQDQQPDTVDAPQNT²⁰, and P2 (¹⁶²EHAYPNASHPWDEDVMPDL¹⁸⁰--that are conserved among HBoV1-4. To confirm epitope immunogenicity, we immunized mice with the immunodominant P1 and P2 peptides identified in our screen and found that they elicited high titer antibodies in mice. These two antibodies could only recognize the VP2 of HBoV 1-4 in Western blot assays, rather than those of the two other parvoviruses human parvovirus B19 and human parvovirus 4 (PARV4. Based on our findings, we evaluated epitope-based peptide-IgM ELISAs as potential diagnostic tools for HBoVs IgM antibodies. We found that the P1+P2-IgM ELISA showed a higher sensitivity and specificity in HBoVs IgM detection than the assays using a single peptide. CONCLUSIONS/SIGNIFICANCE: The identification of the conserved B-cell epitopes among human bocavirus species contributes to our understanding of immunological cross-reactivities of HBoVs, and provides important insights for the development of HBoV diagnostic tools.

Flow cytometric analysis of platelet cyclooxygenase-1 and -2 and surface glycoproteins in patients with immune thrombocytopenia and healthy individuals.

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Rubak, Peter; Kristensen, Steen D; Hvas, Anne-Mette

2017-06-01

Immature platelets may contain more platelet enzymes such as cyclooxygenase (COX)-1 and COX-2 than mature platelets. Patients with immune thrombocytopenia (ITP) have a higher fraction of immature platelets and can therefore be utilized as a biological model for investigating COX-1 and COX-2 platelet expression. The aims were to develop flow cytometric assays for platelet COX-1 and COX-2 and to investigate the COX-1 and COX-2 platelet expression, platelet turnover, and platelet glycoproteins in ITP patients (n = 10) compared with healthy individuals (n = 30). Platelet count and platelet turnover parameters (mean platelet volume (MPV), immature platelet fraction (IPF), and immature platelet count (IPC)) were measured by flow cytometry (Sysmex XE-5000). Platelet COX-1, COX-2, and the glycoproteins (GP)IIb, IX, Ib, Ia, and IIIa were all analyzed by flow cytometry (Navios) and expressed as median fluorescence intensity. COX analyses were performed in both whole blood and platelet rich plasma (PRP), whereas platelet glycoproteins were analyzed in whole blood only. ITP patients had significantly lower platelet count (55 × 10 9 /L) than healthy individuals (240 × 10 9 /L, p platelet count and IPC (both p-values Platelet COX-1 expression was higher in ITP patients than healthy individuals using whole blood (p COX-1 platelet turnover and COX-1 expression (all p-values platelet turnover and COX-1 and COX-2 expressions (all p-values platelet turnover in ITP patients (all p-values 0.14, rho = 0.11-0.28). In conclusion, ITP patients expressed higher COX-1 and platelet glycoprotein levels than healthy individuals. COX-1 and platelet glycoproteins demonstrated positive correlations with platelet turnover in ITP patients. In healthy individuals, COX-1 and COX-2 expression correlated positively with platelet turnover. PRP was more sensitive compared with whole blood as regards determination of COX. Therefore, PRP is the recommended matrix for investigating COX-1 and COX-2 in

Sequence analysis of the 5′ third of glycoprotein C gene of South American bovine herpesviruses 1 and 5

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Traesel, C.K.; Bernardes, L.M. [Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil); Spilki, F.R. [Laboratório de Microbiologia Molecular, Universidade Feevale, Novo Hamburgo, RS (Brazil); Weiblen, R.; Flores, E.F. [Setor de Virologia, Departamento de Medicina Veterinária Preventiva, Universidade Federal de Santa Maria, Santa Maria, RS (Brazil)

2015-03-06

Bovine herpesviruses 1 (BoHV-1) and 5 (BoHV-5) share high genetic and antigenic similarities, but exhibit marked differences in tissue tropism and neurovirulence. The amino-terminal region of glycoprotein C (gC), which is markedly different in each of the viruses, is involved in virus binding to cellular receptors and in interactions with the immune system. This study investigated the genetic and antigenic differences of the 5′ region of the gC (5′ gC) gene (amino-terminal) of South American BoHV-1 (n=19) and BoHV-5 (n=25) isolates. Sequence alignments of 374 nucleotides (104 amino acids) revealed mean similarity levels of 97.3 and 94.2% among BoHV-1 gC (gC1), respectively, 96.8 and 95.6% among BoHV-5 gC (gC5), and 62 and 53.3% between gC1 and gC5. Differences included the absence of 40 amino acid residues (27 encompassing predicted linear epitopes) scattered throughout 5′ gC1 compared to 5′ gC5. Virus neutralizing assays testing BoHV-1 and BoHV-5 antisera against each isolate revealed a high degree of cross-neutralization between the viruses, yet some isolates were neutralized at very low titers by heterologous sera, and a few BoHV-5 isolates reacted weakly with either sera. The virus neutralization differences observed within the same viral species, and more pronounced between BoHV-1 and BoHV-5, likely reflect sequence differences in neutralizing epitopes. These results demonstrate that the 5′ gC region is well conserved within each viral species but is divergent between BoHV-1 and BoHV-5, likely contributing to their biological and antigenic differences.

Adipokine zinc-α2-glycoprotein regulated by growth hormone and linked to insulin sensitivity.

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Balaz, Miroslav; Ukropcova, Barbara; Kurdiova, Timea; Gajdosechova, Lucia; Vlcek, Miroslav; Janakova, Zuzana; Fedeles, Jozef; Pura, Mikulas; Gasperikova, Daniela; Smith, Steven R; Tkacova, Ruzena; Klimes, Iwar; Payer, Juraj; Wolfrum, Christian; Ukropec, Jozef

2015-02-01

Hypertrophic obesity is associated with impaired insulin sensitivity and lipid-mobilizing activity of zinc-α2-glycoprotein. Adipose tissue (AT) of growth hormone (GH) -deficient patients is characterized by extreme adipocyte hypertrophy due to defects in AT lipid metabolism. It was hypothesized that zinc-α2-glycoprotein is regulated by GH and mediates some of its beneficial effects in AT. AT from patients with GH deficiency and individuals with obesity-related GH deficit was obtained before and after 5-year and 24-month GH supplementation therapy. GH action was tested in primary human adipocytes. Relationships of GH and zinc-α2-glycoprotein with adipocyte size and insulin sensitivity were evaluated in nondiabetic patients with noncancerous cachexia and hypertrophic obesity. AT in GH-deficient adults displayed a substantial reduction of zinc-α2-glycoprotein. GH therapy normalized AT zinc-α2-glycoprotein. Obesity-related relative GH deficit was associated with almost 80% reduction of zinc-α2-glycoprotein mRNA in AT. GH increased zinc-α2-glycoprotein mRNA in both AT of obese men and primary human adipocytes. Interdependence of GH and zinc-α2-glycoprotein in regulating AT morphology and metabolic phenotype was evident from their relationship with adipocyte size and AT-specific and whole-body insulin sensitivity. The results demonstrate that GH is involved in regulation of AT zinc-α2-glycoprotein; however, the molecular mechanism linking GH and zinc-α2-glycoprotein in AT is yet unknown. © 2014 The Obesity Society.

Clinical Control of HIV-1 by Cytotoxic T Cells Specific for Multiple Conserved Epitopes.

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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

Microarray glycan profiling reveals algal fucoidan epitopes in diverse marine metazoans

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Asunción Salmeán, Armando; Hervé, Cécile; Jørgensen, Bodil

2017-01-01

Despite the biological importance and pharmacological potential of glycans from marine organisms, there are many unanswered questions regarding their distribution, function, and evolution. Here we describe microarray-based glycan profiling of a diverse selection of marine animals using antibodies...... raised against fucoidan isolated from a brown alga. We demonstrate the presence of two fucoidan epitopes in six animals belonging to three phyla including Porifera, Molusca, and Chordata. We studied the spatial distribution of these epitopes in Cliona celata ("boring sponge") and identified...

Glycoprotein expression by adenomatous polyps of the colon

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Roney, Celeste A.; Xie, Jianwu; Xu, Biying; Jabour, Paul; Griffiths, Gary; Summers, Ronald M.

2008-03-01

Colon cancer is the second leading cause of cancer related deaths in the United States. Specificity in diagnostic imaging for detecting colorectal adenomas, which have a propensity towards malignancy, is desired. Adenomatous polyp specimens of the colon were obtained from the mouse model of colorectal cancer called adenomatous polyposis coli-multiple intestinal neoplasia (APC Min). Histological evaluation, by the legume protein Ulex europaeus agglutinin I (UEA-1), determined expression of the glycoprotein α-L-fucose. FITC-labelled UEA-1 confirmed overexpression of the glycoprotein by the polyps on fluorescence microscopy in 17/17 cases, of which 13/17 included paraffin-fixed mouse polyp specimens. In addition, FITC-UEA-1 ex vivo multispectral optical imaging of 4/17 colonic specimens displayed over-expression of the glycoprotein by the polyps, as compared to non-neoplastic mucosa. Here, we report the surface expression of α-L-fucosyl terminal residues by neoplastic mucosal cells of APC specimens of the mouse. Glycoprotein expression was validated by the carbohydrate binding protein UEA-1. Future applications of this method are the development of agents used to diagnose cancers by biomedical imaging modalities, including computed tomographic colonography (CTC). UEA-1 targeting to colonic adenomas may provide a new avenue for the diagnosis of colorectal carcinoma by CT imaging.

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

DEFF Research Database (Denmark)

Hansen, Christian Skjødt; Østerbye, Thomas; Marcatili, Paolo

2017-01-01

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....... 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...

Determination of critical epitope of PcMab-47 against human podocalyxin

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Shunsuke Itai

2018-07-01

Full Text Available 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.

Determination of critical epitope of PcMab-47 against human podocalyxin.

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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.

Epitope Mapping of Metuximab on CD147 Using Phage Display and Molecular Docking

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Bifang He

2013-01-01

Full Text Available Metuximab is the generic name of Licartin, a new drug for radioimmunotherapy of hepatocellular carcinoma. Although it is known to be a mouse monoclonal antibody against CD147, the complete epitope mediating the binding of metuximab to CD147 remains unknown. We panned the Ph.D.-12 phage display peptide library against metuximab and got six mimotopes. The following bioinformatics analysis based on mimotopes suggested that metuximab recognizes a conformational epitope composed of more than 20 residues. The residues of its epitope may include T28, V30, K36, L38, K57, F74, D77, S78, D79, D80, Q81, G83, S86, N98, Q100, L101, H102, G103, P104, V131, P132, and K191. The homology modeling of metuximab and the docking of CD147 to metuximab were also performed. Based on the top one docking model, the epitope was predicted to contain 28 residues: AGTVFTTV (23–30, I37, D45, E84, V88, EPMGTANIQLH (92–102, VPP (131–133, Q164, and K191. Almost half of the residues predicted on the basis of mimotope analysis also appear in the docking result, indicating that both results are reliable. As the predicted epitopes of metuximab largely overlap with interfaces of CD147-CD147 interactions, a structural mechanism of metuximab is proposed as blocking the formation of CD147 dimer.

Optimal selection of epitopes for TXP-immunoaffinity mass spectrometry.

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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.

Monoclonal antibodies to molluskan hemocyanin from Concholepas concholepas demonstrate common and specific epitopes among subunits.

Science.gov (United States)

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.

THE ROLE OF P-GLYCOPROTEIN IN RATIONAL PHARMACOTHERAPY IN CARDIOLOGY

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A. V. Shulkin

2015-09-01

Full Text Available On the basis of the analysis of published data the role of P-glycoprotein, carrier protein, in rational pharmacotherapy in cardiology was shown on the example of its substrates – digoxin, antiplatelet agents and anticoagulants. Determination of C3435T polymorphism of multidrug resistance gene (MDR1, encoding P-glycoprotein, in pharmacotherapy with digoxin, antiplatelet drugs (clopidogrel tikagrelol, prasugrel and anticoagulants (dabigatran etexilate, rivaroxaban, edoxaban is not feasible in routine practice. Drug in- teractions have clinical implications for the efficacy and safety of pharmacotherapy in coadministration of these drugs with P-glycoprotein substrates, inducers and inhibitors.

Molecular fingerprinting of complex grass allergoids: size assessments reveal new insights in epitope repertoires and functional capacities.

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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

Structures and Functions of Pestivirus Glycoproteins: Not Simply Surface Matters

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Fun-In Wang

2015-06-01

Full Text Available Pestiviruses, which include economically important animal pathogens such as bovine viral diarrhea virus and classical swine fever virus, possess three envelope glycoproteins, namely Erns, E1, and E2. This article discusses the structures and functions of these glycoproteins and their effects on viral pathogenicity in cells in culture and in animal hosts. E2 is the most important structural protein as it interacts with cell surface receptors that determine cell tropism and induces neutralizing antibody and cytotoxic T-lymphocyte responses. All three glycoproteins are involved in virus attachment and entry into target cells. E1-E2 heterodimers are essential for viral entry and infectivity. Erns is unique because it possesses intrinsic ribonuclease (RNase activity that can inhibit the production of type I interferons and assist in the development of persistent infections. These glycoproteins are localized to the virion surface; however, variations in amino acids and antigenic structures, disulfide bond formation, glycosylation, and RNase activity can ultimately affect the virulence of pestiviruses in animals. Along with mutations that are driven by selection pressure, antigenic differences in glycoproteins influence the efficacy of vaccines and determine the appropriateness of the vaccines that are currently being used in the field.

Broadly reactive human CD8 T cells that recognize an epitope conserved between VZV, HSV and EBV.

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Christopher Chiu

2014-03-01

Full Text Available Human herpesviruses are important causes of potentially severe chronic infections for which T cells are believed to be necessary for control. In order to examine the role of virus-specific CD8 T cells against Varicella Zoster Virus (VZV, we generated a comprehensive panel of potential epitopes predicted in silico and screened for T cell responses in healthy VZV seropositive donors. We identified a dominant HLA-A*0201-restricted epitope in the VZV ribonucleotide reductase subunit 2 and used a tetramer to analyze the phenotype and function of epitope-specific CD8 T cells. Interestingly, CD8 T cells responding to this VZV epitope also recognized homologous epitopes, not only in the other α-herpesviruses, HSV-1 and HSV-2, but also the γ-herpesvirus, EBV. Responses against these epitopes did not depend on previous infection with the originating virus, thus indicating the cross-reactive nature of this T cell population. Between individuals, the cells demonstrated marked phenotypic heterogeneity. This was associated with differences in functional capacity related to increased inhibitory receptor expression (including PD-1 along with decreased expression of co-stimulatory molecules that potentially reflected their stimulation history. Vaccination with the live attenuated Zostavax vaccine did not efficiently stimulate a proliferative response in this epitope-specific population. Thus, we identified a human CD8 T cell epitope that is conserved in four clinically important herpesviruses but that was poorly boosted by the current adult VZV vaccine. We discuss the concept of a "pan-herpesvirus" vaccine that this discovery raises and the hurdles that may need to be overcome in order to achieve this.

Epitope mapping of the alpha-chain of the insulin-like growth factor I receptor using antipeptide antibodies.

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Delafontaine, P; Ku, L; Ververis, J J; Cohen, C; Runge, M S; Alexander, R W

1994-12-01

Insulin-like growth factor I (IGF I) is an important mitogen for vascular smooth muscle cells (VSMC). The IGF I receptor (IGF IR) is a heterotetramer composed of two cross-linked extracellular alpha-chains and two membrane-spanning beta-chains that contain a tyrosine-kinase domain. It has a high degree of sequence similarity to the insulin receptor (IR), and the putative ligand-specific binding site has been localized to a cysteine-rich region (CRR) of the alpha-chain. To obtain insights into antigenic determinants of the IGF IR, we raised a panel of site-specific polyclonal antibodies against short peptide sequences N-terminal to and within the CRR. Several antibodies raised against linear epitopes within the CRR bound to solubilized and native rat and human IGF IR by ELISA, did not cross-react with IR, but unexpectedly failed to inhibit 125I-IGF I binding. A polyclonal antibody directed against a 48-amino acid synthetic peptide, corresponding to a region of the CRR postulated to be essential for ligand binding, failed to react with either solubilized, reduced or intact IGF IR. Three antibodies specific for the N-terminus of the alpha-chain reacted with solubilized and native IGF IR. One of these, RAB 6, directed against amino acids 38-44 of the IGF IR, inhibited 125I-IGF I binding to rat aortic smooth muscle cells (RASM) and to IGF IR/3T3 cells (overexpressing human IGF IR) by up to 45%. Immunohistochemical analysis revealed strong IGF IR staining in the medial smooth muscle cell layer of rat aorta. These findings are consistent with a model wherein conformational epitopes within the CRR and linear epitopes within the N-terminus of the alpha-chain contribute to the IGF I binding pocket. These antibodies should provide a valuable tool to study structure-function relationships and in vivo regulation of the IGF IR.

The POM monoclonals: a comprehensive set of antibodies to non-overlapping prion protein epitopes.

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Magdalini Polymenidou

Full Text Available PrP(Sc, a misfolded and aggregated form of the cellular prion protein PrP(C, is the only defined constituent of the transmissible agent causing prion diseases. Expression of PrP(C in the host organism is necessary for prion replication and for prion neurotoxicity. Understanding prion diseases necessitates detailed structural insights into PrP(C and PrP(Sc. Towards this goal, we have developed a comprehensive collection of monoclonal antibodies denoted POM1 to POM19 and directed against many different epitopes of mouse PrP(C. Three epitopes are located within the N-terminal octarepeat region, one is situated within the central unstructured region, and four epitopes are discontinuous within the globular C-proximal domain of PrP(C. Some of these antibodies recognize epitopes that are resilient to protease digestion in PrP(Sc. Other antibodies immunoprecipitate PrP(C, but not PrP(Sc. A third group was found to immunoprecipitate both PrP isoforms. Some of the latter antibodies could be blocked with epitope-mimicking peptides, and incubation with an excess of these peptides allowed for immunochromatography of PrP(C and PrP(Sc. Amino-proximal antibodies were found to react with repetitive PrP(C epitopes, thereby vastly increasing their avidity. We have also created functional single-chain miniantibodies from selected POMs, which retained the binding characteristics despite their low molecular mass. The POM collection, thus, represents a unique set of reagents allowing for studies with a variety of techniques, including western blotting, ELISA, immunoprecipitation, conformation-dependent immunoassays, and plasmon surface plasmon resonance-based assays.

Allergen and Epitope Targets of Mouse-Specific T Cell Responses in Allergy and Asthma

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Véronique Schulten

2018-02-01

Full Text Available Mouse allergy has become increasingly common, mainly affecting laboratory workers and inner-city households. To date, only one major allergen, namely Mus m 1, has been described. We sought to identify T cell targets in mouse allergic patients. PBMC from allergic donors were expanded with either murine urine or epithelial extract and subsequently screened for cytokine production (IL-5 and IFNγ in response to overlapping peptides spanning the entire Mus m 1 sequence, peptides from various Mus m 1 isoforms [major urinary proteins (MUPs], peptides from mouse orthologs of known allergens from other mammalian species and peptides from proteins identified by immunoproteomic analysis of IgE/IgG immunoblots of mouse urine and epithelial extracts. This approach let to the identification of 106 non-redundant T cell epitopes derived from 35 antigens. Three major T cell-activating regions were defined in Mus m 1 alone. Moreover, our data show that immunodominant epitopes were largely shared between Mus m 1 and other MUPs even from different species, suggesting that sequence conservation in different allergens is a determinant for immunodominance. We further identified several novel mouse T cell antigens based on their homology to known mammalian allergens. Analysis of cohort-specific T cell responses revealed that rhinitis and asthmatic patients recognized different epitope repertoires. Epitopes defined herein can be formulated into an epitope “megapool” used to diagnose mouse allergy and study mouse-specific T cell responses directly ex vivo. This analysis of T cell epitopes provides a good basis for future studies to increase our understanding of the immunopathology associated with MO-allergy and asthma.

Importance of the short cytoplasmic domain of the feline immunodeficiency virus transmembrane glycoprotein for fusion activity and envelope glycoprotein incorporation into virions

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Celma, Cristina C.P.; Paladino, Monica G.; Gonzalez, Silvia A.; Affranchino, Jose L.

2007-01-01

The mature form of the envelope (Env) glycoprotein of lentiviruses is a heterodimer composed of the surface (SU) and transmembrane (TM) subunits. Feline immunodeficiency virus (FIV) possesses a TM glycoprotein with a cytoplasmic tail of approximately 53 amino acids which is unusually short compared with that of the other lentiviral glycoproteins (more than 100 residues). To investigate the relevance of the FIV TM cytoplasmic domain to Env-mediated viral functions, we characterized the biological properties of a series of Env glycoproteins progressively shortened from the carboxyl terminus. All the mutant Env proteins were efficiently expressed in feline cells and processed into the SU and TM subunits. Deletion of 5 or 11 amino acids from the TM C-terminus did not significantly affect Env surface expression, fusogenic activity or Env incorporation into virions, whereas removal of 17 or 23 residues impaired Env-mediated cell-to-cell fusion. Further truncation of the FIV TM by 29 residues resulted in an Env glycoprotein that was poorly expressed at the cell surface, exhibited only 20% of the wild-type Env fusogenic capacity and was inefficiently incorporated into virions. Remarkably, deletion of the TM C-terminal 35 or 41 amino acids restored or even enhanced Env biological functions. Indeed, these mutant Env glycoproteins bearing cytoplasmic domains of 18 or 12 amino acids were found to be significantly more fusogenic than the wild-type Env and were efficiently incorporated into virions. Interestingly, truncation of the TM cytoplasmic domain to only 6 amino acids did not affect Env incorporation into virions but abrogated Env fusogenicity. Finally, removal of the entire TM cytoplasmic tail or deletion of as many as 6 amino acids into the membrane-spanning domain led to a complete loss of Env functions. Our results demonstrate that despite its relatively short length, the FIV TM cytoplasmic domain plays an important role in modulating Env-mediated viral functions

T-cell recognition is shaped by epitope sequence conservation in the host proteome and microbiome

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Bresciani, Anne Gøther; Paul, Sinu; Schommer, Nina

2016-01-01

or allergen with the conservation of its sequence in the human proteome or the healthy human microbiome. Indeed, performing such comparisons on large sets of validated T-cell epitopes, we found that epitopes that are similar with self-antigens above a certain threshold showed lower immunogenicity, presumably...... as a result of negative selection of T cells capable of recognizing such peptides. Moreover, we also found a reduced level of immune recognition for epitopes conserved in the commensal microbiome, presumably as a result of peripheral tolerance. These findings indicate that the existence (and potentially...

Topology characterization of a benzodiazepine-binding beta-rich domain of the GABAA receptor alpha1 subunit.

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Xu, Zhiwen; Fang, Shisong; Shi, Haifeng; Li, Hoiming; Deng, Yiqun; Liao, Yinglei; Wu, Jiun-Ming; Zheng, Hui; Zhu, Huaimin; Chen, Hueih-Min; Tsang, Shui Ying; Xue, Hong

2005-10-01

Structural investigation of GABAA receptors has been limited by difficulties imposed by its trans-membrane-complex nature. In the present study, the topology of a membrane-proximal beta-rich (MPB) domain in the C139-L269 segment of the receptor alpha1 subunit was probed by mapping the benzodiazepine (BZ)-binding and epitopic sites, as well as fluorescence resonance energy transfer (FRET) analysis. Ala-scanning and semiconservative substitutions within this segment revealed the contribution of the phenyl rings of Y160 and Y210, the hydroxy group of S186 and the positive charge on R187 to BZ-binding. FRET with the bound BZ ligand indicated the proximity of Y160, S186, R187, and S206 to the BZ-binding site. On the other hand, epitope-mapping using the monoclonal antibodies (mAbs) against the MPB domain established a clustering of T172, R173, E174, Q196, and T197. Based on the lack of FRET between Trp substitutionally placed at R173 or V198 and bound BZ, this epitope-mapped cluster is located on a separate end of the folded protein from the BZ-binding site. Mutations of the five conserved Cys and Trp residues in the MPB domain gave rise to synergistic and rescuing effects on protein secondary structures and unfolding stability that point to a CCWCW-pentad, reminiscent to the CWC-triad "pin" of immunoglobulin (Ig)-like domains, important for the structural maintenance. These findings, together with secondary structure and fold predictions suggest an anti-parallel beta-strand topology with resemblance to Ig-like fold, having the BZ-binding and the epitopic residues being clustered at two different ends of the fold.

Usefulness of the nonself-self algorithm of HLA epitope immunogenicity in the specificity analysis of monospecific antibodies induced during pregnancy

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Rene J Duquesnoy

2015-05-01

Full Text Available Background HLAMatchmaker is a program to analyze the epitope specificities of HLA antibodies. It considers each HLA allele as a string of eplets. Intralocus and interlocus comparisons between donor and recipient alleles offer a structural assessment of compatibility and an analysis of allele panel reactivity patterns can generate information about epitope specificities of HLA antibodies. However, HLAMatchmaker cannot always generate conclusive interpretations of reactivity patterns of all monospecific antibodies which by definition recognize single epitopes. Hypothesis We have therefore developed a new antibody analysis approach that utilizes the nonself-self algorithm of HLA epitope immunogenicity. It is based in the concept that HLA antibodies originate from B-cells with immunoglobulin receptors to self HLA epitopes on one given allele and which can be activated by epitopes defined by a few nonself residue differences whereas the remainder of the structural epitope of the immunizing allele consists of self residues. Methods Three human monoclonal class I antibodies from HLA typed women sensitized during pregnancy were tested in Ig-binding assays with single alleles on a Luminex platformFindings Three new HLA epitopes were identified; they are defined by combinations of nonself and self residues for one allele of the antibody producer. Conclusion The nonself-self paradigm of HLA epitope immunogenicity offers a second approach to analyze HLA antibody specificities.

Quantitative mass spectrometric analysis of glycoproteins combined with enrichment methods.

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Ahn, Yeong Hee; Kim, Jin Young; Yoo, Jong Shin

2015-01-01

Mass spectrometry (MS) has been a core technology for high sensitive and high-throughput analysis of the enriched glycoproteome in aspects of quantitative assays as well as qualitative profiling of glycoproteins. Because it has been widely recognized that aberrant glycosylation in a glycoprotein may involve in progression of a certain disease, the development of efficient analysis tool for the aberrant glycoproteins is very important for deep understanding about pathological function of the glycoprotein and new biomarker development. This review first describes the protein glycosylation-targeting enrichment technologies mainly employing solid-phase extraction methods such as hydrizide-capturing, lectin-specific capturing, and affinity separation techniques based on porous graphitized carbon, hydrophilic interaction chromatography, or immobilized boronic acid. Second, MS-based quantitative analysis strategies coupled with the protein glycosylation-targeting enrichment technologies, by using a label-free MS, stable isotope-labeling, or targeted multiple reaction monitoring (MRM) MS, are summarized with recent published studies. © 2014 The Authors. Mass Spectrometry Reviews Published by Wiley Periodicals, Inc.

A Method for Determining the Content of Glycoproteins in Biological Samples

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Yang Gao

2016-11-01

Full Text Available The glycoprotein purified from the mycelium extract of Tremella fuciformis was marked with iodine through the iodine substitution reaction. The content of iodine, which is indicative of the amount of the marked tremella glycoprotein (ITG, was detected with Inductively coupled plasma mass spectrometry (ICP-MS. The method was found to be stable, sensitive, and accurate at detecting the content of iodine-substituted glycoprotein, and was used in the quantitative analysis of biological samples, including blood and organs. Different biological samples were collected from rats after oral administration of ITG, and were tested for iodine content by ICP-MS to calculate the amount of ITG in the samples. The results suggested that ICP-MS is a sensitive, stable, and accurate method for detection of iodinated glycoproteins in blood and organs.

Glycoprotein of the wall of sycamore tissue-culture cells.

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Heath, M F; Northcote, D H

1971-12-01

1. A glycoprotein containing a large amount of hydroxyproline is present in the cell walls of sycamore callus cells. This protein is insoluble and remained in the alpha-cellulose when a mild separation procedure was used to obtain the polysaccharide fractions of the wall. The glycoprotein contained a high proportion of arabinose and galactose. 2. Soluble glycopeptides were prepared from the alpha-cellulose fraction when peptide bonds were broken by hydrazinolysis. The soluble material was fractionated by gel filtration and one glycopeptide was further purified by electrophoresis; it had a composition of 10% hydroxyproline, 35% arabinose and 55% galactose, and each hydroxyproline residue carried a glycosyl radical so that the oligosaccharides on the glycopeptide had an average degree of polymerization of 9. 3. The extraction of the glycopeptides was achieved without cleavage of glycosyl bonds, so that the glycoprotein cannot act as a covalent cross-link between the major polysaccharides of the wall. 4. The wall protein approximates in conformation to polyhydroxyproline and therefore it probably has similar physicochemical properties to polyhydroxyproline. This is discussed in relation to the function of the glycoprotein and its effect on the physical and chemical nature of the wall.

MuPeXI: prediction of neo-epitopes from tumor sequencing data

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Bjerregaard, Anne-Mette; Nielsen, Morten; Hadrup, Sine Reker

2017-01-01

Personalization of immunotherapies such as cancer vaccines and adoptive T cell therapy depends on identification of patient-specific neo-epitopes that can be specifically targeted. MuPeXI, the mutant peptide extractor and informer, is a program to identify tumor-specific peptides and assess...... their potential to be neo-epitopes. The program input is a file with somatic mutation calls, a list of HLA types, and optionally a gene expression profile. The output is a table with all tumor-specific peptides derived from nucleotide substitutions, insertions, and deletions, along with comprehensive annotation...

Global identification of prokaryotic glycoproteins based on an Escherichia coli proteome microarray.

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Zong-Xiu Wang

Full Text Available Glycosylation is one of the most abundant protein posttranslational modifications. Protein glycosylation plays important roles not only in eukaryotes but also in prokaryotes. To further understand the roles of protein glycosylation in prokaryotes, we developed a lectin binding assay to screen glycoproteins on an Escherichia coli proteome microarray containing 4,256 affinity-purified E.coli proteins. Twenty-three E.coli proteins that bound Wheat-Germ Agglutinin (WGA were identified. PANTHER protein classification analysis showed that these glycoprotein candidates were highly enriched in metabolic process and catalytic activity classes. One sub-network centered on deoxyribonuclease I (sbcB was identified. Bioinformatics analysis suggests that prokaryotic protein glycosylation may play roles in nucleotide and nucleic acid metabolism. Fifteen of the 23 glycoprotein candidates were validated by lectin (WGA staining, thereby increasing the number of validated E. coli glycoproteins from 3 to 18. By cataloguing glycoproteins in E.coli, our study greatly extends our understanding of protein glycosylation in prokaryotes.

Thyroid hormone upregulates zinc-α2-glycoprotein production in the liver but not in adipose tissue.

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Rafael Simó

Full Text Available Overproduction of zinc-α2-glycoprotein by adipose tissue is crucial in accounting for the lipolysis occurring in cancer cachexia of certain malignant tumors. The main aim of this study was to explore whether thyroid hormone could enhance zinc-α2-glycoprotein production in adipose tissue. In addition, the regulation of zinc-α2-glycoprotein by thyroid hormone in the liver was investigated. We performed in vitro (HepG2 cells and primary human adipocytes and in vivo (C57BL6/mice experiments addressed to examine the effect of thyroid hormone on zinc-α2-glycoprotein production (mRNA and protein levels in liver and visceral adipose tissue. We also measured the zinc-α2-glycoprotein serum levels in a cohort of patients before and after controlling their hyperthyroidism. Our results showed that thyroid hormone up-regulates zinc-α2-glycoprotein production in HepG2 cells in a dose-dependent manner. In addition, the zinc-α2-glycoprotein proximal promoter contains functional thyroid hormone receptor binding sites that respond to thyroid hormone treatment in luciferase reporter gene assays in HepG2 cells. Furthermore, zinc-α2-glycoprotein induced lipolysis in HepG2 in a dose-dependent manner. Our in vivo experiments in mice confirmed the up-regulation of zinc-α2-glycoprotein induced by thyroid hormone in the liver, thus leading to a significant increase in zinc-α2-glycoprotein circulating levels. However, thyroid hormone did not regulate zinc-α2-glycoprotein production in either human or mouse adipocytes. Finally, in patients with hyperthyroidism a significant reduction of zinc-α2-glycoprotein serum levels was detected after treatment but was unrelated to body weight changes. We conclude that thyroid hormone up-regulates the production of zinc-α2-glycoprotein in the liver but not in the adipose tissue. The neutral effect of thyroid hormones on zinc-α2-glycoprotein expression in adipose tissue could be the reason why zinc-α2-glycoprotein is not

Thyroid hormone upregulates zinc-α2-glycoprotein production in the liver but not in adipose tissue.

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Simó, Rafael; Hernández, Cristina; Sáez-López, Cristina; Soldevila, Berta; Puig-Domingo, Manel; Selva, David M

2014-01-01

Overproduction of zinc-α2-glycoprotein by adipose tissue is crucial in accounting for the lipolysis occurring in cancer cachexia of certain malignant tumors. The main aim of this study was to explore whether thyroid hormone could enhance zinc-α2-glycoprotein production in adipose tissue. In addition, the regulation of zinc-α2-glycoprotein by thyroid hormone in the liver was investigated. We performed in vitro (HepG2 cells and primary human adipocytes) and in vivo (C57BL6/mice) experiments addressed to examine the effect of thyroid hormone on zinc-α2-glycoprotein production (mRNA and protein levels) in liver and visceral adipose tissue. We also measured the zinc-α2-glycoprotein serum levels in a cohort of patients before and after controlling their hyperthyroidism. Our results showed that thyroid hormone up-regulates zinc-α2-glycoprotein production in HepG2 cells in a dose-dependent manner. In addition, the zinc-α2-glycoprotein proximal promoter contains functional thyroid hormone receptor binding sites that respond to thyroid hormone treatment in luciferase reporter gene assays in HepG2 cells. Furthermore, zinc-α2-glycoprotein induced lipolysis in HepG2 in a dose-dependent manner. Our in vivo experiments in mice confirmed the up-regulation of zinc-α2-glycoprotein induced by thyroid hormone in the liver, thus leading to a significant increase in zinc-α2-glycoprotein circulating levels. However, thyroid hormone did not regulate zinc-α2-glycoprotein production in either human or mouse adipocytes. Finally, in patients with hyperthyroidism a significant reduction of zinc-α2-glycoprotein serum levels was detected after treatment but was unrelated to body weight changes. We conclude that thyroid hormone up-regulates the production of zinc-α2-glycoprotein in the liver but not in the adipose tissue. The neutral effect of thyroid hormones on zinc-α2-glycoprotein expression in adipose tissue could be the reason why zinc-α2-glycoprotein is not related to weight

Epitope-based peptide vaccine design and target site depiction against Ebola viruses: an immunoinformatics study.

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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.

3,3′,4,4′,5-Pentachlorobiphenyl Inhibits Drug Efflux Through P-Glycoprotein in KB-3 Cells Expressing Mutant Human P-Glycoprotein

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Hiroshi Fujise

2004-01-01

Full Text Available The effects on the drug efflux of 3,3′,4,4′,5-pentachlorobiphenyl (PCB-126, the most toxic of all coplanar polychlorinated biphenyls (Co-PCBs, were examined in KB-3 cells expressing human wild-type and mutant P-glycoprotein in which the 61st amino acid was substituted for serine or phenylalanine (KB3-Phe61. In the cells expressing P-glycoproteins, accumulations of vinblastine and colchicine decreased form 85% to 92% and from 62% to 91%, respectively, and the drug tolerances for these chemicals were increased. In KB3-Phe61, the decreases in drug accumulation were inhibited by adding PCB-126 in a way similar to that with cyclosporine A: by adding 1 μM PCB-126, the accumulations of vinblastine and colchicine increased up to 3.3- and 2.3-fold, respectively. It is suggested that PCB-126 decreased the drug efflux by inhibiting the P-glycoprotein in KB3-Phe61. Since there were various P-glycoproteins and many congeners of Co-PCBs, this inhibition has to be considered a new cause of the toxic effects of Co-PCBs.

Prediction of conserved sites and domains in glycoproteins B, C and D of herpes viruses.

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Rasheed, Muhammad Asif; Ansari, Abdur Rahman; Ihsan, Awais; Navid, Muhammad Tariq; Ur-Rehman, Shahid; Raza, Sohail

2018-03-01

Glycoprotein B (gB), C (gC) and D (gD) of herpes simplex virus are implicated in virus adsorption and penetration. The gB, gC and gD are glycoproteins for different processes of virus binding and attachment to the host cells. Moreover, their expression is necessary and sufficient to induce cell fusion in the absence of other glycoproteins. Egress of herpes simplex virus (HSV) and other herpes viruses from cells involves extensive modification of cellular membranes and sequential envelopment, de-envelopment and re-envelopment steps. Viral glycoproteins are important in these processes, and frequently two or more glycoproteins can largely suffice in any step. Hence, we target the 3 important glycoproteins (B, C and D) of eight different herpes viruses of different species. These species include human (HSV1 and 2), bovine (BHV1), equine (EHV1 and 4), chicken (ILT1 and MDV2) and pig (PRV1). By applying different bioinformatics tools, we highlighted the conserved sites in these glycoproteins which might be most significant regarding attachment and infection of the viruses. Moreover the conserved domains in these glycoproteins are also highlighted. From this study, we will able to analyze the role of different viral glycoproteins of different species during herpes virus adsorption and penetration. Moreover, this study will help to construct the antivirals that target the glycoproteins of different herpes viruses. Copyright © 2018 Elsevier Ltd. All rights reserved.

Characterization of CD4 T Cell Epitopes of Infliximab and Rituximab Identified from Healthy Donors

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Moustafa Hamze

2017-05-01

Full Text Available The chimeric antibodies anti-CD20 rituximab (Rtx and anti-TNFα infliximab (Ifx induce antidrug antibodies (ADAs in many patients with inflammatory diseases. Because of the key role of CD4 T lymphocytes in the initiation of antibody responses, we localized the CD4 T cell epitopes of Rtx and Ifx. With the perspective to anticipate immunogenicity of therapeutic antibodies, identification of the CD4 T cell epitopes was performed using cells collected in healthy donors. Nine T cell epitopes were identified in the variable chains of both antibodies by deriving CD4 T cell lines raised against either Rtx or Ifx. The T cell epitopes often exhibited a good affinity for human leukocyte antigen (HLA-DR molecules and were part of the peptides identified by MHC-associated peptide proteomics assay from HLA-DR molecules of dendritic cells (DCs loaded with the antibodies. Two-third of the T cell epitopes identified from the healthy donors stimulated peripheral blood mononuclear cells from patients having developed ADAs against Rtx or Ifx and promoted the secretion of a diversity of cytokines. These data emphasize the predictive value of evaluating the T cell repertoire of healthy donors and the composition of peptides bound to HLA-DR of DCs to anticipate and prevent immunogenicity of therapeutic antibodies.

Optimal selection of epitopes for TXP-immunoaffinity mass spectrometry

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Joos Thomas

2010-06-01

Full Text Available Abstract Background 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. Results 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. Conclusions 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.

Increased platelet expression of glycoprotein IIIa following aspirin treatment in aspirin-resistant but not aspirin-sensitive subjects

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Floyd, Christopher N; Goodman, Timothy; Becker, Silke; Chen, Nan; Mustafa, Agnesa; Schofield, Emma; Campbell, James; Ward, Malcolm; Sharma, Pankaj; Ferro, Albert

2014-01-01

Aims Aspirin is widely used as an anti-platelet agent for cardiovascular prophylaxis. Despite aspirin treatment, many patients experience recurrent thrombotic events, and aspirin resistance may contribute to this. We examined the prevalence of aspirin resistance in a healthy population, and investigated whether the platelet proteome differed in aspirin-resistant subjects. Methods Ninety-three healthy subjects received aspirin 300 mg daily for 28 days. Before and at the end of treatment, urine was taken to determine 11-dehydrothromboxane B2, and blood was taken to measure arachidonic acid (AA)-induced aggregation of platelet-rich plasma and to interrogate the platelet proteome by mass spectrometric analysis with further confirmation of findings using Western blotting. Results In two of the 93 subjects, neither AA-induced aggregation nor urinary 11-dehydrothromboxane B2 was effectively suppressed by aspirin, despite measurable plasma salicylate concentrations, suggesting the presence of true aspirin resistance. Despite no detectable differences in the platelet proteome at baseline, following aspirin a marked increase was seen in platelet glycoprotein IIIa expression in the aspirin-resistant but not aspirin-sensitive subjects. An increase in platelet glycoprotein IIIa expression with aspirin resistance was confirmed in a separate cohort of 17 patients with stable coronary artery disease on long term aspirin treatment, four of whom exhibited aspirin resistance. Conclusions In a healthy population, true aspirin resistance is uncommon but exists. Resistance is associated with an increase in platelet glycoprotein IIIa expression in response to aspirin. These data shed new light on the mechanism of aspirin resistance, and provide the potential to identify aspirin-resistant subjects using a novel biomarker. PMID:25099258

Glycoprotein and proteoglycan techniques

International Nuclear Information System (INIS)

Beeley, J.G.

1985-01-01

The aim of this book is to describe techniques which can be used to answer some of the basic questions about glycosylated proteins. Methods are discussed for isolation, compositional analysis, and for determination of the primary structure of carbohydrate units and the nature of protein-carbohydrate linkages of glycoproteins and proteoglycans. High resolution NMR is considered, as well as radioactive labelling techniques. (Auth.)

HIV-1 envelope glycoprotein

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Caulfield, Michael; Cupo, Albert; Dean, Hansi; Hoffenberg, Simon; King, C. Richter; Klasse, P. J.; Marozsan, Andre; Moore, John P.; Sanders, Rogier W.; Ward, Andrew; Wilson, Ian; Julien, Jean-Philippe

2017-08-22

The present application relates to novel HIV-1 envelope glycoproteins, which may be utilized as HIV-1 vaccine immunogens, and antigens for crystallization, electron microscopy and other biophysical, biochemical and immunological studies for the identification of broad neutralizing antibodies. The present invention encompasses the preparation and purification of immunogenic compositions, which are formulated into the vaccines of the present invention.

RB4CD12 epitope expression and heparan sulfate disaccharide composition in brain vasculature.

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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.

Identification of linear B-cell epitopes on goose parvovirus non-structural protein.

Science.gov (United States)

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.

Identification of two novel rabbit hemorrhagic disease virus (RHDV) B cell epitopes and evaluation of its immunoprotection against RHDV.

Science.gov (United States)

DeSheng, Kong; HuaiRan, Liu; JiaSen, Liu; Zuo, Yu; Qian, Jiang; DongChun, Guo; XiaoLiang, Hu; FengJie, Wang; QianQian, Huang; LianDong, Qu

2015-07-01

The VP60 protein of rabbit hemorrhagic disease virus (RHDV) is a structural protein with important roles in viral replication and assembly. In this study, we immunized BALB/c mice with the RHDV-TP strain. Six monoclonal antibodies (mAbs) were selected and characterized by enzyme-linked immunosorbent assay, Western blotting, and indirectly immunofluorescence analysis (IFA). All six mAbs (AD4, AG10, BC9, BE8, BH3, and DE2) had positive reactions with recombinant VP60 as analyzed by IFA, but only two (AG10 and DE2) reacted with denatured RHDV by Western blotting. Fifty-four partially overlapping fragments of the VP60 gene were expressed with His or Glutathione S-transferase (GST) tags to identify the epitopes recognized by AG10 and DE2. These two epitopes were located at the C-terminal of VP60 and were longer (64 and 53 amino acids, respectively) than normal B cell epitopes. However, both AG10 and DE2 also interacted with RHDV2 VP60 expressed in insect cells. Amino acid alignments of the AG10 and DE2 epitope regions between RHDV and RHDV2 VP60 indicated several mutations, suggesting that the epitopes recognized by the mAbs AG10 and DE2 were discontinuous. Epitope immunogenicity was evaluated by inoculating specific pathogen-free rabbits with saline, purified DE2 epitope, or RHDV inactive vaccine. Rabbits immunized with the DE2 epitope developed high levels of RHDV-specific antibodies but no cellular immune response and died after challenge with RHDV-HYD isolate. Despite their lack of neutralizing activity, these mAb reagents and epitopes may have useful clinical applications and will be valuable tools in further studies of the structure and function of the RHDV VP60 protein.

Identifikasi epitop dari Streptococcus mutans terhadap sekretori Imunoglobulin A saliva (The identification of Streptococcus mutans epitopes to secretory Immunoglobulin A saliva

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Anita Yuliati

2005-09-01

Full Text Available S. mutans is one of the etiology agent of dental caries, these bacteria have a surface protein of about 185 kDa named Ag I/II. The secretory of sIgA saliva to Ag I/II of S.mutans has shown to be able to prevent colonization in human oral cavity. Peptides derived from the 824 to 853 residues of the P region of antigen I/II S. mutans related to the pathogenesis of dental caries. The aim of this study was to identify the overlapping sequence of amino acids (epitope derived from the 624 to 853 residues of P of antigen I/II S. mutans to sIgA saliva on caries and caries-free subject in a observational cross sectional study. The P region of antigen I/II S.mutans was cut into 22 peptides of 9 mer sequences with an overlapping of 8 mer and an offset of 1 mer, synthesized on polyethylene pins and tested for the reactivity with an ELISA indirect method to sIgA saliva on caries and caries-free subject. The results of this study showed that amino acid sequences with TPPVKP (832–837 and TAPTKPTY (838–845 were reactive to sIgA saliva on caries and caries-free subject. The conclusion of this study was that the overlapping common sequence of amino acid (epitopes corresponding to TPPVKP (832–837 and TAPTKPTY (838–845 was identified as caries marker epitopes in human.

[Chemical modification of allergen leading to changes in its epitopic activity].

Science.gov (United States)

Babakhin, A A; Gushchin, I S; Andreev, S M; Petrukhina, A I; Viler, A V; Stokinger, B; Nolte, G; Dubuske, L M; Khaitov, R M; Petrpv, R V

1999-01-01

Modification of a model allergen ovalbumin (OA) with succinylation led to a decrease of its allergenicity measured by passive cutaneous anaphylaxis reaction, RAST inhibition assay and basophil histamine release. Modified OA stimulated OA-specific T-cell hybrid 3DO-548 to produce IL-2 at the same level as in case of non-modified OA. Modified OA did not induce anti-OA IgE, but did induce anti-OA IgG antibodies. This approach to chemical modification of allergen-selective blockade of B-cell epitopes while not affecting T-cell epitopes suggests new opportunities in creation of safe and effective allergovaccines.

Crystallization and preliminary X-ray analysis of Chandipura virus glycoprotein G

International Nuclear Information System (INIS)

Baquero, Eduard; Buonocore, Linda; Rose, John K.; Bressanelli, Stéphane; Gaudin, Yves; Albertini, Aurélie A.

2012-01-01

Chandipura virus glycoprotein ectodomain (Gth) was purified and crystallized at pH 7.5. X-ray diffraction data set was collected to a resolution of 3.1 Å. Fusion in members of the Rhabdoviridae virus family is mediated by the G glycoprotein. At low pH, the G glycoprotein catalyzes fusion between viral and endosomal membranes by undergoing a major conformational change from a pre-fusion trimer to a post-fusion trimer. The structure of the G glycoprotein from vesicular stomatitis virus (VSV G), the prototype of Vesiculovirus, has recently been solved in its trimeric pre-fusion and post-fusion conformations; however, little is known about the structural details of the transition. In this work, a soluble form of the ectodomain of Chandipura virus G glycoprotein (CHAV G th ) was purified using limited proteolysis of purified virus; this soluble ectodomain was also crystallized. This protein shares 41% amino-acid identity with VSV G and thus its structure could provide further clues about the structural transition of rhabdoviral glycoproteins induced by low pH. Crystals of CHAV G th obtained at pH 7.5 diffracted X-rays to 3.1 Å resolution. These crystals belonged to the orthorhombic space group P2 1 2 1 2, with unit-cell parameters a = 150.3, b = 228.2, c = 78.8 Å. Preliminary analysis of the data based on the space group and the self-rotation function indicated that there was no trimeric association of the protomers. This unusual oligomeric status could result from the presence of fusion intermediates in the crystal

Synthetic glycopeptides and glycoproteins with applications in biological research

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Ulrika Westerlind

2012-05-01

Full Text Available Over the past few years, synthetic methods for the preparation of complex glycopeptides have been drastically improved. The need for homogenous glycopeptides and glycoproteins with defined chemical structures to study diverse biological phenomena further enhances the development of methodologies. Selected recent advances in synthesis and applications, in which glycopeptides or glycoproteins serve as tools for biological studies, are reviewed. The importance of specific antibodies directed to the glycan part, as well as the peptide backbone has been realized during the development of synthetic glycopeptide-based anti-tumor vaccines. The fine-tuning of native chemical ligation (NCL, expressed protein ligation (EPL, and chemoenzymatic glycosylation techniques have all together enabled the synthesis of functional glycoproteins. The synthesis of structurally defined, complex glycopeptides or glyco-clusters presented on natural peptide backbones, or mimics thereof, offer further possibilities to study protein-binding events.

Promiscuous prediction and conservancy analysis of CTL binding epitopes of HCV 3a viral proteome from Punjab Pakistan: an In Silico Approach

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Idrees Muhammad

2011-02-01

Full Text Available Abstract Background HCV is a positive sense RNA virus affecting approximately 180 million people world wide and about 10 million Pakistani populations. HCV genotype 3a is the major cause of infection in Pakistani population. One of the major problems of HCV infection especially in the developing countries that limits the limits the antiviral therapy is the long term treatment, high dosage and side effects. Studies of antigenic epitopes of viral sequences of a specific origin can provide an effective way to overcome the mutation rate and to determine the promiscuous binders to be used for epitope based subunit vaccine design. An in silico approach was applied for the analysis of entire HCV proteome of Pakistani origin, aimed to identify the viral epitopes and their conservancy in HCV genotypes 1, 2 and 3 of diverse origin. Results Immunoinformatic tools were applied for the predictive analysis of HCV 3a antigenic epitopes of Pakistani origin. All the predicted epitopes were then subjected for their conservancy analysis in HCV genotypes 1, 2 and 3 of diverse origin (worldwide. Using freely available web servers, 150 MHC II epitopes were predicted as promiscuous binders against 51 subjected alleles. E2 protein represented the 20% of all the predicted MHC II epitopes. 75.33% of the predicted MHC II epitopes were (77-100% conserve in genotype 3; 47.33% and 40.66% in genotype 1 and 2 respectively. 69 MHC I epitopes were predicted as promiscuous binders against 47 subjected alleles. NS4b represented 26% of all the MHC I predicted epitopes. Significantly higher epitope conservancy was represented by genotype 3 i.e. 78.26% and 21.05% for genotype 1 and 2. Conclusions The study revealed comprehensive catalogue of potential HCV derived CTL epitopes from viral proteome of Pakistan origin. A considerable number of predicted epitopes were found to be conserved in different HCV genotype. However, the number of conserved epitopes in HCV genotype 3 was

The glycoproteins of Marburg and Ebola virus and their potential roles in pathogenesis.

Science.gov (United States)

Feldmann, H; Volchkov, V E; Volchkova, V A; Klenk, H D

1999-01-01

Filoviruses cause systemic infections that can lead to severe hemorrhagic fever in human and non-human primates. The primary target of the virus appears to be the mononuclear phagocytic system. As the virus spreads through the organism, the spectrum of target cells increases to include endothelial cells, fibroblasts, hepatocytes, and many other cells. There is evidence that the filovirus glycoprotein plays an important role in cell tropism, spread of infection, and pathogenicity. Biosynthesis of the glycoprotein forming the spikes on the virion surface involves cleavage by the host cell protease furin into two disulfide linked subunits GP1 and GP2. GP1 is also shed in soluble form from infected cells. Different strains of Ebola virus show variations in the cleavability of the glycoprotein, that may account for differences in pathogenicity, as has been observed with influenza viruses and paramyxoviruses. Expression of the spike glycoprotein of Ebola virus, but not of Marburg virus, requires transcriptional editing. Unedited GP mRNA yields the nonstructural glycoprotein sGP, which is secreted extensively from infected cells. Whether the soluble glycoproteins GP1 and sGP interfere with the humoral immune response and other defense mechanisms remains to be determined.

Expression of Hemagglutinin–Neuraminidase and fusion epitopes of Newcastle Disease Virus in transgenic tobacco

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Amir Ghaffar Shahriari

2016-07-01

Conclusion: Developments in genetic engineering have led to plant-based systems for recombinant vaccine production. In this research, tobacco plant was used to express F and HN epitopes of NDV. Our results indicate that for the production of recombinant vaccine, it is a novel strategy to use concatenated epitopes without their genetic fusion onto larger scaffold structure such as viral coat protein.

Identification of one B-cell epitope from NS1 protein of duck Tembusu virus with monoclonal antibodies.

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Jinfeng Ti

Full Text Available This study describes the identification of one linear B-cell epitope on TMUV NS1 protein with monoclonal antibody (mAb 3G2 by indirect enzyme-linked immunosorbent assay (ELISA. In this study, NS1 protein was expressed in prokaryotic expression system and purified. One mAb against NS1 protein was generated from Balb/c mice immunized with recombinant protein NS1. A set of 35 partially-overlapping polypeptides covering the entire NS1 protein was expressed with PGEX-6P-1 vector and screened with mAb 3G2. One polypeptide against the mAb was acquired and identified by indirect ELISA and western-blot. To map the epitope accurately, one or two amino acid residues were removed from the carboxy and amino terminal of polypeptide sequentially. A series of truncated oligopeptides were expressed and purified. The minimal determinant of the linear B cell epitope was recognized and identified with mAb 3G2. The accurate linear B-cell epitope was 269DEKEIV274 located in NS1 protein. Furthermore, sequence alignment showed that the epitope was highly conserved and specific among TMUV strains and other flavivirus respectively. The linear B-cell epitope of TMUV NS1 protein could benefit the development of new vaccines and diagnostic assays.

Epitope mapping of the domains of human angiotensin converting enzyme.

Science.gov (United States)

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.

Three-dimensionally Functionalized Reverse Phase Glycoprotein Array for Cancer Biomarker Discovery and Validation

OpenAIRE

Pan, Li; Aguilar, Hillary Andaluz; Wang, Linna; Iliuk, Anton; Tao, W. Andy

2016-01-01

Glycoproteins have vast structural diversity which plays an important role in many biological processes and have great potential as disease biomarkers. Here we report a novel functionalized reverse phase protein array (RPPA), termed polymer-based reverse phase GlycoProtein Array (polyGPA), to specifically capture and profile glycoproteomes, and validate glycoproteins. Nitrocellulose membrane functionalized with globular hydroxyaminodendrimers was used to covalently capture pre-oxidized glycan...

Multi-level Strategy for Identifying Proteasome-Catalyzed Spliced Epitopes Targeted by CD8+ T Cells during Bacterial Infection

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Anouk C.M. Platteel

2017-08-01

Full Text Available Proteasome-catalyzed peptide splicing (PCPS generates peptides that are presented by MHC class I molecules, but because their identification is challenging, the immunological relevance of spliced peptides remains unclear. Here, we developed a reverse immunology-based multi-level approach to identify proteasome-generated spliced epitopes. Applying this strategy to a murine Listeria monocytogenes infection model, we identified two spliced epitopes within the secreted bacterial phospholipase PlcB that primed antigen-specific CD8+ T cells in L. monocytogenes-infected mice. While reacting to the spliced epitopes, these CD8+ T cells failed to recognize the non-spliced peptide parts in the context of their natural flanking sequences. Thus, we here show that PCPS expands the CD8+ T cell response against L. monocytogenes by exposing spliced epitopes on the cell surface. Moreover, our multi-level strategy opens up opportunities to systematically investigate proteins for spliced epitope candidates and thus strategies for immunotherapies or vaccine design.

Filamentous fungi as production organisms for glycoproteins of bio-medical interest

NARCIS (Netherlands)

Maras, M.; Die, I. van; Contreras, R.; Hondel, C.A.M.J.J. van den

1999-01-01

Filamentous fungi are commonly used in the fermentation industry for large scale production of glycoproteins. Several of these proteins can be produced in concentrations up to 20-40 g per litre. The production of heterologous glycoproteins is at least one or two orders of magnitude lower but

Co-treatment by docetaxel and vinblastine breaks down P-glycoprotein mediated chemo-resistance

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Mahsa Mohseni

2016-03-01

Results: Combination treatment of the cells with docetaxel and vinblastine decreased the IC50 values for docetaxel from (30±3.1 to (15±2.6 nM and for vinblastine from (30±5.9 to (5±5.6 nM (P≤0.05.               P-glycoprotein mRNA expression level showed a significant up-regulation in the cells incubated with each drug alone (P≤0.001. Incubation of the cells with combined concentrations of both agents neutralized P-glycoprotein overexpression (P≤0.05. Adding verapamil, a P-glycoprotein inhibitor caused a further increase in the percentage of apoptotic cells when the cells were treated with both agents.  Conclusion:Our results suggest that combination therapy along with P-glycoprotein inhibition can be considered as a novel approach to improve the efficacy of chemotherapeutics in cancer patients with high P-glycoprotein expression.

Interaction of an immunodominant epitope with Ia molecules in T-cell activation

DEFF Research Database (Denmark)

Adorini, L; Sette, A; Buus, S

1988-01-01

The amino acid sequence corresponding to residues 107-116 of hen egg-white lysozyme (HEL) has been identified as containing an immunodominant T-cell epitope recognized in association with the I-Ed molecule. The immunodominance of this epitope in HEL-primed H-2d mice was demonstrated by analysis o......-120)-peptide was found to be immunogenic in H-2d mice. Thus, a single semiconservative substitution drastically reduces binding capacity and abolishes immunogenicity, suggesting that a strict correlation exists between binding of a peptide to Ia molecules and its immunogenicity....

Identification of NY-BR-1-specific CD4(+) T cell epitopes using HLA-transgenic mice.

Science.gov (United States)

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.

Availability of the B beta(15-21) epitope on cross-linked human fibrin and its plasmic degradation products

Science.gov (United States)

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.

In Silico Perspectives on the Prediction of the PLP's Epitopes involved in Multiple Sclerosis.

Science.gov (United States)

Zamanzadeh, Zahra; Ataei, Mitra; Nabavi, Seyed Massood; Ahangari, Ghasem; Sadeghi, Mehdi; Sanati, Mohammad Hosein

2017-03-01

Multiple sclerosis (MS) is the most common autoimmune disease of the central nervous system (CNS). The main cause of the MS is yet to be revealed, but the most probable theory is based on the molecular mimicry that concludes some infections in the activation of T cells against brain auto-antigens that initiate the disease cascade. The Purpose of this research is the prediction of the auto-antigen potency of the myelin proteolipid protein (PLP) in multiple sclerosis. As there wasn't any tertiary structure of PLP available in the Protein Data Bank (PDB) and in order to characterize the structural properties of the protein, we modeled this protein using prediction servers. Meta prediction method, as a new perspective in silico , was performed to fi nd PLPs epitopes. For this purpose, several T cell epitope prediction web servers were used to predict PLPs epitopes against Human Leukocyte Antigens (HLA). The overlap regions, as were predicted by most web servers were selected as immunogenic epitopes and were subjected to the BLASTP against microorganisms. Three common regions, AA 58-74 , AA 161-177 , and AA 238-254 were detected as immunodominant regions through meta-prediction. Investigating peptides with more than 50% similarity to that of candidate epitope AA 58-74 in bacteria showed a similar peptide in bacteria (mainly consistent with that of clostridium and mycobacterium) and spike protein of Alphacoronavirus 1, Canine coronavirus, and Feline coronavirus. These results suggest that cross reaction of the immune system to PLP may have originated from a bacteria or viral infection, and therefore molecular mimicry might have an important role in the progression of MS. Through reliable and accurate prediction of the consensus epitopes, it is not necessary to synthesize all PLP fragments and examine their immunogenicity experimentally ( in vitro ). In this study, the best encephalitogenic antigens were predicted based on bioinformatics tools that may provide reliable

[Construction and characterization of an epitope-mutated Asia 1 type foot-and-mouth disease virus].

Science.gov (United States)

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.

New insights into non-conventional epitopes as T cell targets: The missing link for breaking immune tolerance in autoimmune disease?

Science.gov (United States)

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.

Glycoproteins and sialyl transferase of human B lymphoblastoid cell lines

International Nuclear Information System (INIS)

Lui, S.W.L.; Ng, M.H.

1980-01-01

We used two radiolabeling methods to study glycoproteins on the surface of lymphoblastoid cells. One of the methods affects tritiation of residues which are oxidized with galactose oxidase and the other causes tritiation of neuraminic acid residues. This approach was shown to allow a better resolution of cell surface glycoproteins than if either method were used alone. Glycoproteins of B 1 - 19 cells which harbor the Epstein-Barr virus genomes were compared with those of its parental cell line, BJAB, which does not harbor the viral genomes. These studies did not reveal a unique viral protein. A 28,000 mol. wt. glycoprotein was found to be the most prominent neuraminic acidlabeled product of B 1 - 19 cells and also of the two other cell lines, Raji and Ly38, which harbor the EBV genomes. A similar molecular weight species from BJAB cells identified by galactose oxidase labeling might be deficient in neuraminic acid residues as it was poorly labeled by the periodate oxidation method. The neuraminic acid content and level of sialyl transferase of BJAB cells were found to be lower than those of the other cell lines studied. (auth.)

Effects of chronic ethanol administration on hepatic glycoprotein secretion in the rat

International Nuclear Information System (INIS)

Sorrell, M.F.; Nauss, J.M.; Donohue, T.M. Jr.; Tuma, D.J.

1983-01-01

The effects of chronic ethanol feeding on protein and glycoprotein synthesis and secretion were studied in rat liver slices. Liver slices from rats fed ethanol for 4-5 wk showed a decreased ability to incorporate [ 14 C]glucosamine into medium trichloracetic acid-precipitable proteins when compared to the pair-fed controls; however, the labeling of hepatocellular glycoproteins was unaffected by chronic ethanol treatment. Immunoprecipitation of radiolabeled secretory (serum) glycoproteins with antiserum against rat serum proteins showed a similar marked inhibition in the appearance of glucosamine-labeled proteins in the medium of slices from ethanol-fed rats. Minimal effects, however, were noted in the labeling of intracellular secretory glycoproteins. Protein synthesis, as determined by measuring [ 14 C]leucine incorporation into medium and liver proteins, was decreased in liver slices from ethanol-fed rats as compared to the pair-fed controls. This was the case for both total proteins as well as immunoprecipitable secretory proteins, although the labeling of secretory proteins retained in the liver slices was reduced to a lesser extent than total radiolabeled hepatic proteins. When the terminal sugar, [ 14 C]fucose, was employed as a precursor in order to more closely focus on the final steps of hepatic glycoprotein secretion, liver slices obtained from chronic ethanol-fed rats exhibited impaired secretion of fucose-labeled proteins into the medium. When ethanol (5 or 10 mM) was added to the incubation medium containing liver slices from the ethanol-fed rats, the alterations in protein and glycoprotein synthesis and secretion caused by the chronic ethanol treatment were further potentiated. The results of this study indicate that liver slices prepared from chronic ethanol-fed rats exhibit both impaired synthesis and secretion of proteins and glycoproteins, and these defects are further potentiated by acute ethanol administration

Identification, isolation, and N-terminal sequencing of style glycoproteins associated with self-incompatibility in Nicotiana alata.

Science.gov (United States)

Jahnen, W; Batterham, M P; Clarke, A E; Moritz, R L; Simpson, R J

1989-05-01

S-Gene-associated glycoproteins (S-glycoproteins) from styles of Nicotiana alata, identified by non-equilibrium two-dimensional electrophoresis, were purified by cation exchange fast protein liquid chromatography with yields of 0.5 to 8 micrograms of protein per style, depending on the S-genotype of the plant. The method relies on the highly basic nature of the S-glycoproteins. The elution profiles of the different S-glycoproteins from the fast protein liquid chromatography column were characteristic of each S-glycoprotein, and could be used to establish the S-genotype of plants in outbreeding populations. In all cases, the S-genotype predicted from the style protein profile corresponded to that predicted from DNA gel blot analysis using S-allele-specific DNA probes and to that established by conventional breeding tests. Amino-terminal sequences of five purified S-glycoproteins showed a high degree of homology with the previously published sequences of N. alata and Lycopersicon esculentum S-glycoproteins.

High-throughput sequencing enhanced phage display enables the identification of patient-specific epitope motifs in serum

DEFF Research Database (Denmark)

Christiansen, Anders; Kringelum, Jens Vindahl; Hansen, Christian Skjødt

2015-01-01

of the bioinformatic approach was demonstrated by identifying epitopes of a prominent peanut allergen, Ara h 1, in sera from patients with severe peanut allergy. The identified epitopes were confirmed by high-density peptide micro-arrays. The present study demonstrates that high-throughput sequencing can empower phage...

A dominant EV71-specific CD4+ T cell epitope is highly conserved among human enteroviruses.

Directory of Open Access Journals (Sweden)

Ruicheng Wei

Full Text Available CD4+ T cell-mediated immunity plays a central role in determining the immunopathogenesis of viral infections. However, the role of CD4+ T cells in EV71 infection, which causes hand, foot and mouth disease (HFMD, has yet to be elucidated. We applied a sophisticated method to identify promiscuous CD4+ T cell epitopes contained within the sequence of the EV71 polyprotein. Fifteen epitopes were identified, and three of them are dominant ones. The most dominant epitope is highly conserved among enterovirus species, including HFMD-related coxsackieviruses, HFMD-unrelated echoviruses and polioviruses. Furthermore, the CD4+ T cells specific to the epitope indeed cross-reacted with the homolog of poliovirus 3 Sabin. Our findings imply that CD4+ T cell responses to poliovirus following vaccination, or to other enteroviruses to which individuals may be exposed in early childhood, may have a modulating effect on subsequent CD4+ T cell response to EV71 infection or vaccine.

Epitope design of L1 protein for vaccine production against Human Papilloma Virus types 16 and 18.

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Baidya, Sunanda; Das, Rasel; Kabir, Md Golam; Arifuzzaman, Md

2017-01-01

Cervical cancer accounts for about two-thirds of all cancer cases linked etiologically to Human Papilloma Virus (HPV). 15 oncogenic HPV types can cause cervical cancer, of which HPV16 and HPV18 combinedly account for about 70% of it. So, effective epitope design for the clinically relevant HPV types 16 and 18 would be of major medical benefit. Here, a comprehensive analysis is carried out to predict the epitopes against HPV types 16 and 18 through "reverse vaccinology" approach. We attempted to identify the evolutionarily conserved regions of major capsid protein (L1) as well as minor capsid protein (L2) of HPV and designed epitopes within these regions. In this study, we analyzed about 49 and 27 sequences of HPV L2 and L1 proteins respectively. Since we found that the intertype variability of L2 is higher than for L1 proteins, our analysis was emphasized on epitopes of L1 of HPV types 16 and 18. We had selected HLA-A*0201, DRB1*1501, DQB1*0602, DRB1*0401 and DQB1*0301 alleles for the prediction of T cell epitopes of L1 of HPV 16 and 18. Finally, we reported that predicted epitope sequences EEYDLQFIFQLCKITLTA, and RHGEEYDLQFIFQLCKITLTA of L1 protein of HPV 16, and LPDPNKF, PETQRLVWAC, PVPGQYDA, YNPETQRLVWAC, DTGYGAMD, PVPGQYDATK, KQDIPKVSAYQYRVFRV, RDNVSVDYKQTQLCI and YSRHVEEYDLQFIF of L1 protein of HPV 18 could be therapeutic tools for vaccine design against HPV.

Recognition of Porphyromonas gingivalis gingipain epitopes by natural IgM binding to malondialdehyde modified low-density lipoprotein.

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S Pauliina Turunen

Full Text Available 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.

Epitope structure and binding affinity of single chain llama anti-β-amyloid antibodies revealed by proteolytic excision affinity-mass spectrometry.

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Paraschiv, Gabriela; Vincke, Cécile; Czaplewska, Paulina; Manea, Marilena; Muyldermans, Serge; Przybylski, Michael

2013-01-01

ß-Amyloid (Aß) immunotherapy has become a promising strategy for reducing the level of Aß in brain. New immunological approaches have been recently proposed for rapid, early diagnosis, and molecular treatment of neurodegenerative diseases related to Alzheimer's Disease (AD). The combination of proteolytic epitope excision and extraction and mass spectrometry using digestion with various proteases has been shown to be an efficient tool for the identification and molecular characterization of antigenic determinants. Here, we report the identification of the Aβ epitope recognized by the variable domain of single chain llama anti-Aβ-antibodies, termed Aβ-nanobodies, that have been discovered in the blood of camelids and found to be promising candidates for immunotherapy of AD. The epitope recognized by two Aβ-specific nanobodies was identified by proteolytic epitope extraction- and excision-mass spectrometry using a series of proteases (trypsin, chymotrypsin, GluC-protease, and LysC-protease). Matrix-assisted laser desorption ionization--mass spectrometric analysis of the affinity--elution fraction provided the epitope, Aβ(17-28), in the mid- to carboxy-terminal domain of Aβ, which has been shown to exert an Aß-fibril inhibiting effect. Affinity studies of the synthetic epitope confirmed that the Aβ(17-28) peptide is the minimal fragment that binds to the nanobodies. The interactions between the nanobodies and full length Aβ(1-40) or Aβ-peptides containing or lacking the epitope sequence were further characterized by enzyme linked immunosorbent assay and bioaffinity analysis. Determinations of binding affinities between the Aβ-nanobodies and Aβ(1-40) and the Aβ(17-28) epitope provided K(D) values of approximately 150 and 700 nmol, respectively. Thus, the knowledge of the epitope may be highly useful for future studies of Aβ-aggregation (oligomerization and fibril formation) and for designing new aggregation inhibitors. Copyright © 2012 John Wiley

Distribution of some pectic and arabinogalactan protein epitopes during Solanum lycopersicum (L.) adventitious root development.

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Sala, Katarzyna; Malarz, Katarzyna; Barlow, Peter W; Kurczyńska, Ewa U

2017-01-25

The adventitious roots (AR) of plants share the same function as primary and lateral roots (LR), although their development is mainly an adaptive reaction to stress conditions. Regeneration of grafted plants is often accompanied by AR formation thus making the grafting technique a good model for studying AR initiation and development and their means of emergence. Pectins and arabinogalactan proteins (AGP) are helpful markers of particular cellular events, such as programmed cell death (PCD), elongation, proliferation or other differentiation events that accompany AR development. However, little is known about the distribution of pectins and AGPs during AR ontogeny, either in the primordium or stem tissues from which AR arise or their correspondence with these events during LR formation. AR were developed from different stem tissues such as parenchyma, xylem rays and the cambium, depending on the stem age and treatment (grafting versus cutting) of the parental tissue. Immunochemical analysis of the presence of pectic (LM8, LM19, LM20) and AGP (JIM8, JIM13, JIM16) epitopes in AR and AR-associated tissues showed differential, tissue-specific distributions of these epitopes. Two pectic epitopes (LM19, LM20) were developmentally regulated and the occurrence of the LM8 xylogalacturonan epitope in the root cap of the AR differed from other species described so far. AGP epitopes were abundantly present in the cytoplasmic compartments (mainly the tonoplast) and were correlated with the degree of cell vacuolisation. JIM8 and JIM13 epitopes were detected in the more advanced stages of primordium development, whereas the JIM16 epitope was present from the earliest division events of the initial AR cells. The comparison between AR and LR showed quantitative (AGP,) and qualitative (pectins) differences. The chemical compositions of adventitious and lateral root cells show differences that correlate with the different origins of these cells. In AR, developmental changes in the

HIV-1 integrase inhibitors are substrates for the multidrug transporter MDR1-P-glycoprotein

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Cara Andrea

2007-03-01

Full Text Available Abstract Background The discovery of diketoacid-containing derivatives as inhibitors of HIV-1 Integrase (IN (IN inhibitors, IINs has played a major role in validating this enzyme as an important target for antiretroviral therapy. Since the in vivo efficacy depends on access of these drugs to intracellular sites where HIV-1 replicates, we determined whether the IINs are recognized by the multidrug transporter MDR1-P-glycoprotein (P-gp thereby reducing their intracellular accumulation. To address the effect of IINs on drug transport, nine quinolonyl diketo acid (DKA derivatives active on the HIV-1 IN strand transfer (ST step and with EC50 ranging from 1.83 to >50 μm in cell-based assays were tested for their in vitro interaction with P-gp in the CEM-MDR cell system. IINs were investigated for the inhibition and induction of the P-gp function and expression as well as for multidrug resistance (MDR reversing ability. Results The HIV-1 IINs act as genuine P-gp substrates by inhibiting doxorubicin efflux and inducing P-gp functional conformation changes as evaluated by the modulation of UIC2 mAb epitope. Further, IINs chemosensitize MDR cells to vinblastine and induce P-gp expression in drug sensitive revertants of CEM-MDR cells. Conclusion To our knowledge, this is the first demonstration that HIV-1 IINs are P-gp substrates. This biological property may influence the absorption, distribution and elimination of these novels anti HIV-1 compounds.

Tomato bushy stunt virus (TBSV), a versatile platform for polyvalent display of antigenic epitopes and vaccine design

International Nuclear Information System (INIS)

Kumar, Shantanu; Ochoa, Wendy; Singh, Pratik; Hsu, Catherine; Schneemann, Anette; Manchester, Marianne; Olson, Mark; Reddy, Vijay

2009-01-01

Viruses-like particles (VLPs) are frequently being used as platforms for polyvalent display of foreign epitopes of interest on their capsid surface to improve their presentation enhancing the antigenicity and host immune response. In the present study, we used the VLPs of Tomato bushy stunt virus (TBSV), an icosahedral plant virus, as a platform to display 180 copies of 16 amino acid epitopes of ricin toxin fused to the C-terminal end of a modified TBSV capsid protein (NΔ52). Expression of the chimeric recombinant protein in insect cells resulted in spontaneous assembly of VLPs displaying the ricin epitope. Cryo-electron microscopy and image reconstruction of the chimeric VLPs at 22 A resolution revealed the locations and orientation of the ricin epitope exposed on the TBSV capsid surface. Furthermore, injection of chimeric VLPs into mice generated antisera that detected the native ricin toxin. The ease of fusing of short peptides of 15-20 residues and their ability to form two kinds (T = 1, T = 3) of bio-nanoparticles that result in the display of 60 or 180 copies of less constrained and highly exposed antigenic epitopes makes TBSV an attractive and versatile display platform for vaccine design.

Identification of a novel canine distemper virus B-cell epitope using a monoclonal antibody against nucleocapsid protein.

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Yi, Li; Cheng, Yuening; Zhang, Miao; Cao, Zhigang; Tong, Mingwei; Wang, Jianke; Zhao, Hang; Lin, Peng; Cheng, Shipeng

2016-02-02

Canine distemper virus (CDV) is a member of the genus Morbillivirus within the family Paramyxoviridae and has caused severe economic losses in China. Nucleocapsid protein (N) is the major structural viral protein and can be used to diagnose CDV and other morbilliviruses. In this study, a specific monoclonal antibody, 1N8, was produced against the CDV N protein (amino acids 277-471). A linear N protein epitope was identified by subjecting a series of partially overlapping synthesized peptides to enzyme-linked immunosorbent assay (ELISA) analysis. The results indicated that (350)LNFGRSYFDPA(360) was the minimal linear epitope that could be recognized by mAb 1N8. ELISA assays revealed that mouse anti-CDV sera could also recognize the minimal linear epitope. Alignment analysis of the amino acid sequences indicated that the epitope was highly conserved among CDV strains. Furthermore, the epitope was conserved among other morbilliviruses, which was confirmed with PRRV using western blotting. Taken together, the results of this study may have potential applications in the development of suitable diagnostic techniques for CDV or other morbilliviruses. Copyright © 2015 Elsevier B.V. All rights reserved.

Oligomannose-Rich Membranes of Dying Intestinal Epithelial Cells Promote Host Colonization by Adherent-Invasive E. coli

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Tetiana Dumych

2018-04-01

Full Text Available A novel mechanism is revealed by which clinical isolates of adherent-invasive Escherichia coli (AIEC penetrate into the epithelial cell layer, replicate, and establish biofilms in Crohn's disease. AIEC uses the FimH fimbrial adhesin to bind to oligomannose glycans on the surface of host cells. Oligomannose glycans exposed on early apoptotic cells are the preferred binding targets of AIEC, so apoptotic cells serve as potential entry points for bacteria into the epithelial cell layer. Thereafter, the bacteria propagate laterally in the epithelial intercellular spaces. We demonstrate oligomannosylation at two distinct sites of a glycoprotein receptor for AIEC, carcinoembryonic antigen related cell adhesion molecule 6 (CEACAM6 or CD66c, on human intestinal epithelia. After bacterial binding, FimH interacts with CEACAM6, which then clusters. The presence of the highest-affinity epitope for FimH, oligomannose-5, on CEACAM6 is demonstrated using LC-MS/MS. As mannose-dependent infections are abundant, this mechanism might also be used by other adherent-invasive pathogens.

Evaluation and use of in-silico structure-based epitope prediction with foot-and-mouth disease virus.

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Daryl W Borley

Full Text Available Understanding virus antigenicity is of fundamental importance for the development of better, more cross-reactive vaccines. However, as far as we are aware, no systematic work has yet been conducted using the 3D structure of a virus to identify novel epitopes. Therefore we have extended several existing structural prediction algorithms to build a method for identifying epitopes on the appropriate outer surface of intact virus capsids (which are structurally different from globular proteins in both shape and arrangement of multiple repeated elements and applied it here as a proof of principle concept to the capsid of foot-and-mouth disease virus (FMDV. We have analysed how reliably several freely available structure-based B cell epitope prediction programs can identify already known viral epitopes of FMDV in the context of the viral capsid. To do this we constructed a simple objective metric to measure the sensitivity and discrimination of such algorithms. After optimising the parameters for five methods using an independent training set we used this measure to evaluate the methods. Individually any one algorithm performed rather poorly (three performing better than the other two suggesting that there may be value in developing virus-specific software. Taking a very conservative approach requiring a consensus between all three top methods predicts a number of previously described antigenic residues as potential epitopes on more than one serotype of FMDV, consistent with experimental results. The consensus results identified novel residues as potential epitopes on more than one serotype. These include residues 190-192 of VP2 (not previously determined to be antigenic, residues 69-71 and 193-197 of VP3 spanning the pentamer-pentamer interface, and another region incorporating residues 83, 84 and 169-174 of VP1 (all only previously experimentally defined on serotype A. The computer programs needed to create a semi-automated procedure for carrying out

The Occurrence of Sequences Identical with Epitopes from the Allergen Pen a 1.0102 Among Food and Non-Food Proteins

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Minkiewicz Piotr

2015-03-01

Full Text Available The presence of common epitopes among tropomyosins of invertebrates, including arthropods, e.g. edible ones, may help to explain the molecular basis of cross-reactivity between allergens. The work presented is the first survey concerning global distribution of epitopes from Pen a 1.0102 in universal proteome. In the group of known tropomyosin epitopes, the fragment with the sequence ESKIVELEEEL was found in the sequence of channel catfish (Ictalurus punctatus tropomyosin. To date, this is the first result suggesting the presence of a complete sequential epitope interacting with gE in vertebrate tropomyosin. Another fragment with the sequence VAALNRRIQL, a major part of the epitope, was found in 11 fish, 8 amphibians, 3 birds, 19 mammalians and 4 human tropomyosin sequences. Identical epitopes are common in sequences of invertebrate tropomyosins, including food and non-food allergens annotated in the Allergome database. The rare pentapeptide with the DEERM sequence occurs in proteins not sharing homology with tropomyosins. Pathogenic microorganisms are the most abundant category of organisms synthesizing such proteins.

Indirect recognition of HLA epitopes in solid organ transplantation

NARCIS (Netherlands)

Geneugelijk, C.C.A.

2017-01-01

Alloreactivity due to HLA mismatches between donor and recipient remains the major limiting factor in successful graft outcome after solid organ transplantation. However, the immunogenicity of individual HLA mismatches is highly variable. Therefore, epitope-based HLA matching may be a sophisticated

A Novel Method for Detection of Glycoproteins on Sodium Dodecyl Sulphate Polyacrylamide Gel Using Radio-Iodinated Tyrosine

DEFF Research Database (Denmark)

Nalla, Amarnadh; Draz, Hossam M.; Dole, Anita

2009-01-01

The aim of this study is to develop a novel method for detection of glycoproteins on polyacrylamide gel. In this method, radio-iodinated-tyrosine (125I-tyrosine) was conjugated to glycoprotein by schiff's base mechanism on the sodium dodecyl sulfate- polyacrylamide gel. Ovalbumin and Concanavalin...... of glycoproteins using 125I-tyrosine selectively detected ovalbumin. Present results showed that MPD enhanced glycoprotein detection method can be used as a sensitive tool for the detection of glycoproteins on polyacrylamide gel...

Spatial Localization of the Ebola Virus Glycoprotein Mucin-Like Domain Determined by Cryo-Electron Tomography

OpenAIRE

Tran, Erin E. H.; Simmons, James A.; Bartesaghi, Alberto; Shoemaker, Charles J.; Nelson, Elizabeth; White, Judith M.; Subramaniam, Sriram

2014-01-01

The Ebola virus glycoprotein mucin-like domain (MLD) is implicated in Ebola virus cell entry and immune evasion. Using cryo-electron tomography of Ebola virus-like particles, we determined a three-dimensional structure for the full-length glycoprotein in a near-native state and compared it to that of a glycoprotein lacking the MLD. Our results, which show that the MLD is located at the apex and the sides of each glycoprotein monomer, provide a structural template for analysis of MLD function.

P-glycoprotein targeted nanoscale drug carriers

KAUST Repository

Li, Wengang; Abu Samra, Dina Bashir Kamil; Merzaban, Jasmeen; Khashab, Niveen M.

2013-01-01

Multi-drug resistance (MDR) is a trend whereby tumor cells exposed to one cytotoxic agent develop cross-resistance to a range of structurally and functionally unrelated compounds. P -glycoprotein (P -gp) efflux pump is one of the mostly studied drug

Endoplasmic reticulum targeting sequence enhances HBV-specific cytotoxic T lymphocytes induced by a CTL epitope-based DNA vaccine

International Nuclear Information System (INIS)

Xu Wei; Chu Yiwei; Zhang Ruihua; Xu Huanbin; Wang Ying; Xiong Sidong

2005-01-01

CD8 + T cells play a critical role in protective immunity against Hepatitis B Virus (HBV). Epitope-based DNA vaccines expressing HBV-dominant CTL epitopes can be used as candidate vaccines capable of inducing cytotoxic T Lymphocytes (CTL) responses. A plasmid DNA encoding a CTL epitope of HBV core antigen, HBc 18-27 , was constructed. Intramuscular immunization of C57BL/6 mice with this DNA vaccine resulted in successful induction of HBV-specific CTL responses. In order to promote transportation of the peptide into endoplasmic reticulum (ER) to bind to MHC class I molecules for optimal class I antigen presentation, an ER targeting sequence (ERTS) was fused with the C 18-27 encoding gene. ERTS fusion significantly enhanced specific CD8 + T cell responses in terms of CTL cytolysis as well as IFN-γ secretion. This enhancement was correlated with promoted epitope presentation on target cell surface. We report here an enhanced immunogenicity of an epitope-based DNA vaccine using an ER targeting signal sequence, which has significant implications for future design of therapeutic HBV vaccine

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

OpenAIRE

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

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. T...

76 FR 51374 - Direct Discovery of HLA Associated Influenza Epitopes Isolated From Human Cells for Vaccine and...

Science.gov (United States)

2011-08-18

... direct-discovery technology for use in FDA laboratories. C. Eligibility Information The technology...] Direct Discovery of HLA Associated Influenza Epitopes Isolated From Human Cells for Vaccine and... technology to molecularly characterize peptide epitopes that are processed and presented on soluble HLA...

Prediction of CD8+ Epitopes in Leishmania braziliensis Proteins Using EPIBOT: In Silico Search and In Vivo Validation.

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Angelo Duarte

Full Text Available Leishmaniasis is caused by intracellular Leishmania parasites that induce a T-cell mediated response associated with recognition of CD4+ and CD8+ T cell Line 1Lineepitopes. Identification of CD8+ antigenic determinants is crucial for vaccine and therapy development. Herein, we developed an open-source software dedicated to search and compile data obtained from currently available on line prediction algorithms.We developed a two-phase algorithm and implemented in an open source software called EPIBOT, that consolidates the results obtained with single prediction algorithms, generating a final output in which epitopes are ranked. EPIBOT was initially trained using a set of 831 known epitopes from 397 proteins from IEDB. We then screened 63 Leishmania braziliensis vaccine candidates with the EPIBOT trained tool to search for CD8+ T cell epitopes. A proof-of-concept experiment was conducted with the top eight CD8+ epitopes, elected by EPIBOT. To do this, the elected peptides were synthesized and validated for their in vivo cytotoxicity. Among the tested epitopes, three were able to induce lysis of pulsed-target cells.Our results show that EPIBOT can successfully search across existing prediction tools, generating a compiled list of candidate CD8+ epitopes. This software is fast and a simple search engine that can be customized to search over different MHC alleles or HLA haplotypes.

Pep-3D-Search: a method for B-cell epitope prediction based on mimotope analysis.

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Huang, Yan Xin; Bao, Yong Li; Guo, Shu Yan; Wang, Yan; Zhou, Chun Guang; Li, Yu Xin

2008-12-16

The prediction of conformational B-cell epitopes is one of the most important goals in immunoinformatics. The solution to this problem, even if approximate, would help in designing experiments to precisely map the residues of interaction between an antigen and an antibody. Consequently, this area of research has received considerable attention from immunologists, structural biologists and computational biologists. Phage-displayed random peptide libraries are powerful tools used to obtain mimotopes that are selected by binding to a given monoclonal antibody (mAb) in a similar way to the native epitope. These mimotopes can be considered as functional epitope mimics. Mimotope analysis based methods can predict not only linear but also conformational epitopes and this has been the focus of much research in recent years. Though some algorithms based on mimotope analysis have been proposed, the precise localization of the interaction site mimicked by the mimotopes is still a challenging task. In this study, we propose a method for B-cell epitope prediction based on mimotope analysis called Pep-3D-Search. Given the 3D structure of an antigen and a set of mimotopes (or a motif sequence derived from the set of mimotopes), Pep-3D-Search can be used in two modes: mimotope or motif. To evaluate the performance of Pep-3D-Search to predict epitopes from a set of mimotopes, 10 epitopes defined by crystallography were compared with the predicted results from a Pep-3D-Search: the average Matthews correlation coefficient (MCC), sensitivity and precision were 0.1758, 0.3642 and 0.6948. Compared with other available prediction algorithms, Pep-3D-Search showed comparable MCC, specificity and precision, and could provide novel, rational results. To verify the capability of Pep-3D-Search to align a motif sequence to a 3D structure for predicting epitopes, 6 test cases were used. The predictive performance of Pep-3D-Search was demonstrated to be superior to that of other similar programs

Cysteine-rich secretory protein 3 is a ligand of alpha1B-glycoprotein in human plasma

DEFF Research Database (Denmark)

Udby, Lene; Sørensen, Ole E; Pass, Jesper

2004-01-01

Human cysteine-rich secretory protein 3 (CRISP-3; also known as SGP28) belongs to a family of closely related proteins found in mammals and reptiles. Some mammalian CRISPs are known to be involved in the process of reproduction, whereas some of the CRISPs from reptiles are neurotoxin...

Epitope analysis of anti-myeloperoxidase antibodies in patients with ANCA-associated vasculitis.

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Shen-Ju Gou

Full Text Available OBJECTIVE: Increasing evidences have suggested the pathogenic role of anti-neutrophil cytoplasmic antibodies (ANCA directing myeloperoxidase (MPO in ANCA-associated vasculitis (AAV. The current study aimed to analyze the association between the linear epitopes of MPO-ANCA and clinicopathological features of patients with AAV. METHODS: Six recombinant linear fragments, covering the whole length amino acid sequence of a single chain of MPO, were produced from E.coli. Sera from 77 patients with AAV were collected at presentation. 13 out of the 77 patients had co-existence of serum anti-GBM antibodies. Ten patients also had sequential sera during follow up. The epitope specificities were detected by enzyme-linked immunosorbent assay using the recombinant fragments as solid phase ligands. RESULTS: Sera from 45 of the 77 (58.4% patients with AAV showed a positive reaction to one or more linear fragments of the MPO chain. The Birmingham Vasculitis Activity Scores and the sera creatinine were significantly higher in patients with positive binding to the light chain fragment than that in patients without the binding. The epitopes recognized by MPO-ANCA from patients with co-existence of serum anti-GBM antibodies were mainly located in the N-terminus of the heavy chain. In 5 out of the 6 patients, whose sera in relapse recognize linear fragments, the reactivity to linear fragments in relapse was similar to that of initial onset. CONCLUSION: The epitope specificities of MPO-ANCA were associated with disease activity and some clinicopathological features in patients with ANCA-associated vasculitis.

Homology building as a means to define antigenic epitopes on dihydrofolate reductase (DHFR) from Plasmodium falciparum

DEFF Research Database (Denmark)

Alifrangis, Michael; Christensen, Inge T; Jørgensen, Flemming S

2004-01-01

in the gene coding for Pf-DHFR. Furthermore, we wanted to study the potential use of homology models in general and of Pf-DHFR in particular in predicting antigenic malarial surface epitopes. METHODS: A homology model of Pf-DHFR domain was employed to define an epitope for the development of site...

Glycoproteins of axonal transport: affinity chromatography on fucose-specific lectins

Energy Technology Data Exchange (ETDEWEB)

Gustavsson, S.; Ohlson, C.; Karlsson, J.O.

1982-03-01

Rapidly transported fucose-labeled glycoproteins from axons of rabbit retinal ganglion cells were solubilized with nonionic detergents. The solubilized components were subjected to affinity chromatography on three different fucose-specific lectins. A recently characterized fucose-specific lectin from Aleuria aurantia bound reversibly approximately 60% of the applied protein-bound radioactivity. The lectins from Lotus tetragonolobus and Ulex europaeus bound are very small proportions of the labeled rapidly transported glycoproteins.

Fine-mapping of immunodominant linear B-cell epitopes of the Staphylococcus aureus SEB antigen using short overlapping peptides.

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Zhuo Zhao

Full Text Available Staphylococcal enterotoxin B (SEB is one of the most potent Staphylococcus aureus exotoxins (SEs. Due to its conserved sequence and stable structure, SEB might be a good candidate antigen for MRSA vaccines. Although cellular immune responses to SEB are well-characterized, much less is known regarding SEB-specific humoral immune responses, particularly regarding detailed epitope mapping. In this study, we utilized a recombinant nontoxic mutant of SEB (rSEB and an AlPO4 adjuvant to immunize BALB/c mice and confirmed that rSEB can induce a high antibody level and effective immune protection against MRSA infection. Next, the antisera of immunized mice were collected, and linear B cell epitopes within SEB were finely mapped using a series of overlapping synthetic peptides. Three immunodominant B cell epitopes of SEB were screened by ELISA, including a novel epitope, SEB205-222, and two known epitopes, SEB97-114 and SEB247-261. Using truncated peptides, an ELISA was performed with peptide-KLH antisera, and the core sequence of the three immunodominant B cell epitopes were verified as SEB97-112, SEB207-222, and SEB247-257. In vitro, all of the immunodominant epitope-specific antisera (anti-SEB97-112, anti-SEB207-222 and anti-SEB247-257 were observed to inhibit SEB-induced T cell mitogenesis and cytokine production from splenic lymphocytes of BALB/c mice. The homology analysis indicated that SEB97-112 and SEB207-222 were well-conserved among different Staphylococcus aureus strains. The 3D crystal structure of SEB indicated that SEB97-112 was in the loop region inside SEB, whereas SEB207-222 and SEB247-257 were in the β-slice region outside SEB. In summary, the fine-mapping of linear B-cell epitopes of the SEB antigen in this study will be useful to understand anti-SEB immunity against MRSA infection further and will be helpful to optimize MRSA vaccine designs that are based on the SEB antigen.

In Silico Prediction of T and B Cell Epitopes of Der f 25 in Dermatophagoides farinae

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Xiaohong Li

2014-01-01

Full Text Available The house dust mites are major sources of indoor allergens for humans, which induce asthma, rhinitis, dermatitis, and other allergic diseases. Der f 25 is a triosephosphate isomerase, representing the major allergen identified in Dermatophagoides farinae. The objective of this study was to predict the B and T cell epitopes of Der f 25. In the present study, we analyzed the physiochemical properties, function motifs and domains, and structural-based detailed features of Der f 25 and predicted the B cell linear epitopes of Der f 25 by DNAStar protean system, BPAP, and BepiPred 1.0 server and the T cell epitopes by NetMHCIIpan-3.0 and NetMHCII-2.2. As a result, the sequence and structure analysis identified that Der f 25 belongs to the triosephosphate isomerase family and exhibited a triosephosphate isomerase pattern (PS001371. Eight B cell epitopes (11–18, 30–35, 71–77, 99–107, 132–138, 173–187, 193–197, and 211–224 and five T cell epitopes including 26–34, 38–54, 66–74, 142–151, and 239–247 were predicted in this study. These results can be used to benefit allergen immunotherapies and reduce the frequency of mite allergic reactions.

Affinity study on bovine serum albumin's peptides to amphiphilic gold nanoparticles: A test of epitopes and non-epitopes

Science.gov (United States)

Yuan, Ming; Li, Wanrong; Yang, Mingming; Huang, Xiufeng; Bai, Zhijun; Liu, Yushuang; Cai, Weijun; Wang, Yuqin; Zhang, Feng

2017-09-01

It is an inevitable event that nanoparticles (NPs) will encounter proteins/peptides in nano-medicine, so it has been significant to know their interaction mechanism before in vivo applications. Previously, a 105-amino-acid sequence had been reported as the binding site between bovine serum albumin (BSA) and amphiphilic polymer coated gold nanoparticles (AP-AuNPs) along with a mortise-tenon joint hypothesis. This article tested the affinity difference between two epitope peptide sequences such as: LGEYGFQNALIVR (S1), DAFLGSFLYEYSR (S2) and one non-epitope peptide sequence as: FDEHVKLVNELTEF (S3). With the photoluminescent amino acid residues, the fluorescence quenching method based on the nanometal surface energy transfer (NSET) principle was able to study the thermodynamics of the current binding system. The binding constants (Ka) were determined and followed the order as: Ka-S1 > Ka-S2 >> Ka-S3. Moreover, Hill constants indicated that cooperativity only presented in the interactions of AP-AuNP with either S1 or S2, but not for S3. Moreover, gel electrophoresis, surface plasmon resonance, atomic force microscopy and three dimensional fluorescence microscopy were all also used to comprehensively analyse the binding interaction mechanism. These results further provided useful information to better understand the mortise-tenon joint, which might find applications to nanofabrication and biomedicine.

Development of a radioimmunoassay for 'Tamm-Horsfall-like' glycoprotein in serum and cerebrospinal fluid

International Nuclear Information System (INIS)

Hartmann, L.; Bringuier, A.-F.; Schuller, E.

1983-01-01

Affinity chromatography purification was combined with a radioimmunoassay for 'Tamm-Horsfall-like' glycoprotein. This enabled serum comcentrations to be established and to demonstrate its presence in cerebrospinal fluid for the first time. This assay method used in different circumstances suggests a multifocal synthesis. Nevertheless, urinary Tamm-Horsfall glycoprotein so far must be distinguished from the serum or cerebrospinal fluid Tamm-Horsfall-like glycoprotein. (Auth.)

IMMUNOCAT—A Data Management System for Epitope Mapping Studies

Directory of Open Access Journals (Sweden)

Jo L. Chung

2010-01-01

Full Text Available To enable rationale vaccine design, studies of molecular and cellular mechanisms of immune recognition need to be linked with clinical studies in humans. A major challenge in conducting such translational research studies lies in the management and integration of large amounts and various types of data collected from multiple sources. For this purpose, we have established “IMMUNOCAT”, an interactive data management system for the epitope discovery research projects conducted by our group. The system provides functions to store, query, and analyze clinical and experimental data, enabling efficient, systematic, and integrative data management. We demonstrate how IMMUNOCAT is utilized in a large-scale research contract that aims to identify epitopes in common allergens recognized by T cells from human donors, in order to facilitate the rational design of allergy vaccines. At clinical sites, demographic information and disease history of each enrolled donor are captured, followed by results of an allergen skin test and blood draw. At the laboratory site, T cells derived from blood samples are tested for reactivity against a panel of peptides derived from common human allergens. IMMUNOCAT stores results from these T cell assays along with MHC:peptide binding data, results from RAST tests for antibody titers in donor serum, and the respective donor HLA typing results. Through this system, we are able to perform queries and integrated analyses of the various types of data. This provides a case study for the use of bioinformatics and information management techniques to track and analyze data produced in a translational research study aimed at epitope identification.

IMMUNOCAT-a data management system for epitope mapping studies.

Science.gov (United States)

Chung, Jo L; Sun, Jian; Sidney, John; Sette, Alessandro; Peters, Bjoern

2010-01-01

To enable rationale vaccine design, studies of molecular and cellular mechanisms of immune recognition need to be linked with clinical studies in humans. A major challenge in conducting such translational research studies lies in the management and integration of large amounts and various types of data collected from multiple sources. For this purpose, we have established "IMMUNOCAT", an interactive data management system for the epitope discovery research projects conducted by our group. The system provides functions to store, query, and analyze clinical and experimental data, enabling efficient, systematic, and integrative data management. We demonstrate how IMMUNOCAT is utilized in a large-scale research contract that aims to identify epitopes in common allergens recognized by T cells from human donors, in order to facilitate the rational design of allergy vaccines. At clinical sites, demographic information and disease history of each enrolled donor are captured, followed by results of an allergen skin test and blood draw. At the laboratory site, T cells derived from blood samples are tested for reactivity against a panel of peptides derived from common human allergens. IMMUNOCAT stores results from these T cell assays along with MHC:peptide binding data, results from RAST tests for antibody titers in donor serum, and the respective donor HLA typing results. Through this system, we are able to perform queries and integrated analyses of the various types of data. This provides a case study for the use of bioinformatics and information management techniques to track and analyze data produced in a translational research study aimed at epitope identification.

Spatial localization of the Ebola virus glycoprotein mucin-like domain determined by cryo-electron tomography.

Science.gov (United States)

Tran, Erin E H; Simmons, James A; Bartesaghi, Alberto; Shoemaker, Charles J; Nelson, Elizabeth; White, Judith M; Subramaniam, Sriram

2014-09-01

The Ebola virus glycoprotein mucin-like domain (MLD) is implicated in Ebola virus cell entry and immune evasion. Using cryo-electron tomography of Ebola virus-like particles, we determined a three-dimensional structure for the full-length glycoprotein in a near-native state and compared it to that of a glycoprotein lacking the MLD. Our results, which show that the MLD is located at the apex and the sides of each glycoprotein monomer, provide a structural template for analysis of MLD function. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Herpes simplex virus glycoprotein D relocates nectin-1 from intercellular contacts.

Science.gov (United States)

Bhargava, Arjun K; Rothlauf, Paul W; Krummenacher, Claude

2016-12-01

Herpes simplex virus (HSV) uses the cell adhesion molecule nectin-1 as a receptor to enter neurons and epithelial cells. The viral glycoprotein D (gD) is used as a non-canonical ligand for nectin-1. The gD binding site on nectin-1 overlaps with a functional adhesive site involved in nectin-nectin homophilic trans-interaction. Consequently, when nectin-1 is engaged with a cellular ligand at cell junctions, the gD binding site is occupied. Here we report that HSV gD is able to disrupt intercellular homophilic trans-interaction of nectin-1 and induce a rapid redistribution of nectin-1 from cell junctions. This movement does not require the receptor's interaction with the actin-binding adaptor afadin. Interaction of nectin-1 with afadin is also dispensable for virion surfing along nectin-1-rich filopodia. Cells seeded on gD-coated surfaces also fail to accumulate nectin-1 at cell contact. These data indicate that HSV gD affects nectin-1 locally through direct interaction and more globally through signaling. Copyright © 2016 Elsevier Inc. All rights reserved.

Use of two predictive algorithms of the world wide web for the identification of tumor-reactive T-cell epitopes.

Science.gov (United States)

Lu, J; Celis, E

2000-09-15

Tumor cells can be effectively recognized and eliminated by CTLs. One approach for the development of CTL-based cancer immunotherapy for solid tumors requires the use of the appropriate immunogenic peptide epitopes that are derived from defined tumor-associated antigens. Because CTL peptide epitopes are restricted to specific MHC alleles, to design immune therapies for the general population it is necessary to identify epitopes for the most commonly found human MHC alleles. The identification of such epitopes has been based on MHC-peptide-binding assays that are costly and labor-intensive. We report here the use of two computer-based prediction algorithms, which are readily available in the public domain (Internet), to identify HL4-B7-restricted CTL epitopes for carcinoembryonic antigen (CEA). These algorithms identified three candidate peptides that we studied for their capacity to induce CTL responses in vitro using lymphocytes from HLA-B7+ normal blood donors. The results show that one of these peptides, CEA9(632) (IPQQHTQVL) was efficient in the induction of primary CTL responses when dendritic cells were used as antigen-presenting cells. These CTLs were efficient in killing tumor cells that express HLA-B7 and produce CEA. The identification of this HLA-B7-restricted CTL epitope will be useful for the design of ethnically unbiased, widely applicable immunotherapies for common solid epithelial tumors expressing CEA. Moreover, our strategy of identifying MHC class I-restricted CTL epitopes without the need of peptide/HLA-binding assays provides a convenient and cost-saving alternative approach to previous methods.